1. Electron and photon induced damage to biomolecular systems M. Folkard Gray Cancer Institute, PO Box 100, Mount Vernon Hospital, Northwood HA6 2JR, UK folkard@gci.ac.uk

2. Ionising radiations damage biomolecules (including DNA) by breaking bonds. Bond-breaks occur either: - Directly, by direct ionisation of the biomolecule - Indirectly, through the ionisation of water, and the formation of damaging reactive radicals Radiation damage of biomolecules

3. Ionizing radiation damages ALL biomolecules similarly We now know that the most radiation-sensitive biomolecule in living tissue is DNA Consequently, it is damage to DNA that leads to all observed macroscopic biological effects

4. repair mis-repair mutation viable cell not repaired cancer cell death Radiation damage of biomolecules

5. Physical10 -20 - 10 -8 sionisation, excitation Timescale of events: Early boil. hours - weekscell death, animal death Late boil. yearscarcinogenesis Radiation damage of biomolecules Chemical10 -18 - 10 -9 sfree radical damage 10 -3 s - hourschemical repair

6. Nevertheless, the effectiveness of an ionising radiation critically depends both on its type (i.e. photon, particle) and on its energy Therefore, these differences arise solely because radiations of different quality and type produce different patterns of ionisation Radiation damage of biomolecules For the same dose, both the quality and the number of ionisations produced by ALL ionising radiations is the same

7. Biological effectiveness: radiation type Energetic X-rays

8. 1 Gy ~ 1000 tracks per cell ~ 100,000 ionisations per cell Biological effectiveness: radiation type

9.  -particles 1 Gy ~ 3 - 4 tracks per cell ~ 100,000 ionisations per cell Biological effectiveness: radiation type

10. Millar et al. Biological effectiveness: radiation type C3H 10T1/2 cells 10 20 30 0 0 2 4 6 transformants / 10 4 surviving cells 250 kVp X-rays 4 He 2 dose / Gy

11. 10 1 10 0 10 -1 10 -2 10 -3 10 -4 0 4 8 12 surviving fraction dose / Gy V79 cells energetic X-rays 1.5 keV Al K X-rays Prise, Folkard & Michael, 1989 0.28 keV C K X-rays Goodhead and Nikjoo, 1989 Biological effectiveness: radiation quality

12. The primary factor that determines biological effectiveness is ionisation density -  -particles and low-energy X-rays are densely ionising - energetic X-rays are sparsely ionising Biological effectiveness In general, densely ionising radiations are more effective than sparsely ionising radiations

13. 2  m 200 nm 20 nm 2 nm Biophysical Models of radiation damage - Develop a mathematical model of the cell and radiation track-structure

14. 200 nm energetic X-rays Biophysical Models of radiation damage Breckow & Kellerer, 1990  e-e-

15. 20 nm 1.5 keV Al K X-rays Biophysical Models of radiation damage Nikjoo, Goodhead, Charlton, Paretzke, 1989 1.5 keV X-ray e-e- e-e-

16. 2 nm 0.28 keV C K X-ray Biophysical Models of radiation damage 0.28 eV X-ray Nikjoo, Goodhead, Charlton, Paretzke, 1989 e-e-

17.  - particle Biophysical Models of radiation damage  -particle  e-e- 2 nm

18. photon single-strand break DNA Damage

19. double-strand break e-e- photon DNA Damage

20. complex damage Locally multiply damaged sites (LMDS) DNA Damage

21. The track-structure models are very good at mapping the pattern of ionizations relative to the DNA helix The next key step is to map the pattern of breaks in the DNA helix For this, we need to know the amount of energy deposited through ionisation, and the amount of energy required to produce strand-breaks

22. 1 MeV electrons 100806040200 Energy E / eV Frequency per eV liquid water DNA most probable E loss: 23 eV Re-drawn from; LaVerne and Pimblott, 1995 DNA Damage Theoretical spectrum of energy depositions by energetic electrons

23. 100 keV electrons 300 eV electrons 2 nm 10 -5 10 -6 10 -7 10 -8 10 -9 3002001000 Energy E / eV Freq. Events >E per target / Gy Re-drawn from; Nikjoo and Goodhead, 1991 Frequency of energy depositions >E in a 2 nm section of the DNA helix Most energy depositions ~few 10’s eV Few energy depositions >200 eV DNA Damage

24. Questions: How much energy is involved in the induction of single- and double-strand breaks by ionizing radiations? What is the minimum energy required to produce: 1) a single-strand break 2) a double-strand break

25. DNA Damage 0 1 2 100200 300400 probability of break energy in DNA / eV SSB DSB Nikjoo et al calculated the probability of SSB and DSB, based on data for strand breaks from I 125 decays Minimum energy to produce SSB ~20 eV Minimum energy to produce DSB ~50 eV Re-drawn from; Nikjoo, Charlton, Goodhead, 1994

26. ionising synchrotrons characteristic X-ray sources vacuum tubes linacs gas discharge sources isotope sources Energetic photon sources typical cluster size 1 eV1 keV1 MeV1 GeV ultra-violetsoft X-rays X- and  -rays photon energy / eV

27. Measurement of DNA damage Use Plasmid DNA (circular double-stranded molecules of DNA, purified from bacteria) i.e. pBR322 (4363 base-pairs) Un-damaged DNA (supercoiled) linear double-strand break relaxed single-strand break

28. relaxed linear supercoiled Measurement of DNA damage These forms can be easily separated by gel-electrophoresis

29. energy / eV SEYA, LiF, MgF window TGM, polyimide window SEYA, aluminium window 1010050200 10 12 10 11 10 10 9 photons s -1 cm -1 Experiments using the Daresbury Synchrotron

30. window electrometer valve pump VUV grid sample sample ‘wobbler’ Experiments using the Daresbury Synchrotron ‘dry’ DNA irradiator

31. SSB induction in ‘dry’ DNA 150 eV photons % supercoiled DNA Photons / cm 2 0 1x10 13 2x10 13 3x10 13 1 10 100

32. 0 1x10 14 2x10 14 3x10 14 8 eV 10 100 1 0.0 2.0x10 13 4.0x10 13 6.0x10 13 8.0x10 13 11 eV 1 10 100 0 1x10 13 2x10 13 3x10 13 150 eV 1 10 100 0.0 1.0x10 15 2.0x10 15 10 100 7 eV 1 % supercoiled DNA Photons / cm 2 SSB induction in ‘dry’ DNA

33. 150 eV photons DSB induction in ‘dry’ DNA % linear DNA 0 1x10 13 2x10 13 3x10 13 0 5 10 15 Photons / cm 2

34. 0.0 2.0x10 13 4.0x10 13 6.0x10 13 8.0x10 13 0 4 8 12 11 eV 0 1x10 13 2x10 13 3x10 13 0 5 10 15 150 eV 8 0 2x10 14 0 2 4 6 8 eV 1x10 14 3x10 14 % linear DNA 0.0 1.0x10 15 2.0x10 15 0 2 4 6 8 7 eV Photons / cm 2 DSB induction in ‘dry’ DNA

35. 51050100200 SSB DSB Quantum Efficiency /  Photon Energy / eV 10 -5 10 -4 10 -3 10 -2 10 -1 10 -0 ~20-fold Q.E. for SSB & DSB (dry plasmid) Prise, Folkard et. al, 1995, Int. J. Radiat. Biol. 76, 881-90. SSB thresholdDSB threshold

36. Observations 0.0 2.0x10 13 4.0x10 13 6.0x10 13 8.0x10 13 0 4 8 12 11 eV 0.0 2.0x10 13 4.0x10 13 6.0x10 13 8.0x10 13 11 eV 1 10 100 37% % supercoiled % linear photons / cm 2 The 37% ‘loss of super- coiled’ level represents an average of one ssb per plasmid. At an equivalent dose, about 4% dsb produced Induction of dsb is linear with dose, and has non- zero initial slope Therefore dsbs are NOT due to the interaction of two (independent) ssbs

37. Free radical damage of DNA photon H 2 O H 2 O + + e - H + + OH

38. 020 scale / mm VUV ‘DNA in solution’ VUV irradiator MgF DNA in 50  m gap

39. ‘DNA in solution’ VUV irradiator

40. ionising synchrotrons gas discharge sources Energetic photon sources 1 eV1 keV1 MeV1 GeV ultra-violetsoft X-rays X- and  -rays photon energy / eV Useful region for ‘solution irradiator’

41. 110 130 150 170 190 0 20 40 60 80 100 120 140 Wavelength / nm Output Peak at 147 nm ( = 8.5 eV) RF-excited Xenon Lamp VUV spectrum

42. source (Xenon lamp) VUV irradiator (lamp) concave grating monochromator

43. VUV irradiator (lamp)

44. DNA damage yields in solution: 0481216 10 100 % supercoiled DNA Dose / Gy 50 SSB 0481216 0 2 4 6 8 % linear DNA Dose / Gy DSB 7 eV photons

45. 7eV 10 100 % supercoiled DNA Dose / Gy 50 SSB 024681012 0 2 4 6 8 10 12 14 16 024681012 Dose / Gy % linear DNA DSB 8.5 eV photons DNA damage yields in solution:

46. 10 100 % supercoiled DNA Dose / Gy 50 SSB 024681012 0 2 4 6 8 10 12 14 16 024681012 Dose / Gy % linear DNA DSB 8.5 eV photons DNA damage yields in solution:

47. 10 100 % supercoiled DNA Dose / Gy SSB 024681012 0 2 4 6 8 10 12 14 16 024681012 Dose / Gy % linear DNA DSB 8.5 eV photons 50 + 1mM Tris (OH radical scavenger) DNA damage yields in solution:

48. 024681012 Dose / Gy 8.5 eV 0 2 4 6 8 10 12 14 16 % linear DNA no scavenger scavenger Observations At all dose levels, the addition of a radical scavenger reduces the number of induced dsb The OH mediated damage is linear with dose This suggests that a single OH radical can produce a dsb

49. Are the strand-breaks due to (non-ionizing) UV damage? It is possible that ssb and dsb are caused by contaminating UV radiation UV-induced DNA damage consists mostly of the formation of pyrimidine dimers Addition of T4 endonuclease V converts pyrimidine dimers to strand-breaks

50. SSB DSB 02 1 10 50 100 4681012 % supercoiled Dose / Gy 02 4 4681012 8 16 20 Dose / Gy % linear no T4 with T4 +T4 endonuclease V DNA damage yields in solution: 8.5 eV photons

51. Mechanisms for ssb and dsb induction at low-energies Boudaiffa et al. have demonstrated that ssb and dsb can be induced in DNA by electrons with energies as low as 5 eV, through the process of ‘electron attachment’ Resonant Formation of DNA Strand Breaks by Low-Energy (3 to 20eV) Electrons. Science 287, 1658-1660 (2000). B. Boudaiffa, P. Cloutier, D. Hunting, M.A. Huels et L. Sanche. “This finding presents a fundamental challenge to the traditional notion that genotoxic damage by secondary electrons can only occur at energies above the onset of ionization…”

52. Mechanisms for ssb and dsb induction at low-energies Incident electron energy / eV 05101520 0 2 4 6 8 0 1 2 DNA breaks / incident electron (x10 -4 ) DSBs SSBs

53. Mechanisms for ssb and dsb induction at low-energies Below 15 eV, electrons can attach to molecules and form a ‘resonance’ e - + RH RH * transient molecular anion (TMA) RH * R + H _ electron autodetachment, or dissociation DSB induction occurs when fragmentation components react with the opposite strand This can induce an SSB

54. K.M. Prise G.C. Holding D. Cole C. Turner S. Gilchrist B Vojnovic B.D. Michael F.A. Smith B. Brocklehurst C.A. Mythen A.Hopkirk M. Macdonald I.H. Munro Acknowledgments GCI other

55.  The action spectra for ssb and dsb induced in dry DNA are similar, indicative of a common precursor. Conclusions  DNA in solution irradiated with 7 eV, or 8.5 eV photons gives a linear (or linear-quadratic) dsb induction, indicative of a single-event mechanism.  Addition of tris suggests that a single OH radical has a significant probability of inducing a dsb.

56. 701.9x10 -5 9.4x10 -7 20 71--------- 8.0*03.2x10 -5 6.4x10 -7 50 8.0* 11.0x10 -5 3.9x10 -7 26 8.502.4x10 -5 1.5x10 -6 16 8.511.2x10 -5 4.2x10 -7 29 Co 60 02.2x10 -5 6.7x10 -7 33 Co 60 1 8.7x10 -6 4.3x10 -7 20 E/eVtris/mM ssb / Gy -1 bp -1 dsb/ Gy -1 bp -1 ssb/dsb synchrotron * DNA damage yields in solution:

57. % supercoiled DNA 0102030 1 10 50 100 Dose / Gy 0102030 0 2 4 6 8 10 12 % linear DNA Dose / Gy SSB no tris 1mM tris no tris 1mM tris Co 60  -rays (+ 1mM tris) DNA damage yields in solution:

58. 6.06.57.07.58.08.59.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 yield ferric ions / photon energy / eV Water radical yields by Fricke dosimetry Watanabe, R., Usami, N., Takakura, K., Hieda, K. and Kobayashi, K., 1997, Radiation Research, 148, 489-490. dsb/ Gy -1 bp -1 0.0 2x10 -7 4x10 -7 6x10 -7 8x10 -7 1x10 -6 2x10 -6 DSB

59. 6.06.57.07.58.08.59.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 yield ferric ions / photon energy / eV Water radical yields by Fricke dosimetry Watanabe, R., Usami, N., Takakura, K., Hieda, K. and Kobayashi, K., 1997, Radiation Research, 148, 489-490. ssb/ Gy -1 bp -1 0.0 2x10 -5 1x10 -5 SSB