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_at_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
Radiation damage of biomolecules
5
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
Biological effectiveness radiation type
Energetic X-rays
9
Biological effectiveness radiation type
a-particles
10
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
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-rays are 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
Biophysical Models of radiation damage
- Develop a mathematical model of the cell and
radiation track-structure
14
Biophysical Models of radiation damage
energetic X-rays
200 nm
15
Biophysical Models of radiation damage
1.5 keV AlK X-rays
20 nm
16
Biophysical Models of radiation damage
0.28 keV CK X-ray
17
Biophysical Models of radiation damage
a - particle
18
DNA Damage
single-strand break
19
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
100
80
60
40
20
0
Energy E / eV
23
DNA Damage
Frequency of energy depositions gtE in a 2 nm
section of the DNA helix

• Most energy depositions few 10s eV

• Few energy depositions gt200 eV

Re-drawn from Nikjoo and Goodhead, 1991
24
Questions

• How much energy is involved in the induction of
  single- and double-strand breaks by ionizing
  radiations?

25
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
Energetic photon sources
27
Measurement of DNA damage
Use Plasmid DNA (circular double-stranded
molecules of DNA, purified from bacteria) i.e.
pBR322 (4363 base-pairs)
28
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
100
50
200
energy / eV
30
Experiments using the Daresbury Synchrotron
dry DNA irradiator
31
SSB induction in dry DNA
150 eV photons
32
SSB induction in dry DNA
33
DSB induction in dry DNA
150 eV photons
34
DSB induction in dry DNA
35
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
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
Free radical damage of DNA
38
DNA in solution VUV irradiator
39
DNA in solution VUV irradiator
40
Energetic photon sources
synchrotrons
gas discharge sources
41
RF-excited Xenon Lamp
VUV spectrum
Peak at 147 nm ( 8.5 eV)
Output
110
130
150
170
190
Wavelength / nm
42
VUV irradiator (lamp)
43
VUV irradiator (lamp)
44
DNA damage yields in solution
7 eV photons
45
DNA damage yields in solution
8.5 eV photons
46
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
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
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
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
51
Mechanisms for ssb and dsb induction at
low-energies
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
DSBs
2
1
0
DNA breaks / incident electron (x10-4)
SSBs
Incident electron energy / eV
53
Mechanisms for ssb and dsb induction at
low-energies

• Below 15 eV, electrons can attach to molecules
  and form a resonance

• This can induce an SSB

• DSB induction occurs when fragmentation
  components react with the opposite strand

54
Acknowledgments
GCI
other
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
• Hopkirk
• M. Macdonald
• I.H. Munro

55
Conclusions

• The action spectra for ssb and dsb induced in dry
  DNA are similar, indicative of a common precursor.

• 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
DNA damage yields in solution
E/eV tris/mM ssb / Gy-1bp-1 dsb/
Gy-1bp-1 ssb/dsb
7 0 1.9x10-5 9.4x10-7 20 7 1 --- --- --- 8.0
0 3.2x10-5 6.4x10-7 50 8.0
1 1.0x10-5 3.9x10-7 26 8.5 0 2.4x10-5 1.5x10-
6 16 8.5 1 1.2x10-5 4.2x10-7 29 Co60 0 2.2x10-
5 6.7x10-7 33 Co60 1 8.7x10-6 4.3x10-7 20
synchrotron

57
DNA damage yields in solution
Co60 g-rays ( 1mM tris)
100
12
SSB
50
10
8
6
supercoiled DNA
linear DNA
10
4
2
no tris
no tris
1mM tris
1mM tris
0
1
0
10
20
30
0
10
20
30
Dose / Gy
Dose / Gy

58
Water radical yields by Fricke dosimetry
Watanabe, R., Usami, N., Takakura, K., Hieda, K.
and Kobayashi, K., 1997, Radiation Research, 148,
489-490.
3.0
2.5
2.0
yield ferric ions / photon
1.5
1.0
0.5
0.0
energy / eV
59
Water radical yields by Fricke dosimetry
Watanabe, R., Usami, N., Takakura, K., Hieda, K.
and Kobayashi, K., 1997, Radiation Research, 148,
489-490.
3.0
SSB
2x10-5
2.5
2.0
yield ferric ions / photon
ssb/ Gy-1bp-1
1.5
1x10-5
1.0
0.5
0.0
0.0
energy / eV