Radiation Biology Handbook for Teachers and Students PDF
Document Details
Uploaded by ImpressedBigfoot
Tehran University of Medical Sciences
2010
Tags
Summary
This handbook is a comprehensive resource for teachers and students of radiation biology. It covers the radiobiology of normal tissues and tumours, radiation protection, and related clinical aspects. The resource is designed to address the knowledge gaps in radiobiology for teachers in specific countries who do not have easy access to appropriate books and reports.
Full Transcript
Radiation Biology: A Handbook for Teachers and Students VIENNA, 2010 T R A I N I N G C O U R S E S E R I E S 42 RADIATION BIOLOGY: A HANDBOOK FOR TEACHERS AND STUDENTS TRAINING CO...
Radiation Biology: A Handbook for Teachers and Students VIENNA, 2010 T R A I N I N G C O U R S E S E R I E S 42 RADIATION BIOLOGY: A HANDBOOK FOR TEACHERS AND STUDENTS TRAINING COURSE SERIES No. 42 The following States are Members of the International Atomic Energy Agency: AFGHANISTAN GHANA NORWAY ALBANIA GREECE OMAN ALGERIA GUATEMALA PAKISTAN ANGOLA HAITI PALAU ARGENTINA HOLY SEE PANAMA ARMENIA HONDURAS PARAGUAY AUSTRALIA HUNGARY PERU AUSTRIA ICELAND PHILIPPINES AZERBAIJAN INDIA POLAND BAHRAIN INDONESIA PORTUGAL BANGLADESH IRAN, ISLAMIC REPUBLIC OF QATAR BELARUS IRAQ REPUBLIC OF MOLDOVA BELGIUM IRELAND ROMANIA BELIZE ISRAEL RUSSIAN FEDERATION BENIN ITALY SAUDI ARABIA BOLIVIA JAMAICA BOSNIA AND HERZEGOVINA JAPAN SENEGAL BOTSWANA JORDAN SERBIA BRAZIL KAZAKHSTAN SEYCHELLES BULGARIA KENYA SIERRA LEONE BURKINA FASO KOREA, REPUBLIC OF SINGAPORE BURUNDI KUWAIT SLOVAKIA CAMBODIA KYRGYZSTAN SLOVENIA CAMEROON LATVIA SOUTH AFRICA CANADA LEBANON SPAIN CENTRAL AFRICAN LESOTHO SRI LANKA REPUBLIC LIBERIA SUDAN CHAD LIBYAN ARAB JAMAHIRIYA SWEDEN CHILE LIECHTENSTEIN SWITZERLAND CHINA LITHUANIA SYRIAN ARAB REPUBLIC COLOMBIA LUXEMBOURG TAJIKISTAN CONGO MADAGASCAR THAILAND COSTA RICA MALAWI THE FORMER YUGOSLAV CÔTE D’IVOIRE MALAYSIA REPUBLIC OF MACEDONIA CROATIA MALI TUNISIA CUBA MALTA TURKEY CYPRUS MARSHALL ISLANDS UGANDA CZECH REPUBLIC MAURITANIA UKRAINE DEMOCRATIC REPUBLIC MAURITIUS UNITED ARAB EMIRATES OF THE CONGO MEXICO UNITED KINGDOM OF DENMARK MONACO GREAT BRITAIN AND DOMINICAN REPUBLIC MONGOLIA NORTHERN IRELAND ECUADOR MONTENEGRO EGYPT MOROCCO UNITED REPUBLIC EL SALVADOR MOZAMBIQUE OF TANZANIA ERITREA MYANMAR UNITED STATES OF AMERICA ESTONIA NAMIBIA URUGUAY ETHIOPIA NEPAL UZBEKISTAN FINLAND NETHERLANDS VENEZUELA FRANCE NEW ZEALAND VIETNAM GABON NICARAGUA YEMEN GEORGIA NIGER ZAMBIA GERMANY NIGERIA ZIMBABWE The Agency’s Statute was approved on 23 October 1956 by the Conference on the Statute of the IAEA held at United Nations Headquarters, New York; it entered into force on 29 July 1957. The Headquarters of the Agency are situated in Vienna. Its principal objective is “to accelerate and enlarge the contribution of atomic energy to peace, health and prosperity throughout the world’’. RADIATION BIOLOGY: A HANDBOOK FOR TEACHERS AND STUDENTS INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 2010 COPYRIGHT NOTICE All IAEA scientific and technical publications are protected by the terms of the Universal Copyright Convention as adopted in 1952 (Berne) and as revised in 1972 (Paris). The copyright has since been extended by the World Intellectual Property Organization (Geneva) to include electronic and virtual intellectual property. Permission to use whole or parts of texts contained in IAEA publications in printed or electronic form must be obtained and is usually subject to royalty agreements. Proposals for non-commercial reproductions and translations are welcomed and considered on a case-by-case basis. Enquiries should be addressed to the IAEA Publishing Section at: Sales and Promotion, Publishing Section International Atomic Energy Agency Vienna International Centre PO Box 100 1400 Vienna, Austria fax: +43 1 2600 29302 tel.: +43 1 2600 22417 email: [email protected] http://www.iaea.org/books For further information on this publication, please contact: Applied Radiation Biology and Radiotherapy Section International Atomic Energy Agency Vienna International Centre PO Box 100 1400 Vienna, Austria email: [email protected] RADIATION BIOLOGY: A HANDBOOK FOR TEACHERS AND STUDENTS IAEA, VIENNA, 2010 IAEA-TCS-42 ISSN 1018-5518 © IAEA, 2010 Printed by the IAEA in Austria March 2010 FOREWORD Knowledge of the radiobiology of normal tissues and tumours is a core prerequisite for the practice of radiation oncology. As such the study of radiobiology is mandatory for gaining qualification as a radiation oncologist in most countries. Teaching is done partly by qualified radiobiologists in some countries, and this is supplemented by teaching from knowledgeable radiation oncologists. In low and middle income (LMI) countries the teachers are often radiation oncologists and/or medical physicists. In Europe, a master’s course on radiobiology is taught jointly by a consortium of five European Universities. This is aimed at young scientists from both Western and Eastern Europe, training in this discipline. Recently the European Society for Therapeutic Radiology and Oncology (ESTRO) initiated the launch of a radiobiology teaching course outside Europe (Beijing, 2007; Shanghai, 2009). Radiation protection activities are governed by many regulations and recommendations. These are based on knowledge gained from epidemiological studies of health effects from low as well as from high dose radiation exposures. Organizations like the International Commission on Radiological Protection (ICRP) have put a lot of effort into reviewing and evaluating the biological basis to radiological protection practices. Personnel being trained as future radiation protection personnel should have a basic understanding of the biological and clinical basis to the exposure limitations that they are subject to and that they implement for industrial workers and the public at large. It is for these reasons that aspects of Radiobiology related to protection issues are included in this teaching syllabus. In LMI countries, many more teachers are needed in radiobiology, and the establishment of regional training centres or special regional training courses in radiobiology, are really the only options to solve the obvious deficit in knowledge of radiobiology in such countries. Radiobiology teaching courses organized or sponsored by the IAEA are oversubscribed, and the students themselves confirm the great need for this type of teaching. Requests have been received from a number of countries in all regions asking for the IAEA to help organize radiobiology teaching. More qualified professionals are also needed for this exercise. Already there are some initiatives e.g. an IAEA project produced in 2007 a distance-learning course in the Applied Sciences of Oncology (ASO) for Radiation Oncologists (also available on the IAEA-website since 2008) including 10 modules in radiobiology. This handbook for teachers and students was formulated based on the recommendations of a Consultants Meeting on International Syllabus for Radiobiology Teaching held 12-14 December 2005 in Vienna, Austria. Whilst this information is available in various books and other reports, it is summarized and collated here so that the whole document has a degree of completeness. This should be helpful in particular to those countries that do not have easy access to appropriate books and reports. Comments and suggestions on this syllabus as a teaching tool were sought from committees of the ESTRO and ASTRO (American Society for Therapeutic Radiology and Oncology). This handbook is written in two parts: (a) Teaching programme including a common basic radiobiology education and teaching programme for radiation oncologists, radiation therapy technologists, diagnostic radiologists, radiation biologists, medical physicists, radiation protection officers and other disciplines involved in radiation activities. This will take 1 week of teaching (30 hours), including a practical or tutorial session at the end of each day. This is followed by a further week of advanced teaching for radiation oncologists, and a further 3 days for radiation protection personnel. (b) Minimal Essential Syllabus for Radiobiology and two extra modules for radiation oncologists and radiation protection personnel, respectively. For each discipline, the basic module and an extra module would constitute the minimum essential syllabus and teaching requirements. It is hoped that this handbook for teachers and students will fulfil the needs of the Member States and serves the basis for regulatory requirements in these countries. The IAEA officer responsible for this publication is J. Wondergem of the Division of Human Health. CONTENTS 1. TEACHING PROGRAMME........................................................................................... 1 2. MINIMUM ESSENTIAL SYLLABUS FOR RADIOBIOLOGY................................. 13 2.1. Introduction...................................................................................................... 13 2.2. Physics and chemistry of radiation interactions with matter............................ 13 2.2.1. Sources of ionizing radiation................................................................ 13 2.2.2. Types of ionizing radiation................................................................... 14 2.2.3. Particulate radiations............................................................................. 19 2.2.4. Linear energy transfer........................................................................... 20 2.2.5. Radiation dose and units....................................................................... 21 2.2.6. Principles of radiation dosimetry.......................................................... 22 2.2.7. Direct and indirect effects..................................................................... 23 2.3. Molecular and cellular radiobiology................................................................. 26 2.3.1. Radiation lesions in DNA..................................................................... 26 2.3.2. Major types of DNA repair................................................................... 26 2.3.3. Damage recognition and signalling...................................................... 27 2.3.4. Consequences of unrepaired DNA damage: chromosome damage...... 27 2.3.5. Radiobiological definition of cell death................................................ 28 2.3.6. Suvival curves and models................................................................... 28 2.3.7. Cell cycle effects................................................................................... 29 2.3.8. Relative biological effectiveness (RBE)............................................... 30 2.3.9. Cellular repair exemplified in survival curves...................................... 30 2.3.10. Cellular hyper-radiosensitivity (HRS) and induced repair (IRR)......... 31 2.3.11. Other molecular targets: bystander (epigenetic) effects....................... 31 2.3.12. Radiation sensitisers............................................................................. 31 2.3.13. Radiation protectors.............................................................................. 32 2.4. Tumour radiotherapy........................................................................................ 33 2.4.1. Tumour growth..................................................................................... 33 2.4.2. Tumour response to irradiation............................................................. 33 2.4.3. Dependence of tumour control on dose and tumour size...................... 34 2.4.4. Dose fractionation effects..................................................................... 35 2.4.5. Predicting the radiation response of tumours....................................... 35 2.4.6. Tumour hypoxia.................................................................................... 36 2.5. Normal tissue response to radiotherapy............................................................ 38 2.5.1. Cellular and tissue response.................................................................. 38 2.5.2. Acute tissue responses.......................................................................... 39 2.5.3. Late tissue responses............................................................................. 39 2.5.4. Predicting normal tissue response......................................................... 40 2.5.5. Therapeutic ratio................................................................................... 41 2.5.6. Whole body irradiation......................................................................... 41 2.6. Radiobiological basis of radiation protection................................................... 42 2.6.1. Health consequences after total body irradiation from radiation accidents................................................................................................ 42 2.6.2. Long term radiation risks from low radiation doses............................. 45 2.6.3. Radiation-induced cancer in the A-bomb survivors............................. 46 2.6.4. Epidemiological studies in other radiation-exposed populations......... 47 2.6.5. Mechanisms of radiation-induced cancer............................................. 51 2.6.6. Radiation effects in the developing embryo and fetus.......................... 51 2.6.7. Radiation-induced heritable diseases.................................................... 52 References to Section 2................................................................................................... 55 3. EXTRA MODULE FOR RADIATION ONCOLOGISTS............................................ 57 3.1. Introduction...................................................................................................... 57 3.2. Physics.............................................................................................................. 57 3.2.1. Brachytherapy, radionuclides, and radioimmunotherapy..................... 58 3.2.2. Charged particles and high LET radiotherapy...................................... 58 3.2.3. Boron neutron capture therapy (BNCT)............................................... 59 3.3. Molecular and cellular biology......................................................................... 59 3.3.1. Techniques............................................................................................ 59 3.3.2. Cell signaling........................................................................................ 63 3.3.3. Oncogenes and tumour suppressor genes............................................. 67 3.4. The cell cycle.................................................................................................... 69 3.4.1. Cycle-dependent kinases and cyclins................................................... 70 3.4.2. Activation of Cdks by binding to cyclins............................................. 71 3.4.3. Inhibitors of cyclin-dependent kinases................................................. 71 3.5. DNA damage and repair................................................................................... 72 3.6. Tumour growth and cell kinetics...................................................................... 75 3.6.1. Tumour growth..................................................................................... 75 3.6.2. Cell kinetics.......................................................................................... 76 3.6.3. Cell proliferation in normal tissues....................................................... 78 3.7. Cell death mechanisms..................................................................................... 79 3.8. In vitro and in vivo assays for cell survival...................................................... 81 3.9. Repair of radiation damage............................................................................... 84 3.10. Tumour biology and host/tumour interactions................................................. 88 3.11. Radiobiology of normal tissue damage............................................................ 94 3.11.1. Acute tissue responses.......................................................................... 95 3.11.2. Late tissue responses............................................................................. 96 3.11.3. Whole body irradiation......................................................................... 98 3.11.4. Retreatment tolerance........................................................................... 99 3.11.5. Volume effects...................................................................................... 99 3.11.6. Therapeutic ratio (or Index)................................................................ 100 3.12. Time-dose-fractionation................................................................................. 100 3.12.1. Repair.................................................................................................. 101 3.12.2. Repopulation....................................................................................... 101 3.12.3. Redistribution/recruitment.................................................................. 102 3.12.4. Reoxygenation.................................................................................... 103 3.12.5. Time and dose relationships............................................................... 103 3.12.6. Isoeffect curves................................................................................... 104 3.12.7. The linear quadratic equation and models for isoeffect...................... 105 3.12.8. Altered fractionation schedules.......................................................... 106 3.13. Predictive assays............................................................................................. 107 3.13.1. Predicting the response of tumours..................................................... 107 3.13.2. Predicting normal tissue response....................................................... 108 3.14. Combined radiation and drug treatments........................................................ 109 3.15. Clinical radiobiology of common cancers...................................................... 114 3.16. Second cancers in radiotherapy patients......................................................... 114 3.17. Summary......................................................................................................... 117 References to Section 3................................................................................................. 118 4. EXTRA MODULE FOR RADIATION PROTECTION PERSONNEL..................... 120 4.1. Introduction.................................................................................................... 120 4.2. Radiation accidents and environmental radiation exposure........................... 120 4.2.1. Dose estimation................................................................................... 120 4.3. Diagnosis and medical management of radiation syndromes........................ 122 4.3.1. LD-50 (lethal dose 50)........................................................................ 122 4.3.2. Radiation syndromes........................................................................... 123 4.3.3. Medical management of radiation accidents...................................... 123 4.3.4. Methods of triage for treatment after a radiation accident.................. 127 4.4. Radiation carcinogenesis................................................................................ 128 4.4.1. Mechanisms of carcinogenesis........................................................... 128 4.4.2. Epidemiological evidence for radiation carcinogenesis..................... 129 4.4.3. The A-bomb Survivor Life-Span Study, cancer mortality and cancer incidence.................................................................................. 129 4.4.4. The Chernobyl accident...................................................................... 132 4.4.5. Patients treated for benign diseases.................................................... 133 4.4.6. Radon exposure of hard rock miners or in homes.............................. 135 4.5. Heritable radiation effects.............................................................................. 136 4.6. Effects on the developing embryo.................................................................. 140 4.7. The system for radiation protection................................................................ 141 4.7.1. The derivation of risk coefficients and organ weighting factors from epidemiological data.................................................................. 142 4.7.2. Dose limits.......................................................................................... 145 4.7.3. Risk-benefit considerations in breast cancer screening using mammography.................................................................................... 146 References to Section 4................................................................................................. 148 CONTRIBUTORS TO DRAFTING AND REVIEW........................................................... 151 1. TEACHING PROGRAMME A. MINIMUM ESSENTIAL MODULE FOR RADIOBIOLOGY (1 WEEK/30 HOURS) Day 1 (including practical/tutorial 1) Introduction Physics and Chemistry of radiation interaction with matter a) Interactions of electromagnetic radiations with matter, photoelectric effect, compton scatter, pair production, dependence on photon energy, dependence on Z (atomic number) of absorbing material, distribution of energy deposition (scale), half value layer b) Interactions of particles with matter, electrons, energy dependence, alpha particles, neutrons c) Linear energy transfer (LET)/Relative biologic effectiveness (RBE) d) Definition of dose; gray (Gy) e) Principles of dosimetry Ionization chambers, Themoluminescent dosimetry (TLD) f) Radiation Chemistry of water g) Formation and reaction of free radicals with oxygen, scavengers: Direct/Indirect effects of radiation on macromolecules Concept of chemical restitution/competition Day 2 (including initiating practical/tutorial 2 and 3) Molecular cellular radiobiology a) Types of radiation lesions to deoxyribonucleic acid (DNA) and repair: base damage, single strand breaks (SSB), double strand breaks (DSB), mechanisms of repair, molecular role of e.g. protein53 (p53), ataxia teleangiectasia mutated gene (ATM) b) Effects on chromosomes – use in biodosimetry c) Radiobiological definition of cell death and cell survival d) Manifestations of radiation-induced cell death (apoptosis, necrosis, mitotic catastrophe, senescence) e) Survival curves and models, clonogenicity (main criterion), limitations of determination of cell numbers at a fixed time f) Cell cycle: sensitivities in different phases, and cell cycle checkpoints 1 g) RBE – cell survival – change in slope and shoulder of survival curve, dependence of RBE on dose h) Cellular repair: sub lethal damage repair (SLDR)/potential lethal damage repair (PLDR) cell survival, half time of repair i) Dose rate effects: dependence on repair and proliferation j) Chemical modifiers Oxygen effect: radiation sensitizers/protectors k) Other cellular targets, e.g. membranes, mitochondria l) Bystander effects at low doses Day 3 (a.m.) (continuing of practicals/tutorials 2 and 3) Tumour radiobiology including tumour growth and micro-environmental effects a) Tumour growth characteristics; e.g. exponential growth b) Dependence of tumour cure probability on dose, tumour size, fractionation, overall treatment time c) Tumour stem cells/clonogenic tumour cell inactivation. Poisson statistics of tumour cure. d) Time factor in radiotherapy e) Palliative radiotherapy (tumour growth delay) Day 4 (including practical/tutorial 4) Normal tissue effects a) Concept of damage manifested early versus late: underlying mechanisms e.g. oxidative stress and cell kinetics b) Early effects: Clinical manifestation Time course and dose response, latency Hypoplasia due to cell killing Interacting factors: inflammation, cytokines Dose/dose-rate/time/fractionation dependence c) Late effects: Clinical manifestation 2 Time course and dose response, latency Dependence on fraction size Chronic inflammatory responses Micro vascular injury fibrosis Consequential late effects d) Whole body exposure Radiation syndromes Day 5 a.m. Radiation Carcinogenesis a) A-bomb survivors: leukaemia, solid tumours, dose dependence, dependence on age at exposure, concept of relative versus absolute risk b) Mechanisms of multistage carcinogenesis. In vitro transformation, animal models, radiation-induced mutations c) Dose response relationship, dose-rate and latency in humans, organ dependence, estimation of radiation risk d) Definition of Sievert (Sv), organ weighting factors Day 5 a.m. Radiation Effects in Utero a) Types of injury b) Dependence on stage of pregnancy c) Protection of the embryo d) Dose response for mental retardation Radiation Induced heritable damage a) Mutations b) Doubling dose c) Risk estimation, single gene disorders and multi-factorial diseases 3 Practicals/Tutorials a) Dosimetry with ionization chambers; shielding b) Chromosome aberrations in irradiated lymphocytes (0-3 Gy) – dicentrics and micronuclei c) Data analysis for cell survival curves; scoring colonies d) Data analysis of in vivo fractionation studies: skin, Gastro-intestinal tract, kidney, spinal cord. B. EXTRA MODULE FOR RADIATION ONCOLOGISTS (40 HOURS – INCLUDING 10 HOURS PRACTICALS) Day 1 (including practical/tutorial) Introduction Physics a) Dosimetry in radiotherapy b) Depth doses for photons, electrons, protons and heavy particles (concept of Bragg peak), particle therapy c) Isodose curves (fraction doses adding up, contrast with isoeffect curves, not linear), dose volume histograms d) Boron Neutron Capture Therapy (BNCT), requirement for preferential boron uptake in tumour, concern re-vascular uptake, poor characteristics of penetration of thermal neutron beams e) Physics of radioimmunotherapy, use of different isotopes, problems of tissue distribution, dose calculations Molecular and cellular Biology a) Principles of some common techniques e.g. immunoblotting, microarrays, proteomics (2-D gels) b) Techniques to modify gene expression c) DNA/Chromatin structure and function; (De)-methylation, (De)-Acetylation e) Regulation of transcription, translation and post-translational modification, e.g. glycosylation, meristylation f) Cell signalling – signalling cascades Receptor/ligand interactions; phosphorylation/dephosphorylation reactions g) Oncogenes and Tumour suppressor genes 4 h) Mechanisms of action of some signal-transduction therapeutic Agents e.g. Epidermal growth factor receptor (EGFR) inhibitors, Ras inhibitors, Farnesyltransferase inhibitors (FTI). i) Radiation effects on cell signalling, e.g. EGFR pathway The cell cycle (and signal transduction pathways) a) Cell cycle description b) Methods to determine cell cycle parameters, e.g. flow cytometry – DNA staining and bromo deoxyuridine (BrdU) c) Control of cell cycle: cyclins, cyclin dependent kinases (CDKs), cyclin dependent kinase inhibitors (CDKIs), role of p53 d) Radiation-induced cell cycle checkpoints Day 2 (including practical/tutorial) Cell death mechanisms a) Radiobiological definition of cell death (loss of reproductive ability- reproductive death), abortive cell divisions after irradiation b) Apoptosis – Developmental and stress induced, morphological and biochemical features, molecular pathways c) Necrosis – Morphological, pathological, and biochemical features d) Mitotic Catastrophe – Morphology e) Cell senescence and radiation-induced differentiation DNA damage and repair a) Types of lesions and frequency per cell per Gy b) Multiple damaged sites (clustered damage) c) Types and Molecular mechanisms of DNA repair: Base damage Single strand breaks Double-strand breaks: homologous recombination repair (HR), non-homologous end-joining (NHEJ) Repair of cross-links Mutations affecting repair (ATM etc) Molecular responses to DNA damage (p53, ATM, etc) 5 d) Principles of assay techniques – elution, electrophoresis including comets, repair foci, plasmid-based assays Other molecular targets a) Membranes (Oxidative damage, lipid peroxidation, sphingomyelinase activation in endothelial cells). b) Activation of stress response genes, radiation induced signal transduction Cell survival curves a) Colony formation assays versus cell viability assays b) Dose-survival relationships c) Linear-quadratic model; two component exponential model, definition of survival curve parameters d) Sub-lethal and potentially lethal damage repair, half time of repair and incomplete repair, effect of unequal fraction size on repair e) Dose rate and fractionation effects f) Oxygen effect – level, time scale, mechanisms g) LET versus OER and RBE; Radio-sensitizers, protectors h) Low dose hypersensitivity, induced radio-resistance, mechanisms i) Bystander effects, mechanisms Day 3 (including practical/tutorial) Tumour biology and host/tumour interactions a) Growth kinetics of experimental tumours and cancer in patients, impact of tumour pathology, tumour progression, metastatic spread b) Vasculature, angiogenesis and tumour microenvironment c) Hypoxia – Oxygen measurement techniques, radiobiological-hypoxic fractions, acute/transient (perfusion-limited) versus chronic (diffusion-limited) hypoxia d) Mechanism of reoxygenation, hypoxic cell radiosensitisers, bioreductive agents e) Methods of correction of hypoxia-associated radioresistance in tumours: high LET radiotherapy, hypoxic cell radiosensitizers, increased oxygen concentration in breathing air, correction of anaemia f) Tumour response assays – tumour cure 50 (TCD50), threshold dose (TD50), in vivo/in vitro colonies, tumour regrowth delay, (TGD), in vitro tumor models (e.g. spheroids), human tumour Xenografts and isogeneic/ transgenic mouse tumours 6 g) Differences between tumour types h) Virally-associated cancers, molecular and biological basis to induction and radiation response of virally-associated cancers e.g. human papiloma virus (HPV) and cervix cancer, Epstein-Barr virus (EBV) and nasopharynx cancer, HBV and liver cancers, HIV and the Acquired Immuno-Deficiency Syndrome (AIDS)-defining and associated malignancies Day 4 (including practical/tutorial) Radiobiology of Normal Tissue damage a) Early normal tissue damage: Pathogenesis in critical normal tissues (skin, G-I tract mucosa, bladder, bone marrow), kinetics/latency cell turnover and stem cell function, role of inflammation, cytokines, reactive oxygen species Dose response. b) Late normal tissue damage: Pathogenesis in critical normal tissues (Lung, heart, central nervous system (CNS), skin, kidney, liver, G-I tract, bladder, salivary gland) kinetics/latency cell turnover Role of inflammation, cytokines, reactive oxygen species Microvascular damage, fibrosis, ischaemia and atrophy Functional vs. structural damage Growth factors and stimulated regeneration (including stem cells) Concept of normal tissue tolerance Over-reacting patients - radiosensitivity syndromes Concept of functional subunits – parallel and serial organisation c) Second cancers in radiotherapy patients d) Conditioning for bone marrow transplantation Time-Dose Fractionation a) The 5 Rs of fractionated radiotherapy (Repair, Repopulation, Radiosensitivity, Redistribution, Reoxygenation) b) Isoeffect curves c) Linear-quadratic (LQ) parameters, biological effective dose (BED), linear-quadratic equivalent dose (LQED) 7 d) Residual injury and re-treatment e) Accelerated repopulation in tumours and normal tissues, time factor in radiotherapy f) Therapeutic ratio g) Concept of tumour control probability (TCP) and normal tissue complication probability (NTCP) models h) Modified Fractionation (Hyper-, Hypo-, Accelerated. Concomitant boost) i) Radiobiology of resource-sparing protocols, e.g. for palliative treatments Brachytherapy a) Radiobiological principles b) Half time of repair c) Dose distribution d) Volume specification Volume Effects a) Isoeffect versus iso-tolerance b) Radiobiological interpretation of dose-volume histograms c) Volume considerations of functional versus structural damage d) Conformal and intensity modulated radiation therapy (IMRT) techniques Day 5 (including practical/tutorial) Principles of combined radiation and drug treatments a) Spatial cooperation versus interactive effects b) Different toxicities in tumour and normal tissues c) Possible mechanisms of interaction d) Principles of clinical use including concurrent and sequential treatments, role of chemotherapy in consequential late radiation toxicity, late cardiac effects e) Tumour micro-environmental effects in chemotherapy Biological and novel therapies a) Biological therapies and their mechanism of action b) Novel targets for anti-cancer drugs including vasculature and cell signal control and oncogene products 8 c) Bioreductive drugs, antibody-directed enzyme prodrug therapy (ADEPT) d) Photodynamic therapy e) Gene therapy, gene-directed enzyme prodrug therapy (GDEPT), radiation-induced gene expression including molecular switching techniques f) Radioimmunotherapy and targeted radiotherapy Day 6 (including practical/tutorial) Predictive Assays a) Rationale for normal tissues and tumours – intrinsic radiosensitivity, surviving fraction at 2Gy (SF2), cell kinetics, and hypoxia b) Molecular, subcellular, cellular and non-invasive tests c) Results to date d) Future possibilities, e.g. gene expression profiling Clinical Radiobiology of common cancers a) Radiobiological issues in the treatment of the common cancers such as cervix, head and neck, lung, breast, prostate b) Resistance mechanisms and clinical radiobiology c) Cervix cancer, SF2, Hypoxia, Repopulation, Brachytherapy and external beam treatments, BED, LQED calculations d) Head and neck cancer, optimum fractionation schedules, volume effects –Morbidity scoring scales, salivary gland sparing, role of brachytherapy e) Lung cancer e.g. biological imaging of target volume using positron emission tomography (PET), accelerated radiotherapy. Radiochemotherapy schedules f) Breast cancer e.g. role of hypofractionation and brachytherapy, cardiac effects, antiestrogens and radiation toxicity g) Prostate cancer e.g. role of hypofractionation and brachytherapy, dose escalation, biochemical relapse Practicals/Tutorials DNA Laboratory techniques: practical demonstrations of some of the techniques from the above lectures e.g. comet assay, micronuclei, flow cytometry (DNA analysis), gel electrophoresis Survival curves in practice: practical session on the shapes of survival curves, and their importance in various clinical scenarios 9 Analysis of scoring of normal tissue damage: LENT/SOMA versus RTOG/EORTC scoring systems, head and neck squamous cell carcinoma (HNSCC), Cervix Ca LQ model: BED, LQED, / ratio values: a) Fractionation calculations in practice b) Physical dose distribution and biological response distribution c) Combined brachy/teletherapy treatments; compensations for interruptions in treatment d) Importance of treating all fields per day e) Influence of radiation source decay with respect to repair half-time and dose effectiveness f) Clinical impact of errors in dose delivery Critical reading of relevant literature C. EXTRA MODULE FOR RADIATION PROTECTION PERSONNEL (20 HOURS - 1 DAY ACCIDENTS, 1 DAY CARCINOGENESIS, 1 DAY REMAINDER) Day 1 (including practical/tutorial) Introduction Environmental radiation exposure and radiation accidents Dose estimation: a) Retrospective dose estimation for past exposures: e.g. for A–bomb survivors, populations exposed by the Chernobyl accident, the Techa River pollution, the Semipalatinsk test site. b) Radioecology: atmospheric dispersion, deposition (wet and dry), uptake in food chain, dose commitment from internal and external exposure. Relevant radioisotopes (Cs, I, Sr) c) Biological dosimetry in accidental exposures: Stable and unstable chromosome aberrations (lymphocytes, haemoglobin and glycophorin-A (GPA) mutations) Diagnosis and medical management of radiation syndromes a) Lethal dose-50 (LD-50): laboratory experiments and human estimates b) Radiation syndromes (Neurovascular, Heamatopoeietic, Cutaneous and G-I tract syndromes) c) Diagnosis and medical management of radiation Accidents: Radiobiological rationale for therapeutic strategies such as barrier nursing, bone marrow stem cell transplantation, cytokine treatment d) Methods of triage for treatment after a radiation accident: 10 Acute symptoms (vomiting, diarrhoea, hair loss, nausea) Laboratory tests (Lymphocytes count and granulocyte count) Day 2 (including practical/tutorial) Radiation Carcinogenesis a) Molecular mechanisms of multistage carcinogenesis: Initiation, promotion, progression Activation of oncogenes (i.e. genetic rearranged) Inactivation of suppressor genes (e.g. p53), loss of heterozygosity (LOH), polymorphisms Genomic instability, mini and microsatellites Genetic susceptibility to radiation-induced cancer (e.g. Retinoblastoma (Rb) gene) b) Epidemiological evidence for radiation carcinogenesis: Epidemiological methods, cohort studies and case control studies Bomb survivor life-span studies: mortality and cancer incidence – design of study, results, dose response, latency, absolute vs. relative risk) Patients treated for benign diseases such as ankylosing spondylitis, mastitis, tinea capitis Tuberculosis patients undergoing multiple fluoroscopy Radon exposure of hard-rock miners or in homes, interaction with smoking The influence of age at exposure and gender on incidence and latency Dose-response relationships for radiation-induced leukaemia and cancers, particularly at low doses. Limitations of epidemiological studies The influence of dose rate Absolute vs. relative risk models Life time risk extrapolations Heritable effects a) Methods to determine radiation-induced rates of single gene mutations b) Doubling dose at low dose, low dose rate irradiation c) Critical germ cell stages for heritable radiation damage 11 d) Factors affecting the risk of heritable radiation damage: mutational component, potential recoverability correction factor e) Risk estimation for single gene disorders and multifactorial diseases Effects on the developing embryo a) Intrauterine death, congenital malformations, and neonatal death, microcephaly, severe mental retardation, growth retardation b) Dependence on gestational age of radiation effects on the embryo or foetus c) Dose dependence of risk of severe mental retardation after exposure in weeks 8-15 and weeks 16-25, evidence for thresholds d) Protection of the embryo in diagnostic radiology and from occupational exposure Day 3 (including practical/tutorial) Radiation protection a) Effective and committed dose, definition of sievert (Sv), organ weighting factors, linear no-threshold (LNT) model b) Dose limits for occupational and public exposures and their justification. c) Dose limits for stochastic and deterministic effects 12 2. MINIMUM ESSENTIAL SYLLABUS FOR RADIOBIOLOGY 2.1. Introduction This is expected to comprise 1 week of teaching of around 30 hours including discussion periods, practical sessions, tutorials, and revision using distance-learning and other texts in the student’s own time. 2.2. Physics and chemistry of radiation interactions with matter 2.2.1. Sources of ionizing radiation Ionizing radiations may be emitted in the decay process of unstable nuclei or by de-excitation of atoms and their nuclei in nuclear reactors, X ray machines, cyclotrons and other devices. During radioactive decay gamma rays are often produced alongside other types of radiation such as or rays. When a nucleus emits an or particle, the daughter nucleus is sometimes left in an excited state which, after de-excitation, returns to a lower energy level by emitting a γ ray in much the same way that an atomic electron can jump to a lower energy level by emitting visible light. Both natural background radiation from cosmic and terrestrial sources, and man-made radiations, cause ionization of atoms or molecules, which may cause injury to cells. Living organisms are continuously exposed to ionizing radiations from natural radiation. In addition, exposures occur as a result of human activities and medical practices. Radiations are broadly categorized into natural and man-made sources (Table 2.1). More than 90 % of radiation exposure to man occurs from natural sources e.g. cosmic rays, and terrestrial sources that comes from radionuclides in the earth’s crust, air, food and water and the human body itself. Man-made radiation exposure to populations occurs mainly from medical uses of radiation and radioisotopes in health care, occupational sources in the generation of electricity from nuclear power reactors, industrial uses of nuclear techniques, and in the past from nuclear weapons testing. Use of ionizing radiation in medical diagnosis and therapy is widespread and constantly increasing due to useful newer health care applications. It is widely accepted that diagnostic radiation exposures can be significantly reduced by adequate safety measures and optimization of nuclear-based procedures and practices. 13 TABLE 2.1 AVERAGE ANNUAL EFFECTIVE DOSE OF IONIZING RADIATION TO INDIVIDUALS* (*as in year 2000) Source Dose (mSv) Range (mSv) Natural background External exposure Cosmic 0.4 0.3 – 1.0 Terrestrial 0.5 0.3 – 0.6 Internal Exposure Inhalation (mainly radon) 1.2 0.2 – 10. Ingestion 0.3 0.2 – 0.8 Total 2.4 1 - 10 Man-made (artificial) Medical 0.4 0.04 – 1.0 Nuclear Testing 0.15 – decreasing trend Chernobyl accident 0.002 0.04 – decreasing trend Nuclear power production 0.0002 Decreasing trend Total 2.8 1 - 10 2.2.2. Types of ionizing radiation Ionizing radiation may be divided into directly and indirectly ionizing for the understanding of biological effects. Most of the particulate types of radiation are directly ionizing i.e. individual particles with adequate kinetic energy can directly disrupt the atomic structure of the absorbing medium through which they pass producing chemical and biological damage to molecules. In contrast, electromagnetic radiations, namely, X and γ rays, are indirectly ionizing because they do not produce chemical and biological damage themselves but produce secondary electrons (charged particles) after energy absorption in the material. Fig. 2.1 Schematic of the electromagnetic spectrum (Hall and Giaccia, 2006). 14 2.2.2.1. Electromagnetic radiation Electromagnetic radiation includes radiowaves, microwaves, visible light, ultra violet light, X rays and γ rays (Figure 2.1). These waves are essentially characterized by their energy which varies inversely with the wavelength. They can be thought of as moving packets of energy (quanta) and in this form are called photons. The quantum of energy associated with the waves progressively increases from radiowaves with least energy to X and with highest energy, and X and γ ray photons have the ability to eject an electron from its orbit in an atom (are ionizing radiations). Ionization is the process of removing one or more electrons from atoms by the incident radiation leaving behind electrically charged particles (an electron and a positively charged ion) which may subsequently produce significant biological effects in the irradiated material (Figure 2.2). The ionized or excited atom or molecule may either fragment producing free radicals or return to the parent state. If the energy transferred by ionizing radiation to the atom is insufficient to eject orbital electrons, the electrons may be raised from lower to higher orbitals and the atom is said to be excited. Other radiations of the electromagnetic spectrum fall short of the energy required to remove an electron from an atom and they are called non-ionizing radiations. Non-ionizing radiations are generally considered harmless to biological tissues at levels below those that cause heating effects, although there remain controversies in this area and research is ongoing. Cellular phones, radar, infrared, radiowaves, microwaves, visible light, ultrasound fall into this category. Because of the longer wavelengths and, therefore, smaller energy per quanta, they are not known to cause significant chemical changes in atoms or molecules of the medium. However, the exact demarcation between ionizing and non-ionizing radiation parts of the spectrum is somewhat arbitrary because some molecules can be ionized with very little energy, and far- UV radiation can behave similarly to X and γ rays. Fig. 2.2 Direct versus indirect action (Hall and Giaccia, 2006). 15 2.2.2.2. Interactions of electromagnetic radiation When electromagnetic radiation travels through matter, it can be transmitted without transferring any energy or its intensity may be reduced by interaction with the traversed material. The attenuation occurs due to individual photon interactions with the atoms encountered. Biological effects arise when electromagnetic radiations, mainly X rays or γ rays, are either scattered or absorbed by the atoms of tissues/organs. Quantum theory considers electromagnetic radiation as streams of packets/bundles of energy called photons. The energy of a photon of electromagnetic radiation is given by Planck’s equation, where E = h = hc/ E is the energy of the photon, h is Planck’s constant, and is the frequency of the photon. The energy of a photon is directly related to its frequency and inversely to wavelength,. Wave velocity is obtained by the product of frequency and wavelenth, c = , where c is the velocity of light. Biological effects of radiation arise when ionizing radiation interacts with an organism/tissue and leaves some energy behind. The process by which electromagnetic photons are absorbed in matter depends on their energy and the atomic number of the absorbing material. Photons passing through matter transfer their energy through the following three main processes: photoelectric absorption, Compton scattering, and pair production (Figure 2.3). Fig. 2.3 Dominant types of interactions as a function of the atomic number Z of the absorber and the energy of the photon radiation (Podgorsak, 2005). 16 2.2.2.3. Photoelectric absorption In photoelectric absorption, the photon interacts with a bound inner shell electron in the atom of the absorbing medium and transfers its entire energy to the electron ejecting it from the occupied atomic shell. The incident photon disappears and the energy transferred is used to overcome the binding energy of the electron and the remainder appears as kinetic energy of the resulting photoelectron. Thus, the kinetic energy of the ejected photoelectron equals the energy of the incident photon minus the binding energy of the electron. Kinetic Energy (electron) = h – E b where h is the energy of incident photon, and Eb is the binding energy of the electron. The ejected photoelectron travels a certain distance within the absorber and loses its energy through secondary ionizations. In this way, the entire photon energy of the incident photon is deposited in the tissue irradiated. As a result, an atom that participated in photoelectric interaction is left ionized. The vacancy created due to ejection of the electron is instantly filled by an electron from an outer orbital of the same atom, emitting the balance of energy as a photon between the respective orbits with characteristic low energy. The photoelectric effect is the dominant energy transfer mechanism for X and γ ray photons having energies below 50 keV in biological tissues, but it is much less important at higher energies. (An electron volt is a measure of energy which is the kinetic energy gained by an electron passing through a potential difference of one volt. 1 eV = 1.602 x 10 –19 Joules). 2.2.2.4. Compton scattering The process of energy deposition called the Compton Effect occurs when the incident photon interacts with the outer orbital electron whose binding energy is very low compared with that of the incident photon. In this interaction, the incident photon transfers energy to an atomic electron causing its ejection from the atom. The photon is scattered with the remainder of the original energy in a different direction to that of the incident photon. Compton scatter thus causes ionization of the absorbing atom due to loss of an electron. The scattered electron (a secondary charged particle) travels some distance in matter and eventually loses energy by further ionization and excitation events to become part of the material. The probability of Compton scattering decreases with increasing photon energy. It is the principal absorption mechanism for X and γ rays in the intermediate energy range of 100 keV to 10 MeV. This range is in the therapeutic radiation range, and it also forms most of the γ radiation present in a nuclear explosion. 2.2.2.5. Pair production When a photon of high energy ( >1.02 MeV) interacts with atoms of the medium, the incident photon can be spontaneously converted into the mass of an electron and positron pair by interaction of the Coulomb force in the vicinity of the nucleus. The oppositedly charged particles are emitted in opposite directions to each other and cause damage as secondary charge particles. A positron is the anti-matter equivalent of an electron and it has the same mass as an electron, but it has a positive charge equal in strength to the negative charge of an electron. The energy of the interacting photon in excess of the equivalent rest mass of the two particles (1.02 MeV) appears as the kinetic energy of the pair and the recoil nucleus. The positron has a very short lifetime and, at the end of its range, it combines with a free electron. The entire mass of these two particles is then converted into two γ photons each of 0.51 MeV 17 energy emitted in opposite directions. The secondary electrons (or positrons) produced in any of these three processes frequently have enough energy to produce many further ionizations up to the end of their range. 2.2.2.6. Dependence of absorption on atomic number The radiation energy deposition depends on the energy of the radiation and the atomic number (Z) of the absorbing material. The mass absorption coefficient of photoelectric absorption varies directly with the third power of the atomic number of the absorber (Z3). The effective atomic number of bone is about twice that of soft tissues, and the probability that a photon will be absorbed in bone is about six times that in an equal thickness of soft tissues. Bone is mainly comprised of calcium whereas soft tissues are comprised of low atomic number elements such as carbon, hydrogen and oxygen. On the other hand, the mass absorption coefficient for the Compton process is nearly independent of atomic number. Compton and photoelectric effects are vital for appropriate applications in X- ray diagnosis and cancer therapy. In radiotherapy, high-energy photons in the range of 1-10 MeV are preferred because absorbed dose is nearly the same in bone and soft issues whereas low energy photons are preferred in diagnosis because of the much desired large contrast in absorption of these tissues. 2.2.2.7. Half value layer When an electromagnetic radiation like X or γ rays passes through matter, its intensity is gradually reduced or attenuated with increasing depth due to the energy deposition interactions. This results in a decrease of photons, mainly due to photoelectric absorption and Compton scattering processes. The probability for absorption in a layer of material is proportional to the mass density. For a monoenergetic beam of photons, a constant fraction decreases as the beam travels through each unit of thickness in the absorber. This results in an exponential decrease in intensity with an increase in the thickness represented by the following equation; I (x) = I0 e- µx where I (x) = the intensity at thickness x, I0 = is the initial intensity on the surface of the absorber, = n× is the absorption coefficient measured in cm 1, n = the number of atoms per cm3 in the material, = the absorption cross section in cm2, and x = the thickness of material in cm. The thickness of absorber that reduces the photon intensity to one half is called the half value layer (HVL). Absorption of the beam depends on the mass and thickness of the absorber and the energy of the beam. Low energy photons are much more likely to be absorbed than high energy photons, for example the first 1.5 cm of water absorbs 40 % of 50 kVp X rays. The probability that a photon will interact with an orbital electron is optimum when its energy equals the binding energy of electron in the encountered atom. The total absorption coefficient of aluminium (Atomic No. 13) for γ rays plotted against photon energy shows that mostly Compton scattering dominates. In contrast, the total absorption coefficient of lead (atomic number 82) for γ rays, plotted against photon energy shows that the photoelectric effect dominates at low energies and pair production dominates above 5 MeV. Lead is often used to protect the body from radiation exposure because of its suitable HVL properties. 18 2.2.3. Particulate radiations Particulate radiations (e.g. α, β particles (electrons), protons, neutrons, ions), also produce their effects by causing ionization and excitation processes randomly in the atoms or molecules of the traversed material. The passage of charged particles, electrons and positively charged ions, causes intense damage (energy deposition) to molecules along the path in living tissue due to strong electrostatic interactions between the travelling particle and the electrons of the atoms of the medium. 2.2.3.1. Charged elementary particles Protons with one unit mass and one positive charge, cause less damage than α particles (helium nuclei) because the rate of deposition of energy varies inversely in proportion to the velocity of the particle and directly in proportion to the square of the charge. At the same energy, α particles have lower velocity because of their higher mass and carry twice the charge of a proton. Radioactive materials often release α particles and because they are a highly ionizing form of particulate radiation they usually have low penetration. They quickly lose their energy and they penetrate only a few tens of microns in body tissue. They can be fully absorbed by a sheet of paper. Beta particles (β, electrons) are also emitted by radioactive nuclei, as well as being displaced from atoms and molecules by X and γ rays as discussed above. They carry a single negative charge but their path in absorbing materials such as tissue is erratic due to their light mass (approx 1/2000 that of a proton). High energy electrons ionize much less efficiently than α particles because of their lower mass (and resulting higher velocity) and lower charge. Therefore, they penetrate tissues to a greater depth than α particles. Generally, beta particles do not penetrate further than the skin of the human body. 2.2.3.2. Uncharged particles Neutrons (n) are uncharged particles with a mass very similar to that of a proton and are an indirectly ionizing radiation because without a charge they cannot participate in electrostatic interactions. At the same mass and energy, neutrons are more penetrating than are charged particles. Although neutrons do not interact strongly with electrons of atoms in the traversed material and do not directly ionize atoms, they do cause a density of ionization that is, far greater than in the case of X rays. Neutrons interact with the atomic nuclei of the medium and they lose energy by different interaction processes depending on their energy (velocity) and the mass of the encountered nucleus. In soft tissues, because of the abundance of protons with mass equal to that of neutrons, fast neutrons (>1 MeV) mostly lose energy by elastic scattering through collision processes producing high energy recoil protons, which in turn deposit energy by electrostatic interactions with electrons in the tissue as described above. Neutrons begin to interact by inelastic scattering at energies above 6 MeV, and fast neutrons may interact with carbon and oxygen nuclei producing α particles, recoil protons and heavy nuclear particles. Fast neutrons can be made into thermal neutrons via a process called moderation. In reactors, typically heavy water, light water, or graphite are used to moderate neutrons. Thermal neutrons have a much larger effective cross-section than fast neutrons, and, therefore, can be absorbed more easily by any atomic nuclei with which they collide, creating a heavier and often unstable isotope of the irradiated element. Most fission reactors use a neutron moderator to slow down, or thermalize the neutrons that are emitted by nuclear fission so that they are more easily captured, causing further fission. This ability of neutrons to produce radioactive 19 nuclei (neutron activation) which then produce ionizing radiation by their decay can be used to analyse the atomic composition of certain materials. 2.2.3.3. Ions The nuclei of carbon, neon, silicon, argon atoms form charged ions when one or more orbital electrons have been stripped off. These can be accelerated to hundreds of MeV energies in special accelerator facilities. High energy charged ions offer special advantages in cancer radiotherapy because of the energy distribution along their track which has a high peak at its end (the Bragg peak). This allows the possibility of depositing high energy densities at depth in tissue but these facilities are as yet very limited on account of high costs and sophisticated technical requirements. 2.2.4. Linear energy transfer When ionizing radiations traverse through matter, they lose energy gradually through various interaction processes along the length of their path. For a particular absorber, the rate of loss of energy depends on the energy and type of radiation as well as the density of the material (Table 2.2). The density of energy deposition in a material such as tissue is called the Linear Energy Transfer (LET) of the radiation. It is defined as the average energy deposited per unit length of track of radiation and the unit is keV/ µm. Note that the LET varies along the length of the track of charged particles because as the charged particle deposits energy in tissue it slows down. The rate of transferring energy (-dE/dX, loss of energy per unit distance) increases as this occurs, such that there is a peak of energy deposition at the end of the track (the Bragg peak). LET essentially indicates the quality of different types of radiation and is important because the biological effect of a radiation (its relative biological effectiveness, RBE) depends on its average LET. Charged particles generally have higher LET than X and γ rays because of their greater energy deposition along the track. Radiations are categorized into low and high LET radiations with particulate radiations usually being high LET radiations whereas X and γ rays are low LET radiations due to their sparse ionizations (Table 2.2). In general the RBE of a radiation increases with its LET up to a value of about 100 keV/µm and above this value starts to decline due to energy deposition in excess of that needed to cause the biological effect (overkill). Energy loss events are essentially randomly distributed along the track of the photon or charged particle. For low LET radiations the energy deposition events along the track of the photon are sparse relative to the dimensions of biomolecules such as DNA with the result that photons may pass through such a molecule without depositing any energy. For such radiations the amount of energy deposited in a region of the track similar in dimensions to biological molecules also various widely from a few eV up to 100s of eV. For high LET radiation the energy loss events are much more closely spaced and significant energy will be deposited along all parts of the track similar in dimension to biomolecules. 20 TABLE 2.2 TYPICAL LET VALUES OF IONIZING RADIATION Radiation Linear Energy Transfer, KeV/µm Co- 60 γ rays 0.2 250 kVp X rays 2.0 10 MeV protons 4.7 150 MeV protons 0.5 14 MeV neutrons 12 2.5 MeV α particles 166 2 GeV Fe ions 1000 (Hall and Giaccia, 2006) 2.2.5. Radiation dose and units The biochemical changes produced by ionizing radiations are the fundamental events leading to radiation damage in tissues. Radiation is measured either as exposure or as absorbed dose. The absorbed dose is the amount of energy absorbed in a system and generally regarded as the best way to quantify the irradiation absorption. 2.2.5.1. Exposure The radiation exposure is a measure of radiation based on its ability to produce ionization in air under standard temperature and pressure, and is the quantity indicated by many radiation detectors such as ionization (eg Geiger-Muller) chambers. The (S.I.) unit for exposure is Coulombs/kg in air (or Roentgen R in old units: 1 R = 2.58 x 10-4 C/kg air). The unit of exposure is only defined for air and cannot be used to describe dose to tissue. Nevertheless ionization chambers are widely used to calibrate medical radiation devices and conversion factors to calculate absorbed dose from exposure have been carefully documented for different radiation energies and tissues. 2.2.5.2. Absorbed dose The amount of energy absorbed per mass is known as radiation dose. Radiation dose is the energy (Joules) absorbed per unit mass of tissue and has the (S.I.) units of gray (1 Gy = 1 J/ kg). In the past the rad (radiation absorbed dose) was used, where 100 rad = 1 Gy (1 rad = 1 cGy). Various types of radiation dose units are used in radiobiology and Table 2.3 presents some of the frequently used dose units for measuring these radiation quantities. 2.2.5.3. Equivalent dose As discussed above the biological effectiveness (RBE) of each type of radiation varies greatly depending largely on LET. For radiation protection and occupational exposure purposes the term ‘equivalent dose’ is used to compare the biological effectiveness of different types of radiation to tissues. The (S.I.) dose equivalent (HT) in sievert (Sv) is the product of the 21 absorbed dose (DT) in the tissue multiplied by a radiation weighting factor (WR), often called the quality factor. TABLE 2.3 SUMMARY OF RADIATION DOSES AND UNITS Dose SI Unit Old unit Conversion factor Exposure C/kg air Roentgen 1 R = 2.58 x 10-4 C/kg air Absorbed dose gray (Gy) rad 100 rad = 1 Gy Equivalent dose sievert (Sv) rem 100 rem = 1 Sv Equivalent dose is expressed as a summation to include the effects of irradiation of tissue by more than one type of radiation. In the past the unit rem (radiation equivalent man) was used to compare doses received by different types of radiations (100 rem = 1 Sv). The quality factor for low LET radiations is 1 so that for low LET radiations 1 Sv = 1 Gy. HT= WR x DT 2.2.5.4. Effective dose Effective Dose is used to estimate the risk of radiation in humans. It is sum of the products of equivalent doses to each organ/tissue (HT) and the tissue weighting factor (WT) (Tabel 6). E = W T x HT The unit of effective dose is the Sievert (Sv). 2.2.5.5. Collective dose Collective dose is defined as the dose received per person in Sv multiplied by the number of persons exposed per year i.e. man-sievert per year. This unit is generally used for protection purposes and in population response calculations. 2.2.6. Principles of radiation dosimetry Absorption of radiation in material produces many changes, which form the basis to dose measurements based on physical, chemical and biological effects. Different detectors have been used to develop dosimeters for ionizing radiation and some of them are used to measure relative dose distributions for therapeutic electron and photon beams. A few of them are used for measurements of absolute or reference absorbed dose called primary standards. Detectors can be divided broadly into three categories: those that measure directly the quantity of energy absorbed, detectors that measure ionization and those that quantify free radicals formed in the absorbing medium. Secondary chemical dosimeters are widely used commercially and have proved beneficial to clinical and scientific communities for both research and applications in photon radiation dosimetry. Among the most popular dosimeters are the Fricke chemical dosimeter, thermo- luminescence dosimeters (TLD) and ion chambers or diode dosimeters. These dosimeters are each characterized by their own merits and are useful in particular conditions of operation. The fundamental requirement for a suitable dosimeter is the linearity of response as a function of radiation dose within a wide dosage range. 22 2.2.6.1. Chemical dosimeters The Fricke chemical dosimeter is based on chemical change by absorption of radiation and used to measure, X, γ and electron doses. The principle consists of the chemical change of ferrous ions (Fe +2) into ferric ions (Fe +3) by absorption of radiation energy. Measurement is accomplished by optical absorption of ferric ions, which has a high extinction coefficient allowing determination of concentration changes. The major drawback is the unreliability in the presence of undesirable impurities. The method is highly unstable in air especially after irradiation but is relatively cost effective. The measurements are highly linear with increasing dose up to more than 150 Gy. 2.2.6.2. Thermoluminescence dosimeters (TLD) Thermoluminescence is based on generation of trapped electrons by exposure of lithium fluoride to radiation. The measurement of dose consists of measuring the luminescence induced by thermal treatment after radiation exposure. The light emitted is proportional to radiation dose. Lithium fluoride chips provide good spatial information but require careful calibration and rather laborious read-out. In addition, TLD are oxygen sensitive which imposes a limitation. The method is not as cost effective as the Fricke dosimeter, it lacks ease of preparation and the measurements become nonlinear at absorbed doses above 10 Gy. Optically stimulated thermoluminescence (OSL) is used in another device based on aluminum oxide and this requires no processing. It was originally developed for radiation therapy but is now also used for diagnostic purposes. 2.2.6.3. Ionization chambers Ionization chambers consist of an air-filled chamber containing two electrodes to which a voltage is applied. They measure the current flow which occurs due to the ionization of the air molecules exposed to radiation. They are capable of giving instant readings with good accuracy. The chambers are easy to use but are poor in providing spatial information. Diode dosimeters are based on the principle of ion collection formed by radiation incident in the chamber. Measurement consists of collection of ions on the cathode, formed by exposure to radiation, but this technique requires intricate circuitry and is not cost effective. Ion chamber performance depends on the voltage applied for charge collection. 2.2.6.4. Film dosimetry Special radiographic films have been developed for verification of dose in radiotherapy practice. This has proved useful for measuring dose profiles but the method has limited accuracy and dose range for determination of absolute radiation doses. 2.2.7. Direct and indirect effects The physical interactions of ionizing radiation leads to loss of energy of radiation and production of ionization and excitation of atoms and molecules which may convert into free radicals in pico to femto seconds after physical interaction with atoms (10-13 to -15 s). These radicals react with neighbouring molecules and produce secondary DNA or lipid radicals by reaction with another neighbouring molecule. Chain reactions may also occur, particularly in lipids, and may play a role in damage to cell membranes. Free radicals are fragments of molecules having unpaired electrons, which have high reactivity with cellular molecules and, therefore, have a short life. They can be detected by fast measuring techniques like pulse radiolysis and flow electron spin resonance (ESR). 23 Free radicals are generated in great number by ionizing radiation due to the process of energy absorption and breakage of chemical bonds in molecules. These are known to play a major role in radiation effects on biological tissues and organisms. These radicals are highly reactive and found in a number of biological processes, metabolism, oxidation, reduction, and pathological diseases and cancer induction. Both electromagnetic and particulate radiations act on cells to cause free radicals and subsequent molecular damage through direct as well as indirect actions. When ionizing radiation energy is deposited in a macromolecule that is important for the biological effect observed (often DNA for cell killing), it is called a direct effect of radiation. Alternatively, photons may be absorbed in the water of an organism causing excitation and ionization in the water molecules. The radicals formed after passage of radiation and water radiolysis, namely the hydrated electron (eaq-), the hydrogen atom (H.) and the hydroxyl radical (.OH) contribute in causing damage to biological systems. A compound with a high rate constant of reaction can scavenge primary free radicals of water radiolysis. Free radicals of biomolecules can be restituted by hydrogen donating compounds, such as thiols and cysteine. Alternatively, they can be fixed by reaction with oxygen or oxygen mimicking compounds, which makes them permanently damaged. This is called ‘the oxygen effect’, which forms the basis of increasing molecular and cellular damage in the presence of oxygen. These chemical reactions form the basis of searching for compounds which can sensitize cell/tissue damage or protect them against radiation, and which are of direct relevance to radioprotection and cancer radiotherapy. 2.2.7.1. Direct effects Ionizing radiation (IR) can act on biological molecules (RH, representative of hydrocarbons) causing ionization and excitation. One or more chemical bonds may be broken giving atoms or molecules with unpaired electrons, which are very reactive and have a short life. The formation of these radicals occurs in the picosecond time range after the passage of the photons. The bond may be repaired or cross-linking may occur due to radical-radical reactions. These free radicals may also react with oxygen, and in the case of lipids may initiate chain reactions (see below). IR + RH → R +H Both H·and R·radicals can react with another molecule e.g. DNA, lipids, proteins. R + R’H → R’ · + RH Radicals can produce cross linking reactions. R + R · →R - R · It is estimated that about one third of biological damage by γ radiation is caused by direct effects. This process becomes more dominant with high LET radiation, such as neutrons or α particles. 2.2.7.2. Indirect Effects - Water Radiolysis The absorption of energy depends on the abundance of material in the path of the radiation. Water is the most predominant molecule in living organisms (about 80 % of the mass of a living cell is water). Therefore, a major proportion of radiation energy deposited will be absorbed in cellular water. A complex series of chemical changes occurs in water after 24 exposure to ionizing radiation. This process is called water radiolysis. The understanding of chemical changes in water is essential in studies of radiation effects on living cells. Interaction of radiation with water causes ionization and excitation process producing short- lived H2O+ radical-cations, fast electrons, and electronically-excited water molecules (H2O+). H2O+ ions and excited water molecules are unstable and decompose within 10-13 s to form OH and H radicals IR + H2O → H2O+ + e- H2O + H2O+→ H3O+ +OH IR + H2O → H2O*→H2O + photon emitted or H2O*→OH + H The hydroxyl radical has an unpaired electron and is a highly reactive oxidizing agent. It can diffuse a short distance and react with critical target molecules producing another radical. This can react with water forming an anion which rapidly dissociates to give a hydrogen atom (H · ). The ejected secondary electrons may interact with a water molecule to form hydroxyl ions and a hydrogen atom (a hydrogen radical), or they may lose energy by a sequence of interactions with the medium until they attain thermal energies after about 10-11 s. The thermalized electrons are then solvated by dielectric interactions with neighbouring water molecules to form e-aq i.e. e-aq is a free electron in a solvent cavity surrounded by a sheath of orientated water dipoles. It reacts with a proton to give a hydrogen atom (H · ): e- + H2O→H2O-→ OH- + H e-aq + H+ → H e-aq is the strongest known reducing species at pH 7.0. In oxygenated solutions, e-aq is converted to O2-, which is a strong oxidizing agent and the precursor of hydrogen peroxide: e-aq+ O2 → O2- These primary water radicals (eaq, OH, H ) have high reactivity towards molecules of cells, DNA, lipids and other subcellular constituents. In oxygenated solutions, hydrogen atoms can react with oxygen to give hydroperoxyl free radicals (HO2 ): H + O2 → HO2 The relative yields of the water radiolysis products depend on the pH and LET of the radiation. The concentration of these radicals are expressed in terms of a G value which is defined as the number of radicals or molecules produced per 100 eV of energy absorbed in the medium. Typical G-values are Ge-aq = 2.6, GOH·= 2.6, GH·= 0.6. 2.2.7.3 Free radical scavengers Certain compounds with a high rate constant of reaction may scavenge the primary radicals of water radiolysis (e.g. dimethylsulphoxide). Hydroxyl radicals can also be scavenged by a 25 number of –SH containing compounds as a moiety in their chemical structure. The hydrated electron can be efficiently scavenged by oxygen producing a number of oxygen-centered radicals. Scavenging of hydroxyl radicals forms one basis for development of radioprotectors. Amifostine (WR 2721), an aminothiol, is one of the well-known protectors which has potential application in radiotherapy. Thiol compounds may also donate hydrogen atoms to radical sites on other biological molecules such as DNA but scavengers act primarily against the indirect effect induced by water radicals. Hence they have reduced efficacy for high LET radiation for which the direct effect plays a more prominent role in biological damage such as cell killing. 2.3. Molecular and cellular radiobiology 2.3.1. Radiation lesions in DNA Radiation causes a wide range of lesions in DNA such as single strand breaks in the phosphodiester linkage, double strand breaks on opposing sites or displaced, base damage, protein-DNA crosslinks and protein-protein crosslinks involving nuclear proteins such as histones and non-histone proteins. The presence of histones and DNA in a 1:1 weight ratio makes histones prime candidates for crosslinks. The number of DNA lesions generated by irradiation is large, but the number giving rise to cell kill is extremely small. The numbers of lesions induced in the DNA of a cell by a dose of 1-2 Gy are approximately: base damages > 1000; single strand breaks (ssb) ~1000; double strand breaks (dsb) ~40. Dsb play a critical role in cell killing, and there are experimental data showing initially-produced dsb correlate with radiosensitivity and survival at low dose, and unrepaired or mis-repaired dsb to correlate with survival after higher doses. Increasing evidence suggests the importance of complex dsb lesions after high LET irradiation. Knowledge of radiation track structure has been used to explain the wide variation and wide distribution of lesions in DNA. The importance of clusters of energy deposition events (ionizations and excitations) at track termini of secondary electrons resulting in multiple closely-spaced lesions (multiply damaged sites) within a range of 20 nm, has been recognised as important for cell killing and in regard to the ability of cells to repair such lesions. 2.3.2. Major types of DNA repair There are multiple enzymatic mechanisms of DNA repair in cells that act on different types of lesions. For double strand breaks there are two primary repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ repair operates on blunt ended DNA fragments resulting from broken phosphodiester linkages. There is a requirement for Ku70/Ku80 repair proteins to recognize the lesion termini, binding of the Ku-heterodimer to DNA-PK (protein kinase), and activation of the XRCC4 ligase enzyme by this complex for final religation of the fragments after enzymatic “cleaning up” of the broken ends of the DNA molecule, by a variety of other recruited proteins, so that ligation can occur. Repair by NHEJ operates throughout the cell cycle but dominates in G1/S-phases. The process is error prone because it does not rely on sequence homology. Dsb repair by homologous recombination (HR) utilizes sequence homology with an undamaged copy of the broken region and hence can only operate in late S- or G2- phases of the cell cycle. It starts by nucleolytic resection of blunt ends, binding of NBS/MRE11/rad50 protein complex to the DNA termini, followed by strand exchange facilitated by attachment of rad51/XRCC2 protein. Then there is DNA synthesis of the missing nucleotides on the undamaged templates and ligation. This creates a complex strand crossover between the damaged and undamaged strands known as a Holliday junction, which is finally resolved before the repair process is complete. Other DNA repair mechanisms such as base excision repair (BER), mismatch repair (MR) and nucleotide 26 excision repair (NER) respond to damage such a base oxidation, alkylation, and strand intercalation. 2.3.3. Damage recognition and signalling A first step in recognition of radiation damage (strand breaks) to DNA is ATM binding to DNA termini. This induces kinase activity in ATM which phosphorylates and activates the CHK kinases, which in turn phosphorylate p53. As a result p53 is released from MDM-2 and is stabilized to induce p21, which inhibits the cyclin-dependent kinase cyclinE-CDC-2 controlling the G1-S transition in the cell cycle. The resultant G1 arrest (G1 block) after irradiation ensures that the damaged DNA is not replicated before repair. Tumours showing mutant p53 or p53 null status, as the result of p53 destruction by viral protein E6, fail to initiate a G1 arrest and may not restitute damaged DNA before replication. But even p53 mutant cells display a G2 arrest and may exercise repair options (or induce apoptosis) and thus prevent mitotic propagation of defective DNA in M phase. Repair signalling starting at ATM proceeds via downstream activation of BRCA1, c-Abl, NBS1 and RAD 51 to initiate DNA repair. An alternative response to DNA damage is induction of apoptosis initiated by p53, although this occurs extensively after irradiation only in a few specific cell types, such as cells of hematopoietic lineages, endothelial cells, germ cells and oligodendrocytes. C-Abl, BID and the proapoptotic factor BAX (in the Bcl-2 family of proteins) respond to sequential phosphorylation cascades starting with ATM. 2.3.4. Consequences of unrepaired DNA damage: Chromosome damage Mutations from low dose exposure influence base pairing, coding, transcription and gene expression. Chromosome analysis in mitotic spreads (karyotyping), micronucleus formation and fluorescent in situ hybridisation (FISH) can detect unrepaired DNA damage in chromatids by a variety of DNA damaging agents including radiation. Aberrant chromosomes arise when broken ends rejoin with other broken ends to generate rings, dicentrics, translocations and other chromosome aberrations. Dicentric chromosome aberrations arise post replication from the joining of 2 broken chromatids in different chromosomes and can be use as a marker for radiation exposure. Acentric fragments and dicentrics are unstable aberrations and may not survive past the next mitosis, implicating loss of genetic material which may signal death in diploid cells. In polyploidy cells such losses may be of lesser consequence Micronuclei contain acentric fragments and may be detected by stimulating lymphocytes (or certain other cell types) into division followed by treatment with cytochasin B, which allows nuclear division but stops cellular division. The micronucleus assay, although somewhat less sensitive, is a simple and effective alternative to chromosome analysis. The use of the micronucleus assay has been studied for the purpose of radiosensitivity testing of patients using lymphocytes, but limitations exist due to assay variability. 2.3.5. Radiobiological definition of cell death Cells are generally regarded as having been “killed” by radiation if they have lost reproductive integrity, not by whether they physically survive in the population. Loss of reproductive integrity can occur by apoptosis, necrosis, mitotic catastrophe or by induced senescence. Although all but the last of these mechanisms ultimately results in physical loss of the cell this may take a significant time to occur, e.g mitotic catastrophe may not happen until several divisions have taken place. Apoptosis or programmed cell death is a strong feature in embryological development and in lymphocyte turnover. Previously, this early form of cell death was called interphase cell death. Apoptosis can be identified by microscopy and 27 typical shrinkage of cellular morphology, condensation of chromatin, nucleosome laddering indicating chromatin degradation, cell membrane blebbing, activation of caspases and release of cytochrome c. Exposed phosphatidyl serine in the cell wall permits binding of annexin V and assessment of apoptosis by flow cytometry. The characteristics of apoptosis (which is non-inflammatory) are in contrast to those of necrosis, typified by cell edema, poor staining of nuclei, increase of membrane permeability, shut down of cell metabolism, and an accompanying inflammatory response. Senescence or replicative senesence (RS) is observed when cells stop dividing, and this differs from the behaviour of stem cells and tumour cells which do not show these limitations. Senescent cells are somewhat edematous and show poor cell-cell contact, increased polyploidy, decreased ability to express heat shock proteins, and shortening of telomeres. Apoptosis occurs in particular cell types after low doses of irradiation e.g. lymphocytes, serous salivary gland cells, and certain cells in the stem cell zone in testis and intestinal crypts. Reproductive cell death is a result of mitotic catastrophe which can occur in the first few cell divisions after irradiation, and it occurs with increasing frequency after increasing doses. Cells that fail to divide successfully after irradiation can also undergo apoptosis at that stage. Cellular necrosis generally occurs after high radiation doses. A rapid fall of cell numbers after irradiation is likely to be due to apoptosis but may also occur by mitotic catastrophe in rapidly proliferating populations. Whether apoptosis reflects overall cell killing in tumour cell inactivation by radiation is currently unresolved and may only be the case for certain types of tumour cells. 2.3.6. Suvival curves and models The accepted gold standard for measuring the radiosensitivity of a cell population is the retention of reproductive integrity or mitotic intactness i.e. the ability of a cell to undergo more than 5-6 cell divisions (and produce a viable colony containing at least 50 cells). This is referred to as cell survival and percent survival after irradiation is calculated by correcting for the ‘plating efficiency’ of unirradiated cells, this often being less than 100% due to the true proportion of colony-forming cells in those plated being low, or potential influences of the media, pH, temperature and cell specific factors. Measurements of apoptosis or MTT or SRB vital dye staining growth assays are often used instead of a colony assay for measuring radiosensitivity for reasons of simplicity, shorter assay time, and operation in multiwells permitting large number of parameters to be tested e.g. a range of growth inhibiting drugs. Major disadvantages of these approaches are the narrow range of doses and survivals that can be used, greater assay variability, and, particularly, that the assays do not test mitotic viability. Hence these assays rely on the (often unfounded) assumption that there is a clear relationship between apoptosis or cellular growth and cell survival over a wide range of doses and survival levels. Survival curves are best shown as a semilog plot of survival against irradiation dose, generally in the dose range of 1 – 10 Gy for single cells. The most common model used today is the linear-quadratic model, fitted using a second-order polynomial, with the constants and describing the decline of survival (S) with increasing dose (D). S = e –( D + D2 ) Equal cell kill of linear and quadratic components is achieved when dose D = /. For high LET irradiation the quadratic component is small or non-existent. An older model is the single hit/ single target model described by S = e – D/Do. 28 Do is effectively the reciprocal of (above) and represents the dose which reduces survival to e-1 or 37 %. The linear relationship is consistent with data from some bacteria but it does not apply in eukaryotic cells (except at high LET), which show shouldered survival curves that can be accommodated by a single-hit multitarget model described by: S = 1- [1 - e- (D/Do)]n. This is reliable at high dose but not at low dose, because it does not describe accurately the ‘shoulder’ region at low doses, even if another single-hit term is added. For practical purposes, there are merits of using survival at 2 Gy (SF2), because this is a dose fraction using commonly in radiotherapy. 2.3.7. Cell cycle effects Renewing cells in a growing population (e.g. skin, gut, bone marrow, tumour cells or cells in culture), but not when resting in Go phase, participate in the cell cycle. Replication of the genome occurs in S-phase and mitotic propagation to daughter generations occurs in G2/M phases. Typical cell generation times are 10 – 40 hours with the G1 phase taking about 30 %, S-phase 50 %, G2 phase 15 % and M-phase 5 % of the cell cycle time, although G1 phase time may vary and be much longer in slowing proliferating populations. In interphase the majority of cells are in G1 or Go. There are checkpoints at the G1/S and G2/M boundaries that monitor the fidelity of genomic processing. Binding of cyclins to cyclin dependent kinases activates the kinase complex to negotiate the checkpoints: cyclin B1/ p34 CDC-2 for G2/M transition, cyclin D1/cdk-4 for M/G1 transition, cyclin E/cdk-2 for G1/S and cyclinA/cdc-2 for S/G2 transition. Drugs that abrogate cell cycle blocks e.g. caffeine and pentoxifylline, are radiosensitizing by rapidly re-establishing the B1/p34 CDC-2 pair, promoting early mitotic progression before complete recovery and directly inhibiting HR repair in G2. In p53 mutants (i.e. in most tumours) and in cells of p53 null status arising from p53 destruction after viral infection (by the HPV E6 protein), p21 induction is abolished and p21 controlled inhibition of G1/S transition cannot occur. In the absence of the G1 block, cells enter a block at G2/M. Most tumour cells being p53 mutant hence would display altered checkpoint expression and limited repair routes with opportunities for ther
