Radiobiology Textbook [1st ed. 2023] 3031188098, 9783031188091

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Sarah Baatout Editor

Radiobiology Textbook

123

Radiobiology Textbook

Sarah Baatout Editor

Radiobiology Textbook

Editor Sarah Baatout Institute of Nuclear Medical Applications Belgian Nuclear Research Centre (SCK CEN) Mol, Belgium

ISBN 978-3-031-18809-1    ISBN 978-3-031-18810-7 (eBook) https://doi.org/10.1007/978-3-031-18810-7 Belgian Nuclear Research Centre (SCK CEN) © The Editor(s) (if applicable) and The Author(s) 2023. This book is an open access publication. Open Access  This book is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this book are included in the book's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the book's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Foreword

I feel immensely delighted and consider it an honour to write the foreword for the Radiobiology Textbook edited by Prof Sarah Baatout. I went through the textbook with utter curiosity and found it irresistible to stop reading from beginning to end. Indeed, the book will prove a boon and treasure of knowledge for radiobiology researchers, physicians, clinicians, environmentalists, nuclear workers, industry professionals/managers, and radiation technology developers.

New Discoveries and Early Excitements With the discoveries of X-ray in 1895 and radioactivity in 1898, unusual excitement was witnessed among scientists and researchers all over the world. It was commonly perceived that a new revolution had arrived in science, which might prove a panacea for every enigma. Besides researchers and academicians, the general public was highly enthusiastic and saw the emergence of new discoveries as an auspicious signal to humankind. Interestingly, physicians were quick to show the courage and enthusiasm to apply newly discovered radiation for treating cancer. That was a great medical challenge at that point in time. It was remarkable learning that X-radiation could kill living cells, including cancer cells, and had the potential to provide marked relief to cancer patients. In fact, X-radiation and radiation emitted from radioactive materials like radium became a public curiosity and an object of fun for those who wanted to have new experiences such as visualizing bones in the body and using radium lipsticks. In early years, both the scientists and common people were unaware and unmindful of the harmful effects of radiation. However, over a short span of time, it became known that radiation researchers suffered from harmful effects of radiation such as induction of cancer.

Radiobiology Was Born It soon became apparent that understanding the mechanisms of biological effects of ionizing radiation like X-ray and gamma ray was important, and the field of radiobiology was born. Scientists also realized that setting safety standards for radiation was most urgent. Since radiation cannot be seen, tasted, or smelled, scientists began studying the interaction of radiation with matter, including radiation effects on living systems. Early studies showed that radiation could kill living cells, including tumour cells. How radiation kills living cells became the main focus of radiobiological researchers. Those who engaged themselves in radiobiology research came from diverse backgrounds, such as physics, chemistry, and biological sciences (life science, zoology, microbiology, etc.). Researchers from specific disciplines started intense investigations on physical effects (radiation physics), chemical effects (radiation chemistry), and biological effects (radiobiology) of radiation. One of the most significant contributions of radiobiological research was the discovery of the oxygen effect, which emphasized free radical production mechanisms in the radiation action on biological and chemical systems. Experiments on cellular colony formation showed that, in the presence of oxygen, more cell death occurred for the same irradiation dose. v

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Further radiobiological studies then laid the foundations for setting the safety standards and regulations for radiation exposure. The international radiation research community established organizations of experts, and the International Commission on Radiation Protection (ICRP) was formed in 1928 to provide recommendations and guidance about protecting humans against the risks of ionizing radiation. The United Nations Scientific Committee on Effects of Atomic radiation (UNSCEAR) was then formed in 1955 to determine the level and effects of ionizing radiation from atomic bombs and nuclear accident exposures. The same year, the US Academy of Sciences Committee on Biological Effects of Ionizing Radiation (BEIR) was established to determine and guide risks of radiation exposure on living organisms. The International Atomic Energy Agency (IAEA), created in 1957, aimed to guide and advise on safe radiation dose regulations for workers and the public. With the intensive use of X-rays and gamma rays in medical practice, radiation has now become a household word amongst the public. In fact, medical science has acquired an extraordinary capability to diagnose and treat human diseases by radiation-based devices and protocols. Against this backdrop, the need for a comprehensive textbook was made clear by researchers and clinicians. This Radiobiology Textbook is designed to meet the demands of radiation and medical professionals, provides a thorough description of radiobiology, and stimulates young talents to engage in research and accept the challenges of advancing knowledge to serve humankind. More radiobiological research is needed to answer and explain several controversial issues, such as the dose-response curve, the observed differences in individual radiosensitivity, the radiation resistance of cancer cells, and many other questions. The radiation effect on somatic cells can be immediate or delayed, but radiation genetic effects are displayed only years and centuries later, something that needs to be further investigated in the future.

How Radiation Kills Living Cells/Tissues Fundamentally, radiobiologists aim to understand the effects of radiation on cells, tissues, organs, and organisms, for animals, plants, microbial systems, and eventually humans. In this context, the discovery of DNA structure, as double-stranded helix with nucleotides as the basic units by Watson and Crick in the 1950s, propelled radiobiological studies on the mechanism of radiation-induced cell death. Radiological studies showed that radiation can kill exposed cells by damaging the DNA in the nucleus, which if not repaired prove fatal for cells. Since tumour cells divide faster than normal cells, it was hypothesized that radiation could kill these cells more efficiently. However, due to hypoxia in the tumour core, tumour cells showed resistance to radiation, leading to disappointment amongst radiation therapists. Therefore, research was undertaken to develop sensitizers of tumour cells to radiation, oxygen being the best radiosensitizer. These developments in radiobiological concepts and understanding of radiation cell killing mechanisms sustained the active research excitement in radiobiology. The medical field witnessed revolutions in caring for and treating cancer patients by using newer radiation technologies. Today, more than 40% of cancer patients are treated by radiation for therapeutic and palliative procedures. The technology consists of carefully targeting radiation beams and certain radiopharmaceuticals to destroy cancer cells while minimizing the damage to nearby healthy cells. Radiobiological studies in the 1920s helped design patient treatment protocols in what is popularly called fractionated radiotherapy.

Limitations in Radiotherapy Radiation acts equally on normal and tumour cells. Therefore, radiation therapy of cancer patients is limited by any toxicity towards normal cells. The next goal of radiobiology was to inflict selective damage on a tumour whilst sparing normal cells. Based on radiobiological

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effectiveness of different cell types to the same dose of radiation, particle radiation therapy and ion beam therapies were being developed to improve the radiotherapy for patients. In addition, the rapidly increasing applications of radiation in research, industry, medicine, biotechnology, and the environment required more intensified radiobiological studies. Today, the public’s major radiation exposures arise from medical applications such as diagnostic X-rays and CT scans to diagnose diseases, and cancer radiotherapy, including treatment of injuries. Therapeutic drugs with radioactive material attached, known as radiopharmaceuticals, are also routinely used in clinics to diagnose and treat some diseases. These procedures are a valuable tool to help doctors save lives through quick and accurate diagnosis.

The Book Contents This Radiobiology Textbook is a comprehensive, advanced, and up-to-date volume, carefully designed and meticulously compiled by experts and practicing radiobiologists in the field drawn from reputed universities and institutes across the world. Both the experts and contributors to each chapter have remained focused to create an outstanding book useful to young radiation researchers, mid-careerists, accomplished scientists and radiation researchers in biology, biotechnology, medicine, environment, industry, and workers in nuclear power plants. There are 12 chapters written by international specialists, followed by a thorough review from experts in the respective fields. A notable and marked feature of the book is the coverage of a wide range of relevant radiobiology topics and applications. To make learning easy and enjoyable, and to enable the basic principles and core concepts to be grasped, each chapter has been designed to provide rich and up-to-date contents together with the learning objectives, chapter summary, a few exercises, key references, and suggested future readings. It is hoped that learners find the book smooth reading and a gradual building of their knowledge repository, stimulating curiosity for a deeper insight to the subject. The book begins with a brief account of the history of radiobiology, followed by the chapter on basic concepts in radiation biology, which covers basic mechanisms of radiation damage to cellular molecules, direct and indirect effects, and low-dose radiation effects with relevance to health and environment. Chapter 3 on the molecular radiation biology describes molecular details of radiation-induced lesions in DNA, types of DNA damage and mechanisms of DNA damage repair, mis-repair, and consequences to the life of cells. The following chapter on mechanistic, modelling, and dosimetry radiation biology covers the basic principles of radiation dosimetry, micro-dosimetry, dose-response and related issues. The chapter on clinical radiation biology for clinical oncology makes it attractive reading for radiation therapists and nuclear medicine physicians but will also hopefully stimulate interest of basic researchers as well as tumour therapy professionals. The objective of treating cancer patients effectively by radiation involves understanding the radiation damage mechanisms of tumour and normal tissues and the prediction of radiation response. Going over the contents of Chap. 6 provides the required specialized knowledge on clinical radiation oncology modalities such as external and internal (brachytherapy) radiation treatments, high LET therapy, and rationale of dose fractionation. Chapter 7 describes individual radiosensitivity and biomarkers for disease and treatment outcomes in therapies. Radiobiology has a crucial role in situations of nuclear plant accidents and mass exposures expected from terrorist groups. The chapter on radiobiology of accidental, public, and occupational exposures deals with the radiation accident scenario, radiation health effects, radiation risks and bio-dosimetry aspects to provide safety to workers and general public. The chapter on environmental radiobiology is most timely and relevant, describing the mobility and distribution of radionuclides in water, air, and soil with the safety and environmental perspectives. Studies on radiation effects on non-human organisms such as plants and microbial systems to measure, assess, and monitor the impacts of radiation exposures are equally important. A most fascinating chapter in this book describes various aspects of space radiobiology, which is a futuristic and young branch of radiobiology to which bright curious minds are expected to be attracted and to engage in

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radiobiological research. The last but one chapter concerns radioprotectors, radiomitigators, and radiosensitization, which are topics of practical importance to ensure human and environmental safety and strategies for protection. The last chapter covers ethical, legal, and social issues of radiation exposure, which re-defines the values of ethics in the radiation research field and addresses legal and social aspects of professional and public concern. The much-publicized negative aspects of radiation technology (radiophobia) are misconceived perceptions that need to be corrected by considering the diagnostic and therapeutic power and future promise of radiobiological research and applications. Without doubt, both radiation research professionals and curiosity-driven general readers will find the book stimulating, interesting, and informative.

Perspectives and Future Scope in Radiobiology I must re-emphasize that with the ever-increasing applications of radiation technology in health and society, environment, industry, space research, and nuclear power, the radiobiology textbook of this high quality and with the coverage of frontline topics in the field is invaluable and highly warranted. The wide range of topics covered in this book with updated knowledge will prove a boon to researchers, policy makers, academicians, clinicians, and industry professionals. It is hoped that the book will arouse renewed interest among young students and will prove useful to beginners as well as senior researchers in radiobiological research and applications. More importantly, the book will prove a good reference and will help catapult future advances in radiation science and technology especially in the understanding of biological effects of radiation on living cells, tissues, and organs relevant to human health. Kaushala Prasad Mishra Radiation Biology and Health Sciences Division Bhabha Atomic Research Center Mumbai, India Nehru Gram Bharti University Prayagraj, India Asian Association for Radiation Research Mumbai, India

Preface

Welcome to the Radiobiology Textbook, which was built upon the expertise of 126 international specialists, at the forefront of various aspects of radiobiology, to bring the reader the latest and most comprehensive update in the field. Radiobiology is the branch of biology concerned with the effect of ionizing radiation on organisms. It is also a field of clinical and basic medical science that involves the study of the health effects of radiation, and the application of biology in radiological techniques and procedures for treatment and diagnostics. Multidisciplinary radiobiological research forms the scientific basis of various disciplines such as radiation protection, radiotherapy, and nuclear medicine. The goal of radiobiological research is to understand better the effects of radiation exposure at the cellular and molecular levels in order to determine the effects on health. Therefore, radiobiology encompasses various disciplines including biology, clinical applications, pharmacy, environmental and space life sciences, which make radiobiology overall a broad and rather complex topic. Throughout this textbook, we tried to organize the information from the multifaceted fields of radiobiology to enable the reader to see the Big Picture. To accomplish this synthesis of the information, unifying themes were necessary. These themes are represented by the various chapters. This textbook aims to provide a solid foundation to those interested in the basics and practice of radiobiology science, and its relevance to clinical applications, environmental radiation research, and space research. It is intended to be a learning resource to meet the needs of students, researchers or any citizen, with an interest in this rapidly evolving discipline who is eager to learn more about radiobiology, but it is also a teaching tool with accompanying teaching materials to help educators. This book offers a unique perspective to students and professionals, covering not only radiation biology but also radiation physics, radiation oncology, radiotherapy, radiochemistry, radiopharmacy, nuclear medicine, space radiation biology and physics, environmental radiation protection, nuclear emergency planning, radiation protection, molecular biology, bioinformatics, and DNA repair.

The Contributors The world is a better place thanks to those people who want to help others. What makes it even better are the people who share their expertise to mentor and educate future professionals. We have invited some of the leading writers and thinkers in the field of radiobiology to provide, in this textbook, an overview of the major considerations associated with the topic of radiobiology. This textbook is an international endeavour, which started during the worldwide COVID pandemic and gathered 126 experts from all over the world. It includes leading radiation biologists, physicists, and clinicians from all over the world. Many contributors to this textbook regularly teach this material at both national and international levels and have many years’ experience of explaining, elaborating, and clarifying complex theoretical and practical concepts in their particular field of radiobiology. Each contributor has a unique expertise and set of competences related to radiobiology, always with a critical and open mind. Where needed, ix

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they did not hesitate to address the challenges, the pitfalls, the limitations, and the beauty of the various aspects of radiobiology.

Various Chapters and Themes The textbook is organized into chapters that can be used to support student/reader preparation in any type of educational arrangement. The chapters are intended to be complete in themselves and as such can be read independently and out of sequence. This resource is intended to provide readers with a high-level view of the most relevant topics related to radiobiology. It is also intended to include all content a learner would need about a particular subject area within radiobiology. Furthermore, this textbook combines the best attributes of many different educational formats into one single resource that best supports the learning environment of the reader interested in the subject of radiobiology. The textbook intends to cover all sub-disciplines involved in radiobiology. With its 12 chapters, it provides a comprehensive review of the history of radiation biology, the development of therapeutic evidence, and the basic concepts, an understanding of the molecular mechanisms induced by radiation as well as clinical, environmental, and space radiobiology. It deepens our knowledge of individual radiation sensitivity and biomarkers and gives a complete update on the use of potential radioprotectors, radiomitigators, and radiosensitizers. Finally, it discusses the legal, epistemological, ethical, and social concerns regarding radiation exposure. A brief description of each chapter is given below: Chapter 1, entitled “History of Radiation Biology”, describes the discovery of X-rays in 1895 by Wilhelm Röntgen and of radioactivity by Pierre and Marie Curie shortly after. It details the early observations of radiation effects that promoted the early development of radiotherapy. It then presents the first evidence of radiation epidemiology and radiation carcinogenesis. Chapter 2 (Basic Concepts of Radiation Biology) reviews basic radiation biology and associated terminology to impart a better understanding of the importance of the basic concepts of interactions of ionizing radiation with living tissue. The chapter familiarizes the reader with basic and important radiation biology concepts, the use of radioactivity and its applications, the various types of interactions of radiation with living tissue, and possible effects from that exposure. It then focuses on theoretical dose–response curves and how they are used in radiation biology, and discusses stochastic versus non-stochastic effects of radiation exposure, and what these terms mean in relation to both high- and low-dose radiation exposure. Finally, a part dedicated to targeted and non-targeted effects, as well as low-dose radiation effects, ends the chapter. Chapter 3 concerns molecular radiation biology, which has become a powerful discipline and tool for detailed investigations into biological mechanisms of modern radiobiology. The chapter reviews the types of radiation-induced lesions in DNA, the types of DNA damage repair pathways as well as the importance of chromatin architecture in DNA damage and repair. It also describes the cytogenetic, oxidative stress and clonogenic cell survival methods, as well as the impact of radiation on cell cycle progression, cell death mechanisms, telomere shortening, and on the connectivity between cells. Finally, it highlights omic changes (genetics, lipidomics, proteomics, and metabolomics) as well as the involvement of specific pathways and the epigenetic factors modified by radiation. In Chapter 4 (Mechanistic, Modeling, and Dosimetric Radiation Biology), the principles of radiation dosimetry are explained and the relationship of track structure to early DNA damage and the importance of microdosimetry are addressed. The chapter establishes the relation between target theory and dose-response models. Chapters 5 (Clinical Radiobiology for Radiation Oncology) and 6 (Radiobiology of Combining Radiotherapy with Other Cancer Treatment Modalities) are both clinical chapters. Chapter 5 is dedicated to the principles of tumour radiotherapy, the therapeutic window and

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therapeutic ratio, tumour growth and tumour control, and the 6Rs concept. The next part of the chapter reviews the principles of dose fractionation, whole body irradiation, and the impact of tumour hypoxia. Tumour resistance and progression, and the role of tumour microenvironments are also considered and discussed. Chapter 5 finishes with sections dedicated to normal tissue damage, response to radiotherapy, the importance of stem cells and the microbiota in radiotherapy, as well as radiomics. Chapter 6 reviews the various conventional and alternative radiation schemes and analyses the various radiotherapy modalities in combination with other cancer treatment modalities (e.g. chemotherapy, targeted therapy, hormone therapy, and hyperthermia). Specific sections are dedicated to brachytherapy, radionuclide therapy, charged particle therapy, and the use of nanoparticles in cancer therapy. Chapter 7 addresses individual radiation sensitivity and biomarkers. From general considerations and classification of biomarkers, it then moves on to the collection of samples for radiation studies and the existing predictive assays. It then reviews the variation of radiation sensitivity as a function of age, biological sex, and genetic syndromes. The chapter ends with a perspective on personalized medicine. Chapter 8 provides in-depth coverage of radiobiology in accidental, public, and occupational exposures, reviewing the various radiation exposure scenarios, the long-term health effects of low-dose radiation in exposed human populations, and the problem posed by radon. A technical part of the chapter is dedicated to triage methods used after a radiation accident and to the available biodosimetry techniques. Chapter 9 (Environmental Radiobiology) provides an overview of the behaviour and fate of radioelements in the environment. It then reviews the impact of ionizing radiation on nonhuman biota (plants, invertebrates, vertebrates, microorganisms) and discusses the specific case of NORM (naturally occurring radioactive materials) contamination. Chapter 10 (Space Radiobiology) starts with a thorough review of the history of space radiation studies, followed by a description of the space radiation environment. It continues with a description of the impact of space travel on human health. It then reviews the various models (animals, plants, small organisms, microorganisms) sent to space and the biological changes induced by space radiation. It then focusses on space radiation resistance and gives a thorough description of the irradiation tests with ground-based facilities similar to the space environment. The authors of Chapter 11 present a review of radioprotectors, radiomitigators, and radiosensitizers, as well as internal contamination by radionuclides and possible treatment. It provides an exhaustive overview of molecules and the mechanisms able to intervene in the biological effects of ionizing radiation and discusses their potential clinical use in radiotherapy or in the field of radiation protection following accidental exposure to radiation and/or nuclear emergencies. Finally, Chapter 12 explores the ethical, social, epistemological, and legal considerations relevant to radiobiology. The chapter provides an overview of the basic principles relevant to each aspect whilst discussing contentious topics and potential future developments, along with more in-depth analysis where relevant.

Didactical and Pedagogical Approach To write such a textbook, a strong didactical and pedagogical approach was crucial. To be effective, a textbook must be readable, challenging, and also exciting to the reader. Special care was taken to make the reader read, the teacher teach, and the student study this textbook, and to motivate and maintain their interest through the textbook. To make complex concepts or material easily understood, we provided the readers with thorough explanations, free of unnecessary terminology.

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The general title Radiobiology Textbook illustrates the intention to provide detailed information about the entire field of radiobiology for anyone who is studying or is interested in this field. The textbook includes a table of contents in which all topics are listed with page references to enable the reader to locate topics quickly within a chapter. The standard chapter outline begins with an introduction giving a brief overview statement about what the reader can expect from the chapter and how the book can be used to teach a course. The learning objectives are intended to give a clear list of the educational scope and aims of the chapter. Before starting to read or study a chapter, the reader is encouraged to scan the introduction and learning objectives to understand the relationship of the material to be read. A set of keywords at the start of each chapter highlights the most significant words used specifically as an index to the content of the chapter. The textbook is also enriched with high-resolution images, graphs, figures, and high-quality supporting illustrations, to make it as clear, didactical, and appealing as possible to the reader. In all figures, for which we used a consistent colour code for all chapters, particular attention was paid to aid understanding, summarizing, and visualizing of the concepts detailed in the text. Simplicity was the most important consideration in figures, to help the reader grasp and interpret clearly and quickly. The easy access to the complex ideas presented in the figures and in the text is one of the important hallmarks of this textbook. Many figures in this textbook are true pieces of art meant to teach, but also to astonish with their beauty, the different aspects covered by radiobiology. Various types of graphs (bar charts, pie charts, histograms, plots, line graphs) are also used to display quantitative relationships between variables. Where needed, the text has been enhanced with tables to help summarize existing literature, present the results of epidemiological studies, or convey specific variables or statistical data on a particular domain. Tables have also been used as an alternative to numerical or listed data in order to make the text more readable, accessible, and understandable. In some cases, published figures, graphs, or tables have been used. Where needed, the necessary copyright permission was obtained. In each chapter, textboxes have been added to draw the reader’s attention to the section highlights, and these will be helpful to remember the most important topics covered within the chapter. These textboxes are embedded within the text narrative and summarize the content of the chapter at a glance, and enable the reader to rapidly scan and preview the content and direction of a chapter at a high concept level before beginning the detailed reading. Abbreviations have been used with moderate frequency in the textbook. These allow concepts that would otherwise require many words, were they to be written out completely, to be communicated quickly and effectively. Each nonstandard abbreviation is defined clearly when it is first introduced in the chapter and then used consistently throughout the chapter. The exercises and self-assessment at the end of each chapter allow the reader to evaluate and test their understanding of the chapter’s material but also to apply what they have learnt. The exercises are aimed at requiring the reader to use critical thinking skills. The questions are tied directly to the concepts taught in the chapters and are meant to help the reader determine whether they have mastered the important concepts of the chapter. The questions cover important information presented in the chapter. Answers are provided for each exercise. Recent reviews of publications in radiobiology suggest that the volume of research literature has been on the rise. Therefore, a careful analysis of the literature in the field from major databases (such as Web of Science, PubMed/Medline) was conducted ensuring highly relevant material is cited in this textbook. The list of references provided at the end of each chapter summarizes the main publications in the field addressed within each topic. Supplemental information in the section “further reading” is also included as appropriate at the end of each chapter. This is intended for readers who wish to deepen their knowledge and understanding. The “further reading” sections helps to illustrate, clarify, and apply the concepts encountered in the chapter.

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An index at the end of the textbook offers the reader an informative and balanced picture of the textbook’s contents, and serves as a concise and useful guide to all pertinent terms used in the textbook. These terms are presented as an alphabetically ordered list of the main entries. The textbook is open access as a support to worldwide education. It is targeted at an international audience, but in particular at those countries facing challenges in accessing educational material. The creation of this open access resource was also intended to address one of the predominant challenges in education, namely the cost of textbooks. The most commonly required textbooks in undergraduate and graduate education remain traditional and disciplinebased. In the absence of an integrated resource, students are requested to purchase and juggle preparation materials from many different discipline-based textbooks. With no fee required for readers to access or download this textbook, we hope to achieve the highest level of accessibility and to contribute to a better and more widespread knowledge of radiobiology as a discipline, as well as to facilitate efficient and focused learning by the reader.

Reviewing This textbook has been reviewed extensively. As it contains an important amount of information, the editor and authors have taken the utmost care to ensure accuracy and minimize potential errors or omissions. Each chapter has been cross-reviewed by authors of other chapters, after which each chapter was reviewed by more than 20 external experts, all renowned in their field of competence. We hope that each reader will feel gratified by the knowledge gathered from this textbook and that the textbook will become the radiobiologist’s trusted companion. Prof. Sarah Baatout Institute of Nuclear Medical Applications Belgian Nuclear Research Centre, SCK CEN Mol, Belgium Gent University (UGent), Ghent, Belgium Catholic University of Leuven (KULeuven), Leuven, Belgium United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) Vienna, Austria The European Radiation Research Society, Brussels, Belgium

Acknowledgements

As editor, and on behalf of all the contributors, I am pleased to provide readers the required theoretical and practical tools at a time when all areas of radiobiology are expanding rapidly. One hundred twenty-six international experts contributed to this textbook, sharing their endless expertise. Each one has contributed in significant ways and strived to help readers learn. Each one has provided their extraordinary insights into the complex subject of radiobiology. They have been an inspiration and foundation for this textbook. Without their expertise, support, and dedication, this book would not exist. It took just more than one year to write this textbook from the day of the kick-off meeting at the height of the COVID pandemic. During this period, online meetings dedicated to each chapter were held every 3 weeks to discuss the progress of the writing of each chapter and to review the content of each chapter. I would therefore like to express my immense gratitude upon the completion of this tremendous collaborative work and would like to thank each contributor warmly for their time, their energy but also the wonderful friendship, kindness, and teamwork that made each meeting and each part of the written text such a wonderful and constructive experience. The figure below indicates the geographical distribution of the contributors according to their country of employment.

Of all the contributors, I have particularly appreciated the dedication of Dhruti Mistry (for making most of the beautiful figures of this book), Alexandra Dobney (for taking care so patiently of all the copyright permission issues), Kristina Viktorsson and Judith Reindl (for checking all issues related to plagiarism). The contact points for each chapter (Yehoshua Socol, Ans Baeyens, Judith Reindl, Giuseppe Schettino, Peter Sminia, Vidhula Ahire, Liz Ainsbury, Christine Hellweg, Ruth Wilkins, Joana Lourenço, Alegría Montoro, and Alexandra Dobney) have played a crucial role in the coordination and the finalization of the writing of each chapter. The list of references per chapter required special support and help from Nathalie Heynickx, Silvana Miranda, Ans Baeyens, Ruth Pereira, Anne-Sophie Wozny, Cristian Fernandez, and Kristina Viktorsson, which I would also particularly like to acknowledge. xv

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Acknowledgements

Thank you also to Olivier Guipaud, Tom Boterberg, Bjorn Baselet, Nicholas Rajan, Abel Gonzalez, and Hussam Jassim for different aspects related to the reviewing, the coordination of specific written parts, and help with the figures or the guiding of the younger experts of the textbook. More than 20 external reviewers willingly agreed to carefully review parts of the textbook and we would like to thank them for taking the time and effort needed to review this textbook. Their insightful recommendations and suggestions were very helpful in evaluating the quality of the writing and the relevance of each section of the textbook. I would also like express my gratitude to SCK CEN general management, legal department, and communication department for their support throughout the process of the preparation of the textbook and for covering all the publishing costs related to this textbook in order to make it open access. A special thanks to the staff of the radiobiology unit who were extremely supportive during this endeavour. Figures were made thanks to the use of Biorender software. I would also like to thank the publishers and authors of the published data used in this textbook for having allowed their published figures, tables, and graphs to be used for free. As editor, I also greatly appreciated the dedication and professionalism of the editorial and publishing staff of Springer for their wonderful support in producing this textbook, in particular that of Antonella Cerri, Saraniya Vairamuthu, Kripa Guruprasad, and Parvathy Devi Gopalakrishnan. The final words of thanks are for my family. To my brother Akim, my father Sammy, my mother-in-law Brenda, and my father-in-law John who are no longer with us, but who were always wonderful supporters. To my Mum, Elise, teacher with a deep respect for education at all levels, for her generosity, never ending support, and kindness. To my rather wonderful husband, Andrew, a patient and uncommonly discerning critic, for his love, support, and encouragement in this adventure. To our children, Alexandra and William, for their love, kindness, unconditional support, and honesty, and for making it all worthwhile. Prof. Sarah Baatout Institute of Nuclear Medical Applications Belgian Nuclear Research Centre, SCK CEN Mol, Belgium Gent University (UGent), Ghent, Belgium Catholic University of Leuven (KULeuven), Leuven, Belgium United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) Vienna, Austria The European Radiation Research Society, Brussels, Belgium

Contents

1 History  of Radiation Biology�������������������������������������������������������������������������������������   1 Dimitrios Kardamakis, Sarah Baatout, Michel Bourguignon, Nicolas Foray, and Yehoshua Socol 2 Basic  Concepts of Radiation Biology������������������������������������������������������������������������  25 Ans Baeyens, Ana Margarida Abrantes, Vidhula Ahire, Elizabeth A. Ainsbury, Sarah Baatout, Bjorn Baselet, Maria Filomena Botelho, Tom Boterberg, Francois Chevalier, Fabiana Da Pieve, Wendy Delbart, Nina Frederike Jeppesen Edin, Cristian Fernandez-Palomo, Lorain Geenen, Alexandros G. Georgakilas, Nathalie Heynickx, Aidan D. Meade, Anna Jelinek Michaelidesova, Dhruti Mistry, Alegría Montoro, Carmel Mothersill, Ana Salomé Pires, Judith Reindl, Giuseppe Schettino, Yehoshua Socol, Vinodh Kumar Selvaraj, Peter Sminia, Koen Vermeulen, Guillaume Vogin, Anthony Waked, and Anne-Sophie Wozny 3 Molecular Radiation Biology�������������������������������������������������������������������������������������  83 Judith Reindl, Ana Margarida Abrantes, Vidhula Ahire, Omid Azimzadeh, Sarah Baatout, Ans Baeyens, Bjorn Baselet, Vinita Chauhan, Fabiana Da Pieve, Wendy Delbart, Caitlin Pria Dobney, Nina Frederike Jeppesen Edin, Martin Falk, Nicolas Foray, Agnès François, Sandrine Frelon, Udo S. Gaipl, Alexandros G. Georgakilas, Olivier Guipaud, Michael Hausmann, Anna Jelinek Michaelidesova, Munira Kadhim, Inês Alexandra Marques, Mirta Milic, Dhruti Mistry, Simone Moertl, Alegría Montoro, Elena Obrador, Ana Salomé Pires, Roel Quintens, Nicholas Rajan, Franz Rödel, Peter Rogan, Diana Savu, Giuseppe Schettino, Kevin Tabury, Georgia I. Terzoudi, Sotiria Triantopoulou, Kristina Viktorsson, and Anne-Sophie Wozny 4 Mechanistic,  Modeling, and Dosimetric Radiation Biology����������������������������������� 191 Giuseppe Schettino, Sarah Baatout, Francisco Caramelo, Fabiana Da Pieve, Cristian Fernandez-Palomo, Nina Frederike Jeppesen Edin, Aidan D. Meade, Yann Perrot, Judith Reindl, and Carmen Villagrasa 5 Clinical  Radiobiology for Radiation Oncology ������������������������������������������������������� 237 Peter Sminia, Olivier Guipaud, Kristina Viktorsson, Vidhula Ahire, Sarah Baatout, Tom Boterberg, Jana Cizkova, Marek Dostál, Cristian Fernandez-­Palomo, Alzbeta Filipova, Agnès François, Mallia Geiger, Alistair Hunter, Hussam Jassim, Nina Frederike Jeppesen Edin, Karl Jordan, Irena Koniarová, Vinodh Kumar Selvaraj, Aidan D. Meade, Fabien Milliat, Alegría Montoro, Constantinus Politis, Diana Savu, Alexandra Sémont, Ales Tichy, Vlastimil Válek, and Guillaume Vogin

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6 Radiobiology  of Combining Radiotherapy with Other Cancer Treatment Modalities��������������������������������������������������������������������������������������������������������������������� 311 Vidhula Ahire, Niloefar Ahmadi Bidakhvidi, Tom Boterberg, Pankaj Chaudhary, Francois Chevalier, Noami Daems, Wendy Delbart, Sarah Baatout, Christophe M. Deroose, Cristian Fernandez-Palomo, Nicolaas A. P. Franken, Udo S. Gaipl, Lorain Geenen, Nathalie Heynickx, Irena Koniarová, Vinodh Kumar Selvaraj, Hugo Levillain, Anna Jelínek Michaelidesová, Alegría Montoro, Arlene L. Oei, Sébastien Penninckx, Judith Reindl, Franz Rödel, Peter Sminia, Kevin Tabury, Koen Vermeulen, Kristina Viktorsson, and Anthony Waked 7 Individual  Radiation Sensitivity and Biomarkers: Molecular Radiation Biology������������������������������������������������������������������������������������������������������������������������� 387 Elizabeth A. Ainsbury, Ana Margarida Abrantes, Sarah Baatout, Ans Baeyens, Maria Filomena Botelho, Benjamin Frey, Nicolas Foray, Alexandros G. Georgakilas, Fiona M. Lyng, Inês Alexandra Marques, Aidan D. Meade, Mirta Milic, Dhruti Mistry, Jade F. Monaghan, Alegría Montoro, Ana Salomé Pires, Georgia I. Terzoudi, Sotiria Triantopoulou, Kristina Viktorsson, and Guillaume Vogin 8 Radiobiology  of Accidental, Public, and Occupational Exposures������������������������� 425 Ruth Wilkins, Ana Margarida Abrantes, Elizabeth A. Ainsbury, Sarah Baatout, Maria Filomena Botelho, Tom Boterberg, Alžběta Filipová, Daniela Hladik, Felicia Kruse, Inês Alexandra Marques, Dhruti Mistry, Jayne Moquet, Ursula Oestreicher, Raghda Ramadan, Georgia I. Terzoudi, Sotiria Triantopoulou, Guillaume Vogin, and Anne-Sophie Wozny 9 Environmental Radiobiology������������������������������������������������������������������������������������� 469 Joana Lourenço, Carmel Mothersill, Carmen Arena, Deborah Oughton, Margot Vanheukelom, Ruth Pereira, Sónia Mendo, and Veronica De Micco 10 Space Radiobiology����������������������������������������������������������������������������������������������������� 503 Christine Elisabeth Hellweg, Carmen Arena, Sarah Baatout, Bjorn Baselet, Kristina Beblo-Vranesevic, Nicol Caplin, Richard Coos, Fabiana Da Pieve, Veronica De Micco, Nicolas Foray, Boris Hespeels, Anne-Catherine Heuskin, Jessica Kronenberg, Tetyana Milojevic, Silvana Miranda, Victoria Moris, Sébastien Penninckx, Wilhelmina E. Radstake, Emil Rehnberg, Petra Rettberg, Kevin Tabury, Karine Van Doninck, Olivier Van Hoey, Guillaume Vogin, and Yehoshua Socol 11 Radioprotectors,  Radiomitigators, and Radiosensitizers��������������������������������������� 571 Alegría Montoro, Elena Obrador, Dhruti Mistry, Giusi I. Forte, Valentina Bravatà, Luigi Minafra, Marco Calvaruso, Francesco P. Cammarata, Martin Falk, Giuseppe Schettino, Vidhula Ahire, Noami Daems, Tom Boterberg, Nicholas Dainiak, Pankaj Chaudhary, Sarah Baatout, and Kaushala Prasad Mishra 12 Ethical,  Legal, Social, and Epistemological Considerations of Radiation Exposure ��������������������������������������������������������������������������������������������������������������������� 629 Alexandra Dobney, Abel Julio González, Deborah Oughton, Frances Romain, Gaston Meskens, Michel Bourguignon, Tim Wils, Tanja Perko, and Yehoshua Socol

Contents

Contributors

Ana  Margarida  Abrantes  Institute of Biophysics, Faculty of Medicine, iCBR-CIMAGO, Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal ESTESC-Coimbra Health School, Instituto Politécnico de Coimbra, Coimbra, Portugal Vidhula Ahire  Chengdu Anticancer Bioscience, Ltd., Chengdu, China J. Michael Bishop Institute of Cancer Research, Chengdu, China Niloefar  Ahmadi  Bidakhvidi Department of Nuclear Medicine, University Hospitals Leuven, Leuven, Belgium Elizabeth  A.  Ainsbury  Radiation, Chemical and Environmental Hazards Directorate, UK Health Security Agency, Oxford, UK Carmen Arena  Department of Biology, University of Naples Federico II, Naples, Italy Omid  Azimzadeh Section Radiation Biology, Federal Office for Radiation Protection, Oberschleißheim, Germany Sarah Baatout  Institute of Nuclear Medical Applications, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Ans Baeyens  Radiobiology, Department of Human Structure and Repair, Ghent University, Ghent, Belgium Bjorn  Baselet Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Kristina  Beblo-Vranesevic Radiation Biology Department, Astrobiology, Institute of Aerospace Medicine, German Aerospace Center, Cologne, Germany Maria  Filomena  Botelho Institute of Biophysics, Faculty of Medicine, iCBR-CIMAGO, Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal Clinical Academic Center of Coimbra, Coimbra, Portugal Tom  Boterberg Department of Radiation Oncology, Ghent University Hospital, Ghent, Belgium Particle Therapy Interuniversity Center Leuven, Department of Radiation Oncology, University Hospitals Leuven, Leuven, Belgium Michel Bourguignon  University of Paris Saclay (UVSQ), Paris, France Valentina  Bravatà  National Research Council (IBFM-CNR), Institute of Bioimaging and Molecular Physiology, Cefalù (PA), Italy Laboratori Nazionali del Sud, INFN-LNS, National Institute for Nuclear Physics, Catania, Italy xix

xx

Marco  Calvaruso National Research Council (IBFM-CNR), Institute of Bioimaging and Molecular Physiology, Cefalù (PA), Italy Laboratori Nazionali del Sud, INFN-LNS, National Institute for Nuclear Physics, Catania, Italy Francesco P. Cammarata  National Research Council (IBFM-CNR), Institute of Bioimaging and Molecular Physiology, Cefalù (PA), Italy Laboratori Nazionali del Sud, INFN-LNS, National Institute for Nuclear Physics, Catania, Italy Nicol  Caplin Human Spaceflight and Robotic Exploration, European Space Agency, Noordwijk, The Netherlands Francisco Caramelo  University of Coimbra, Coimbra, Portugal Pankaj Chaudhary  The Patrick G. Johnston Centre for Cancer Research, Queen’s University Belfast, Belfast, United Kingdom Vinita  Chauhan Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, ON, Canada Francois Chevalier  UMR6252 CIMAP, Team Applications in Radiobiology with Accelerated Ions, CEA-CNRS-ENSICAEN-Université de Caen Normandie, Caen, France Jana Cizkova  Department of Radiobiology, Faculty of Military Health Sciences, University of Defence, Hradec Králové, Czech Republic Richard Coos  Laboratory of Analysis by Nuclear Reactions, University of Namur, Namur, Belgium Noami  Daems Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Nicholas Dainiak  Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, United States of America Fabiana Da Pieve  Royal Belgian Institute for Space Aeronomy, Brussels, Belgium European Research Council Executive Agency, European Commission, Brussels, Belgium Wendy Delbart  Nuclear Medicine Department, Hôpital Universitaire de Bruxelles (H.U.B.), Brussels, Belgium Veronica De Micco  Department of Agricultural Sciences, University of Naples Federico II, Naples, Italy Christophe  M.  Deroose Department of Nuclear Medicine, University Hospitals Leuven, Leuven, Belgium Alexandra Dobney  Queen Mary University of London, London, United Kingdom Caitlin Pria Dobney  Department of Physics, University of Toronto, Teddington, Canada Marek  Dostál Department of Radiology and Nuclear Medicine, Faculty of Medicine, Masaryk University and University Hospital Brno, Brno, Czech Republic Department of Biophysics, Faculty of Medicine, Masaryk University, Brno, Czech Republic Nina Frederike Jeppesen Edin  Department of Physics, University of Oslo, Oslo, Norway Martin Falk  Department of Cell Biology and Radiobiology, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic Cristian Fernandez-Palomo  Institute of Anatomy, University of Bern, Bern, Switzerland

Contributors

Contributors

xxi

Alžběta  Filipová Department of Radiobiology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic Nicolas Foray  Inserm Unit 1296 “Radiation: Defense, Health, Environment”, Centre LéonBérard, Lyon, France Giusi  I.  Forte National Research Council (IBFM-CNR), Institute of Bioimaging and Molecular Physiology, Cefalù (PA), Italy Laboratori Nazionali del Sud, INFN-LNS, National Institute for Nuclear Physics, Catania, Italy Agnès  François  Radiobiology of Medical Exposure Laboratory, Institute for Radiological Protection and Nuclear Safety (IRSN), Fontenay-aux-Roses, France Nicolaas A. P. Franken  Department of Radiation Oncology, Amsterdam University Medical Centers, Location University of Amsterdam, Amsterdam, The Netherlands Center for Experimental and Molecular Medicine (CEMM), Laboratory for Experimental Oncology and Radiobiology (LEXOR), Amsterdam, The Netherlands Cancer Center Amsterdam, Cancer Biology and Immunology, Amsterdam, The Netherlands Sandrine  Frelon  Health and Environment Division, Research Laboratory of Radionuclide Effects on Environment, Institute for Radiological Protection and Nuclear Safety (IRSN), Saint-Paul-Lez-Durance, France Benjamin  Frey  Translational Radiobiology, Department of Radiation Oncology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Udo  S.  Gaipl  Translational Radiobiology, Department Universitätsklinikum Erlangen, Erlangen, Germany

of

Radiation

Oncology,

Lorain  Geenen Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Department of Radiology and Nuclear Medicine, Erasmus Medical Center, Rotterdam, The Netherlands Mallia  Geiger Radiobiology of Medical Exposure Laboratory, Institute for Radiological Protection and Nuclear Safety, Fontenay-aux-Roses, France Alexandros  G.  Georgakilas DNA Damage Laboratory, Physics Department, School of Applied Mathematical and Physical Sciences, National Technical University of Athens (NTUA), Athens, Greece Abel Julio González  Argentine Nuclear Regulatory Authority, Buenos Aires, Argentina Olivier Guipaud  Radiobiology of Medical Exposure Laboratory, Institute for Radiological Protection and Nuclear Safety (IRSN), Fontenay-aux-Roses, France Michael  Hausmann Kirchhoff Institute for Physics, University Heidelberg, Heidelberg, Germany Christine  Elisabeth  Hellweg Radiation Biology Department, Institute of Aerospace Medicine, German Aerospace Center, Cologne, Germany Boris  Hespeels Namur Research Institute for Life Sciences, Institute of Life-Earth-­ Environment, Research Unit in Environmental and Evolutionary Biology, University of Namur (UNamur-LEGE), Namur, Belgium Anne-Catherine  Heuskin Namur Research Institute for Life Sciences, Laboratory of Analysis by Nuclear Reactions, University of Namur, Namur, Belgium

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Nathalie Heynickx  Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Department of Molecular Biotechnology, Ghent University, Ghent, Belgium Daniela Hladik  Bundesamt für Strahlenschutz, Oberschleißheim, Germany Alistair  Hunter Radiobiology Section, Department of Radiation Oncology, University of Cape Town and Groote Schuur Hospital, Cape Town, South Africa Hussam Jassim  Radiotherapy Department, General Najaf Hospital, Kufa, Najaf, Iraq Department of Medical Physics, University Al-Hilla College, Babylon, Iraq Karl Jordan  School of Physics, Clinical and Optometric Sciences, Technological University Dublin, Dublin, Ireland Munira Kadhim  Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, United Kingdom Dimitrios  Kardamakis Department of Radiation Oncology, University of Patras Medical School, Patras, Greece Irena  Koniarová  Department of Radiation Protection in Radiotherapy, National Radiation Protection Institute, Prague, Czech Republic Jessica  Kronenberg Radiation Biology Department, Institute of Aerospace Medicine, German Aerospace Center, Cologne, Germany Felicia  Kruse Radiation, Chemical and Environmental Hazards Directorate, UK Health Security Agency, Oxford, United Kingdom Hugo  Levillain  Medical Physics Department, Hôpital Universitaire de Bruxelles (H.U.B.), Université Libre de Bruxelles, Bruxelles, Belgium Joana Lourenço  CESAM & Department of Biology, University of Aveiro, Aveiro, Portugal Fiona M. Lyng  Center for Radiation and Environmental Science, Technological University Dublin, Dublin, Ireland Inês  Alexandra  Marques Institute of Biophysics, Faculty of Medicine, iCBR-CIMAGO, Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal Clinical Academic Center of Coimbra, Coimbra, Portugal Aidan D. Meade  School of Physics and Clinical and Optometric Sciences, Faculty of Science, Technological University Dublin, Dublin, Ireland Sónia Mendo  CESAM & Department of Biology, University of Aveiro, Aveiro, Portugal Gaston Meskens  Science and Technology Studies Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Centre for Ethics and Value Inquiry, Ghent University, Ghent, Belgium Anna Jelínek Michaelidesová  Nuclear Physics Institute, Czech Academy of Sciences, Czech Technical University, Faculty of Nuclear Sciences and Physical Engineering, Prague, Czech Republic Czech Academy of Sciences, Rez, Czech Republic Mirta  Milic Mutagenesis Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia Fabien  Milliat Radiobiology of Medical Exposure Laboratory, Institute for Radiological Protection and Nuclear Safety, Fontenay-aux-Roses, France

Contributors

Contributors

xxiii

Tetyana  Milojevic Space Biochemistry Group, Department of Biophysical Chemistry, University of Vienna, Vienna, Austria Luigi  Minafra National Research Council (IBFM-CNR), Institute of Bioimaging and Molecular Physiology, Cefalù (PA), Italy Laboratori Nazionali del Sud, INFN-LNS, National Institute for Nuclear Physics, Catania, Italy Silvana  Miranda  Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Faculty of Bioscience and Engineering, Ghent University, Ghent, Belgium Kaushala  Prasad  Mishra  Radiobiology Unit, Bhabha Atomic Research Center, Mumbai, Maharashtra, India Dhruti  Mistry Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Simone  Moertl  Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Oberschleißheim, Germany Jade  F.  Monaghan Center for Radiation and Environmental Science, Technological University Dublin, Dublin, Ireland Alegría Montoro  Radiological Protection Service, University and Polytechnic La Fe Hospital of Valencia, Valencia, Spain Laboratorio de Dosimetría Biológica, Servicio de Protección Radiológica Hospital Universitario y Politécnico la Fe, Valencia, Spain Jayne  Moquet Radiation, Chemical and Environmental Hazards Directorate, UK Health Security Agency, Oxford, United Kingdom Victoria  Moris Research Unit in Environmental and Evolutionary Biology (URBE), University of Namur (UNamur-LEGE), Namur, Belgium Laboratory of Molecular Biology and Evolution (MBE), Department of Biology, Université Libre de Bruxelles, Brussels, Belgium Carmel Mothersill  Faculty of Science, McMaster University, Hamilton, Canada Elena  Obrador Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain Arlene L. Oei  Department of Radiotherapy Oncology, Amsterdam UMC, Location University of Amsterdam, Amsterdam, The Netherlands Center for Experimental and Molecular Medicine (CEMM), Laboratory for Experimental Oncology and Radiobiology (LEXOR), Amsterdam, The Netherlands Cancer Center Amsterdam, Cancer Biology and Immunology, Amsterdam, The Netherlands Ursula Oestreicher  Bundesamt für Strahlenschutz, Oberschleißheim, Germany Deborah Oughton  Norwegian University of Life Sciences (NMBU), Ås, Norway Sébastien  Penninckx Medical Physics Department, Hôpital Universitaire de Bruxelles (H.U.B.), Université Libre de Bruxelles, Bruxelles, Belgium Ruth Pereira  GreenUPorto—Sustainable Agrifood Production Research Centre/Inov4Agro, Department of Biology, Faculty of Science of the University of Porto, Campus de Vairão, Vila do Conde, Portugal Tanja Perko  Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium University of Antwerp, Antwerp, Belgium

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Yann  Perrot Institut de Radioprotection et Sûreté Nucléaire, Fontenay-aux-Roses Cedex, France Ana Salomé Pires  Institute of Biophysics, Faculty of Medicine, iCBR-CIMAGO, Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal Clinical Academic Center of Coimbra, Coimbra, Portugal Constantinus  Politis  Department of Oral and Maxillofacial Surgery, University Hospitals Leuven, Leuven, Belgium Department of Imaging and Pathology, OMFS IMPATH Research Group, Faculty of Medicine, KU Leuven, Leuven, Belgium Roel  Quintens Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Wilhelmina E. Radstake  Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Faculty of Bioscience and Engineering, Ghent University, Ghent, Belgium Nicholas  Rajan Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Raghda Ramadan  Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Emil  Rehnberg Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Department of Molecular Biotechnology, Ghent University, Ghent, Belgium Judith  Reindl Section Biomedical Radiation Physics, Institute for Applied Physics and Measurement Technology, Department for Aerospace Engineering, Universität der Bundeswehr München, Neubiberg, Germany Petra  Rettberg Radiation Biology Department, Astrobiology, Institute of Aerospace Medicine, German Aerospace Center, Cologne, Germany Franz Rödel  Department of Radiotherapy and Oncology, Goethe University, Frankfurt am Main, Germany Peter Rogan  Departments of Biochemistry and Oncology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada Frances Romain  University of Manchester, Manchester, United Kingdom Diana Savu  Department of Life and Environmental Physics, Horia Hulubei National Institute of Physics and Nuclear Engineering, Magurele, Romania Giuseppe Schettino  National Physical Laboratory, Teddington, United Kingdom University of Surrey, Guilford, United Kingdom Vinodh  Kumar  Selvaraj  Department of Radiation Oncology, Thanjavur Medical College, Thanjavur, India Alexandra Sémont  Radiobiology of Medical Exposure Laboratory, Institute for Radiological Protection and Nuclear Safety, Fontenay-aux-Roses, France Peter Sminia  Department of Radiation Oncology, Amsterdam University Medical Centers, Location Vrije Universiteit/Cancer Center Amsterdam, Amsterdam, The Netherlands Yehoshua Socol  Jerusalem College of Technology, Jerusalem, Israel

Contributors

Contributors

xxv

Kevin  Tabury Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Department of Biomedical Engineering, University of South Carolina, Columbia, SC, USA Georgia I. Terzoudi  Health Physics, Radiobiology and Cytogenetics Laboratory, Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, National Centre for Scientific Research “Demokritos”, Athens, Greece Ales Tichy  Department of Radiobiology, Faculty of Military Health Sciences, University of Defence, Hradec Králové, Czech Republic Sotiria Triantopoulou  Health Physics, Radiobiology and Cytogenetics Laboratory, Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, National Centre for Scientific Research “Demokritos”, Athens, Greece Vlastimil  Válek Department of Radiology and Nuclear Medicine, Faculty of Medicine, Masaryk University and University Hospital Brno, Brno, Czech Republic Karine Van Doninck  Research Unit in Environmental and Evolutionary Biology (URBE), University of Namur (UNamur-LEGE), Namur, Belgium Université Libre de Bruxelles, Molecular Biology and Evolution, Brussels, Belgium Margot  Vanheukelom  Biosphere Impact Studies, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Olivier  Van Hoey Research in Dosimetric Applications Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Koen  Vermeulen Institute of Nuclear Medical Applications, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Kristina  Viktorsson Department of Oncology/Pathology, Karolinska Institutet, Solna, Sweden Carmen  Villagrasa Institut de Radioprotection et Sûreté Nucléaire, Fontenay-aux-Roses Cedex, France Guillaume  Vogin Centre Francois Baclesse, University of Luxembourg and Luxembourg Institute of Health, Luxembourg, Luxembourg Centre François Baclesse, National Radiotherapy Center of Luxembourg, Esch-sur-Alzette, Luxembourg Anthony  Waked Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Laboratory of Nervous System Disorders and Therapy, GIGA Neurosciences, Université de Liège, Liège, Belgium Ruth  Wilkins Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, ON, Canada Tim Wils  KU Leuven, Leuven, Belgium Anne-Sophie Wozny  Cellular and Molecular Radiobiology Lab, UMR CNRS 5822, Lyon 1 University, Oullins, France Department of Biochemistry and Molecular Biology, Lyon-Sud Hospital, Hospices Civils de Lyon, Pierre-Bénite, France

xxvi

Reviewers Gersande  Alphonse Laboratoire de radiobiologie Cellulaire et Moléculaire, Faculté de médecine Lyon-Sud, IP2I CNRS UMR 5822, Oullins, France Adayabalam Balajee  Oak Ridge Associated Universities, Oak Ridge, TN, United States of America Marc  Benderitter Department of Research in Radiobiology and Regenerative Medicine, Institut de radioprotection et de sûreté nucléaire (IRSN), Fontenay-aux-Roses, France Virginie  Chapon Aix Marseille Université, CEA, CNRS, BIAM, Saint Paul-Lez-Durance, France Eric Deutsch  Radiotherapy Department, Gustave Roussy Institute, Paris, France Nicolaas A. P. Franken  Amsterdam Medical Center, Amsterdam, The Netherlands Anna Friedl  Department of Radiation Oncology, LMU University Hospital Munich, Munich, Germany Almudena  Real  Gallego Radiation Protection of the Public and the Environment Unit, CIEMAT, Madrid, Spain Stephanie  Girst Institute of Applied Physics and Metrology, Department of Aerospace Engineering, Universität der Bundeswehr München, Neubiberg, Germany Eric Grégoire  Institut de radioprotection et de sûreté nucléaire (IRSN), Fontenay-aux-Roses, France Makoto Hosono  Department of Radiology, Kindai University Faculty of Medicine, OsakaSayama, Osaka, Japan Nagraj G. Huilgol  Nanavati Hospital, Mumbai, India Marek Janiak  Dept. of Radiobiology and Radiation Protection, Military Institute of Hygiene and Epidemiology, Warsaw, Poland Wook Kang Keon  Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, South Korea Laure Marignol  Radiobiology, Discipline of Radiation Therapy, School of Medicine, Trinity College, Dublin, Ireland Kaushala Prasad Mishra  Radiation Biology and Health Sciences Division, Bhabha Atomic Research Center, Mumbai, India Nehru Gram Bharti University, Prayagraj, India Asian Association for Radiation Research, Mumbai, India Judith  Reindl Institute of Applied Physics and Metrology, Department of Aerospace Engineering, Universität der Bundeswehr München, Neubiberg, Germany Vassiliki Rizomilioti  University of Patras, Patras, Greece Viacheslav Soyfer  Tel Aviv Sourasky Medical Center, Ichilov Hospital, Tel Aviv, Israel Simon Stuttaford  Castletown Law, Edinburgh, Scotland, United Kingdom

Contributors

Contributors

xxvii

Walter  Tinganelli  Clinical Radiobiology Group, Biophysics Department, Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, Germany

GSI

Nathalie Vanhoudt  Unit for Biosphere Impact Studies, SCK CEN, Belgian Nuclear Research Centre, Mol, Belgium Moshe Yanovskiy  Jerusalem College of Technology, Jerusalem, Israel

1

History of Radiation Biology Dimitrios Kardamakis, Sarah Baatout, Michel Bourguignon, Nicolas Foray, and Yehoshua Socol Nothing in life is to be feared, it is only to be understood. Now is the time to understand more, so that we may fear less. —Marie Curie.

In November 1895, Wilhelm Conrad Roentgen discovered X-rays; in March 1896, Henri Becquerel discovered natural radioactivity; and in December 1898, Marie and Pierre Curie produced polonium and later radium. Almost immediately after these discoveries, radiation biology, defined as the study of the effects in biological systems of exposure to radiation, began (Fig. 1.1).

A plethora of clinical observations, initially on the skin, contributed to a better knowledge of the biological effects of ionizing radiation. The first molecular and cellular mechanistic models of the radiation action were proposed in the 1930s and 1940s and then after the discovery of the DNA structure in the 1950s. It is noteworthy that the first theories unifying molecular and cellular features of irradiated human cells emerged in the 1980s during which the first quantitative features of human radiosensitivity were pointed out [1–4]. These great discoveries at the turn of the twentieth century initiated a new era in human history. Especially, medicine has greatly profited from their applications in diagnosis and treatment of various diseases, revolutionizing our understanding of diseases. The discoveries had a vast impact on society in general and on healthcare in particular. In this chapter, we present the main landmarks in the history of X-rays and, more generally, of ionizing radiation. Brief biographies of the pioneers in this field are presented in a chronological description of the whole field and emphasis is placed on the continuity in the development of the application of ionizing radiation to human life.

D. Kardamakis Department of Radiation Oncology, University of Patras Medical School, Patras, Greece e-mail: [email protected]

N. Foray Inserm Unit 1296 “Radiation: Defense, Health, Environment”, Centre Léon-Bérard, Lyon, France e-mail: [email protected]

S. Baatout Institute of Nuclear Medical Applications, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium e-mail: [email protected]

Y. Socol (*) Jerusalem College of Technology, Jerusalem, Israel e-mail: [email protected]

Learning Objectives

• To learn about the lives and scientific achievements of the pioneers in radiation • To understand the logic behind the applications of ionizing radiation in modern times • To understand the progression of the scientific knowledge of the physiological and biological effects of ionizing radiation

1.1 Introduction

M. Bourguignon University of Paris Saclay (UVSQ), Paris, France

© The Author(s) 2023 S. Baatout (ed.), Radiobiology Textbook, https://doi.org/10.1007/978-3-031-18810-7_1

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2 Fig. 1.1  Milestones of radiation biology

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1.2 Early Observations of Radiation Effects 1.2.1 The Discovery of X-Rays and Radioactivity By the end of the nineteenth century, “Newtonian” physics had explained nearly all the phenomena involving mass, speed, electricity, and heat. However, some questions remained unanswered, notably the origin of the luminescence phenomena observed either in glass vacuum tubes subjected to a high voltage (e.g., the Crookes tubes—Fig. 1.2) or on certain ores [4]. In both cases, one of the major questions was their inducibility vis-à-vis the sunlight. The German physicist Wilhelm Conrad Roentgen addressed the first challenge by putting some opaque boxes on the Crookes tube, while the Frenchman Henri Becquerel focused on the second one by studying light emitted by uranium ores in the darkness. The two series of experiments became legendary and led to two Nobel prizes in physics [4]. In November 1895, Wilhelm Conrad Röntgen (Roentgen) (1845–1923) detected electromagnetic radiation of a sub-­ nanometer wavelength range, today known as X- or Roentgen rays. For this discovery, he was awarded the first Nobel Prize in Physics in 1901. Although he investigated these X-rays and learned much about their interactions with matter, for a

Fig. 1.2 Crookes, or cathode ray, tube. (Source: Wikimedia. Reproduced with permission)

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long time, he was not entirely convinced that he had made a real discovery [5] (Box 1.1). Box 1.1 Wilhelm Conrad Röntgen

• Wilhelm Conrad Röntgen (1845–1923) experimented with Crookes tubes and in November 1895 detected electromagnetic radiation of a sub-­ nanometer wavelength range (X-rays). • He earned the first Nobel Prize in Physics in 1901.

Roentgen was born in Lennep, Rhineland, Germany [6]. When he was 3 years old, his family moved to the Netherlands. He was an average student in the primary and secondary school, and in November 1865, he enrolled in the polytechnical school of Zurich, graduating as a mechanical engineer in 1868. After that, Roentgen remained at the University of Zurich as a postgraduate student in mathematics having August Kundt, an expert in the theory of light, as a mentor. Roentgen’s first experiments in Zurich concerned the properties of gases and proved to be important for his subsequent discoveries. His doctoral thesis “Studies on Gases” led to his being awarded a PhD degree in 1869 and being appointed as an assistant to Kundt. In 1870, Roentgen, following Kundt, returned to Germany to the University of Wurzburg (Bavaria). In the autumn of 1893, he was elected Rector at the University of Wurzburg, having 44 publications and being highly respected by his colleagues and the larger academic community. Richard I. Frankel gives an excellent description of the life of W. C. Roentgen as a scientist and describes in detail the events leading up to his groundbreaking discovery. On November 8, 1895, after experimenting with cathode rays produced in tubes developed by Johann Hittorf and William Crookes, Roentgen made his discovery. He repeated and expanded his work and gave the first description of the physical and chemical properties of X-rays. He demonstrated that these rays could penetrate not only glass and air but also other materials, including various metals. However, a thin sheet of lead completely blocked them. Roentgen inferred that the radiation he observed was in fact rays because it traveled in straight lines and created shadows of the type that would be created by rays (Fig. 1.3). While studying the ability of lead to stop the rays, Roentgen held a small piece of this metal between his thumb and index finger and placed it

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Fig. 1.3  Left: Wilhelm Conrad Röntgen (1845– 1923), a portrait by Nicola Perscheid, circa 1915. Right: The first roentgenogram—the hand of Röntgen’s wife after its irradiation with X-rays (Dec 22, 1895)

in the path of the rays. He noted that he could distinguish the outline of the two digits on the screen and that the bones appeared as shadows darker than the surrounding soft tissue. Roentgen continued his work over the next weeks, during which he made additional images and showed that the rays darkened a photographic plate. In his manuscript entitled “Uber eine neue Art von Strahlen” (“On a New Kind of Rays”) submitted to the Physikalisch-Medizinische Gesellschaft in Wurzburg on December 28, 1895, he used the term “X-rays” for the first time [5]. Roentgen did not leave any autobiography, so all information regarding people and events which had an influence on his work comes from his biographers. Scientists whose work had greatly influenced Roentgen were the physicist August Kundt (1839–1894), the physicist and mathematician Rudolf Clausius (1822–1888), and the physicist and physician Hermann Ludwig Ferdinand von Helmholtz (1821–1894), all three of German origin. Of importance is his lifelong friendship with the physicist Ludwig Zehnder who served as Roentgen’s chief assistant and became an occasional co-author. It is worth mentioning the relationship between Roentgen and his contemporary German experimental physicist Philipp Lenard (1862–1947), director of the Physical Institute at Heidelberg University. Lenard (Fig. 1.4) first published the results of his experiments on cathode rays in 1894 and was awarded for this the Nobel Prize in Physics in 1905. Prior to Roentgen’s discovery, the two scientists exchanged several letters regarding the aspects of the cathode ray research, and Roentgen referenced Lenard in his initial publications on

Fig. 1.4  Philipp Eduard Anton von Lenard (1862–1947)

X-rays and used Lenard’s modified tube for his experiments (Box 1.2).

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Box 1.2 Philipp Lenard

• Philipp Lenard (1862–1947) was awarded the Nobel Prize in Physics in 1905 for “his work on cathode rays.” • However, Lenard became extremely embittered by not winning the Prize in 1901. He became one of Adolf Hitler’s most ardent supporters, eventually becoming “Chief of Aryan Physics” under the Nazi regime. • After World War II, he was not sentenced (for his prominent role in the Nazi regime) only due to his old age.

However, when Roentgen alone was awarded the Nobel Prize in 1901 “in recognition of the extraordinary services he has rendered by the discovery of the remarkable rays subsequently named after him,” Lenard became extremely embittered, and for the rest of his life, he insisted that he had shown Roentgen the way to his discovery. Lenard became one of the early adherents of the National Socialism and one of Adolf Hitler’s most ardent supporters, eventually becoming “Chief of Aryan Physics” under the Nazi regime. In 1933, he published a book called “Great Men in Science” in which he failed to mention not only Jews, such as Einstein or Bohr, but also non-Aryans like Marie Skłodowska-Curie and even Roentgen. When World War II ended, Lenard’s prominent role in the Nazi regime led to his arrest, but due to his old age, instead of being sentenced to prison, he was sent to live in a small German village, where he died at the age of 83 [7, 8]. A few months after the discovery of X-rays, radioactivity was described. Antoine-Henri Becquerel (1852–1908) (Fig. 1.5) was a member of a distinguished family of four generations of physicists, all being members of the French Académie des Sciences. Becquerel’s initial research was in phosphorescence, the emission of light of one color followFig. 1.5  Left: Henri Becquerel (1852–1908), circa 1905. Right: Becquerel’s photographic plate exposed to a uranium salt

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ing a body’s exposure to the light of another color. In early 1896, following Röntgen’s discovery, Becquerel “began looking for a connection between the phosphorescence he had already been investigating and the newly discovered X-rays” [9] and initially thought that phosphorescent materials, such as some uranium salts, might emit penetrating X-ray-like radiation, but only when illuminated by bright sunlight. By May 1896, after a series of experiments with non-phosphorescent uranium salts, he correctly concluded that the penetrating radiation came from the uranium itself, even without any external excitation. The intensive study of this phenomenon led Becquerel to publish seven papers in 1896 only. Becquerel’s other experiments allowed him to figure out what happened when the “emissions” entered a magnetic field: “When different radioactive substances were put in the magnetic field, they deflected in different directions or not at all, showing that there were three classes of radioactivity: negative, positive, and electrically neutral” [10] (Box 1.3).

Box 1.3 Henri Becquerel

• Henri Becquerel (1852–1908) discovered radioactivity in 1896 while studying phosphorescent uranium salts. • Later in the same year, upon experimenting with non-phosphorescent uranium salts, he concluded that the penetrating radiation came from the uranium itself. • He was awarded the Nobel Prize in Physics in 1903.

Interestingly, radioactivity could have been discovered nearly four decades earlier. In 1857, the photographic investor Abel Niépce de Saint-Victor (1805–1870) observed that uranium salts emitted radiation that darkened photographic emulsions. Later in 1861, he realized that uranium salts produced invisible radiation. In 1868, Becquerel’s father

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Edmond published a book entitled “La lumière: ses causes et ses effets (Light: Its causes and its effects),” where he mentioned that Niépce de Saint-Victor had observed that some phosphorescent objects could expose photographic plates even when unexposed to sunlight. It is known that “gamma rays” emitted from radioactive materials were first observed in 1900 by the French chemist and physicist Paul Ulrich Villard (1860–1934). Villard investigated radiation from radium salts impinging onto a photographic plate from a shielded container through a narrow aperture. He used a thin layer of lead that was already known as alpha-absorber [11]. He was able to show that the remaining radiation consisted of a second and third type of rays. The second type was deflected by a magnetic field similar to the known “canal rays” and could be identified with beta rays described by Ernest Rutherford. The third type, however, was very penetrating and had never been identified before [12]. Being a modest man, he did not suggest a specific name for the type of radiation he had discovered, and in 1903, it was Rutherford who proposed that Villard’s rays should be called gamma rays [13]. It is of great importance to read the following notes written by Becquerel on 2 March 1896: “I will insist particularly upon the following fact, which seems to me quite important and beyond the phenomena which one could expect to observe: The same crystalline crusts (of potassium uranyl sulfate), arranged the same way with respect to the photographic plates, in the same conditions and through the same screens, but sheltered from the excitation of incident rays and kept in darkness, still produce the same photographic images. Here is how I was led to make this observation: among the preceding experiments, some had been prepared on Wednesday the 26th and Thursday the 27th of February, and since the sun was out only intermittently on these days, I kept the apparatuses prepared and returned the cases to the darkness of a bureau drawer, leaving in place the crusts of the uranium salt. Since the sun did not come out in the following days, I developed the photographic plates on the 1st of March, expecting to find the images very weak. Instead, the silhouettes appeared with great intensity …” Becquerel used an apparatus to show that the radiation he discovered was different from X-rays in the way that the new radiation emitted by radioactive materials was bent by the magnetic field so that the radiation was charged. When different radioactive substances were put in the magnetic field, their radiation was either not deflected or deflected in different directions. Becquerel discovered therefore three classes of radioactivity emitting negative, positive, and electrically neutral particles [14]. A story like that of “Roentgen and Lenard” has developed between “Becquerel and Thompson.” In London,

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Professor of Physics Silvanus Thompson (1851–1916), the founding President of the Roentgen Society, had been experimenting with uranium nitrate and at the end of January 1896 (a few weeks before Becquerel) found that when the uranium salt was exposed to sunlight while placed on a shielded photographic plate, film blackening appeared beneath the uranium. Thompson delayed writing a communication to the Royal Society and so he lost the paternity of radioactivity! Becquerel was awarded the 1903 Nobel Prize for Physics jointly with Pierre Curie (1859–1906) and Marie Curie (1867–1934) “in recognition of the extraordinary services he has rendered by his discovery of spontaneous radioactivity.” He received one-half of the Prize with the Curies receiving the other half [15]. The physicist Ernest Rutherford (1871–1937) is often credited as the father of nuclear physics. In his early work, he developed the concept of radioactive materials’ half-life; discovered the radioactive element radon; named the radiation types alpha, beta, and gamma; and classified them by their ability to penetrate different materials. The abovementioned experiments were performed at McGill University in Montreal, Quebec, Canada (Fig.  1.6). In 1903, Rutherford and Frederick Soddy published the “Law of Radioactive Change” to account for all their experiments with radioactive materials. Though the Curies had already suggested that radioactivity was an intra-atomic phenomenon, the idea of the atoms of radioactive substances breaking up was principally new. Until then, atoms had even been assumed to be indivisible (Greek: a-tom), and it was Rutherford and Soddy who demonstrated that radioactivity involved spontaneous disintegration of “radioactive” atoms into other elements. The results of this work provided the basis for the Nobel Prize in Chemistry awarded to Rutherford in 1908 “for his investigations into the disintegration of the elements, and the chemistry of radioactive substances” [16] (Box 1.4).

Box 1.4 Ernest Rutherford

• Ernest Rutherford (1871–1937) is known as the father of nuclear physics. He was the first to suggest the existence of nuclei. • He developed the idea that radioactivity involved spontaneous disintegration of atoms. • In 1908, he was awarded the Nobel Prize in Chemistry “for his investigations into the disintegration of the elements, and the chemistry of radioactive substances.”

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Fig. 1.6  Left: Ernest Rutherford (1871–1937). Right: Rutherford in his laboratory at McGill University (Canada), 1905. (Reproduced with permission)

Fig. 1.7  Marie and Pierre Curie in their Laboratory, circa 1904

Pierre Curie (1859–1906) was a French physicist and a pioneer in crystallography and radioactivity. In 1900, he became Professor at the Faculty of Sciences, University of Paris, and in 1903, he received the Nobel Prize in Physics together with his wife Marie (Fig.  1.7), which they shared with Henri Becquerel. Notably, Marie had been Pierre’s assistant at the City of Paris Industrial Physics and Chemistry Higher Educational Institution (ESPCI Paris).

The term “radioactivity” was coined by Marie Curie, who together with her husband Pierre extracted uranium from pitchblende (uraninite). To their surprise, the leftover ore was more radioactive than pure uranium, and they assumed that other radioactive elements were present in the ore, a hypothesis which resulted in the discovery of the new elements, polonium and radium. However, 4 years of processing tons of the uranium ore had to pass before they isolated enough polonium and radium to determine their chemical properties. It should be noted that one ton of pitchblende contains only about 0.15 g of radium. Pierre Curie and his student Albert Laborde discovered nuclear energy by identifying the continuous emission of heat from radium particles. Incidentally, as early as 1913, H.  G. Wells coined the term “atomic bomb”—a bomb of unprecedented power based on the use of nuclear energy— appearing in his novel “The World Set Free.” It should be mentioned, however, that “his” atomic bomb had nearly nothing in common with the actual atomic bomb created three decades later. The curie (Ci) became the unit of radioactivity, originally named as such by the Radiology Congress in 1910, clearly in honor of Pierre Curie. Corresponding to the activity of about 1  g of radium, the Ci is not a SI unit, and in 1964, it was formally replaced by the becquerel (Bq, this time to honor

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Henri Becquerel), a SI unit which corresponds to one disintegration per second (Box 1.5).

Box 1.5 Pierre and Marie Curie

• Pierre Curie (1859–1906) and his wife Marie Salomea Skłodowska-Curie (1867–1934) discovered the elements radium and polonium. • The term “radioactivity” was coined by Marie Curie. • Pierre Curie discovered nuclear energy by identifying the continuous emission of heat from radium particles. • In 1903, Pierre and Marie Curie were awarded the Nobel Prize in Physics (together with H. Becquerel) for the discovery of radioactivity. • In 1913, H. G. Wells coined the term “atomic bomb” mentioned in his novel “The World Set Free.”

Marie Salomea Skłodowska-Curie, also known as Madame Curie (1867–1934), was a Polish physicist and chemist. She was the first woman to win the Nobel Prize (1903) and the first person to win it twice (1911) in two different scientific fields (physics and chemistry). In July 1898, Pierre and Marie Curie published a joint paper announcing the existence of a new element they named “polonium,” and in December of the same year, they proclaimed the existence of another element, “radium.” Between 1898 and 1902, the Curies published a total of 32 scientific papers including one on the radiobiological effects of “radium rays” on normal and tumor cells [17]. Noteworthy, Mr. and Mrs. Curie did not patent their discoveries and benefited little from the increasingly profitable application of radium for the therapy of various ailments. During World War I, the radiologist Antoine Béclère persuaded Marie Curie to use X-rays for the diagnosis of wounded soldiers on the front lines. She gave her full support to this project and, using her authority as a Nobel Prize winner, organized the Mobile Radiology Units (Fig. 1.8), 20 of which were installed in the first year of the war. She also designed needles containing “radium emanation” to be used for sterilizing infected tissues. The half-life of radium 226 is 1600 years, which is very much shorter than that of uranium (4.5 × 109 years), so radiation of the former is much more intense. Hence, for the study of radioactivity, radium was much more convenient than the very weakly radioactive uranium. The rays emitted by radium proved also to be an excellent tool for exploring the microscopic structure of matter; radium

Fig. 1.8  Marie Curie in a mobile military X-ray unit during the Great War (WWI), circa 1915

became to be used for this purpose already at the end of 1901 (Box 1.6).

Box 1.6 Maria Salomea Skłodowska-Curie

• Marie Salomea Skłodowska-Curie (1867–1934) was the first woman to win a Nobel Prize (1903 in physics) and the first person to win the Nobel Prize twice (1911 in chemistry). • During the Great War (WWI), she focused on the use of radiation to diagnose wounded soldiers. She developed and organized mobile X-ray units, 20 of which she installed in the first year of the war.

While uranium was the first radioactive element to be discovered, radium was much more popular, as it was a spontaneously luminous material that emitted an incredible quantity of radiation. The popularity of radium is shown in a novel by Maurice Leblanc, “The Island of Thirty Coffins,” published in 1919 where a central role is played by a stone “shivering with radium, from where goes steadily a bombardment of invigorating and miraculous atoms.” The research that led to the discovery of radium in 1898 was performed despite considerable difficulties, including inadequate lab and lack of funding. However, Pierre Curie managed to get uranium ore from Bohemia, which at the time belonged to Austria. The help of the Austrian Government, which gave one ton of pitchblende, as well as the help of the chairman of the Austrian Academy of Sciences, was gratefully acknowledged in a letter by Marie Curie, who wrote: “The preparation of radium has been very expensive. We thank the Académie des sciences [...].” After 2 years, however, the Curies became famous, and the situation had improved considerably.

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The collaboration between Pierre and Marie Curie is exemplary in many ways. These two people really complemented each other, as Pierre was dreamy and imaginative, ready to undertake various difficult projects, and Marie was full of energy pursuing her goals. Sadly, Marie Curie died at the Sancellemoz Sanatorium in Passy (Haute-Savoie), France, of aplastic anemia, presumably from exposure to radiation during her scientific research, compounded by her exposure to X-rays in the field radiology units during World War I. Immediately after the discovery of radium and polonium by Marie and Pierre Curie, the latter examined the possibility to use radium as a powerful therapeutic tool [18, 19]. First successful results were obtained in patients with lupus vulgaris, a form of tuberculosis of the skin. For patients with lesions situated in deeper organs, radium salts were used. In 1904, John MacLeod at Charing Cross Hospital designed one of the first glass radium applicators to treat throat cancer [20], and in 1917, Benjamin Barringer used needles containing radium salts for treating prostate cancer [21]. After World War I, a number of technological devices were proposed to treat a wide spectrum of tumors. This therapeutic approach was initially called curietherapy in Europe and brachytherapy in the USA [22]. Along with the first medical applications of X-rays or radium, the first radiation-induced tissue reactions were also observed. In the first decade of the nineteenth century, three major applications of X-rays were developed, namely radiography and radiotherapy, mainly against skin diseases such as lupus rather than cancers, as well as radiation-induced hair removal. From a number of these applications, numerous adverse tissue reactions directly due to radiation have

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been described. In this period, the term “radiodermatitis” was proposed [2]. In 1906, the participants of a Congress of Radiologists organized in Lyon (France) concluded that some patients may show some unexpected skin reactions probably due to radiation [23]. In 1911, the radiologist Léon Bouchacourt, based on the results of the application of radiation treatment for hypertrichosis to a couple of young people, published a paper with a premonitory title: “About the sensitivity to Roentgen Rays of the skin of different individuals and, for a given individual, of the different part of the body” [24, 25]. In this paper, Bouchacourt suggested not only that each individual may show a specific sensitivity to radiation but also that some tissues/organs may be characterized by a specific response to radiation [2]. It is clear that the radiation-­ induced adverse tissue reactions were documented very early and that the notion of individual radiosensitivity is an old concept [25].

1.2.2 Recognition of the Acute Injury The toxicity of X-rays became apparent soon after their discovery by Roentgen (Fig. 1.9). Hair loss has been recognized by May 1896, and skin toxicity was noted a few months later. Early X-ray images required exposures of as long as 80 min, and thus early X-ray workers were among the most severely affected. Dr. Hall-Edwards, the British physician responsible for the first clinical X-ray “photograph” in England in early 1896, developed cancer of the hands from radiation exposure incurred while holding patients’ extremities on photographic plates. In 1896, a commercial demonstrator at Bloomingdale Brothers store in New York, whose X-ray machine ran con-

Fig. 1.9  Radiation injury. (Sources: left—Finzi [26], right) https://wellcomecollection.org/works/g94c5mtb

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tinuously for 2–3 h a day, reported the development of dry skin, followed by changes like a strong sunburn and later scaliness of the skin. He also noted the cessation of fingernail growth and loss of hair from the involved portions of the skin (Box 1.7).

Box 1.7 Radiation Poisoning

• Acute radiation effects (radiodermatitis. etc.) were observed almost immediately after the discovery of ionizing radiation. • In spite of this, the so-called mild radium therapy was extensively misused.

By chance, Roentgen had conducted virtually all his experiments in a zinc box, which gave better definition of the X-ray beam. He had also added a lead plate to the zinc and thus, fortuitously, protected himself from the radiation that he discovered [5]. In 1902, Guido Holzknecht (1872–1931) devised a color dosimeter (“chromoradiometer”) based on the discoloration of crystals after exposing them to X-rays. Holzknecht, like a number of other physicians in the early days of radiology, died from the consequences of radiation “poisoning,” and his name is displayed on the Monument in honor of the X-ray and Radium Martyrs of All Nations erected in Hamburg, Germany [27]. However, these injuries were not initially attributed to X-ray exposures. Nevertheless, formal action to protect from the harmful effects of radiation was required, and in March 1898, a Committee of Inquiry was established by the British Roentgen Society to “investigate the alleged injurious effects of Roentgen rays” [28]. The Committee mentioned explicitly the known adverse effects: skin inflammation, loss of hair, and more it urged collecting information on various effects of X-rays. Right from the first days of the use of radiation, the press reported on the death of “radiological” personnel from cancer, and so European countries and the USA established radiation protection Committees [29]. In 1925, the “First International Congress of Radiology” was organized in London, and it was decided to establish the “International X-ray Unit Committee.” Hence, the ancestor of the “International Commission on Radiation Units and Measurements (ICRU)” was born [30, 31]. Exposure to radium also caused acute injuries. Two incidents are worth mentioning. The first cases of radium “poisoning” were recorded among girls painting the luminous watch dials in the Radium Luminous Materials Company, New Jersey, USA (“the radium girls”). The luminous paint was a mixture of radium salts with zinc sulfide. The work-

Fig. 1.10  A bottle of Radithor—one of the most famous varieties of radium-infused water commercially available in the USA in the 1920s

ers swallowed and inhaled the paint, and this resulted in the death of 18 out of 800 employed workers between 1917 and 1924 [32]. The causes of death were either cancer (probably osteosarcoma of the jaw) or aplastic anemia, necrosis of the jaw, and spontaneous fractures [33, 34]. But it was the death of the wealthy American iron and steel industrialist Eben Byers in 1932 which put an end to the so-called mild radium therapy. His death was attributed to the enormous quantities of Radithor (Fig.  1.10) that he had consumed. Radithor, produced in the Bailey Radium Laboratories in New Jersey and advertised in the newspapers as “Science to cure all the ­living dead,” was commercially available in the USA.  Each bottle contained 1  μCi of 226Ra and 1  μCi of 228 Ra in 16.5 mL of liquid. Byers started drinking Radithor in 1927 and stopped by 1930 when his teeth started to fall out (it was estimated that he had emptied between 1000 and 1500 bottles). Eventually, he died from sarcoma of the upper and lower jaws [35]. This event was probably the reason why the era of the “mild radium therapy” came to an end [36] (Box 1.8).

Box 1.8 Radium Misuse

• Radium was extensively misused before World War II via consumption of various radium-containing products. • The first cases of radium “poisoning” were recorded among the “radium girls” painting the luminous watch dials. • The death of the American millionaire Eben Byers in 1932 seems to be the event that ultimately led to cessation of radium misuse.

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1.2.3 The Law of Bergonié and Tribondeau The so-called fundamental Law of Bergonié and Tribondeau put forward in 1906 postulated that normal tissues appear to be more radiosensitive if their cells are less differentiated, have a greater proliferative capacity, and divide more rapidly. Various data suggest that this law applies to tumors as well. Heinrich Ernst Albers-Schönberg, Jean Alban Bergonié, Claudius Regaud, and Louis Tribondeau made significant contributions to our knowledge of the biological effects of ionizing radiation. Between 1895 and 1908, they studied histological features of irradiated gonads in numerous animal models. Although the law of Bergonié and Tribondeau that links radiosensitivity with proliferation is not generally applicable, the enormous efforts these scientists made to fight cancer by using ionizing radiation should be acknowledged (Box 1.9).

Box 1.9 The Law of Bergonié and Tribondeau

• The “law of Bergonié and Tribondeau” was formulated in 1906 and postulated that normal tissues appear to be more radiosensitive if their cells are less differentiated, have a greater proliferative capacity, and divide more rapidly. • The law of Bergonié and Tribondeau has not been verified. However, it has facilitated the advances in radiation biology and understanding of the relationship between cell proliferation and tissue radiosensitivity.

Fig. 1.11  Bergonié, Tribondeau, and Regaud

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In 1906, Jean Bergonié and Louis Tribondeau published a communication to the French Academy of Sciences about the link between cellular proliferation and response to radiation. According to Bergonié and Tribondeau [37], “X rays act on cells inasmuch efficiently as cells have a greater reproductive activity, their karyokinetic fate is longer, their morphology and function are at least definitively fixed.” While they never used the term “radiosensitivity,” this article has with time been read as “cells are inasmuch radiosensitive as they grow fastly” and is still considered as a founding law of radiation oncology. Today, however, there is evidence that this “law” can be contradicted by numerous counterexamples. An epistemological analysis of the archives of Claudius Regaud, another pioneer of radiation biology and a contemporary of Bergonié and Tribondeau, sheds new light on this law [38]. Let us now briefly review some important facts about the life and work of these three French scientists. Jean Alban Bergonié (1857–1925) (Fig.  1.11) was a physicist and a medical doctor. His expertise in the two areas allowed him to use electrical currents in medical therapy and to develop many new devices based on the discovery of X-rays and radium. In 1911, because of his hitherto intense use of X-rays in the therapy of patients, he developed dermatitis on the right index, and in 1922, his hand (and thereafter his arm) was amputated. Ultimately, he died from lung cancer in 1925 [39]. Of note, Bergonié funded the Journal Archives d’Électricité Médicale where he wrote that X-rays were discovered “simply thanks to the invention of the Crookes tube some 15 years earlier” [39]. In 1906, he expressed the opinion that “there are two

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error types that may affect the medical application of X-rays: (1) the uncertainties in the assessment of radiation dose, … and (2) the differences in the sensitivity of the patients” [23]. Louis Tribondeau (1872–1918) (Fig.  1.11) was born in Sète in Southern France and in 1890 joined the Health Corps of the French Navy. Tribondeau was one of the first histologists who described the microscopic features of tuberculous epididymitis. But he became famous thanks to his staining techniques for bacteriology. In 1918, he died from the Spanish flu [39]. Born in Lyon, France, Claudius Regaud (1870–1940) (Fig.  1.11) studied medicine in Lyon and attended the microbiology lectures at the Pasteur Institute [40]. In 1893, he worked in Lyon in the laboratory of Joseph Renaut, an eminent histologist, known for his staining technique based on mercury. In Renaut’s laboratory, Regaud improved the staining technique of Ehrlich (methylene blue) and developed his own staining method based on ferric hematoxylin, which reveals mitochondria and cytoplasm [40–42]. In 1912, Regaud became head of the Biology Section of the new Radium Institute of Paris, where Marie Curie headed the Physics Section. During World War I, he served as head of an Army Hospital. Not only did he organize the emergency services very effectively, but he also managed multidisciplinary meetings between surgeons, radiologists, hygienists, nurses, and other staff. From 1918 until 1939, he treated thousands of cancer patients and developed a method of fractionated radiotherapy. He died of pneumonia in December 1940 [40]. On August 5, 1895, Regaud presented the new improvements on his staining technique at the Congress of Neurology in Bordeaux [41]. Tribondeau and Bergoni also attended the sessions and had probably read the papers by Regaud in which the histology of the rodent reproductive system was described in detail based on his new staining technique. After the discovery of X-rays by Roentgen in December 1895, two German scientists, H.  E. Albers-Schönberg and H. Frieben, began to study the effects of this type of radiation on spermatogenesis by irradiating testicles of rabbits and guinea pigs [39, 43, 44]. In Bordeaux, Bergonié undertook to reproduce the experiments of the two Germans. As a physicist, he was able to build irradiation devices but, owing to his limited knowledge of histology, he asked Tribondeau for his technical savoir faire [39]. Between 1904 and 1905, Bergonié and Tribondeau published their first observations about irradiated testicles of rats having used Regaud’s staining technique [45]. They emphasized the role of spermatogonia as pluripotent cells and as the most radiosensitive cells of the reproductive system. However, since the experiments involved irradiation with X-rays, interpretation of the data remained ambiguous.

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Regaud realized that there might be misinterpretations of his own technique. Unlike Bergonié, Regaud was a histologist and not a physicist and was helped by Thomas Nogier, a specialist in medical physics. Regaud and Nogier replicated the experiments of Bergonié and Tribondeau using rat models, single exposures, and Regaud’s staining technique [46]. In 1908, Regaud claimed that in young rats, spermatogonia are less radiosensitive than in the adult animals although proliferation rates of these cells are similar in the two groups of rats [47]. However, according to Regaud and Lacassagne [48], Bergonié and Tribondeau generalizations were “imprudently” based on the studies of rat testes. In 1925, Regaud did not hesitate to write about the law of Bergonié and the Tribondeau-Bergonié’s eulogy that “Actual law as so many people believe it? No. But nice formula of the first approximation” [49]. These days, several oncology lectures still cite Bergonié and Tribondeau’s law as a founding principle of radiotherapy according to which tumors are more radiosensitive than healthy tissues due to the higher proliferation rate of the former. In this erroneous claim, three kinds of errors were made: 1. Tumors are not necessarily more radiosensitive than normal tissues. 2. Proliferation rate is not necessarily correlated with the cellular death rate after irradiation. 3. Radiosensitivity and cancer susceptibility to irradiation are two different notions [50]. The link between proliferation rate and radiosensitivity is far from obvious, and the law of Bergonié and Tribondeau should have been modified as follows: “the faster cells proliferate, the faster cell death will appear.” Besides, reviews about the Tpot (the potential doubling time parameter) have shown that the yield of cell death clearly does not correlate with proliferation rate [51, 52]. For example, fibroblasts from ataxia telangiectasia are hyper-radiosensitive, while their proliferation rate is lower than that of fibroblasts from healthy patients [53]. When fibroblasts are transformed by the Simian Virus 40 (SV40), the cells become unstable and their proliferation rate increases while they are less radiosensitive than their non-transformed counterparts [54]. Other counterexamples of the law of Bergonié and Tribondeau are as follows: the Li-Fraumeni syndrome (caused by the p53+/− mutations) confers radioresistance associated, however, with impaired cell cycle arrests, instability, and cancer proneness. Similarly, some highly proliferating tumors may be very radioresistant [55]. To conclude, despite its popularity, the law of Bergonié and Tribondeau has not been fully validated. Yet, it has made a significant contribution to the advances in radiation biology and the relationship between proliferation and radiosensitivity.

1  History of Radiation Biology

1.2.4 Early Optimism and Pessimism The report of the discovery of “mysterious rays” (X meaning unknown) created a great sensation and spread rapidly in many countries: The first report in the press of Roentgen’s feat appeared in Vienna on January 5, 1896, and days later in Germany, England, and the USA [56]. Of all the properties of X-rays, their ability to make the “invisible visible” was the most fascinating and remained for several years the principal topic for their use in the imaging of anatomical and technical objects (Fig. 1.12). The first X-ray machines were large, loud, sparkling, and smelly devices, prone to causing accidents and injury. Such bizarre and sometimes mind-boggling presentations solidified the current public perception of X-rays as a fantastically powerful and yet controversially useful tool. As one of the symbols of the new scientific medicine, X-rays have largely lived up to the public’s expectations of a technological panacea, which was reinforced by the spectacle of their generation and their undeniable effects on the body. This “domestication” of X-ray machines highlighted their failure as modern heroic medicine, while reinforcing at the same time the emerging understanding of radiation as a Fig. 1.12  Cartoon from “Life,” February 1896. The New Roentgen Photography. “Look pleasant, please”

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“subtle, cumulative, and insidious threat” [57, 58] (Box 1.10).

Box 1.10 X-rays Sensation

• The report of the discovery of “mysterious rays” created a great sensation and spread rapidly in many countries. • As one of the icons of the new scientific medicine, X-rays bore much of the public’s expectations for a technological panacea.

In addition to the discovery of X-rays, the year 1895 also saw the death of Louis Pasteur. After a plethora of controversies, the “microbial” theory developed by Pasteur triumphed at the end of the nineteenth century to such an extent that nearly all the diseases were believed to originate from a microbial etiology [59]. This was also the case with cancer, a disease that was already well known, but much less frequent than tuberculosis or diphtheria. The so-called parasitic theory of cancer suggested that tumors arise as a result of infection of tissues by microorganisms. This theory opposed the

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“cellular” theory, which explained carcinogenesis as due to the transformation of one or more cells. Hence, early after the discovery of X-rays, the first experiments involving both X-rays and microbes revealed the biocidal properties of X-rays [60]. In this historical context, Victor Despeignes, a hygienist and physician in a village of Savoy, Les Echelles, France, in February 1896 was visited by a man of 52, who suffered from pain in his abdomen [3, 60] and had been diagnosed with stomach cancer. Convinced by the works of his former colleagues of the Medical Faculty of Lyon, who in March 1896 demonstrated the curative effects of X-rays in patients with tuberculosis [61], in July 1896, Despeignes performed the first anticancer radiotherapeutic trial by irradiating his patient’s tumor with X-rays in two daily sessions. However, although the therapy led to a significant decrease of the tumor volume, the patient died 22 days after the beginning of the treatment. Despeignes described all these observations in two articles in the Lyon Medical Journal [3, 60, 62−64]. The reconstitution of the radiotherapy of Despeignes suggested that his patient did not suffer from a stomach cancer, a rather radioresistant neoplasm, but from gastric lymphoma, possibly the mucosa-associated lymphoid tissue (MALT) lymphoma of a high-grade Burkitt type, which is very radiosensitive. Unfortunately, following the opposition or reservations of his colleagues vis-à-vis the therapeutic properties of X-rays, Despeignes discontinued further trials with X-rays [3, 60]. Emil Grubbe (1875–1960), who received his medical degree in 1898, was allegedly the first American to use X-rays as a treatment for cancer. According to his own report, on January 26, 1896, he treated in Chicago a woman with breast cancer and, the following day, a man suffering from ulcerating lupus [65]. However, the validity of these statements remains questionable for many reasons. Firstly, no death certificates or medical records of Grubbe’s patients have been found. Secondly, these treatments were not described in any peer-reviewed publications. Grubbe did not describe any clinical features potentially resulting from these treatments [65]. In August 1896, Leonhard Voigt irradiated in Germany a cancer of the nasopharynx, but, as in Grubbe’s case, the records of this treatment cannot be validated [65]. The first radiation treatment considered to be successful was given in 1897 in Germany by Eduard L. Schiff to a patient suffering from erythematous lupus [66, 67]. While the X-rays generated by the Crookes tubes manufactured in the first two decades of the twentieth century were too “soft” to fully permeate the tumorous tissue, the later technological advances permitted Claudius Regaud and Antoine Lacassagne to p­ erform in the 1930s the first series of anti-

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cancer radiotherapy at the Curie Institute in Paris, France [2] (Box 1.11).

Box 1.11 Radiology

• Counterintuitively for the modern reader, ionizing radiation was initially used mostly for treatment rather than for diagnosis. • Development of diagnostic radiology remained slow till the outbreak of the Great War (WWI) in 1914.

The development of diagnostic radiology remained slow until about 1914, when two incidents precipitated its growth: the invention in 1913 of a new type of the cathode tube by the American physicist W.  D. Coolidge (1873–1975) and the beginning of the Great War (World War I) associated with the need for medical assistance to the wounded soldiers. Beginning from the 1920s, X-rays were used regularly for the detection of pulmonary tuberculosis. Before that, the “radiologists” were almost no more than “photographers.” “Thanks to” tuberculosis, the “photographers” became skilled diagnosticians and thus the medical specialty of radiology emerged. Noteworthy, the Roentgen Society founded in London in November 1897 was in 1927 renamed the British Institute of Radiology; in 1931, the section of Radiology was established at the Royal Society of Medicine; and in 1934, the British Association of Radiologists was founded (5  years later, it was renamed the Faculty of Radiologists). At that period, radiology was faced with two problems: First, physicians regarded radiology as an intruder in their territory and contrasted the “dead photograph” with the “living sound” of auscultation, and second, the images obtained were of poor quality because all the anatomical structures were superimposed. To overcome this latter problem, B. G. Ziedses des Plantes (1902–1933) built the first machine for planigraphy, in which the X-ray tube and the film moved together around the plane of interest allowing to reconstruct an arbitrary number of planes from a set of projections. He also designed the subtraction method to improve images after the injection of contrast agents [68]. The history of radiation therapy (radiotherapy) can be traced back to experiments made just after the discovery of X-rays, when it was shown that exposure to ionizing radiation may lead to cutaneous burns. In 1902, several physicians began the systematic use of radiation for the treatment of malignant tumors. The increased use of electrotherapy and escharotics (the medical application of caustic substances) inspired doctors to use radiation for the treatment

1  History of Radiation Biology

of nearly any disease—lupus, basal cell carcinoma, epithelioma, tuberculosis, arthritis, pneumonia, and chronic ear infections (https://www.cdc.gov/nceh/radiation/nri/ patientinfo.htm; [4, 69, 70]). Active use of ionizing radiation for treatment of various diseases continued until the early 1960s. Since then, radiation therapy has been used nearly exclusively in cancer therapy. Two factors contributed to phasing out of radiotherapy for non-oncological purposes: the growing awareness of the radiation-induced carcinogenesis and the development of efficient drugs, primarily, antibiotics (Box 1.12).

Box 1.12 Radiation Therapy

• Ionizing radiation was successfully used for the treatment of numerous diseases until the early 1960s. • Since then, radiation therapy has been used almost exclusively in cancer therapy. • Two factors contributed to phasing out of radiotherapy for non-oncological purposes: the growing awareness of the radiation-associated carcinogenesis and the development of efficient drugs.

Until 1920, patients with cancer were treated mainly by surgeons who assumed that the mechanism of radioactivity involved a “caustic effect.” At that time, when the sources of X-rays produced “weak” radiation, capable of only superficial penetration, it was logical that it was dermatologists who strived to use X-rays in therapy. The crucial experiments performed by Robert Kienböck (1871–1953) entailed the proof that an X-ray dose, rather than electric phenomena, was the active agent causing biological effects when “illuminating the skin using Roentgen tubes” [71]. In the 1910s and 1920s, radiobiology was at its infancy, based mainly on empirical observations of the effects of radiation on the skin. The technical progress made with the Coolidge tubes and the higher voltage that these tubes could be operated with introduced the techniques of the “deep X-ray treatment.” The first radiotherapy textbook titled “Treatment of Cancer by Radium” was authored by surgeon Sir Stanford Cade and appeared in 1928 [72]. At the same time, the Scottish radiotherapist Ralston Paterson (1897–1981) who used X-rays for the treatment of lung cancer wrote, “In cases of true primary carcinoma of the lung, surgery as yet offers little hope of relief … A group of nineteen patients treated by high-voltage roentgen rays is reported. All died within ten months, all but three within four months. This brief period of survival is the

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same as that in a group of cases in which there was no treatment. Although life is not prolonged, roentgen-ray treatment in all, but advanced cases give marked temporary palliation” [73]. In 1929, the pioneer Swedish radiotherapist Gösta Forssell (1876–1950) delivered the tenth Mackenzie Davidson Memorial Lecture and summarized the current state of radiotherapy [74, 75]. Figure 1.13 shows a table from Forssell’s summary. In 1896, less than a year after the discovery of X-rays, Walter Levitt wrote on modern developments in X-ray therapeutic techniques and stressed that it is Leopold Freund from Vienna to whom “belongs the credit of having carried out the first X-ray treatment.” Freund had noticed that epilation was one of the most constant effects of the exposure to X-rays, and when a patient with a hairy mole on the face came to him for advice, he conceived the idea of treating it with X-rays [76]. At about the same time, Robert McWhirter from Edinburgh wrote on the radiosensitivity in relation to radiation intensity. Frank Ellis from the Sheffield National Radium Centre during his long life (1905–2006) also contributed to the development of radiotherap; in June 1939, he reported on the radiosensitivity of malignant melanoma [77, 78]. Other publications of this period on the use of radium include illustrations of masks holding the radium needles applied to the skin (Fig. 1.14) and tubes containing radium for the internal use in cervical cancer [79]. Concurrently, the late effects of radiation on the skin were studied and reported in detail, and plastic surgery was applied to the treatment of radiodermatitis and radionecrosis [26, 80]. At this gestational period, the pioneers of radiotherapy did not really know (a) what doses to use and how to measure them and (b) what are the advantages and disadvantages of using single or fractionated doses of X-rays. The concept of fractionation of the X-ray treatment was introduced by Claudius Regaud from the Foundation Curie in Paris and his brilliant collaborator Henri Coutard at the first International Congress of Radiology held in 1925 in London. Still, well into the 1930s, most radiotherapists were not convinced that fractionated therapy was superior to the single-dose schedule. With the establishment of the fractionation as standard treatment, radiotherapy ceased to rely solely on clinical observation, without rigid, preconceived planning, and began to be based on detailed physical modeling and dosimetry, to avoid as much as possible the irradiation of healthy tissues. This required a very close cooperation between ­radiotherapist and radiophysicists and led to the birth of two new disciplines, radiobiology and medical physics [81].

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Only Radiotherapy. (Results obtained at Radiumhemmet.)

Cases treated between

1910–1915

Total No. of cases.

. .

Total No. of cases treated

1916–1921

Carcinoma labii . Total No. of cases treated Operable cases without glandular metastases

.

Operable prim. tumours Local recurrences

. .

Inoperable prim. tumours

. . . . . .

Ca. linguae without apparent metastases Carcinoma cervicis uteri Total No. of cases examined Total No. of cases treated Operable and border-line cases 1913–1921

. .

. .

. . . .

207

142

69%

182

142

78%

. . . .

66 52

45

68%

45

86%

. . . .

. . . . . .

113 68

21 21

18% 31%

. . . .

. . . .

29

16

55%

19

4

21%

20

1

5%

11

6

60%

. . . . . .

. . . . . .

. . . . . .

. . . . . .

790

163

20·6%

737

163

21·1%

188

76

40·4%

. .

. .

. . . .

46 25

20 15

43·5%

. . . .

. . . .

. . . .

238 154

58 28

24% 18%

Carcinoma corporis uteri Total No.of cases examined (all treated) Operable and border-line cases

1910–1922

Percentage.

Carcinoma oris (Ca. linguae; ca. subling.; ca. mandib.; ca buccae) . . Total No. of cases treated Cases without glandular metastases

1914–1923

Number.

Carcinoma cutis Operable cases without glandular metastases

1910–1917

Cases cured.

Sarcomata . . Primary tumours Recurrences after operation

. .

Fig. 1.13  Summary of the effects of radiotherapy of cancer performed in Sweden between 1910 and 1923 [75]

60%

1  History of Radiation Biology

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Wintz who preferred to apply high doses in a short period of time (intensive radiotherapy) [4]. Particularly, Henri Coutard suggested that high doses per fraction should be avoided due to the damage they caused to the connective tissues [83]. Coutard applied the concept of fractionated radiotherapy with treatment courses protracted over several weeks. With this strategy, Coutard managed to cure patients with various head and neck malignancies that are difficult to treat even today. It should be noted that the French radiotherapist was among the first to recognize that tumors of different histologies vary in their sensitivity to radiation. These observations led to the conclusion that radiation oncologists should protract the treatment duration to spare healthy tissues while increasing the dose per fraction to kill a tumor. Obviously, the current standard fractionation scheme of 1.8–2 Gy per fraction five times per week originated from individual observations of patients and empirical experience rather than from a purely scientific basis [84].

1.3.2 Cure with Fractionated Treatment Fig. 1.14  Mask to hold the radium needles for treatment of skin cancer [79]

1.3 Development of Fractionation in Radiotherapy 1.3.1 Early Fractionation As mentioned above, Victor Despeignes in his historical attempts applied a bi-fractionated radiotherapy based on the hypothesis that the dose should not be too high to spare healthy tissues. Fractionated treatments can be traced back to the first trials performed by Leopold Freund in 1896  in Vienna, Austria. Today, Freund is considered the founder of medical radiology and radiotherapy [3, 82]. During the first decade of the twentieth century, many different anticancer strategies involving ionizing radiation were applied to treat various tumors. However, the energy of X-rays provided by the available tubes was limited to some tens of kilovolts, and therefore the radiation penetration into the body was very limited. Between the 1920s and 1930s, pioneers from the “French school” at the Institut Curie in Paris led by Henri Coutard, Claudius Regaud, and Juan A. del Regato showed that hypofractionation might lead to severe tissue reactions and promoted the hyperfractionated regimen by spreading the delivery of the dose over a longer period of time. In 1911, Claudius Regaud showed that a ram’s testes could be sterilized without causing major burns to the scrotal skin if three irradiations were delivered 15 days apart. This practice was opposed to the “German school” led by Holzknecht and

The technological race to produce the highest X-ray energies permitted the cure of the deepest tumors and helped in extending the application of hyperfractionated treatments to various cancers. For instance, the first electrostatic generator, developed by Robert van de Graaff in 1929, permitted the installation at the Huntington Memorial Hospital Boston, MA, USA, of a 2 MV irradiator dedicated to radiotherapy, and the first treatments with 60Co source began there in 1951. Two years later, the first 4 MV double-gantry linear accelerator (linac) was installed at the Newcastle Hospital in the UK [4] (Box 1.13).

Box 1.13 Evolution of Radiation Therapy

• The first fractionated radiation treatment was performed in 1896. • Accelerator-based therapy has been performed since 1929 (with 2 MV electrostatic accelerator). • Treatments with the 60Co source emerged in 1951.

With these technological advances, the early and late post-radiotherapy tissue reactions were more and more accurately documented and standard current hyperfractionated treatments were progressively defined for all types of tumors. In 1967, Frank Ellis developed the so-called Strandqvist’s concept and suggested a formula defining the nominal standard dose (NSD) [85, 86]. Many variant formulas derived from the original one have since been devised [87]. Unfortunately, while the NSD formula has had a significant influence on clinical practice and was successful in predict-

18

ing isoeffective regimens for the early effects, it dramatically failed in the prediction of severe late effects after the large-­ dose fractions. Progressively, the use of the parameters of the linear quadratic (LQ) model permitted a better approach to guide clinicians in their choice of the dose fractionation regimen [88]. Today, the generally accepted model explaining both early and late effects consists of four independent processes that are thought to occur between fractions and favor the survival of normal tissues over cancers: (a) repair of sublethal cellular damage, (b) redistribution of tumor cells from radioresistant (late S phase) into radiosensitive (G2M) portions of the cell cycle, (c) reoxygenation of the hypoxic (and hence radioresistant) portions of tumors, and (d) migration of normal cells into the irradiated healthy tissues close to the tumor to repopulate them with new functional cells. Recently, the debate about dose hypofractionation has been relaunched with the advent of stereotactic technologies that permit targeting the tumor with great precision, limiting therefore the exposure of healthy tissues surrounding the tumor. Particularly, anticancer treatments with stereotactic radiosurgery (SRS) and stereotactic body radiation therapy (SBRT) are based on the combination of a high-precision tumor targeting with hypofractionation [89]. Cyberknife (Accuray Incorporated, Sunnyvale, CA, USA) is one of the most recent and innovative techniques developed for the SBRT. It is a robotic system delivering many (usually a hundred) independent and noncoplanar beams converging onto the tumor with sub-millimetric accuracy under continuous X-ray image guidance [90]. Studies have shown the efficiency and safety of the SRS and SBRT techniques in many instances, including some involving the Cyberknife. Still, however, owing to the lack of a clear radiobiological mechanistic model that will define objective criteria, no consensus about the total dose, dose per fraction, and treatment duration has been achieved [89].

1.4 Development of the Therapeutic Ratio In 1936, the German radiologist Hermann Holthusen (1886– 1971) considered the effect of a radiation dose on the probability of controlling tumor and the development of normal tissue complications [91]. By 1975, this concept was formalized and further developed. Nowadays, the ultimate objective of radiation therapy is to control tumors without causing excessive normal tissue toxicity. The term “therapeutic ratio” defines the relationship between the tumor control probability (TCP) and the likelihood of normal tissue damage—normal tissue complication probability (NTCP). The difference

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Fig. 1.15  Therapeutic window

between TCP and NTCP is called a “therapeutic ratio” or “therapeutic window” (Fig. 1.15) (Box 1.14).

Box 1.14 Therapeutic Window

• The ultimate objective of radiation therapy is to control tumor growth without causing excessive damage to normal tissues. • Tumor control probability (TCP) and normal tissue complication probability (NTCP) depend differently on the radiation dose. • The difference between TCP and NTCP is called “therapeutic ratio” or “therapeutic window.

Clinical studies have validated the benefit of contemporary irradiation techniques for improving the therapeutic ratio. Large meta-analyses have shown that concurrent radioand chemotherapy improves local control in many types of cancer. Clinical trials using molecularly targeted therapies have not yielded satisfactory results yet. The notable exception is head and neck squamous cell carcinoma treated with combined radiotherapy and cetuximab. Noticeably, irradiation of normal tissue should not be viewed only as a source of toxicity, because both the abscopal and bystander effects (discussed in Chap. 2) suggest that such irradiation may also result in therapeutic outcomes [92–95]. Today, clinical strategies enhancing the efficacy and decreasing the toxicity of radiotherapy, i.e., increasing the overall therapeutic window, are of paramount importance and there is demand for novel radiation sensitizers that are expected to scale up the window. This is especially important for tumors characterized by high probability of recurrence, such as locally advanced lung carcinoma, and head

1  History of Radiation Biology

and neck and gastrointestinal tumors. Molecular target therapies with identified mechanisms of action should be given top priority. Examples include targeting cell survival and proliferation signaling such as the EGFR and PI3K/ AKT/mTOR pathways, DNA repair genes including PARP and ATM/ATR, angiogenic growth factors, epigenetic regulators, and immune checkpoint proteins. By manipulating various mechanisms of tumor resistance to ionizing radiation, targeted therapies hold significant value to increase the therapeutic window of radiotherapy. Furthermore, the use of novel nanoparticle-based therapies, such as nanoparticle delivery of chemotherapies, metallic (high-Z) nanoparticles, and nanoparticle delivery of targeted therapies, may improve the therapeutic window by enhancing the tumor response to ionizing radiation and/or reducing normal tissue toxicity [96].

1.5 Radiation Epidemiology and Radiation Carcinogenesis Radiation effects can be divided into early and late outcomes. Another classification is into deterministic and stochastic effects. The most common radiation-induced deterministic injuries include skin burns and cataracts. Since these effects occur after absorption of high doses of radiation, they can be easily avoided by adherence to the rules of radiological protection. The most important stochastic effect of significant irradiation is malignancy. Data suggest an elevated risk from medical radiation [97], especially with the highest exposures [98]. As mentioned earlier, biological effects caused by X-rays and radium were noted very soon after the discoveries of Roentgen, Becquerel, and the Curies. Early pathologies, such as radiation dermatitis and hair loss (epilation, alopecia), led to the birth of radiobiology and prompted scientists to follow up patients for long periods of time to study late effects of irradiation as well. While radiosensitivity reactions require rather high doses, exposure to ionizing radiation may also induce cancer [50]. The first radiation-induced cancer was reported by Frieben in 1902 on his own hand [99]. Cancers, but also leukemia, were mainly diagnosed in the pioneers of radiation. Hence, the incidence of radiation-induced cancers among clinicians manipulating X-ray tubes increased drastically [13]. Before the Second World War, a cohort of hundreds of female workers (“the radium girls”—see Sect. 1.2.2) in watch factories in New Jersey, Illinois, and Connecticut between 1917 and 1924 contracted some radiation-induced tumors probably due to self-luminous paintings containing radium [32]. This episode had a major

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societal, ethical, and legal impact in the USA and in the world. This period was contemporary with the organization of the first world congresses of radiology from which the International X-ray and Radium Protection Committee (IXRPC) arose and the first radiation protection recommendations were proposed [13]. Regarding epidemiology, radio-induced cancers were observed historically in pioneers of ionizing radiation, later in patients from various medical cohorts [97], and then in the atomic bomb survivors [100]. In the 1920s, the American geneticist, Hermann Joseph Muller, who irradiated fruit flies (Drosophila melanogaster) with large doses of X-rays, discovered radiationinduced mutations [101]. At that time, geneticists were convinced that no mechanism for gene repair existed and therefore that mutagenic damage was cumulative. From their point of view, no tolerant dose could ever be set, and the safety level should only be weighed against the cost of achieving it [102]. In 1946, Muller was awarded the Nobel Prize for his discovery, and in his Nobel Prize Lecture, he argued that the dose-­response for radiation-induced mutations was linear and that there was “no escape from the conclusion that there is no threshold dose” [103]. This statement may be ethically questionable since Muller was already aware of counterevidence when he delivered his lecture [104]. After the Hiroshima and Nagasaki bombings, geneticists were concerned that exposure to radiation from the nuclear fallout would likely have devastating consequences on the gene pool of the human population. Later (at the end of the 1950s), after no radiation mutagenesis was found in the A-bomb survivors’ descendants [105], carcinogenesis became the main concern. During the next decades, there was considerable controversy and both logical and circular arguments were exchanged. It has been said that among scientists, “the data to support the linearity at low dose perspective were generally viewed as lacking, but the fear that they may be true was a motivating factor” [102]. • The linear no-threshold (LNT) model for radiation risk assessment gradually gained ground after Muller’s Nobel lecture. In 1956, the ICRP officially abandoned the tolerance level concept (that was in use since 1931) and substituted LNT for it. The latter model suggests that any radiation exposure presents carcinogenic risk and that the risk is proportional to the absorbed dose of radiation. Formally, LNT has been introduced and remains a practical operational model only for radiation protection. Alas, contrary to the plethora of the existing evidence [106], this hypothesis has acquired de facto the status of a scientific theory and remains the driving

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force of the prevailing radiophobia in the society (Box 1.15).

evaluation of the entire field of thyroid cancer epidemiology [115] (Box 1.16).

Box 1.15 LNT

Box 1.16 Secondary Cancers

• The linear no-threshold (LNT) model for radiation risk assessment was introduced following Muller’s discovery of radiation-induced mutations in 1927. • Evidence supporting LNT is inconclusive at very low doses.

• As a rule, secondary cancers appear near the high-­ dose treatment volume; this is a major argument supporting their radiation origin. • Cancer patients in general are at a high risk for developing secondary neoplasms. Radiotherapy is probably responsible for only 8% of the secondary cancers. • The primary carcinogenic factors—genetic, lifestyle, and environmental—increase the risk of the radiation-induced and secondary cancer. Individual radiosensitivity may play a crucial role. • The relative risk of radio-induced cancer is organ dependent. It has been assumed that the thyroid is by far the most radiosusceptible organ; however, the recently acknowledged problem of thyroid cancer overdiagnosis requires re-evaluation of the entire field of thyroid cancer epidemiology.

Over the last decades, the attitude to risk associated with ionizing radiation has become more sensible. We now know that exposures to low doses of radiation initiate cellular and intercellular changes leading to stress-induced adaptive responses and metabolic alterations. Furthermore, repair mechanisms preventing the accumulation of damage—also of non-radiogenic origin—were also discovered [107]. Consequently, it became obvious that while high doses of ionizing radiation certainly cause harm, low doses can be beneficial for human health; such an effect is called hormesis [108], but the circumstances in which hormesis might occur in humans are not known. Recently, the so-called secondary neoplasms which appear in patients treated with radiotherapy for a primary tumor have become the focus of interest in the studies of radiation-induced cancer [109]. It is still not clear whether secondary cancers are triggered by radiation or other factors. Characteristic features of these cancers are as follows: • •

•

•

Various epidemiological studies indicate an association between cancer and previous exposure to ionizing radiation even at rather low doses. Most studies do not consider the potential medical exposures of people, as in the case of the A-bomb survivor studies. Although these studies do not establish a link between exposure to ionizing radiation and cancer, the existence of a dose-effect relationship, when it can be established, is in favor of a possible link. The risk As a rule, they appear near the high-dose treatment vol- evaluation thus requires that dosimetry should be precisely ume, which supports their radiation origin [110]. and accurately monitored. These epidemiological observaCancer patients are at a high risk in general for develop- tions give consistency to the linear no-threshold (LNT) ing secondary malignancies [111]. It has been estimated ­relationship, which has been used for regulatory purposes in that radiotherapy is responsible for only about 8% of the radiological protection, although, as mentioned above, it has secondary cancers [112]. no indisputable scientific basis [116]. The usual confounding factors of carcinogenesis (genetic, Radiation-induced carcinogenicity stems from the fact lifestyle, environmental, etc.) increase the risk of the sec- that ionizing radiation is one of the causes of the DNA ondary and radiation-induced cancer. Individual radiosen- lesions. Each DNA insult when unrepaired, particularly in sitivity may play a major role [3]. persons with an abnormal DNA damage response (DDR), The relative risk of radio-induced cancer is organ depen- contributes to the overall DNA dysfunction and paves the dent, the thyroid being by far the most radiosusceptible way to oncogenesis [117]. Abnormal DDR has been reported organ [113]; however, the recently acknowledged prob- following low-dose exposures to X-rays [118]. However, lem of thyroid cancer overdiagnosis [114] demands re-­ multiple repair and defense mechanisms operating at the

1  History of Radiation Biology

molecular, cellular, tissue, and organismal levels may assure the effective elimination of potentially carcinogenic cells and may make the LNT model irrelevant to the biological reality [107]. To conclude, the responsibility of high-dose ionizing radiation in the stochastic appearance of cancers is certain. However, it is very likely that there are no radio-induced cancers at low doses and low dose rates in the sense that they would be due to the sole ionizing radiation. However, low doses of ionizing radiation and of other genotoxic stressors (exposomes) should not be examined independently from each other (Box 1.17). Box 1.17 Radio-Induced Cancers

• High-dose ionizing radiation can be associated with the stochastic appearance of cancers. • It is likely that exposures to low doses of ionizing radiation are not alone responsible for radio-­ induced cancers. • Low doses of ionizing radiation and other genotoxic stressors should not be examined independently from each other.

1.6 Exercises and Self-Assessment Q1. Who made and when were made the major discoveries in the field of ionizing radiation? Q2. What is the basis for conclusion about the carcinogenic effects of ionizing radiation? Q3. (Open question) How was ionizing radiation misused in the first third of the twentieth century? What were the main events that led to cessation of the misuse? Q4. (Open question) What were the main stages in the development of radiation therapy?

1.7 Exercise Answers QA1. Wilhelm Roentgen, Henry Becquerel, Pierre and Marie Curie, and Ernest Rutherford laid the foundations of understanding the ionizing radiation from 1895 until the beginning of the Great War (1914). QA2. (a) Historical observations (b)  Epidemiologic studies, especially with the cohort of atomic bomb survivors of Hiroshima and Nagasaki (c) Basic understanding of the cellular mechanism regarding DNA insults and DNA damage response

21

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Further Reading Berger H. The mystery of a new kind of rays. Scotts Valley: CreateSpace; 2012. Bernier J, editor. 1895–1995: Radiation oncology: a century of progress and achievement. ESTRO: Lambert; 1995. Mould FM.  Radium history mosaic. Warsaw: Nowotwory Journal of Oncology; 2007. Thomas AMK, Isherwood I, Wells PNT. The invisible light—100 years of medical radiology. Oxford: Blackwell Science; 1995. Weinberg RA.  The biology of cancer. New  York: Garland Science; 2014.

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Basic Concepts of Radiation Biology Ans Baeyens, Ana Margarida Abrantes, Vidhula Ahire, Elizabeth A. Ainsbury, Sarah Baatout, Bjorn Baselet, Maria Filomena Botelho, Tom Boterberg, Francois Chevalier, Fabiana Da Pieve, Wendy Delbart, Nina  Frederike Jeppesen Edin, Cristian  Fernandez-Palomo, Lorain Geenen, Alexandros G. Georgakilas, Nathalie Heynickx, Aidan D. Meade, Anna Jelinek Michaelidesova, Dhruti Mistry, Alegría Montoro, Carmel Mothersill, Ana Salomé Pires, Judith Reindl, Giuseppe Schettino, Yehoshua Socol, Vinodh Kumar Selvaraj, Peter Sminia, Koen Vermeulen, Guillaume Vogin, Anthony Waked, and Anne-Sophie Wozny

A. Baeyens (*) Radiobiology, Ghent University, Ghent, Belgium e-mail: [email protected]

T. Boterberg Department of Radiation Oncology, Ghent University Hospital, Ghent, Belgium

A. M. Abrantes Institute of Biophysics, Faculty of Medicine, iCBR-CIMAGO, Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal

Particle Therapy Interuniversity Center Leuven, Department of Radiation Oncology, University Hospitals Leuven, Leuven, Belgium e-mail: [email protected]

ESTESC-Coimbra Health School, Instituto Politécnico de Coimbra, Coimbra, Portugal e-mail: [email protected]

F. Chevalier UMR6252 CIMAP, Team Applications in Radiobiology with Accelerated Ions, CEA-CNRS-ENSICAEN-Université de Caen Normandie, Caen, France e-mail: [email protected]

V. Ahire Chengdu Anticancer Bioscience, Ltd., and J. Michael Bishop Institute of Cancer Research, Chengdu, China E. A. Ainsbury Radiation, Chemical and Environmental Hazards Directorate, UK Health Security Agency, Oxford, UK e-mail: [email protected] S. Baatout · D. Mistry · K. Vermeulen Institute of Nuclear Medical Applications, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium e-mail: [email protected]; [email protected] B. Baselet Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium e-mail: [email protected] M. F. Botelho · A. S. Pires Institute of Biophysics, Faculty of Medicine, iCBR-CIMAGO, Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal Clinical Academic Center of Coimbra, Coimbra, Portugal e-mail: [email protected]; [email protected]

F. Da Pieve Royal Belgian Institute for Space Aeronomy, Brussels, Belgium European Research Council Executive Agency, European Commission, Brussels, Belgium W. Delbart Nuclear Medicine Department, Hôpital Universitaire de Bruxelles (H.U.B.), Brussels, Belgium e-mail: [email protected] N. F. J. Edin Department of Physics, University of Oslo, Oslo, Norway e-mail: [email protected] C. Fernandez-Palomo Institute of Anatomy, University of Bern, Bern, Switzerland e-mail: [email protected] L. Geenen Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Department of Radiology and Nuclear Medicine, Erasmus Medical Center, Rotterdam, The Netherlands

© The Author(s) 2023 S. Baatout (ed.), Radiobiology Textbook, https://doi.org/10.1007/978-3-031-18810-7_2

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A. G. Georgakilas DNA Damage Laboratory, Physics Department, School of Applied Mathematical and Physical Sciences, National Technical University of Athens (NTUA), Athens, Greece e-mail: [email protected] N. Heynickx Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Department of Molecular Biotechnology, Ghent University, Ghent, Belgium e-mail: [email protected] A. D. Meade School of Physics, Clinical and Optometric Sciences, Faculty of Science, Technological University Dublin, Dublin, Ireland e-mail: [email protected]

G. Schettino National Physical Laboratory, Teddington, UK e-mail: [email protected] Y. Socol Jerusalem College of Technology, Jerusalem, Israel e-mail: [email protected] V. K. Selvaraj Department of Radiation Oncology, Thanjavur Medical College, Thanjavur, India P. Sminia Department of Radiation Oncology, Amsterdam University Medical Centers, Location Vrije Universiteit/Cancer Center Amsterdam, Amsterdam, The Netherlands e-mail: [email protected]

A. J. Michaelidesova Nuclear Physics Institute of the Czech Academy of Sciences, Rez, Czech Republic

G. Vogin Centre Francois Baclesse, University of Luxembourg and Luxembourg Institute of Health, Luxembourg, Luxembourg e-mail: [email protected]

Czech Technical University, Faculty of Nuclear Sciences and Physical Engineering, Prague, Czech Republic e-mail: [email protected]

A. Waked Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium

A. Montoro Radiological Protection Service, University and Polytechnic La Fe Hospital of Valencia, Valencia, Spain e-mail: [email protected]

Laboratory of Nervous System Disorders and Therapy, GIGA Neurosciences, Université de Liège, Liège, Belgium e-mail: [email protected]

C. Mothersill Faculty of Science, McMaster University, Hamilton, Canada e-mail: [email protected]

A.-S. Wozny Cellular and Molecular Radiobiology Lab, UMR CNRS 5822, Lyon 1 University, Oullins, France

J. Reindl Section Biomedical Radiation Physics, Institute for Applied Physics and Measurement Technology, Universität der Bundeswehr München, Neubiberg, Germany e-mail: [email protected]

Department of Biochemistry and Molecular Biology, Lyon-Sud Hospital, Hospices Civils de Lyon, Pierre-Bénite, France e-mail: [email protected]

Learning Objectives

• To understand what radiation is, how the different types of radiation differ, and how the energy is transferred to matter • To describe the natural and artificial sources of ionizing radiation to which we are exposed • To understand the principles of radioactive decay, the production of artificial radioactive isotopes, and some important aspects of their environmental and clinical applications • To describe the different dose quantities and units used to describe radiation • To understand the concept of linear energy transfer (LET) and ionization clustering and how these are

used to describe the relative biological effectiveness (RBE) • To understand how ionizing radiation induces biological effects following energy deposition within biological tissues • To understand the different types of health effects following different ionizing radiation doses and exposure scenarios • To explain the factors influencing the results of low doses and introduction of the concept of targeted and non-targeted radiation effects

2  Basic Concepts of Radiation Biology

2.1 Physical and Chemical Aspects of Radiation Interactions with the Matter 2.1.1 Matter and Energy There exists a wide variety of different types of particles in nature. These vary across those more commonly known, such as the constituents of atoms like electrons spinning around nuclei and protons and neutrons inside the nuclei. Particles generated through other particles’ decay and those which are the carriers of the fundamental electromagnetic, strong and weak nuclear, and gravitational force are also incredibly important in nature. In physical science, a particle is characterized either as a localized entity which can be described by its own physical characteristics such as volume, density, and mass or as a wave, the latter being a less intuitive concept. Such dual nature of particles is named the wave-particle duality. The de Broglie wavelength associated with a particle is inversely proportional to its momentum, p, through the Planck constant, h:

λ=

h h h = ( photons ) = ( particles with mass ) . (2.1) p E/c m⋅v

When particles interact with objects much larger than the wavelength of the particles themselves, they show negligible interference effects. To get easily observable interference effects in the interaction of particles with matter, the longest wavelength of the particles and hence the smallest mass possible are needed. The wavelengths of high-speed electrons are comparable to the spacings between atomic layers in crystals. Therefore, this effect was first observed with electrons as diffraction, a characteristic wave phenomenon, in 1927 by C.J.  Davisson and L.H.  Germer [1] and independently by G.P. Thomson [2]. Such experiments established the wavelike nature of electron beams, providing support to the underlying principle of quantum mechanics. Thomson’s experiment of a beam of electrons that can be diffracted just like a beam of light or a water wave is a well-known case taught in basic courses of quantum mechanics [3]. For electromagnetic radiation for energies E = hc/λ of a few keV, the wavelength λ becomes comparable with the atomic size. At this energy range, photons can be practically considered as particles with zero mass and momentum p = E/c. Indeed, despite photons having no mass, there has long been evidence that electromagnetic radiation carries momentum. The photon momentum is, however, very small, since p  =  h/λ and h is very small [6.62606957  ×  10−34 (m2 kg/s)], and thus it is generally not observed. Nevertheless, at higher energies, starting from hard X-rays (which have a small wavelength and a relatively large momentum), the

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effects of photon momentum can eventually be observed. They were observed by Compton, who was studying hard X-rays interacting with the lightest of particles, the electron. On a larger scale, photon momentum can have an effect if the photon flux is considerable and if there is nothing to prevent the slow recoil of matter due to the impinging and conservation of the total momentum. This may occur in deep space (a quasi-vacuum condition), and “solar” sails with low mass mirrors that would gradually recoil because of the impinging electromagnetic radiation are actually being investigated and tested to actually take spacecraft from place to place in the solar system [4–6]. While for photons the concept of wavelength is more intuitively directly related to the phenomena and excitations they can trigger in matter, for particles with mass (massive particles), the wavelength is usually too small to have a practical impact on our observation of interaction phenomena. Nevertheless, depending on the phenomenon or on the specific aspect one is looking at, it may be more convenient to consider the particles either as localized entities or in terms of waves. Understanding the phenomenon of the passage of charged particles, in particular protons and other hadrons, heavy ions, electrons, and neutral particles, such as neutrons and photons, in matter has been a tempting and fascinating topic since the early development of quantum mechanics. The study of the passage of a particle through matter requires knowledge of the many interactions that govern the response of the target to the incoming (strong or weak) particle in the target itself. The number of these interactions is daunting, especially for the case of high-energy particles. In principle, to understand the types of possible particle-matter interactions and thus the response of the matter to radiation, it is more appropriate to consider the speed of the particle rather than the energy. The energy is less meaningful as the high energy of a heavy ion may be associated mostly to its mass, rather than purely to its speed. It is nevertheless common also to refer to the kinetic energy of the particle when looking at the induced interactions a particle can have when traveling through matter, distinguishing the particles with different mass. The interaction of a massive particle with matter can be understood by looking at Fig. 2.1, where the particle’s kinetic energy is plotted against the de Broglie wavelength, and the relevant dimensions of a nucleon, nucleus, electron orbitals, and water molecule (O–H distance) are reported. At high-projectile kinetic energies in the region of 1–10 GeV (reported are the cases of a proton, a neutron, and a 12C ion), the wavelength of the projectile is similar to the size of the nucleon, and hence the projectile is able to interact directly with the components of the single nucleons (quarks, gluons) in the nucleus of the target atom. At slightly lower kinetic energies (~1  MeV–1  GeV), the

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Fig. 2.1  Plot of the projectile kinetic energy vs. the de Broglie wavelength. The sizes of a nucleon, uranium nucleus, lead orbitals and water molecule are also reported. (Courtesy of Dr. Marc Verderi, Laboratoire Leprince-Ringuet, CNRS/ IN2P3, Ecole Polytechnique, Institut Polytechnique de Paris, France)

wavelength of the projectile becomes comparable to that of the nucleus of uranium, and thus the projectile can interact with the nucleons, but not with the constituents of the nucleons. This can cause fragmentation of the nucleus and generation of secondary species and decay particles that are emitted in the de-excitation of the nucleus, which is brought in an excited state by the impacting particle. Descending in kinetic energy, the wavelength of the incoming radiation on the order of the entire nucleus means that the impacting particle can interact with the entire nucleus but not with the nucleons. Further lower in energy and at increased wavelength, the incoming radiation has a wavelength of similar size to the electronic orbitals (reported here are lead orbitals), and still further of similar size to a water molecule, thus entering the regime of molecule-dominating behavior. It is thus clear that when spanning large energy windows, many different physical interactions take place with the target, which probe the different units of matter which are considered as elemental for different sub-disciplines of physics. It has to be stressed that in its path through matter, the primary particle can generate several secondary particles, such as electrons, by ionization and/or decay particles of excited nuclei in nuclear inelastic collisions. In the latter case, “daughter nuclei” are generated, which also act as projectiles interacting within the system. In the case of biological targets, primary radiation can generate ions, electrons, excited molecules, and molecular fragments (free radicals) that have lifetimes longer than approximately 10−10  s. The new species in turn travel and diffuse and start chemical

reactions, the evolution of which is a main contributor to the effects at biological level. Nowadays, apart from the well-known fields of the high-­ energy physics and nuclear science, radiation science is important in numerous sub-disciplines, such as ion beam therapy [7, 8], radiation protection in medicine [9] and nuclear facilities [10], development of risk assessment models for nuclear accidents [11], or radiation protection in deep space manned missions [12–14]. Apart from the effects on humans, parallel streams of research exist for the studies on radiation effects induced in plants, seeds, and animals, for the survival and adaptation around the Chernobyl site and even for the effects on small biological molecules of interest in studies on the search of life on other planets or their moons [15–19] (Box 2.1).

Box 2.1 Description of Particle Interactions

• The appropriateness of a description of particles as localized entities or as waves depends on the wavelength of the particle, the characteristics of the probed dimension of the target system, and the resulting phenomenon (change in the state of the target) which we are interested in. • There exists a wide range of interactions that particles can induce in matter, from the interactions with quarks and gluons in high-energy collisions to excitations of electrons and vibrations in molecules which dominate at lower energies.

2  Basic Concepts of Radiation Biology

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Fig. 2.2  The electromagnetic spectrum (Created with BioRender)

2.1.2 Electromagnetic Radiation Electromagnetic radiation transfers energy without any atomic or molecular transport medium. According to the wave-particle duality of quantum physics, electromagnetic radiation can be described either as a wave or as a beam of energy quanta called photons. To understand how electromagnetic radiation interacts with matter, we need to think of electromagnetic radiation as photons, and it is the energy of each photon, which determines how it interacts with matter. Figure  2.2 shows the spectrum of electromagnetic radiation. It is divided into radio waves, microwaves, infrared, (visible) light, ultraviolet (UV), and X- and γ-rays depending on the frequency and energy of the individual photons. Depending on the photon energy, the photon interaction with an atom can result in ionization, where an electron gets enough energy to leave the molecule/atom; excitations, where the electron gets the exact energy needed to move from an inner electron shell to an outer shell; or changes in the rotational, vibrational, or electronic valence configurations (Box 2.2).

Box 2.2 Ionizing Radiation

• It is not the total energy but the energy per photon which determines how the radiation interacts with matter. • Ionizing radiation is the radiation with enough energy per photon to kick out one atomic electron.

Radiation can be divided into ionizing and nonionizing radiation. Ionizing radiation carries more than 10 eV, which is enough energy to break chemical bonds. Unlike ionizing

radiation, nonionizing radiation does not have enough energy to remove electrons from atoms and molecules.

2.1.2.1 Nonionizing Electromagnetic Radiation The UV spectrum is in the range of 3.1–124 eV. Even though the high-energy UV (UVC) can be ionizing, this is absorbed in the atmosphere and does not reach the Earth. Only UVA (3.10–3.94  eV) and UVB (3.94–4.43  eV) are transmitted through the atmosphere. UVB radiation has the energy to excite DNA molecules in skin cells. This can result in aberrant covalent bonds forming between adjacent pyrimidine bases, producing pyrimidine dimers. Most UV-induced pyrimidine dimers in DNA are removed by the process known as nucleotide excision repair, but unrepaired pyrimidine dimers have the potential to lead to mutations and cancer. UVA can induce production of reactive oxygen and reactive nitrogen species (ROS, RNS), which happens through interaction with chromophores such as nucleic acid bases, aromatic amino acids, NADH, NADPH, heme, quinones, flavins, porphyrins, carotenoids, 7-­dehydrocholesterol, eumelanin, and urocanic acid [20]. ROS can induce ionizations in DNA.  In summary, the UV light that reaches the Earth (UVA and UVB) has too low photon energies to induce direct ionization but can cause DNA instability through excitation (Box 2.3). Box 2.3 Characteristics of UV—Radiation

• Ionizing UV radiation (UVC) is absorbed in the atmosphere. • UVB can induce pyrimidine dimers in DNA. • Both UVA and UVB can induce ROS, which in turn can induce DNA damage.

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2.1.2.2 Ionizing Electromagnetic Radiation An X-ray photon is emitted from an electron that is either slowed down or moves from one stationary state to another in an atom; a γ-photon is sent out by disintegration of an atomic nucleus. Except for the origin, from the physical perspective, there is no difference between X-ray and γ-photon radiation. A photon can interact with matter by three different processes depending on its energy and the atomic number of the elements of the matter. In the photoelectric effect, an atomic electron absorbs all the energy of the incoming photon and is emitted from the atom. Note that the photoelectric effect cannot occur with an electron that does not belong to an atom. This is because both energy and momentum need to be conserved, which cannot be achieved without an atom carrying the rest momentum. The Compton effect implies, just like the photoelectric effect, that an electron is knocked out from an atom by transfer of energy from the photon. However, for the Compton effect, a secondary photon is also emitted, which preserves the momentum (Fig.  2.3). Therefore, the process may also apply to a nonatomic, or free, electron. The amount of energy transferred from the incident wave to the electron depends on the scatter angle as follows: λ ′ − λ = λc (1 − cos θ ) , (2.2) h where λc = is a constant denoted “the Compton me c wavelength for electrons” which equals the wavelength of a photon having the same energy as the rest-mass energy of the electron. Notice that maximum energy transfer to the electron is obtained with a scatter angle of 180° (backscatter), but it is not possible to transfer all the energy of the incoming photon to the electron (conservation of momentum).

A. Baeyens et al.

Fig. 2.4  A typical example of a sequence of energy deposits. The energy of an original 1.25 MeV photon is deposited in five subsequent Compton processes with a final energy deposition in the form of a photoelectric process. The figure shows the mean range in water (dotted arrows) for the incoming photon and the reduced-energy photons emitted for each Compton process. The scale shown in the bottom left only applies to photons. The electron mean range is much shorter starting at about 2 mm going down to about 36 μm in the last Compton scattering (which is still larger than a typical cell diameter) (Created with BioRender)



Fig. 2.3  The Compton process. The incident photon (γ-ray) interacts with an electron initially at rest resulting in a scattered photon (at angle θ) and electron (at angle Φ). The energy (E) and momentum (p) of the photon and electron before and after (marked with ′) scattering are given in the figure (Created with BioRender)

As seen in Fig. 2.4, depending on the incoming photon energy, there will be a series of Compton processes, each with emission of an electron, followed by a photoelectric process in the end. The result of such a Compton track is an energy distribution of secondary electrons with many low-­energy electrons but also a few with high energy. The high-­energy electrons are important for the dose distribution in the irradiated material, because they transport energy away from the place of the primary photon interaction and deposit their energy further into the irradiated material. Pair production occurs by the incoming photon interacting with the nuclear forces in the irradiated material resulting in an electron-positron pair. The rest energy of the two newly formed particles is 1.022 MeV, so the incoming photon must have higher energy than this for the process to occur. In body tissues and cells, more than 20  MeV in photon energy is required for pair production to dominate over the Compton processes. The Compton process dominates in biological material for energies relevant for medical use of photons. However, the cross section (an expression of the probability of interaction) for each process also depends on the atomic number Z. The cross section is proportional to Z4 for photoelectric effect, Z for Compton effect, and Z2 for pair production.

2  Basic Concepts of Radiation Biology

Thus, the higher the effective atomic number, the lesser the importance of the Compton effect (Box 2.4).

Box 2.4 Interaction of Photon with Matter

• Electromagnetic radiation can ionize atoms/molecules through three different processes (photoelectric effect, Compton process, and pair production) depending on the photon energy and atomic number of the elements involved. • The Compton process dominates in biological material for energies relevant for medical use of photons, but a Compton track ends with the photoelectric effect.

2.1.3 Particle Radiation As described above, in physics, a particle is considered to be an object, which can be described through its properties including volume, density, and mass. In the context of particle radiation, two types of particles are defined: charged particles, such as electrons, protons, α-particles, or other ions and uncharged particles such as neutrons. In general, particle radiation can interact with matter through a number of different processes, where the frequency of occurrence depends on the particles’ mass, velocity, and charge. In the first type of the process called electronic interaction, the particle interacts with electrons in the atomic shell, and in the second, called nuclear interaction, the particle interacts with the atomic nuclei. All interactions can be considered as collisions between two masses, which can be either elastic or inelastic. There are three types of electronic or Coulomb interactions, which can occur with or without energy loss from the incident particle. Elastic scattering of the particle in the atomic shell occurs with only neglectable energy transfer, as only the energy which needs to be transferred is that which is necessary to fulfill energy and momentum conservation. In this case, the incident particle is scattered and changes its direction. The two inelastic electronic processes are shown in Fig. 2.5 (left). The particle described through its atomic number z, its mass m, and its energy E is interacting with an atom Fig. 2.5  Visualization of the electronic interactions (left) and the nuclear interaction (right) of a particle with atomic number z, mass m, and energy E with matter with atomic number Z, mass number A, and density ρ (Created with BioRender)

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of the matter characterized by the atomic number Z, the mass number A, and the density of the matter ρ. In the inelastic collision, the particle transfers energy to the hit electron. If sufficient energy is transferred, the electron will leave the atom, thus ionizing it. When the transferred energy is higher, the electron gets additional kinetic energy and can then itself act as particle radiation. If the energy is lower and fits the energy difference between two electron shells (the defined energies at which electrons “orbit”), the electron is excited, which means lifted to the higher shell. After a certain time, the electron falls back while emitting a photon with the energy corresponding to the energy difference between the shells. In nuclear interactions, again three types can be defined. Firstly, elastic nuclear scattering, also called nuclear coulomb scattering, describes the elastic collision of a particle with the atomic nucleus. Here, the particle does not lose energy and only a deflection occurs (Fig. 2.5). In inelastic nuclear scattering, the particle is deflected and emits light, the so-called bremsstrahlung. Lastly, an interaction with the target nuclei itself is possible inducing nuclear reactions.

2.1.3.1 Charged Particle Radiation Charged particle radiation describes high-energy massive particles such as electrons, protons , and other ions. These particles interact with matter through the described electronic or nuclear interactions. In each interaction, only a small amount of the total energy is transferred, and although the whole process of interaction is statistical in its nature, one can say that the particles stop more or less uniformly at a certain distance called the range. Furthermore, in each interaction, a certain angular deflection happens, which causes the particle to travel in a crooked path, and which effectively causes the incident particle beam to widen, while traversing a medium. The types of interactions can be described through the occurring energy loss and deflection of particle radiation in matter. Ionizations and excitations, which occur in the electronic interactions, can be differentiated into soft and hard collisions. Interactions of the charged particle with the electrons in the outer atomic shell are called soft collisions, as the energy transfer is low (a few eV). The electrons, which are ionized, have a low energy and therefore emit all the energy in close proximity to the point of interaction. These soft collisions are responsible for approximately 50% of the total

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energy transfer of a particle. As the energy transfer of a single collision is very low, the particle velocity decrease is also low. But as a lot of these interactions occur, the slowing is, although of statistical nature, on average happening continuously. For particles which have a very high energy and thus velocity, the Cherenkov effect can occur. This effect describes the emittance of light, when a particle flies through matter with a velocity larger than the speed of light in this corresponding matter. This light is called Cherenkov radiation and can be seen as blue in the cooling water of nuclear reactors. The Cherenkov effect does not play a role in the effects of particle radiation on biological matter. Coulomb interactions with the electrons of the inner shells are called hard collisions. Here, the electrons produced in ion-

izations have a higher energy and larger deflection angles compared to the ones from soft collisions. These electrons are called δ-rays, and they transfer their energy via soft collisions to the matter, thus spreading the energy distribution of an incident particle up to several μm distance to the incident particle track. This effect plays a major role in the microdosimetry. Electronic interactions are the main contributors to the energy loss for high ion energies (see Fig.  2.6) but have a negligible deflection per collision. Energy loss through elastic nuclear scattering as described above is only an important contribution to the total energy loss for ion energies below approximately 0.01 MeV/u. Here, the ions are already close to stopping and have a remaining range in the order of nanometers. For high ion energies

a

b

c

d

Fig. 2.6 (a) Energy loss for protons (purple) and carbon (blue) ions depends on ion type and ion energy. For lower energies, the nuclear energy loss (dotted lines) starts to get an influence. At energies above ~0.0005 MeV/u for protons and ~0.005 MeV/u for carbon ions, the electronic energy loss is dominant (dashed lines) and the nuclear energy loss can be even neglected for higher energies. E/A is the energy divided by mass number. (b) Energy loss for a proton with initial energy of Ein = 200 MeV with a range in water of 256 mm on the left and for a carbon ion with initial energy of Ein = 375 MeV/u with a range in water of

251 mm on the right: at the end of range at a path length, the energy loss is increasing and rapidly goes to zero when the ion stops. The curve shape for the carbon ion is the same as for the proton but with a higher energy loss at all times. Energy losses are calculated via SRIM (SRIM—The Stopping and Range of Ions in Matter, J. Ziegler, http://www.srim.org/). (c) Stopping power of electrons depending on electron energy simulated using estar (https://physics.nist.gov/PhysRefData/Star/Text/ESTAR. html). (d) Energy loss of electrons in adipose tissue with penetration depth (inspired by Hazra et al. 2019) (licensed under CC-BY-­4.0) [26]

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2  Basic Concepts of Radiation Biology

(E > several 100 MeV/u), elastic and inelastic nuclear scattering are again mainly responsible for deflection but also for energy loss through emission of bremsstrahlung. There are also other mechanisms possible, happening quite rarely at the energies used in society, but which should be mentioned here [21, 22]. These are direct interactions with the nuclei, namely transfer reactions like stripping or pickup, where nucleons are transferred from or to the incident particle. Also charge exchange can happen, which is a combination of stripping and pickup, where a neutron of the particle is exchanged with a proton of the atom or vice versa. Also, fragmentation can occur, where the incident particle and/or the atomic nucleus break up into (more than two) fragments. And finally, fusion reactions can occur, where the incident particle is fused into the atomic nucleus and both together form a new nucleus. Energy Loss and Range The exact energy loss during an interaction is described through the so-called stopping power S and is made up of the collision Scol and the radiation Srad stopping power [23]:

S=

dE = Scol + S rad . dx

(2.3)

The collision stopping power is the energy loss through collisions along the track in matter. For high energies of the impacting particles, the collisional stopping power can be described by the known Bethe–Bloch formula, which is based on perturbation theory and can also incorporate relativistic corrections. For protons or heavier ions, the collision power is



1 Z  dE  2 2 Scol =  ⋅ z2 ⋅ 2  = ρ ⋅ 4π ⋅ re ⋅ mo c ⋅ d ⋅ x u A β  col (2.4) ⋅Rcol ( β ) . For electrons or positrons, this is



1 Z  dE  2 2 Scol =  ⋅ z2 ⋅ 2  = ρ ⋅ 2π ⋅ re ⋅ mo c ⋅ u⋅A β  dx col (2.5) ∗ . β ⋅Rcol ( )

Scol ∝ ρ ⋅



(2.6)

The radiation stopping power does not play a role for protons and heavier particles, due to their heavy masses, but for electrons, which are more than three orders of magnitudes lighter. The radiation stopping power for electrons is 1 2 1 Z  dE     = ρ ⋅ ⋅ re ⋅ α ⋅ ⋅ Etot ⋅  Rrad , n + Rrad , e  . (2.7) d x u A Z   rad   2



S rad = 

With Etot the total energy of the electron and α the fine-­ structure constant. Again, dimensionless rest functions occur describing the cross sections for interactions with nuclei Rrad, n and electrons in the atomic shell Rrad, e. For quantification in radiobiology, the detailed description of the stopping power is not used, as it would be too complicated, and the perturbation parts only contain a small correction. Conventionally, the linear energy transfer dE LET = is used instead. The LET only takes electronic dx interactions into account. The difference between LET and electronic stopping lies in their origin. The electronic stopping is focused on the energy loss of the impacting particle, and it has a negative sign as it acts as a friction force. The LET has a positive sign, and it is the energy that the target sees deposited in itself; this “positive amount of energy” creates the nonequilibrium dynamics, which are the first radiation-­induced effects. The LET and the electronic stopping are equal for big samples, which is the case in radiobiology. Therefore, the LET is the same as the electronic stopping, which can be looked up in programs such as pstar, astar, or SRIM [24, 25]. For protons and heavier ions at energies larger than ~0.01 MeV/u, the electronic energy loss is the dominant process, as can be seen in Fig. 2.6, whereas for low ion energies, the nuclear energy loss becomes dominant, validating the use of LET as the most appropriate measurement quantity for radiobiologically relevant energies of >1 MeV. The energy loss has a peak at 2

This formula includes the properties of the particle energy, charge number, and velocity characterized by moc2, z2, and β2 and the properties of the matter density ρ, charge number Z, and mass number A. re is the classical electron radius and u ∗ the atomic mass unit. The terms Rcol(β) and Rcol ( β ) are called rest function for heavier particles or electrons and positrons, respectively. These are dimensionless quantities, which contain the complex energy and matter-dependent cross sections for collision stopping. In practical use, especially in radiobiology, it is just important to know some proportionalities:

Z 2 1 ⋅ z ⋅ 2 . A v



v ≈ z3 ⋅

2 c keV ≈ z 3 ⋅ 25 . 137 u

(2.8)

For even higher ion energies, the energy loss decreases again. For a single collision, considering a maximum energy ΔEmax which can be transferred through electronic interactions is

∆Emax ≈ 4

me E. m

(2.9)

With me being the electron mass, m the ion mass, and E the ion energy. For protons, this maximum energy transfer

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per collision is ΔEmax, p ≈ 0.2 % Ep. For carbon ions, it is even lower at ΔEmax, C ≈ 0.02 % EC. Therefore, thousands of collisions are necessary before an ion stops, and the more energy it has lost, the slower it gets and therefore the interactions get closer together. If one looks at the energy loss of an ion depending on the path length traveled in a target medium, a unique distribution is visible (Fig. 2.6b). The energy loss at the entrance is low and only slightly increasing with depth. Just in the last millimeters or even below, the energy loss sharply increases. After the peak, an even sharper decrease is visible until the ion stops only shortly after reaching the peak energy loss. This distribution is called the Bragg curve. Due to this distribution, a range of the particle can be defined, which is the average distance the ion travels before it stops. Due to the statistical nature of the interactions, the range can only be given as an average quantity. The ion range can be calculated as [23]:



R ( Ekin ) =

Ekin

∫ 0

−1

 dE  −  dE.  dx 

(2.10)

For example, for protons with therapy-relevant energies between approx. 10  MeV and 200  MeV, the range can be approximated to mc 2  E  R p ≅ 19 µ m  .  with E =  MeV  1− β 2 1.8



(2.11)

The unique energy loss distribution, with a peak energy loss just at the end of range, gives particles a great advantage in tumor therapy compared to photons, as the tissue behind the tumor will not get irradiated at all, as explained in Chap. 6. For low-energy electrons, the collision stopping power is the dominant process, whereas for higher energies, the radiation stopping power gets dominant (Fig. 2.6c). The energy loss distribution with penetration depth is due to the contribution of the radiation stopping power different to protons and heavier ions (Fig. 2.6d). There is no clear range visible, but after a small buildup, the maximum is reached, followed by a decrease, and with higher depth the energy loss will be zero; this is when the electron has stopped. The possible penetration depth and especially the maximum of energy loss are dependent on energy. This is relevant for therapy, where low-energy electrons are used to irradiate skin tumors, whereas for deeper lying tumors, higher energies are necessary (Box 2.5).

Box 2.5 Characteristics of Charged Particles

• Charged particles transfer their energy mainly through coulomb interactions with electrons and nuclei of the atoms of the matter. • The energy loss of the particle can be described by the Bethe–Bloch formula of the stopping power. • For ions, only collision stopping power plays a role, and for electrons also radiation stopping power. • Ions have a defined range, where energy loss follows the Bragg curve.

Scattering and Deflection The interaction of particles with matter is not only responsible for energy loss but also for a deflection of the incident particle. For the coulomb interactions with electrons, only negligible deflection occurs. The nuclear Coulomb interactions also give small deflections per collision. Furthermore, Rutherford scattering with the atomic nucleus can occur. Taking all the interactions into account, significant deflection of particles is common. This process is called multiple small-­ angle scattering. Additionally, the Rutherford scattering can lead to single largeangle scattering events, but this effect is very rare. The scattering of single ions leads to widening of the incident beam of particles with penetration depth. Due to the dominance of the multiple small-angle scattering, the lateral profile of the beam can be approximated by a Gaussian distribution. It is important to know that for larger lateral distances, the Gaussian distribution no longer holds, as the large-angle scattered ions are deflected in this region. But as already mentioned, this is a rare process and does not have an influence on the beam size. The lateral spread defined as the σ of the Gaussian distribution is z 32 , with Ekin the kinetic energy of the particle, z the σ∝ x Ekin charge, and x the distance traveled (Box 2.6).

Box 2.6 Scattering of Particles

• Coulomb interactions are responsible for scattering of the particle. • Multiple coulomb scattering leads to a deflection of the particle. • Single Rutherford scattering with the atomic nuclei leads to large deflections, but these are very rare. • An incident particle beam will have a Gaussian energy distribution profile in the lateral direction due to the statistical nature of scattering.

2  Basic Concepts of Radiation Biology

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Fig. 2.7  Quark structure of proton and neutron, with binding gluons shown (Created with BioRender)

2.1.3.2 Neutron Radiation The existence of the neutron as a component of the atom was first proposed by Rutherford in 1911, though it was Chadwick who in 1932 detected the particle as a result of experiments involving gamma irradiation of paraffin [27]. Advances in particle physics have led to our current understanding of hadronic matter which includes neutrons, such that the quark model of the neutron envisages the particle as consisting of two down quarks and an up quark (udd), as shown in Fig. 2.7. The neutron differs from the proton (uud) by a single quark such that it has almost identical mass (mn = 939.6 MeV/c2, mp = 938 MeV/c2) though the neutron has zero charge. It also differs further in that, while the proton is thought to be stable (current T1/2 of ~1038 years), the free neutron is unstable with a mean lifetime of approximately 879.6  s. While electrically neutral, the neutron does have a magnetic moment of approximately −1.93 ⌠N, where that for the proton is approximately 2.79 ⌠N (and where ⌠N is the nuclear magneton). As the neutron is a fermion, it has a spin of ½ [28]. Early experiments with neutrons relied upon their production in prototype nuclear reactors. Here, neutrons were classified according to their energies as thermal (E ~ 0.038 eV, on average associated with a Maxwell–Boltzmann distribution of particles at room temperature), slow (E  10 MeV), or relativistic (with energies producing velocities of 0.1 c or above) [29]. Exploration of neutron interactions with matter has revealed that they have very complex energy cross sections, which vary substantially with the target material. However, the interactions may be broadly classified as elastic or inelastic interactions, with elastic collisions having a greater cross section at high neutron energies [29]. In elastic interactions, the neutron collides, typically, with a target nucleus, transferring some of its kinetic energy to the nucleus, which then recoils. It may be demonstrated that the maximum energy Q that a neutron of energy En and mass M may transfer to a recoil nucleus of mass m is given by [29]. Q=

4mMEn

. 2 ( M + m)

(2.12)

In general, one may observe a cosine-squared spatial distribution of recoil energies for nuclei, Q, from which the original energy of the neutron beam may be estimated [29]:

Q = En cos 2 θ .

(2.13)

In inelastic scattering events, either the neutron can promote the nucleus of element X to an excited state, from which the nucleus itself decays by re-emitting the neutron with different energy and momentum [(n,n′) reactions], or, for neutrons with energy below 0.5  MeV, the nucleus absorbs (“captures”) the incident neutron, causing it to transmute to a new elementary state, Y, generally with the emission of some product projectile, b, such as a proton, alpha particle, or gamma ray. The latter nuclear reactions are written as

X ( n,b ) Y ,

(2.14)

where examples include 9Be(n,γ)10Be and 75As(n,γ)76As (radiative capture reactions). The development of sources of neutrons for industrial purposes has been a highly complex undertaking. Spallation sources of neutrons, where a material is bombarded with a projectile particle and then emits a beam of neutrons, have existed for some time. However, these systems require acceleration of a projectile beam, which renders them costly from an energy-input perspective, though they produce highly intense beams which are useful in the imaging of materials, as well as for both breeding and burning of nuclear fuel. Most neutron beams are produced via collimation and focusing of neutron beams from nuclear reactors, for similar applications to those already highlighted, and importantly for therapeutic applications in medicine. The development of Wolter mirrors and lenses has provided the means to direct and focus beams of neutrons in a highly precise manner allowing for controlled therapeutic applications.

2.2 Sources and Types of Ionizing Radiation Humans are continuously exposed to low levels of ionizing radiation from the surroundings as they carry out their normal daily activities; this is known as background radiation,

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which is present on Earth at all times [30]. In addition, we are exposed to ionizing radiation from artificial sources during medical examinations and treatments, during processing and using radioactive materials, and during operation of nuclear power plants or accelerators (Figs.  2.8 and 2.9). Below we provide a summary of the possible scenarios of exposure to natural and artificial radiation.

2.2.1 Natural Background Radiation Natural radiation is all around us, and we receive it from the atmosphere, rocks, water, plants, as well as the food we eat (Fig. 2.8). Naturally occurring radioactive materials are presFig. 2.8  Natural sources of ionizing radiation and their pathways (Figure from European Commission, Joint Research Centre—Cinelli, G., De Cort, M. & Tollefsen, T., European Atlas of Natural Radiation, Publication Office of the European Union [41]) (licensed under CC-BY-4.0)

Fig. 2.9  Worldwide average annual human exposure to ionizing radiation (from UNSCEAR (2008) Sources and effects of ionizing radiation) (Created with BioRender)

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ent in the Earth’s crust; the floors and walls of our homes, schools, or offices; and food. Radioactive gasses are also present in the air we breathe. Our muscles, bones, and other tissues contain naturally occurring radionuclides [31]. Hence, our lives have evolved, and our bodies have adapted to the world containing considerable amounts of ionizing radiation. As per the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR), terrestrial radiation, inhalation, ingestion, and cosmic radiation are the four foremost sources of public exposure to natural radiation. 1. Terrestrial Radiation: One of the major sources of natural radiation is the Earth’s crust, where the key contributors are the innate deposits of thorium, uranium, and

2  Basic Concepts of Radiation Biology

potassium. These minerals are called primordial radionuclides and are the source of terrestrial radiation. These deposits discharge small quantities of ionizing radiation during the process of natural decay, and these minerals are found in building materials. Therefore, humans can get exposed to natural radiation both outdoors and indoors. These radiation levels can fluctuate substantially depending on the location. Traces of radioactive materials can be found in the body where nonradioactive and radioactive forms of potassium and other elements are metabolized in the same way [32]. 2. Inhalation: Humans are exposed to inhalation of radioactive gasses that are formed by radioactive minerals found in soil and bedrock. For example, uranium-238, during its decay, produces radon (222Rn) which is an inert gas and thorium produces thoron (220Rn). These gasses get diluted to harmless levels when they traverse the Earth’s atmosphere. However, at times, these gasses escape through cracks in the building foundations, are trapped, and accumulate inside buildings where they are inhaled by the occupants (indoor living) [30]. 3 . Ingestion: Vegetables and fruits are grown in the soil and groundwater, which usually contain radioactive minerals. We ingest these minerals and subsequently are exposed to internal natural radiation. Carbon-14 and potassium-40 are naturally occurring radioactive isotopes which possess similar biological characteristics as their nonradioactive isotopes. These radioactive and nonradioactive elements are used not only in building our bodies but also in maintaining them. Therefore, such natural radioisotopes recurrently expose us to radiation [30]. 4 . Cosmic Radiation: Space is permeated by radiation, not only of electromagnetic type but also constituted by ionizing particles with mass. The electromagnetic radiation in space spans all wavelengths, from infrared to visible, from X-ray to gamma rays. In general, however, “space radiation” mostly refers to corpuscular radiation, which has three main sources: (a) Galactic Cosmic Rays (GCRs): The GCRs constitute the slowly varying, low-intensity, and highly energetic radiation flux background in the universe, mostly associated with explosions of distant supernovae. The GCR spectrum consists of approximately 87% hydrogen ions (protons) and 12% helium ions (α-particles), with the remaining 1–2% of particles being HZE (high charge Z and energy) nuclei. The energies are between several tenths and 10 × 10 GeV/nucleon and more. GCRs directly hit the top of the Earth’s atmosphere, generating secondary particle showers. However, some direct

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GCRs and generated secondary particles infiltrate the Earth’s atmosphere reaching the ground. Such radiation gets absorbed by humans, and it thus constitutes a source of natural radiation exposure. Since at higher altitude the amount of atmosphere shielding us from incoming radiation is less, the higher we go in altitude, the higher dose we receive. For example, those living in Denver, Colorado (altitude of 5280  ft  =  about 1610  m), receive a higher annual radiation dose from cosmic radiation than someone living at sea level (altitude of 0  ft) [32]. GCR ions are a major health threat to astronauts for missions beyond the near-­Earth environment and for interplanetary travel [33]. For Mars, the thin atmosphere combined with the absence of a planetary magnetic field essentially offers very little shielding from the incoming GCRs [34, 35]. Also, GCRs directly reach the surface of airless bodies such as the Moon [36]. (b) Radiation from the Sun: This consists of both low-­ energy particles flowing constantly from the Sun (the solar wind) and of solar energetic particles (SEPs), originating from transient intense eruptions on the Sun [37]. The solar wind is stopped by the higher layers of the atmosphere of our planet (and other celestial bodies with an atmosphere). SEPs come as huge injections and are composed predominantly of protons and electrons. Typical proton energies range from 10 to 100 of MeV.  They are generally quite efficiently stopped in the Earth’s atmosphere, but some direct SEPs and their high flux of secondaries could eventually be dangerous for high-altitude/latitude flights and their crew [38] and for astronauts of the International Space Station (ISS) in extravehicular activities. Finally, SEPs can be a strong concern also for astronauts during interplanetary travel, such as a trip to Mars, even inside the spacecraft [39], or for humans on the surface of the Moon. (c)  Trapped Radiation: This consists of GCRs and SEPs and their secondaries trapped by the Earth’s magnetic field into the Van Allen radiation belts. Such belts comprise a stable inner belt of trapped protons and electrons (energies are between keV and 100 MeV) and a less stable outer electron belt. The inner Van Allen belt comes closest to the Earth’s surface, down to an altitude of 200 km, in a region just above Brazil. This area is named the South Atlantic Anomaly [40]. An increased flux of energetic particles exists in this region and exposes

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orbiting human missions to higher-than-usual levels of radiation (Box 2.7).

Box 2.7 Sources of Natural Radiation

The natural radiation to which we are continually exposed has its sources in: • Cosmic radiation (the portion of it reaching the ground) • Radiation from radioactive elements in rocks • Radioactive gasses, generally at harmless concentration in the air but that can potentially also get trapped in building walls • Food, grown in soil and groundwater, which can contain radioactive minerals

2.2.2 Artificial Radiation Sources Nuclear power stations/plants use uranium to drive a fission reaction that heats water to produce steam. The latter drives turbines to produce electricity. During their normal activities, nuclear power plants release small amounts of radioactive elements, which can expose people to low doses of radiation. The water that passes through a reactor is processed and filtered to remove these radioactive impurities before being returned to the environment. Nonetheless, minute quantities of radioactive gasses and liquids are ultimately released to the environment. Such releases must be continuously monitored and are under the legislative framework of international organizations dealing with nuclear energy, such as the European Atomic Energy Community (EURATOM), established by one of the Treaties of Rome in 1958. Similarly, uranium mines and fuel fabrication plants release some radioactivity that contributes to the dose of the public [42]. The eventual release of radioactive materials should also be monitored and kept under established levels during the decommissioning of a nuclear power plant, from the shutdown of the reactor to the operation of radioactive waste facilities, and also including the short- and intermediate-term storage of spent nuclear waste to the transport to and storage in long-term geological disposal areas. Technologically enhanced naturally occurring radioactive materials (TENORM): All minerals and raw materials contain radionuclides, commonly denoted as naturally occurring radioactive materials (NORM). When concentrations of radionuclides are increased by technological processes, the term technologically enhanced NORM (TENORM) is applicable. Coal-fired power stations, for example, emit an amount of radioactivity compared to or even higher (especially in the past) than nuclear power

plants. Just for example, US coal-fired electricity generation in 2013 gave rise to 1100 tonnes of uranium and 2700 tonnes of thorium in coal ash. Other TENORM industries include oil and gas production, metallurgy, fertilizer (phosphate) manufacturing, building industry, and recycling [43]. Accelerators: The operation of accelerators, such as the Large Hadron Collider (LHC) at CERN for fundamental high-energy physics experiments, results in the production of radiation, in particular protons, because of the nuclear interactions between high-energy beams and accelerator components. Thus, the radiation levels around accelerators must be monitored continuously to ensure the protection and safety of the workers and of the public [44]. Radionuclide production facilities: Radionuclides are used worldwide in (a) medical imaging, fundamental to make correct diagnoses and provide treatments, in which radionuclides are injected into patients at low doses for functional imaging to detect diseases, and (b) therapy, in which radionuclides bound to other molecules or antibodies can be guided to a target tissue, for a local treatment of cancer. Facilities that produce radionuclides and facilities in which radionuclides are processed are reactors and particle accelerators. Radionuclides used in imaging and therapy are often beta or alpha emitters, or both. Thus, the facilities, reactors, and particle accelerators can present radiation hazards to workers and must be properly controlled and monitored, as is the case with the subsequent processing of radioactive material. Among the 238 research reactors in operation in 2017, approximately 83 were considered useful for regular radioisotope production [45]. Approximately 1200 cyclotrons worldwide were used to some extent for radioisotope production in 2015 [46]. The facilities must ensure the application of the requirements of the IAEA [47] (2014) intended to provide for the best possible protection and safety measures. Hospitals: Daily, healthcare workers and patients are exposed to various diagnostic and therapeutic radiation sources [48, 49]. The radiation environment in different hospital departments (nuclear medicine, diagnostic radiology, radiotherapy, …) can be generated by different sources. Hospitals providing radionuclide-based treatments need to protect the staff involved and keep their dose within the acceptable levels. Similarly, the discharged patient must be monitored and measurements for protection purposes must be taken to keep dose to the public within acceptable levels. This may require hospitalization with isolation during the first hours or days of treatment [50, 51]. Waste should be minimized and segregated, and packages labeled and stored for decaying. Measures should also be in place for patients’ household waste related to, for example, urine. In a radiology department, the radiation emitted during fluoroscopic procedures is responsible for the greatest radiation dose to the medical staff. Radiation from diagnostic imaging modalities, such as mammography, computed tomography, and nuclear medical imaging, is a minor contributor to the cumula-

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tive dose incurred by healthcare personnel [52]. In radiotherapy departments, photons and electrons are mainly produced by linear accelerators. Rarely, cobalt sources are used to produce radiation. With the current safety regulations, radiotherapy staff will get almost no dose during normal operation. The same is true for modern brachytherapy machines, which are almost all after loading machines avoiding direct contact between the radioactive source and the operator. Ion radiotherapy facilities: Most currently existing ion radiotherapy facilities use protons, with new facilities now being built for the acceleration of other ions, such as carbon. They are mostly cyclotrons or synchrotrons. For such facilities, the major issue is the massive production of neutrons. Ionizing radiation results from the passage of such neutrons through matter and from the radioactivity induced in exposed materials. In accelerator facilities, radioactivity is produced in the very material components, such as their beam delivery/ shaping components, as well as in all the structural components and other materials in the facility. Induced radioactivity in treated patients could also reach considerable levels. Nuclear bombs: Nuclear weapons have an explosive power deriving from the uncontrolled fission reaction of plutonium and uranium. This yields a large number of radioactive substances (isotopes) that are blown into the atmosphere. These radioactive isotopes gradually fall back to Earth. If a weapon is exploded near the Earth surface, radioactive fallout is formed in the vicinity of the burst point in a matter of tens of minutes to a couple of days (depending on the burst yield and the distance to the burst point); if a weapon is detonated aboveground (e.g., in Hiroshima and Nagasaki, the bombs exploded about 500 m above the ground level), local fallout is not formed but the radionuclides fall worldwide over a period of many years. Gamma-ray and neutron exposures leading to increased cancer incidence have been studied in the survivors of the atomic bombings in Japan since 1950 (the so-called Life Span Study, LSS, cohort), and currently all potentially suitable risk estimates are built on the excess risk from the LLS study [53]. Interestingly, the numerous tests of nuclear weapons performed by many countries since after World War II and the ensuing fallout have contributed minimally to the overall background radiation exposure (Box 2.8).

Box 2.8 Sources of Artificial Radiation

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2.3 Direct and Indirect Effects of Radiation The interaction of ionizing radiation (IR) with matter leads to biological damage that can impair cell viability. Biological damage induced by IR arises from either direct or indirect action of radiation. Direct effects occur when IR interacts with critical target molecules such as DNA, lipids, and proteins, leading to ionization or excitation, which causes a chain of events that ultimately leads to the alteration of biomolecules. Indirect effects occur when IR interacts with water molecules, the major constituent of the cell. This reaction, called water radiolysis, generates high-energy species known as reactive oxygen species (ROS) that are highly reactive toward critical targets (cell macromolecules) and, when associated with reactive nitrogen species (RNS), lead to damage to the cell structure. Mechanism and critical targets for ionizing radiation to produce biological damage through direct and indirect effects are shown in Fig.  2.10. Damages to cell macromolecules may be multiple and are detailed in Chap. 3.

2.3.1 Direct Effects of Radiation Direct effects occur when the ionization takes place within a critical target with relevance to cell functions, such as DNA, lipids, and proteins. These effects are produced by both high and low linear energy transfer (LET) radiation. However, it is the predominant mode of action of high LET radiation such as alpha particles and neutrons, comprising about two-thirds of the radiation effects. When critical molecules in the cell are directly hit by radiation, their molecular structure may be altered resulting in their functional impairment. While molecules from all cell organelles (including mitochondria, endoplasmic reticulum, or Golgi apparatus) may be hit, the nuclear DNA molecule has always been seen as the most critical target (because, unlike proteins, lipids, and carbohydrates, only a single copy of DNA is present in a cell) and was, therefore, the most thoroughly studied. The DNA damage produced by radiation includes base alterations, DNA–DNA cross-links, single- or double-strand breaks (SSB or DSB), or complex damages (described in Chap. 3).

Artificial radiation sources are: • Medical and radionuclide production facilities, accelerators for ion beam cancer therapy • Technologically enhanced naturally occurring radioactive materials (TENORM) • Nuclear power plants • Accelerators for purely fundamental research in physics

2.3.2 Indirect Effects of Radiation Indirect damages produced by IR in the cell macromolecules are mediated by ROS (resulting from water radiolysis) and by RNS (formed following the reaction of O2 with endogenous nitric oxide). The indirect effects contribute to about two-thirds of the damages induced by low LET radiation (X-rays, gamma-rays, beta particles), which is explained by

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Fig. 2.10  Mechanism and critical targets for ionizing radiation to produce biological damage through direct and indirect effects (Created with BioRender)

the fact that they are more sparsely ionizing compared to high LET radiation. When radiation deposits energy in a biological tissue, it takes time until perceiving that an effect has occurred. The succession of the generation of events determines the four sequential stages that translate into the biological effects. These stages, with very different duration, are physical, physicochemical, chemical, and biological [54–56]. The physical stage is very transient, lasting less than 10−16–10−15  s, during which energy (kinetic if particles, or electromagnetic if waves) is transferred to the electrons of atoms or molecules, determining the occurrence of ionization and/or excitation. It is at this stage that ions are formed, which will initiate a sequence of chemical reactions that end up in a biological effect. In the case of water radiolysis (decomposition of water molecules due to IR), the ions H2O+ and e− are formed, as well as the excited water molecule (H2O*) [54–56]. Very soon (10−12 s) after the formation of these ions, the physicochemical stage begins, with their diffusion in the medium and consequent intermediate formation of oxygen and nitrogen radical species, i.e., atoms, molecules, or ions that have at least one unrepaired valence electron and hence are very reactive chemically. Following the example of

water radiolysis, it is at this stage that H· + HO·, H2 + 2HO, HO· + H3O+, HO· + H2 + OH−, and e−aq are formed [55, 56], but also superoxide anion (O2·−) and hydrogen peroxide (H2O2). Peroxynitrite anion (ONOO−) is also formed following the reaction of O2·− with endogenous nitric oxide (NO). Together with peroxynitrous acid (ONOOH), nitrogen dioxide (NO2·), dinitrogen trioxide (N2O3), and others, they are referred to as RNS. The activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the mitochondrial electron transport chain (ETC), or the nitric oxide synthase by IR can also contribute to ROS/RNS generation. In the next chemical stage, the formed radicals and ions recombine and interact with critical cellular organic molecules (DNA, lipids, proteins), inducing structural damages that will translate into disruption of the function of these molecules. Within the DNA molecule, possible chemical reactions with nitrogenous bases, deoxyribose, or phosphate group may result in breaks and recombinations with the consequent formation of abnormal molecules. Among ROS, OH, which has a strong oxidative potential, is a main contributor to cell damages. The chemical stage can last from 10−12 s to a few seconds [55, 56]. ROS and RNS have also been largely implicated in

2  Basic Concepts of Radiation Biology

the so-called non-targeted effects of IR (further discussed in Sect. 2.8.2). Finally, the biological phase occurs, as a consequence of the spreading of chemical reactions involving various biological processes. The existence of more or less effective ­cellular damage repair mechanisms is responsible for the more or less belated appearance of biological effects and explains the possible long duration of this stage: from a few minutes to decades, depending on the type of radiation, the dose and dose rate, and the radiosensitivity of the irradiated tissue. Differences in tissue radiosensitivity can be partially explained by the cellular antioxidant capacity, which may vary between cell types. Indeed, to counteract oxidative insults, cells have evolved several defense mechanisms that consist of enzymatic and nonenzymatic systems. When the amount of ROS/RNS exceeds the antioxidant capacity of the cells, a state of oxidative stress arises, characterized by a decreased pool of antioxidants and modifications in nucleic acids, lipids, and proteins. Oxidative stress can persist for much longer and extend far beyond the primary targets as well as can be transmitted to progeny of the inflicted cells. Responsible for this seems to be the continuous production of ROS and RNS, which can last for months.

2.3.3 Biological Damages Induced by Direct and Indirect Effects of Radiation on Cell Organelles Virtually all cell molecules and organelles may be damaged by IR, with consequences for the cell function depending on the impact of the damage inflicted. According to the radiobiology paradigm, a nucleus is regarded as the main target of IR due to the genetic information contained in the DNA. Therefore, damages to this molecule are considered the most critical ones for cell survival. While efficient repair mechanisms exist to preserve the genome integrity, IR may break bonds in purine and pyrimidine nitrogenous bases in the DNA (which may lead to mutations), SSBs or DSBs, cross-linking, and complex damages. Among these lesions, DSBs and complex damages are the most serious due to the difficulty of their repair. A thorough description of DNA lesions is provided in Chap. 3. Mitochondria can also be subject to radiation damage, both directly and indirectly. These organelles may represent more than 30% of the total cell volume, and the mitochondrial circular DNA can suffer strand breaks, base mismatches, or even deletions of variable length. In this context, mitochondria constitute a major target of IR [57]. Besides the DNA, changes in mitochondrial morphology have also

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been observed [58]. Absorption of IR may lead to the enlargement of mitochondria and the increase in length and number of branches of the cristae [58, 59], rupture of the outer and inner membranes, as well as vacuolization and loss of the matrix. These alterations are accompanied by the decreased activity of the respiratory chain, with special emphasis on complexes I, II, and III, which are systematically referred to as especially sensitive to the direct effects of IR. Additionally, there is a decrease in the respiratory capacity driven by succinate and the ATP synthase, with a consequent impact on oxidative phosphorylation. The radiation-induced decrease in the rate of oxidative phosphorylation can recover over time, depending on the cell type [60, 61]. The electrons in the respiratory chain can leak during their transport and reduce oxygen molecules leading to the formation of superoxide anions, which are precursors of most ROS. Upon irradiation, the level of ROS produced in the mitochondria greatly increases, although under physiological conditions, it is already high. Irradiation may also cause morpho-functional changes in the endoplasmic reticulum (ER). After exposure to IR, ER dilates, vesicles appear, and its cisternae break into fragments. In the case of rough endoplasmic reticulum, irradiation induces degranulation accompanied by transformation of the membrane-bound ribosomes into free organelles [59, 62]. Likewise, irradiation may also disorganize the structure of the Golgi apparatus due to the induced fragmentation and rearrangement of its cisterns. In view of the effects of IR on the endoplasmic reticulum-Golgi apparatus complex, the ensuing alterations in the synthesis and maturation of proteins in the irradiated cells come as no surprise. Lysosomes may also increase in number and volume in the irradiated cells, which is accompanied by upregulation of the enzymatic activity in these organelles [58, 59] (Box 2.9).

Box 2.9 Direct and Indirect Effects of Radiation

• Direct effects predominate after exposure to high LET radiation (e.g., alpha particles, neutrons). • Exposure to low LET radiation (e.g., X-rays, gamma rays, beta particles) induces mostly indirect effects. • Indirect effects are mediated by ROS/RNS produced during and after the radiolysis of water. • Apart from nuclear DNA, other cellular molecules and organelles may be altered by IR, including mitochondrial DNA, plasma membrane lipids, endoplasmic reticulum, Golgi apparatus, and lysosomes.

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2.4 Radioactivity and Its Applications

Bq, where 1 Bq = 1 decay per second. Therefore, 1 Ci = 3.7 × 1010 Bq (Box 2.10).

Radiation and radioactivity have been existing ever since the Earth was formed and long before life started to evolve. All living organisms on Earth are continuously exposed to both natural and artificial radioactivity, and without it, life in the present form would have not evolved. Since the first experiments with radioactivity, our understanding of this phenomenon has increased, and consequently, today radioactivity has numerous applications important to human life and health.

2.4.1 Radioactive Decay 2.4.1.1 Natural Radioactivity The rate of decay of a radioactive source is proportional to the amount of the substance that is present at any given instant. Therefore, if the number of radioactive nuclei in a sample is N, then we may say the following: dN ∝N dt ⇒ −dN ∝ N ⋅ dt , ∴ − dN = λ ⋅ N dt −



(2.15)

where λ is the decay constant, which describes the rate of decay for a particular radioactive isotope. If we integrate both sides of Eq. (2.15), we get the following more familiar equation:

N = N 0 e − λt .

(2.16)

If we let the variable T1/2 be the “half-life of the substance,” i.e., the time taken for the activity of the substance to reduce from its initial value to half of its initial value, then we may modify Eq. (2.16) as



N0 = N 0 e − λT1/ 2 2 ln 2 0.693 ∴T 1 = = λ λ 2

(2.17)

The activity, A, of a given sample of a radioactive substance, i.e., the number of decays per second (in Bq), is given by the following equation:

A ( t ) = λ ⋅ N ( t ) ,

(2.18)

where calculations based on activities may be performed using Eqs. (2.2) and (2.3) above with the values of A inserted instead of N. The radioactivity of a sample is quoted in terms of the units of Curies, Ci (the radioactivity of a gram of 226 Ra), where 1  Ci =3.7 × 1010 decays per second. This is more commonly quoted in terms of the S.I. unit the Becquerel,

Box 2.10 The Activity of a Radioactive Substance

• The activity (A) of a radioactive substance is given in becquerel (1  Bq is the number of decays per second). • The radioactivity of a sample can also be expressed in curies (Ci), where 1 Ci = 3.7 × 1010 Bq.

2.4.1.2 Radioactive Equilibrium In nature, the abundance of the isotopes of certain radioactive nuclei depends on the abundance of their precursors, and the rate at which these precursors decay. Hence, the rate of production of each daughter nuclide of a certain radioactive isotope depends upon the rate at which its parent nuclide decays. All naturally occurring radioactive nuclides that are located below plutonium, 239Pu, in the periodic table are produced from the decay of just four parent (progenitor) isotopes: thorium (4n series), neptunium (4n  +  1 series), uranium/radium (4n  +  2), and actinium (4n  +  3). Each of these nuclides then has a decay series or chain (see example in Fig. 2.11) with associated rates of decay at each step that determine the abundance of all other radionuclides in the universe. The neptunium series is not observed in nature at the present time as 237Np, and all of its daughter nuclides have decayed since the birth of the universe, although the product of the series, bismuth 209Bi, is observed as a stable isotope in nature, pointing to the existence of the series at one time in the past. Each decay series begins with a radioactive isotope and ends with a stable daughter product. The parent isotopes of the isotopes at the beginning of the thorium, neptunium, and actinium series are produced as follows: Th series: 252Cf → 248Cm → ® 244Pu → ® 240U → ® 240Np → ® 240Pu → ® 236U Np series: 249Cf → ® 245Cm → ® 241Pu → ® 241Am → ® 237Np Ac series: 239Pu → ® 235U If we consider a hypothetical decay series as in Fig. 2.12, the three daughter isotopes of isotope A (namely isotopes B, C, D) are produced at different rates, each dependent on the decay constants of the isotope that is their parent. Say only N0 atoms of A exist at time t = 0; then N A = N 0 e−λ t (2.19) A



dN B = λ A N A − λB N B dt

(2.20)



dN C = λB N B − λC N C . dt

(2.21)

2  Basic Concepts of Radiation Biology

43

Fig. 2.11 Uranium, 238U/ radium, 226R (4n + 2) decay series. Radioactive decay series. (2020, September 8). [Retrieved August 16, 2021, from https://chem.libretexts. org/@go/page/86256 (open-source CC-BY textbook)]

From Eqs. (2.19) and (2.20):

(2.22)

Multiplying across by eλB t e λB t .



⇒



(

d N B e λB t dt

) = N λ e( 0

A

λB − λ A ) t

.

Box 2.11 Natural Radioactivity



N B e λB t = N 0

λA e( λB − λA )t − 1  λB − λ A 

And multiplying across by e − λB t gives

(2.24)

dN B t + λB N B e λ B t = N 0 λ A e ( λ B − λ A ) dt

Integrating both sides then gives

λ A − λAt (e ) λB λ ∴ N B = N0 A . λB N B = N0

dN B + λB N B = λA N 0 e − λAt . dt

N B = N0

λA ( e−λAt − e−λBt ) . λB − λ A

(2.23)

If the parent is very much shorter lived than the daughter, i.e., if λA  >  λB, we then have radioactive equilibrium (Fig. 2.12a). If the parent is longer lived than the daughter, then λA 50–60 Gy would allow for a more appropriLQ model). ate dental management: more aggressive in SQ7. Tumor oxygen status, the degree of repopulation or the >60 Gy zone and far less aggressive in the other proliferation rate and intrinsic radiosensitivity. areas of the jaw, improving the quality of life of these SQ8. Proteomics, genomics, epigenomics, genomics, or patients. The fewer extractions in highly irradiated transcriptomics, used for measuring proteins, DNA/ areas, the lesser the risk for ORN. chromatin, DNA, or RNA and transcription, SQ18. The principal function of stem cells is to maintain respectively. tissue homeostasis including continuous regenera SQ9. Alternative (d). OER is 1 for high LET radiation like tion and associated constant number of cells. α- particles. SQ19. Bone marrow stem cells. SQ10. Alternative (c). The presence of the nitro group in SQ20. The answer is displayed in Fig. 5.29. In brief, CSC second position, increases electron affinity and may have (1) Increased DNA repair capacity which radiosensitization. allows them to handle IR-induced DNA DSBs; (2)

298

Increased signaling networks that block IR-induced cell death including deficient pro-apoptotic signaling and increased anti-apoptotic signaling; (3) CSCs have slow proliferation and may therefore not be so sensitivity to IR-induced DNA damage. SQ21. Studies showed evidence of the existence of bidirectional effects of RT on the tumor and on the intestinal microbiota. In prospective clinical studies, a reduction of the fecal microbial diversity during and after pelvic RT was measured in patients suffering from intestinal complications. Also, RT-induced modification of microbiota diversity and composition can modify the host immune response and in turn the effectiveness of the anticancer treatment themselves including RT.

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5  Clinical Radiobiology for Radiation Oncology M.  Textural features of hypoxia PET predict survival in head and neck cancer during chemoradiotherapy. Eur J Nucl Med Mol Imaging. 2020;47(5):1056–64. https://doi.org/10.1007/ s00259-019-04609-9. 329. Giannini V, Mazzetti S, Bertotto I, Chiarenza C, Cauda S, Delmastro E, Bracco C, Di Dia A, Leone F, Medico E, Pisacane A, Ribero D, Stasi M, Regge D.  Predicting locally advanced rectal cancer response to neoadjuvant therapy with 18F-FDG PET and MRI radiomics features. Eur J Nucl Med Mol Imaging. 2019;46(4):878–88. https://doi.org/10.1007/s00259018-4250-6. 330. Xiong J, Yu W, Ma J, Ren Y, Fu X, Zhao J.  The role of PET-based radiomic features in predicting local control of esophageal cancer treated with concurrent chemoradiotherapy. Sci Rep. 2018;8(1):9902. https://doi.org/10.1038/s41598-018-28243-x. 331. Gnep K, Fargeas A, Gutiérrez-Carvajal RE, Commandeur F, Mathieu R, Ospina JD, Rolland Y, Rohou T, Vincendeau S, Hatt M, Acosta O, de Crevoisier R.  Haralick textural features on T2 -weighted MRI are associated with biochemical recurrence following radiotherapy for peripheral zone prostate cancer. J Magn Reson Imaging. 2017;45(1):103–17. https://doi.org/10.1002/jmri. 25335. 332. van Dijk LV, Langendijk JA, Zhai TT, Vedelaar TA, Noordzij W, Steenbakkers RJHM, Sijtsema NM.  Delta-radiomics features during radiotherapy improve the prediction of late xerostomia. Sci Rep. 2019;9(1):12483. https://doi.org/10.1038/ s41598-019-48184-3. 333. Cunliffe A, Armato SG 3rd, Castillo R, Pham N, Guerrero T, Al-Hallaq HA. Lung texture in serial thoracic computed tomography scans: correlation of radiomics-based features with radiation therapy dose and radiation pneumonitis development. Int J Radiat Oncol Biol Phys. 2015;91(5):1048–56. https://doi.org/10.1016/j. ijrobp.2014.11.030. 334. Hettal L, Stefani A, Salleron J, Courrech F, Behm-Ansmant I, Constans JM, Gauchotte G, Vogin G. Radiomics method for the differential diagnosis of radionecrosis versus progression after fractionated stereotactic body radiotherapy for brain oligometastasis. Radiat Res. 2020;193(5):471–80. https://doi.org/10.1667/ RR15517.1.

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Further Reading Coppes RP, Dubrovska A.  Targeting stem cells in radiation oncology. Clin Oncol (R Coll Radiol). 2017;29(6):329–34. https:// doi.org/10.1016/j.clon.2017.03.00. Donlon NE, Power R, Hayes C, Reynolds JV, Lysaght J. Radiotherapy, immunotherapy, and the tumour microenvironment: turning an immunosuppressive milieu into a therapeutic opportunity. Cancer Lett. 2021;502:84–96. https://doi.org/10.1016/j.canlet.2020.12.045. Horsman MR, Overgaard J.  The impact of hypoxia and its modification of the outcome of radiotherapy. J Radiat Res. 2016;57(Suppl 1):i90–8. https://doi.org/10.1093/jrr/rrw007. Huang Y, Fan J, Li Y, Fu S, Chen Y, Wu J.  Imaging of tumor hypoxia with radionuclide-labeled tracers for PET.  Front Oncol. 2021;11:731503. https://doi.org/10.3389/fonc.2021.731503. Joiner MC, Van der Kogel AJ, editors. Basic clinical radiobiology. 5th ed. CRC Press; 2018. McBride WH, Schaue D.  Radiation-induced tissue damage and response. J Pathol. 2020;250(5):647–55. https://doi.org/ 10.1002/path.5389. Reda M, Bagley AF, Zaidan HY, Yantasee W. Augmenting the therapeutic window of radiotherapy: a perspective on molecularly targeted therapies and nanomaterials. Radiother Oncol. 2020;150:225–35. https://doi.org/10.1016/j.radonc.2020.06.041. Sebestyén A, Kopper L, Dankó T, Tímár J. Hypoxia signaling in cancer: from basics to clinical practice. Pathol Oncol Res. 2021;27:1609802. https://doi.org/10.3389/pore.2021.1609802. Van de Guchte M, Mondot S, Doré J. Dynamic properties of the intestinal ecosystem call for combination therapies, targeting inflammation and microbiota, in ulcerative colitis. Gastroenterology. 2021;161(6):1969–1981.e12. https://doi.org/10.1053/j. gastro.2021.08.057. Vogin G.  Description and management of radiotherapy-induced long-term effects. In: Rauh S, editor. Survivorship care for cancer patients. Cham: Springer; 2021. https://doi.org/ 10.1007/978-3-030-78648-9_13.

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6

Radiobiology of Combining Radiotherapy with Other Cancer Treatment Modalities Vidhula Ahire, Niloefar Ahmadi Bidakhvidi, Tom Boterberg, Pankaj Chaudhary, Francois Chevalier, Noami Daems, Wendy Delbart, Sarah Baatout, Christophe M. Deroose, Cristian Fernandez-Palomo, Nicolaas A. P. Franken, Udo S. Gaipl, Lorain Geenen, Nathalie Heynickx, Irena Koniarová, Vinodh Kumar Selvaraj, Hugo Levillain, Anna Jelínek Michaelidesová, Alegría Montoro, Arlene L. Oei, Sébastien Penninckx, Judith Reindl, Franz Rödel, Peter Sminia, Kevin Tabury, Koen Vermeulen, Kristina Viktorsson, and Anthony Waked V. Ahire (*) Chengdu Anticancer Bioscience, Ltd., Chengdu, China J. Michael Bishop Institute of Cancer Research, Chengdu, China N. Ahmadi Bidakhvidi · C. M. Deroose Department of Nuclear Medicine, University Hospitals Leuven, Leuven, Belgium Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, KULeuven, Leuven, Belgium e-mail: [email protected]; christophe. [email protected] T. Boterberg Department of Radiation Oncology, Ghent University Hospital, Ghent, Belgium Particle Therapy Interuniversity Center Leuven, Department of Radiation Oncology, University Hospitals Leuven, Leuven, Belgium e-mail: [email protected] P. Chaudhary Patrick G. Johnston Center for Cancer Research, Queen’s University Belfast, Northern Ireland, United Kingdom e-mail: [email protected] F. Chevalier UMR6252 CIMAP, Team Applications in Radiobiology with Accelerated Ions, CEA-CNRS-ENSICAEN-Université de Caen Normandie, Caen, France e-mail: [email protected] N. Daems Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium W. Delbart Nuclear Medicine Department, Hôpital Universitaire de Bruxelles (H.U.B.), Brussels, Belgium e-mail: [email protected]

S. Baatout Radiobiology Unit, Belgian Nuclear Research Centre (SCK CEN), Mol, Belgium Institute of Nuclear Medical Applications, Belgian Nuclear Research Center (SCK CEN), Mol, Belgium e-mail: [email protected] C. Fernandez-Palomo Institute of Anatomy, University of Bern, Bern, Switzerland e-mail: [email protected] N. A. P. Franken · A. L. Oei Department of Radiation Oncology, Amsterdam University Medical Centers, Location University of Amsterdam, Amsterdam, The Netherlands Center for Experimental and Molecular Medicine (CEMM), Laboratory for Experimental Oncology and Radiobiology (LEXOR), Amsterdam, The Netherlands Cancer Biology and Immunology, Cancer Center Amsterdam, Amsterdam, The Netherlands e-mail: [email protected] U. S. Gaipl Translational Radiobiology, Department of Radiation Oncology, Universitätsklinikum Erlangen, Erlangen, Germany e-mail: [email protected]; [email protected] L. Geenen Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Department of Radiology and Nuclear Medicine, Erasmus Medical Center, Rotterdam, The Netherlands N. Heynickx Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Department of Molecular Biotechnology, Ghent University, Ghent, Belgium e-mail: [email protected]

© The Author(s) 2023 S. Baatout (ed.), Radiobiology Textbook, https://doi.org/10.1007/978-3-031-18810-7_6

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312 I. Koniarová Department of Radiation Protection in Radiotherapy, National Radiation Protection Institute, Prague, Czech Republic e-mail: [email protected] V. K. Selvaraj Department of Radiation Oncology, Thanjavur Medical College, Thanjavur, India H. Levillain · S. Penninckx Medical Physics Department, Hôpital Universitaire de Bruxelles (H.U.B.), Bruxelles, Belgium e-mail: [email protected]; [email protected] A. J. Michaelidesová Nuclear Physics Institute of the Czech Academy of Sciences, Rez, Czech Republic Faculty of Nuclear Sciences and Physical Engineering, Prague, Czech Republic e-mail: [email protected] A. Montoro Laboratorio de Dosimetría Biológica, Servicio de Protección Radiológica Hospital Universitario y Politécnico la Fe, Valencia, Spain e-mail: [email protected] J. Reindl Section Biomedical Radiation Physics, Institute for Applied Physics and Measurement Technology, Universität der Bundeswehr München, Neubiberg, Germany e-mail: [email protected]

Learning Objectives

• To understand the biological rationale and characteristics of conventional and alternative fractionation schemes used in clinical RT practice and get insight into the biological aspects (acceptability of high dose fractions, optimal dose-time) of hypofractionation regimen. • To understand the definition and radiobiologic principles of Stereotactic Body Radiation Therapy (SBRT)/hypofractionation/boron neutron capture therapy (BNCT); and learn about their treatment planning and associated applications in clinical settings. • To understand the basic concept of combining RT with various other treatment modalities that can enhance the effect of radiation by specifically targeting cancer cells or the immune system as well as for minimizing the adverse effects on normal cells. • To understand the principles and clinical applications of both diagnostic and therapeutic radiopharmaceuticals.

V. Ahire et al. F. Rödel Department of Radiotherapy and Oncology, Goethe University, Frankfurt am Main, Germany e-mail: [email protected] P. Sminia Department of Radiation Oncology, Amsterdam University Medical Centers, Location Vrije Universiteit/Cancer Center Amsterdam, Amsterdam, The Netherlands e-mail: [email protected] K. Tabury Radiobiology Unit, Belgian Nuclear Research Centre (SCK CEN), Mol, Belgium Department of Biomedical Engineering, University of South Carolina, Columbia, SC, United States of America e-mail: [email protected] K. Vermeulen Institute of Nuclear Medical Applications, Belgian Nuclear Research Centre SCK CEN, Mol, Belgium e-mail: [email protected] K. Viktorsson Department of Oncology/Pathology, Karolinska Institutet, Stockholm, Sweden e-mail: [email protected] A. Waked Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium Laboratory of Nervous System Disorders and Therapy, GIGA Neurosciences, Université de Liège, Liège, Belgium e-mail: [email protected]

• To grasp the different methods of spatial RT fractionation and how tissue is spared by using these methods. • To learn basic principles of brachytherapy and understand the principles, treatment course and planning, application in clinical setting as well as the theory behind personalized radioembolization/ selective internal radiotherapy (SIRT). • To study the basic concepts and clinical applications of diagnostic/therapeutic radiopharmaceuticals and high linear energy transfer (LET) carbon ion irradiation. • To get an overview of nanotechnology and how it can improve treatment of cancer as well as challenges of translating it into clinical settings. • To acquire an understanding of the risk factors involved in acquiring secondary tumors after RT.

6  Radiobiology of Combining Radiotherapy with Other Cancer Treatment Modalities

6.1 Physics Radiotherapy (RT) relies on the effect of ionizing radiation (IR) to biological matter, i.e., cells. The radiation is transferring its energy to atoms and molecules present in the cells, which lie in the path of the radiation, and therefore ionizing them. These ionizations, i.e., the removal of electrons from the atom, lead to the breaking of chemical bonds in the molecules. If these ionizations occur in the cell nucleus, the DNA, carrier of the human genome, is damaged. In RT, the capability of radiation to damage the genome is exploited to kill tumor cells. The most important quantity to define the damage, which is caused, is the dose dE (6.1) dM i.e., the energy transferred from the ion to the matter (dE) by unit mass (dM). In general, one can say that the higher the dose, the larger the damage and the higher the probability of killing a cell. However, the same physical dose of different types of radiation can cause different damage in the cells. Various types of radiation are utilized for RT. These types of radiation can be distinguished by the socalled depth dose distribution, which is the dose which is transferred to matter along the path of radiation as shown in Fig. 6.1. Electron radiation transfers most of its energy just after it interacts with matter, i.e., tissue, making it suitable for the treatment of tumors close to the skin. If one uses electrons with higher energy, such as the shown 250 MeV electrons, the dose peak can be shifted deeper into the tissue. However, this comes with the disadvantage that the maximum range is also longer, resulting in more dose to the normal tissue beyond the tumor. Furthermore, such electron beams are quite complicated to produce. For photon beams used in RT, the dose increases in the so-called build-up region until it reaches the maximal dose and then gradually decreases. The D=

Fig. 6.1  Comparison of the relative depth dose distribution of 15 MeV electrons (green), 250 MeV electrons (purple), 2 MeV photons (red), 150 MeV protons (dark blue), and 250 MeV/u carbon (turquoise) and cobalt 60 (orange)

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depth of the maximal dose can be a few μm (for kV beams, i.e., beams with particle energy in the kilovolt regime) or several mm or cm (for MV (megavolt) beams). In contrast to electrons and photons, particles such as protons or high linear energy transfer (LET) carbon ions show a totally different dose distribution depth. The ions deliver a low dose when entering tissue. With depth this transfer is slowly increasing, while the ion gets slower. With further energy loss and decreasing speed, the dose drastically increases and reaches a maximum just before the ion stops in the tissue. This unique dose distribution is called the Bragg curve in honor to the physicist William Henry Bragg, who discovered this behavior in 1904 [1]. To widen the treatment depth range, a spread-out Bragg peak (SOBP) is created by varying the energy of the incident proton beam. As a result, a uniform dose can be delivered to the tumor. The radiobiological impact of particles with high LET is higher compared to photons, and it increases dramatically in the distal edge and fall-off. The uncertainty in relative biological effectiveness (RBE) of ion beams is still a limitation in its clinical application and should be considered during the treatment planning as a part of the process leading to a robust treatment plan. A detailed description about the physical and biological interactions of radiation to biological matter and the consequences for the biological effect can be found in Chaps. 2 and 3.

6.2 Conventional and Alternative Radiation Schemes

Box 6.1 Conventional and Alternative Radiation Schemes

• Typical conventionally fractionated irradiation schemes use 2  Gy fractions, 5 fractions per week for 3–7 weeks, depending on the tumor type. • Alternative radiation schemes, i.e., either smaller or larger sized fractions, multiple fractions per day, or different overall treatment time should be based on the various biological processes and response characteristics of both the normal and malignant tissues in the exposed volume.

When using radiation for cancer treatment purposes, the total radiation dose is generally applied in a regimen with multiple small fractions, aiming to reach tumor kill while sparing adjacent normal, healthy tissues, and organs. Most tumors are treated with a conventional fractionation regimen, which is characterized by daily fractions of 1.8–2 Gy, 5 days per week, for a duration of 3–7 weeks,

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Table 6.1  Characteristics of radiotherapy treatment regimen and involved radiobiological processes. (Reproduced with permission from [2]) Radiation treatment regimen Total dose (Gy) Fraction size (Gy) Number of fractions per day Treatment (days per week) Overall treatment time (weeks) Radiobiological reasoning—note the 6 Rs of Radiobiology

Conventional fractionation 70 1.8–2 1

Hyperfractionation ≥70 100 mGy) will also vary according to the age of exposure. Dogs exposed to doses higher than 7 Gy showed soft tissue cancers when exposed in utero but not when exposed as young adults [46]. In rodent species, there were limited carcinogenic effects on animals that were exposed to doses between 0.1 and 1 Gy [138]. Birds The effects of radiation exposure in birds are apparently similar to the ones observed in small mammals [45]. The LD50 for wild birds is in the same range as small mammals (5–12 Gy). For poultry, the LD50 determined experimentally for mortality is of 7–11 Gy in 3–4-day-old individuals when irradiation lasts for less than 1 h and of 12–20 Gy when irradiated for 24 h. Egg production is affected in white leghorn chicken at a total absorbed dose of 4–8  Gy and at higher doses, effects are more severe and long lasting [45]. A limited number of experiments performed in artificially incubated chicken embryos showed a LD50 of 12–13 Gy, which apparently indicates a higher radioresistance than adults [46]. In white leghorn chickens, eggs hatchability is affected at a total absorbed dose of 8  Gy, but the progeny is unaffected [48]. The International Commission on Radiation Protection also reported dose ranges for which long-term effects on developing embryos were reported (100– 1000 mGy/day), reduced reproductive success (1–10 mGy/ day) and increased morbidity (10–100  mGy/day) [139]. Recently, it was reported a decrease in species abundance at a dose range of (from 0.3 to 97 μGy/h) in the Fukushima exclusion zone, which is consistent with the dose ranges reported for increased morbidity and decreased reproductive success [140]. The existing knowledge on DNA damage/alterations on birds exposed to ionizing radiation results from the evaluation of effects of radioactive environmental contamination resulting from the Fukushima and Chernobyl accidents [141]. Reptiles and Amphibians The information gathered so far for reptiles and amphibians suggest that their radiosensitivity is similar to that of mammals and birds. The LD50 values recorded for frogs, salamanders, turtles and snakes vary between 2 and 24 Gy [46]. The main cause of death identified was damage to the hematopoietic system [46]. In two separate experiments performed on lizards, two very different LD50 doses ranges were obtained (10–12 and 17–22 Gy). The possible reasons for this marked

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difference are associated with the fact that these values may vary according to radiation type and quality, the dose rate to which the organisms were exposed and their maintenance conditions at the laboratory [46]. An acute exposure to 50 Gy caused temporary sterility in males, but recovery was well in process after 48  days post irradiation and irradiation of gonads in males and females to an absorbed dose of 4.5 Gy leads to a substantial decrease in the production of offspring [46]. Regarding amphibians, different life stages showed different radiosensitivities. For adult toads, the LD50 value is of 24 Gy, for juveniles it is of 10 Gy and for tadpoles it is of 17 Gy [46, 139]. The life stage more sensitive to radiation exposure was the fertilized egg with an LD50/40 (LD50 after 40 days of exposure) of 0.6 Gy [33]. There is evidence that the exposure of male toads to 3–20  Gy caused a reduced survival and increased induction of abnormalities to the offspring [46, 139]. Although these LD50 values for amphibians seem slightly higher than the ones recorded for mammals, time after exposure optimal for the recording of LD50 values seem to be an important factor [33]. Reptiles and amphibians are poikilothermic organisms; therefore, their metabolism is quite variable and different from mammals and birds [33]. A study performed on 4 species of amphibians showed that if the assay period was extended a decrease in the LD50 to values that ranged between 0.8 and 7 Gy would be recorded [33]. Chronic irradiation exposure (5.5 years duration) of common side blotched lizard, western whiptail, long nosed leopard lizard and long nosed lizard showed that at ranges from 285 to 570 μGy/h, radiation exposure caused lack of reproduction, female ovaries regression and some degree of male sterilization [46]. Regarding the induction of DNA damage, it was observed by Ulsh and co-authors [142] that the exposure of turtles from the species Trachemys scripta to 0–8 Gy 137Cs gamma radiation, given at a dose rate of 0.55  Gy/h induced the occurrence of significant levels of chromosome translocations in lymphocytes. Studies on the induction of DNA alterations in amphibians and reptiles have been performed in Fukushima and Chernobyl exclusion zones, as well as in areas contaminated with NORM. Aquatic Vertebrates Among non-mammalian aquatic organisms, fish are the most sensitive to the exposure to ionizing radiation [45, 46]. Although these organisms are also poikilothermic (as amphibians and reptiles), and therefore, apparently more radioresistant than mammals, there is a substantial overlap in radiosensitivities [46]. Until now, there is no substantial data on effects of ionizing radiation on marine mammals, however, there is no reason to believe that their radiosensitivity is substantially different from that of terrestrial mammals. Data

9  Environmental Radiobiology

on acute exposures exist mainly for bony and freshwater fishes, with a small number of studies on cartilaginous and marine and anadromous species. The LD50 determined for six marine species after 40–50  days of exposure was of 9–23  Gy [46, 139]. Fish developing embryos are, however, more sensitive than adults, as for silver salmon their LD50 after 50 days of exposure is of 0.30  Gy at hatching and 0.16  Gy at a post-hatching larval stage of 90 days [46]. A study performed on sharks (Triakis scyllium and Heterodontus japonicus) exposed to 20  Gy showed that mortality occurred after 20  days of exposure, due to hematopoietic and gastrointestinal damage [33]. This suggests that the radiosensitivity of cartilaginous fish may be similar to that of teleost fish. Regarding reproduction, an acute exposure to 10  Gy reduced the total number of germ cells at all developmental stages of medaka fish (Oryzias latipes) [46]. A similar radiosensitivity was found in rainbow trout, with an induction of more than 50% sterility in organisms exposed late in embryonic development [46]. This leads to the conclusion that as in mammals, the newly hatched fry and the primordial gonads in fish embryos are more sensitive to the acute radiation exposure than in adult fish [46]. Irradiation of mature medaka fish at acute doses of 5–10 Gy only induced temporary sterility, being completely recovered at 60  days after irradiation [46]. On the other hand, chronic irradiation of males from the fish species Ameca splendens for 5.4 days at a dose rate from 137Cs gamma rays of 7300 mGy/h disrupted spermatogenesis and render the animals sterile at an accumulated dose of 9.7 Gy (8 weeks of exposure) [46]. There was 60–70% recovery, 236  days after irradiation [46]. Another freshwater fish, the guppy (Poecilia reticulata), when exposed to gamma dose rates from 1700 to 13,000 mGy/h showed a significant reduction in fecundity, but no negative effects on survival and sex ratio, as well as no significant higher incidence of abnormalities in the offspring were observed [33, 136]. The marine fishes Pleuronectes platessa and the eelpout (Zoarces viviparus) exposed to 240 and 2000 mGy/h gamma radiation, respectively, showed a significant reduction of testes when compared to the control [136]. There are some findings also on the effects of the exposure to ionizing radiation in the immune system of these organisms. A significant reduction in the humoral immune response in the rainbow trout (Oncorhynchus mykiss) exposed to tritium beta-particles for 20 days at a dose rate as low as 8.3–83 mGy/h during embryogenesis was evidenced through a reduction in antibody titer following a specific challenge [46]. Regarding DNA damage there are very few studies on which some conclusion can be taken on this matter. On a study on medaka fish, at larval stages, there was a significant induction of vertebral anomalies after irradiation at dose

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rates from 137Cs gamma rays higher than 18,000 mGy/h and also to beta particles from 3H at dose rates higher than 35,000 mGy/h [46]. There is also a report on the occurrence of minor morphological abnormalities in the operculum of salmons exposed to a gamma radiation dose rate of 200 mGy/h that may affect latter survival [136].

9.4 The Particular Case of NORM Contamination Anthropogenic activities of concern related to the environmental release of natural uranium isotopes (mainly 238U and 235 U) and other radionuclides from their decay chains, namely 226Ra and 223Ra, 222Rn, and 210Po, include mainly the production of phosphate fertilizers, uranium mining and milling and the incorrect disposal of tailings, uranium conversion and enrichment, the production of uranium fuel, production of coal, oil and gas, extraction of rare earths, extraction and purification of water, extraction of minerals for building materials and the generation of geothermal energy [3, 143]. All of these industrial activities increase the concentration of these elements in all environmental matrices, thereby posing a risk to human and non-human biota as many of them have not been regulated for NORM release [3, 143]. Another important issue is the fact that the contaminated areas that result from these anthropogenic activities do not only present high levels of certain natural radionuclides, like 226Ra, 222Rn, and 210Po but also other important stressors, namely metals like manganese, zinc, iron, aluminum, etc. [143]. These are usually multiple exposure scenarios, which contain several kinds of contaminants that may act synergistically and increase the risk of the occurrence of biological effects on human and non-human biota and even of modifying the susceptibility of cells/organisms to the biological effects of ionizing radiation exposure [144].

9.4.1 Chronic Exposure and Interaction with Uranium and Metals The accumulation of small amounts of radionuclides and metal over long periods is translated in chronic exposure to radiation. Naturally contaminated sites harbor a diversity of microbial species that become resistant or tolerant to these contaminants by bioaccumulating radionuclides and metals either by biosorption to their cell surfaces and biomolecules or by internalization into their cells. Briefly, under environmental conditions, chronic IR effects are very complex, particularly when compared to those from laboratory exposures because (1) radiation emitted by the different radionuclides present has different biological effects, (2) radiation from the

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same location is absorbed differently by different microorganisms, (3) abiotic factors (e.g., temperature, nutrients, pH, other stressors) are present and can interfere with radiation, (4) cooperation/interaction between microbial communities, including diversity and/or abundance can all be modulated by radiation [62]. Regarding uranium, probably the most well studied radionuclide, and for which a lot of information is available, interaction with microbial cells involving solubility by biomineralization (bioprecipitation) depends on all the above factors and also on the presence of affinity groups generated by microorganisms’ cell metabolism, like hydroxides, phosphates, and carbonates. Uranium toxicity is both chemical and radiological. In the environment, uranium exists in its reduced insoluble form U(IV), and/or the oxidized form U(VI), which is soluble and toxic. Microorganisms interact with uranium by changing its redox state, aerobically, through oxidation (biolixiviation), or anaerobically by reduction. In order to do that, microorganisms need to be highly tolerant to uranium and to radiation. Other processes of microbial interaction with metals, involve biosorption, where contaminants passively concentrate through binding to cell structure constituents (e.g., lipopolysaccharides, teichoic acids, peptidoglycan), and biomineralization, which leads to the formation of biominerals using organic phosphate sources and phosphatases. Unless disturbance occurs, NORM sites have a characteristic microbiome, which is specific for a given site, but may share common microbial genera and species, regardless of location and/or chemical contamination. It includes nitrate-­ reducing bacteria that tolerate acidic and low-nutrient conditions, while being highly resistant to metals. Members of the Proteobacteria (Alpha-, Beta-, Delta- and Gamma- proteobacteria), Acidobacteria, Actinobacteria, Bacteroidetes, and Firmicutes are generally associated with uranium transformation and are therefore found in these environments. Most represented bacterial genus include Geobacter, Thiobacillus, Arthrobacter, Bacillus, Actinobacteria, Desulfovibrio, and Microbacterium. Most of the studies are focused on bacteria and bacterial communities. Although little information exists regarding fungi, they are particularly resistant to radiation and thus play a role in the process of detoxification of radionuclides. For instance, an isolate of the genus Paecilomyces, was found to detoxify U(VI) through bioprecipitation of the metal, and the reduction was promoted by phosphate. Also, the yeast S. cerevisiae was able to reduce U(VI) toxicity by biomineralization [60]. Accordingly, the survival, abundance, and maintenance of a given species or community diversity depend on its adaptability to the existing conditions. Furthermore, several studies suggest that in those radionuclide-rich natural sites, resistance to high levels of chronic IR may occur among taxa that tolerate a wide range of environmental conditions and, therefore, have an advantage over other more sensitive species [62].

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9.4.2 Effects of NORM and Metals on Eukaryotes 9.4.2.1 Invertebrates There have been some studies in aquatic organisms, namely in Daphnia magna, Daphnia longispina, and Moinodaphnia macleayi at NORM sites [145, 146]. When testing several percentages of a uranium mine effluent containing metals and radionuclides from 238U and 235U decay chains, the Antunes et al. [145] study recorded an EC5011 for daphnids immobilization at 50.4% for D. magna and at 28.4% for D. longispina, showing that D. magna was less sensitive than D. longispina. However, regarding fertility, D. magna was more sensitive than D. longispina, as this last species did not show significant effects in the offspring produced at effluent concentrations lower than 30.38%. Regarding M. macleayi, when a natural population of these organisms, living adjacent to a uranium mine in Australia, was challenged with a concentration of uranium ranging from 0 to 700 μg/L, it was shown that this population comparing to other populations tested, was the one that presented the highest sensitivity as it evidenced the lowest NOECs and LOECs.12 It was shown that although this population lived in a water containing already considerable amounts of uranium, there was no tolerance to higher levels of uranium, when compared to the other tested populations. This probably shows that it was an already very stressed population that suffered “genetic erosion” [147] and because of that, it had lower capacity to deal with additional stresses, such as a single high dose of uranium. When D. magna was exposed to uranium and to a uranium mine effluent [148, 149], significant genotoxic effects (DNA strand breaks) were detected in neonates and 50 (blue line), and >100 MeV (green line) was measured by the primary Geostationary Operational Environmental Satellite (GOES) satellite of the Space Weather Prediction Center (SWPC). CO Colorado, MeV Mega electron volt, NOAA National Oceanic and Atmospheric Administration, s second, sr steradian, UTC Coordinated

Universal Time. Reprinted with permission under terms of the Creative Commons Attribution License [28]. (b) Distribution in the energy of proton fluences for major past SEPs events (free space). (Reprinted with permission from: The space radiation environment: an introduction. Schimmerling W. https://three-jsc.nasa.gov/concepts/ SpaceRadiationEnviron.pdf. Date posted: 2-5-2011)

Table 10.1  Comparison of GCR and SPE Spatial distribution Composition Temporal variations Energy Origin Flux density Biological effects

GCR Isotropic beyond terrestrial influence (no preferred direction of arrival) Protons (~87%) and helium ions (~12%) with the remainder consisting of HZE (1–2%) Chronic Extending to at least 1017 eV in some cases (much greater maximum than solar particles) Theories only; supernova explosions, neutron stars, pulsars, or other sources Relatively low: about 2 particles/cm2/s of all energies Primarily genotoxic and mutagenic with some vital cell destruction

SPE Non-isotropic at onset, later becoming diffused through the solar system Mostly protons Acute About 1010 eV highest recorded Active regions of flares on the Sun, CMEs Very high: may be as high as 106 particles/cm2/s Primarily acute damages, possible sudden illness, incapacitation, or death

Adapted from https://msis.jsc.nasa.gov/sections/section05.htm#_5.7_RADIATION

10.3.1.3 Solar Wind The solar wind is a continuous flow of plasma from the Sun’s corona, mainly consisting of protons, electrons with a small percentage of He ions, with kinetic energies between 0.5 and 10 keV. There are also some trace amounts of heavy ions and atomic nuclei such as C, N, O, Ne, Mg, Si, S, and Fe. Their energy results from the high temperature of the Sun’s corona and allows them to escape the Sun’s gravity. The flux of the solar wind varies over time, solar longitude and latitude, together with its temperature, density, and speed. At distances of more than a few solar radii from the Sun, the solar wind reaches supersonic speeds of 250–750 km/s [32]. At much greater distances, about 75–90 astronomical units (1

au is the distance Sun-Earth), the so-called “termination shock,” interactions of the local interstellar medium with the solar wind slow it down to subsonic speed. There are different classes of solar wind [30]: (a) The long-lived solar wind high-speed streams, representatives of the inactive or “quiet” Sun. Sources for such streams are coronal holes usually located above inactive parts of the Sun, where “open” magnetic field lines prevail, e.g., around activity minima at the polar caps; (b) A slow wind stream from more active near-equatorial regions on the Sun, often associated with “closed” magnetic structures. Sharp boundaries exist between these two solar wind streams (in longitude as well as in latitude),

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and their main properties differ significantly according to the location and magnetic properties at the source; (c) Another slow solar wind stream emerging during high solar activity, from active regions distributed over large parts of the Sun, in a highly turbulent state. It is highly variable and usually contains a significant fraction (about 4%) of alpha particles; (d) The solar wind disturbances superimposed on the ambient solar wind in case of CMEs. They exhibit unusually high percentages of alpha particles (up to about 30%). The Earth’s magnetosphere deflects the solar wind, causing most of the solar wind to flow around and beyond us. Nevertheless, a small number of particles from the solar wind reach the upper atmosphere and ionosphere. This may produce phenomena such as aurora and geomagnetic storms, the latter occurring when large inflation of the magnetosphere, due to an increased pressure of the contained plasma, distorts the geomagnetic field. In space missions, the solar wind has no impact on astronauts, as it is efficiently stopped by the spacecraft shielding and also by appropriate astronaut suits, because of the small range in a matter of the low speed-solar wind particles. However, if not appropriately shielded, the solar wind particles may affect the human body during eventual EVAs in deep space or on the surface of airless bodies, such as the Moon.

10.3.1.4 Trapped Radiation Trapped radiation particles are produced mainly by the interaction of GCRs and SEPs with the Earth’s atmosphere and are trapped by its magnetic field into the Van Allen radiation belts. These comprise: (a) A stable inner belt of trapped protons and electrons with energies between some keV and 100 MeV that is centered at a height between 300 and 1000 km above the Earth and reaches up to a height of around 10,000 km. (b) A less stable outer electron belt, comprising mainly high-energy (0.1–10 MeV) electrons and which extends from an altitude of about 10,000–40,000  km (see Fig. 10.7 for a schematic representation). In the radiation belts, the energetic particles move along Earth’s magnetic field lines, via the combination of three types of motion: a fast rotation (or “gyration”) around magnetic field lines, typically thousands of times each second; a back-and-forth bouncing along the stronger magnetic fields in the northern and southern hemispheres, typically lasting 1/10 s; a slow drift around the magnetic axis of the Earth (the drift is eastward for electrons and westwards for ions), such drift is from the current field line to its neighbor, with the particle keeping roughly the same distance from the axis. A

Fig. 10.7  Radiation belts of the Earth. (Figure from Van Allen radiation belt. Reprinted with permission from Wikipedia. Author Booyabazooka at English Wikipedia, https://commons.wikimedia.org/ wiki/File:Van_Allen_radiation_belt.svg)

typical time to complete a full circle around the Earth is a few minutes. In the area above the southeastern part of South America and the South Atlantic, the inner radiation belt approaches the surface of Earth down to a few hundreds of kilometers (South Atlantic Anomaly, SAA). This is caused by the tilt and shift of the axis of the dipole-like magnetic field of the Earth with respect to its axis of rotation [33]. The dip of magnetic lines leads to an increased particle flux within this region. The dose rate experienced by the astronauts on the ISS has a considerable contribution from trapped protons in the inner Van Allen belt because the ISS orbit with an altitude of about 400 km passes through this belt at the SAA (roughly 50% of the total dose rate) [34].

10.3.2 Radiation Environment in Low Earth Orbit (LEO) A low Earth orbit (LEO) is an Earth-centered orbit close to our planet with an altitude ranging from 160 to 2000 km. Thus, the ISS, which flies at an altitude of around 400 km, is also in such an orbit, with an orbital inclination (the tilt of the orbital plane with respect to the equatorial plane, which helps to understand an orbit’s orientation with respect to the equator) of 51.6° and an orbiting period of 90–93 min. Consequently, in 24 h the ISS makes 16 orbits of Earth and travels through 16 sunrises and sunsets. The environment of these altitudes is extreme and characterized by microgravity, high vacuum, meteoroids, extremes of temperature, ionospheric plasma, space debris, and UV as well as ionizing radiations. The radiation sources are GCR, trapped radiation, and SEP events. The GCR environment accounts for about 50% of the total dose rate, the other 50% being induced by trapped protons of the inner belt, the only component of the inner belts that reaches energies and intensities to be important for effects on astronauts inside the ISS [35]. Other orbits, such

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as Medium Earth Orbits (2000–35,786 km), Geostationary orbits (35,786 km), and High Earth Orbits (over 35.786 km), are exposed to different sub-components of the trapped radiation, some may not pose any danger. On board the ISS, astronauts encounter SPE events as a transient increase in dose rates. As mentioned above, the GCR flux is modulated by the solar cycle. At the ISS altitude, the GCR flux is also modified by the geomagnetic field, besides the modulation due to the solar activity. This field removes particles with lower energies (~few GeV/nucleon), but particles of higher energies are unaffected [36]. At low altitudes, the trapped radiation is also modulated by solar activity: at solar maximum, because of the increase in UV radiation, the upper atmosphere expands, leading to the loss of trapped protons at low altitudes. Furthermore, the inner radiation belt is mainly filled by decaying neutrons created by incoming GCR particles and the GCR flux is inversely proportional to solar activity [37]. Therefore, at solar maximum, a lower proton flux is present, leading to a smaller radiation hazard compared to the solar minimum [36, 38]. The interaction of energetic protons and HZE nuclei with spacecraft structures produces an additional intravehicular radiation field. This secondary radiation includes mainly, protons, neutrons, photons (X-rays and gamma rays), leptons (e.g., electrons and positrons), mesons (e.g., charged pions) and a great number of lighter and heavier nuclear isotopes (ions) [39, 40]. This happens in LEO and is of high concern in particular for the deep Space phase of a mission (see below), as the spacecraft would not be protected by the Earth’s atmosphere and magnetic field.

10.3.3 Radiation Environment Beyond LEO (Deep Space, Moon, Mars) 10.3.3.1 Deep Space Radiation challenges for astronautic missions beyond LEO, such as travel to the Moon or Mars, come from SEP events, GCR and intravehicular secondary radiation (Fig. 10.2). The solar wind particles, also constantly present in deep Space, do not contribute to the radiation dose induced in crews inside a spacecraft, as they are efficiently stopped even by thin shielding thicknesses. Similar to the case of the LEO scenario, most GCRs are not efficiently stopped by regular depths of spacecraft shielding. The intravehicular radiation field is constituted by the ensemble of secondary radiation mentioned above. Adding more shielding would increase to a considerable extent the weight at launch and would not reduce the GCR-induced absorbed dose to zero. As the only modulation of GCR in deep space is provided by the shielding of the heliospheric field during solar maximum, the idea of carrying out missions to Mars during solar maximum has been considered a

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viable option. If one considers that during a 180-day trip at the solar maximum peak a crew would also likely receive a total SEP-contributed dose equivalent, a round trip to Mars would result in a total dose equivalent of 560 ±180 mSv,1 higher than the estimation based on the data from the Radiation Assessment Detector (RAD) onboard the Mars Science Laboratory NASA mission [41] which was on cruise during solar minimum [42]. The above estimate for the radiation exposure is substantially lower than the accepted safe upper limit for 30–60-years old nonsmoking females and males (above 1500 mSv—see Fig. 10.8). However, inaccuracy and limitations of the models and unpredictability of SEP events must be considered.

10.3.3.2 Airless Bodies: The Moon The Moon is about 380,000 km away from Earth and is the next endeavor for space missions beyond LEO.  Although some areas of the Moon have a weak magnetic field, the Moon does not have a global magnetic field like on Earth and no atmosphere. Consequently, its surface is not shielded from radiation. The solar wind particles get stopped in the first millimeters or, maximally, centimeters of the lunar regolith, while GCR and SEP can impact the lunar surface also resulting in the production of backscattered secondary particles. The total amount of radiation that astronauts will be exposed to is influenced strongly by solar activity, their whereabouts on the Moon surface with respect to local magnetic fields, and the type and amount of radiation shielding used in spacecraft, Moon vehicles, and habitats. Recently, the Lunar Lander Neutrons and Dosimetry experiment aboard China’s Chang’E 4 lander revealed that radiation levels on the Moon’s surface are 200–1000 times more than that on Earth’s surface and 2.6 times more than what astronauts onboard the ISS are exposed to Zhang et al. [44]. Efficiency of the radiation shielding by lava tubes on the Moon appears promising to reduce the dose rates considerably [45]. 10.3.3.3 On Mars Mars does not possess a global magnetic field, and it has only a thin atmosphere with its surface pressure less than 1% of that at Earth’s surface. Therefore, high-energy GCRs can reach the surface, although still a considerable portion of them will induce hadronic-electromagnetic-muon cascades in the atmosphere, causing fragmentation/spallation and ionization showers of downward secondaries. All these particles can then induce further reactions in the planet’s regolith, which generate a backscattered, albedo radiation component,

Sievert (Sv) denotes the equivalent dose as measure for biological and medical relevant quantification of dose in radiation protection. For a detailed explanation please refer to Chap. 2. 1 

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Fig. 10.8  Relative radiation exposure of varying duration during medical procedures (green), specific space missions (purple), and on various celestial bodies (blue). The astronaut yearly and career limits are given in red boxes. For comparison, some facts on radiation exposure of the general population and occupational exposure limits (US) are indicated (gold). (Reprinted with permission from Iosim et al. [43])

giving overall complex spectra including both primaries and (downward and upward) secondaries at the surface [46–48]. SEP events can increase the dose rate and dose equivalent at the Martian surface and constitute a danger for EVA on Mars. Only protons impinging the top of the atmosphere with energy above ~200 MeV do actually reach the ground, and thus SEPs events with high flux contribution at high energy constitute the biggest hazard for explorers on Mars if they are not in a habitat or otherwise sufficiently shielded.

For the solar wind, despite the thin character of Mars’ atmosphere, the upper layers of the latter are able to stop such radiation. Underground solutions for Mars habitats, shielded from the radiation by the regolith, are being investigated [49]. Overall, to contextualize radiation doses in space, a comparison of these doses to doses received during medical interventions is shown in Fig. 10.8. It is important to emphasize that being exposed to a hefty radiation dose within a

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short time (minutes to hours) will be more health-threatening than the same dosage over a longer duration of months of years. Yet, although the health effects of acute radiation exposure are well studied, less is known about the effects of chronic exposure.

10.3.4 Space Radiation Shielding Ionizing radiation exposure is one of the most critical health risks for astronauts. Inside the ISS, astronauts are exposed to an effective dose rate of the order of 20 μSv/h, which is about 100 times higher than on the Earth’s surface. Beyond LEO in deep space, the protection of the Earth’s atmosphere and magnetic field disappears, leading to an effective dose rate of the order of 75 μSv/h. Also, on the surface of the Moon or Mars, there is only limited protection and astronauts are exposed to respectively about 30 and 25 μSv/h. It is estimated that astronauts will accumulate during a Mars mission a total effective dose of the order of 1 Sv, leading to an extra risk for cancer of the order of a few percent up to more than 10% depending on sex and age [50]. Furthermore, on their way through deep space or on the surface of the Moon or Mars, astronauts can receive such high doses during intense solar storms that immediate health effects or even a deadly outcome are possible (see Sect. 10.4.2). Therefore, it is clear that astronauts need to be protected against ionizing radiation in space.

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The only technology that can currently be used in practice to reduce the radiation level in spacecraft is to use shielding materials for stopping part of the radiation. The heavy ion impinging on the shielding material is the projectile, and the shielding material is the target. A multitude of interactions can occur when the projectile hits the target, including fragmentation of the projectile or target. For comparison of different materials, the area density as mass per unit area in g/ cm2 is used (for example, an 1 cm thick plate of Al with the density of 2.7 g/cm3 has an area density of 2.7 g/cm2). In current spacecraft, one makes most use of constructive materials such as aluminum. Unfortunately, such materials are not the most efficient for radiation shielding in space (see Chap. 4). The interaction of energetic GCRs with heavier elements such as aluminum results in the breakup of these heavier elements and the creation of secondary cosmic radiation such as energetic heavy ions and neutrons. Therefore, when using aluminum for shielding, the effective dose rate first increases as function of the shielding thickness before it starts to decrease and this decrease is quite flat as attenuation of heavy ions is nearly in balance with the build-up of light particles (Fig. 10.9 Left). Materials consisting of lighter elements such as hydrogen have a higher stopping power per unit of mass for charged radiation particles as they attenuate their fluence via projectile fragmentation. They also minimize the build-up of neutrons and other target fragments. Radiation protection of astronauts can thus be further optimized by making use of

Fig. 10.9  Calculated dose equivalent rate in LEO (51.6° inclination, 390 km altitude) as a function of shielding thickness given as area density for different shielding materials: (left) GCR, (right) Van Allen trapped protons. (Data used with permission from Dietze et al. [37])

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lighter shielding materials or, for instance, also by making strategic use of the necessary stock of water as additional shielding. Figure 10.9 shows the calculated dose equivalent rate for a LEO orbit similar to that of ISS (51.6° inclination, 390 km altitude) as a function of shielding thickness for different shielding materials. At standard temperature and pressure, based on a density of 1000 kg/m3, the water column required to reach an area density of 20 g/cm2 would have a height of 20 cm. For 20 g/cm2 aluminum, a material thickness of 7.4 cm is derived from the density at room temperature of 2.7 g/cm3. At the same area density of 20 g/cm2, the shielding effect of water is much more pronounced than the one of aluminum. The thickness of the two materials is different, but they would contribute to the same extent to the mass budget of the spacecraft which is critical for leaving the Earth surface during launch. The left and right plots show the results for respectively GCRs and Van Allen protons. These plots clearly show that hydrogenous materials are much more efficient for radiation shielding in space. In spacecraft it is unfortunately not possible to reduce the effective dose rate to the dose rate on Earth’s surface. With limited shielding, a large part of the energetic protons and electrons from SEPs and the Van Allen protons can be stopped. However, GCRs have such high energies that about 1000 g/cm2 of shielding is required to reduce the effective dose rate to the level on Earth’s surface. Due to mass constraints in spacecraft, only shielding of the order of a few 10 g/cm2 is possible. In spacecraft, astronauts can thus be protected against sudden very high and potentially deadly doses from solar storms, but they will be unavoidably chronically exposed to the ever-present GCRs leading to an increased risk for late effects. It is clear that with current technology additional radiation exposure in spacecraft is unavoidable. However, for future manned missions to the Moon or Mars during which astronauts will stay on the surface for a longer time it will be necessary to strongly reduce their radiation exposure during their stay. This is possible because on the surface of the Moon or Mars, we can make use of the present soil material to provide adequate shielding. A few meters of soil material should suffice to reduce the effective dose rate level to similar levels as on Earth’s surface. This can be done by building igloos or by living in caves or lava tubes. Besides shielding by using materials to block the radiation, it is in principle also possible to make use of strong electromagnetic field for shielding. Several research groups are investigating this possibility. However, the required mass and energy consumption of such systems makes the concept practically impossible with current technology (Box 10.1).

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Box 10.1 Highlights

(a) GCRs are the constantly present highly energetic radiation in space, they are mostly constituted by protons, with a smaller contribution from alpha particles and HZE particles. They generate particle showers in the atmosphere, although a small portion of direct GCRs can eventually reach the ground. (b) SEP events are more probable during solar maximum, but they can actually also occur during solar minimum. (c) Trapped radiation is constituted by GCRs and solar protons trapped in the Van Allen belts. Trapped radiation is a concern for ISS-like missions, especially because of the flux accumulated during different orbits in the SAA, or also missions on other orbits crossing one or the other belt.

10.3.5 Mathematical Modelling the Space Radiation Environment and Induced Doses 10.3.5.1 Transport of Radiation Through Matter: Deterministic and Monte Carlo Methods The modeling of the radiation environment at or inside a spacecraft, at different altitudes in the atmosphere or at the surface/subsurface of a planet, a moon, or a small body allows to obtain the relevant dosimetric quantities for the assessment of the health risks incurred by humans due to radiation [51–53], as well as to estimate the half-lives of biomolecules in search-for-life studies [54, 55]. The transport of radiation through matter is described by the time-independent Linear Boltzmann Transport Equation, which allows to treat atomic and nuclear collisions. The Boltzmann transport equation (10.1) describes the flux ni(r, E, Ω, t) of several types of particles i, possessing different energies E, and moving in different directions Ω by considering the particle balance in a small volume V. It thus gives the average space-time distribution of the expected energy-momentum behavior of the particle beam, transported and scattered across the target, where each interaction is characterized by its own differential crossd 2σ . The Boltzmann equation reads as section d ΩdW follows:

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∫ dr

∂ni ( r,E ,Ω,t ) ∂t

= − ∫ dAj ( r,E ,Ω,t ) ⋅ aˆ − N ∫ drni ( r,E ,Ω,t ) v ( E ) σ ( E ) S V     unscattered particles

particles scattered out

d 2σ + N ∫ dr ∫ dE ′ ∫ d Ω′ni ( r,E ′,Ω′,t ) v ( E ′ ) V d Ω′′dW ′′   particles scattered in

(10.1)

d 2σ sec,i + N ∫ dr ∫ dE ′ ∫ d Ω′∑n j ( r,E ′,Ω′,t ) v ( E ′ ) + dr Qsource ( r,E ,Ω,t ) V V  d Ω′′dW ′′ ∫ j   source production of secondaries

In this equation: • the first term is the time-dependent flux change, due to particles escaping from the system boundaries, or disappearing by an absorption reaction or radioactive decay; • on the right-hand side, the unscattered term represents the flux change due to translation without change of energy and direction (free flight); • the particles scattered out are those exiting a “cell” (a unit volume in the phase space, the latter comprising both space and time variables); • the particles scattered in are those entering a “cell” from a “cell” at a previous point in the phase space; • the production of secondaries represents the effect of collisions; • the source term can be external (e.g., a particle beam irradiating the target volume), or internal (e.g., neutrons from fission reactions in the volume). In particular for high-energy particles, the number of interactions that must be described in order to find the solution to this equation is daunting, including ionization, excitation, spallation/fission/fragmentation, production of positron-emitting nuclei, and de-excitation through gamma rays. A solution to the problem can be attained via two different approaches: 1. Deterministic methods. These are deterministic approaches based on approximations to the Boltzmann equation and often on the reduction to a 1D problem via the use of the straight-ahead approximation, according to which the secondary particles from nucleon-nucleus collisions are emitted in the direction of the incident nucleon [37]. They rely on models for the relevant quantities in the transport calculation and use the continuous slowing down approximation (CSDA). Deterministic codes such as NASA’s HZETRN [56] and BRYNTRN [57] follow such an approach and require relatively low computational resources to perform calculations and the calcula-

 tion time is relatively short. This is due to the fact that deterministic codes do not consider all products of reactions and neglect their correlation, e.g., the coefficients used in the Boltzmann equation are related to relatively simple one-particle quantities. Thus, correlations on event-by-event basis are not considered and particle scattering at an angle is ignored [58]. Last, such methods can only be applied to restricted geometries and restricted interaction models. 2. Monte Carlo method. Monte Carlo (MC) is a stochastic method, exploiting random numbers to (a) “generate” an initial particles’ “cocktail”; (b) track them in arbitrary geometries; (c) accumulate the contribution of each track to a statistical estimator of the desired physical observables [59]. Step-by-step particles’ transport is simulated according to the statistical model of their interactions. Quantities (such as step lengths, event type, energy losses, and deflections) are sampled via generation of random values according to a given probability distribution. Indeed, in MC codes, the MC method deals with sampling from suitable stochastic distributions, with large samplings allowing to solve the integrations of multidimensional integrals. In the context of space environment, the main interest is in high-energy particles whose scattering is generally lowangle. Therefore, it is reasonable to approximate multiple scatterings by a single continuous step, taking into account overall energy loss and direction change. This approach is known as the condensed-history technique. For example, ionization and excitation energy losses are described as continuous processes, i.e., they are continuously distributed along a particle step, if the loss is lower than a chosen threshold, together with their fluctuations. Several MC codes are used nowadays throughout the world, such as Geant4 [60], FLUKA [61], and PHITS [62]. MC codes provide a detailed treatment of the threedimensional transport of ions and neutral particles (see Chap. 4).

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10.3.5.2 Practical Steps in the Modelling of the Space Radiation Environment and Induced Doses An overview of the different steps for calculation of the radiation environment of a celestial body is given in Fig. 10.10. Input Spectra The input spectrum for GCRs can be chosen among different existing models that account for the variations of GCR particle fluxes due to variations in solar activity and in the large-scale heliospheric magnetic field throughout the solar cycle. The ISO 15390 model (ISO-15390 2004) [63] accounts for solar cycle variations in the GCR intensities on the basis of 12-month averages of the sunspot number. Changes in the large-scale heliospheric magnetic field are usually taken proportional to the corresponding changes in the Sun’s magnetic field, considering also solar cycle. More accurate models describe the spectra of GCR beyond the heliospheric modulation region. The CREME96 [64] and its updated version CREME2009 (https://creme.isde. vanderbilt.edu/) are based on a semi-empirical model [65] where the particle spectrum is calculated as a product of a function describing the LIS and a function describing the modulation according to solar activity. GCR particle spectra are described in the energy range from 10 to 105 MeV/ Fig. 10.10  Scheme for Monte Carlo (MC) calculations of the radiation environment at a planet/ celestial body, here in particular Mars. GCRs galactic cosmic rays, SEPs solar energetic particles, p+ protons, He2+ ions helium ions

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nucleon, from H up to Ni nuclei from the year 1760 to present. The Badhwar–O’Neill 2010 (BON2010) [66] uses, instead of an empirical description of the modulated GCR. As in the CREME model, a physical approach to describe the GCR propagation in the heliosphere due to diffusion, convection, and adiabatic deceleration. The BON2010 model exploits data from the International Sunspot Number (ISN) and considers time lag of GCR flux relative to the solar activity. The ISN is calibrated with GCR measurements from the Advanced Composition Explorer (ACE) and the Interplanetary Monitoring Platform-8 (IMP-8). The BurgerUsoskin model [67] is limited to GCR He and H ions assuming a constant ratio of the two types of ions. The reconstruction of the modulation parameter is based on neutron monitor count rates. The DLR model by Matthia et al. [68] describes the GCRs spectra of nuclei based on a single parameter, which is derived from measurements of the ACE spacecraft and from Oulu neutron monitor count rates for different solar modulation conditions. SEP proton spectra are often considered from historical events, then parameterized by double power law fits in kinetic energy to event-accumulated integral fluence measured by the Geostationary Operational Environment Satellites and/or ground-based neutron monitor data [69].

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Input spectra for both GCR and SEP events can often be retrieved via user-friendly tools, such as the SPENVIS online tool (https://www.spenvis.oma.be/) that is actually a collection of modules that allow for calculations of the radiation environment and radiation-induced effects via MC simulations in Geant4, or the On-Line Tool for the Assessment of Radiation in Space (OLTARIS) which operates on top of the deterministic code HZETRN (https:// oltaris.nasa.gov/). Atmospheric Model For Earth, more than 99.99% of its atmosphere’s mass is contained in the lower atmospheric layers below about 100 km. This region is mainly composed of N2, O2, and Ar which account for about 75%, 23%, and 1.3% by mass, respectively. The exact mass fraction of each constituent depends on the altitude. The water content in the atmosphere is highly variable but small, with the hydrogen fraction only reaching the order of 10–5% even in cloudy conditions [70]. Composition, density, temperature, and pressure vertical profiles can be obtained, for example, from the empirical atmospheric model 1 NRLMSISE-00 [71], which includes total mass density from satellite accelerometers and from orbit determination covering 1981– 1997. For Mars, vertical profiles for pressure, density, temperature, and chemical composition of the atmosphere are often constructed exploting databases like MCD (Mars Climate Database http://www-mars.lmd.jussieu.fr) [46, 49]. Data can be extracted for specific locations, a specific day/night time, and season. The surface elevation and topology are extracted from the Mars Orbiter Laser Altimeter (MOLA) aboard Mars Global Surveyor. The fields (temperature, wind, density, pressure, radiative fluxes, etc.) are stored on a 5° × 5°, longitude-latitude grid from the surface to 120 km (and above) are averaged and stored 12 times a day, for 12 Martian “seasons.” Surface and Subsurface For Earth, the soil is often considered to consist of 50%Vol solids (of which 75%Vol SiO2 and 25%Vol Al2O3) and a scalable amount of H2O. Studies show that the neutron environment strongly depends on soil moisture (and air humidity) [72]. The composition of the surface and subsurface of Mars can either be chosen to model specific scenarios, for example, a default basaltic composition (SiO2 51.2%, Fe2O3 9.3%, H2O 7.4%) [73] or more/less hydrated compositions to study the possibility of underground shielding habitats [49], or it can be taken from data from the Gamma Ray Spectrometer aboard Mars Odyssey [46]. The dosimetric quantities at the Martian surface do not depend strongly on the regolith composition, although some differences due to hydration and Fe-content can affect neutrons and gamma rays spectra [49].

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Propagation MC particle transport codes strongly rely on the availability of physics models and database of cross sections. A schematic view of the downward and upward main particles that need to be considered is shown in Fig.  10.11. In the open source Geant4 code [60], hadronic models are: (1) datadriven, which mainly deals with the detailed transport of low-energy neutrons and isotope production, (2) parametrized models which include fission, capture, elastic, and inelastic scattering reactions; (3) theoretical models for high energies, above several 10–100 MeV, where experimental cross-section data are scarce. For electromagnetic physics, the basic processes for electrons, positrons, photons, and ions, such as Compton scattering, photoelectric effect, pair production, muon-pair production for photons, ionization, δ-electron production, Bremsstrahlung, Čerenkov radiation, and annihilation, are considered. Additionally, processes involving the atomic shell structure such as Rayleigh scattering are also considered. Special process classes handle muon interactions like Bremsstrahlung, capture, and annihilation. Multiple scattering models provide corrections for path lengths and lateral displacements of multiple scattered charged particles. In order to decrease the computational time and resources, a certain production cutoff in the range is set for electrons, positrons, and photons, which is translated to energy below which the particle then loses its remaining kinetic energy continuously along the track and no secondary particles are produced. Target In principle, the proper approach to calculate the absorbed dose and dose equivalent rates is to use. Such standardized phantom has been defined by the International Commission on Radiation Units (ICRU) and it is given by the ICRU sphere, a 30 cm-diameter sphere with a density of 1 g/cm3 and a mass composition of 76.2% O, 11.1% C, 10.1% H, and 2.6% N,

Fig. 10.11  Schematic view of the particle showers (main particles are plotted here) generated in the downward propagation of primary GCRs particles through the Martian atmosphere and of the backscattered particles [74]

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which reflects the composition of tissue. Still, in recent times more human-like phantoms have been used [75]. However, such complexity is not always necessary, and sometimes other spheres of water or water slabs have been used [76]. Apart from running the MC (or deterministic) codes in standalone mode, several tools such as the previously mentioned SPENVIS online system (https://www.spenvis.oma.be/), OLTARIS (https://oltaris.nasa.gov/), and the EXPACS/PARMA code (https://phits.jaea.go.jp/ expacs/) based on PHITS can be used to run a combination of the steps described above, resulting in a punctual estimation of doses at a specific location on a body or altitude in an atmosphere or in radiation maps covering several regions. For human exploration of Mars and other bodies, the quantities of interest are the absorbed dose corrected by the relative biological effectiveness (RBE) factor (to estimate the risk for acute effects or death due to high doses for Solar Energetic Particle events) and the Effective Dose and Dose Equivalent to respectively estimate the risks to long-term effects induced by exposure to GCRs and to compare with measurements from radiation detectors. Space Agencies implement the ALARA principle [77] which ensures that mission operations are designed to keep the radiation risks as low as reasonably achievable. Although the different agencies use common limits for deterministic effects on the ISS, different career radiation exposure limits (for stochastic effects) for astronauts in LEO missions exist and no specific limits for interplanetary missions are issued (only those for LEO exist).

10.3.5.3 Harmonization of Risk Models for Stochastic Effects: The Problem of Radiation Quality Factors Harmonization of risk models requires improvements in modeling radiation sources, in the accuracy of radiation transport codes, and the development of new realistic quality factors based on the features of the variegated radiation field in Space. As already mentioned in Chap. 2, the approach commonly used for estimating risk from high linear energy transfer (high-LET) radiations is based on multiplying the induced

absorbed dose (in units of gray) by a so-called quality factor, or RBE factor (always greater than one, usually below 20) representing the enhancement of effectiveness of the highLET radiation. Such increased effectiveness comes from available evidence on the RBE of the radiations from both laboratory and theoretical studies (Sects. 10.4 and 10.5). As previously shown, RBE varies with LET. It depends also on other factors and may be different, e.g., for particular chromosome aberrations, mutations, or different tumor types. Also, RBE may vary in different biological systems. Furthermore, low-LET dose response is usually nonlinear while high-LET response tends to be more linear. However, for radiation protection purposes, the use of RBE for low-dose exposure to radiation with different LET was superseded by the adoption of radiation weighting factor, wR, by the International Commission on Radiological Protection (ICRP) [78], to convert absorbed dose (measured in Gy) to equivalent dose (measured in Sv) in a tissue and to effective dose (measured also in Sv) in the body. ICRP recommends wR = 1 for photons of all energies, electrons, and leptons. The value wR = 2 is recommended for protons and charged pions, and wR = 20 for α-particles, heavy charged particles, and fission fragments [78] (see Table  10.2). However, the adoption of specific values for such weighting factors, based on the judgment from the available data on RBE, was accompanied by a recognition of the simplistic description and of the limited accuracy that the systematic application of this set of values for wR would have brought. Thus, quality factors, Q(LET), defined as a continuous function of the LET of the radiation, were later introduced in order to give broadly similar results for measured radiation fields [78] (see Table 10.2). Such quality factors are nowadays used in the risk assessment model by the European Space Agency and were also used in the previous risk assessment model by NASA. Nevertheless, this specification of Q in terms of the LET alone suffers from the limitations already highlighted in Chap. 1, about the fact that the sole LET cannot fully describe the effectiveness of radiation in inducing biological damage. Indeed, even simply from the perspectives of the first-stage radiation-induced effects, without mentioning the complex dependencies of the RBE on phenomena related to the chemi-

Table 10.2  Radiation weighting factors and quality factors Radiation type Photons Electrons and muons Protons and charged pions Alpha particles, fission fragments, heavy ions Neutrons  For LET  100 keV/μm

Radiation weighting factor (wR) 1 1 2 20 A continuous function of neutron energy

Quality factor (Q(LET))

1 Q = 0.32L–2.2 Q = 300L–1/2

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cal and biological steps, it remains the fact particles with different charge and different velocity may have the same LET and still inducing different final biological effects. The variation in the effectiveness of radiation in inducing different final biological effects has thus its root in the differences in track structures between particles that have the same LET but different charge and velocity, as highlighted in Chap. 1. Differences can be particularly large for the HZE particles encountered in space, methods used on Earth are inadequate for space travel, as, among other reasons, the ICRP radiation quality description does not represent HZE radiobiology correctly. The key difference between (a) the quality factor used by NASA [79] for the projection of risk from space exposures and (b) the quality factor recommended by the ICRP (Q(LET)) for operational radiation protection on Earth is consideration of track structure (Box 10.2). Box 10.2 Modeling

(a) The Boltzmann equation describes the transport of radiation in matter; it can be solved via analytical (deterministic) or via numerical (Monte Carlo) methods. (b) The different steps for setting up a calculation of the radiation environment are input radiation spectra, definition of the parameters describing the atmosphere, with dependence on the altitude, definition of the regolith composition, definition of the physics model to be used according to the different energy ranges, definition of the target where the scoring of the absorbed dose will be done.

10.4 Human Health and Organs at Risks for Space Travel The space environment is hostile to the health of astronauts in several ways. The confinement in the restricted space of spacecraft for shorter or longer periods exposes the crew to sometimes severe behavioral problems. Microgravity can lead to osteoporosis, a modification of the electrolyte compartments, sarcopenia, cardiac arrhythmias, dysthemeral rhythm disorganization, vestibular deconditioning, relative immunosuppression, and postural hypotension on return [80]. Finally, the space radiation environment is very different and much more hostile than that encountered on Earth. Add a temperature amplitude of 300 °C on the spacecraft’s surface and the almost absolute vacuum conditions that astronauts must consider during extravehicular excursions. Finally, let us point out the disturbances secondary to the return to the ground: neurological, vestibular, cardiovascular reconditioning, etc.

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10.4.1 Radiation Exposure During Space Missions The constant flux of galactic cosmic rays (GCR) causes astronauts’ chronic low-dose whole-body exposure during space missions. The primary GCR particles interact with the spacecraft hull, so that astronauts are—like patients—exposed to secondary radiation from nuclear interactions between the incident radiation and the shielding of the spacecraft. Due to mass limitations for launching spacecraft, complete shielding of GCR is not feasible. Compared to an astronaut suit for extravehicular activities, the shielding of the spacecraft by aluminum and other materials strongly reduces the skin dose and also, but to a much lower extent, the whole-body dose. On the microscopic level, due to the physical characteristics of particle radiation, very high doses can be reached, leading to permanent damage (see Sect. 10.4). In LEO, traversal of the SAA of the inner radiation belt contributes to the accumulated dose during, e.g., a mission on the ISS.  Human phantom experiments on the ISS (MATROSHKA experiment series) allowed the quantification of the effective dose rate which was 690–720 μSv/day during extravehicular activities and lower inside the ISS amounting to 550–570 μSv/day [81, 82]. Therefore, astronauts accumulate effective doses of around 100 mSv during a 6-months ISS mission. The variations of the accumulated dose depend on solar activity and the flight altitude of ISS, with higher doses during lower solar activity and increasing flight altitude. For a 1000day Mars mission, a total effective dose of galactic cosmic radiation of about 1 Sv is expected [83, 84], which is quite considerable and exceeds terrestrial lifetime radiation exposure limits, which amount to 400 mSv in the European Union. Risks of cancer and degenerative diseases are associated with this chronic GCR exposure (Fig. 10.12). Solar Particle Events (SPE) emanating from the Sun (Sect. 10.3.1.2) result in increased proton fluxes that may reach the spacecraft or a celestial body surface. In LEO, protection by the Earth’s magnetic field is still sufficient to protect from deadly SPE, but in free space or on planets or moons without magnetic field and atmosphere, high doses might be accumulated within hours or days in situations of insufficient shielding, e.g., in a spacesuit. Above a certain threshold, acute effects will occur (Fig. 10.12). In contrast to GCR, shielding of SPE protons is feasible in special compartments of the spacecraft, which can be surrounded by more material. Astronauts can protect themselves from an SPE in such a radiation shelter until the proton flux normalizes.

10.4.2 Acute Effects Deterministic effects appear for acute global exposures classified as medium, high, and very high (0.2 to more than 10 Sv) by UNSCEAR [85].

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Fig. 10.12  Possible health effects of space radiation exposure

Under exceptional conditions of insufficient shielding during spaceflight, the exposure to mostly protons during a large solar particle event (SPE), the whole-body dose can reach several Gy or the skin dose even tens of Gy and thereby cause the acute radiation syndrome (ARS, see Chap. 2, Sect. 2.7.2). Such situations in the event of a solar flare of exceptional intensity can occur in LEO in areas of weakness of the Van Allen belts, extravehicular exit, and exit on extraterrestrial soil in a spacesuit or an insufficiently shielded vehicle. The total dose is delivered over a short period of time: generally, instantaneously but by definition over less than 4 days. The acute effects affect rapidly renewing tissues which are particularly radiosensitive (bone marrow, digestive epithelium, germ cells, skin). The classic “radiation sickness” or prodromal syndrome (headache, dizziness, nausea, bone marrow hypoplasia) occurs for an exposure of 0.5–1 Gy. A dose of 3–4 Gy kills 50% of exposed individuals in 1 month [86]. Unlike the desired partial exposure of patients undergoing radiotherapy, solar flares are unpredictable, which seriously complicates mission planning for astronauts.

10.4.2.1 Chronic and Late Effects: Cancer and Degenerative Diseases For several decades, NASA has collected data concerning acute and chronic morbidity and mortality in US astronauts in the NASA’s Longitudinal Study of Astronaut Health [87].

One main aim is to determine whether astronauts’ occupational space radiation exposure is associated with an increased risk of cancer or other diseases. The cohort is made up of 312 astronauts selected by NASA since 1959. Employees at the NASA Johnson Space Center in Houston, Texas, served as the control group. In January 2003, just before the explosion of the Columbia shuttle, 29 deaths (9.3%) were counted in the group of astronauts versus 17 (1.8%) in the control group. Note 20 accidental deaths among astronauts (versus 2 in the matched group). No other cause reached the threshold of significance. Compared to the control group at matched age, astronauts had a higher specific mortality rate (SMR) from cancer. This difference was not significant. However, both groups had a lower specific mortality rate than the general population. Fourteen cases of cancer have been described in astronauts (not counting 33 cases of non-melanoma skin cancer), which represents a relative risk of 1.59 compared to the Air Force pairings but of 0.54 compared to the cohort of NCI (general population), which ultimately remains insignificant. A later study found that standardized mortality rates for astronauts were significantly below US white male population rates [88]. During a Mars exploration mission, each cell nucleus of an astronaut would be crossed by a proton or a secondary electron every 2 days, and by a heavier ion every month [89].

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Due to their strong ionizing power, these ions appear to be the main vector of carcinogenic risk despite their low fluence. The interval between irradiation and tumor appearance has been shown in rats to be shortened compared to conventional radiation [90, 91]; fewer events would be needed in the promotion of carcinogenesis induced by high-LET particles. Particle mass, energy, and charge can influence the cancer risk of an HZE particle. The linear no-threshold (LNT) model used to predict the risk of cancer mortality in astronauts sent on interplanetary missions relies on data from atomic bomb survivors extrapolated to this particular population, to these types of particles, and to the dose rates encountered in the space environment. Though nearly universally used by public bodies to assess cancer risk, LNT is far from being a scientific consensus and its application for low dose rates is rather controversial—see Chap. 2. For cancer risk estimation, age at exposure, attained age, sex- and tissue-specific mortality and incidence, and latency has to be considered. Also, an important question is whether the additional cancer risk induced by space radiation exposure is independent of other cancerogenic events (excess absolute risk, EAR), or whether the risk depends on other cancer risks (excess relative risk, ERR). Table 10.3 summarizes the LNT-estimated carcinogenic risk under different exposure conditions. The confidence interval includes epidemiological, physical, and biological uncertainties. The maximum acceptable risk for an astronaut dying from cancer is typically set at 3% [50]. Besides the calculated increased cancer risk for astronauts, cataracts might be triggered or promoted by space radiation exposure. Astronauts exposed to a dose of more than 8 mSv exhibit earlier and more frequent cataracts (in a study that identified 295 astronauts paired with as many US Air Force pilots) [92].

Table 10.3  Doses and LNT-based estimates for cancer mortality risk following space missions

Absorbed dose (Gy) Moon Mission 0.06 (180 days) Mars Orbit 0.37 Mission (600 days) Mars Mission 0.42 (1000 days)

Effective dose (Sv) 0.17 1.03

1.07

Risk of death by cancer (%) [IC95%] Male 40 Female 40 y.o. y.o. 0.68 0.82 [0.20–2.40] [0.24–3.00] 4.00 4.90 [1.00– [1.40–16.20] 13.50] 4.20 5.10 [1.30– [1.60–16.40] 13.60]

10.4.2.2 Chromosomal Aberrations and Biodosimetry Due to the densely distributed ionizations around a heavy ion’s path through a cell nucleus, severe DNA damage (Sect. 10.5.3) possibly leading to chromosomal aberrations (Sect. 10.5.2) can be induced. Therefore, chromosome damage induced in vivo was identified early as a sensitive biodosimeter [93, 94] that integrates radiation exposure in quality and quantity and also the individual radiosensitivity [95]. Peripheral blood lymphocytes are accessible by venipuncture and the chromosomal aberration test can be performed with these cells before and after flight. In order to determine the effects of space radiation on astronauts, chromosomal aberrations were quantified already in Gemini astronauts before and after the spaceflight [96]. In some astronauts, a small increase was observed after the flight which did not correlate with flight duration (1–14 days), extravehicular activities, or diagnostic radioisotope injections [96]. Missions with a duration of up to 3 weeks did not result in an increase of the aberrations above background; after missions of 6 months or longer, a rise was clearly observed [95, 97–104], but dose estimation based on the cytogenetic analysis varied strongly [95]. Here, the interindividual variability of the translocations’ half-life in peripheral blood lymphocytes has to be considered [105]. Also, the basal aberration frequency and the reaction toward ionizing radiation varies from individual to individual [106– 108]. Furthermore, the effects of multiple space missions might not be additive [109, 110]. Prediction of dicentrics frequencies for a Mars mission assume values 10–40× above background in peripheral lymphocytes [111]. For detection of reciprocal translocations, multicolor fluorescence in situ hybridization (mFISH) was first applied to members of the Mir-18 crew [112]. In search of a specific marker of heavy ion exposure, complex chromosome interchanges were suggested and analyzed in blood lymphocytes of astronauts [113, 114]. High-resolution multicolor banding (mBAND) of chromosome 5 can visualize intrachromosomal exchanges—long-term missions to the ISS did not increase this parameter [115]. Such inversions were only recently found in three astronauts during a 6-months ISS mission [116]. Complex chromosomal rearrangements occur very rarely in astronauts therefore their use as biomarker is limited [93]. Over the years, different cytogenetic or chromosomal signatures that allow reconstruction of absorbed dose and radiation quality were suggested, such as insertions [117], inversions [118], and complex chromosome interchanges, but up to now, no consensus for a biomarker of exposure to highLET radiation has been reached [119] (see Sect. 8.7). The relevance of the telomere elongation that was first observed during the 1-year ISS mission and its fast shortening after return to Earth [120], which was now also found during 6-months missions [116], for assessment of space

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radiation risk is currently unclear. The telomere changes are considered as an integrative biomarker for effects of the spaceflight environment [121].

10.4.2.3 Light Flashes Before the first human went to space, in 1952, Professor Cornelius A. Tobias made the famous prediction that cosmic radiation can cause unusual light sensations by interaction with the visual system. The Apollo-11 astronaut Edwin (Buzz) Aldrin was first reported to have perceived light flashes during the Moon mission [122]. This initiated a series of investigations already during the following Apollo missions [123], and later on Mir, Skylab, Apollo-Soyuz Test Project (ASTP), Shuttle missions, and on the ISS.  They started with observation sessions and nuclear emulsion plates (Apollo light flash moving emulsion detector, ALFMED). The observations were later combined with sophisticated particle detectors in the Silicon Eye (SilEye-1 and -2) experiments on Mir [124], and Alteino-SilEye-3 and Anomalous Long-Term Effects on Astronauts (ALTEA) experiments on ISS, which included also an electroencephalograph. The observations of the Apollo astronauts resulted in an average event rate of one light flash event in ~3 min [123]. In LEO, when passing through the SAA, the light flash rates are very high [125], and outside the SAA, light flash frequency is higher in the polar parts of the orbit than in equatorial latitudes [126]. The number of light flashes perceived in LEO varies on average between one every minute up to one every 7 min on Mir [127] or every 20 min [128, 129] dependent on the orbital height, the inclination, the shielding of the spacecraft and solar activity [130]. So, in conclusion, contrarily to the usual statement that we have no senses to perceive ionizing radiation, when closing their eyes, most space travelers can “see” the exposure to galactic cosmic rays and trapped radiation as mostly colorless light flashes or phosphenes in the form of spots, stars, streaks, or diffuse clouds of light [125]. About 15–20 min of dark adaptation is required [123] so that they are usually perceived before falling asleep. This light flash phenomenon is explained by a visual sensation that is produced by the interaction of highly energetic heavy ions with the retina of the eye [131, 132] or possibly with visual centers in the brain or the optic nerve after penetration of the spacecraft walls and the eye or head. The interaction might be direct or indirect via Cherenkov radiation in vitreous humor which is emitted as light when the charged particle passes through it with a velocity higher than the speed of light in the vitreous humor [133]. The probability of a heavy ion to cause a light flash has been estimated to be around 1%—with increasing probability with increasing LET—and for protons to be below 0.001% in LEO [127]. A deleterious effect of the flashes on vision is not suspected, but some astronauts report that their sleep was disturbed by light flashes.

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10.5 Biomolecular Changes Induced by Space Radiation Ionizing radiation, which exists primarily in the form of high-energy, charged particles make up space radiation. The radiation environment in space is characterized by a high complexity due to different sources and a higher number of particle species, and a broad energy range. Galactic cosmic radiation (GCR), solar particle events (SPE), and, in LEO, trapped radiation are the naturally occurring sources of space radiation. The exposure to GCR occurs at a low dose rate on the organismal level, but strong cellular effects might be triggered in case of a “hit” by an energetic particle, especially high Z and high energy (HZE) particles or heavy ions. HZE particles make up only 1% of GCR therefore only small hit frequencies are expected in the human body that could be responsible for late effects [134]. First evidence of biological effects of HZE particles was found in mice after a highaltitude balloon flight when the coat of black mice locally turned grey [135]. Single particle effects on different dormant biological systems under spaceflight conditions were proven by means of the Biostack experiments on the Apollo-16 and -17 missions [10, 136]. In this experimental system, biological systems and detector foils were stacked onto each other to allow assignment of heavy ion hits to the biological systems. Heavy ion hits were detected in plastic foils (cellulose nitrate, polycarbonate), silver chloride crystals, and nuclear emulsions. The biological systems were immobilized on the foils with water-soluble polyvinyl alcohol and included Bacillus subtilis spores, seeds of the thale cress Arabidopsis thaliana, roots of the field bean Vicia faba, eggs of the brine shrimp Artemia salina, insect eggs (stick insect, Carausius morosus and rice weevil, Tribolium confusum), and protozoa cysts (Colpoda cucullus). The outgrowth of B. subtilis after germination was significantly reduced after an HZE particle hit [137, 138]. During the development of brine shrimp eggs that were hit by a single particle, abnormalities appeared at the extremities, the thorax, and the abdomen [139] and the eggs showed the most sensitive reaction toward HZE particles compared to the other biological systems in Biostack [137, 140]. Developmental abnormalities were also found in hit insect eggs [141]. The total dose for the Biostack experiments was quite low (5.8–7.5 mGy), and ~0.03 mGy was allocated to the HZE particles, whereby it has to be considered that the local dose in a hit cell can be much higher than the total dose. These experiments were continued in LEO using the Free Flyer Biostack Experiment (LDEF—Long Duration Exposure Facility) [142], EURECA—European Retrievable Carrier [143–146], and the biosatellites COSMOS 1887 and 2004 [147, 148] and refined, so that synergistic effects of

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HZE particle hits and microgravity in the developmental disorders of C. morosus were revealed. These intriguing results showing strong deleterious effects of single particle traversals and even an enhancement by other spaceflight environmental effects initiated a multitude of biological experiments in space and at heavy ion accelerators (see Sect. 10.9) in order to quantify the biological effectiveness of HZE particles, to understand the underlying mechanisms and to develop countermeasures. A variety of experimental models are used for these experiments (see Sects. 10.5–10.7, and 10.8.2). The uncertainties in risk assessment for cancer and non-cancer effects in the central nervous system and other organ systems for astronauts are still unacceptably high therefore further investigations into the biological effects of HZE particles are necessary. The experimental approaches shown in Box 10.3 below take the low dose rate but strong biological effects in case of a particle hit into account.

Box 10.3 Experimental Approaches for HZE Particle Effects

Natural GCR exposure • Correlation of biological effects with single particle hits by combination of biological model and detector foil, e.g., Biostack; can be combined with 1xg reference centrifuge to determine contribution microgravity effects • Correlation of light flashes with HZE particles that traverse astronauts’ eyes • Dose accumulation over weeks or months by storing dormant or freeze-dried or deep-frozen cells or small organisms in space, subsequent reactivation and measurement of radiation damage or response • Determination of spaceflight effects by exposure of, e.g., fruit flies, rodents, or other organisms on satellites or high-altitude balloons

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human cells by alpha particles with an LET up to 100 keV/ μm was already observed in the 1960s [149], indicating an RBE for cell killing up to 7. Since then, survival data after heavy ion exposure were collected for many mammalian cell types including primary cells and tumor cell lines using the colony forming ability (CFA) test which is described in Chap. 2. This was less driven by space radiation research but by tumor therapy research to identify suitable ions and to determine the cell killing RBE for treatment planning. The shoulder observed in the dose response curves for cell killing by low-LET radiation disappears in high-LET survival curves, resulting in purely exponential dose–effect relationships and indicating the lack of repair capacity after heavy ion exposure [150] (Fig. 10.13). Clonogenic cell survival data for more than 1100 experiments comparing the effects of ion irradiation to photon irradiation are available in a database established by the GSI biophysics group [151]. The database is called Particle Irradiation Data Ensemble (PIDE, www.gsi.de/bio-pide). The maximal RBE for cell killing (10% survival level) was observed in the LET range of 100–200 keV/μm with values of 2–7 [151]. This large variation in RBE is explained by the influence of particle species and energy in addition to LET, of cell type and other experimental factors. At LETs above ~200 keV/μm, more energy is deposited in a cell traversed by a particle than is required to kill the cell and more hits per cell cannot produce more cell death as any hit will kill the cell, resulting in a decrease of RBE that is called “overkill effect.” The clonogenic survival data integrate cell death by various modes such as mitotic catastrophe, apoptosis, necrosis,

Exposure to selected HZE particles • Exposure of a variety of biological systems at heavy ion accelerators or microbeam facilities to selected heavy ions (singe particle at defined energy or mixture of particles of defined energies) and analysis of the biological response

10.5.1 Cellular Survival, Cell Death, and Proliferation As described in Chap. 2, radiation quality is an important factor influencing the cell death response. It can affect the extent and mode of cell death. A stronger cell killing of

Fig. 10.13  Survival of mammalian cells after exposure to low linear energy transfer (LET) and high-LET radiation. Low-LET radiation includes photons, electrons, positrons, protons, and more. High-LET radiation encompasses heavy ions, and, depending on energy, also He ions and neutrons

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autophagy, and other mechanisms (see Chap. 2) and permanent cell cycle arrest, possibly accompanied by cellular senescence. As for low-LET radiation, it depends on the cell or tissue type whether a cell population is prone to ionizing radiation-induced apoptosis [152]. Apoptosis might occur at higher rates after high-LET radiation exposure compared to low-LET irradiation with a maximum at a LET of ~100 keV/ μm [153]. The consequences of heavy ion-induced cell death for the organism can be that transformation of a heavily damaged cell is prevented thereby protecting from cancer. This effect also limits the number of cells with mutations (see Sect. 10.5.4) or chromosomal aberrations at a LET >200 keV/μm (see Sect. 10.5.2) and cellular transformation (see Sect. 10.5.5). On the other hand, deleterious effects might occur such as depletion of stem cell pools or loss of terminally differentiated cells with no or low regeneration potential that might affect the functionality of a tissue or organ. For some microorganisms, growth and viability were measured during space missions. A 14-days exposure of Escherichia coli on the Space Shuttle or 140-d exposure on Mir did not result in any differences in viability and mutations frequencies in comparison to ground controls [154, 155]—the same was the case in Saccharomyces cerevisiae [156]. Using repair-deficient E. coli mutants, DNA polymerase, and 3′→5′ exonuclease were identified as the most important enzymes for GCR-induced DNA damage in E. coli [157]. Also, the slime mold Dictyostelium discoideum did not grow differently and did not show differences in the mutation frequency in the spores during a 7-days Shuttle flight [158, 159], but the number of spores per fruiting body was reduced [160].

10.5.2 Chromosomal Aberrations Chromosomal aberrations are alterations in DNA structure that become microscopically visible after following a chromosome staining protocol [161] (see Chap. 2). They can result from mis-rejoining of DNA ends from ionizing radiation-induced DNA double strand breaks (DSB), from lack of repair leading to terminal deletions and incomplete exchanges or from chromosome mis-segregation [162, 163]. They are exquisitely and quantitatively sensitive to ionizing radiation. Symmetrical resolution of the DNA DSB can lead to chromosomal interchanges resulting in translocations which are usually nonlethal. Asymmetrical resolution produces among other dicentrics (chromosomes with two centromeres) and acentric fragments, mostly contained within micronuclei; also, during the repair process, DNA sections can be lost, producing a deletion [164]. Ionizing radiation can also induce quadriradials (U-type by asymmetrical resolution, X-type by symmetrical resolution). Complex and asymmet-

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ric aberrations such as dicentrics usually lead to cell death (lethal aberrations) [163]. They are determined during metaphase or by chemically induced Premature Chromosome Condensation (PCC, see Chap. 2) during interphase [165], usually in lymphocytes or fibroblasts, providing data on a cell-by-cell basis. Their dose–response relationship follows a curvature. While dicentrics and acentric fragments can be detected with a GIEMSA staining, mFISH is required for interchromosomal translocations and mBAND for intrachromosomal translocations (see Sect. 10.3.4). Inversions can be detected by Directional Genomic Hybridization (dGH) [166]. Chromosomal aberrations are of high interest in space radiation biology as they are an early-stage effect and regarded as a surrogate endpoint for cancer risk as many human cancers are linked to them and all “clastogens2” are both mutagenic and carcinogenic. For carcinogenesis, the surviving cells with chromosomal aberrations are relevant. The fraction of these cells depends on LET, track structure, and fractionation (Box 10.4). HZE particles have a very high efficiency in inducing

Box 10.4 Factors Influencing Induction of Chromosomal Aberrations by Ionizing Radiation

Dose rate Fractionation Linear energy transfer (LET) Track structure Cell nuclear geometry (e.g., spherical or flat)

chromosomal aberrations—the RBE in comparison to lowLET radiation was estimated to reach 30–35 during interphase [163, 167, 168]. Furthermore, high-LET α-particles at low fluences (1 track per cell nucleus) were more efficient in inducing complex aberrations in human peripheral blood lymphocytes than X-rays [117]. Complex chromosome aberrations are defined as aberrations that involve three or more breaks in at least two chromosomes. Here, the particle track structure comes into play [169]. Delta rays move out of the primary particle track, producing further ionizations that can induce damage. This damage might interact with other breaks generated by either a separate track or delta rays emanating from it (intratrack action). The range of the delta rays is proportional to the specific energy of its corresponding primary particle. Higher energy particles would have a greater chance of track interaction than their lower energy A “clastogenic” agent directly causes DNA strand breaks or disturbs normal DNA-related processes resulting in insertion, deletion, or rearrangement of chromosome sections. 2 

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counterparts because of the longer range of the delta rays where breaks can be close in space and time at high doses and dose rates as they are produced by multiple tracks (intertrack action). The breakpoints induced by delta rays add up to those produced in the primary particle track. Hence, the number of exchange breakpoints and their spatial arrangement are important determinants for the formation of complex exchanges. For example, the number of breakpoints per cell was higher for 56Fe ions (1.1 GeV/n) and α-particles (0.9 MeV/n) in comparison to 137Cs γ-rays. In spherical cell nuclei, one particle traversal is sufficient to produce two breakpoints, e.g., in a lymphocyte [170, 171]. In summary, HZE particles produce more breakpoints per track and more highly complex exchanges compared to low-LET radiation [118] (Fig. 10.14). These complex aberrations partly disappear between the first and second cell division after radiation exposure, but some are transmissible and might be stable through several cell generations.

10.5.3 DNA Damage and Repair Kinetics As other radiation qualities, protons, α-particles, and HZE particles can induce various types of DNA damage by direct ionization or indirectly through radiolysis of intracellular water (see Chap. 2). Among base damage, loss of bases, DNA-DNA and DNA-protein crosslinks, single strand breaks (SSBs), and double strand breaks (DSBs), DNA DSBs are the most severe DNA lesion. Unrepaired DNA

Fig. 10.14  As a heavy ion travels through a mammalian cell nucleus, a multiple of ionizations is produced, damaging a chromosome arranged in its nuclear territory several times. Delta rays emanating from the primary track can induce further damage. Therefore, traversal of highLET radiation through a cell nucleus can produce many breakpoints in chromosomes

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DSBs are at the center of biological effects such as cell killing and chromosomal aberrations and are trailblazers of the majority of early and late effects induced by ionizing radiation exposure [163, 172, 173]. What makes particle radiation special is the multitude of ionizations localized along the particle’s path through the cell. The spatial distribution of direct DNA damage differs strongly for low- and high-LET radiation, with a diffuse distribution for the former and clusters for the latter. Such clusters of different damage (base lesions, abasic sites, SSB, DSB, etc.) within a few helical turns of DNA are called complex DNA damage (Fig. 10.15) (formerly: multiply damaged sites or clustered DNA damage) [164, 174, 175]. Although the contribution of direct action to the biological effectiveness of high-LET radiation is larger than indirect action [176], reactive oxygen species (ROS) generated by radiolysis can also play a part in the overall radiation effects. As the lifetime and diffusion range of ROS are small, only radicals produced in DNA’s vicinity are relevant for DNA damage induction and increase in its complexity. With increasing LET, the contribution of direct effects rises, and the indirect effects drop. Low-LET radiation and endogenous ROS rarely induce complex DNA damage [163]. The detection of GCR-induced DNA damage succeeded in HeLa cells during the Shuttle and Mir missions [177–179]. In human lymphoblastoid cells that were stored at –80 °C for

Fig. 10.15  Comparison of ionizations (grey dots) in a DNA molecule that are induced by electrons as an example of low-LET radiation and by a high-LET α-particle. The ionizations produced by the α-particle are located densely along the track, with some secondary electrons (δ rays) generated while traversing the cell. This spatial distribution goes along with a higher probability of simultaneously breaking both DNA strands thereby producing a double strand break (DSB), and also further damage to bases and single strand breaks (SSB) in close proximity which is then called complex DNA damage

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several months on ISS—in total 134 days at an average dose rate of 0.7  mSv/day, one particle track per 100 cells was detected by means of immunofluorescence staining of γH2AX after return to the ground (see below) [180]. Such tracks were also observed in human fibroblasts that were cultivated for 14 days on the ISS [181].

10.5.3.1 Repair of HZE Particle-Induced Double Strand Breaks and Complex Damage Various DNA damage repair pathways ensure genome integrity and stability in uni- and multicellular organisms. The current understanding is that multiple repair pathways have to be coordinated to repair complex DNA damage making it very challenging, that short fragments might be lost during repair and that multiple breakpoints in the DNA ribose-phosphate backbone can favor complex genomic rearrangements [164, 182]. The damage might still persist at DNA replication because of repair delays that were observed after HZE particle exposure. If repair of complex lesions is completed, its fidelity might be lower when compared to simple DNA damage [183–185]. After 56Fe ion (1 GeV/n) exposure, 14% of the damage remained unrepaired compared to 5% after γ-ray or α-particle exposure [171]. In vivo, persistent DNA DSBs were found even 1 month after exposure to iron ions [186]. Growth arrest, cell death, or senescence are possible consequences of such unrepaired DNA damage [164], while mutations and chromosomal aberrations are key steps in cellular transformation and tumorigenesis. DSBs are mainly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in eukaryotes (see Chap. 2). DNA DSB repair follows biphasic kinetics with a faster velocity in the beginning and lower speed at later timepoints. The phosphorylated form of the histone variant H2AX (γH2AX) [187, 188] as a marker of DNA DSB is often applied to microscopically visualize DSB induced by high-LET radiation exposure, sometimes in combination with antibodies binding to 53BP1 or other DSB repair proteins or to oxidative base damage [189]. After immunofluorescence staining, fluorescent foci indicate γH2AX and 53BP1 accumulation around DNA DSB. Groundbased experiments performed at heavy ion accelerators allow quantification of DNA damage induction and DNA repair by one ion with a specific energy or, since lately, several ion species with specific energies hitting the cells from one direction (see Sect. 10.10.4). They are usually performed additionally with low-LET radiation for comparison. For example, in human fibroblasts, repair of DSB induced by carbon ions was slower than those induced by proton or helium ion irradiation and the size of the repair foci increased with increasing LET [190]. Larger repair foci that persist longer are a common finding when exposure to heavy ions and X-rays are compared [191, 192]. One day after exposure to 1 GeV/n iron ions, 30–40% of the 53BP1 and γH2AX foci

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still remained indicating the extent of residual damage [193]. The slow repair kinetics and incompleteness of repair of DNA damage induced by high-LET radiation [190, 191, 194] are consistent findings of experiments with mammalian cells at heavy ion accelerators. Also, there are some hints that high-LET radiation inhibits c-NHEJ and shifts toward errorprone alternative nonhomologous end joining repair and microhomology-mediated end joining, resulting in a lowered fidelity of repair for days or weeks [195]. Other studies have shown that the repair of complex DNA requires DNA resection for processing at the DNA ends in G1 and G2 cells and forces the pathway choice toward resection-dependent HR [196, 197]. As mentioned above, to repair complex DNA damage, other repair pathways might be involved such as base excision repair (BER) and/or nucleotide excision repair NER [198]. Oxidative base damage such as 8-oxoguanine can be restituted by BER starting with damage recognition and removal by a DNA glycosylase and final steps by polymerase and ligase proteins [172]. NER is responsible for the repair of larger helix-distorting lesions. In summary, DNA damage complexity increases with increasing LET, resulting in less effective DNA repair, a higher rate of residual lesions, genomic instability, and enhanced cell killing [174].

10.5.3.2 Effects of Other Spaceflight Environmental Factors Such as Microgravity on DNA Repair The results of the Biostack experiments raised the question of whether microgravity or other spaceflight environmental factors affect DNA repair processes, as explained hereinafter. The advanced Biostack experiments included an inflight 1g control on a centrifuge, allowing the separation of effects of microgravity and of all other environmental factors. In this experiment, eggs of the stick insect Carausius morosus were exposed in space and the HZE particle hits were traced back to the eggs by means of particle track detector foils. Back on Earth, the insects were allowed to hatch. When the eggs were hit by an HZE particle under microgravity, more abnormalities were observed compared to hits during centrifugation at 1g, indicating additive or even synergistic damaging effects of cosmic radiation and microgravity [144]. Therefore, DNA repair and radiation response under microgravity were examined in further spaceflight experiments using a 1g centrifuge inflight control and in groundbased simulation using clinostats or random positioning machines. For determining subtle differences in DNA repair capacity or kinetics, a high level of DNA damage has to be induced. For this purpose, the dose rates of GCR in LEO on the Space Shuttle or on ISS are too low; therefore, DNA damage has to be induced by irradiation on ground in a metabolically inactive state, by irradiation in space using an arti-

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ficial radiation source brought in LEO or by incubation with chemicals. When radiation damage was already induced on ground using a radiation source, cooled cells were brought to space and activated there to repair their DNA under microgravity [199, 200]. Alternatively, DNA DSB were induced by bleomycin [201] or restriction enzymes [202] during spaceflight. Often, no or only small interactions were found [203, 204]. In yeast however DNA DSB repair was delayed under microgravity, suggesting additive effects of radiation and microgravity [205, 206]. In human fibroblasts and Bacillus subtilis, microgravity did not influence the repair of DNA SSB and DSB [200, 207]. Also, ligase activity [204] and DNA replication [208] were not affected. The expression of genes involved in the DNA damage response was altered under microgravity [209–211]. Besides these gene expression changes, a growth-stimulating effect of microgravity was observed in many ground-based and space experiments that might contribute to the microgravity effects on the DNA damage response [209]. In ground-based experiments, limitations of various microgravity simulators have to be considered [212], especially the generation of shear forces [213] as possible confounders. For microorganisms, such as bacteria, it has also to be considered whether they are motile because of, e.g., flagella or not, and the effect of microgravity can be most likely attributed to changes in the medium surrounding the microbes [214]. Animal experiments addressing the question of DNA repair under spaceflight conditions are scarce. After a 14-day spaceflight, the level of the tumor suppressor p53, which acts as a transcription factor in the DNA damage response, was increased in the muscle of mice compared to ground control mice [179]. Experiments with the nematode Caenorhabditis elegans during the Shenzhou-8 mission revealed changes in the expression of four microRNAs and of 4.2% of the genes involved in the DNA damage response after 16.5 days of microgravity when compared to the inflight 1g control [215]. Hindlimb unloading is used in rodent models to simulate on ground the head-ward fluid shift that occurs in microgravity. After 21 days of hindlimb unloading and low-dose irradiation of mice, some genes involved in DNA repair, chromatin organization, and cell cycle were differentially expressed in the spleen compared to control mice [216].

10.5.3.3 Future Space Experiments Space experiments are the only way to unambiguously identify the effects of real microgravity on biological systems, here the enzymatic repair of radiation-induced DNA damages. The opportunities to perform experiments with actively metabolizing organisms in space are rare and usually have a long lead time from the acceptance of an experiment proposal to the execution of the experiment in space. The Biolab facility in the Columbus module of the ISS provides many possibilities for biological experiments on

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microorganisms, cells, tissue cultures, small plants, and small invertebrates in LEO (https://www.esa.int/Science_ Exploration/Human_and_Robotic_Exploration/Columbus/ Biolab). However, experiments on the ISS are subjected to limitations such as up- and download mass, up- and download temperature conditions, availability of a suitable facility in space, data downlink, number of sample replicates, appropriate control experiments in space and on ground. The Biolab facility will be used for LUX-in-Space (ESA AO LSRA-2014-026, Team Coordinator: P.  Rettberg), the first space experiment where the whole series of events from DNA damage induction in metabolically active cells to the different steps of enzymatic repair reactions will take place in real microgravity and the repair kinetics will be monitored by optical measurements in situ. The effects of microgravity will be clearly separated from other spaceflight factors by comparison with parallel samples on an onboard 1g centrifuge in the Biolab facility and in a parallel ground control experiment with identical samples in flight-identical hardware. Due to safety issues, ESA decided to apply UV radiation for DNA damage induction. It causes defined types of DNA damage, e.g., cyclobutane pyrimidine dimers, which are among those also induced by ionizing radiation. Bacteria serve as model organisms possessing the same type of nucleotide excision repair as all other living organisms including humans. The capability of bacterial cells to counteract radiation damage by activating genes involved in DNA repair will be assessed using a bioluminescent reporter gene operon under the control of the SOS regulon, known as the SOS LUX assay. The DNA repair kinetics will be followed by bioluminescence and optical density measurements. For the space experiment, TripleLux Part C preparatory work was already performed successfully to adapt the SOS LUX assay to the space conditions provided by the Biolab facility on the ISS.  This experiment was canceled later by ESA due to a lack of available resources at that time and it is a predecessor of LUX-in-Space [217, 218]. The launch of LUX-in-Space is scheduled for 2023/2024. Biosentinel will be the first deep-space experiment investigating the repair of DNA damage induced by space radiation (Principal Investigator: Sharmila Bhattacharya). It is a further development of NASA’s biological CubeSats, small satellites with different payloads that were already flown successfully in LEO. Biosentinel will first follow a trajectory of cis-lunar flyby and, for 6–12 months, enter a heliocentric orbit. The organism under investigation is the budding yeast Saccharomyces cerevisiae. These eukaryotic cells are robust, desiccation resistant, were already flown in space before, and have similarities to cells of higher organisms such as humans. Cells from a radiation-resistant yeast wildtype strain and a radiation-sensitive Δrad51D mutant will be uploaded in a dry form. After different periods of time, during which the cells will accumulate radiation-induced DNA damage, the

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cells will be activated by the addition of nutrient medium and their growth and metabolic activity will be measured optically. In parallel, another Biosentinel payload will be flown in the ISS, in addition to the corresponding ground reference experiment. The launch is scheduled for 2022 as a secondary payload of NASA’s Artemis-1 mission [219, 220].

10.5.4 Mutagenesis Mutations as a deleterious outcome of erroneous repair of space radiation-induced DNA damage are of special interest in radiation risk assessment as they can initiate the multi-step carcinogenic process [163, 182] and they can be responsible for genetic effects in the offspring if they occur in the germline. Mutations can be detected in cells that survived irradiation and are, as chromosomal aberrations, late endpoints of radiation-induced DNA damage. For improving space radiation risk assessment, the dependence of mutation induction by radiation of different linear energy transfer (LET) was examined in different biological systems: Mutation induction by heavy ions was determined in many organisms including bacteria (E. coli, B. subtilis), yeast (S. cerevisiae), Neurospora, Drosophila, C. elegans, M. musculus, plants, and mammalian cell systems including human fibroblasts and lymphoid cells. These were mostly ground-based experiments at heavy ion accelerators. The hypoxanthine guanine phosphoribosyl transferase (HPRT, EC 2.4.2.8) gene (mutations on the single copy X-chromosome in male-derived cells) in human diploid fibroblasts was used in early studies of LET dependency of mutation induction. A maximum of around 7 times more mutations compared to low-LET radiation was observed for helium ions or heavier ions with a LET of 100–300 keV/μm [221]. The number of mutations per single track through a mammalian cell nucleus increases with LET, reaching saturation at around 100 keV/μm [222]. The induction of mutations in the X-linked HPRT locus in Chinese hamster cells by accelerated heavy ions reached a local maximum in the LET range of 80–100 keV/μm [223]. Studies on mutation induction in autosomes became possible by means of AL human-hamster hybrid cells having one copy of human chromosome 11. In these hybrid cells, neutrons of various energies were more efficient in inducing mutations in the a1 locus on chromosome 11 compared to gamma rays; the RBE reached up to 30 at the 0.1% survival level [224]. The autosomal thymidine kinase gene (TK1) locus in human cells allowed investigation of the loss of heterozygosity (LOH) which can occur via deletion or allelic recombination and it revealed a higher peak of mutations at a lower LET (~50–100 keV/μm) compared to the HPRT mutations (up to 15× compared to ~5×). As for other biological endpoints, LET is not the only determinant of the biologi-

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cal efficiency of an HZE particle. The track structure means the energy deposition pattern varies for different ion species of the same LET. Such an effect of ion species was observed for mutation induction at the HPRT locus in human fibroblast-like cells—the RBE for mutation induction determined in this system was between 3.6 and 7 for carbon and neon ion beams in the LET range of 60–120 keV/μm compared to 137 Cs gamma rays [225]. Besides mutations observed in the direct hit cells, bystander mutagenesis can contribute to the overall mutation rate after particle exposure as it was observed, for example, after alpha particle exposure [226]. An experiment on the ISS designed to detect mutations in human cells that were induced by natural galactic rays made use of the frozen storage as described in Sect. 10.5.3. Frozen human lymphoblastoid TK6 cells were stored for 134 days in the Kibo module of the ISS and accumulated a dose of 72 mSv. After analysis on ground, a tendency for higher mutation frequency at the TK locus was observed in the flight samples compared to ground control [227]. Earlier experiments on Mir for 40 days with a model system based on Saccharomyces cerevisiae and Escherichia coli also revealed two to threefold higher mutation frequencies in some flight samples compared to ground samples, with a predominance of large deletions that might be caused by high-LET radiation [228].

10.5.5 Transformation If mutations occur in tumor suppressor genes and inactivating them, or proto-oncogenes and activating them, cells can be transformed and lose growth control including anchoragedependent growth. It can be seen as a surrogate marker for the carcinogenic potential of a radiation quality in question. Transformation can only occur in cells that survived the radiation exposure. In vitro, transformation of mammalian cells is determined by their ability to grow anchorage independently in soft agar. The soft agar test was applied to different cell types after exposure to HZE particles at heavy ion accelerators in order to determine their potential for transformation, usually in comparison to low-LET radiation. Already in the 1980s, it was shown that HZE particles are more effective in transforming mammalian cells than lowLET radiation: In mouse embryonic cells (C3H10T1/2), the effectivity of transformation increased up to 10 with a LET ~200 keV/μm [229] while Hei et  al. observed a plateau at LETs of 80–120 keV/μm [230]. In Golden hamster embryo cells, 14N ions (LET 530 keV/μm) and 4He ions (36 and 77 keV/μm) were ~3× more effective in inducing cellular transformation than gamma or X-rays [231]. Later, a maximal RBE for neoplastic transformation was found at a LET of ~100 keV/μm, reaching a maximum of seven [232]. In

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human bronchial epithelial cells, iron and silicon ions (LET 151 and 44 keV/μm, respectively) were more efficient in inducing transformation than gamma rays from a 137Cs source especially when these cells were oncogenically progressed by stable transfection of mutant oncogenes [233].

10.5.6 Cell Cycle Changes Cell cycle arrests play a central role in the DNA damage response of dividing cells. Before the cell enters the next cell cycle phase, e.g., from G1 to S phase or from S to G2/M phase, they allow repair of damaged DNA (Chap. 2). They can therefore protect from cell death, mutations or chromosomal aberrations. Concerning the special radiation qualities present in space that are prone to induce complex DNA damage which might persist longer, stronger, or longer cell cycle arrests might be induced in comparison to low-LET radiation. Early experiments observing mitotic delay by timelapse microscopic cinematography already gave hints that accelerated neon ions produce a stronger delay compared to Co-60 gamma rays [234]. High-LET radiation produces stronger and more persistent blocks in the G2 phase of the cell cycle than low-LET radiation [235]. In synchronous V79 Chinese hamster cells, the cell cycle delays per particle traversal increased with increasing LET and were primarily due to blocks in S and G2/M phase of the cell cycle [236]. Permanent arrest in the G1 phase can also be induced by high-LET radiation [237]. The relative biological efficiency of heavy charged particles with a LET in the range of 100– 330 keV/μm for inducing cell division delays was 3.3–4.4 [236] and the percentage of mitotic cells as indication of an arrest at the early G2/M checkpoint decreased with increasing LET [238]. The cell cycle regulating protein p21 (CDKN1A) accumulates in nuclear foci rapidly after heavy ion exposure of fibroblasts [239]. Besides this, expression levels of cell cycle regulatory proteins might be affected to a higher extent by high-LET radiation compared to low-LET radiation [237], for example, after iron ion exposure p21 expression was much higher compared to gamma rays and persisted 10 days after irradiation [193].

10.5.7 Gene Expression Similar to studies with low-LET radiation, gene expression studies after high-LET radiation developed from a focus on single genes (mRNA and protein level by Northern Blot, RT-PCR, real-time RT-qPCR, Western Blot) to arrays of multiple genes, microarrays [240] and detection of the levels of all mRNAs present in cell populations or even single cells by RNA sequencing. After exposure to ionizing radiation, signal transduction pathways can result in the activa-

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tion of transcription factors. These transcription factors bind to binding sites in their target genes’ promoters which are specific for them (usually short palindromic DNA motifs) [241]. Also, besides promoter or enhancer activation via transcription factor binding, epigenetic mechanisms can be responsible for (persistent) gene expression changes and are therefore the focus of mechanistic research (see Sect. 10.5.9). In addition to spaceflight experiments, a huge amount of gene expression data from ground-based exposure to neutrons, protons, and different heavy ions for different experimental model systems exists. NASA GeneLab (https:// genelab.nasa.gov/) offers a repository for space-related omics data, among others transcriptomics and proteomics from experiments with model organisms, cells, cell lines, and tissues. Currently, a comprehensive picture of gene expression changes is difficult to paint due to the multiple influencing factors that range from the model system (e.g., gut epithelial cells and human bronchial epithelial cells, tissue, animal model) to the methods, cell cycle phase, radiation qualities, doses, kinetics of exposure, timepoint after exposure, and additional spaceflight environmental factors (such as simulation of microgravity effects by hindlimb unloading). The interpretation of the data is complicated by the fact that in the majority of the heavy ion accelerator experiments, the dose is acutely applied within minutes, while exposure during long-term space missions is protracted over several months. The emerging view is that heavy ions, especially iron ions are capable to induce a stress response persisting for several weeks in addition to an early transient response. This early response can encompass p38MAPK and TP53 activation and expression of its target genes, whereby the cell cycle regulator gene CDKN1A can also be expressed TP53-independently. In tissues, long-term changes in the expression of genes involved in inflammatory and free-radical scavenging pathways occur after iron ion exposure and these changes involve transcription factors such as signal transducer and activator of transcription 3 (STAT3), GATA binding protein 4 (GATA4), Nuclear Factor κB (NF-κB) and nuclear factor of activated T cells 4 (NFATc4) [242]. In human cells, NF-κB was strongly activated by heavy ions, its activation depended on LET [243] and the expression of several chemo- and cytokines was increased [244].

10.5.8 Telomeres and Aging HZE particles are potent inducers of senescence, more potent than gamma rays. Senescence-associated changes in the tumor microenvironment may induce invasion and stemness of tumor cells. Senolytics can be applied to eliminate senescent cells and thereby deplete senescent stromal

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cells with tumor supportive roles. Shortening of telomeric sequences can lead to telomere fusions and contributes the chromosome instability after heavy ion exposure [245]. Furthermore, accumulation of short telomeres eventually triggers apoptosis or senescence. Unlike normal somatic cells, germline, stem, and tumor cells avoid the latter through a high expression of telomerase. Due to natural telomere shortening during cell division, telomere length is highly linked to aging [246]. Considering the environmental radiation exposure during spaceflight, with higher levels of HZE particles compared to on Earth, NASA investigated the effect of spaceflight on telomere length in the twin study. The twin study examined molecular- and physiological differences of twin astronauts, one spending a year onboard the ISS and the other on Earth [120]. Telomere lengths of peripheral blood mononuclear cells (PBMCs), collected from peripheral blood samples taken preflight from both twins were of similar length. However, during spaceflight, the space twin’s telomere length increased significantly, while the Earth twin’s telomers remained stable during the study. Once returning to Earth, the increased telomere length diminished within 48 h and the number of short telomeres increased compared to preflight [116]. While an unexpected finding, increased telomere length has recently been associated with other biological functions such as DNA damage response, cell cycle kinetics, and mitochondrial stress [247]. Indeed, chromosome aberrations (inversions and translocations) were more frequent during spaceflight and inversion frequencies of the space twin remained elevated postflight, consistent with ionizing radiation exposure inflight. Furthermore, DNA damage repair pathways were upregulated in several circulating immune cells, suggesting increased genomic instability due to ionizing radiation during spaceflight [121]. Similar results (increased telomere length and chromosomal aberrations) were also seen in astronauts during a 6-month spaceflight mission. While telomerase activity likely is responsible for the increased telomere length inflight, the actual contributing mechanism is still unknown. However, astronauts returning from 1 year and 6 month missions showed elevated telomerase activity upon return to Earth [116].

10.5.9 Epigenetics Persistent gene expression and functional changes induced by space radiation exposure could be caused by changes in the epigenome. Changes in the DNA methylation profile and in the histone code encompassing methylation and acetylation of histones could therefore contribute to high-LET carcinogenesis and degenerative diseases and could represent possible prophylactic or therapeutic targets.

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For example, in immortalized human bronchial epithelial cells, hypermethylation at CpG sites occurred early after Fe-56 ion exposure and persisted a long time [248]. Longterm epigenetic reprogramming after such exposure was also observed in hematopoietic progenitor and stem cells [249]. High levels of DNA methylating enzymes were also found in the hippocampus of Si-28 ion irradiated mice that developed cognitive impairment [250]. In addition to heavy ion exposure experiments, combined exposure to simulated microgravity and chronic low-dose irradiation or spaceflight experiments using small animals or cell cultures and astronaut data reveal alterations in the methylome and histone modification status after combined exposure to spaceflight environmental factors such as microgravity and space radiation. The lasting imprint of high-LET radiation exposure on the epigenome might allow monitoring the cumulative biological impact of space radiation exposure [248].

10.6 Small Animal Experimental Models and Biological Changes of Space Radiation 10.6.1 Importance of the Use of Animals in Research and Their Particular Use in Space The use of small animal models in research is debatable, but still essential to provide general information on cellular and molecular mechanisms, to develop new drugs and treatments. They are mainly used in fundamental scientific research, for the advancement and development of new diagnostic tests and treatment for diseases, for education of researchers as well as in safety assessments of drugs and chemicals. Animals are a useful research subject for a variety of reasons. Only in living organisms, it is possible to study complex physiological processes. Furthermore, the environment of the experiment can be perfectly controlled (e.g., diet, light, housing, etc.). Also, they have a shorter life cycle so studies can be conducted throughout a whole lifespan or across generations. Animals are biologically very similar to humans and often suffer from similar health problems. In fact, mice share more than 85% of protein-encoding genes with humans—Why Mouse Matters, from the National Human Genome Research Institute (https://www.genome. gov/10001345/importance-of-mouse-genome). Animal experiments can cause harm to the animal thus ethical review processes have been established around the world [251]. With respect to this, the 3R´s principle by [252] ensure the reduction of animal numbers, refining the test methods to lower the harm to the animal to a minimum and

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replace animal experiments with alternative methods, when possible (Box 10.5).

Box 10.5 Russell and Burch’s The Principles of Humane Experimental Technique was First Published in 1959

The aim of the principle is to improve the treatment of laboratory animals and at the same time advance the quality of scientific studies. Replacement: Includes methods that avoid or replace the use of animals such as computer/mathematical models (in silico), cell culture models (in vitro), or relative replacement (e.g., invertebrates, such as fruit flies and nematode worms). Reduction: With improved experimental design, modern imaging, or sharing data and resources, the total number of animals needed can be minimized. Refinement: Modification in the experiment, which minimize pain, suffering, and distress and allow general improvement of animal welfare (e.g., improvement in the research animal housing conditions, analgesia, and anesthesia for pain relief).

The animals that are most used for terrestrial research are mice, fish, and rats. Since the beginning of space exploration also animals have been used in space programs. Similarly, to how microgravity and cosmic radiation can affect human health, animals are also affected. This is why during an early space mission, at the beginning of 1940, animals were used to investigate various biological processes and the effects of space flights on living organisms. On the 20th of February 1947 the first living organism, fruit flies, were sent to space with the V2 rocket. The dog Laika was the most famous and first mammal which was sent to an orbital spaceflight around the Earth (Fig.  10.16) onboard of the Soviet Spacecraft Sputnik 2 on 3rd November 1957 [253]. Since then, a variety of animals have been sent into space including rodents, ants, cats, monkeys, spiders, and jellyfishe. Nowadays the effect of space conditions on animals, including microgravity and radiation, can also be studied to a certain degree on Earth with the help of clinostats, particle accelerator, and X-ray machines. However, all factors of the complex space environment cannot be simulated simultaneously on Earth.

10.6.2 Acute Effects 10.6.2.1 Acute Radiation Syndrome In case of a large SPE and insufficient shielding, the acute radiation syndrome (ARS, see Chap. 2) might be induced, endangering astronauts’ health and mission success. To

Fig. 10.16  On 3 November 1957 Laika was the first living mammal that was sent to space onboard the satellite Sputnik 2

understand the pathogenesis of ARS induced by protons and develop therapeutic approaches for space missions, experiments with different animal models including rodents, minipigs, and non-human primates were performed. Whole-body doses up to 2 Gy are expected when astronauts are exposed to large SPE in free space with insufficient shielding. In this dose range, effects on the immune system (see Sect. 10.6.2.3) dominate the syndrome. As the skin dose can be 5–10 higher, the skin might be damaged (see Sect. 10.6.2.2).

10.6.2.2 Skin Effects Forming the barrier between the outside environment and the inside of the body, the skin is a vital organ. Different skin layers provide the skin with tensile strength and keep a proper barrier function to prevent body water loss, regulate the immune defense and temperature, and protect against ultraviolet damage. The outermost layer, the epidermis, is built mostly out of layers of keratinocytes that differentiate and migrate toward the skin surface. A balance between the proliferation of keratinocytes and shedding of dead cells at the surface of the skin regulates the thickness of the epidermal layer. Below the epidermis lays the dermal skin layer which is mostly composed of connective tissue. Skin’s tensile strength and elasticity are provided by Collagen type I and III, and elastic fibers. Fibroblasts are the major provider synthesizing these proteins. Furthermore, they play a major part in skin wound healing by migrating to the side of the wound, recruiting other cells, and remodeling the extracellular matrix (ECM) to restore the injured skin [254]. The skin receives greatest dose and greatest number of stopping particles, particularly during solar flares [255]. SPE events during EVA could lead to higher skin dose than to internal organs. Furthermore, simulations of SPEs has shown

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that the total skin dose for astronauts performing EVAs is estimated to be up to 32 Gy (for SPE simulation of August 1972) [31]. Radiation-induced skin injuries can be distinguished by several phases depending on the condition of exposure [256]. Early skin reaction is shown by erythema within a few hours after irradiation. After several weeks, inflammatory damage, erythema, loss of epidermal cells, moist desquamation, hyperpigmentation, edema/hyper-proliferation, and epilation can be observed. Late effects can develop after several months and include dermal atrophy, necrosis, and problems related to the deterioration of the skin vasculature. Skin problems, such as burns and slower wound healing, combined with a deprived immune system increase the risk of infections and hinder recovery from ARS [31]. Because of morphological similarities between (mini) pigs and human skin, these animals have been widely used to better understand the skin reaction to ionizing radiation. Furthermore, rodent models such as mouse, rat, or guinea pig have also been studied for ionizing radiation effects on skin. Using porcine models, researchers have been able to indicate skin toxicity after exposure to a simulated SPE radiation resembling the energy and fluence profile of a SPE documented in 1989 [257]. Hyperpigmentation of minipig irradiated skin was observed 7 days after irradiation and lasted throughout the entire observation period. These observations were supported by an increase in melanin deposition found in the stratum granulosum. Further observations of increased proliferation, parakeratosis (an accelerated keratinocytic turnover) and increased amount of melanophages, are thought to be an indication of an inflammatory skin response after irradiation. Other studies exposed minipigs to doses ranging from 5 to 25  Gy of electrons [258]. In agreement with previous mentioned study, a dose-dependent hyperpigmentation of the skin was observed as well as an increase in melanin deposition. Furthermore, in the highest dose exposed group of 25 Gy, skin wounds and ulcers developed 19 days after irradiation on body parts that received the highest dose (tail, ears, and legs). In addition, hair loss in the form of alopecia was observed along the dorsum of these pigs. Low dose rate exposure of skin to low doses of photons, seem to mostly induce oxidative stress and ECM alterations as observed in a mouse model [259]. Skin gene expression changes related to oxidative stress and extracellular matrix (ECM) have been found after whole-body γ-ray exposure. At low dose rates, genes involved in the formation of reactive oxygen species (ROS) were significantly upregulated at doses of 0.25 Gy. Furthermore, dose rate effects were also found in ECM gene expression profiles. Enhanced expression of genes encoding ECM structural components were found after low dose rate exposure.

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10.6.2.3 Acute Effects of Proton Radiation Exposure in the Immune System The immune system consists of a variety of cells, processes, and chemicals that combine efforts to protect the body from foreign microbes, viruses, cancer cells, and toxins [260]. Dysfunction of the human immune system has been shown during [261] and even after space flight [262]. Among the causes of this immune dysfunction, an altered distribution of the cellular components and altered cytokine profiles [263], as well as cytoskeleton alterations and gene expression dysregulation [264] has been shown in many immune cells. When human lymphocytes are subjected to simulated cosmic radiation in vitro they show chromosomal damage, depending on the type of radiation shielding. The adverse effects of space radiation on the immune system is one of the major concerns for space flight. The vast majority of the cellular components that constitute the immune system are highly sensitive to ionizing radiation [265]. It is still not clear if space radiation has a synergistic effect in combination with microgravity, principally in long duration missions and in the context of the immune system. As mentioned, in vitro models have been widely used for studying the effects of space radiation on several cellular types. However, the complexity of most systems—such as the case of the immune system—require approaches that will better mimic physiologic conditions, either in ground-based studies or inflight campaigns. Several animal models that recreate some of the conditions of space flight have been developed for use on Earth. For immunology studies, murine models remain one of the most commonly used small animal model in space radiobiology. Rats exposed to 56-Fe (5  GeV/n) to total doses of 0, 1, 2, and 4  Gy showed a decrease in their lymphocytes, particularly B cells. In another study, mice were irradiated with total (single) doses of 0, 0.5, 2, and 3 Gy with 56-Fe ions. Red blood cell (RBC) counts diminished proportionally to the dose. All three major types of leukocytes also decreased [266]. Sanzari et al. [267] directed a series of radiation experiments using Yucatan minipigs. The animals were exposed to beams comprised of Solar Particle Events (SPE)-like protons, 155 MeV, and electrons, 6 and 12 MeV, with dose profiles that mimic SPE radiation. Their findings suggest that, based on the magnitude of the decrease and the time required to reach the lowest leukocyte counts after irradiation, the proton SPE radiation had more impact on the count than electron SPE radiation, with lymphocytes being the most sensitive type of leukocytes. After proton SPE radiation at skin doses >5 Gy, certain populations of leukocytes (neutrophils) had lasting effects following the irradiation (up to 90 days) [267]. For studying the intricate function of the immune system and how it responds to acute exposures of space radiation, small animal models are essential since they can showcase the network of phenomena. Adding up to the already chal-

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lenging task of pinpointing the alterations occurring in the irradiated immune system we must find a way of adding the following to the equation: isolation, altered circadian rhythms, psychologic stress, and, of course, altered gravity levels. Chronic and late effects of space radiation exposure encompass increased cancer risk, early cataract formation, and a possibly increased risk for degenerative diseases of several organ systems such as the cardiovascular and the central nervous system).

10.6.2.4 Cancer Animal models of cancer induction by space radiation play a crucial role in the determination of the radiation risk associated with a space mission. Firstly, they provide with information about the RBE of different space radiation components such as HZE particles for cancer induction in different organs when compared to a low-LET radiation quality, such as gamma rays or X-rays. The Radiation Quality Factor is derived from the RBE data for cancer induction by HZE particles to scale from gamma radiation to the mixed field of GCR in space radiation cancer risk models. If the RBE is above 1, a higher cancer risk can be assumed for space radiation compared to well-known terrestrial low-LET radiation qualities. Secondly, experiments with high and low dose rates are the basis to estimate the dose and dose rate effectiveness factor (DDREF) to scale from acute to chronic radiation exposure and thereby account for dose rate effects. As animal experiments with exposure at low dose rates are rarely feasible at heavy ion accelerators because of restricted beam time access, dose-rate effect experiments were performed so far at neutron facilities. Furthermore, animal models give insight into the mechanisms of cancerogenesis by HZE particle exposure, e.g., the role of non-targeted effects, and thereby allow to identify potential molecular targets for effective countermeasures (Box 10.6). Box 10.6 Mouse strains

Inbred mouse strains are produced by at least 20 generations of brother-sister mating and they are traceable to a single founding pair. The individuals of an inbred strain are genetically nearly identical to each other and experimental results are highly reproducible. Examples: CBA mouse (cross of Bagg albino and DBA), C57BL/6 mouse (with black coat), BALB/c (Bagg albino) mouse. Outbred strains provide genetic diversity and are effectively wildtype in nature with as little inbreeding as possible. Mating of at least two strains led to the generation of the first filial generation (F1) hybrid mice.

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The first animal experiment with HZE particles to determine cancer induction by single ion exposure used mice and focused on the induction of tumors of the exocrine Harderian gland which is located between eye and ear [268–271]. In these experiments, tumor prevalence was determined by sacrificing mice at a predetermined timepoint after exposure and the number of mice with tumors was counted or the number of tumors per mouse was counted. As this gland does not exist in humans, other animal models were developed and applied. Two different approaches predominate: either wildtype rodents, e.g., inbred, F1 hybrid, or outbred mice, or genetically altered rodent models are exposed to HZE particles at a heavy ion accelerator. Multiparent outbreeding strategies can reduce the strong effects of the genetic background that limit gene-environment interactions in studies with inbred, genetically homogeneous animals [272]. To consider sex-specific cancer types, optimally, both sexes are included [272]. After whole-body irradiation of wild-type rodents, they were followed up over the lifespan of the animals for tumor induction. Alternatively, rats were followed up by palpation until first tumor (time-to-cancer incidence), with an additional follow-up until death. After necropsy, histology was performed to determine the number and types of cancer, e.g., mammary tumors [273, 274]. Here, high numbers of animals are required to detect the increase of cancer incidence above the background cancer rates. Therefore, genetically altered mouse models were developed in order to lower the number of mice and to mimic a specific cancer induction and promotion pathway, mostly for lung, gastrointestinal [275, 276] or liver cancer (hepatocellular carcinoma) [277, 278]. Using a genetically radio-sensitized model implies an assumption about the mechanisms of radiation-induced cancerogenesis—genetically engineered mice carry some, but not all mutations, needed to generate cancer. The rationale behind this approach is to consider somatic mutations in cancer genes such as NOTCH1 and TP53 that might be already present in astronauts when they depart for their first space missions as the number of mutations in the epithelium increases with age [279]. In risk models, development of leukemia (leukemogenesis) and induction of solid tumors are considered separately because of different latency periods after radiation exposure and dose–response relationships. Leukemogenesis is highly relevant for space missions because of its short latency in humans. The CBA mouse strain is susceptible to radiationinduced acute myeloid leukemia (AML) [280] which is explained by a deletion in chromosome 2 (PU.1) that can occur 1 month after irradiation. A point mutation in the second copy of the PU.1 gene causes a differentiation block in the myeloid cells which favor autocrine growth stimulation. In this model, the RBE of iron ions for induction of AML was 1, meaning that the risk of AML induction by high-LET iron ions and low-LET radiation is comparable. As only surviving

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cells can be transformed into a cancer cell, a higher mutation and chromosomal aberration rate induced by HZE particles can be compensated by cell death from collateral damage after an HZE ion traversed a myeloid cell [277]. RBE for other cancer types can be much different as the effectiveness of HZE ions in inducing a specific cancer type depends on the mechanism responsible for the tumorigenesis in that particular cancer. For example, in the same mouse strain that was used for the AML studies, hepatocellular carcinoma (HCC) was induced by HZE particles with an RBE of up to 74 [278]. Concerning solid tumors, a special focus in the studies so far was to evaluate the stage of tumors that can be induced by HZE particles and on detailed studies on lung cancer, gastrointestinal cancer, and brain tumors (Box 10.7).

Box 10.7 Tumor Types Observed After HZE Particle Exposure of Outbred Mice Are Similar to Those Arising Spontaneously or After Gamma Irradiation

Pituitary adenoma, osteosarcoma, Harderian gland tumor, soft tissue sarcoma, thyroid adenoma, ovarian Granulosa cell tumor, mammary adenocarcinoma, histiocytic sarcoma, hemangiosarcoma, hepatocellular carcinoma (HCC), pulmonary adenocarcinoma, small cell lung cancer, myeloid leukemia, (thymic) lymphoma (T cell, B cell), brain tumors, e.g., gliomas [272].

The lung has the highest susceptibility to radiationinduced carcinoma incidence and mortality, based on analysis of human populations exposed to radiation (Life Span Study of atomic bomb survivors). A minimum of five genetic changes convert immortalized human lung epithelial cells to malignant tumors. For lung carcinogenesis, BALB/cByJ or C57/BL6 mice or the K-rasLA1 mouse model [281] were used. In C57/BL6 mice, lung tumors occurred in irradiated mice but not in controls and all were adenocarcinomas, with no significant differences between males and females and for dose fractionation (dividing a radiation dose into multiple fractions, see Chap. 4) versus single dose were found. Incidence of lung tumors was higher in high-LET-irradiated mice than in X-ray-irradiated mice, with an RBE above 6 for all investigated HZE particles (Fe, Si, and O ions) [282]. In the pathogenesis of gastrointestinal tumors, for instance, colorectal cancer and hepatocellular carcinoma (HCC), inflammation plays a crucial role. Animal experiments revealed that heavy ion radiation triggers a pro-inflammatory state which can be associated with late colonic tumors. Furthermore, premalignant polyps with mutations in

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the Adenomatous polyposis coli (APC) gene could be already present in middle-aged astronauts. In the small intestine, the formation of a few polyps and later adenomas and even adenocarcinomas can result from truncation of the APC gene at codon 1638 [283]. Therefore, a mouse model with a chainterminating mutation by a mutation to a stop codon or a frameshift (see Chap. 4) in one allele of the APC gene was developed for colon cancer research (Apc1638N/+). APC mutant mouse models show a good correlation with carcinogens implicated in human colorectal cancer. Delayed genomic instability in APC1638N/+ mice paves the way to gastrointestinal tumorigenesis. In this model, no evidence for dose-rate effects with HZE particle exposure was found [275], indicating that the carcinogenic potential of HZE particles is independent of the dose rate. Also, genetically altered mouse models for the formation of brain tumors are used in space radiation research as already experiments from the 1970s indicated that charged particles can induce glioblastomas: Monkeys (Macaca mulatta) irradiated with high-energy protons (55 MeV, penetration depth ~2.5 cm) surviving 2 years or longer developed glioblastomas [284]. Here, the focus is on the loss of tumor suppressors such as cyclin dependent kinase inhibitor 2A (Cdkn2a or Ink4Arf), phosphatase and tensin homolog (Pten), and TP53  in astrocytes and on oncogene activation (e.g., epidermal growth factor receptor variant III, EGFRvIII) after irradiation. Iron and silicon ions were much more potent tumor inducers in “preinitiated” astrocytes than gamma rays [285]. The animal studies with single beam irradiations show that the efficiency of HZE particles to induce cancer is related to ion energy, LET with a peak RBE below 100 keV/ μm, sex of the animals, and depends on the tumor type [275]. The RBE for cancer induction was recently determined to range from 5 to 16 [286], representing a snapshot that will be further updated as not all available data were included. Currently, based on the results of single beam irradiations, multiple beam experiments with up to 33 ion beams are performed at the NASA Space Radiation Laboratory (NSRL) using the GCR simulator in order to understand whether the effects of the different GCR components act in an additive or even in a synergistic manner in cancer induction.

10.6.2.5 Cataract According to recent epidemiological evidence, radiationinduced cataract (see Chap. 2) occurs with a threshold absorbed dose of 0.5 Gy (0–1 Gy) of sparsely ionizing radiation, meaning that a cataract can arise after any ionizing radiation dose no matter how low if the remaining lifespan is long enough for its appearance. The 1 Sv GCR dose to be expected for a 1000-day Mars mission [83, 84] means that even the upper limit of the cataract-induction threshold dose confidence interval will be reached during a human Mars exploration mission. In astronauts, epidemiological data sug-

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gest a higher risk for the development of cataracts in case of missions in LEO with high inclination [287]. Due to its germinative zone in the lens epithelium, the eye lens is a radiation-sensitive organ. These cells are actively proliferating during lifetime and finally differentiate into transparent lens fibers. In case cells are damaged, they cannot be eliminated from the lens which is covered by a capsule and not vascularized. Exposure of the eye lens to ionization radiation is thought to result in sub-capsular cortical lens opacification via various steps, starting with genetic damage of lens epithelial cells via changes in cell cycle control, apoptosis, differentiation, or other pathways controlling lens fiber cells’ differentiation, and cellular disorganization. Due to higher local dose and different patterns of cellular energy deposition from high-LET components of GCR, higher efficiency in the induction of lens-damaging effects is assumed than for low-LET radiation. Therefore, animal experiments were mostly performed to determine the RBE of HZE particles to induce lens opacification and to detect possible dose rate effects. In rats, the RBE reached 50–100 for HZE particles within LET above 80 keV/μm [288] and fractionation of exposure did not reduce the cataractogenic effect [289]. Neutrons as secondary particles occurring in spacecraft and on planetary or moon surfaces had also a high RBE for cataract-induction in rats [290]. To determine the role of genetic predispositions, mice that are heterozygous for Ataxia telangiectasia mutated protein (ATM) were exposed to HZE particles and cataract formation was followed. ATM plays a central role in the DNA damage response (DDR). Heterozygosity for the ATM gene predisposes carriers for early onset time and progression of cataracts even without exposure to ionizing radiation [291]. Also after gamma ray and 1 GeV/n iron ion exposure, cataracts appear earlier in ATM heterozygous animals compared to wild-type mice and the RBE for HZE particle induced cataract formation ranged from 4 to 200, whereby the highest values were found for the lowest dose (10 mGy) and RBE decreases with increasing dose [292, 293]. In conclusion, HZE particles present in GCR and neutrons as part of the secondary radiation field are highly cataractogenic and the mechanisms such as long-term changes in gene expression, complex DNA damage, and chromosomal aberrations in eye lens epithelial cells (LECs) are still under investigation.

10.6.2.6 Cardiovascular System Exposure to space hazards, including microgravity and heavy ion exposure can cause harmful effects on the cardiovascular system during spaceflight. Upon entering microgravity, cephalad fluid shifts cause increased stroke volume and cardiac output. Furthermore, the cephalad fluid shift is also hypothesized to cause visual impairments due to increased cranial pressure [294]. During flight, mean arterial pressure is decreased, together with central venous pressure. Furthermore, decreased systemic vascular resistance, results

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from increased cardiac output, systemic arterial vasodilation, and decreased arterial pressure [295]. Other effects of microgravity exposure include hypovolemia, cardiac arrhythmia, cardiac atrophy, and orthostatic intolerance. Believed to be caused by fluid shifts and movement of interstitial water from the legs to the head, the fluid reduction and eventually hypovolemia results in a reduced number of red blood cells [296]. Moreover, cardiac atrophy occurs as a result of decreased metabolic demand and oxygen uptake during microgravity conditions. Together, cardiac deconditioning, i.e., hypovolemia, cardiac atrophy, and decreased cardiac output, causes a decreased exercise capacity and orthostatic intolerance post-flight [297]. While effects related to microgravity exposure during spaceflight are fairly well-known (albeit underexplored), impacts of the cardiovascular system from space radiation and heavy ion exposure during spaceflight are less known. Furthermore, studies from space analogs focusing on radiation effects have shown several effects on the cardiovascular system. Mice exposed to heavy ions show myocardial remodeling, resulting in hypertrophy and cardiac fibrosis [186]. Additionally, accelerated development of atherosclerosis has been found in mice after heavy ion exposure. Leading to a greater prevalence of myocardial infarction [298]. Both in vivo and in vitro models during space flight as well as using space analogs have been used to investigate underlying mechanisms of space-induced CVD. Important mechanisms include endothelial dysfunction, cellular apoptosis, cellular senescence, inflammation, and reactive oxygen species production [297].

10.6.2.7 Central Nervous System Exposure to heavy ion, especially during long-term space mission, can also affect the central nervous system (CNS). The CNS is part of the nervous system and is composed of the brain and the spinal cord. It is responsible for perceiving any exterior information, transmitting, and subsequently processing it. Responsible for signal transmission are neurons, whereas glial cells (oligodendrocytes, microglia, or astrocytes) have diverse function such as the trophic support of neurons. As neurons are terminally differentiated and have a very restricted regeneration potential, damaged cells will usually not be replaced and thus damage might accumulate over months or years. Acute CNS effects of ionizing radiation exposure are only observed after exposure to very high doses and can be expected in spaceflight only during very large Solar Particle Events (SPE) in case of insufficient shielding. Thus, for more than 20 years, possible effects of chronic low-dose exposure of the CNS to galactic cosmic rays (GCR) are discussed and a decrease in CNS performance of astronauts is suspected, which was also further evidenced in animal studies [299, 300]. Normally rodent animal experiments are performed at heavy ion accelerators simulating space radiation at doses below 1 Gy in a relatively short time, revealing impairment in

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cognitive performance, reduction of dendrites, reduced neurogenesis, and increased neuroinflammation [301–303]. As these effects can be seen even months after irradiation, late effects are possible even after exposure to lower doses [304]. Whereby it has to be considered that in these heavy ion accelerator experiments, the dose can only be applied in a short time, and prolonged exposure over several weeks or months mimicking the real situation in spaceflight is not often possible. With the use of new, low dose rate neutron irradiation facilities, it is now possible to expose rodents to a chronic low-dose as expected during space flights [305]. Also, mice that were irradiated with this chronic neutron irradiation (for 6 months) resulted in diminished hippocampal neuron excitability, a region which is essential for memory and learning, and disrupted hippocampal and cortical long-term potentiation. In addition, mice showed severe impairments in memory and learning tasks as well as distress behaviors [305]. One limit of experiments at the accelerator is that only radiation exposure of a few single radiation types can be studied, while in space, radiation exposure consists of a complex radiation mixture. It is still unclear if humans’ brains are affected to the same extent, but chronic low-dose radiation may cause problems for astronauts regarding decision-making processes or performance [306] (Box 10.8).

Box 10.8 Section Highlights

Since their discovery by Antonie van Leeuwenhoek in 1702, bdelloid rotifers and tardigrades have remained intriguing organisms. Their tolerance to desiccation at any stage of their life and their ability to survive a variety of stresses (e.g., low and high temperatures, absence of oxygen, vacuum, high level of ionizing radiation, etc.), makes them good candidates to study extreme resistance mechanisms in the context of space research. Tardigrades have a long history of space astrobiology experiments being among the first animals exposed to space vacuum and radiation. Recent experiments performed onboard of the ISS used bdelloid rotifers and tardigrades to study the adaptation to microgravity and cosmic radiation during spaceflight.

10.6.3 Tiny and Extremely Resistant: Why Bdelloid Rotifers and Tardigrades Are Animal Model Systems for Space Exploration? 10.6.3.1 Bdelloids and Tardigrades, Small Animals to Study Desiccation, Radiation Tolerance and Limit of Life Bdelloid rotifers (Fig. 10.17) and tardigrades (Fig. 10.18) are among the smallest animals on Earth: most species are

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less than 1mm in size and contain ~1000 cells. Despite their small size, these animals have complete nervous, muscular, digestive, excretory, and reproductive systems. Mainly living in semi-terrestrial environments, such as lichens and mosses, most (but not all) bdelloid and tardigrade species are able to enter and survive complete desiccation (see Box 10.9 for definition) at any stage of their life cycle. When water starts to evaporate, these animals begin to contract their muscles and their body to adopt a “tun” shape allowing an optimal desiccation resistance [307–310]. This proper contraction of the body, followed by a specific organization of internal structures is a key step in enabling a successful recovery of desiccated animals after rehydration [308, 309]. The desiccation resistance and recovery rate vary between species [311–314]. The survival rate depends on the length of the desiccation period, the relative humidity, temperature, and animal age. Tardigrades desiccated over 10–20 years, within dry mosses stored at room temperature, were successfully rehydrated confirming their desiccation resistance for periods [315] [316]. While being frozen, these animals were shown to survive over 30 years of desiccation [317]. Bdelloid rotifers have also been shown to survive long periods of desiccation, up to 9 years [318]. As for tardigrades, cold temperatures seem to extend the capacity of desiccated bdelloids to cope with the long duration of metabolic arrest. In a recent publication by Shmakova et al. bdelloid rotifer specimens were recovered from frozen permafrost soil 24,000 years old [319]. If no data are still available for tardigrades, studying old permafrost samples may reveal other records of small animals’ life preservation. For example, some nematodes were described to successfully recover after melting from 30 to 40,000 years old samples [320].

Box 10.9 Desiccation or Drought Tolerance?

Desiccation tolerance must be differentiated from drought tolerance. Many organisms are able to tolerate drought as a reduction in water availability in the environment for longer or shorter times. However, a reduced number is able to survive a loss of 90% or more of their body water content. Complete desiccation is reached when the water content decreases below 10% of the dried mass, not enough to form a monolayer around macromolecules, preventing enzymatic reactions and therefore metabolism.

Mostly found in habitats where physical parameters can change unpredictably, tardigrades and bdelloid rotifers were described to be able to cope with a wide range of physical extremes besides desiccation and freezing, such as UV radia-

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Fig. 10.17  Overview of the bdelloid rotifer Adineta vaga life cycle. Bdelloid rotifers live in limno-terrestrial habitats like mosses and lichens. Adapted to these environments, they can be desiccated at any stage of their life cycles including egg stage. When they are exposed to desiccation, adults adopt a “tun” shape allowing optimal desiccation resistance. Adineta vaga is about 200–250 μm long. (Credits B. Hespeels)

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Fig. 10.18  Morphology of adult tardigrades and eggs. (a, b) Lateral and dorsal views of Echiniscus testudo. (c) Dorsal view of Paramacrobiotus areolatus. (d, e) Global morphology of eggs laid by

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Macrobiotus kamilae and P. areolatus. Pictures were captured using scanning electron microscopy. (Illustration kindly provided by Daniel Stec and reprinted with his permission)

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tion, high temperatures (exceeding 100 °C for a few minutes), high pressure or deep space vacuum [317, 321–327]. Among others, bdelloid rotifers and tardigrades were described to be highly resistant to low- and high-LET [328] radiation. In 2008, it was demonstrated for the first time that two bdelloid rotifer species, A. vaga and Philodina roseola, were resistant to ionizing radiation while being hydrated, surviving up to 1200 Gy of gamma radiation with fecundity (i.e., the total number of daughters produced by irradiated animals) and fertility (i.e., the capacity to produce at least one daughter) showing a dose response [329]. Later, it was demonstrated that desiccated bdelloid rotifers survive doses >5000  Gy of X-ray and proton radiation. These levels of radiation exposure were contrasting the Lethal Dose 50 (LD50) (i.e., dose required to kill 50% of the irradiated population) of mammalian cells which range from 2 to 6  Gy after X-ray irradiation. Similarly, desiccation-resistant tardigrades were described to survive high dose of X-ray and gamma ray (LD50 ranging between 3000 and 6000 Gy) (reviewed in [322]). Unexpectedly, radio-resistance of hydrated and desiccated tardigrades appeared to be more tolerant to high-LET radiation. For example [330], LD50 of the eutardigrade Richtersius coronifer was approx. 10,000 Gy. A major difference in comparison with bdelloid rotifers was that, despite a high survival after irradiation, most tardigrades were unable to produce fertile eggs for doses >100 Gy [322]. As an example, the tardigrade Hypsibius dujardini treated with gamma radiation had an estimated LD50/48 h for survival of ∼4200 Gy, and doses above 100 Gy dramatically impaired the production and hatching of laid eggs [331].

individuals require small vessels. (3) Distribution: rotifers and tardigrades are readily found in nature and are easily cultivated under controlled conditions. (4) Life span: rotifers and tardigrades have short life cycles that can be studied in a reasonable time period. (5) Reproductive mode: all bdelloid rotifers and some tardigrade species reproduce parthenogenetically. This reproduction system offers two key advantages: a rapid expansion of the population, and a high degree of reliability, as the genome is fully transmitted to the offspring. Therefore, the use of clonal lines reduces the biological variability noise in biological experiments. (6) Extremotolerance: both bdelloid rotifers and tardigrades were described to be able to deal with a high number of DNA DSBs and various stressors encountered by astronauts during space flight. Small extremotolerant animals can provide new perspectives in the adaptation of life to the space environment and ultimately lead to enhancing radio-resistance. For both clades, radiation resistance and radiation-sensitive species can be used in comparative experiments. (7) Storage: as most tardigrades and bdelloids survive desiccation and freezing, they can be stored easily before and after scientific experiments with limited impact on their biology and the scientific output. (8) Desiccation resistance: the desiccated state of tardigrades and bdelloid rotifers correlates with increased resistance to stresses, including deep space vacuum and extreme temperatures. These multiple properties and advantages for space experiments make bdelloid rotifers and tardigrades good candidates to test the limits of life during space exposure. An overview of space experiments involving tardigrades and rotifers is presented in the next two sub-sections.

10.6.3.2 Small Animals and Space Research As an alternative to other animal models, the use of rotifers and tardigrades was proposed for space research. Indeed, these animals may contribute to better understanding damage and consequences induced by exposure to radiation and/ or microgravity. How these organisms may respond and adapt to these stresses pave the road to the discovery of new molecules or candidate genes. Ultimately research outputs may be used to improve health span and protect astronauts or individuals subjected to radiation during space flights or medical treatments. The use of rotifers and tardigrades as space research models was proposed because of the following aspects. (1) Complexity: they are Metazoans (multicellular animals), containing tissues and organs, having a complete gut and a complex muscular structure, yet being very simple animals. Rotifers and tardigrades are however made up of about 1000 cells, while a human is made up of several millions of cells. This simplification allows to disentangle complex problems through easier approaches. (2) Miniaturization: rotifers and tardigrades are small; experiments performed with numerous

10.6.3.3 Tardigrades, Pioneer Animals of Astrobiology Field In September 2007, tardigrades were exposed to LEO within the Biopan-6 experimental platform provided by the European Space Agency (ESA) (“Tardigrades in Space,” TARDIS.  FOTON-M3 mission). During 10 days at LEO (258–281  km above sea level) samples of desiccated adult eutardigrades of the species Richtersius coronifer, Milnesium tardigradum, Echiniscus testudo, and Ramazzottius oberhaeuseri were exposed to space vacuum, cosmic radiations, and two different UV-radiation spectral ranges [323]. It was demonstrated that tardigrades were able to survive space vacuum and cosmic radiation with a survival rate ranging between 70% and 80%. Any impact on the reproductive capacities of exposed animals was reported. However, samples exposed to full solar radiation experienced high mortality. A small fraction of survivors died a few days post-rehydration without the production of any viable offspring. By filtering UV and restricting the exposure of desiccated tardigrades only to UVA and UVB, a significant part of desiccated tardigrades was able to be reactivated and was

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able to reproduce. Since the fertility of descendant generations of M. tardigradum was not impacted, it was suggested that survivors were able to repair a priori the damages induced by the spaceflight and did not transfer them to future generations [332]. In parallel to the TARDIS experiment (Fig.  10.19), two other experiments were launched onboard of the FOTON-M3: the (1) RoTaRad mission (Rotifers, Tardigrades, and Radiation) and (2) Tarse project (Tardigrade Resistance to Space Effects). RoTaRad experiment confirmed that desiccated tardigrades stored under controlled atmosphere were able to survive while being exposed to a combination of cosmic radiations and microgravity. However, the survival rates were reduced during this experiment likely due to the applied desiccation protocol [333]. With the Tarse project focusing on the eutardigrade Macrobiotus richtersi species, hydrated and desiccated individuals were exposed to the space environment for 12 days. In both states, microgravity and radiation had no effect on the survival rate, reproductive capacity, and DNA integrity of exposed animals. Despite the absence of visible morphological changes, it was nevertheless reported that the activity of key antioxidant proteins (including catalase and superoxide dismutase) was decreased during spaceflight. The amount of Heat Shock Proteins 70 and 90, known to be involved in stress resistances of tardigrades, did not differ after this short-term exposure to spaceflight. A few years later, the TARDIKISS experiments (Tardigrades In Space) were launched with the last Space Shuttle mission (STS-134 2011) [334]. During this 16-days mission, the enzyme activity of key antioxidants was investigated in desiccated tardigrades from the two species Paramacrobiotus richtersi and Ramazzottius oberhaeuseri [334]. Supporting the idea that desiccated animals were a

Fig. 10.19  View of TARDIS experiment. (a) View of the exobiology Biopan platform containing TARDIS experiment. For 12 days in September 2007, approximately 3000 water bears were launched in space during the Foton-M3 mission. Reprinted with permission from

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weakly affected by microgravity and cosmic radiation, comparative data analysis between flight and ground samples showed no significant differences in the enzymatic activity of antioxidants. In June 2021, a fifth experiment was launched onboard of the ISS to investigate the short-term and multigenerational survival of tardigrades. The aim of the Cell Science-04 experiment (CS-04) was to evaluate the transcriptomic response of hydrated tardigrades cultured on the ISS using a dedicated cell culture system (Bioculture System, developed at NASA Ames Research Center). For this experiment, the tardigrade Hypsibius exemplaris was used as model species. Scientists are currently evaluating the ability of these animals to survive onboard of ISS for short and long periods of time (up to four generations). In parallel, the transcriptomic responses of these animals are being investigated to follow the evolution of the expression profiles of tardigrades in a microgravity environment. A progressive adaptation of tardigrades onboard of ISS may lead to a better understanding of the molecular responses involved in gravity sensing and will help expand research to secure astronaut’s health for future space missions. Among others, tardigrades were described to express several antioxidant proteins to face desiccation and radiation stresses [322, 335]. In particular, tardigrades were described to express specific proteins binding to DNA and protecting their genome from ROS induced by desiccation and ionizing radiation [336, 337].

10.6.3.4 Bdelloid Rotifers, a New Model Species for Space exploration How microgravity and cosmic radiation may affect desiccated bdelloid rotifers was tested for the first time in 1997. b

ESA. (b) Details of the sample holder containing the tardigrades Richtersius coronifer. Tardigrades on the top level were exposed to the Sun and were optionally protected with filters. (Image kindly provided by K. Ingemar Jönsson and reprinted with his permission)

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For their first exposure to space, dry samples of Macrotrachela quadricornifera were transported onboard of the space shuttle STS-81 for a total of 10 days. The data revealed a similar survival rate and reproductive fitness for ground controls and flight samples [312]. Since desiccated rotifers appeared to be protected from the impact of microgravity and cosmic radiation, at least in this short-term exposure experiment, researchers started to investigate the consequences of space flight on hydrated bdelloids. In absence of gravity, it has been hypothesized that the distribution of cytoskeletal elements or yolk granules in the egg cytoplasm is impacted. This abnormal organization of the cytoskeleton could impact the rotifer reproduction. Therefore, researchers first investigate the capacity of bdelloid rotifers to complete their embryological development under microgravity was initially investigated. Pre-flight experiments were performed under hyper-gravity environment (up to 20 g) and under simulated microgravity (as low as 0.0001 g) using a 3D random positioning machine (3DRPM). Results showed that the rotifer development remained constant regardless of the treatment experienced,

Fig. 10.20  View of Rob1 hardware used to culture hydrated A. vaga individuals onboard of ISS (December 2019). Top left: Rob1 hardware after its assembly at the launch site at Kennedy Space Center. Rob1 hardware is a passive hardware containing five culture bags containing hydrated specimens of A. vaga. Hardware enables gas exchanges between rotifer cultures and the outside through a permeable membrane. Top right: View of the culture bags assembled inside Rob1 hardware. Culture bags, loaded with 10,000 A. vaga individuals each, are made of Teflon and ensure an optimal gas exchange between the culture medium and the outside. Bags are waterproof and avoid any leakage of the medium (composed of mineral water and sterile lettuce juice) or rotifers. Reprinted with permission of Marc Guillaume. Bottom left: View of ESA astronaut Luca Parmitano loading two Rob1 hardware on KUBIK. KUBIK is a small incubator, temperaturecontrolled, with removable inserts designed for self-contained microgravity experiments. (Reprinted with permission of NASA)

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except for some minor modifications in early embryos experiencing 20 g with no subsequent impact on the development. This first investigation suggests that bdelloid rotifers continue embryological development despite changes in g-force. Unfortunately, no data from flight experiment development associated with embryological development of bdelloid rotifers exposed to space environment was released post-flight. Twenty years later, the bdelloid rotifer A. vaga was sent onboard of the ISS for two independent experiments. In December 2019, two autonomous hardware, each containing five culture bags loaded with 10,000 individuals, were transported onboard of ISS.  Hydrated animals were exposed to launch conditions and exposed to 12 days of microgravity. At the same time, a ground reference experiment was implemented on Earth to compare the biological responses of rotifers to space conditions on ISS. The aim of this first experiment (RoB1, Fig.  10.20) was to compare the transcriptomic responses of hydrated A. vaga samples exposed to space environment with the ground control samples. Preliminary results

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Fig. 10.21  View of one Rob2 hardware used onboard of the ISS (left) and Astronauts checking the correct rehydration of A. vaga individuals. Sixteen pieces of hardware were sent to ISS, each containing 40,000

dry rotifers. Once onboard, rotifers were automatically rehydrated and cultivated 11 days before their fixation and download to Earth. (Reprinted with permission of Boris Hespeels and NASA)

confirmed the successful maintenance of hydrated bdelloid individuals on ISS, without additional food or oxygen supply and without astronaut intervention. All the replicates (ten) of the autonomous A.vaga cultures survived and reproduced on ISS with no visible impact on the morphology in spaceexposed samples. While it is well documented that astronauts experience DNA damage when exposed to cosmic radiation, accumulating DNA mutations and/or genomic rearrangements [163], the combined effect of cosmic radiation and microgravity on the living organism is still debated. It is suspected that microgravity reduces the efficiency of DNA repair and increases cancer risk [207, 215, 338]. Several studies using simulated microgravity highlighted a decrease in DNA repair efficiency. However, no effects of spaceflight on the cellular capacity to repair artificially induced DNA was observed (see Moreno-Villanueva et al. [209] for review). In order to obtain more insights, bdelloid rotifers have been used as a model system to evaluate their DNA repair efficiency of induced DNA breaks in space environment as compared to Earth samples. By the end of 2020, desiccated and irradiated A. vaga individuals were sent onboard of ISS. Before launch, desiccated animals were irradiated with 500 Gy of X-ray or proton radiation. Onboard, bdelloids were rehydrated and cultivated for different time periods to (1) follow the putative DNA repair process occurring post-rehydration and (2) investigate whether these irradiated rotifers still produce offspring under microgravity. In addition, half of the samples were exposed to simulated gravity using a centrifuge on ISS. Finally, a ground experiment was conducted in parallel

at the launch site at Kennedy Space Center for comparison. Data generated by this second space experiment (entitled RoB2, see Fig. 10.21) will enable: first, to compare the DNA repair kinetic of rehydrated bdelloids post irradiation in 1G, μG, and simulated 1G; second, to compare the radiation responses of rehydrated rotifers after exposure to low LET or high LET; and third, to compare the DNA repair efficiency in space and on Earth by isolating eggs or juveniles from the exposed samples and use whole genome sequencing to compare the genomic structure of these animals pre- and postexposure. This space experiment with bdelloid rotifers will contribute to our understanding of DNA repair process activity in space, in the presence or absence of microgravity. Moreover, studying the molecular processes involved in the DDR process of A. vaga will be of huge interest for future space travel. In general, the ongoing rotifer space experiments will contribute to a better understanding of the mechanisms involved in the protection and repair of damages induced by radiation. They pave the road to the discovery of new molecules or candidate genes that could ultimately be used to improve health span and protect astronauts or individuals subjected to radiation during space flights or medical treatments. This research is also of fundamental importance for the understanding of extreme biology and the questions raised on the origin of life and its ability to spread through outer space. A third experiment, supported by ESA, is under preparation to evaluate whether rotifers can survive full space exposure, outside ISS, as was previously reported for tardigrades.

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10.7 Plant Experimental Models and Biological Changes of Space Radiation 10.7.1 Plants vs. Animal Models Long space exploration missions, settlement on orbital stations, or future planetary settlement (e.g., on Mars) will require further development of Life Support Systems (LSS). The LSS are able to regenerate a great amount of essential resources for survival and represent an ideal solution since it is not technically and economically feasible in long space missions to transport a large amount of consumables from the Earth [339– 341]. Bioregenerative Life Support Systems (BLSS) are an artificial closed ecosystem characterized by the same structure as a terrestrial ecosystem: producers (plants), consumers (humans/animals), and decomposers (microorganisms). Among biological components within BLSS, higher plants would have the same role on Earth as producers. Through photosynthesis, plants would utilize carbon dioxide produced by space crew and provide oxygen and fresh food. Moreover, they would use nutrients derived from human wastes and guarantee water purification by transpiration. Furthermore, plant cultivation in space also would provide psychological support against isolation [342, 343]. Fig. 10.22  A comparison among different responses of Plants (P) and Mammals (M) to ionizing radiation. (Reprinted with permission from Arena et al. [346])

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Each organism in Space is subjected to several factors which are potential constraints for biological life. Among the environmental factors (e.g., altered gravity, interaction between microgravity and fluid-dynamics, modified conditions of pressure, temperature, confinement, etc.) limiting plant growth in space, ionizing radiation influences severely the development of organisms at molecular, morpho-structural, and physiological levels [163, 182, 344]. Indeed, ionizing radiation is considered one of the main constraints for the long permanence of humans in Space. All organisms in extraterrestrial environments are subject to higher levels of ionizing radiation than on Earth and, notwithstanding the large number of studies aimed at understanding the effect of ionizing radiation on animals, the knowledge on plant reaction is limited. Available information is limited to horticultural model crops which are candidate for fresh food production in BLSS.  Moreover, most experiments are based on the irradiation of dry seeds and data from irradiation tests using other biological models (e.g., seedlings, adult plants, actively growing tissues) are scanty. In Fig. 10.22, a comparison of the responses of plants and mammals to ionizing radiation exposure is shown. Generally, plants are more resistant than mammals. Ionizing radiation is known to have differential effects on plant growth, develop-

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ment, and reproduction, ranging from detrimental outcomes at high doses, harmful consequences at intermediate levels, and stimulatory effects at very low doses. This phenomenon is called “hormesis.” Particularly, low doses of ionizing radiation have been reported to stimulate seed germination and root growth [345, 346]. However, ionizing radiation can also induce dwarf growth that is a desirable trait under conditions of limited volume availability in missions on orbital stations or during exploration traveling. The increased radioresistance of plants is still a debated issue since it can be associated with a genetic basis, but it can also reflect biochemical and biomolecular mechanisms of shelter from genotoxic damage. The severity of the effects of ionizing radiation on plants is dependent upon several factors including radiation-related parameters (e.g., dose, LET) and organism-related traits (e.g., species, cultivar, physiological status, and structural properties, as well as plant genome organization including the polyploidy) [345, 347].

10.7.2 Biological Changes from Genetics to Organogenesis In adult plants, in the case of organs at complete development, resistance to stressors can be often ascribed to integrated mechanisms of adaptation operating at morpho-structural and eco-physiological levels since the limits of major metabolic and physiological processes are dictated by the plant’s structure [348, 349]. Growth, reproduction, and, ultimately, survival of plants in Space depend on photosynthesis which is strongly responsive to ionizing radiation acting on the various components of the photosynthetic apparatus, such as pigment–protein complexes responsible for light absorption, electron transport carriers, and enzymes of carbon reduction cycle [345]. Ionizing radiation leads to several detrimental effects in photosynthetic apparatus, such as loss of functionality of photosystem II (PSII) and generation of free radicals causing photosynthetic membranes’ oxidation [350–352]. Changes in the total antioxidant pool and in the distribution of phenolic compounds in leaf tissues were observed in plants exposed to very high doses of X-rays, namely 50 and 100 Gy [353]. However, chronic exposure to low doses of ionizing radiation seems to enhance the activity of some antioxidant enzymes, providing plants with a radio-resistance [354, 355]. Moreover, the degree of plasticity of leaf cytological and anatomical traits in response to environmental changes can be responsible for enhancing or constraining processes such as light interception and gas exchanges, definitely affecting photosynthesis. Similarly, the correct functioning of the whole water transport system throughout the plant is

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responsible for water supply up to the leaves, necessary for efficient photosynthesis. The ability of xylem to transport water efficiently depends on the morphological features of its conduits and on the ultra-structural properties of conduit cell walls, whose main components can be differently affected by ionizing radiation. Apart from a few findings mainly related to specific ultrastructural modifications occurring on irradiated seeds, the effect of cosmic radiation on organ/tissue organization, especially in relationship with eco-physiological traits, is still poorly explored. Moreover, most of the studies regard experiments with low-LET ionizing radiation [346, 355], and only a few data are available on the effects of chronic radiation exposure on plants in general, mainly deriving from nuclear accidents as Chernobyl in Ukraine (1986) and Fukushima in Japan (2011).

10.8 Eukaryotic Cell Experimental Models and Biological Changes of Space Radiation 10.8.1 Definition of Eukaryotes Regarding the complexity of their cells, all living organisms can be classified into two groups-prokaryotes and eukaryotes. Compared to prokaryotes, eukaryotic cells are highly organized and contain a cell nucleus. Prokaryotes are bacteria and archaea, while protists, plants (see Sect. 10.5.8), animals (see Sect. 10.5.7), and fungi (see Sect. 10.5.9) are eukaryotes. In the following the effect of space radiation on in vitro models (conducted in a cell culture dish) and ex vivo models (experiments outside a living body) will be described.

10.8.2 Definition of In Vitro Models In vitro models used in science, are very important, as they provide insight into cells. With this, the function of primary cells and cell lines of various origin (vertebrates including human, insects, and mussels) can be studied.

10.8.3 Definition of Ex Vivo Models Ex vivo models or tissue explants allow studying complex functions and interactions of different cells within an organ. For these experiments, the living tissues are directly removed from a living organism or can be generated by means of pluripotent stem cells and cultivated under controlled conditions.

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10.8.4 3D Cell Culture Models 10.8.4.1 Definition 3D Cultures In comparison to cells in monolayer cultures (2D), cells in 3D cultures react completely differently. The biggest disadvantages of 2D cultures are the unnatural contact with a plastic or glass surface, the flat morphology of the cells on the growth surface that restricts intercellular contacts and the lack of an extracellular matrix which surrounds cells in vivo. These conditions modify the metabolism and functioning of cells and often result in the loss of the specific differentiation of a cell. The structure, function, and composition of organs and tissues can thus be better studied in 3D cell culture systems. They enable cell–cell and cell–extracellular matrix interactions in a three-dimensional space. 3D cultures are a very helpful tool before performing whole-animal studies. They can further be used to study the understanding of how processes in tissues are affected by spaceflight conditions, including space radiation and microgravity, which otherwise cannot be investigated in animal or human subject studies. There are many different models of 3D cell cultures, including organoids, ex vivo tissue, or slice cultures, which are explained in the following. Furthermore, it is possible to create these models with 3D bioprinting, which have then a structure which closely resembles the organization of tissue or organs. In fact, the European Space Agency (ESA) recently summarized the capability science requirements for 3D bioprinting on the ISS to support medical treatment on long-term space missions. In all given examples two or more cell types can be cocultured, closely simulating the situation in organs or tissues, e.g., investigation of cellular differentiation processes in tissues, nerve-muscle function, tissue regeneration and repair, vascular tissue function, brain tissue homeostasis and aging, immune system processes or cardiac muscle function. 10.8.4.2 Organoids Human organoids, derived from stem cells or progenitor cells, are tiny self-organized organ-specific 3D cultures, recreating the physiological and cytoarchitecture of human organs. With this, the model reflects the in  vivo situation much better than single cell cultures. For research purposes, it is feasible to create organoids that resemble the brain, kidney, lung, intestine, stomach, and liver. Organoids will help to study the effect of space radiation on the overall response of organs, including cellular heterogeneity, cell-matrix interactions, cell-cell interactions, morphology, and functional changes [356, 357], which cannot be studied in in  vitro systems. One major disadvantage compared to in vivo systems is the lack of microenvironment. The effects of microgravity on human brain organoids were tested on the ISS during the Space Tango-human Brain investigation in 2019 (NASA). Of special interest was the

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effect on the brain cells including survival, migration, metabolism, and the formation of neuronal networks (Muotri, unpublished).

10.8.4.3 Spheroids Spheroids are also 3D cell cultures, but in comparison to organoids, they form simple clusters into sphere-like formation, but they cannot self-assemble or regenerate. Whereby the cellular functions inside spheroids are closely correlated to the size, uniformity is especially important for reproducible results. To guarantee this, several methods for culturing are available such as hanging drops, scaffolds, liquid overlay technique, and hydrogels [358]. Nowadays spheroids are highly used to study the microenvironments of tumors or their response to radiotherapy. Already in 2016, the SPHEROIDS project was launched on the ISS.  Here, endothelial cells, which under simulated microgravity form small, rudimentary blood vessels, were exposed to real microgravity for 12 days on the ISS. The formation of spheroids under space conditions and under simulated microgravity on Earth were similar [359], underlining the important role of microgravity in spheroids formation. Differences between the three types of cultures are summarized in Fig. 10.23. 10.8.4.4 Organotypic Slice Cultures Organotypic slice cultures are tissue samples that are cut in thinly, about 300 μm, thick slice and are then cultivated on semipermeable insert. Most common are organotypic slice cultures that originate from different parts of the brain (e.g., hippocampus, cerebellum, or cortex) and can be kept in cultures for long term, while slices originating from liver tumors can only be kept in culture for a short time [360, 361]. Also,

Cell types Derived from Morphology Represent Example

Monolayer Single cells

Spheroid Mulple cells types

Donor Paents, Animals Non-natural, flaen Replicate only ssues, but not organs MEF, ESCs, iPSCs

Cell line monoculture Simple cell clusters Single ssue of an organ Muscle spheroids, lung epithelial spheroids, Tumor spheroids

Fig. 10.23  Difference between the different cultures

Organoid Epithelial and mesenchymal cells Stem cells More complex cell clusters Mulple ssues of an organ or “Mini-organs” Organoids for brains, guts, lungs, hearts

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this 3D culture has the advantage that the composition and architecture of the extracellular matrix as well as the tissue are preserved. During analysis of the slices, it has to be considered that every slice, even from the same organ, has a partly different composition, cell counts, and viability, limiting the reproducibility of results produced by this method.

10.8.4.5 Organ Cultures Organ cultures were developed from tissue and slice cultures. By using organ cultures, it is possible to study the functions of an organ in various conditions and states in an in  vitro organ. Hereby, the entire organs or only a part of the organ are excised from the body and cultured. Also, with this method, the 3D structure of the tissue of choice is preserved. For space exploration, the eye lens is of special interest, because it is amongst the most radiosensitive tissues in the human body. Ionizing radiation can cause a posterior subcapsular cataract [287, 362, 363]. Whole lenses and lens epithelial cells in culture enable the study of early mechanisms of space radiation-induced cataractogenesis and of the relative biological efficiency of different space radiation components to induce early changes. With regard to the human lens in anatomy and size, the porcine eye is very similar. Thus, it is used to study the radiation response in the whole organ. Translation to the human eye lens can be enabled by using human-transformed epithelial cells or lens epithelial cells from donor patients. As the viability of eye lenses in cultures is limited to a few weeks, studies on radiation-induced fullblown cataract formation usually require animal experiments over their lifespan (Sect. 10.5). In addition to that, the microgravity environment on the ISS suits perfectly to 3D print tissue cultures and later maybe entire organs. Compared to conditions on Earth where scaffolds or matrices are needed to form organoids, in space cells can easily self-organize into their precise structure. On the one hand, the bioprinted tissue could be used in the future to treat injured astronauts [364] and on the other hand the technique can be transferred to Earth and then be applied to the field of regenerative medicine for organ transplantations [365]. In July 2019, the 3D BioFabrication Facility (BFF— see Fig. 10.24) has arrived onboard of the ISS, with this it is now possible to study 3D bioprinting of different human tissues in space. Also, here real microgravity has the benefit that printed structures will not collapse, enabling also the printing of soft human tissue (NASA) (Box 10.10).

Box 10.10 Highlights

• Several cell cultures system can be studied under space conditions • The microgravity environment on the ISS suits perfectly to print 3D tissue cultures

Fig. 10.24 NASA’s 3D BioFabrication Facility BFF. (Image JSC2019E037579, Credits NASA)

10.8.5 Omics Approaches in Space Life Sciences Understanding the effects of the space environment on microorganisms has witnessed recently considerable progress (whereas the main factors are microgravity, radiation, and vacuum). However, explicit knowledge of molecular mechanisms responsible for survival and adaptation in space is still missing. Space environment affects a variety of physiological features of microorganisms. The above features include metabolism, motility and proliferation rate, division of cells, and also virulence and biofilm production (Fig.  10.25) [366]. Molecular-level understanding of the above effects in space-exposed microorganisms is still lacking. It is believed that omics-based approach, together with classical phenotyping and physiological measurements, will be a useful toolbox for understanding mechanisms of microbial survivability in the harsh conditions of outer space. “Omics” stands for genomics, transcriptomics, proteomics, metabolomics, and more. Systems biology is an interdisciplinary approach in biomedical research aiming at understanding the biological system at the organism, tissue, and cell level. Systems biology incorporates the results of –omics techniques, genome-scale metabolic and regulatory biomathematical models to understand molecular interactions, evolution, functional and phenotypical diversity, and molecular adaptation. The omics-based approach integrates various pieces of biological information from genomes, mRNA, and proteins to metabolites [367]. The –omics-based approach has recently opened a window for a deep insight into molecular machinery implicated in the survivability of space-exposed microorganisms by revealing expression, metabolic functioning, and regulation of the genes and proteins encoded by the genomes of “space travelers.” The diverse biological activities of microorgan-

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Fig. 10.25  Molecular response experienced by microorganisms in the outer space environment revealed with the help of global and integrative –omics approaches of systems biology that have been recently used to study microorganisms exposed to real and simulated space conditions. (Reprinted with permission from Milojevic et al. [366])

isms in space are affected by metabolic alterations caused in turn by genetic regulations (Fig. 10.26). It has been demonstrated by means of –omics-based approaches that exposed microbes switch to “energy saving mode.” Research identified some global regulatory molecules that drive molecular response of a few space-exposed microorganisms [366–369]. Various kinds of stress responses (e.g., general, osmotic, and oxidative) experienced by microorganisms in conditions of real and simulated outer space have been deciphered via – omics-assisted analyses [366]. Various genes with altered expression after microbs’ exposure to real and simulated outer space environment (Fig.  10.25) have been identified [366]. State-of-the-art –omics technologies have been successfully used to understand molecular mechanisms responsible for alterations of microbial virulence in space conditions (Fig. 10.27) [366]. Space exposure imposes stresses that affect microbial survival rates and may lead to certain discrepancies in –omicsassisted analysis of returned/exposed microorganisms. The composition of the cultivation medium influences the microbial space response [369], e.g., by providing specific antioxidants presented in rich medium, which may protect microbial cells against ionizing radiation. The majority of space experiments have been performed on satellites, where microorganisms are cultivated in environment protected from all factor but microgravity [366]. Direct exposure to real space environment outside the ISS followed by investigation with – omics techniques was performed on a few microbial species only [367, 370, 371]. Therefore, in order to broaden our

knowledge of molecular mechanisms of microbial survivability in outer space, there is an urgent need for further experiments with direct exposure. Often, a multi-omics postflight analysis has the problem of a limited number of microbiological samples exposed to the space environment. Therefore, the researchers should critically assess the design of outerspace experiments to provide a sufficient number of independent biological samples in order to enable statistically significant results in processing the –omics data. It is also extremely important to avoid artifacts: due to very high sensitivity of the –omics techniques of occasional occurrence of uncontrolled conditions, stress-related artifacts cannot be ruled out. In this context, it is highly desired to develop novel approaches for the efficient extraction of DNA, RNA, proteins, and metabolites simultaneously from the minimal amount of microbial cells [367, 372]. Furthermore, the absence of detailed reports regarding the environmental conditions during space exposure and corresponding ground control experiments is, unfortunately, a frequent reality that requires a critical reassessment of research planning. Providing a full record of controlled parameters (like temperature, humidity, and pressure profiles) during flight, simulated, and control experiments is highly desired to achieve a comprehensive and artifacts-free analysis of the effects of the space environment on the physiology and molecular machinery of microorganisms. It has been proposed that in future space experiments, detailed metabolomic analysis of exposed microorganisms should be performed in addition to the proteotranscriptomic profiling. This novel approach has provided already plenty of

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Fig. 10.26  Stress responses experienced by microorganisms in outer space real and simulated conditions, revealed with –omics-assisted investigations. Proteins and genes of stress responses with altered abunFig. 10.27  Molecular alterations underlying microbial pathogenicity, virulence, and biofilm formation in the outer space environment, resolved with –omics-assisted investigations [366]

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dance and expression after exposure of microorganisms to the outer space real and simulated environment [366]

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new findings on fine molecular networks regulating the space response [367]. Recent works (e.g., the NASA twins’ study) used a multi-omics, systems biology analytical approach to analyze biomedical profiles of astronauts [120]. Results of performed targeted and untargeted metabolomics combined with proteomics effectively revealed the biomedical responses of a human body during a year-long spaceflight indicating mitochondrial stress as a consistent phenotype of spaceflight [120, 373]. Finally, the combination of molecular data with a genome-scale metabolic reconstruction of the respective species should be implemented, delivering the space-induced microbiome signatures [366].

10.9 Space Radiation Resistance 10.9.1 Health Risk Reduction from Space Radiation Exposure in Humans Humans have all evolved in an environment containing a persistent low level of constant exposure to different endogenous and exogenous mutagenic agents, and consequently have developed many cellular mechanisms for either DNA protection or repair (see Chap. 2). However, when humans travel into space, these naturally evolved cellular mechanisms might not be enough as many major health threats from space radiation has been identified, e.g., central nervous system injury, cardiovascular diseases, immune dysfunction, cancer development, and premature aging. To reduce the risk of humans in space, there are some possible interventions which can limit the effects of space radiation. A dedicated review can be found elsewhere [374]. One way of reducing the health risk from space radiation exposure in humans is selecting more radioresistant humans during the selection campaigns of space agencies. The most used way is to perform in vitro adaptive response studies, in which cells collected from the candidates are used to measure their response to a fixed dose of ionizing radiation. While the results of these studies are not necessarily used during candidate selection, they hold great value in selecting the right people that will be more protected against space radiation. Another strategy would be to pharmacologically hamper the processes underlying the molecular (side) effects of space radiation exposure. Examples are the application of radioprotectors and geroprotectors, as well as supplementation with antioxidants or antioxidative capacity increasing compounds (see Chap. 11). While these pharmaceuticals hold great promise, many of them are still under investigation and not allowed to be used on humans. An alternative method to elevate humans’ natural radiation protection capacity is inducing a hibernating or hyposta-

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sis state. It is well-known that natural hibernators become more radioresistant during their inactive state. The reason for this has not yet been fully elucidated. It is probably due to several factors related to slower cell metabolism and increased tissue hypoxia. In recent years, a technique has been developed that allows hibernation to be reproduced even in those animals that would not usually be able to hibernate, such as rats. This technique is nowadays known as synthetic hibernation or synthetic torpor [375]. Although this research has a big potential to limit radiation-associated risks in space, it is quite far from practical use yet. Another futuristic method is the use of deuterium, the stable isotope of hydrogen. As carbon-deuterium bonds need more energy to break than normal carbon-hydrogen bonds, the necessary energy to break the hydrogen bonds between DNA bases would be higher, making deuterated DNA less sensitive than normal DNA to DNA damage following ionizing radiation exposure. However, a lot of issues have to be solved before deuterium could be applied in humans: lack of evolutionary adaption to catabolize organic compounds containing deuterium, consequent slower rate of vital metabolic reactions, and their potential toxic effects. Nevertheless, it has been shown that deuterated food or water intake helps to increase life or health spans from numerous model organisms. Gene therapy stands for the use of genetic modifying techniques in order to achieve a therapeutic effect. In the context of radiation and radiation protection, this has been studied for several radioresistance mechanisms making these techniques interesting for deep space missions, where radiation protection concerns arise [374] One of the strategies for gene therapy in radioresistance is the overexpression of endogenous antioxidants, for example, magnesium superoxide dismutase (MnSOD) that acts as a scavenger for reactive oxygen species produced after the interaction of radiation with the cell [376]. Another angle in which gene therapy can be useful for improving radioresistance is by enhancing the DNA damage repair such as the overexpression of certain repair proteins that are normally active in repairing the damage in the DNA strands after radiation exposure [377]. A promising approach takes its inspiration from extremophiles and their impressive radioresistance capabilities, in concrete, the tardigrades, a microscopic animal that is capable of surviving in extreme conditions. A protein identified in these organisms, termed damaged suppressor (Dsup), has been made to be expressed in human cell lines, reducing the number of DNA strand breaks and preserving cellular proliferative abilities after high doses of radiation [337].

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10.9.2 Mechanisms in Extremophiles 10.9.2.1 What is an Extremophile? Extreme conditions in a natural environment are only extreme from a human point of view. Extremophiles can only live under these conditions and depend on them. Often organisms living under these conditions are called “extremophiles” or “polyextremophiles” since most of them are coping with different extremes in their natural environment [378]. 10.9.2.2 Which Adaptations/Mechanisms Are Known? Plenty of different (poly) extremophiles in natural and human-made extreme environments exist in natural and human-made harsh environments. Examples include anaerobes, (hyper-) thermophiles, psychrophiles, halophiles, acidophiles, xerophiles, and piezophiles. For all the named organismic groups, cellular adaptation mechanisms are known that protect the cells themselves or enable them to live under extreme conditions in their natural habitat. In addition to the intracellular protection mechanisms, general protection mechanisms like spore formation are wellknown. For example, the spore of the Bacterium Bacillus subtilis is characterized by a thick layer of peptidoglycan, a low water content inside the cell, a DNA conformation changed from B to A, and the presence of α/β-type small acid-soluble spore proteins which accumulate within the spore. In general, spores are more tolerant to inactivating physical stresses, like radiation as vegetative cells [379, 380]. Spore formation is known to be an answer to changing conditions in the environment that is used by microorganisms and fungi; special forms like the anhydrobiotic state are also observed in other eukaryotic cells like tardigrades, nematodes, and rotifers [381]. Spore formation and the anhydrobiotic state, as well as intracellular adaptation mechanisms, are relevant for possible survival after exposure to ionizing (space) radiation [323, 382]. In addition to spore formation, biofilm growth by the production of extracellular polymeric substances (EPS) also leads to a higher ionizing radiation tolerance [383]. Besides the named cellular adaptation mechanisms, there are also different intracellular adaptation mechanisms possible to cope with extreme environmental stresses. As described before, ionizing radiation exposure does not only lead to direct effects and intracellular damage, such as DNA strand breaks, it also leads to indirect effects, like ROS production. Hyperthermophilic Archaea, like Pyrococcus furiosus, are partly tolerant to the indirect effects of ionizing radiation, due to mechanisms protecting the DNA from the influence of ROS [384]. In these Archaea, DNA binding proteins play a major role as they bind and protect the DNA thereby limiting the accessibility of the DNA to ROS.  In addition, increased expression of different enzymes like superoxide dismutase and the glutathione peroxidase can also reduce the level of intracellular ROS.

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For the Bacterium Deinococcus radiodurans as well as for the Archaeon Halobacterium salinarum special intracellular Mn/Fe ratios are described: they demonstrate an intracellular accumulation of high amounts of manganese along with low iron levels, which contribute to their high radiation tolerance [385, 386]. This special Mn/Fe ratio was not found in radiation-tolerant anaerobic microorganisms. It is proposed that the low levels of IR-generated ROS under anaerobic conditions combined with highly constitutively expressed detoxification systems in these anaerobes are key to their radiation resistance and circumvent the need for the accumulation of Mn-antioxidant complexes in the cell [387]. Furthermore, polyploidy or the presence of several DNA copies within one single cell has been discussed to contribute to tolerance to desiccation and therefore also to ionizing radiation [388]. Halophilic organisms have different strategies to cope with a high salt concentration in their natural habitat. One option is the intracellular accumulation of salt or other compatible solutes [389]. It is also known that compatible solutes can contribute to the tolerance to ionizing radiation in halophilic microorganisms [389]. Additionally, protective mechanisms such as membrane pigments, including carotenoids, melanin, scytonemin, and bacterioruberin were found to be important in ionizing radiation protection in different organisms through the scavenging of hydroxyl radicals [390, 391].

10.9.2.3 How Relevant Are These Adaptations/ Mechanisms for Space Radiation? In general, there is no direct adaptation of microorganisms to space conditions or space radiation known as all organisms evolved on Earth. Nevertheless, there have been and still are space experiments ongoing where the adaptability of different organisms is investigated during exposure to space conditions. In this context, we speak about the side effects of other tolerances or resistances which enable the organisms to endure space stressors. In general, organisms which are tolerant to desiccation developed mechanisms to repair the DNA which is damaged during the desiccation process. The same repair mechanisms can also be used to repair DNA damage caused by other stressors, such as ionizing radiation. One prominent example is the desiccation and radiation tolerance of the microorganism Deinococcus radiodurans. This organism uses the same cellular adaptation and repair strategies after exposure to drought and ionizing radiation exposure. However, not all desiccation-tolerant organisms are tolerant to (ionizing) radiation exposure [392]. The same is true for other repair machineries, where no direct correlation between hyper-/thermophilic organisms or the ability to produce compatible solutes and radiation tolerance could be identified [393, 394]. In addition, some microorganisms (e.g., Ignicoccus hospitalis) demonstrate a high survival rate after ionizing radiation exposure but possess a repair mechanism which is not known up to now [395].

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10.10 Irradiation Experiments at GroundBased Facilities for Simulation of the Space Environment 10.10.1 Low Dose Rate Irradiation Facilities As mentioned at the beginning of this chapter, protons account for nearly 87% of the total flux of the galactic cosmic radiation (GCR), helium ions—for approximately 12%, and the remaining heavy ions, or high-Z elements (HZE),— for less than 1%. However, the relative distribution of the effective dose is quite different. Multiplying the abundance by Z2 provides an estimate of the contribution to the dose. One should further consider the quality factor of the biological effectiveness of the corresponding radiation. As a result, HZE particles contribute approximately 89% of the total dose equivalent (mSv) in free space. Among the HZE particles in GCR, iron is the largest contributor (26%) to the effective dose [396].

10.10.2 Low Dose Rate Particle Irradiation Facilities Low dose rate irradiation is usually provided in a laboratory either by X-ray machines or radioactive sources, and neither mimics GCR well. The X-rays are low-energy radiation and do not mimic the penetrating capability of GCR.  Usual radioactive sources emit α-, β-, and γ-radiation. While α-particles are helium nuclei abundant in GCR, the energy of Fig. 10.28  Schematic view of the SNAKE (Superconducting nanoprobe for (kern) particle physics experiments) setup, including linear particle accelerator (orange), focusing unit (superconducting magnetic lens) and detection system with the particle detector and ultrafast high-voltage switch

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typical α-particles is 4–9  MeV as compared with above 1000 MeV in GCR. As a result, α-particles cannot penetrate the thinnest screen (even the skin) and cannot be used for GCR simulation. γ-Radiation has a much stronger penetration capability, and γ-emitters are used [397]. However, the biological effects of γ-radiation and ions are different. As for the β-radiation, in the sense of GCR simulation it combines the drawbacks of α and γ: having low penetrating capability, its biological effects are similar to γ and far from high-energy ions. A partial solution has been found by using a unique artificial isotope Californium-252, which exhibits exceptionally high neutron emission. 252Cf is used, e.g., at the ESA test station and the new facility in Japan [398].

10.10.3 Low Energy Particle Irradiation Facilities Although low-energy charged particles (up to about 20 MeV) do not reproduce the characteristics found in the GCR spectrum, low-energy facilities are widely available and are useful to help in the screening and the design of experiments that will be further carried out on higher energy accelerators (see next subsection). Several accelerator types can be used to produce such low-energy beams, but electrostatic tandem accelerators are probably the most widely used. A schematic representation of such accelerator is given in Fig. 10.28. The first part consists of an ion source, which can produce any negative ion

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(with one extra electron) from hydrogen to uranium. The produced negative ion beam is then extracted from the source and guided to the main tube. The acceleration is carried out in two stages hence the name “tandem accelerator.” First, the negative ions are attracted to the positive high-voltage “terminal” located in the center of the tube. Then, negative ions can be stripped of part of their electrons (usually 2–3) in the stripper channel, turning to positive ions. These positive ions are repelled by the positive terminal voltage to the end of the tube, which is at ground potential. High-energy ions are focused by (usually superconducting) magnets and deflected into one of the beamlines, according to the particle energy, mass, and charge. Regarding the beam size, two configurations are used: microbeam and broad beam. The initial accelerated ion beam is always a microbeam with a diameter often below 5 μm. Microbeams are a useful tool in studying the bystander effect (described in detail in Chap. 2). Indeed, such a beam permits to irradiate selectively one or more cells inside a population. This offers the possibility to either target the cell nucleus, the conventional target in radiobiology, the cytoplasm, or organelles. It also provides the advantage of knowing precisely the dose delivered to the cells and the number of particle shoots being determined in advance. In the context of space radiation, where the flux of Fig. 10.29  Aerial view and general layout of the NASA Space Radiation Laboratory (NSRL) facility in Upton, NY, USA. EBIS electron beam ion source. (Satellite view courtesy Google Earth)

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high-mass particles is very low and the occurrence of a single shoot-in through a cell is very high, the bystander effect is a topic of crucial importance. Indeed, it is observed through a variety of endpoints: reduction in cell survival, double strand break induction, micronuclei, mutations, and expression of apoptosis, inflammation, and cell cycle-related genes. Broad beams can be produced either by using scattering foils, by scanning microbeam, or by defocusing them. Beam homogeneity is controlled by plastic scintillators or siliconbased detectors.

10.10.4 High-Energy Particle Irradiation Facilities The importance of accelerator-based studies was acknowledged by NASA decades ago. After preliminary research at the existing accelerators, it was decided to build a dedicated beamline. In 2003, the NASA Space Radiation Laboratory (NSRL) was commissioned at the Brookhaven National Laboratory (BNL). The NSRL layout is presented in Fig. 10.29. The facility is capable of supplying particles from protons (p) to gold (Au). Available beam energies range from 50 to 2500 MeV for protons and 50 to 1500 MeV per nucleon for ions between helium (He-2) and iron (Fe-56; Z  =  26).

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Heavier ions with atomic numbers up to Z = 79 (Au) are limited to approximately 350–500 MeV per nucleon (https:// www.bnl.gov/nsrl/). The choice of Fe-56 ions is justified by a sharp decline in abundance for ions heavier than iron [396] while the chosen energy is around the peak of the galactic cosmic radiation spectrum. Moreover, the linear energy transfer is about 140 keV/μm, around the peak of effectiveness for late radiation effects [399]. The three key areas developed together to ultimately provide the GCR simulator at NSRL are illustrated in Fig. 10.30. Several important results have been obtained at NSRL. We can mention, for example, the observation that, despite being high-LET particles, heavy ions are not more effective than γ-radiation in the induction of leukemia in mice [400]. Another example is the discovery of specific types of brain damage caused by heavy ions [401], types that had not been known from X-ray studies. The basic idea of a high-energy accelerator is illustrated schematically in Fig. 10.31. Each accelerating section itself consists of a sequence of resonant cavities in which the RF (radio frequency) electromagnetic field is oscillating. Ions Fig. 10.30  Three key areas developed to provide the GCR simulator at NSRL. (Source: Simonsen et al. [396], reproduced with permission)

Fig. 10.31  General layout of a linear high-energy particle accelerator. RF radio frequency

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traverse RF cavities subsequently; the timing of the passage of each cavity is synchronized with the direction and phase of the electric field—therefore, each ion is accelerated from cavity to cavity. In case of a linear accelerator (LINAC), the accelerating sections are positioned adjacently along a straight line. In case of a synchrotron (like EBIS in Fig. 10.31), the accelerating sections are positioned along a circumference, while the charged particle beam is bent between the sections by a magnetic field. The resonant frequency is usually either about 1  GHz (L-band of the RF spectrum) or about 3 GHz (S-band). The electromagnetic power for feeding the RF cavities is generated usually by a high-power klystron. The accelerating cavities can be made either of normalconducting metal (“warm” cavities usually made of copper) or of superconductor (usually, niobium). In the last case, cryogenic cooling to liquid-helium temperature is needed. Accelerating gradients are usually in the range of 10–30 MeV/m, e.g., the 3-km long SLAC accelerator commissioned back in the 1960s, accelerating electrons to the energy of 50 GeV, has an average accelerating gradient of about 17 MeV/m (with 3-GHz copper cavities).

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Output of RF-driven particle accelerator necessarily consists of single bunches. Such bunches are called micropulse, and their duration is just a fraction of the oscillating field period. For example, for the S-band with period of about 300 ps = (3 GHz)–1, the typical micropulse duration is below 20 ps. The train of micropulse is called “macropulse.” While there is no theoretical limit for macropulse length, practically it is limited by the driving klystron pulse length: For S-band normal-conducting linacs the typical value is 5–20 μs, for L-band superconducting linacs—much longer (1 ms and more). Particle beams are collimated and bent “magnetic lenses”—magnetic fields created by electromagnets or permanent magnets. These magnetic devices, governing the charged particle beam propagation, are called electron-optic (or ion-optic) devices. Such devices have some similarities to classical light optics in terms of mathematics, but in general comprise a separate field of knowledge. The reader interested in learning the field of particle beam optics is referred to the classical textbook of Reiser [402].

10.10.5 Space Environment Simulation Platforms Although ionizing radiations were identified as the main showstopper to exploration mission, there are additional stresses in the space environment. While most of the factors below are not relevant to astronauts, they are important in studying extremophiles. 1. Low pressure: The pressure varies from 10–1 Pa near Earth atmosphere to 10–14 Pa in deep space. Due to the degassing, pressure around the ISS is higher than in deep space ranging from 10−7  Pa in the Ram direction (e.g., front of the ISS relative to flight direction) to 10−4 Pa in the Wake direction (e.g., rear of the ISS relative to flight direction) [403]. 2. Cold temperature: The low-pressure environment previously described drastically increases the molecular mean free path in space resulting in low heat transfer. Consequently, the temperature in deep space ranges from 3 to 4 K (–270 to –260°C) [404]. 3. Solar radiation: Highly energetic phenomena are occurring in the Sun leading to the emission of high-intensity electromagnetic radiations. By moving away from the Sun, the emitted solar radiations are spread out over a large surface area reducing the solar irradiance with increased Sun-object distance. ISS, located at one astronomical unit distance from the Sun, is exposed to an approximate 1400 W/m2 heat flux. The associated electromagnetic spectrum extends from X-rays to radio waves with a higher proportion in the visible (47%), infrared (45%), and ultraviolet (7%) ranges [405].

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4. Day and night cycles: As ISS orbits around the Earth and the latter around the Sun, the stress exposure has a cyclic temporal behavior with two main periods. The “day and night” cycle caused by the rotation around the Earth has a period of 91  min resulting in fluctuation in total irradiance and temperature of more than 1000 W/m2 and 5–10 °C. The second cycle (period of approximately 1 month) is due to the position change of ISS orbital plane relative to the Sun. In this case, greater variations of temperature (up to 60 °C) with a maximal temperature of about 50 °C were reported [406]. To study the biological impact of the space environment, modules outside the ISS are an ideal environment to expose biological samples to LEO, where the conditions strongly differ from the ones encountered on Earth. However, the poor availability of these facilities stimulated the creation of exposure chambers on Earth capable of reproducing this LEO environment. The Laboratory for Analysis by Nuclear Reactions (LARN, University of Namur, Belgium, https:// www.unamur.be/en/sci/physics/ur-en/larn-en) has developed an exposure module to simulate the aforementioned conditions on the ground for extended periods of time (several months). To this end, biological samples are placed into a vacuum chamber and exposed to various constraints. A cooling system located underneath the sample tray and an electromagnetic source reproducing the solar spectrum are controlled by a monitoring system capable to simulate the slow and fast cycles described above. In addition, a variety of neutral density filters and cut-off waveband filters enables to create multiple irradiance conditions within the same experiment, in order to investigate what part of the UV-visible spectrum is the most deleterious. A similar facility also exists at DLR Cologne and has been recurrently used for pre-flight test programs and mission ground reference experiments for several astrobiological long-term space missions [405].

10.11 Exercises and Self-Assessment Q1. Which types of radiation exist in space? Q2. Are astronauts fully protected from radiation by spacecraft walls? Q3. Can you describe the difference between the radiation environment on Earth (on ground) and the one at the surface of Mars? Q4. What do we know now about specific health problems of astronauts? Q5. What is the role of plants in Bioregenerative Life Support Systems (BLSS)? Q6. What is the 3R Principle?

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Q7. What kind of chronic effects on the CNS (central nervous system) were observed? Q8. What is the difference between organoids and spheroids? Q9. From which cells organoids can be cultured? Q10. What is the main reason why bioprinting and other 3D cultures can be better cultured in Space? Q11. What parameters should be considered if we would like to simulate conditions on Moon/Mars surface or in deep space (including stress unrelated to radiation)? Q12. What is the interest to study the biological effects of low energy charged particles in the context of space radiation exposure?

10.12 Exercise Answers SQ1. Galactic Cosmic Rays, Solar Energetic Particles, Trapped radiation, and the solar wind SQ2. No, the high penetrating character of GCRs and the cascades of secondary particles generated by the passage of GRCs ions through the spacecraft walls create an intravehicular field which is of high concern for the health risk of astronauts SQ3. On Earth we are protected by both the atmosphere and the magnetic field: GCRs hitting the top of the atmosphere create particle showers but only a few of such secondaries (and very few of direct GCR ions) reach the ground. SEP are mostly shielded by the atmosphere and are of concern only for extreme events and mostly for high latitude/high-altitude flights for eventual biological risks, or on ground for infrastructures. On Mars, the very thin atmosphere offers very little shielding, and the exposure to GRCs, their secondary particles, and SEP is a concern. SQ4. Though there are many concerns, the present evidence does not provide a conclusive answer. Astronauts as a cohort are not less healthy than US Air Force pilots, e.g., and much healthier than the general public (due to selection). The LNT-estimated cancer death risk for prolonged missions is considerable, but applicability of LNT for low dose rates is questionable. SQ5. Plants in LSS remove carbon dioxide and provide oxygen, help water purification, can recycle wastes of the astronauts, and provide fresh food for the crew. SQ6. Reduction (first R) of animal numbers, Refining (second) the test methods to lower the harm to the animal to a minimum, and Replace (third) animal experiments with alternative methods, when possible

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SQ7. Reduction of dendrites, reduced neurogenesis and increased neuroinflammation, diminished hippocampal neuron excitability SQ8. Spheroids are also a 3D cell cultures, but in comparison to organoids, they form simple clusters into sphere-like formation, but they cannot self-assemble or regenerate. SQ9. Pluripotent stem cells SQ10. Microgravity SQ11. The particular profile of radiation spectrum can vary according to the location of interest. The ISS environment benefits from some protection granted by the Earth magnetic field. This is not the case for deep space or Mars, where the full spectrum of galactic cosmic rays should be considered. Solar proton events should be included, can vary in magnitude and their extent also depends on the distance to the sun. Beside radiation, day and night cycle can impact temperature conditions, which lead to biological effects on some model organisms. Pressure value and the presence or not of atmosphere should also be considered. SQ12. Although the vast majority of particles in the GCR spectrum have very high energy, low energy charged particles are still produced in shielding materials and arise from fragmentation of heavier energetic nuclei. These low-energy particles often traverse shielding and remain of concern.

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10  Space Radiobiology tion limits of life: Ignicoccus hospitalis between replication and VBNC.  Arch Microbiol. 2021;203(4):1299–308. https://doi. org/10.1007/s00203-020-02125-1. 396. Simonsen LC, Slaba TC, Guida P, Rusek A. NASA’s first groundbased Galactic Cosmic Ray Simulator: enabling a new era in space radiobiology research. PLOS Biol. 2020;18(5):e3000669. https://doi.org/10.1371/journal.pbio.3000669. 397. ESA.  Materials & Electrical Components Laboratory. ESA. 2021. Retrieved Dec 2021 from https://www.esa. int/Enabling_Support/Space_Engineering_Technology/ Materials_Electrical_Components_Laboratory. 398. Takahashi A, Yamanouchi S, Takeuchi K, Takahashi S, Tashiro M, Hidema J, Higashitani A, Adachi T, Zhang S, Guirguis FNL, Yoshida Y, Nagamatsu A, Hada M, Takeuchi K, Takahashi T, Sekitomi Y.  Combined environment simulator for low-dose-rate radiation and partial gravity of moon and Mars. Life (Basel). 2020;10(11):274. https://doi.org/10.3390/life10110274. 399. Durante M, Golubev A, Park W-Y, Trautmann C.  Applied nuclear physics at the new high-energy particle accelerator facilities. Phys Rep. 2019;800:1–37. https://doi.org/10.1016/j. physrep.2019.01.004. 400. Michael W, Joel SB, Helle B-O, Ray FA, Paula CG, Eugene JE, Christina MF, Fitsum H, Christine LRB, Brad C, Matthew AC, Robert LU. Incidence of acute myeloid leukemia and hepatocellular carcinoma in mice irradiated with 1 GeV/nucleon 56Fe ions. Radiat Res. 2009;172(2):213–9. https://doi. org/10.1667/RR1648.1. 401. Parihar VK, Allen BD, Caressi C, Kwok S, Chu E, Tran KK, Chmielewski NN, Giedzinski E, Acharya MM, Britten RA, Baulch JE, Limoli CL.  Cosmic radiation exposure and persistent cognitive dysfunction. Sci Rep. 2016;6(1):34774. https://doi. org/10.1038/srep34774. 402. Reiser M.  Theory and design of charged particle beams. John Wiley & Sons; 2008. 403. Rabbow E, Rettberg P, Barczyk S, Bohmeier M, Parpart A, Panitz C, Horneck G, von Heise-Rotenburg R, Hoppenbrouwers T, Willnecker R, Baglioni P, Demets R, Dettmann J, Reitz G. EXPOSE-E: an ESA astrobiology mission 1.5 years in space. Astrobiology. 2012;12(5):374–86. https://doi.org/10.1089/ ast.2011.0760. 404. Haefer RA. Vacuum and cryotechniques in space research. Vacuum. 1972;22(8):303. https://doi.org/10.1016/0042-207x(72)93789-X. 405. Rabbow E, Parpart A, Reitz G.  The planetary and space simulation facilities at DLR Cologne. Micrograv Sci Technol. 2016;28(3):215–29. https://doi.org/10.1007/s12217-015-9448-7.

569 406. Rabbow E, Rettberg P, Barczyk S, Bohmeier M, Parpart A, Panitz C, Horneck G, Burfeindt J, Molter F, Jaramillo E, Pereira C, Weiss P, Willnecker R, Demets R, Dettmann J, Reitz G.  The astrobiological mission EXPOSE-R on board of the International Space Station. Int J Astrobiol. 2015;14(1):3–16. https://doi.org/10.1017/ S1473550414000202.

Further Reading Airbus Space Systems. University of Zurich and Airbus grow miniature human tissue on the International Space Station ISS. In: Airbus Newsroom. Aug 2021. https://www.airbus.com/en/newsroom. Cekanaviciute E, et al. Central nervous system responses to simulated galactic cosmic rays. Int J Mol Sci. 2018;19(11):3669. European Space Agency. 3D bioprinting for space. In: ESA media. Nov 2018. https://www.esa.int/ESA_Multimedia. European Space Agency. Upside-down 3D-printed skin and bone, for humans to Mars. In: ESA enabling and support. Jul 2019. https:// www.esa.int/Enabling_Support. Furukawa S, et  al. Space radiation biology for “Living in Space”. Biomed Res Int. 2020;2020:4703286. Gray T. A brief history of animals in space. In: NASA history archives. Aug 2004. https://history.nasa.gov/animals.html. Hellweg CE, Berger T, Matthiä D, Baumstark-Khan C.  Radiation in space: relevance and risk for human missions. Springer International Publishing; 2020. Horneck G, et  al. Space microbiology. Microbiol Mol Biol Rev. 2010;74(1):121–56. Limoli C. Space brain: the adverse impact of deep space radiation exposure on the brain. In: Space physiology: to Mars and beyond, vol. 117. The Physiology Society; 2020. Nelson GA.  Space radiation: central nervous system risks. In: Young LR, Sutton JP, editors. Handbook of bioastronautics. Cham: Springer; 2021. Senatore G, Mastroleo F, Leys N, Mauriello G. Effect of microgravity & space radiation on microbes. Fut Microbiol. 2018;13:831–47. Sgobba T, Kanki B, Clervoy J-F, Sandal GM. Space safety and human performance. Butterworth-Heinemann; 2018. Sims J. Why astronauts are printing organs in space. In: BBC Future. Jun 2021. https://www.bbc.com/future. The Royal Museums Greenwich. What was the first animal sent into space? In: Royal Museums Greenwich Stories. 2022. https://www. rmg.co.uk/stories

Open Access  This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons. org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

Radioprotectors, Radiomitigators, and Radiosensitizers

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Alegría Montoro, Elena Obrador, Dhruti Mistry, Giusi I. Forte, Valentina Bravatà, Luigi Minafra, Marco Calvaruso, Francesco P. Cammarata, Martin Falk, Giuseppe Schettino, Vidhula Ahire, Noami Daems, Tom Boterberg, Nicholas Dainiak, Pankaj Chaudhary, Sarah Baatout, and Kaushala Prasad Mishra

Learning Objectives • To understand how radioprotectors, radiomitigators, and radiosensitizers work in increasing the effect of radiotherapy (RT) through enhanced apoptosis of cancer cells while simultaneously reducing or diminishing the effect on normal cells. • To review the characteristics of an ideal radioprotector and to understand mechanisms by which natural or synthetic compounds can prevent or avoid the damage associated with low or high doses of ionizing radiation (IR). • To learn how radiomitigators can reduce the damage caused by IR and contribute to the repair/regeneration of damaged tissues even when they are administered after exposure.

A. Montoro (*) Radiological Protection Service, University and Polytechnic La Fe Hospital of Valencia, Valencia, Spain e-mail: [email protected] E. Obrador Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain e-mail: [email protected] D. Mistry · N. Daems · S. Baatout Institute of Nuclear Medical Applications, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium e-mail: [email protected] G. I. Forte · V. Bravatà · L. Minafra · M. Calvaruso · F. P. Cammarata Institute of Bioimaging and Molecular Physiology, National Research Council (IBFM-CNR), Cefalu, Italy National Institute for Nuclear Physics, Laboratori Nazionali del Sud, INFN-LNS, Catania, Italy e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]

• To understand the mechanisms underlying cancer cell radioresistance and how radiosensitizers (natural or synthetic) are able to sensitize cancer cells. • To learn about the radiosensitization phenomenon and the associated molecular mechanisms. The combined action of these molecules with radiation offers a new strategy for enhanced IR cytotoxicity in cancer cells together with reducing normal tissue toxicity.

M. Falk Department of Cell Biology and Radiobiology, Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic e-mail: [email protected] G. Schettino National Physical Laboratory (NPL), Teddington, United Kingdom e-mail: [email protected] V. Ahire Chengdu Anticancer Bioscience, Ltd., J. Michael Bishop Institute of Cancer Research, Chengdu, China T. Boterberg Department of Radiation Oncology, Ghent University Hospital, Ghent, Belgium Particle Therapy Interuniversity Center Leuven, Department of Radiation Oncology, University Hospitals Leuven, Leuven, Belgium e-mail: [email protected] N. Dainiak Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, United States of America P. Chaudhary The Patrick G. Johnston Centre for Cancer Research, Queen’s University Belfast, Belfast, Northern Ireland, United Kingdom e-mail: [email protected] K. P. Mishra Radiobiology Unit, Bhabha Atomic Research Center, Mumbai, Maharashtra, India

© The Author(s) 2023 S. Baatout (ed.), Radiobiology Textbook, https://doi.org/10.1007/978-3-031-18810-7_11

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By the end of this chapter, readers are expected to understand the importance of applying current knowledge in the development of new synthetic or natural radioprotectors and radiosensitizers and develop an understanding of their cellular and molecular mechanisms of action.

11.1 Introduction Radiation protection aims to reduce unnecessary radiation exposure with the intention to minimize the harmful effects of radiation on human health. With increasing use of radiation technologies and radioisotopes in medicine and industry, the risk of radiological and nuclear accidents escalates, affecting human health. Nuclear power plants and industrial accidents pose a serious threat to public health. Emergency preparedness in an event of nuclear terrorism and nuclear warfare requires the use of existing radiomodifiers and public health measures such as sheltering in place and the use of personal protective equipment (PPE). New approaches are urgently needed for protecting the persons working in a radiation field, first responders, and general population in the form of safe, effective, and easily accessible radioprotective agents. Cellular exposure to IR induces genomic instability or mutations predisposing to carcinogenesis and/or cell death. Upon exposure, radiation induces DNA damage, lipid peroxidation, oxidation of thiol groups located in the plasma membrane and membranes of the cellular organelles, DNA strand breaks, and base alterations in cells, tissues, and organs. These changes may trigger a series of cellular responses, including activation of DNA damage repair path-

Fig. 11.1  Classification of radiomodifiers with their biological properties

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ways, signal transduction responses, gene transcription, and immune and proinflammatory responses. Triggering these pathways helps to recover damaged cells or eliminate the dysfunctional cells. However, they may also result in the development of tissue toxicities. The radiation research program of the National Cancer Institute (NCI) has proposed the following pharmacological classification of agents with IR response modification properties according to the timing of administration (Fig. 11.1): A radioprotective agent/drug prevents harmful effects of radiation exposure while a radiosensitizing agent makes tumor cells more susceptible to radiation, in order to maximize the effect of radiotherapy while having less effect on normal tissues. Radiomitigators can attenuate IR damages even when they are delivered at the same time or after radiation exposition.  The use of radiation-effect modulators (radioprotectors, radiomitigators, and/or radiosensitizers) can mitigate side effects and increase the efficacy of RT in cancer patients (Fig. 11.2).

11.1.1 Radioprotectors The extent of radiation damage to living cells and organisms depends on the type of radiation (alpha (α) particles, beta (β) particles, positrons, X-rays, gamma rays (γ-rays), UV, etc.). Attempts to protect against the damaging effects of radiation were made as early as 1949. Efforts are actively being continued to search for radioprotectors suitable to be used in specific scenarios of radiation exposure. Possible applications of radioprotectors are outlined in Fig. 11.3.

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Fig. 11.2  The use of radioprotectors, radiomitigators, and radiosensitizers before, during, or after irradiation

Fig. 11.3  Various applications of radioprotectors

Over the last few decades, many natural and synthetic compounds have been investigated for their potential as radioprotectors. Natural or synthetic radioprotectors are able to (i) reduce direct or indirect radiation damage, (ii) repair direct and indirect damage once they have occurred, and (iii) facilitate the repair of damaged cells or recover depleted cell populations [1].

It should be stressed that the majority of the compounds discussed below are currently not used in routine clinical practice and are still under preclinical or clinical evaluation. Early development of synthetic radioprotectors focused on thiol compounds (e.g., amifostine) and their derivatives, which have been used in cancer patients, to prevent complications of RT. In addition, they have been thought to be use-

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ful in accidental radiation exposure scenarios [2]. However, the practical applicability of the majority of these synthetic compounds remained limited owing to their limited administration routes, narrow administration window for efficacy, high toxicity at high doses or at recurrent usage, and cost factors as well. Besides thiol compounds, various compounds with different chemical structures are being investigated to develop an ideal radioprotector; there is still an urgent need to identify and develop novel, nontoxic, effective, and biocompatible compounds which can adequately protect normal tissues with no sparing of the tumor cells. An interest has been emerging in developing potential new candidate drugs from natural plants and phytochemicals. Plant products could bridge the gaps in the search for an ideal radioprotector due to its abundance, typically low toxicity, and relatively low cost. Characteristics of an Ideal Radioprotector An ideal radioprotective agent should (a) be efficient in providing multifaceted protection, (b) prevent direct and indirect acute or chronic effects on normal tissue, (c) be easily and comfortably administered without toxicity, (d) cause no or minimal adverse effects on the test organism, (e) have a sufficiently long time window of effectiveness after administration and also have a sufficiently long shelf life, (f) have an acceptable stability profile (both of bulk active product and formulated compound), (g) be compatible with a wide range of other drugs, (h) not protect tumors from IR, and (i) be easily accessible and economical and should not require special handling and transportation temperatures (Box 11.1).

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Box 11.1: Radioprotectors

• Radioprotectors (synthetic compounds, natural plant extracts, and phytochemical derivatives) are designed to lessen the effects of radiation-induced damage in healthy tissues. • Radioprotective drugs are effective when administered prior to or during radiation exposure to reduce the radiation-induced injuries/toxicities. • Safe, novel, nontoxic, and easily accessible radioprotective agents are needed to be developed for human health.

Underlying Mechanisms of Radioprotectors Radioprotectors are diverse and elicit their action by various mechanisms (Fig. 11.4) such as: • Scavenging free radicals (either by suppressing the formation or by detoxifying radiation-induced free radical species). • Inducing hypoxia in cells in order to avoid synthesis of reactive oxygen species (ROS). • Increasing levels of antioxidant defenses such as GSH (reduced glutathione) and/or antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx), thioreductase, catalase (CAT), etc.). • Triggering one or more cellular DNA damage repair pathways. • Impeding cell division or inhibiting apoptotic cell death.

Fig. 11.4  Potential mechanism of action of radioprotectors against cell damage due to IR

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• • • • •

Modulating redox-sensitive genes. Modulating growth factors and cytokine production. Controlling inflammatory response. Chelating or decorporating radionuclides. Promoting tissue regeneration (intestinal or hematopoietic and immunostimulant compounds), gene therapy, and/or stem cell therapy. In most cases, these molecules are administered after exposure to radiation, which is why they should be also considered radiomitigators.

The most common mechanisms of radioprotection are the scavenging of free radicals, repair of DNA damages, inhibition of apoptosis  or inflammation, increase antioxidant defenses, and modulation of growth factors, cytokines, and redox genes. Thus, the management of radiation exposure may require a holistic multimechanistic approach to achieve optimal radiation protection during RT of cancer patients and in cases of nuclear accidents or emergencies [3] (Box 11.2). Box 11.2: Possible Mechanisms of Radioprotectors

• Radioprotectors can be screened for their effective emerging strategies, such as modulation of growth factors, cytokines, redox genes, and tissue renewal. • The radioprotective agents are often antioxidants, which may suppress or scavenge the radiation-­ induced free radicals from the cell. • These compounds are cofactors or can induce/stimulate antioxidants  enzymes (like SOD, GPx, and) activity, which would likely lead to both prevent DNA damage and decrease in lipid peroxidation. • They may have the ability to enhance DNA repair, reduce the postradiation inflammatory response, or even delay cellular division allowing more time for cells to repair the DNA damage or undergo cell death.

Therapeutic Principles to Develop Radioprotectors (Portrayed in Fig. 11.5) Antioxidant Activity Radioprotectors should prevent/suppress the formation of radiation-induced free radicals (most of them are produced during radiolysis with water), thereby inhibiting their reactions with biomolecules, reducing the incidence of DNA strand breaks, and preventing the occurrence of cellular malfunction (more detail in Chap. 2). Since free radicals are short-lived (approximately 10−10 s) and interact rapidly with biomolecules, it is necessary that radioprotectors are present in sufficient concentration in the cellular milieu, at the time of radiation exposure.

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Molecules or compounds which increase the activity or expression of antioxidant enzymes are also considered radioprotectors. Many antioxidants have the potential to act as radioprotectors; however, not all antioxidants offer radioprotection, and this paradox may be explained by the relative activity of a compound when reacting with radiation-induced reactive species compared with those generated under H2O2 ­induced oxidative stress. Conventional antioxidants may not be able to scavenge this less reactive secondary species because either they do not accumulate in proximity to the secondary radicals or they may not have enough kinetic reactivity to scavenge them effectively. Thiols (e.g., amifostine), hydrophilic antioxidants (e.g., GSH), and newly developed cyclic nitroxides have adequate reactivity to effectively scavenge •OH and secondary radicals as well. Molecule-Based Radioprotection or Molecular Radioprotection Molecules or events that play a role late in signaling and IR-induced apoptotic pathways may act as potential targets for post-irradiation interventions. • ATM/ATR is activated by DNA damage and DNA ­replication stress; however, they often work together to signal DNA damage and trigger apoptotic cell death by upregulating proapoptotic proteins such as apoptotic protease-­ activating factor-1 (Apaf-1), phorbol-12-myristate-13-acetate-induced protein 1 (Noxa), and Bcl2associated X (Bax) after IR. • Pifithrin (PFT)-μ (2-phenylethynesulfonamide) directly inhibits p53 binding to mitochondria as well as inactivates the antiapoptotic proteins Bcl-xL and Bcl-2 on the mitochondrial surface, thereby suppressing subsequent release of cytochrome c and apoptosis, whereas PFT-μ reversibly inhibits transcriptionally mediated p53-dependent apoptosis. • Signal transducer and activator of transcription 3 (STAT3) can be activated by various growth factors and protects against IR damage. The protection mediated by STAT3 is attributed to its genomic actions as a transcription factor (such as upregulating genes that are antioxidative, antiapoptotic, and proangiogenic, but suppressing anti-­ inflammatory and antifibrotic genes) and other nongenomic roles targeting mitochondrial function and autophagy. • Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a well-characterized ubiquitous master transcription factor, whose activity is tightly controlled by cytoplasmic association along with its redox-sensitive transcriptional inhibitor Kelch-like ECH-associated protein 1 (Keap1). A well-known mechanism of activation of Nrf2 signaling protects cells against radiation-induced oxidative stress and also maintains cellular reduction-oxidation homeostasis. Upon oxidative stress, Nrf2 dissociates from Keap1

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Fig. 11.5  General therapeutic approaches to develop novel radioprotective agents. IR, directly or indirectly, causes damage to macromolecules such as DNA, lipids, and proteins. As a result, oxidative stress is generated, which either triggers DNA damage repair or induces p53-mediated cell disorders, such as cell cycle arrest and cell apoptosis. When the damage exceeds the cell’s ability to repair itself, the cell appears to follow the death program. The protective activities of poten-

and translocates into the nucleus to activate a series of antioxidant response elements, such as GPx, SOD, CAT, and heme oxygenase-1 (HO-1), increasing total cellular antioxidant capacity (TAC), accompanied by suppressed expression of inflammatory-related genes, avoiding oxidative stress and excessive inflammatory response, which is particularly important in radioprotection. • Heat-shock proteins (HSPs), molecular chaperones, are induced in cells during stress conditions. Importantly, HSPs are cytoprotective and can mediate cell and tissue repair after IR-induced deleterious effects. Higher cytosolic levels of HSPs have been shown to induce radioprotective effects by interfering with apoptotic pathways. • Peroxisome proliferator-activated receptor-γ (PPAR-γ), ligand-activated transcription factors, is a part of the nuclear hormone receptor family. It suppresses IR-­

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tial radioprotectors should target such phases/mechanisms (described in blue dotted box) with the aim to shield the normal cells from harmful insults of irradiation. Inspired from/based on “General principles of developing novel radioprotective agents for nuclear emergency” from Radiation Medicine and Protection (Volume 1, Issue 3, Pages 120–126), by Du et al. 2020, Copyright Elsevier (2022)

induced survival signals and DNA damage responses and enhances IR-induced apoptosis signaling in human cells.

11.1.1.1 Thiol-Containing Molecules In the search for an effective radioprotective agent, the Walter Reed Army Research Institute (USA) screened approximately 4500 compounds from the late 1950s. Cysteine was the first agent to confer radiation protection in mice after total body irradiation (TBI) in 1949. Later, various synthetic compounds with the aminothiol group were developed and proved to be highly effective  in preclinical models [4]. Among them, the most effective was WR-2721 or amifostine, a prodrug activated by alkaline phosphatase to an active sulfhydryl compound WR-1065, and at this moment, it is the only cytoprotective agent specifically approved by the FDA as a radioprotector (Fig. 11.6). The efficacy of amifostine is

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Fig. 11.6  Mechanisms of radioprotection by amifostine

attributed to the free radical scavenging, along with DNA protection and repair, all of which are coupled with the initial induction of cellular hypoxia. At the cellular level, amifostine has significant effects on cell cycle progression and has antimutagenic and anticarcinogenic properties [5]. In fact, amifostine indirectly induces the expression of proteins involved in DNA repair and triggers antiapoptotic pathways [6] and expression of antioxidant enzymes. Some authors have also proposed that it may enhance protective effects by increasing nuclear accumulation and inducing transcription factors related to p53 expression [7]. Moreover, WR-1065 accumulates more rapidly in normal tissues than in malignant cells, because the concentration of membrane-bound alkaline phosphatase tends to be higher on normal cells. Moreover, the lower vascular supply and the acidic environment of many tumors reduce the rate of dephosphorylation of WR-2721 and its uptake. It thus seems to be a really unique molecule that might potentiate radiotherapy (RT) efficacy in two opposite ways at the same time [8]. The US FDA has approved the use of amifostine in pre-

venting/reducing xerostomia (dry mouth) in head and neck cancer patients undergoing RT [5]. It has also been assayed in clinical trials to reduce mucositis, dysphagia, dermatitis, and pneumonitis during radiotherapy of head and neck cancers [9]. However, like other radioprotective aminothiols, the safety profile of amifostine has considerable limitations. Although the side effects such as nausea, vomiting, and hypotension are not life threatening, they can further aggravate the gastrointestinal syndrome. As it will be exposed latter, amifostine has been assessed in combination with other FDA-approved drugs (growth factors, cytokines, vitamin E, metformin, etc) looking for additive or synergistic radioprotective effects to prevent Acute Radiation Syndrome (ARS). Nevertheless, in most of cases none of these novel strategies completely counteracts amifostine’s toxic side effects at the doses needed to be efficacious as radioprotector [5]. Dimethyl sulfoxide (DMSO) has been shown to prevent the loss of proliferative lingual epithelial stem and p­ rogenitor cells upon irradiation by facilitating DNA DSB repair,

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thereby protecting against radiation-induced mucositis without tumor protection. Given its high efficacy and low toxicity, DMSO appears to be a potential treatment option to prevent radiation-induced oral mucositis [10]. GSH (L-γ-glutamyl-L-cysteinyl-glycine) plays a crucial role in the detoxification of reactive oxygen species, H2O2, lipid peroxyl radicals, peroxynitrites through enzymatic reactions, such as those catalyzed by GPxs, glutathione-S-­ transferases (GSTs), formaldehyde dehydrogenase, maleylacetoacetate isomerase, and glyoxalase I [11]. GSH not only protects DNA and other biomolecules against oxidative stress and radioinduced damages, it is also essential to activate DNA repairment mechanisms, to activate proliferation and to avoid radio-induced cell death [12]. In fact, the selective depletion of GSH in cancer cells has been shown to have potent radiosensitizing effects on tumor cells [13]. N-acetylcysteine (NAC) has a powerful antioxidant capacity, preserves GSH cellular levels, and prevents oxidative stress-induced apoptosis. NAC treatment (300  mg/ kg, subcutaneous), starting either 4 h prior to or 2 h after radiation exposure reduced early deaths in abdominally irradiated (X-rays, 20 Gy) C57BL/6 mice, attenuating gastrointestinal syndrome [14]. More recently, preclinical studies have evidenced that NAC can prevent/reduce cardiac, ovarian, renal, and testicular radiation-induced toxicity in rats. Nevertheless, NAC and GSH cannot be used as a radioprotector in cancer patients because they also enhance antioxidant defenses in cancer cells and may increase their metastatic potential [12]. Treatment with erdosteine (a homocysteine derivative) before γ-radiation exposure ameliorated nephrotoxicity and altered kidney function in rats. It is a potent scavenger of free radicals, increases GPx and CAT activity, and reduces oxidized glutathione levels displaying almost normal concentrations with respect to the irradiated group. Moreover, IL-1, IL-6, and TNF-α circulating levels were also significantly improved thus erdosteine provide substantial protection against radiation-induced inflammatory damage as evidenced in the biochemical and histopathological samples [15]. Phosphorothioates and other aminothiols are usually administered shortly before irradiation. They have been hypothesized to act as radioprotectors by one or a combination of the following effects: scavenging radiation-induced free radicals before their reaction with biomolecules; inducing hypoxia; scavenging metals; repairing DNA damage through hydrogen donation to carbon-centered radicals; and stabilizing genome. Moreover, high doses of phosphorothioates administered to mice before radiation have demonstrated anticarcinogenic effects [4]. However, as it happens with other more powerful thiolic radioprotectors (such as amifotine), its use is limited due to undesirable side effects.

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11.1.2 Cyclic Nitroxides (NRs) NRs, like  Tempol, JP4–039, XJB-5-131, TK649.030, or JRS527.084, are stable free radicals containing a nitroxyl group (-NO.) with an unpaired electron. The action of nitroxides to metabolize ROS is ascribed primarily to cyclic oneor two-electron transfer among three oxidation states: the oxoammonium cation, the nitroxide, and the hydroxylamine. Nitroxides undergo a very rapid, one-electron reaction to the corresponding hydroxylamine, which has antioxidant activity. In addition to their ability to neutralize free radicals, NR can easily diffuse through the cell membranes (and have SOD-like activity) (Fig.  11.7), prevent Fenton and Haber-­ Weiss reactions by oxidation of transition metal ions to a higher oxidation state, confer catalase-like activity on heme proteins, and inhibit lipid peroxidation. NRs are able to mitigate TBI-induced hematopoietic syndrome, when are administered before or as late as 72  h after radiation exposition [16]. Gramicidin S-derived nitroxide (JP4–039) is an effective TBI mitigator when  is delivered intravenously up to 72  h after exposure. JP4–039 treatment ameliorated head and neck radiation-induced mucositis and distant marrow suppression in mice [17]. In a comparative study with other four nitroxides, JP4-039  demonstrated the best median survival after radiation exposition [18]. The potential of this type of molecules as radioprotectors and/or mitigators has raised the interest of researchers, and nitroxidic structures has evidenced radioprotective activity. That is the case of nitronylnitroxide radical spin-labeled resveratrol [19].

11.1.3 Antimicrobials Primary experiments performed in the 1960s reported that antibiotic treatment and a single transfusion of allogeneic platelets significantly reduced mortality among monkeys exposed to TBI X-irradiation. Oral administration of streptomycin, kanamycin, neomycin, or gentamicin with drinking water (4  mg/mL) for 2  weeks before supralethal TBI (28.4 Gy) prolonged mean survival in mice (8.2–8.9 days vs. 6.9 for controls) [20]. The efficacy of antibiotics and other antimicrobials (antifungal and antiviral agents) is best explained as a countermeasure for radiation-induced neutropenia and immunosuppression. Tetracycline and ciprofloxacin protected human lymphoblastoid cells, reducing radiation-induced DNA double-­ strand breaks  (DSB) by 33% and 21%, respectively. Their radioprotective efficacy was attributed to the activation of the Tip60 histone acetyltransferase and altered chromatin structure [21]. Tetracycline hydrochloride is a  free radical ­scavenger, protects DNA, and increases survival of C57BL/6 mice by 20% upon a lethal radiation dose of 9 Gy [22].

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Fig. 11.7 Radioprotective properties of cyclic nitroxides include scavenger free radical capacity and SOD-like activity. Adapted from “Nitroxides as Antioxidants and Anticancer Drugs,” by Lewandowski M. and Gwozdzinski K. 2017, Licensed under CC BY 4.0

Mucositis is the most common side effect of RT for head and neck cancers. Preventive measures used in clinical medicine include good oral hygiene, dental and periodontal treatment, avoidance of tobacco products and alcohol, and frequent oral rinsing with a bland mouthwash such as povidone-iodine. Nonabsorbable antibiotic lozenges and/or antifungal topical agents (i.e., bicarbonates and amphotericin B) are also recommended [23]. Minocycline prevented radiation-induced apoptosis and promoted radiation-induced autophagy in primary neurons in  vitro. Minocycline also increases the counts of splenic macrophages, granulocytes, natural killer cells, and lymphocytes, and accelerates neutrophil recovery in C57BL/6 mice exposed to 1-3 Gy 60Co γ-rays. The mechanisms involved in this radioprotective effect were the suppression of cytokines that could prevent hematopoiesis (e.g. macrophage inflammatory protein-1α, TNF-α and INF-γ) and the increased production of IL-1α and β, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-­stimulating factor (G-CSF) [24]. Furazolidone (FZD) is an antimicrobial agent effective on both Gram+ and Gram− bacteria by interfering with bacterial oxidoreductase activity. In vitro, FZD treatment reduced unstable chromosomal aberrations (CAs) (such as acentric and dicentric chromosomes (DC)), chromosome breaks, and radiosensitivity of intestinal epithelial cells. Ma et  al. [25] showed that FZD treatment significantly improved the sur-

vival of lethal dose-irradiated mice, decreased the number of micronuclei (MN), increased the number of leukocytes and immune organ indices, and reversed the apoptosis and autophagy in the small intestine, thus restoring intestinal integrity. Their experiments showed that irradiation resulted in villous ­shortening and crypt dilation accompanied by epithelial atrophy or slough, and even marked edema and inflammatory cell infiltration, and how FZD significantly induced damage recovery. FZD is a clinically used antibiotic with few side effects and has been proposed as an efficacious medical countermeasure (MCM). However, detailed radiation protection activity and clinical applications need to be further studied, because radioprotective efficacy of antibiotics has not yet been tested in clinical trials.

11.1.4 Phytochemicals 11.1.4.1 Plant Extracts Considerable information from in  vivo, ex  vivo, and/or in  vitro studies suggests that crude extracts, fractionated extracts, isolated phytoconstituents, and plant polysaccharides from various plants such as Alstonia scholaris, Centella asiatica, Hippophae rhamnoides, Ginkgo biloba, Ocimum sanctum, Panax ginseng, Podophyllum hexandrum, Amaranthus paniculatus, Emblica officinalis, Phyllanthus amarus, Piper longum, Tinospora cordifolia, Mentha arven-

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sis, Mentha piperita, Syzygium cumini, Zingiber officinale, Ageratum conyzoides, Aegle marmelos, and Aphanamixis polystachya protect against radiation-induced lethality, lipid peroxidation, and DNA damage [26]. From these extracts, polyphenolic and nonpolyphenolic active principles and a range of secondary metabolites (e.g., carotenoids, alkaloids, sulfur compounds), already known for their anticancer properties, have also demonstrated radioprotective potential. Although many have been tested for brevity, this chapter focuses on those with the most promising results in vivo.

11.1.4.2 Polyphenolic Phytochemicals Over the last decades, plant-derived polyphenols have been screened for their potential ability to confer radioprotection. The free radical scavenger potential and antioxidant activity of polyphenols depends, in part, on their ability to delocalize electron distribution, resulting in a more stable phenoxy group. Moreover, intercalation in DNA double helices induces stabilization and condensation of DNA structures making them less susceptible to free radicals’ attack, reducing genotoxic damage induced by IR [27]. They are capable of trapping and neutralizing lipoperoxide radicals and can chelate metal ions (i.e., iron and copper), which play an important role in the initiation of oxidative stress reactions [28, 29]. Polyphenols  radioprotective efficacy is mainly attributed to its (Fig. 11.8) antioxidant and antiinflammatory properties, to their capacity to detoxify free radicals, eliciting DNA repair pathways, stimulating the recovery of hematopoietic and immune functions [28, 29]. In addition to the biochemical scavenger theory, there is also evidence of another potential mechanism by which

Fig. 11.8  Radioprotective and biological properties of polyphenols

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polyphenols activate Nrf2, exhibiting cellular protection against excessive ROS production, oxidative stress, and inflammation as well. Since the chemical features of these natural organic compounds are analogous to phenolic substances, their antioxidant and antiradical/scavenging radical (such as H2O2, 2,2-diphenyl-1-picrylhydrazyl) properties may be correlated positively with the number of hydroxyl groups bonded to the aromatic ring. They can exert their protection against environmental stimuli with the aid of remarkable antioxidant power by balancing the organic oxidoreductase enzyme system, regulating antioxidant-­ responsive signaling pathways, and restoring mitochondrial function. Although topically administered polyphenols may provide strong antioxidant protection, various challenges still exist and are onerous as well: (1) improving the bioavailability of polyphenols more effectively in order to promote their effectiveness is challenging; (2) if the polyphenols are extracted as the medicine or as health supplements, attention should be paid to the activity loss and degradation of polyphenols during the extraction process; (3) the effects cannot be generalized for all kinds of polyphenols, because each polyphenol has its own unique features; and (4) polyphenols have limited water solubility, and so it is important for polyphenols to be involved in rapid metabolism and also prove its chemical stability and solubility under in vivo conditions. To overcome this limitation, Obrador et al. [30] suggested a few feasible options: structural modifications of natural molecules (e.g., in the form of salts) to increase their hydro-­ solubility for intravenous administration or oral formulations to increase their bioavailability (e.g., cocrystals, nanoparti-

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cles, nanozymes). The promising phytochemical, pharmacodynamic, and toxicological research into the properties of polyphenols may serve as potential candidates for radioprotection in the near future. Apigenin exhibits anticancer properties associated with its prooxidant activity, inhibiting tumor growth and inducing cell cycle arrest and apoptosis. Apigenin pretreatment displayed efficacy for radioprotection in TBI Swiss albino mice by reducing cytogenetic alterations and biochemical and hematological changes [31]. Further, when apigenin was administered intraperitoneally at a dose level equal to 15 mg/ kg body, it was found to ameliorate radiation-induced gastrointestinal (GI) damages and restore intestinal crypt-villus architecture [32]. These attributes could be due to its ability to activate the endogenous antioxidants, suppress lipid peroxidation, and modulate inflammatory (NF-κB) and apoptotic signaling mediator/marker (p53, p21, Bax, caspase-3, caspase-9) expression. The in vivo efficacy of apigenin was also evidenced when it was intraperitoneally administered to mice 3 h after receiving γ-rays [33]. A significant reductions in the level of 8-hydroxy-2-deoxyguanosine (8-OH-dG), suppressed expression of NF-κB and NF-κB-regulated proinflammatory cytokines were observed, thus showing the radioprotective potential of apigenin. Curcumin, a yellow pigment of turmeric, is naturally found in the rhizome of Curcuma longa and other Curcuma spp. It is an active immunomodulatory agent which has many scientifically proven health benefits, such as the potential to improve symptoms of anxiety, depression, arthritis, and heart health and prevent Alzheimer’s, cancer, and oxidative and inflammatory conditions. Administration of curcumin in patients undergoing RT has demonstrated a dual action: radioprotection to normal cells through its ability to reduce oxidative stress, scavenge free radicals, inhibit transcription of genes related to oxidative stress, and suppress inflammatory response, as well as radiosensitization in tumor cells [34]. Curcumin, administered before or after a single 50 Gy radiation dose, showed protective effect on radiation-induced cutaneous damage in mice by significantly decreasing mRNA expression of early-responding cytokines (IL-1, IL-6, IL-18, TNF-α, and lymphotoxin-beta) and fibrogenic cytokines [35]. Oral administration of curcumin in mouse before irradiation resulted in a significant rise in activities of GPx and SOD enzymes while declining lipid peroxidation significantly, which indicates increased antioxidant status in mouse exposed to different doses of fractionated γ-radiation [36]. These protective qualities of curcumin may be due to free radical scavenging and upregulation of Nrf2 expression. Ellagic acid (EA), a strong natural antioxidant, has a major protecting role against different diseases associated

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with oxidative stress and inflammation. It also exerts antiangiogenesis effects via down regulation of vascular endothelial growth factor-2 (VEGF-2) signaling pathways in cancer. The amount and duration of EA used play a significant role in suppressing in vivo and in vitro oxidative stresses. In vitro studies [37] displayed high DPPH radical scavenging and lipid peroxidation inhibition activities of EA. It triggered the actions of antioxidant enzymes such as SOD, CAT, and GPx in V79–4 cells; reduced cell proliferation; and induced apoptosis in human osteogenic sarcoma cells as evidenced by chromosomal DNA degradation and apoptotic body appearance. When the human breast cancer cells (MCF-7) were treated with EA (10μM) and exposed with γ-radiation, the rate of apoptotic cell death in sub-G1 phase of cell cycle was high due to decreased mitochondrial membrane potential, upregulated proapoptotic Bax, and downregulated Bcl2, suggesting EA’s role in tumor toxicity to improve cancer radiotherapy [38]. Epicatechin (EC) is a common flavanol found in tea, cocoa, dark chocolates, and red wine. It has the ability to cross the blood-brain barrier and activate brain-derived neurotrophic factor pathways, suggesting its neuroprotective effects. In addition to general antioxidant activities, it aids with the modulation of metabolism of nitric oxide (NO) and other reactive nitrogen species (RNS). To evaluate the radioprotective effects of EC, Swiss albino mice were administered with EC for three consecutive days before exposing them to 5 Gy 60Co γ-irradiation [39]. EC pretreatment ameliorated γ-radiation-mediated alterations in mice, protected the liver and testis from radiation-induced oxidative stress, prevented systemic and cellular stress, and developed inflammation. It may possibly be due to the influence on the endogenous antioxidant defenses system after TBI in mice [40]. Another study [41] intended to investigate the effectiveness of EC in scavenging mitochondrial ROS and mitigating mitochondrial damage as radiation countermeasure agents by using human and mouse cells. It was observed that pre­radiation and postradiation treatments with EC mitigated ROS-mediated mitochondrial damage and IR-induced oxidative stress responses in mice. Also, oral administration of EC significantly enhanced the recovery of mouse hematopoietic cells from radiation injury in vivo, suggesting EC as a potentially viable countermeasure agent which is immediately effective against accidental IR exposure. Epigallocatechin-3-gallate (EGCG) is a natural polyphenolic antioxidant found in a number of plants, predominantly in green tea and black tea and also in small amounts in fruits and nuts. It gets a lot of attention for its potential positive impact on health. It aids weight loss, reduces inflammation, and helps prevent certain chronic conditions, including heart disease, diabetes, and cancers. Pretreatment with

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EGCG significantly enhanced the viability of human skin cells which were irradiated with X-rays and decreased radiation-­induced apoptosis [42]. It was found that EGCG suppressed IR-induced damage to mitochondria via upregulation of SOD2 and induced expression of cytoprotective molecule HO-1  in a dose-dependent manner via transcriptional activation. The therapeutic effects and mechanism of EGCG on radiation-induced intestinal injury (RIII) have not yet been determined; however, Xie et al. recently [43] investigated it both in vitro and in vivo and revealed that treatment with EGCG not only prolonged the survival time of lethally irradiated mice, but also mitigated RIII. Besides, it significantly augmented proliferation and survival of intestinal stem cells and their progeny cells in irradiated mice. Their findings demonstrated that EGCG protected against RIII by reducing the level of IR-induced ROS and DNA damage, inhibiting apoptosis and ferroptosis through activating transcription factor Nrf2-­mediated signaling pathway and its downstream targets comprising antioxidant proteins Slc7A11, HO-1, and GPx4, suggesting that EGCG could be a promising medical countermeasure for the alleviation of RIII. Genistein (GEN), an isoflavonoid compound, is commonly found in soybeans and its products. Mechanistic insights reveal its potential beneficial effects on human diseases such as cancer, by inducing apoptosis and cell cycle arrest. GEN has antiangiogenic, antimetastatic, and antiinflammatory effects. Besides, various studies of GEN have revealed its radioprotective properties by protecting against radiation-induced DNA damage, scavenging free radicals, and altering cell cycle effects. Davis et  al. [44] revealed GEN-induced radioprotection against hematopoietic- acute radiation syndrome (H-ARS) by altering the cell cycle of hematopoietic stem and progenitor cells in a murine model. The extracted GEN displayed protection against IR-induced GI injury and bone marrow toxicity by upregulating the Rassf1a and Ercc1 genes to effectively attenuate DNA damage in a TBI mouse model [45]. Moreover, Song et al. [46] showed that low concentration of GEN (1.5μM) lessened radiation-induced injuries by way of inhibiting apoptosis, alleviating chromosomal and DNA damage, downregulating GRP78, and upregulating HERP, HUS1, and hHR23A.  In contrast, high concentration of GEN (20μM) demonstrated radiosensitizing characteristics in cancer cells. The role of genistein as a radiosensitizer will be further discussed in Sect. 11.4. Naringin, a predominant flavone glycoside, is present in citrus fruits. Manna et  al. [47] demonstrated that pretreatment with naringin significantly prevented γ-radiation (6Gy)-induced intracellular ROS-mediated oxidative DNA damage; inhibited radiation-induced G1/S-phase cell cycle arrest by modulating p53-dependent p21/WAF1, cyclin E, and cyclin dependent kinase 2 (CDK2) activation; and

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reversed the inflammation through downregulating nuclear factor kappa B (NF-kB) signaling pathways and balancing the expression of C-reactive protein, monocyte chemoattractant protein-1 (MCP-1), and iNOS2 at sites of inflammation in murine splenocytes. Besides, naringin pretreatment could effectively deter UVB-mediated DNA damage, alter apoptotic marker expression (Bax, BCl-2, caspase-9, and caspase­3), and potentially modulate NER gene (XPC, TFIIH, XPE, ERCC1, and GAPDH) expression, thereby augmenting DNA repair [48]. Naringenin is present in peppermint and citrus fruits such as oranges, grapefruit, and tangerines. It is endowed with biological effects on human health, which includes a great ability to modulate signaling pathways; efficient impairing of plasma lipid and lipoprotein accumulation; and antiatherogenic and anti-inflammatory effects. To evaluate radioprotective effects of naringenin in  vivo, Swiss albino mice were orally administered 50 mg/kg body weight of naringenin prior to radiation exposure [49], and it protected mice against radiation-induced DNA, chromosomal, and membrane damage. Naringenin pretreatment increased antioxidant status and survival chances, inhibited NF-kB pathway, and downregulated radiation-induced apoptotic proteins (p53, Bax, and Bcl-2) in normal cells resulting in radioprotection at the cellular, tissue, and organism levels. Resveratrol (RV), a natural polyphenol, is produced in several plants in response to stress, injury, and UV radiation. It is present in fruits such as grapes, strawberries, and red wine. It is known for its analgesic, antiviral, cardioprotective, neuroprotective, and antiaging actions. Different doses of RV were administered intraperitoneally to mice prior to total-body γ-irradiation (2 Gy), and it was observed that RV significantly reduced lymphocyte damage in mice caused by γ-radiation due to its ability to scavenge free radicals, restore the levels of intracellular antioxidants (GPx, SOD, CAT activity), and cause cell cycle arrest [50]. RV is also known to have a significant effect in stabilizing p53 and altering proapoptotic and antiapoptotic protein concentration [51]. Zhang et  al. [52] treated with RV  IR-exposed  C57BL/6N mice. RV reduced radioinduced-intestinal injury (upregulating Sirt1 and acetylating p53 expression), improved intestinal morphology, decreased apoptosis of crypt cells, maintained cell regeneration, and ameliorated SOD2 expression, evidencing its radioprotective potential. The role of RV together with pterostilbene as a radiosensitizer will be further discussed in Sect. 11.4. Pterostilbene (PT), is another stilbenoid compound, structurally similar to RV, present in blueberries, grapes, and other similar fruits. It is an active phytonutrient with many biomedical applications in cancer treatment, insulin sensitivity, cardiovascular diseases, aging, and cognition. Moreover, it has a greater bioavailability, efficacy and lower toxicity than RV [53]. Sirerol et al. [54] evidenced that pterostilbene

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reduced chronic UVB irradiation-­induced skin damage and carcinogenesis in hairless mice through maintaining antioxidant defenses, including GSH, CAT, SOD, and GPx. Recently, a combination of natural polyphenols (PT and silibinin) with a NAD+ precursor and a TLR2/6 ligand was shown to protect mice against lethal γ-radiation, increasing long term survival up to 90% of the treated mice [55].

11.1.4.3 Nonpolyphenolic Phytochemicals Caffeic acid/caffeic acid phenethyl ester (CAPE) is found in coffee, tea, chocolate, and colas. It has numerous pharmacological and physiological effects, including cardiovascular, respiratory, renal, and smooth muscle effects, as well as effects on mood, memory, alertness, and physical and cognitive performance. It is essentially regarded as a radiosensitizer by virtue of its inhibition of DNA repair after irradiation. The radioprotective properties of CAPE have also been shown in the bone marrow chromosomes of mice exposed to TBI (1.5 Gy 60Co γ-rays), regardless of its time of administration [56]. Caffeic acid, a known dietary antioxidant, could be used as a supplemental drug which has a dual effect: ameliorating hematopoietic stem cell (HSC) senescence-accompanied long-term BM injury in single (sublethal dose of 5 Gy) TBI and stimulating apoptotic cell death of colon cancer cells in mice [57]. Khayyo et  al. [58] intraperitoneally administered CAPE prior to total-head γ-irradiation and observed that the oxidant stress parameters (total oxidant status, oxidative stress index, and lipid hydroperoxide) were significantly reduced, whereas antioxidant parameters (activity of paraoxonase, arylesterase, total GSH levels) were increased in the rat brain tissue, signifying the protective role of CAPE as an important antioxidant against ROS accumulation induced by total-head irradiation. The role of CAPE as a radiosensitizer will be further discussed in Sect. 11.4.1.7. Sesamol is found in sesame seeds and oil. It has many biological activities and health-promoting benefits such as inducing growth arrest and apoptosis in cancer and cardiovascular cells and enhancing vascular fibrinolytic capacity, antioxidant activity, chemoprevention, antimutagenic, and antihepatotoxic activities. Naturally occurring or synthetic substances of sesamol counteract the damaging effects of oxidation by inhibiting or retarding oxidation reactions. Also, it has the potential to scavenge free radicals and therefore reduces the radiation-induced cytogenetic damage in cells. Kumar et al. [59] investigated its radioprotective potential against radiation-induced genotoxicity in hematopoietic bone marrow of whole-body 𝛾-irradiated (2Gy) mice. ­Pre­administration of 20 mg/kg body weight sesamol reduced the frequency of radiation-induced MN, CAs, and comets (% damaged DNA streak in tail), suggesting its major role in direct scavenging of free radicals to protect bone marrow, spleen, and lymphocytes from radiation-induced cytogenetic damages and genotoxicity. Besides, intraperitoneal pretreat-

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ment of sesamol offered protection to hematopoietic and GI systems against γ-radiation-induced injury in C57BL/6 male mice by inhibiting lipid peroxidation; translocating gut bacteria to spleen, liver, and kidney; enhancing regeneration of crypt cells in GI; reducing the expression of p53 and Bax apoptotic proteins in the bone marrow, spleen, and GI; and alleviating the total antioxidant capacity in spleen and GI tissue [60]. Recently, Majdaeen et al. [61] concluded that regular oral consumption of sesamol extract is more effective than consuming it once before irradiation. 3,3′-Diindolylmethane (DIM), a small-molecule compound and a major bioactive metabolite, is formed by acid hydrolysis of indole-3-carbinol (one of the best characterized components in Cruciferae). It can inhibit invasion, angiogenesis, and proliferation and induce apoptosis in tumor cells by modulating signaling pathways involving AKT, NF-kB, and FOXO3 [62]. Chen et al. [63] investigated the radioprotective effects of DIM in normal tissues using a mouse model approach. It was indicated that treatment with DIM increased the expression of some stress-responsive genes without causing DNA damage, delayed radiation-­ induced cell cycle arrest, and apoptosis. Fan et  al. [64] reported that administration of DIM in a multidose schedule protected rodents against lethal doses of TBI up to 13  Gy. Transcriptomic profiling showed that DIM’s mechanism of radioprotection involved regulation of responses to DNA damage and oxidative stress by inducing ataxia-­telangiectasia mutated (ATM)-driven DDR-like response, enhancing radiation-­induced ATM signaling and NF-κB activation, suggesting its potential role as a MCM in protecting or mitigating adverse effects of RT.

11.1.5 Vitamins With the understanding that free radicals perpetuate a significant amount of the damage caused by IR, vitamins with antioxidant potential (A, C, and E and its derivatives) have been assayed as radioprotectors (Fig.  11.9). Vitamin A and β-carotenes (lutein, lycopene, phytofluene, phytoene, and others) reduced mortality and morbidity in mice exposed to partial or TBI. Dietary vitamin A offered protection in mice subjected to localized radiation exposure focused on the intestine (13  Gy, TBI) and the esophagus (29  Gy) [30]. A single dose of vitamin A injected intraperitoneally 2 h before 2  Gy of γ-radiation exposition, significantly reduced the number of MN in the bone marrow and the genetic damages, due to its capacity to trap free radicals [65]. Carotenoids such as crocin and crocetin (isolated from the dietary herb saffron) have antioxidant, anti-inflammatory, and antiapoptotic effects. In mice bearing pancreatic tumors, crocin significantly reduced tumor burden, radiation-induced toxicity, and hepatic damage and preserved liver morphology [66] while

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Fig. 11.9  Radioprotective effects of vitamins

crocetin also reduced radiation injury in intestinal epithelial cells [67]. Lutein is a pigment classified as a carotenoid, found in plants such as green leafy vegetables (spinach, kale), fruits, corn, egg yolk, and animal fats. While this pigment plays an important role in eye health, lutein supplements also help to prevent colon and breast cancer, diabetes, and heart disease due to its powerful antioxidant potential. In vitro and in vivo lutein was found to scavenge free radicals and inhibit lipid peroxidation by increasing the activity of CAT, SOD, and glutathione reductase [68]. Lutein showed maximum survival in mice treated with 250 mg/kg body weight against a lethal dose of 10 Gy γ-radiation. Pretreatment of lutein maintained near-normal levels of hematological parameters indicating resistance/recovery from the radiation-induced damages [69]. Furthermore, lycopene has the highest antioxidant activity among carotenoids, and it reduces pro­inflammatory cytokine expression such as IL-8, IL-6, and NF-κB. Many preclinical studies evidence its radioprotective efficacy, in particular, if it is administered before or as soon as possible after radiation exposure [70]. Vitamin C is the reduced form of ascorbic acid (AA) and a water-soluble vitamin. The intake of vitamin C decreases the risk of getting cataracts after radiation exposition. AA has low toxicity and cost and is easily available, making it an

attractive radioprotective agent. Administration of AA before γ-irradiation prevents chromosomal damage in bone marrow cells, mainly due to its scavenging activity of ROS, protecting lipid membranes and proteins from oxidative damage. It has also been reported that AA can prevent the adverse effects of TBI by increasing the antioxidant defense systems in the liver and kidney of irradiated animals [71]. Sato et al. [72] demonstrated the significant radioprotective effect of AA on the ARS in special GI syndrome, especially if it is administered before or not later than 24  h after radiation exposition. Vitamin E is an essential fat-soluble nutrient with antioxidant, neuroprotective, and anti-inflammatory properties. Vitamin E family includes eight vitamers, four saturated (α, β, γ, and δ) called tocopherols, and four unsaturated analogs (α, β, γ, and δ) referred to as tocotrienols, which are collectively called tocols, with α-tocopherol being the most abundant in human tissues. Tocols administered subcutaneously 1  h prior to or during 15  min postirradiation improved the 30-day survival in mice, and other tocopherol derivatives, such as α-tocopherol-succinate and α-tocopherol-mono-­ glucoside, have also shown radioprotective effects in  vivo. Moreover, subcutaneous injection of γ-tocotrienol (100– 200 mg/kg) 24 h prior to 60Co γ-irradiation showed a signifi-

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cant protective effect in mice facing radiation doses as high as 11.5 Gy and increased mice survival rate [73]. Preclinical studies have provided evidence that tocotrienols exert radioprotection at least in part via induction of G-CSF, reducing inflammatory response suppressing the expression of TNFα, inducible NO synthase (iNOS), and IL-6 and 8, as well as inhibiting NF-κB signaling [74]. Endothelial cells activated through IR downregulate the expression of thrombomodulin (TM) and increase endothelial surface expression of adhesion molecules, which allow the attachment of immune cells, and thereby contribute to inflammation and activation of the coagulation cascade. The greater efficacy of tocotrienols is attributed to their higher antioxidant potential and its ability to inhibit HMG-CoA reductase activity (decreasing serum cholesterol levels) and increase TM expression in endothelial cells, which result in antipermeability, anti-inflammatory, and antithrombotic response in order to decrease radiation-induced vascular damages. Nevertheless, low bioavailability of tocotrienols is an important limiting factor for their use as radioprotectants, and thus a novel water-soluble liposomal formulation of γ-tocotrienol (GT3) has been developed. GT3 has shown to increase the delivery of γ-tocotrienol in the spleen and bone marrow and offered significant radioprotection in vivo [75]. Despite these promising results, the use of vitamin E derivatives as radioprotectants must be evaluated with caution for their potential toxic effects. More recently, several laboratories have assayed the potential synergistic effect of tocols with other radioprotectants, such as pentoxifylline (PTX) (an antioxidant and anti-inflammatory xanthine derivative, approved by the FDA) which increased survival of mice subjected to 12  Gy 60Co γ-irradiation. Efficacy of PTX and α-tocopherol against radiation-induced fibrosis has been observed in animal models and clinical studies, even though the treatment started after radiation-induced fibrosis manifested clinically. Three clinical trials have evaluated if PTX enhances the radiation-protective properties of γ-tocotrienol, but the results of these studies have not yet been published [74]. At least, two randomized controlled trials provided evidence that dietary supplementation of α-tocopherol and β-carotene during radiation therapy could reduce the severity of treatment adverse effects, but these trials also evidenced that the use of high doses of antioxidants might compromise radiation treatment efficacy. Other combinations like α-tocopherol, acetate and AA showed radioprotective effects and enhanced apoptosis in irradiated cancer cells [76]. Cholecalciferol (D3) and ergocalciferol (D2) are the two forms of vitamin D provided by the food. Exposure to UV radiation of the skin also induces the endogenous synthesis of D3, and for that reason, it is also called the “sunshine vitamin.” D3 and D2 have to undergo a double hydroxylation (in the liver and in the kidney) to form the

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biologically active derivative, that is, calcitriol (1,25-(OH)2vitamin D), an essential hormone in the regulation of phosphocalcic metabolism. In vitro and in vivo studies evidenced the radioprotective efficacy of calcitriol enhancing the expression of genes coding for antioxidant enzymes (such as SODs and GPxs) and metallothioneins which are ROS scavengers [77]. Jain and Micinski [78] showed a positive link between vitamin D and GSH concentrations, as well as reduction in levels of ROS and proinflammatory cytokines, which is undoubtedly beneficial in protecting against IR. Populations of radiologically contaminated areas close to the Chernobyl accident had lower vitamin D blood levels compared to those in the uncontaminated Ukrainian regions [79]. Therefore, oral supplementation with vitamin D during RT or in medical professionals chronically exposed to low IR doses should be taken into consideration also because radiation toxicity can reduce mineral bone density. Recent studies evidence that calcitriol also radiosensitizes cancer cells by activating the NADPH/ROS pathway, which can makes it a promising adjuvant in RT [80].

11.1.6 Oligoelements Many antioxidant/defense enzymes, like SOD and metalloproteins, require trace elements as cofactors. The main oligoelements showing protective effects against radiation-induced DNA damage are zinc (Zn), manganese (Mn), and selenium (Se) [81] (Fig. 11.10). Se is an essential component of selenoenzymes such as GPx, thioredoxin reductase-1 (TR1), and ribonucleotide reductase  (RNR). Se compounds and their metabolites possess a wide range of biological functions including anticancer and cytoprotection effects and modulation of hormetic genes and antioxidant enzyme activities. Exposure to radiation has been associated with a decrease in Se blood levels, and thus administration of seleno-­compounds has emerged as a radioprotective strategy to reduce radiation toxicity. Mechanisms underlying the radioprotection effects include Nrf2 transcription factor activation and the consequent upregulation of the antioxidant-adaptive response in bone marrow stem cells and hematopoietic precursors [82]. 3,3-Diselenopropionic acid (at an IP dose of 2  mg/kg for 5  days prior to γ-TBI) showed radioprotection in mice by decreasing DNA damage and apoptosis [83]. Another recent formulations, poly-vinylpyrrolidone and selenocysteine-­ modified Bi2Se3 nanoparticles, improved the RT efficacy against tumors while exerting radioprotection in normal tissues [84]. Cancer patients, treated orally with Selenium Selenite, experienced a a lower incidence of diarrhoea compared to the placebo group [85]. Selenomethionine also reduces mucositis in patients with advanced head and neck cancer who are receiving cisplatin and radiation therapy (NCT01682031, www.clinicaltrials.gov).

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Fig. 11.10 Radioprotection by oligoelements

Radiation-induced lung pneumonitis is a major  dose-­ limiting side effect of thoracic RT, and the therapeutic options for its prevention are limited. 3,3′-Diselenodipropionic acid (DSePA), a synthetic organoselenium compound, shows moderate GPx-like activity and is an excellent scavenger of ROS. DSePA reduced the radiation-mediated infiltration of polymorphonuclear neutrophils (PMN) and suppressed NF-kB/IL-17/G-CSF/neutrophil axis as well as elevation in levels of proinflammatory cytokines such as IL1-β, ICAM-1 (intercellular adhesion molecule-1), E-selectin, IL-17, and TGF-β in the bronchoalveolar fluid of irradiated mice, thus ameliorating inflammatory responses. Administration of DSePA has shown a survival advantage against TBI and a significant protection to lung tissue against thoracic irradiation [86]. Wang et al. [87, 88] developed a highly efficient radioprotection strategy using a selenium-containing polymeric drug, with low toxicity and long-term bioavailability, The radioprotection activity of (VSe) and N-(2-hydroxyethyl) acrylamide shows more remarkable effects both in cell culture and mice models compared to the commercially available ebselen (organoselenium compound) and also exhibits a much longer retention time in blood (half-life ∼10 h). Crescenti et  al. [89] evaluated in  vivo the tolerance induced by the combination of Se, Zn, and Mn (4 microg/mL each) plus Lachesis muta venom (O-LM) (4 ng/mL) to high doses of TBI (10  Gy, 137Cs source) IR in mice. Mice who received daily O-LM subcutaneous injections, starting

30 days before irradiation, showed a higher number of crypts, enhanced villous conservation, and lack of edema or vascular damage compared to the untreated and irradiated group. O-LM treatment also decreased vascular damage and grade of aplasia of mice bone marrow. O-LM treatment safety and efficacy were tested in a phase I clinical trial, and results indicated that it is an attractive candidate as a radioprotective agent for patients undergoing RT.  Other clinical evidence indicates that Zn supplementation may act as an effective radioprotector in patients during RT. In a randomized clinical study, patients treated with Zn sulfate suffered a lower degree of mucositis compared to the placebo group [90]. Orally administered Zn-carnosine reduced oral mucositis and xerostomia in head and neck cancer patients [91].

11.1.7 Superoxide Dismutase (SOD) Mimetics and Nanoparticles SODs are a group of metalloenzymes that catalyze the dismutation of superoxide radicals (O2˙-) to H2O2 and O2, thus are first line of defense to prevent IR damages. In the event of a radio-nuclear attack or nuclear accident, the skin damage used to be severe. A synthetic SOD/CAT mimetic (EUK-­ 207) administered 48  h after irradiation significatively mitigated radiation dermatitis, suppressed indicators of tissue oxidative stress, and enhanced wound healing [92].

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Clinical applications of SODs mimetics are  limited by their structural instability deficient availability and high cost. Compared with natural enzymes, nanozymes (nanomaterials with enzyme-like activity) are more stable, are economically affordable, and can be easily modified. Due to these characteristics, nanozymes are expected to become effective substitutes for natural enzymes for medical applications. Nanozymes with SOD-like activity have been developed and proved to have a mitigating effect on diseases involving oxidative stress [93]. As shown in Fig. 11.11, after administration, they are internalized by the cells and imitate SOD2 activity in order to inhibit ROS-induced cell damage. Patients treated with RT for cancers of the head, neck, or lung suffer damage to the mucosa of the upper aerodigestive tract. Most of them develop ulcerative forms of mucositis, and severe forms lead to inability to eat solid foods, and in some cases, they cannot drink liquids. Results of clinical trials (now in phase III, e.g., NCT03689712) demonstrated the efficacy of the SOD mimetic, avasopasem manganese (GC4419) [94]. Mn porphyrin-based SOD mimics (MnPs) are reactive with superoxide and with other reactive oxygen, nitrogen, and sulfur species (Fig. 11.12). MnPs have CAT and GPx-­ like activities and peroxynitrite-reducing activity [93]. MnPs administered before and continued after radiation exposure protect from γ-ray, X-ray, and proton beam irradiation dam-

Fig. 11.11  Nanozymes with SOD-like activities

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ages in different animal models, and a few studies indicate that beginning treatment with MnPs after radiation exposure is also effective. In normal tissues, MnPs treatment reduces oxidative stress, NF-κB, and TGF-β signaling pathways and activates Nrf2-dependent pathways. On the contrary, MnPs administration in combination with cancer therapy results in more oxidative stress in cancer cells, which leads to the reduction of NF-κB and HIF-1α and their downstream signaling pathways (Fig. 11.12). These changes are associated with increasing apoptosis and reducing overall cancer growth [95]. BMX-001 is a porphyrin mimetic of the human mitochondrial manganese SOD, with the capacity to cross the blood-brain barrier and protect the brain against IR while acting as a tumor radiosensitizer [96]. It has been assayed as a radioprotector in different clinical trials, e.g., NCT03386500 (patients with recently diagnosed anal cancer), NCT03608020 (cancer patients with multiple brain metastases), NCT02990468 (head and neck cancer), and NCT02655601 (high-grade glioma treated with radiation therapy and temozolomide) [30]. All previous SOD mimetics suppress oxidative stress-­ mediated injuries, supporting the survival of the normal tissue, while promoting apoptotic processes in tumor tissues. The results from the clinical trials will provide us invaluable information on their real clinical utility as radioprotectors.

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Fig. 11.12  Effects of Mn porphyrin-based SOD mimics in normal and cancer cells

11.1.8 Hormonal and Hormonal Mimetic Radioprotectors 11.1.8.1 Catecholamine Agonist Radiation dermatitis is a common side effect of irradiation that limits cancer RT courses. It has already been described how the induction of hypoxia limits the damage associated with radiation, and consequently the option of using vasoconstrictor substances as radioprotectors has been proposed. Topical application of adrenergic vasoconstrictors (epinephrine or norepinephrine) to rat skin before radiation exposition (17.2  Gy) confers 100% protection against radiation dermatitis [97], and similar results were obtained when phenylephrine was topically administered to prevent radiation mucositis. Indralin is an α1-adrenoceptor agonist with vasoconstrictor effects similar to those of epinephrine. Indralin (120 mg/kg)-treated rhesus monkeys survived better (five of six) after being exposed to a lethal TBI 60Co ɣ-irradiation of 6.8 Gy, than nontreated ones (all died). Moreover, less pronounced manifestations of hemorrhagic syndrome, leukopenia, and anemia were also noted [98]. Norepinephrine and α1-adrenoceptor agonists accelerate differentiation of hematopoietic stem cells by blocking their proliferation, thus avoiding, at least, earlier manifestation of radiation injury. A common feature of the radioprotective action of biogenic amines like indralin and aminothiols is the induction of hypoxia, although their

mechanisms of action differ significatively. Norepinephrine and indralin exert their effect through the neurohormonal α1-adrenoceptors, but sulfur-­ containing radioprotectors act directly on tissues. Nevertheless, the use of α-catecholaminergic agonists entails a high risk of increased blood pressure or pressure decompensation in hypertensive patients, which would compromise their widespread use in an accidental emergency radiation exposure.

11.1.8.2 Somatostatin Analogs GI radiation vulnerability to a certain extent can be caused by release of potent pancreatic enzymes into the intestinal lumen after radiation exposure. Therefore, reducing intraluminal proteolytic activity may help attenuate intestinal radiation toxicity. Somatostatin and its analogs (octreotide and pasireotide) inhibit exocrine pancreatic secretions. Octreotide reduces both acute and delayed intestinal radiation injury and diarrhea [99], as it has also been evidenced in a randomized controlled trial in patients who were undergoing radiation therapy to the pelvis (NCT00033605, www.clinicaltrials. gov). Nevertheless, some common side effects such as allergy, nausea, rash, and light-headedness may limit the routine use of octreotide. Moreover, it could also induce hypoglycemia [99] and reduce secretion of GH and IGF1, which could be highly counterproductive for the recovery of damaged tissues.

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SOM230 (pasireotide) is another somatostatin analog under preclinical evaluation as a radioprotector. SOM230 reduced intestinal mucosa injury and increased mouse survival after TBI by inhibiting exocrine pancreatic secretion. Moreover, SOM230 has a 40-fold improved affinity to somatostatin receptor 5 than other somatostatin analogs, and it proved to be beneficial when administered prior to radiation exposure, and also when the treatment started up to 48 h following the exposure [100].

11.1.8.3 Melatonin Several hormones are known to exhibit radioprotective characteristics, and melatonin, N-acetyl-5-methoxytryptamine, is one of them. It is the main secretory product of the pineal gland. Its radioprotective properties are outlined in Figs. 11.13 and 11.14. Melatonin has the ability to neutralize both ROS and NO directly leading to the production of less/nontoxic agents or indirectly increasing the activity of antioxidant enzymes such as SODs, GPx, GR, and CAT at the same time suppressing prooxidant enzymes like xanthine oxidase (XO) [101]. In addition, melatonin induces DNA repair mechanisms, which reduce mutagenic damage and also induction of DNA DSBs, which are lethal events for the cell. Melatonin administration before irradiation with a lethal dose of 60Co γ-rays reversed the upregulation of Bax and p53 proapoptotic genes and elevated Bcl-2, which led to 100% survival and preservation of hematopoietic and GI systems in mice [102]. Inflammation and fibrosis are two degenerative phenomena that are typical pathophysiological processes following RT.  Melatonin via inhibition of NF-kB, COX-2, and iNOS enzymes has the ability to reduce the release of inflammatory cytokines and chemokines. Attenuation of these enzymes’ activities is associated with reduced level of oxidative stress, infiltration of macrophages and lymphocytes, as well as suppression of fibrosis, which prevents radio-induced pneumonitis and lung fibrosis [103], and also heart [104] and brain [105] damage associated with radiation exposition. The physiological concentrations of melatonin in the human blood are approximately much lower during the day

Fig. 11.13  Radioprotective properties of melatonin

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than during the night. Therefore, it seems that radiation therapy with supplementary melatonin leads to more beneficial effects during the nighttime. Melatonin exhibits multiple neutralizing actions to reduce radio-induced damage. Together with its low toxicity and its ability to cross biological barriers, these are all significant properties to consider it for clinical RT applications as well as for mitigation of radiation injury in a possible radiation accident scenario; however, its short half-life in vivo (2 Gy [118]. Neulasta has the advantage that it is administered weekly, compared to daily administrations that is required for Neupogen treatment. GM-CSF increases the differentiation and proliferation of macrophage and granulocyte progenitor cells. When administered as late as 48 h after radiation exposure, GM-CSF reduced the recovery time for neutropenia and thrombocytopenia and decreased the rate of infection [5]. In addition, GM-CSF appears to exhibit an antifibrotic effect in the setting of radiation-induced lung injury (RILI) in experimental animals and humans [122, 123]. Keratinocyte growth factor (KGF), a factor that is produced by mesenchymal cells, protects and repairs epithelial tissues. Early studies suggested that KGF promotes the recovery of the oral mucosa after radiation-induced injury, improves gastrointestinal barrier function, and limits bacterial translocation and subsequent sepsis after irradiation. In clinical studies, Palifermin®, a human recombinant KGF product, reduced the incidence, duration, and severity of oral mucositis and esophagitis in patients treated with chemoradiotherapy and stimulated immune reconstitution following hematopoietic stem cell transplantation [124]. Many cell types release epidermal growth factor (EGF), which promotes the regeneration of hematopoietic stem cells in vivo. EGF was reported to have an additive effect on overall survival with G-CSF (survival of 20% for controls, 67% for EGF, 86% for EGF plus G-CSF) [125]. Fibroblast growth factor (FGF) is found in many tissues throughout the body, and its levels decrease after irradiation. FGF-P is a human recombinant derivative that is capable of activating FGF receptor-1, resulting in protection of the crypts located in the duodenum and improved survival in a GI-ARS mouse model. In addition, platelet counts were found to be higher in FGF-­ P-­treated animals, resulting in decreased hemorrhages and cutaneous ulcerations postirradiation. It has been suggested that FGF-P has the potential to treat radiation-induced skin ulcerations and thermal burns and that it holds potential promise in the management of ischemic wounds and the promotion of tissue engineering and stem cell regeneration [125]. Interleukin-12 (IL-12) has pleiotropic effects on the innate and adaptive immune cells, including stimulation of hematopoiesis. Treatment with HemaMax® (human recombinant IL-12) restored all cell types in bone marrow when administered at 24 and 48 h post-TBI in non-human primates (HNPs) and mice, respectively. Compared to Neupogen, Neulasta, and Leukine, the single administration of HemaMax® is another advantage in the event of a mass casualty incident [126]. A novel, PEGylated IL-11 (Neumega®) is approved to treat thrombocytopenia in cancer patients, but must be injected daily, making its use inconvenient as a

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radiomitigator. To circumvent this problem, another monoPEGylated IL-11 analog (BBT-­059) was designed and demonstrated higher bioavailability and potency in  vivo. In mouse model exposed to high TBI doses, BBT-059 leads to bone marrow cell reconstitution, leading to an accelerated recovery of platelets, erythrocytes, and neutrophils and an increase of survival higher than that obtained with treatment with the PEGylated derivatives of G-CSF and GM-CSF [125]. Erythropoietin is prescribed for the treatment of severe anemia arising from intense chemo- and/or radiation therapies. Erythropoietin and thrombopoietin (TPO) have been used for the victims of radiation exposure in the Tokaimura accident. Romiplostim (Nplate) is a synthetic TPO receptor agonist that preferentially increases platelet generation in bone marrow; contributes to mitigation of radiation-induced thrombocytopenia, anemia, and leukopenia; gives protection; and enhances regeneration of vascular endothelium. Romiplostim has recently received FDA approval to treat acutely irradiated and severely myelosuppressed adult and pediatric patients. More recently, ALXN4100TPO (a TPO receptor agonist) has been shown to stimulate megakaryopoiesis, reduce bone marrow atrophy and radiation-induced mortality in acutely irradiated mice, with the advantage of being less immunogenic than Nplate. Combinations of hematopoietic growth factors and cytokines (G-CSF, GM-CSF, EPO, SCF, and IL-3) have already been used in the treatment of radiological accident victims, but the relative efficacy of this combined treatment is difficult to evaluate due to differing radiation sources, exposure doses, and other circumstances [127]. As explained in detail in Chap. 2, irradiation directly causes ROS overproduction, apoptosis, and/or necrosis, which activate the inflammatory response. In the short term, proinflammatory cytokines, such as IL-1, IL-6, IL-8, IL-33, TNF-α, and TGF-β, help to activate the immune response and bone marrow cellular recovery, but if it is excessive or is maintained for a long time, it can contribute to bystander/ nontargeted effect (damages in tissues that have not been directly exposed), in special autoimmune diseases, fibrosis, and/or cancer initiation and progression. Therefore, the use of cytokines or growth factors capable of increasing the inflammatory response should be carefully evaluated. Moreover, the use of substances that inhibit its release or antagonize its pro­inflammatory effects has been shown to have mitigating effects on the damage caused by IR. Fibrogenic cytokines like TGF-β, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) are involved in radiation-induced fibrosis. TGF-β is able to stimulate ROS and NO production by the immune system, involved in the initiation and progression of chronic oxidative damage after exposure to a high dose of radiation. It is therefore not surprising that combined inhibition of

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TGF-β and PDGF signaling attenuates radiation-induced pulmonary fibrosis associated with decreased pneumonitis and leading to prolonged survival. Inhibition of TGF-β also reduced radiation-induced endothelial vascular damages [113, 114]. Moreover, different phase I/II clinical trials have shown more successful RT response with the combined use of TGF-β inhibitor in metastatic breast cancer patients (LY2157299, NCT02538471). This is of special interest, because the reduction in plasma levels of TGF-β is associated with greater efficacy of RT on different types of cancer and some studies have proposed that attenuation of cytokines by genistein or quercetin ameliorates late effects of IR such as pneumonitis and fibrosis [128]. The necrosis of central nervous system (CNS) tissue is one of brain irradiation’s main risk factors. The same is true for radiation-induced increase of capillary permeability resulting from cytokine release, causing extracellular edema. A recombinant human monoclonal antibody (bevacizumab), which prevents the VEGF from binding to its receptors, reduced brain necrosis in a patient subjected to cranial irradiation and further experiments evidenced its efficacy for the management of edema associated with radiation necrosis [129]. Toll-like receptors (TLRs) play critical roles in basal resistance to IR in animals and multiple radiosensitive tissues. Several TLR ligands had been proved to exert protective roles against IR both in vitro and in vivo, downstream effectors including NF-κB (controller of inflammation, and immune response), interferon regulatory factors, and stress-­ activated protein kinase (Jnk), which in turn results in inhibition of apoptosis, promotion of cell proliferation, regulation of cell cycle, and secretion of cytokines. In cultured cells, TLR2, TLR5, or TLR9 agonists inhibit radiation-induced apoptosis and increase cell survival. CBLB502 (a TLR5 ­agonist) was reported to alleviate bone marrow and intestinal injuries in mice and rhesus monkeys. Activation of TLR4 by its agonist LPS can protect bone marrow damage and lower mice mortality after irradiation. Moreover, some kinds of TLR agonists, such as TLR2/6 coagonist CBLB613, were reported to be more effective in radiomitigation than single-­ TLR agonists. In conclusion, TLRs and their ligands provide novel strategies for radiation protection in nuclear accidents [28, 29, 55].

11.2.2 Cell Therapy Replacement IR is known to be especially damaging on highly proliferative tissues. Cellular sensitivities in approximate descending order from most to least sensitive are lymphocytes, germ cells, proliferating bone marrow and intestinal epithelial cells, and epithelial stem cells. Hematopoietic syndrome (HS) is the dominant manifestation after whole-body doses

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of about 1–6 Gy and consists of a generalized pancytopenia, due to bone marrow stem cell depletion, although, excepting lymphocytes, mature blood cells in circulation are largely unaffected. Patients remain asymptomatic during a latent period as the impediment to hematopoiesis progresses. Risk of infection and sepsis is increased as a result of neutropenia (most prominent at 2–4 weeks) and decreased antibody production. Petechiae and bleeding result from thrombocytopenia, which develops within 3–4  weeks and can persist for months. Anemia develops more slowly because circulating erythrocytes have a longer life span. Clinical management of the HS with risk of sepsis, hemorrhage, and/or acute anemia is related to the standard clinical protocols. Therapy would certainly encompass, but not limited to, the use of antibiotics, blood, and platelet transfusion, although the latter is limited by the recipient’s own immune response. Moreover, aseptic protocols must be rigidly employed. Allogeneic hematopoietic stem cell transplantation can restore bone marrow and immune functions. In the past, stem cells were harvested directly from donor bone marrow in the operating room, but at present, peripheral blood is most used as a source of stem cells for both autologous and allogeneic grafts [130]. Bone marrow stromal cell transplantation has also been shown to renew the irradiated intestinal stem and alleviate radiation-induced GIS [131]. To date, about 50 patients with acute radiation sickness have been treated with allogeneic hematopoietic stem cell transplants, but the median survival time has not yet exceeded 1  month. Despite these results, the efficacy of bone marrow transplantation in patients undergoing RT treatments highlights the need to have mechanisms in place to implement this procedure for patients exposed during a nuclear emergency [132]. Mesenchymal stem cells (MSCs) are nonhematopoietic adult stem cells with self-renewal and multilineage differentiation potential, low immunogenicity, and capacity to restore cell loss in damaged microenvironments. Moreover, MSCs secrete different interleukins, which help in the repair and recovery of cells. Although MSCs were traditionally isolated from bone marrow, cells with MSC-like characteristics are much easier to isolate from a variety of neonatal and adult tissues, including amniotic fluid, umbilical cord, peripheral blood, fat tissue, etc. Treatment with MSCs has shown efficacy in protecting the liver against radiation-induced injury; healing irradiated skin in mice; mitigating radiation-induced GIS, HS, brain injury, and neurological complications of RT; and increasing survival in irradiated mice [102]. Moreover, MSCs have successfully been assayed against radiationinduced pulmonary fibrosis (NCT02277145) and xerostomia (NCT03876197) (www.clinicaltrials.gov) [30]. Nevertheless, despite the extensive use of MSCs in preclinical and ongoing clinical trials, there is a lack of long-term safety in humans. During recent years, it has been demonstrated in animal

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models that MSC-derived extracellular (EVs). EVs can exert the same therapeutic effect of MSC; therefore, EVs can be used as an alternative MSC-based therapy [133]. To cite some examples, EVs inhibit DNA damage and cell death and preserve intestinal function [134] and bone marrow activity providing long-term survival in mice exposed to TBI [135].

11.2.3 Nonsteroidal (NSAIDs) and Steroidal Anti-inflammatory Radiomitigators Radiation initiates many enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) to produce ROS or NO, involved in the activation of inflammatory response. Most NSAIDs, such as aspirin, ibuprofen, indomethacin, diclofenac, and flurbiprofen, are able to inhibit COX-1 and COX-2 enzymes. The protective action COX inhibitors (COXi) is ascertained to the inhibition of the prostaglandin synthesis and directly or indirectly linked with the ability of NSAIDs to arrest cells in the G0 or G1 phase where cells are less sensitive to radiation damage and/or stimulation of the hematopoietic recovery [136]. Both pre- and post-irradiation treatments with sodium diclofenac reduced radiation-induced formation of DC and MN formation in human peripheral blood lymphocytes [137]. Flurbiprofen also showed radioprotection in clinical studies, e.g., delaying the onset of mucositis and reducing its severity after RT in 12 head and neck cancer patients, although the overall severity or duration of mucositis was not improved [138]. A recent meta-analysis of randomized controlled trials indicates that aspirin reduces the overall risk of recurrence and mortality of colorectal cancer and/or colorectal adenomas, which increases the interest in its possible use as a radiomitigator [24, 25]. However, ­nonselective COXi are known to cause undesirable side effects including GI ulcers and bleeding when taken for continued periods of time. Increase of COX-2 gene expression is associated with decreased survival in patients receiving RT [139]. COX-2 selective inhibitors (COXi, as celecoxib, meloxicam, indomethacin) lack the GI toxicity of classical NSAIDs, and therefore, the use of COX-2i like meloxicam has been extensively assayed. Meloxicam administered either before or repeatedly after irradiation exposure has enhanced the recovery of hematopoietic progenitor cells committed to granulocyte-­macrophage and erythroid development in sublethally irradiated mice [140], but the increase in survival was only observed when meloxicam was applied before lethal TBI. Sequential administration of PGE2 and meloxicam was shown to increase hematopoiesis and survival in irradiated mice [141], and meloxicam combined with ibuprofen treatment reduced bone loss after radiation exposure [142]. Radiation pneumonitis is a severe and dose-limiting

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side effect in lung cancer treatment. In this regard, celecoxib was tested in rats after single-dose X-ray irradiation of the right hemithorax and mediastinal region with 20 Gy revealing a dose-dependent protective effect on lipid peroxidation (MDA levels) and histopathological parameters. Celecoxib treatment induced a decrease in severe skin reactions after a high single dose of 50 Gy [136]. Moreover, celecoxib was also found to alleviate radiation-induced brain injury by maintaining the integrity of the BBB (blood-brain barrier) and reducing the inflammation in the rat brain tissues by inhibition of apoptosis in vascular endothelial cells [143]. RIVAD018 is another selective COX-2i which adds to its anti-inflammatory effects the ability to exert antioxidant activity, preventing oxidation of low-density lipoproteins, showing protection on both cellular and vascular models [144]. Several studies have also described that overexpression of COX-2 in cancer cells results in increased tumor angiogenesis, growth, and metastasis; thus, several COX-2 inhibitors have been described as radiosensitizers [136]. Celecoxib restricts neoangiogenesis, leading to a reduction in the survival of hepatocarcinoma and lung and skin cancer cells. In glioblastoma cells, the combined effect of radiation and celecoxib increased tumor cell necrosis, showing a significant reduction in tumor microvascular density compared to irradiation alone [139]. Radiation exposure of skin with high doses (>20  Gy) results in erythema, blistering, and necrosis in sequence. The necrosis generally occurs 10–30  days after exposure, although it may appear earlier in the most severe cases. The earliest administration of systemic and topical anti-­ inflammatory agents reduces the need for surgical excision of the affected tissue. Current therapy might make use of transplanted autologous keratinocytes combined with allogeneic stem cells. Advances in the knowledge of the radiomitigating properties of these compounds may prove to be very useful, particularly for the relatively low cost and toxicity, and specially for their analgesic effects [139]. Steroidal anti-inflammatory drugs such as dexamethasone can be administered after radiation exposure to attenuate fever and inflammatory or pain symptoms or to treat acute pathologies such as pneumonitis. Some authors reported that dexamethasone administration prior or immediately after radiation exposure reduced the risk of cardiac and other tissue fibrosis. Moreover, dexamethasone is often used to manage the inflammatory response in the brain during RT treatment of glioblastoma and other intracranial tumors. The effects of dexamethasone on patient survival however remain controversial because several clinical studies suggest that dexamethasone could potentially restrict effective RT [145].

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11.2.4 Probiotics, Prebiotics, and Fecal Microbiota Transplantation (FMT) Pathologically, acute intestinal epithelium damage is described as dilatation or destruction of crypt cells, decrease in villous height and number, ulceration, severe mucosal and submucosal inflammation, and sepsis associated with a pathogen bacterial translocation. Because of the rapid turnover of intestinal mucosa, the acute-phase symptoms (nausea, vomiting, diarrhea, abdominal pain, and acute mucositis) persist for hours to several months, while other intestinal complications such as obliterative vasculitis, mucosal ulceration, bowel wall thickening or progressive interstitial fibrosis, bowel obstruction, and fistulae formation, with or without fecal incontinence, are late events, often associated with chronic radiation exposition [146]. The reported incidence of severe late chronic radiation enteritis varies between 5 and 15% of patients treated with pelvic RT. Probiotics, prebiotics, and FMT target intestinal microbiota by inhibiting colonization of pathogenic bacteria and restoring microbiome normobiosis. They increase production of mucin in the intestinal epithelial cells and expression of tight junction protein and occludin, thereby enhancing mucus layer function and improving survival of intestinal crypts (Fig. 11.16).  A diverse and healthy commensal intestinal microbiota plays an essential role in GI homeostasis. It has been found that postirradiation enteropathy is associated with low mucosal microbiota diversity, in particular, a decrease of Lactobacillus and Bifidobacterium spp. and an increase in the relative abundance of opportunistic pathogens. Gut

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microbiota dysbiosis aggravates radiation enteritis, reduces the absorbing surface of intestinal epithelial cells, weakens intestinal epithelial barrier function, promotes intestinal inflammation, and contributes to the development of ­mucositis, leading to a persistent diarrhea and bacteremia [147]. Correction of the microbiome by application of probiotics, prebiotics, FMT, and/or antibiotics helps to prevent and treat radiation-induced enteritis [148]. Probiotics are live microorganisms, added to aliments, that have a beneficial role in reducing pathogenic bacteria multiplying without competitors, promoting intestinal immune barrier function, and preventing translocation of harmful bacteria. Preparations containing Bifidobacterium, Lactobacillus, and Streptococcus ameliorated radiation-­ induced gut toxicity, reducing the incidence of diarrhea, and delaying the necessity for rescue treatment with loperamide [147]. Randomized controlled trial evidenced that live Lactobacillus acidophilus plus Bifidobacterium bifidum treatment reduced the incidence of radiationinduced diarrhea and the need for antidiarrheal medication and had a significant benefit on stool consistency [149]. The anti-­inflammatory effect of probiotics has been shown in other pathologies such as ulcerative colitis and Crohn’s disease. The administration of Lactobacillus spp. decreased levels of different colonic inflammatory cytokines such as IL-6, TNF-­α, or NF-κB p65 and recruitment of leukocytes to the colonic mucosa. In mice model, administration of Lactobacillus rhamnosus increased the crypts survival in radiation-induced enteritis by approximately twofold and reduced epithelial cell apoptosis, which depends on intact TLR2 and COX-2 inhibition in

Fig. 11.16  Effect of probiotics, prebiotics, and FMT on the function of the intestinal epithelium and gut microbiome

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mesenchymal stem cells of crypt [150]. Genetically engi- 11.2.5 Angiotensin Axis-Modifying Agents neered species of Lactobacillus plantarum and Lactococcus lactis release SOD inducing anti-­Radiation nephropathy has emerged as a significant compliinflammatory effects and attenuation of enteritis symp- cation in RT and is a potential sequela of radiological terrortoms [151]. Increased production of short-chain fatty ism and radiation accidents. The use of a high-salt diet in the acids is one of the most important probiotic protective immediate post-irradiation period significantly decreases effects implicated in GI and hematopoietic tissue protec- renal injury but is deleterious for the treatment of established tion and increased survival of irradiated mice [152]. disease. FDA-approved drugs that modify the renin-­ Several clinical trials seem to indicate that probiotics angiotensin system are habitually used for the treatment of reduce the incidence of radiotherapy-induced mucositis hypertension and cardiac and/or renal insufficiency. ACEIs [148], even though results are difficult to evaluate, as they constrain angiotensin-converting enzyme (ACEs) and reduce vary in the type of cancer patients recruited, radiotherapy the formation of angiotensin II (AII). Angiotensin receptor modalities used, and type of bacteria used as probiotic blockers (ARBs) impede the function of the angiotensin AT1 [146]. In this regard, choosing the right probiotic can be or AT2 receptors and decrease the actions of AII. crucial, and a recently published systematic review conThe efficacy of ACEIs and ARBs has also been long studcludes that a combination of Bifidobacterium longum, ied for their effects in radiation protection, modulation, or Lactobacillus acidophilus, Bifidobacterium breve, mitigation (Fig.  11.17). Clinical trials have evidenced the Bifidobacterium infantis, and Saccharomyces boulardii potential of ACE inhibitors to reduce radiation-induced could be a good combination of probiotics to reduce inci- pneumonitis and fibrosis (enalapril, NCT01754909, www. dent rates of mucositis or ameliorate its symptoms in clinicaltrials.gov). chemo- or radiotherapy-treated patients [153]. Results of a recent meta-analysis review evidenced that Prebiotics offer a source of enrichment to the microbi- the use of ACEIs, but not ARBs, effectively reduced the inciome, and dietary interventions have demonstrated to reduce dence of radiation pneumonitis in most lung cancer patients. the severity of inflammatory intestinal pathologies and thus That has important clinical implications because lung cancer can potentially serve as a radiomitigative strategy. In fact, a patients receiving thoracic radiation could take an appropriclinical trial (NCT01549782) evidenced that increased con- ate dose of ACEIs to prevent radiation-induced pneumonitis, sumption of certain prebiotics (fiber and plant sugars) was during or after the period of RT, which would greatly improve associated with a reduction in days of diarrhea and improved the quality of life and therapeutic effect. By contrast, even quality of life for irradiated patients [154]. the most expensive ARBs were ineffective [158]. FMT increased the survival rate, elevated peripheral white Five different ACEIs (captopril, lisinopril, enalapril, blood cell counts, and alleviated GI toxicities and intestinal ramipril, and fosinopril), at clinically relevant doses, have epithelial integrity in irradiated mice [155]. Radiation-­ been examined for efficacy as mitigators of radiation-induced induced intestinal edema was strikingly alleviated after nephropathy. Overall, survival in rats is higher after an 8  weeks of FMT of gut microbes from healthy donors, 11–12 Gy TBI when treated with any of the ACEIs captopril, enhancing beneficial bacteria such as Alistipes, enalapril, or fosinopril starting 1 week postirradiation [159]. Phascolarctobacterium, Streptococcus, and Bacteroides All, except fosinopril, effectively abrogated radiation recovery, whereas the abundance of Faecalibacterium nephropathy, with captopril being the most effective [160]. decreased. FMT can reduce the intestinal leakage and Captopril treatment increased survival from thoracic enhance the intestinal functions and epithelial integrity in irradiation to 75% compared with 0% survival in vehiclepatients with chronic radiation enteritis [156]. treated animals, and suppression of inflammation and senesResearchers have long known that administering anti- cence markers, combined with an increase of biotics to irradiated animals can enhance survival by anti-inflammatory factors, was part of the mechanism avoiding opportunistic infections. As previously have involved in its therapeutic effects [161]. Captopril reduced been exposed, antibiotics such as fluoroquinolones and radiation-induced cytokines EPO, G-CSF, and SAA (Nonciprofloxacin also have the advantage of reducing radia- invasive serum amyloid A) in the plasma, mitigated brain tion damage to hematopoietic progenitor cells. Antibiotic microhemorrhage at 21  days postirradiation, and increased cocktail and metronidazole pretreatment are beneficial to EPO levels postirradiation if started prior to radiation expothe reconstruction of gut microbes in irradiated mice. Abx sure. These data suggest that captopril may be an ideal counpretreatment regulates macrophage polarization in the termeasure to mitigate H-ARS following accidental radiation ileum and downregulates the expression of TGF-β1, exposure [162]. A trial of captopril in patients receiving TBI thereby preventing intestinal fibrosis and ultimately demonstrated not only safety, but also efficacy against renal improving the survival of mice with radiation-induced and pulmonary injury [163]. Moreover, prophylactic adminintestinal injury [157]. istration of captopril reduced radiation-induced hypertension

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Fig. 11.17  Role of ACEIs, ARBs, and renin inhibitors in the renin–angiotensin system

and renal failure and mitigated pulmonary endothelial dysfunction and radiation-­ induced pneumonitis and fibrosis. The isoflavone genistein appears to work synergistically with captopril, improving the 30-day survival in mice receiving both drugs from 0 to 95% after 8.25 Gy TBI. The combination therapy reduced anemia and increased the number of circulating hematopoietic cells [164]. In murine models, administration of AT1 receptor antagonist before, during, and after fractionated whole-brain irradiation prevented or reduced cognitive impairment. It is also hypothesized that ARBs may attenuate radiation-induced brain injury by increasing the generation of anti-­inflammatory peptide, angiotensin (1–7). ACEI or AT1 antagonist treatment in hypertensive patients increases blood levels of angiotensin (1–7); prevents oxidative stress, inflammatory cytokine release, and fibrotic events; and also has anticarcinogenic effects, thus having radiomitigating potential as it has been evidenced recently [165]. While other types of antihypertensive drugs are ineffective, ACEIs and AII receptor antagonist type I are effective in

the mitigation of radiation damages. Moreover, some of them also exhibit antitumor effects; thus, there is a strong case for the clinical use of these agents in the treatment of radiation-induced late effects.

11.2.6 Statins The incidence of cardiovascular disease was observed in the atomic bomb survivors, and cardiovascular disease is a known side effect of radiation therapy [166]. Statins (simvastatin, lovastatin, pravastatin, and others) are inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is a rate-limiting enzyme for the synthesis of cholesterol and serves to upregulate low-density lipoprotein (LDL) ­synthesis. Therefore, statins are clinically used to reduce LDL levels in the blood and, consequently, to treat atherosclerosis and hypercholesterolemia. Statins also strongly induce thrombomodulin (TM) expression, which in turn forms a complex with thrombin. Thrombin-TM complexes activate protein C,

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which has anti-inflammatory, anticoagulant, and antioxidant properties. All these beneficial effects may help to attenuating radiation injuries [167]. Radiation exposure (5  Gy X-rays) increased cholesterol levels, and those were reduced by simvastatin treatment [168]. Simvastatin treatment (20 mg/kg/d over 2 weeks) mitigates, to a limited extent, radiation-induced enteric injury (4–8  Gy), as evidenced by improved structural integrity of the mucosa, reduced neutrophil infiltration, decreased thickening of the intestinal wall, and reduced accumulation of collagen I in jejunum and bone marrow in male C57BL/6J mice [169]. Simvastatin also prevented radiation-induced marrow adipogenesis and provided radioprotection to the niche cells [170], and attenuated radiation-induced salivary gland dysfunction in mice [171]. Pathak et al. [167] demonstrated that a single subcutaneous dose of γ-tocotrienol (GT3) rescues mice from lethal radiation doses, and combined treatment (GT3 + simvastatin) provides substantial protection against radiation-induced lethality, hematopoietic injury, and bone marrow damage compared to the single treatment. A combination of statin and ACEI agents has shown efficacy in reducing GI toxicity in patients receiving pelvic RT [172]. Lovastatin treatment of irradiated mice (15 Gy whole-­ lung irradiation), starting immediately after irradiation or 8 weeks post-irradiation (three times a week), demonstrated a reduction in lung tissue lymphocytes and macrophages, decreased collagen content, prevented lung fibrosis, and improved rates of survival [173]. Pravastatin (30 mg/kg body weight given 4 h before irradiation) protected the normal intestine and lung tissues from radiation. The radiomitigating effect of pravastatin was associated with a reduction in the level of radiation-induced DNA DSB.  The pravastatin-treated group showed a significantly lower apoptotic index of the lung and intestinal epithelial cells and reduced the intestinal expression of ataxia-­ telangiectasia mutated and γ-H2A histone family member X (H2AX) after irradiation [174]. Statins are generally well tolerated, and their effect was pronounced for delayed radiation injury and for that reason shows potential as radiomitigators.

11.2.7 Growth Hormone (GH) and Somatomedin C (IGF1) Analogs Long et al. [175] demonstrated that chimeric protein dTMPGH, a tandem dimer formed by thrombopoietin mimetic peptide and GH treatment, increased survival in mice exposed to 60Co γ-ray photons (6 Gy). Meanwhile, dTMPGH treatment accelerated the recovery of bone marrow hematopoiesis, promoted skin wound closure, and mitigated ileum injury. Zinc sulfate and GH administration prevented radiation-induced dermatitis in rats [176], and increased GH/

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IGF1 levels also reduced radio-induced intestinal epithelial cell apoptosis preserving, in the short term, the efficacy of RT on tumors [177]. GH significantly restored follicular development and preserved fertility in female rats exposed to a single TBI of 3.2 Gy [178]. However, in oncology, GH and IGF1 reduce the effectiveness of RT and may frequently cause metastasis and cancer recurrence. Therefore, even if GH/IGF-derived radiomitigative effects are confirmed, further studies of these hormonal treatments would be necessary before translating the results to human clinical trials.

11.2.8 Molecular Hydrogen (H2) Hydrogen can mitigate IR damages through various mechanisms [122, 123]: (a) directly neutralizes hydroxyl radicals and peroxynitrite [179]; (b) indirectly reduces oxidative stress, by upregulating the expression of different endogenous antioxidant enzymes, i.e., SOD, CAT, and GPx; and (c) shows antiapoptotic and anti-inflammatory properties [180]. H2 reduces 8-hydroxy-2′-deoxyguanosine and malondialdehyde levels and increases SOD activity and GSH levels. These findings suggest that the radioprotective effect of H2 is largely due to the inhibition of oxidative stress. In that sense, H2 has demonstrated in vitro radioprotective effects in cells especially sensitive to IR, such as intestinal epithelial cells, hematopoietic precursors, and spermatogonia [180] these protective effect of H2 are not significant when it is administered after radiation [181].  Shin et al. [182] observed that application of H (H2O) to human skin prevented UV-induced erythema and DNA damage, administered even after exposure to RI. Although a lot of in vitro and in vivo research has been done to investigate the potential use of H2 as a radiomitigator, there are scarce clinical data. Kang et  al. [183] performed a placebo-controlled, randomized study to evaluate the validity of ingesting hydrogen-rich water in 49 patients with malignant liver tumors, while they were receiving RT at the same time. Patients drinking H2-rich water had considerably higher quality of life (QOL) scores, notably less appetite loss, and much fewer tasting disorders than patients drinking placebo water, and most importantly, no differences were found in tumor response to RT comparing both groups of patients [183]. In cancer patients, H2 has also shown protective effects against brain, lung, and myocardial injury associated with RT, furthermore preventing side effects like anorexia, taste disorders, or bone marrow damage without compromising the antitumor effects of the treatment [180]. The use of H2 is feasible in the clinical practice because it is stable at normal temperatures; it can be easily administered through various routes such as inhalation, drinking, injection, etc. (Fig. 11.18); it can even cross the blood-brain barrier; has a very favorable tolerability profile; and it shows

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Fig. 11.18  Delivery of hydrogen and its protective and therapeutic opportunities in various systems. Adapted from “Molecular hydrogen: A potential radioprotective agent,” by Hu et al. [122, 123], Licensed under CC BY 4.0

great efficacy as a potential radioprotective agent [122, 123]. Although the human body does not have the enzymes necessary to produce H2, the colonic microbiota can produce about 12  L of H2 per day under physiological conditions. Many results support the idea that upregulation of H2 gas produced by intestinal bacteria could be used as a valid treatment strategy for various diseases. Since there are several methods to supply external H2, it can be easily administered with little or no adverse effects.

11.2.9 Vitamins 1-Methyl nicotinamide (MNA), a derivative of vitamin B3, significantly prolonged survival of mice irradiated at LD30/30 (6.5 Gy), LD50/30 (7.0 Gy), or LD80/30 (7.5 Gy) of γ-rays when the MNA administration started as late as 7  days post-irradiation. Another vitamin B3 derivative, 1-methyl-3-acetylpyridine, was slightly less efficient when it was administered after 7.5 Gy γ-ray exposition. These prosurvival effects might be related to the anti-­inflammatory and/or antithrombotic properties of the vitamin B3 derivatives and do not seem to be mediated by stimulation of hematopoiesis. These results show that MNA may represent a prototype of a radiomitigator because it reduces the severity and/or progression of radiation-induced injuries when applied several hours or days after exposure to high doses of IR [184].

11.3 Internal Contamination by Radionuclides and Treatment After various radiological and nuclear incidents, radioactive materials (radionuclides) may be released in the atmosphere where they could be either inhaled as gas, ingested as particulates, or absorbed through intact skin or subcutaneous tissue [185]. The medical consequences of internal contamination are determined primarily by radiation dose and radiation quality. Deleterious effects include dose-dependent deterministic (i.e., predictable) effects; stochastic (i.e., random) effects such as cancer in tissues where radionuclides are retained for prolonged times, and at a sufficiently high quantity of contamination; multiorgan failure; and death. The radiation quality or specific radionuclide(s) has (have) a characteristic emitted energy (alpha, beta, or gamma/Xray), solubility, radioactive half-life, and biological halflife, which is determined by the time required for a compartment, defined by a body organ or tissue or part of an organ or tissue (see Fig. 11.19) to eliminate half of its radionuclide content. The particle size and chemical composition of the radioactive material impact the site of deposition within the body and route of elimination. Finally, comorbidities such as renal insufficiency, hepatic failure, and pulmonary disorders may impair pathways needed for radionuclide elimination from the body, thereby prolonging exposure [186].

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Fig. 11.19 Biological compartments for radionuclide intake and distribution. Reproduced from Dainiak N and Albanese J, Assessment and clinical management of internal contamination, JRP, 2022, in press, and modified from ICRP, 2015, Occupational Intakes of Radionuclides: Part 1. ICRP Publication 130. Ann. ICRP 44(2)

The  internal contamination with radionuclides involves four metabolic phases: 1. Intake (incorporation) 2. Uptake (absorption into the circulatory system) 3. Retention (deposition) 4. Excretion (decorporation) The excretion of these radionuclides by natural processes can be accelerated using decorporation therapies. This consists of enhancing the action of biological processes through chemical or biological agents, thereby facilitating radionuclide elimination. In the event that radionuclides have been incorporated internally, the objective of the therapy is to reduce the internal dose and thus the risk of biological effects on health. This can be achieved by preventing the incorporation, reducing the absorption and internal deposit of radionuclides, and also promoting their excretion. The decorporation process may have adverse side effects. Therefore, these therapies must be based on risk criteria and applied as soon as possible. The general procedures are intended to reduce or inhibit the absorption of radionuclides from the GI system, the respiratory tract, or the skin and wounds (Fig. 11.19). Some examples of general procedures are the use of emetics, gastric lavage, laxatives, gastric alkalinization, and irrigation if

there are wounds, especially in an emergency scenario. The use of specific drugs to impede the deposition of radionuclides (decorporation agents) in organs or tissues could avoid accumulation and retention of radionuclides and, obviously, is more effective if treatment is started immediately after internal contamination. Decorporation agents can reduce radionuclide absorption, entry, and deposit in organs and tissues and/or accelerate its excretion, finally minimizing the absorbed dose.

11.3.1 Blockers (Metabolic Blocking) Blocking agents work by reducing the absorption of the radionuclide in the body, since they saturate tissues, organs, and metabolic processes using a stable isotope (identical to the nonradioactive element). Among these agents, the best known is potassium iodide (KI), used to prevent the deposit in the thyroid gland of radioactive iodine delivered to the atmosphere as a result of uncontrolled nuclear accident, which can lead to an increased risk of developing thyroid cancers, particularly in infants and young children [187]. KI prevents binding of radioiodine by three mechanisms: a) it will dilute the radioiodine circulating inside the body and available for thyroid uptake; b) it will saturate the active transport mechanism of iodine mediated by the sodium

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iodine symporter (NIS); and c) it will inhibit the organification of iodine, also called Wolff-Chaikoff phenomenon, a mechanism that could lead to a decrease in the synthesis of thyroid hormones and a possible hypothyroidism; but this effect is usually of short duration. This measure only protects the thyroid from radioactive iodine, not other parts of the body. Pharmacologic thyroid blockade by oral KI (50–100 mg in adults) can substantially reduce radioiodine thyroid uptake and was one of the first and urgent protective actions recommended  by the World Health Organization (WHO) (1960–1970s). The recommendations adopted for iodine prophylaxis, in particular those regarding the administration timing, the iodine quantity to be given, and the possible side effects occurring as a result of this measure, are included in the Guide [188]. Although stable iodine is usually considered as the standard for thyroid protection against radioiodine [189], perchlorate can be considered as an alternative, provided that it is administered at equi-effective dosages (1000  mg perchlorate is as effective as 100 mg stable iodine in the aftermath of an acute radioiodine exposure). Perchlorate also protects the thyroid by competition with radioiodine at the NI-symporter site. Considering its simpler protective mechanism and potential advantages in particularly vulnerable subpopulations and its acceptable adverse effects, it seems promising for future studies to focus more closely on perchlorate as an alternative to stable iodine for thyroid protection against radioiodine [187].

11.3.2 Reduced Absorption

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11.3.3 Dilution (Isotopic Dilution) Increasing the intake of liquids, such as water, milk, and tea, or intravenous administration of isotonic saline solution, is a rapid method to increase the excretion of soluble radionuclides. This would be the case of tritium, where ingestion of sufficient liquids reduces the time of permanence in the body [192].

11.3.4 Displacement Displacement shares the same principle as dilution and blocking therapies. However, in this specific case, an element is used that has a different atomic number. Thereby, that element will compete for internal scavenging sites, displacing the radioisotope from a receptor/target. Calcium gluconate, for example, competes with radiostrontium in bone deposition, or stable iodine, which displaces technetium-­ 99m [193]. This method consists of increasing the natural renewal process of the release of radionuclides from organs and tissues, thus reducing deposition and improving the elimination rate by diuresis. As an example, ammonium chloride, which if administered orally, lowers the pH of the blood and increases the elimination of radiostrontium once internalized. Or the use of sodium bicarbonate increases the pH of the blood and favors the removal of uranium [194].

11.3.5 Chelators and Functional Sorbents

Chelating agents are classified as organic or inorganic agents capable of binding to metal ions and forming complex ring Absorption is defined as a movement of material that reaches structures, known as “chelates.” These agents possess atoms the blood regardless of the mechanism. This generally of union or “ligands” that generally form covalent bonds and applies to the entrance in the bloodstream of soluble sub- facilitate the excretion by the kidneys or other organs [186]. stances and material dissociated from particles (NCRP 161). Some examples of this method are the one used to faciliPrussian blue, a nonabsorbable resin (approved by the tate the elimination of plutonium complexes by the kidneys FDA), acts as a laxative agent that promotes the fecal elimi- and the GI. DTPA (diethylenetriaminepentaacetic acid with nation of ingested radiocesium and thallium. The most effec- calcium or zinc) is the chelator with the widest range of tive form of this compound is its colloidal soluble form. This potential use [186]. Other chelators commonly used are compound was used in the Goiânia accident extensively and dimercaptosuccinic acid, dimercaprol, and deferoxamine. successfully for the decorporation of 137Cs. Different silica-­ Different silica-based materials (such as isomers of diphosbased materials have also been tested to capture various phonic acid, hydroxypyridinone, acetamide phosphonic radionuclides of plutonium, americium, uranium, and tho- acid, DTPA, and glycinyl-urea) have also been tested to caprium [190]. ture various radionuclides of plutonium, americium, uraNatural products have also been used to reduce the nium, and thorium [190]. Importantly, factors that can absorption of radionuclides. An example is that orally potentially affect the stability of any chelating agent must ­administered Chlorella algae inhibited the absorption of always be taken into account, i.e., (but not limited to) acidity strontium (90Sr) into the blood and enhanced its fecal elimi- and alkalinity, chemical properties of the agent, its selectivnation [191]. ity, and concentration of competing metals.

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Internal contamination with actinides, whether by inhalation, ingestion, or injuries, represents a serious risk to the health. Some guidelines to assist physicians or other professionals in treating workers or members of the public who may suffer internal contamination with compounds such as plutonium tributyl phosphate, plutonium nitrate, americium oxide, or nitrate can be found in [195]. The use of these types of agents is most effective when administered immediately after exposure to radiation because the radionuclides are still circulating in the body and may not yet have deposited in target organs or cells (liver and bone are examples of preferred targets).

11.3.6 Surgical Excision This method is used for the elimination of a fixed radionuclide contaminant in the body. The surgery must be evaluated carefully, taking into account risks and benefits, and must be carried out with the support and collaboration of radiation protection staff [196]. Occasionally, debridement and excision of the wound may be necessary in order to remove the fixed contamination. It is important that a well-established evaluation is carried out by specialized personnel to support the medical decision, considering the benefits and risks of the surgical procedure. When surgical exploration is necessary, as well as

Fig. 11.20  Isotopes and focal accumulation in the body

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the removal of tissue/foreign material, it should be performed with the help of a radiation protection professional, a radiophysicist who uses a specific probe for wounds. Once the surgical material has been removed, it should be saved for subsequent radioanalysis. There are no contraindications regarding the use of local anesthetics or systemic anesthetic agents.

11.3.7 Lung Lavage (Mechanical) Lung lavage is an invasive medical procedure that involves the same risks as general anesthesia and is only indicated for a limited number of cases. The parameters that are taken into account are the patient’s age, clinical status, existence of comorbidities, radiotoxicity of the contaminant, and dose. This technique will only be used after a meticulous medical and dosimetric evaluation, and in case inhaled and insoluble radioactive particles (plutonium for example) are deposited in the lungs. Other isotopes and focal accumulation are depicted in Fig.  11.20. A flexible bronchoscopy should be performed to enhance bronchoalveolar lavage [197]. This type of bronchoscopy should be performed only if the lung load is high and incorporates a large amount of insoluble inhaled particles, such as alpha particles (α). The objective of this procedure is to avoid deterministic effects for pulmonary doses above 6 Gy-equivalents (Gy-Eq)

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and is stochastic when the committed doses are lower in the lung. All this within a period of 30 days and individualized for each case. The Clinical Decision Guide (CDG) and the IAEA EPR 2018 Guide provide bases that can be used by healthcare providers to treat cases where radionuclides have been deposited internally as explained above. Both guides are useful for medical management of individuals contaminated with radionuclides as a consequence of a nuclear or radiological emergency, or due to an industrial scintigraphy accident, or in patients undergoing treatments with radionuclides. See Annex 1: Contamination by radionuclides and MCMs table.

ER =

11.4 Radiosensitizers Radiotherapy (RT) is a treatment that uses high doses of radiation to kill cancer cells and shrink tumors. Radiosensitizers are chemicals or pharmaceutical agents that increase the cytotoxic effect of IR on cancer cells by accelerating DNA damage and producing free radicals, suppressing the antioxidant mechanism of defenses, or inhibiting the repair of biomolecules, among others. In most cases, radiosensitizers have less effect on normal cells; however, some can also be administered after radiation exposure to treat or reduce the late side effects to healthy tissue. The effectiveness of potential radiosensitizers is measured in terms of the enhancement ratio (ER) (Box 11.4):

Radiation dose required to obtain a given biological effect Radiaation dose required to obtain the same effect in the presence of sensiitizer

Box 11.4: Radiosensitizers • Radiosensitizers specifically target tumor cells and make them more susceptible to IR during RT. • These therapeutic compounds apparently enhance the radiation-induced damage to cancer cells at the molecular level and may also further limit the harmful effects of radiation on normal tissue. • Radiosensitizing agents promote fixation of free radicals by their electron affinity, rendering the molecules incapable of repair. • Their mechanism of action is comparable to the oxygen effect, as biochemical reactions of the damaged molecules preclude the repair of cellular damage.

Characteristics of an Ideal Radiosensitizer For use as an adjunct in RT, an ideal radiosensitizer should not be harmful to healthy tissues and not interfere with other therapies, as well as should be highly efficient on tumor and hypoxic cells. It should also be economically affordable. A radiosensitizer should be nontoxic and should produce an advantage in enlarging the therapeutic window, increasing tumor control probability, and limiting the normal tissue toxicity. This effective gain could result from a selective uptake or absorption rate or half-life of the radiosensitizing molecule in a tumor with respect to normal tissue. Mechanism of Action Radiosensitizers have been developed to modulate the response that occurs during or after the radiation exposure.

At a molecular level, these molecules stimulate the fixing of free radicals generated by radiation. Similarly to the oxygen effect, the biochemical mechanism prevents the repair of damaged molecules. The electron affinity of the radiosensitizers captures independently existing free radicals, rendering the molecules incapable of repair [198]. Although each radiosensitizer has different rationales and limitations, they interact with specific biological targets, i.e., the signaling pathway/cascade (Table 11.1) at diverse levels (Fig. 11.21) from molecules to cells to tissues to organs to a whole organism. The core mechanisms for radiosensitization include: • Inhibiting repair of radiation-induced DNA damage, thereby increasing the degree of radiation-induced apoptosis and DNA damage • Improving cytotoxicity by disrupting the cell cycle and organelle function • Activating and regulating the expression of radiation-­ sensitive genes or silencing genes related to radioresistance Classification Based on the DNA damage and repair mechanisms, radiosensitizers are divided into five groups [199, 200]: (1) reduction of thiols or other intracellular radioprotective molecules; (2) radiolysis of the radiosensitizer, which results in the production of cytotoxic chemicals; (3) inhibitors of repair of biomolecules; (4) thymine analogs incorporated into DNA chain; and (5) oxygen mimetics with electrophilic properties. With the continuous technological innovation, radiosensitizers can be classified into three categories: (1) molecular

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Table 11.1  Potential biological targets at different levels for developing radiosensitizers Levels Reactive oxygen species DNA damage response

Target (molecules/proteins/enzymes involved in signaling pathways/cascades) Targeting mechanisms to generate free radicals Targeting key DDR proteins  • DNA-PKcs  • ATM/ATR  • PARP family  • MRN (MRE11-RAD50-NBS1) complex  • MDC1, Wee1, LIG4, CDK1, BRCA1, CHK1, and HIF-1 Functional organization of Targeting inhibitors of chromatin changes genome (chromatin organization)  • DNA methyltransferase  • Histone acetyltransferase, deacetylase, methyltransferase, demethylase Cellular response to signals Targeting cell cycle proteins  • Blockage of cell cycle checkpoints (G2/M transition)  • Inhibitors of cell survival proteins  • Oncogenes (p53, ras)  • Evading growth suppressors  • Biomechanical effects of microbubbles Tumor microenvironment Targeting  • Prolyl-4-hydroxylases (PDH)  • Oxygen-independent mechanism, including PI3K/AKT and MAPK, or through loss of tumor suppressor protein von Hippel-Lindau (VHL)  • VEGF  • ECM remodeling within tumors Tissue-level effects Targeting  • Inhibitors of angiogenesis (antiangiogenic and/or vascular targeting agents)  • Inhibitors of growth factor signaling  • Anti-VEGF/VEGFR antibodies, antisense suppression of VEGF, VEGFR tyrosine kinase inhibitors, viral-directed targeting of VEGFR signaling  • Blockage of growth factor secretion from dying cells Abbreviations: DDR DNA damage response, DNA-PKcs DNA-­dependent protein kinase, ATM/ATR ataxia–telangiectasia mutated and ATM and Rad3 related, PARP poly[ADP-ribose] polymerase, MDC1 mediator of DNA damage checkpoint protein 1, LIG4 ligase IV, CDK1 cyclin-dependent kinase 1, BRCA1 breast cancer gene 1, CHK1 checkpoint kinase 1, HIF-1 hypoxia-inducible factor-1, ECM extracellular matrix

Fig. 11.21  Development of potential radiosensitizers at different levels. Potential radiosensitizers can be developed focusing on the molecular, cellular, or organismic levels, which may be useful in modulating the radiation effects on cancer cells as well as on normal cells

606 Table 11.2  Small molecules as radiosensitizers Hyperbaric oxygen  A potent radiosensitizer, which promotes toxic and relatively stable free radical formation, useful to effectively enhance the radiosensitivity of the tumors which contain numerous hypoxic cancer cells. NItroxides  The most representative are nitro-containing compounds (such as nitrobenzene, nitroimidazoles, and its derivatives) and nitric oxides (NOs). These are “true radiosensitizers,” having higher electron affinity and better diffusion properties than molecular oxygen. It can theoretically substitute for oxygen in “repairing/ fixing” radiation-induced DNA damage. Carbogen  A mixture of 95% oxygen and 5% carbon dioxide, which improves tumor oxygenation contrasting with hypoxia. Hypoxia-specific cytotoxins  Bioreductive agents, such as aromatic N-oxides, transition metal complexes, quinones (mitomycin C, porfiromycin, and E09), aliphatic N-oxides, and nitro compounds, that selectively radiosensitize the hypoxic cells by virtue of their preferential cytotoxicity. Chemical radiosensitizers  Chemicals targeting a variety of cell signaling pathways, suppressing radioprotective substances, pseudo-substrates, and targeted delivery systems for radiosensitization. Examples are BKM120 (an oral pan-class I PI3K inhibitor), targets of PI3K-Akt pathway, NVP-BEZ235 (a mTOR inhibitor), AMG 232 (an MDM2-p53 interaction), GSH inhibitors, and radiosensitizing nucleosides (5-fluorouracil (FUra), bromodeoxyuridine (BrdUrd), iododeoxyuridine (IdUrd), hydroxyurea, gemcitabine (dFdCyd), fludarabine). Natural radiosensitizers  Natural molecules are safer than synthetic compounds and have anti-inflammatory and antioxidant properties: curcumin, genistein, resveratrol, zerumbone, ursolic acid, etc.

structures of small molecules (Table  11.2); (2) macromolecules with their mechanism of radiosensitivity (Table  11.3); and (3) nanomaterials (Table 11.4) with low cytotoxicity, good biocompatibility, usability, and functionality (Box 11.5).

11.4.1 Nutraceutical Compounds Several nutraceutical chemicals have attracted significant interest in recent decades due to their possible involvement in the prevention and treatment of various illnesses, as well as their favorable effects in boosting human and animal health. In particular, literature data often report their positive effect in combination with chemotherapy in cancer care. Even while intriguing results have been published on this issue at multiple cellular levels, less is known about their role as radiosensitizers. Presence of these compounds during radiation augments their effect by several mechanisms including the lethal reactions of free radicals. Among compounds of various origins that showed radiosensitizer potential, numerous studies have revealed the

A. Montoro et al. Table 11.3  Macromolecules as radiosensitizers Proteins and peptides  Antibody conjugates and cell-penetrating peptides selectively deliver a cytotoxic payload to a tumor and spare most healthy cells. Examples are HER3-ADC (targeting HER3), SYM004, and nimotuzumab (targeting EGFR) and cetuximab (inhibitor of EGFR). miRNAs  Endogenous noncoding microRNAs (miRNAs) can be used as RT sensitization targets. These can be regulatory miRNAs of DNA damage response (DDR) and HR repair factors. siRNAs  Exogenous short interfering RNAs or silencing RNAs (siRNAs), which are noncoding RNA molecules, that can selectively target key mRNAs belonging to pathways involved in the response to radiation, such as DDR, cell cycle regulation, and survival/ apoptosis balance. Oligonucleotides  Small DNA or RNA sequences are able to disturb key mRNA translation. Studies have concerned oligonucleotides targeting the telomerase RNA subunit or telomerase reverse transcriptase (hTERT) or cyclic AMP response element (CRE) decoy oligonucleotide.

important role of molecules of natural origin, when administered in combination to IR. The use of nutraceuticals as sensitizers, in addition to being generally well tolerated, is also easily recovered and less expensive in comparison to synthesized drugs. Their administration reduces the collateral effects frequently associated with medication delivery, and in certain situations, they can help attenuate IR adverse effects through biological processes like those shown in Fig.  11.22. Indeed, in most cases, they show anti-inflammatory and antioxidant properties, which are precious arms to counter the RI side effects on healthy tissues. However, in most cases, they showed direct anticancer activity, as demonstrated by numerous scientific papers. The most studied natural compounds are exposed in Table 11.5 [203].

11.4.1.1 Curcumin Curcumin, the main component in the Indian culinary spice turmeric (Curcuma longa), has been shown to have anticancer potential in several studies. The biological mechanism can be ascribed to cell signaling pathway effects, resulting in the inhibition of cell proliferation and induction of apoptosis. Regarding its radiosensitizing properties evaluated by an in vitro approach, the inhibition of survival and proliferation has been observed on the MCF-7 breast cancer cell line. In addition, the effect of vehicolated curcumin, using solid nanoparticles, combined with X-ray radiation was tested by Minafra and coworkers [204] on the human non­tumorigenic breast epithelial MCF10A cell line and the breast adenocar-

11  Radioprotectors, Radiomitigators, and Radiosensitizers Table 11.4  Nanomaterials as radiosensitizers Noble metal nanomaterials  Nanoparticles, such as gold (Au, Z = 79), silver (Ag, Z = 47), and platinum (Pt, Z = 78), can effectively interact with radiation, emitting secondary electrons which amplify the radiation effects. Heavy metal nanomaterials  Physical dose enhancement methods are comparable for gadolinium (Gd, Z = 64), hafnium (Hf, Z = 72), tantalum (Ta, Z = 73), tungsten (W, Z = 74), and bismuth (Bi, Z = 83) or their stable forms such as oxides, sulfides, and selenides. Examples are gadolinium-based nanoparticles (AGuIX), hafnium oxide (HfO2) nanoparticle (NBTXR3), tantalum pentoxide (Ta2O5) and tantalum oxide (TaOx), bismuth oxide (BiO) nanoparticles, and tungsten oxide nanopowder or nanoparticles (WO3). Ferrite nanomaterials  They can catalyze the reaction of H2O2, generating highly toxic hydroxyl free radicals in the tumor microenvironment with the aim of boosting the radiation therapeutic efficacy. Explored examples are superparamagnetic magnesium ferrite spinel (MgFe2O4) nanoparticles (SPMNPs) and zinc ferrite (ZnFe2O4) nanoparticles Semiconductor nanomaterials  Semiconductor nanosensitizer materials, such as silicon (Si), germanium (Ge), gallium arsenide (GaAs), and semiconductor quantum dots, have unique properties making them great candidates as photosensitizers and radiosensitizers for tumor treatment ([201]; [202]). Explored examples are WO2.9-WSe2-­ PEG semiconductor heterojunction nanoparticles (WSP NPs), titanium peroxide (PAA-TiOx) nanomaterial, copper bismuth sulfide (Cu3BiS3,CBS) nanoparticles, and TiO2 nanotubes. Nonmetallic nanomaterials  Similarly to the metallic nanoparticles’ mechanism of action, nonmetallic nanomaterials can increase oxidative damage. Explored examples are ultrasmall uncapped and amino-silanized oxidized silicon nanoparticles; nanocrystals of underivatized fullerene, C60, (nano-C60); nanodiamonds and carbon nanotubes; and selenium (Se) nanoparticles. Nanostructured substances and drug delivery systems  Chemicals, oxygen carriers, siRNAs, and other radiosensitizing agents are transported via relatively new nano-based delivery systems. Explored examples are the poly(D,L-lactide-co-­ glycolide) (PLGA) nanoparticles containing paclitaxel (a cell cycle-specific radiosensitizer) and etanidazole (a hypoxic radiosensitizer).

Box 11.5: Radiation Sensitizers

• Small molecules are classified based on radiation-­ induced free radicals, pseudo-substrates, and other mechanisms. • Macromolecules such as miRNAs, proteins, peptides, and oligonucleotides have been explored to develop radiosensitizers as they are capable of regulating radiosensitivity. • Promising nanotechnology methods used as radiosensitizers include well-developed nanomaterials with low toxicity, good biocompatibility, and functionalization ease. • Other technologies, such as molecular cloning technology, analysis of molecular structure, and bioinformatics, can speed up the development of new effective radiosensitizing drugs.

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cinoma MCF7 and MDA-MB-231 cell lines. The vehicolated curcumin has been shown to be more effective than the free curcumin on MCF7 and MCF10A, whereas the free molecule resulted to be slightly more effective on MDA-MB-231. The dose-modifying factors (DMFs) were calculated to quantify the radiosensitizing effect, which resulted in 1.78 for MCF7 using vehicolated curcumin and 1.38 with free curcumin on MDA-MB-231 cells. Transcriptomic and metabolomics approach supported this study, revealing the double-positive effect of curcumin as an autophagy enhancer for tumor cells and antioxidant agents [204]. Antiapoptotic signals and block in G2/M cell cycle phase mediated by Bcl-2 were demonstrated in human immortalized prostate adenocarcinoma cells (PC-3) after 5 Gy irradiation combined with 2μM curcumin. Instead, an increased radiosensitivity was observed in HCT116 and HT29 human colorectal cancer cell lines treated with 25μM of curcumin and a single dose of X-ray radiation (10 Gy). Curcumin was also able to decrease COX-2 expression by the inhibition of EGFR phosphorylation both in vitro on the human head and neck squamous cell carcinoma (HNSCC) cell line and in two in vivo models of head and neck tumor. On the human glioblastoma U87MG cell line, the viability was reduced in a dose-dependent manner by 3  Gy of X-ray combined with curcumin at a concentration range of 5–10μM, sustained by the arrest of cell cycle in phase G2/M (which is the most sensitive step to radiation) and the ­inhibition of two master regulators of tumor progression, the MAP kinases ERK and JNK [203]. However, curcumin is an unstable, nonbioavailable compound due to its poor absorption in the GI system. Hence, its therapeutic application is delimited by its pharmacokinetics. Despite promising preclinical studies, no double-blinded placebo-controlled clinical trial, using curcumin as a radiosensitizer, has been successful. The interaction of curcumin with RT on different cancer types has been reviewed by Verma [205]; however, there is still a lack of solid clinical evidence of radiosensitization. For instance, in  vitro and in vivo studies together with clinical bioavailability data do not give evidence for a radiosensitizing effect of curcumin in the treatment of high-grade brain tumors (glioblastoma multiforme). On the other hand, there is limited data on curcumin’s radioprotective function, despite the fact that some clinical trials suggest that curcumin is beneficial for the management of radiation toxicities [205].

11.4.1.2 Resveratrol (RV) and Pterostilbene (PT) The antineoplastic ability of RV encouraged its application also as a radiosensitizer to overcome radioresistance of many cancers. A dose-dependent reduction in the surviving fraction of a non-small cell lung cancer (NSCLC) cell line after ­irradiation with 0–8 Gy of γ-rays in combination with 20μM of RV was

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Fig. 11.22  Radiation therapy and nutraceutical substances may influence signaling pathways involved in migration, inflammatory response, autophagy, and formation of reactive oxygen species (ROS). Adapted

from “Nutraceutical Compounds as Sensitizers for Cancer Treatment in Radiation Therapy,” by [203], Licensed under CC BY 4.0

Table 11.5  Natural compounds related to cancer radiation treatments Natural compounds Curcumin Resveratrol Withaferin A Celastrol Ursolic acid Zerumbone Caffeic acid phenethyl ester Emodin Flavopiridol Berberine Genistein Selenium

Tumor target Colorectal cancer, glioblastoma, head and neck squamous cancer, prostate cancer Breast cancer, glioblastoma, head and neck squamous cancer, melanoma, nasopharyngeal carcinoma, non-small cell lung cancer, prostate cancer Breast cancer, cervical cancer, Ehrlich ascites carcinoma, fibrosarcoma, histiocytic human lymphoma, liver cancer, melanoma, renal carcinoma Lung cancer, prostate cancer Colon carcinoma, gastric adenocarcinoma, non-small cell lung cancer, melanoma, prostate cancer Colorectal cancer, glioblastoma, lung adenocarcinoma, non-small cell lung cancer, prostate cancer Adenocarcinoma, breast cancer, lung cancer, medulloblastoma

Type of treatment X-rays γ-rays, X-rays

Cervical cancer, hepatocellular carcinoma, nasopharyngeal carcinoma, sarcoma Cervix cancer, esophageal adenocarcinoma, esophageal squamous carcinoma, glioma, lung carcinoma, ovarian carcinoma, prostate cancer, zebrafish model Breast cancer, esophageal carcinoma, nasopharyngeal carcinoma, osteosarcoma, prostate cancer Breast cancer, cervical cancer, non-small cell lung cancer Melanoma, glioma, breast cancer

γ-rays, X-rays γ-rays, X-rays

γ-rays, X-rays γ-rays, X-rays γ-rays, X-rays γ-rays, X-rays γ-rays, X-rays

γ-rays, X-rays γ-rays, X-rays γ-rays, X-rays

11  Radioprotectors, Radiomitigators, and Radiosensitizers

observed along with accelerated senescence and cell death following enhanced DNA DSB induced by ROS [203]. However, an increased expression of LC3-II for autophagy response after X-ray and RV treatment (75μM) was demonstrated in SU-2 glioblastoma multiforme cell lines [206]. Also, in GBM, RV showed inhibition of the hypoxia-­ inducible factor HIF-1α, which is responsible for a well-­ known mechanism of radioresistance. Moreover, the interaction of RV with other agents as iododeoxyuridine (IUdR) was also tested and demonstrated the ability to decrease the formation of cancer colonies [203]. In the HNSCC cancer model, suppression of cell proliferation was obtained on a cell line, treated with 100μM of RV combined with 10 Gy of X-ray, also observing the inhibition of STAT3 phosphorylation, a well-known transcription factor driving inflammation and cancer progression. Even the peanut stem extract (PSE), which contains a high amount of RV, has been tested in combination with X-rays, which showed similar radiosensitization effects on radioresistant human prostate cancer cell lines. In this regard, the tumor growth of a prostate cancer xenograft mouse model was reduced with RV and/or PSE (total dose 12 Gy, 5 or 250 mg/ kg, respectively) [203]. RV was also used as a pretreatment (25–150μM) to treat the human NPC CNE-1 cell line with X-ray irradiation (0–6  Gy), revealing the inhibition of the AKT phosphorylated form, a known proproliferative marker. These effects were also confirmed in NPC xenograft models, combining the RV treatment with 4 Gy for 3 days, resulting in a tumor volume reduction. Nevertheless, a key problem is the short RV half-life and low bioavailability under in vivo conditions. In vivo, pterostilbene was proven to be beneficial in the treatment of melanoma and pancreatic cancer. This study demonstrated that PT can be helpful against melanoma by inhibiting the generation of adrenocorticotropic hormone in the brain of a mouse, which impairs the Nrf2-dependent antioxidant defenses of melanoma and pancreatic tumors. This produces tumor growth restraining and tumor sensitization to oxidative stress. In addition, PT has been shown to increase cancer cell death by the induction of lysosomal membrane permeabilization [53].

11.4.1.3 Withaferin A Withaferin A (WA) was the first withanolide to be isolated and extracted from the plant Withania somnifera. WA-induced radiosensitization has been observed in human histiocytic lymphoma, renal carcinoma, and liver, breast, and several other types of cancer. Overall, these studies highlight the effect of combined treatment, mediated by the increase of apoptosis and production of ROS. WA has been shown to suppress cancer cell growth by targeting the intermediate filament protein vimentin, a structural protein of the cell cytoskeleton. In light of its anticancer

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capability, WA was also tested in order to investigate its effect in inducing the radiosensitization of cancer cells. WA’s effects were initially investigated in  vitro and in combination with γ-irradiation on a lung fibroblast cell line, and WA was found to be well tolerated by cells and to mediate a synergistic impact with γ-rays in terms of cell death. Based on these encouraging results, WA was further tested in vivo to assess its effect as a radiosensitizing agent in several cancer models such as the spontaneous murine mammary adenocarcinoma (Ehrlich ascites carcinoma—EAC), a mouse model of fibrosarcoma, and a mouse model of melanoma. Overall, each of the studies demonstrated that the WA and γ-ray combined treatment inhibits tumor growth, increasing tumor-free survival and median survival time of animals [203].

11.4.1.4 Celastrol Celastrol, also known as tripterine, is a triterpenoid derived from the root of the “thunder god wine” plant often found in China and utilized in traditional Chinese medicine for its anti-inflammatory qualities in a variety of conditions, including autoimmune diseases. Moreover, anticancer properties have been revealed, due to its proteasome inhibitory activity and antimetastatic ability. A study has evaluated its radiosensitizer effect on PC-3 cells, both in vitro and in vivo. The in vitro pretreatment with celastrol before irradiation with X-rays resulted in a significant dose-dependent enhancement of IR-induced clonogenic cell killing. This effect was explained by (1) a longer gH2AX activation for a longer time in combined treated cells with respect to the only irradiated ones, thus revealing a DNA repair impairment action by celastrol, and (2) a major expression of apoptosis markers (cleaved PARP and caspase-3). Thus, the same group tested the celastrol radiosensitizer effect on a PC-3 xenograft model. 1  mg/kg of celastrol (5 days/week for 3 weeks) was given to the mice 1 h before irradiation with a single dose of 2  Gy (5  days/week for 2  weeks). The histological analysis showed a significantly increased apoptosis and angiogenesis reduction in the combined treated tumors [203]. Similar effects have been found on the NCI-H460 human lung cancer cell line, combining celastrol with 0–4  Gy of X-rays. Indeed, the EGFR, ErbB2, and survivin irradiation markers were found to be reduced, whereas the celastrol-­ dependent inhibition of HSP90 was observed. Furthermore, celastrol induces a more pronounced ROS generation after irradiation, thanks to its quinone methide moiety [203]. Finally, the effect of celastrol as a radiosensitizer was evaluated on lung cancer with different approaches. Indeed, one research group has identified it as one of the most promising sensitizer candidates among 30 drugs, by an in silico study. Thus, they tested its effect in vitro on A549 and H460 cells, subjected to pretreatment with celastrol and 2–10 Gy

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of dose range. The encouraging results from this in  vitro study were the premises for a preclinical study on a A549 xenograft mouse model. The combined treatment using 2 mg/kg/5 each day and 10 Gy of IR for 12 days produced larger intratumoral necrotic areas [203].

11.4.1.5 Ursolic Acid Ursolic acid (UA) belongs to the family of the pentacyclic triterpenoids. It is generally obtainable from the peel of many fruits, e.g., apples, blueberries, and prunes, and also in many herbs, such as rosemary and thyme. Recently, the following therapeutic properties of UA have been described: anticancer, anti-inflammatory, and antimicrobial, and also its radiosensitization activity in models in in vitro and in vivo studies. For example, in human prostate and colon cancer cells, and in mouse melanoma cells, the UA is able to radiosensitize cells with a significant reduction in cell viability associated with an increase of typical signs of apoptosis cascade, such as cell volume reduction, nuclei fragmentation or condensation, caspase-3 activation (one of the key enzymes involved in the apoptotic pathway), increased levels of cleaved PARP (enzyme involved in DNA repair processes), DNA fragmentation, and also increased ROS generation. In melanoma mouse models, the treatment with UA and IR is able to inhibit tumor growth owing to a downregulation of Bcl-2 and survivin, two known key protein regulators of cell survival [203]. Moreover, UA can also exert a differential effect after exposure of normal or cancer cells to UV, acting as a photosensitizer for the latter and as a photoprotector for normal ones. This action was observed in human melanoma cells and in human retinal pigment epithelium control cells, where induced oxidative stress by ROS production, cell cycle arrest, and cell death induction were evaluated following UA and UV treatments. Furthermore, the UA has a significant radiosensitizing effect in human gastric adenocarcinoma cells, as evidenced by (i) a decrease in the cell survival fraction and otherwise an increase in the number of apoptotic cells (positive to the propidium iodide and annexin V apoptotic markers); (ii) the arrest of the cell cycle (in the G1 and G2/M phases); and (iii) the increase in ROS amount and a decrease of Ki-67-positive proliferating cells [207]. 11.4.1.6 Zerumbone Zerumbone (ZER) is a cyclic ketone and a sesquiterpene compound, a cytotoxic component obtained by steam distillation of the Zingiber zerumbet Smith. ZER is used in food and herbal medicine, and it also has anti-inflammatory, antiproliferative, and antitumor properties, as observed in many tumor types (including breast, pancreas, colon, lung, and skin). In addition, the radiosensitizing effects of ZER on

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tumors, by means of its regulatory activities on DNA DSB repair, cell cycle, and apoptotic pathways, have been highlighted too [203]. ZER was able to significantly increase radiation-induced cell death in human lung adenocarcinoma cells by inhibiting heat-shock proteins (HSP), increasing caspase 3 and PARP cleavage, and inhibiting HSP27 binding to apoptotic molecules such as PKCδ and cytochrome C [203]. In addition, the radiosensitizing effect was also observed in human glioblastoma cells. The same authors showed an IR-induced decrease of cell survival on human prostate cancer cells, associated with a reduced expression of proteins involved in the DNA damage repair pathway, such as γH2AX and ATM [203]. Moreover, in human colon-rectal cancer cells, ZER pretreatment is able to induce apoptosis and enhance radiation-­ induced G2/M arrest and reduction of activation of the DSB DNA repair machinery.

11.4.1.7 Caffeic Acid Phenethyl Esther CAPE is an active component of honeybee propolis, a phenolic compound, and a structural derivative of flavonoids. It was described for its antiviral, bactericidal, anti-­ inflammatory, and antioxidant properties. CAPE compound is also able to change the redox state by perturbing the activation of GSH and to induce apoptosis. Furthermore, it has been shown to be more toxic to cancer cells than normal cells, as well as to amplify the action of RT in a variety of cancers. CAPE has been shown to improve radiation-induced cell cycle arrest and death in human medulloblastoma DAOY cells. In particular, the combined treatment with CAPE and 2 Gy of IR caused an ROS enhancement production, a significant inhibition of NF-kB activity, apoptosis activation, and downregulation of cyclin B1 protein expression. In line with these data, a strong reduction of cell survival, in a concentration-­dependent manner, was described in the same cell line pretreated with CAPE (0.1–10 M) for 24 h before exposure to γ-ray irradiation at various doses (0–8 Gy), associated with cell cycle progression inhibition, by arresting cells in the S phase [203]. The CAPE pretreatment radiosensitizing effect was also shown in mouse CT26 adenocarcinoma cells, using both in  vitro and in  vivo approaches showing decreased cell survival rate and reduced NF-kB activation. CAPEinduced decrease of survival rate was also described in breast and lung cancer cell lines. In particular, in MDA-MB-231 and T47D breast cancer cell lines, CAPE and X-ray combined treatments decreased cell growth and delayed the DNA repair process for up to 60  min after exposure [203].

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11.4.1.8 Emodin carcinoma, by using both in  vitro and in  vivo approaches. Traditional Chinese medicine uses emodin (6-methyl-1,3,8-­ Multiple treatments with docetaxel (10  M), γ-irradiation trihydroxyanthraquinone), a natural phenolic derived from (0–5 Gy), and flavopiridol (120 M) are able to augment radiathe roots and rhizomes of numerous plants (e.g., Polygonum tion effects by inducing cell cycle arrest in the G1 and G2/M cuspidatum and Cascara buckthorn). phases. On the other hand, in esophageal squamous carciEmodin is chemically similar to the mitochondrial ubi- noma cell lines, cell cycle arrest after irradiation was described quinone named DMNQ (2,3-dimethoxy-1,4-­ with the decrease of cyclin D1 and retinoblastoma protein naphthoquinone), an endogenous ROS inductor, as it is able (Rb) levels. Additionally, in the SEG-1 esophageal cancer cell to transfer electrons. It is also known to have antibacterial, line, treatment with flavopiridol 24  h before γ-radiation antiviral, anti-inflammatory, and anticancer effects. The (2–6 Gy) increased radiosensitivity compared to the control, emodin’s antitumor effect has been observed in several types due to inhibition of several CDKs, cell cycle redistribution in of cancer (leukemia, breast, colon, and lung cancer), also in G1 and G2 phases, and induction of apoptosis [203]. combination with RT schedules, although its mechanism of The experimental evidences show that cells containing action still remains unclear. mutated p53 or overexpressed Bcl-2 are more radioresistant Under hypoxic conditions, emodin treatment enhanced than wild type. However, flavopiridol increased the cytotoxic the radiosensitivity of CNE-1 NPC human nasopharyngeal effects of radiation in cells with altered status of p53 and carcinoma cell line. In particular, treatment with 3.9 and Bcl-2, confirming the hypothesis according to which these 7.8 g/mL emodin 24 h before 2 Gy IR induced an increase in two pathways are targeted by radiosensitizer mechanism the apoptosis ratio and cell cycle arrest in the G2/M phase. exerted by flavopiridol [203]. Moreover, the radiosensitizing Moreover, an increase of ROS production in tandem with a effects of flavopiridol were evaluated in  vivo on glioma downregulation of HIF-1 levels (both mRNA and protein) xenograft models using GL261 cells. The interaction of was also described. These data were also confirmed by using γ-radiation (5 Gy), fractionated for 10 days, with flavopiridol CNE-1 xenograft models where a tumor growth delay was (5 mg/kg) resulted in a decrease in cell proliferation, which observed after emodin and IR combined treatments [203]. was mainly mediated by the flavopiridol’s antiangiogenic The radiosensitizing effect of emodin has also been activity, which also inhibited the HIF-1 pathway [203]. observed in the HeLa cervical cancer cell line, where preOn the other hand, as described in OCA-I ovarian carcitreatment with different concentrations of aloe emodin (AE) noma cells, the radiosensitizing action of flavopiridol could before X-ray irradiation (0–10 Gy) leads to decrease in the be sustained also by the downregulation of Ku70 and Ku80 mean lethal dose (D0) in a concentration-dependent manner, proteins, known to be involved in DNA repair mechanisms as well as an enhancement in the percentage of cells in the after radiation exposure, by the redistribution of the cell G2/M phase and a sub-G1 peak at 24, 48, and 72  h, using cycle with a greater accumulation of cells in the two more 50 M and 4 Gy IR. In addition, an increased expression of radiosensitive G1 and G2 phases [203]. cyclin B, γ-H2AX, and alkaline phosphatase (ALP) activity was also described. Similar data regarding a decrease of cell 11.4.1.10 Berberine growth and viability were observed also in human HepG2 Berberine is an alkaloid which can be extracted from the hepatocellular carcinoma cell line treated with 10  Gy of roots of many plants like the barberry, the tree turmeric, and γ-irradiation and AE, under hypoxic conditions. This com- the California poppy. Berberine is used to treat health probbined treatment leads to higher increase in both G2/M and lems like hypercholesterolemia and type 2 diabetes apoptotic populations [203]. mellitus. Berberine works by inhibiting cell cycle progression, 11.4.1.9 Flavopiridol thereby exerting, in  vitro, an antitumor activity in a large Flavopiridol is a flavone originating from the Dysoxylum array of tumors, and its radiosensitizing properties were binectariferum plant commonly used in Indian medicine. investigated on lung, esophageal, and breast cancer cells. This molecule is able to arrest cell cycle by acting on cyclin-­ Since berberine interferes with the expression and activity of dependent kinases (CDKs) during the G1/S or G2/M phases, RAD51, involved in DNA damage repair response, its radiowhich is confirmed in several cancer cell types (chronic lym- sensitizing mechanism is based on hindering DNA damage phocytic leukemia, squamous cancer, breast cancer cells). In recovery after X-ray irradiation. In vitro and in vivo experiaddition, flavopiridol is able to induce the transcriptional mental data has revealed the ability of berberine to inhibit suppression of genes involved in the proliferation pathways, HIF-1α and suppress VEGF.  For example, in an in  vitro to stimulate apoptosis, to inhibit angiogenesis, and to nasopharyngeal carcinoma study, berberine when combined increase the chemotherapeutic effects [203]. with γ-rays demonstrated a reduction of cancer cell proliferThe power of flavopiridol to affect cell radiosensitivity, in ation, viability, and Sp1 decreased expression, a protein tandem with docetaxel, was described in H460 human lung involved in tumor motility and invasion [203].

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11.4.1.11 Genistein As expected, genistein also acts as a radiosensitizing agent, if combined with γ-irradiation, as shown in vitro in cervical cancer cells, where the growth inhibition was associated with survivin downregulation, a prosurvival protein. Again, in cervical neoplasms, genistein enhanced RT effects in multiple ways: by inhibiting G2/M phase of cell cycle; by reducing the expression of two prosurvival proteins, Mcl-1 and AKT; and by triggering cell apoptosis via cytochrome c release, cleavage of caspase-3 and -8, inhibition of Bcl-2, and enhancement of Bax expression. Similar results were also shown on breast and non-small cell lung cancers, where the radiosensitizing ability was associated with the inhibition of Bcl-x, ROS production enhancement, and antioxidant molecule downregulation [203]. 11.4.1.12 BP-C2 BP-C2, a lignin-derived polymer containing benzene polycarboxylic acids complexed with ammonium molybdate, is an antioxidant that promotes the release of prorepair cytokines (IL-4 and IL-10) and suppresses the release of pro­ inflammatory cytokines (TNF-α and IL-6). Orally administered BP-C2 was found to have radioprotective and mitigative activity in H-ARS and GI-ARS [208]. Topical BP-C2 was found to have radiomitigative activity in a cutaneous radiation injury model (CRI-ARS) [209]. 11.4.1.13 Sodium Selenite Several studies have revealed the prooxidant and cytotoxic properties of sodium selenite, with respect to other selenium compounds, recognized for their antioxidant activity. In particular, the effect on natural killer (NK) cell activation is known, as well as the inhibition of the disulfide exchange on cell surface, a remodeling process, which drives cancer to uncontrolled cell division [203]. Schueller et al. [210] tested a 14-day pretreatment of C6 rat glioma cell line with selenite in the range concentration of 2–3.6 mM, before applying 0–20 Gy of γ-rays. The results showed a significant difference between the 0 mM and 3 mM survival curves applying 5  Gy (p  =  0.02) and 10  Gy (p = 0.009). Also, the vehiculated sodium selenite nanoparticles (nano-Se) were tested as radiosensitizers, using the 0–3 mg/mL range concentrations pretreatment, before treating with 0–8 Gy of X-rays. In this case, the authors showed the effect on MCF7 breast cancer cells, observing that combined treatment generated a higher mortality rate of the IR or nano-Se single treatments, inducing block at the G2/M phase of cell cycle, autophagy activation, and ROS generation. Moreover, A375 melanoma cells were subjected to 4-h pretreatment with a selenium nanosystem, using 0–15  mM coated hemocompatible erythrocyte membrane combined with bevacizumab (RBCs@Se/Av) and 2–8  Gy of X-rays. This study showed a strong cell survival reduction, an

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increase in the sub-G1 cell proportion, apoptotic pathway activation, and ROS generation. In addition, as expected by the bevacizumab treatment, decreased VEGF and VEGF2 levels were observed as tumor angiogenesis reduction [203].

11.4.2 Corticosteroids Corticosteroids are a group of hormones, produced by the cortex of the adrenal glands, having the characteristic steroid nucleus and derived from subsequent degradations of the cholesterol side chain. They include numerous molecules with different actions, including sex hormones. However, they are divided into glucocorticoids, such as cortisol which controls the metabolism, and mineralocorticoids, such as aldosterone which controls the concentration of electrolyte and water in the blood. Among these molecules, many are used for their potent anti-inflammatory and immunosuppressive properties, such as corticosterone (C21H30O4) and cortisone (C21H28O5, 17-hydroxy-11-dehydrocorticosterone). In the context of clinical RT, corticosteroids are currently used as mitigators of side effects caused by irradiation [211]. However, some researchers have highlighted the radiosensitizing effects of these molecules, used in the pretreatment phase. Glucocorticoids (GCs), acting on stress pathways, are well known in the treatment of different types of tumors. They have a strong inhibitory action on the pro­inflammatory cytokine production, although their action mechanisms need deeper investigation, if used in combination with IR. An in vitro study has investigated the role of dexamethasone (Dex), a synthetic glucocorticoid, in DNA damage response (DDR) pathway, on three astrocytoma cell lines (CT2A, APP.PS1 L.1, and APP.PS1 L.3). The results showed increased basal levels of γ-H2AX foci, keeping them higher 4  h after irradiation (IR) of the cells, while no effect was shown on the 53BP1 foci formation, compared to untreated cells. The high-level expression of γ-H2AX was reversed by ascorbic acid administration, a strong inhibitor of reactive oxygen species, showing that DEXA induces DNA damage by oxidative stress [203]. In addition, in a preclinical study on rat model, the effect of 1 mg Dex was studied alone or in combination with radioprotective molecules turpentine oil (TO), α2-macroglobulin (α2-M), or amifostine, before the administration of 6.7 Gy (LD50/30) of RI, evaluating survival and blood inflammatory markers. The results showed that Dex alone was lethal for 45% and 55% of control and irradiated rats, respectively. On the other hand, from the combination of pretreatments, it emerged that 1 mg Dex reduced the radioprotective efficacy of TO and Ami to 30% and 40%, respectively, even if, given together, TO and Ami provided 70% protection to rats receiv-

11  Radioprotectors, Radiomitigators, and Radiosensitizers

ing Dex. Instead, TO and α2-M enhanced the rate of survival from 50% to 90% and 100%, respectively [203].

11.4.3 Nanoparticles A crucial question for cancer treatment is how to increase the therapeutic window, enhancing radiation damage in tumors, while preserving the surrounding healthy tissues. One promising strategy is the accumulation of nanoparticles composed of high-Z materials (e.g., gold, palladium, platinum, gadolinium) in the tumor cells. High-atomic-number (Z) compounds have long been used as image contrast agents due to their high X-ray attenuation properties compared to soft tissues. The higher energy absorption of elements such as iodine and barium can enhance the contrast of the organs and tissues in which they are injected. The concentration of the compounds and the radiation doses used for diagnostic applications are usually so low that radiation effects and risks can be neglected. However, the same differential energy absorption principle can be exploited for therapeutic use. Recent developments in nano-manufacturing have provided reasonable and affordable methods to produce high-Z structures with dimensions smaller than 100  nm, which can be loaded in tumor volumes and in tumor cells. Their small size allows the nanostructures to escape the leaky vasculature system of tumor regions, providing a natural method for passive tumor accumulation. The majority of work has been concentrated on gold thanks to its biocompatibility and easy functionalization. The former means that considerable concentrations of gold nanostructures can be administered without toxicity effects, while the latter allows for the development of bespoke products able to accumulate in specific tissues/cells (active accumulation). Gold’s high atomic number (Z = 79) provides excellent radiation absorption contrast as indicated Fig. 11.23  Mass energy absorption coefficient (left-hand-side Y-axis) for gold (purple) and soft tissue (blue) as a function of X-ray energy. Right-hand-side Y-axis indicates the ratio (black)

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in Fig. 11.23. Other materials such as gadolinium (Z = 64) and more recently superparamagnetic iron oxide nanoparticles (SPION, ZFe  =  26) have also been suggested and explored. Early work by Hainfeld [212] demonstrated the potential of high-Z nanostructures to enhance the effect of radiation and improve tumor control in mice treated with kilovoltage X-rays minutes after injection of gold nanoparticles (GNP). In vitro work using a wide range of cell lines and radiation qualities confirmed that the presence of GNP can enhance the effect of radiation by 10–100% [213]. Interestingly, the radiation sensitization observed in in vitro and in vivo work is often significantly greater than that predicted from simple macroscopic dose models. Furthermore, the size, shape, and surface coating of the nanoparticle as well as the radiation quality and cell line have been shown to affect the radiation response observed. The discrepancy between dosimetric and experimental results regarding the radiosensitization effect emphasizes that complex physical, chemical, and biological interactions are involved in high-Z nanoparticle-mediated radiosensitization, which still need to be fully elucidated in order to extrapolate the nanoparticle radiosensitization concept to patient cancer RT. Physical, chemical, and biological mechanisms of nanoparticle radiosensitization are shown in Fig. 11.24.

11.4.3.1 Physical Radiosensitization The physical processes driving the enhancement in radiation effectiveness in the presence of nanoparticles strongly depend on the radiation quality used. For medium-energy X-rays (