Radiation Chemistry; Effects of Radiation On DNA and Chromosomes. Kathryn D. Held, Ph.D. Massachusetts General Hospital Harvard Medical School

Transcription

1 Radiation Chemistry; Effects of Radiation On DNA and Chromosomes Kathryn D. Held, Ph.D. Massachusetts General Hospital Harvard Medical School 1

2 Effects of Radiation On DNA and Chromosomes Introduction to Radiation Chemistry Water Radiolysis DNA Damage Chromosome Aberrations 2

3 Sequences in the Development of Radiobiological Effects Time Event s Absorption of Ionizing Radiation s Physical Events Ionization Excitation s Physicochemical Events Free radical formation Breakage of chemical bonds s Chemical Events Reactions of radicals Minutes to hours Days to months Years Generations Biochemical/Cellular Processes Repair Division delay Chromosome damage Loss of reproductive capacity Tissue Damage CNS, GI, Bone marrow syndromes Late tissue damage Birth defects from in utero exposure Late Somatic Effects Cataracts Carcinogenesis Genetic Effects 3

4 Ionizing Radiation All biological effects produced by ionizing radiation result from the chemical events that occur shortly after the initial deposition of radiation energy. Absorption of IR by matter produces ions and excited molecules. A A + + e - A A* The number of species produced is proportional to dose. 4

5 Ionizing Radiation Free radicals - atoms or molecules that have one or more unpaired electron designated by may be formed by division of a covalent bond R:S R + S may be charged or neutral are generally very reactive 5

6 Direct and Indirect Actions of Ionizing Radiation (from Hall 1994) Low LET 70% 30% 6

7 Effects of Radiation On DNA and Chromosomes Introduction to Radiation Chemistry Water Radiolysis DNA Damage Chromosome Aberrations 7

8 Water Radiolysis Summary H 2 O OH, H, e - aq, H 2, H 2 O 2 OH is the most important biologically. 8

9 Mechanisms of Water Radiolysis Ionization and Excitation H 2 O H 2 O + + e - H 2 O H 2 O* Ion-molecule interaction and dissociation H 2 O + + H 2 O H 3 O + + OH e - + H 2 O H + OH - H 2 O* H + OH Electron hydration e - + (H 2 O) n Spur reactions e - aq H + H H 2 Diffusion OH + OH H 2 O 2 OH + H H 2 O 9

10 G Values (Yields) of Primary Radiolysis Species in Neutral Water -H 2O OH H e - aq H 2 H 2O 2 γ-rays and electrons of MeV 32 MeV α- particles (G-value = moles of material formed or changed by an energy absorption of 1 J) Note: For low LET radiation, the most common radiolysis species are OH and e - aq. With higher LET radiation, yields of radical species decrease and molecular species increase. 10

11 Reactions of OH Oxidation of inorganic compounds OH + M n OH - + M n+1 Addition to free radicals and unsaturated organics OH + CH 2 =CH 2 CH 2 -CH 2 OH Abstraction from saturated organics OH + CH 3 COCH 3 CH 2 COCH 3 + H 2 O 11

12 e - aq and H Reducing species Frequently undergo diffusion-controlled reactions Reactions do not seem to be biologically damaging 12

13 Reactions of Primary Radicals with Oxygen Formation of perhydroxyl and superoxide radicals O 2 + H HO 2 O 2 + e - aq O - 2 O H + HO 2 pk = 4.88 Reactions with organic radicals O 2 + R RO 2 Note: These reactions are thought to be responsible for the Oxygen Effect. 13

14 Radical Scavenging Use of a compound that selectively reacts with certain free radicals Simplifies more complex radiation chemistry 14

15 Radical Scavengers Additive Reaction Active Species Remaining N 2O N 2 O + e - aq + H 2 O OH (H ) OH + OH - + N 2 OH scavengers RH + OH R + H 2 O e - aq (H ) oxygen O 2 + e - aq O 2 - O 2 + H HO 2 OH, O 2 -, HO 2 acid e - aq + H+ H H, OH 15

16 OH Scavengers Decrease Radiationinduced DNA Damage (from Roots and Okada 1972) 16

17 Effects of Radiation On DNA and Chromosomes Introduction to Radiation Chemistry Water Radiolysis DNA Damage Chromosome Aberrations 17

18 DNA is a Primary Target Microbeam experiments show cell nucleus to be more sensitive than cytoplasm. Halogenated base analogues sensitize cells and DNA. Radioisotopes in DNA are more lethal than when in RNA or protein. DNA repair deficient cells are radiation sensitive; drugs that inhibit DNA repair usually are radiosensitizers. Oxygen and LET modify survival, cytogenetic damage and biological activity of DNA in similar manner. 18

19 Reactions of OH with DNA Bases and Sugar 19

20 Reactions of OH with DNA Bases and Sugar Subsequent to radical production in DNA, a multitude of products can be formed. E.g., from thymine alone, more than 30 radiolysis products have been identified, with quite different yields 20

21 Types of DNA Lesions from IR Breaks SSB DSB Base damages Change Loss (abasic sites) Crosslinks DNA-DNA DNA-protein From McMillan and Steel

22 Measurement of DNA Damages Base damages Enzyme sensitivity HPLC, GC-MS, GC-EC Immunological probes Strand breaks Gel electrophoresis (alkaline for SSB; neutral for DSB) Comet assay (alkaline for SSB; neutral for DSB) Foci of DNA repair-related proteins (e.g., γ-h2ax) 22

23 Pulsed Field Gel Electrophoresis 23

24 Comet Assay (Single Cell Gel Electrophoresis) Embed cells in gel and lyse Electrophorese Quantify amount of DNA in tail (damaged) versus head 24

25 Comparison of Assays for Breaks Note: More SSBs than DSBs Breaks usually linear with dose Killing usually shouldered (from Olive 1992) 25

26 Foci of DNA Repair-Related Proteins (e.g., γ-h2ax) as Measure of DNA DSBs (from Bonner 2003) γ-h2ax phosphorylated histone H2A variant X Foci fluorescent blobs representing aggregates of protein recognized by antibodies 26

27 γ-h2ax Foci as a Measure of DSBs (from Rothkamm and Löbrich 2003) (o = foci/cell; = PFGE) slope = 35 DSB/cell/Gy Note: Number of foci increases linearly with dose, with same slope as DSBs measured by PFGE. Even after very low doses, some foci remain at 24 h. 27

28 Foci as Measure of DSBs Caution: γ-h2ax foci have been seen after treatments that do not directly cause DSBs, e.g., hypoxia, hydrogen peroxide Other DNA repair-related proteins also form foci and are being used as surrogates for DSBs, e.g., 53BP1, RAD51 28

29 Number of Radiation-Induced Lesions Type of Lesion Number per Gray Double strand breaks 40 Single strand breaks 1000 Base damages Sugar damages DNA-DNA crosslinks 30 DNA-protein crosslinks 150 Alkali-labile sites Number of Clustered Lesions not yet quantified. 29

30 Energy Deposition Events (spurs, blobs, short tracks) Spurs 100 to 500 ev < 100 ev <5000 ev Blobs Primary >5000 ev Short Tracks Delta rays Branch Tracks (from Mozunder and Magee 1966) 30

31 Energy Deposition Events for Low LET Radiation ENTITY ENERGY DEPOSITED SIZE NUMBER OF WATER MOLECULES PER EVENT ENERGY (%) EVENTS (%) Spur <100 ev 4 nm (diam.) 1100 ~80 95 Blob <500 ev 7 nm (diam.) 6000 ~20 5 Short track ev DNA 2 nm (diam.) Nucleosome disc Thickness 5.7 nm Radius of 5.5 nm Electrons with sufficient energy to form short tracks will also produce spurs and blobs. (from Ward 1988) 31

32 Clustered Lesions (Multiply Damaged Sites) (from Steel 1993) 32

33 Biological Consequences of Clustered Lesions (MDS) Harder to repair accurately than single lesions Unrepaired Block DNA replication Loss of genetic integrity Mispaired May lead to DSBs Deletions could be produced Repair could be completed accurately 33

34 Measured Clustered Lesions Relative Cluster Frequencies in Human Cells DSB 1 Oxidized purines 1 Oxidized pyrimidines 0.9 Abasic sites 0.75 (from Sutherland et al. 2002) 34

35 Chromatin Structure is Important in Radiation Damage to DNA Presence of histones/ chromatin condensation Regionally multiply damaged sites Actively transcribing vs non-transcribing DNA Nuclear matrix attachment sites 35

36 Importance of Histones/Chromatin Condensation Recent studies suggest histone deacetylase (HDAC) inhibitors may be radiation sensitizers. (from Campausen et al. 2004a) (from Campausen et al. 2004b) 36

37 Which DNA lesion is most important biologically? Good correlation between DNA DSBs and cell killing (from Radford 1985) 37

38 Most Important Lesion? To date, most data suggest DSBs Most assays for DSBs will include Clustered Lesions Clustered Lesions may be most important for cell killing 38

39 Biological Consequences DNA damage Accurate repair Misrepair No repair Survival; no mutations Mutations Chromosome aberrations Genomic instability Neoplastic transformation Cell death/inactivation Mitotic Apoptotic Long-term arrest 39

40 Effects of Radiation On DNA and Chromosomes Introduction to Radiation Chemistry Water Radiolysis DNA Damage Chromosome Aberrations 40

41 Chromosome Aberrations Reflect initial DNA damage its repair (or non/misrepair) Two general types Chromosome aberrations G1 irradiation Both sister chromatids involved Chromatid aberrations S or G2 irradiation Usually only one chromatid involved 41

42 Chromosome versus Chromatid Aberrations (from McMillan and Steel 1993) 42

43 Examples of Chromosome Aberrations ring tricentric fragment dicentrics 43

44 Examples of Chromatid Aberrations quadra-radials complex exchange 44

45 Chromosome Aberrations Principal aberrations: Dicentrics Rings Acentric fragments Translocations Anaphase bridges Exchange-type aberrations can be symmetric or asymmetric. Aberrations can be stable or unstable. Dicentrics (e.g., in lymphocytes) are a good biomarker of radiation exposure. 45

46 Micronuclei formation is sometimes used as a surrogate for chromosome aberrations Micronuclei can result from chromosome deletions or fragments 46

47 Techniques Used in Chromosome Analysis Premature Chromosome Condensation (PCC) Fluorescence in Situ Hybridization (FISH; chromosome painting ) Use of FISH-based techniques has made it clear that: radiation-induced chromosome aberrations are more complex than previously realized. complexity of aberrations increases with LET. 47

48 Example of mfish Metaphase chromosomes from lymphocytes of plutonium-exposed individual showing complex rearrangements (from Anderson et al. 2005) 48

49 Dose Response Curve for Chromosome Aberrations is Linear-Quadratic (From Hall 2000) 49

50 Good Correlation Between Chromosome Aberrations and Loss of Clonogenicity (from Hall 2000) 50

51 Biological Consequences DNA damage Accurate repair Misrepair No repair Survival; no mutations Mutations Chromosome aberrations Genomic instability Neoplastic transformation Cell death/inactivation Mitotic Apoptotic Long-term arrest 51

52 Take Home Messages - 1 Indirect action produces most damage from low LET radiation; OH is the most critical water radiolysis species. A plethora of DNA damages are produced by IR. Numerous techniques can be used to measure the damage. Using foci of DNA repair-related proteins to measure DSBs is currently of great interest. IR produces clustered lesions (multiply damaged sites) that are probably most important biologically. 52

53 Take Home Messages - 2 Chromatin structure is important for radiation damage to DNA and its repair. The biological consequences of misrepair or no repair include mutations, aberrations, genomic instability, cell death/inactivation. It is assumed that most cell death results from DNA damage; relationships between loss of clonogenicity, apoptosis or long-term arrest are not straightforward. 53