electron and photon induced damage to biomolecular systems m. folkard gray cancer institute, po box...
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Electron and photon induced damage to biomolecular systems
M. Folkard
Gray Cancer Institute, PO Box 100, Mount Vernon Hospital, Northwood HA6 2JR, UK
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• 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
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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
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repair mis-repair
mutationviable cell
not repaired
cancercell death
Radiation damage of biomolecules
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Physical 10-20 - 10-8 s ionisation, excitation
Timescale of events:
Early boil. hours - weeks cell death, animal death
Late boil. years carcinogenesis
Radiation damage of biomolecules
Chemical 10-18 - 10-9 s free radical damage
10-3 s - hours chemical repair
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• 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
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Biological effectiveness: radiation type Energetic X-rays
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Energetic X-rays
1 Gy ~ 1000 tracks per cell
~ 100,000 ionisations per cell
Biological effectiveness: radiation type
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-particles
1 Gy ~ 3 - 4 tracks per cell
~ 100,000 ionisations per cell
Biological effectiveness: radiation type
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Millar et al.
Biological effectiveness: radiation type
C3H 10T1/2 cells
10
20
30
0
0 2 4 6
tran
sfor
man
ts /
104
sur
vivi
ng c
ells
250 kVp X-rays
4He2
dose / Gy
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101
100
10-1
10-2
10-3
10-4
0 4 8 12
surv
ivin
g fr
actio
n
dose / Gy
V79 cells
energetic X-rays
1.5 keV AlK X-rays
Prise, Folkard & Michael, 1989
0.28 keV CK X-rays
Goodhead and Nikjoo, 1989
Biological effectiveness: radiation quality
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• 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
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2 m200 nm
20 nm
2 nm
Biophysical Models of radiation damage
- Develop a mathematical model of the cell and radiation track-structure
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200 nm
energetic X-rays
Biophysical Models of radiation damage
Breckow & Kellerer, 1990
e-
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20 nm
1.5 keV AlK X-rays
Biophysical Models of radiation damage
Nikjoo, Goodhead, Charlton, Paretzke, 1989
1.5 keV X-ray
e-
e-
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2 nm
0.28 keV CK X-ray
Biophysical Models of radiation damage
0.28 eV X-ray
Nikjoo, Goodhead, Charlton, Paretzke, 1989
e-
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- particle
Biophysical Models of radiation damage
-particle
e-
2 nm
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photo
nsingle-strand break
DNA Damage
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double-strand break
e-
photon
DNA Damage
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complex damage
Locally multiply damaged sites (LMDS)
DNA Damage
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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
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1 MeV electrons
100806040200
Energy E / eV
Fre
quen
cy p
er e
V
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
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100 keV electrons
300 eV electrons
2 nm
10-5
10-6
10-7
10-8
10-9
3002001000
Energy E / eV
Fre
q. E
ven
ts >
E p
er
targ
et /
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
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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
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DNA Damage
0
1
2
100 200 200 300 400
prob
abili
ty o
f br
eak
energy in DNA / eV
SSB
DSB
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
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ionising
synchrotrons
characteristic X-ray sourcesvacuum tubes
linacs
gas discharge sources
isotope sources
Energetic photon sources
typical cluster size
1 eV 1 keV 1 MeV 1 GeV
ultra-violet soft X-rays X- and -rays
photon energy / eV
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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)
lineardouble-strand break
relaxed
single-strand break
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relaxed
linear
supercoiled
Measurement of DNA damage
These forms can be easily separated by gel-electrophoresis
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energy / eV
SEYA, LiF, MgF window
TGM, polyimide window
SEYA, aluminium window
10 10050 200
1012
1011
1010
109
phot
ons
s-1 c
m-1
Experiments using the Daresbury Synchrotron
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window
electrometer
valve
pump
VUV
grid
sample
sample ‘wobbler’
Experiments using the Daresbury Synchrotron
‘dry’ DNA irradiator
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SSB induction in ‘dry’ DNA
150 eV photons%
sup
erco
iled
DN
A
Photons / cm2
0 1x1013 2x1013 3x10131
10
100
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0 1x1014 2x1014 3x1014
8 eV
10
100
1
0.0 2.0x1013 4.0x1013 6.0x1013 8.0x1013
11 eV
1
10
100
0 1x1013 2x1013 3x1013
150 eV
1
10
100
0.0 1.0x1015 2.0x1015
10
100
7 eV
1
% s
upe
rcoi
led
DN
A
Photons / cm2
SSB induction in ‘dry’ DNA
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150 eV photons
DSB induction in ‘dry’ DNA%
line
ar D
NA
0 1x1013 2x1013 3x10130
5
10
15
Photons / cm2
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0.0 2.0x1013 4.0x1013 6.0x1013 8.0x1013
0
4
8
12
11 eV
0 1x1013 2x1013 3x10130
5
10
15
150 eV
8
0 2x1014
0
2
4
68 eV
1x1014 3x1014
% li
nea
r D
NA
0.0 1.0x1015 2.0x1015
0
2
4
6
87 eV
Photons / cm2
DSB induction in ‘dry’ DNA
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5 10 50 100 200
SSB DSB
Qu
ant
um
Effi
cie
ncy
/
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 threshold DSB threshold
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Observations
0.0 2.0x1013 4.0x1013 6.0x1013 8.0x1013
0
4
8
12
11 eV
0.0 2.0x1013 4.0x1013 6.0x1013 8.0x1013
11 eV
1
10
100
37%
% s
uper
coile
d%
line
ar
photons / cm2
• 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
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Free radical damage of DNA
photo
n H2O H2O+ + e-
H+ + •OH
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0 20
scale / mm
VUV
‘DNA in solution’ VUV irradiator
MgF
DNA in 50m gap
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‘DNA in solution’ VUV irradiator
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ionising
synchrotrons
gas discharge sources
Energetic photon sources
1 eV 1 keV 1 MeV 1 GeV
ultra-violet soft X-rays X- and -rays
photon energy / eV
Useful region for ‘solution irradiator’
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110 130 150 170 190
0
20
40
60
80
100
120
140
Wavelength / nm
Ou
tpu
t
Peak at 147 nm ( = 8.5 eV)
RF-excited Xenon Lamp
VUV spectrum
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source (Xenon lamp)
VUV irradiator (lamp)
concave grating monochromator
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VUV irradiator (lamp)
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DNA damage yields in solution:
0 4 8 12 1610
100
% s
upe
rcoi
led
DN
A
Dose / Gy
50
SSB
0 4 8 12 160
2
4
6
8
% li
nea
r D
NA
Dose / Gy
DSB
7 eV photons
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7eV
7eV
10
100
% s
upe
rcoi
led
DN
A
Dose / Gy
50
SSB
0 2 4 6 8 10 12
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Dose / Gy
% li
nea
r D
NA
DSB
8.5 eV photons
DNA damage yields in solution:
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10
100
% s
upe
rcoi
led
DN
A
Dose / Gy
50
SSB
0 2 4 6 8 10 12
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Dose / Gy
% li
nea
r D
NA
DSB
8.5 eV photons
DNA damage yields in solution:
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10
100
% s
upe
rcoi
led
DN
A
Dose / Gy
SSB
0 2 4 6 8 10 12
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Dose / Gy
% li
nea
r D
NA
DSB
8.5 eV photons
50
+ 1mM Tris (•OH radical scavenger)
DNA damage yields in solution:
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0 2 4 6 8 10 12
Dose / Gy
8.5 eV
0
2
4
6
8
10
12
14
16
% li
near
DN
A 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
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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
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SSB DSB
0 21
10
50
100
4 6 8 10 12
% s
upe
rcoi
led
Dose / Gy0 2
4
4 6 8 10 12
8
12
16
20
Dose / Gy
% li
nea
r
no T4 no T4
with T4with T4
+T4 endonuclease V
DNA damage yields in solution:8.5 eV photons
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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…”
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Mechanisms for ssb and dsb induction at low-energies
Incident electron energy / eV0 5 10 15 20
0
2
4
6
8
0
1
2D
NA
bre
aks
/ in
cid
ent
ele
ctro
n (
x10-4
) DSBs
SSBs
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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
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K.M. Prise G.C. HoldingD. ColeC. TurnerS. GilchristB VojnovicB.D. Michael
F.A. SmithB. BrocklehurstC.A. MythenA. HopkirkM. Macdonald I.H. Munro
Acknowledgments
GCI other
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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.
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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
E/eV tris/mM ssb / Gy-1bp-1 dsb/ Gy-1bp-1 ssb/dsb
synchrotron*
DNA damage yields in solution:
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% s
upe
rcoi
led
DN
A
0 10 20 301
10
50
100
Dose / Gy
0 10 20 30
0
2
4
6
8
10
12
% li
nea
r D
NA
Dose / Gy
SSB
no tris 1mM tris
no tris 1mM tris
Co60 -rays (+ 1mM tris)
DNA damage yields in solution:
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6.0 6.5 7.0 7.5 8.0 8.5 9.00.0
0.5
1.0
1.5
2.0
2.5
3.0
yie
ld fe
rric
ion
s / p
hot
on
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
2x10-6DSB
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6.0 6.5 7.0 7.5 8.0 8.5 9.00.0
0.5
1.0
1.5
2.0
2.5
3.0
yie
ld fe
rric
ion
s / p
hot
on
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