Electron and photon induced damage to biomolecular systems

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. Radiation damage of biomolecules • 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

3. Radiation damage of biomolecules • 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 not repaired mutation viable cell cell death cancer Radiation damage of biomolecules

5. Chemical 10-18 - 10-9 s free radical damage 10-3 s - hours chemical repair Radiation damage of biomolecules Timescale of events: Physical 10-20 - 10-8 s ionisation, excitation Early boil. hours - weeks cell death, animal death Late boil. years carcinogenesis

6. Radiation damage of biomolecules • For the same dose, both the quality and the number of ionisations produced by ALL ionising radiations is the same • 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

7. Biological effectiveness: radiation type Energetic X-rays

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

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

10. C3H 10T1/2 cells Biological effectiveness: radiation type 30 4He2 20 250 kVp X-rays transformants / 104 surviving cells 10 0 0 2 4 6 Millar et al. dose / Gy

11. 101 100 10-1 0.28 keV CK X-rays 10-2 10-3 1.5 keV AlK X-rays 10-4 Prise, Folkard & Michael, 1989 Goodhead and Nikjoo, 1989 12 4 8 0 Biological effectiveness: radiation quality V79 cells energetic X-rays surviving fraction dose / Gy

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

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

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

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

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

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

18. photon DNA Damage single-strand break

19. e- photon DNA Damage double-strand break

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

21. DNA Damage • 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. DNA Damage 1 MeV electrons Theoretical spectrum of energy depositions by energetic electrons most probable E loss: 23 eV liquid water Frequency per eV DNA Re-drawn from; LaVerne and Pimblott, 1995 0 20 40 60 80 100 Energy E / eV

23. 10-5 10-6 2 nm 10-7 Freq. Events >E per target / Gy 300 eV electrons 100 keV electrons 10-8 10-9 0 100 200 300 Energy E / eV DNA Damage 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 Re-drawn from; Nikjoo and Goodhead, 1991

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

25. 2 SSB probability of break 1 DSB 0 100 200 200 300 400 energy in DNA / eV DNA Damage Nikjoo et al calculated the probability of SSB and DSB, based on data for strand breaks from I125 decays • Minimum energy to produce SSB ~20 eV • Minimum energy to produce DSB ~50 eV Re-drawn from; Nikjoo, Charlton, Goodhead, 1994

26. ionising photon energy / eV 1 eV 1 keV 1 MeV 1 GeV ultra-violet soft X-rays X- and -rays synchrotrons gas discharge sources characteristic X-ray sources vacuum tubes isotope sources linacs typical cluster size Energetic photon sources

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

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

29. Experiments using the Daresbury Synchrotron 1012 1011 photons s-1 cm-1 SEYA, LiF, MgF window 1010 SEYA, aluminium window TGM, polyimide window 109 10 50 100 200 energy / eV

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

31. 100 % supercoiled DNA 10 1 0 1x1013 2x1013 3x1013 Photons / cm2 SSB induction in ‘dry’ DNA 150 eV photons

32. 100 100 7 eV 8 eV 10 10 1 1 0.0 0 1.0x1015 2.0x1015 1x1014 2x1014 3x1014 % supercoiled DNA 100 100 11 eV 150 eV 10 10 1 1 0.0 0 2.0x1013 4.0x1013 6.0x1013 8.0x1013 1x1013 2x1013 3x1013 Photons / cm2 SSB induction in ‘dry’ DNA

33. 15 10 % linear DNA 5 0 0 1x1013 2x1013 3x1013 Photons / cm2 DSB induction in ‘dry’ DNA 150 eV photons

34. 8 8 eV 6 4 2 0 0 2x1014 1x1014 3x1014 % linear DNA 15 12 8 7 eV 11 eV 150 eV 6 10 8 4 5 4 2 0 0 0 0.0 1.0x1015 2.0x1015 0 0.0 1x1013 2x1013 3x1013 2.0x1013 4.0x1013 6.0x1013 8.0x1013 Photons / cm2 DSB induction in ‘dry’ DNA

35. ~20-fold SSB threshold DSB threshold Q.E. for SSB & DSB (dry plasmid) 10-0 SSB 10-1 DSB 10-2 Quantum Efficiency / F 10-3 10-4 10-5 5 10 50 100 200 Photon Energy / eV Prise, Folkard et. al, 1995, Int. J. Radiat. Biol. 76, 881-90.

36. 37% 12 100 11 eV 11 eV 8 10 4 0 1 0.0 2.0x1013 4.0x1013 6.0x1013 8.0x1013 0.0 2.0x1013 4.0x1013 6.0x1013 8.0x1013 Observations • The 37% ‘loss of super-coiled’ level represents an average of one ssb per plasmid. % supercoiled • At an equivalent dose, about 4% dsb produced • Induction of dsb is linear with dose, and has non-zero initial slope % linear • Therefore dsbs are NOT due to the interaction of two (independent) ssbs photons / cm2

37. H+ + •OH H2O H2O+ + e- photon Free radical damage of DNA

38. MgF VUV DNA in 50mm gap 0 20 scale / mm ‘DNA in solution’ VUV irradiator

39. ‘DNA in solution’ VUV irradiator

40. ionising photon energy / eV 1 eV 1 keV 1 MeV 1 GeV ultra-violet soft X-rays X- and -rays Useful region for ‘solution irradiator’ Energetic photon sources synchrotrons gas discharge sources

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

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

43. VUV irradiator (lamp)

44. 100 8 SSB DSB 6 50 % supercoiled DNA 4 % linear DNA 2 10 0 0 4 8 12 16 0 4 8 12 16 Dose / Gy Dose / Gy DNA damage yields in solution: 7 eV photons

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

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

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

48. 8.5 eV 16 14 no scavenger % linear DNA 12 scavenger 10 8 6 4 0 2 4 6 8 10 12 2 Dose / Gy 0 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. +T4 endonuclease V with T4 with T4 no T4 no T4 DNA damage yields in solution: 8.5 eV photons 100 SSB DSB 20 50 16 % supercoiled 12 10 % linear 8 4 1 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Dose / Gy Dose / Gy