Electron and photon induced damage to biomolecular systems M. Folkard Gray Cancer Institute, PO Box...

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

• 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