Electron and photon induced damage to biomolecular systems
M. Folkard
Gray Cancer Institute, PO Box 100, Mount Vernon Hospital, Northwood HA6 2JR, UK
• 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
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
repair mis-repair
mutationviable cell
not repaired
cancercell death
Radiation damage of biomolecules
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
• 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
Biological effectiveness: radiation type Energetic X-rays
Energetic X-rays
1 Gy ~ 1000 tracks per cell
~ 100,000 ionisations per cell
Biological effectiveness: radiation type
-particles
1 Gy ~ 3 - 4 tracks per cell
~ 100,000 ionisations per cell
Biological effectiveness: radiation type
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
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
• 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
2 m200 nm
20 nm
2 nm
Biophysical Models of radiation damage
- Develop a mathematical model of the cell and radiation track-structure
200 nm
energetic X-rays
Biophysical Models of radiation damage
Breckow & Kellerer, 1990
e-
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-
2 nm
0.28 keV CK X-ray
Biophysical Models of radiation damage
0.28 eV X-ray
Nikjoo, Goodhead, Charlton, Paretzke, 1989
e-
- particle
Biophysical Models of radiation damage
-particle
e-
2 nm
photo
nsingle-strand break
DNA Damage
double-strand break
e-
photon
DNA Damage
complex damage
Locally multiply damaged sites (LMDS)
DNA Damage
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
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
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
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
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
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
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
relaxed
linear
supercoiled
Measurement of DNA damage
These forms can be easily separated by gel-electrophoresis
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
window
electrometer
valve
pump
VUV
grid
sample
sample ‘wobbler’
Experiments using the Daresbury Synchrotron
‘dry’ DNA irradiator
SSB induction in ‘dry’ DNA
150 eV photons%
sup
erco
iled
DN
A
Photons / cm2
0 1x1013 2x1013 3x10131
10
100
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
150 eV photons
DSB induction in ‘dry’ DNA%
line
ar D
NA
0 1x1013 2x1013 3x10130
5
10
15
Photons / cm2
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
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
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
Free radical damage of DNA
photo
n H2O H2O+ + e-
H+ + •OH
0 20
scale / mm
VUV
‘DNA in solution’ VUV irradiator
MgF
DNA in 50m gap
‘DNA in solution’ VUV irradiator
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’
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
source (Xenon lamp)
VUV irradiator (lamp)
concave grating monochromator
VUV irradiator (lamp)
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
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:
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:
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:
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
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
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
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…”
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
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
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
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.
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:
% 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:
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
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