Understanding Radiation Damage to Biomolecules

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