1

Radiation Chemistry;

Effects of Radiation On DNA and

Chromosomes

Kathryn D. Held, Ph.D.

Massachusetts General Hospital

Harvard Medical School

2

Effects of Radiation On DNA and

Chromosomes

• Introduction to Radiation Chemistry

• Water Radiolysis

• DNA Damage

• Chromosome Aberrations

3

Sequences in the Development of Radiobiological Effects

Time Event

10-18

s Absorption of Ionizing Radiation

10-16

s Physical Events

Ionization

Excitation

10-12

s Physicochemical Events

Free radical formation

Breakage of chemical bonds

10-12

– 10-6

s Chemical Events

Reactions of radicals

Minutes to hours Biochemical/Cellular Processes

Repair

Division delay

Chromosome damage

Loss of reproductive capacity

Days to months Tissue Damage

CNS, GI, Bone marrow syndromes

Late tissue damage

Birth defects from in utero exposure

Years Late Somatic Effects

Cataracts

Carcinogenesis

Generations Genetic Effects

4

Ionizing Radiation

• All biological effects produced by ionizing

radiation result from the chemical events that

occur shortly after the initial deposition of

radiation energy.

• Absorption of IR by matter produces ions and

excited molecules.

A A+ + e-

A A*

• The number of species produced is proportional

to dose.

5

Ionizing Radiation

• Free radicals - atoms or molecules that

have one or more unpaired electron

– designated by “•”

– may be formed by division of a covalent

bond

R:S R• + S•

– may be charged or neutral

– are generally very reactive

6

Direct and Indirect Actions of

Ionizing Radiation (from Hall 1994)

Low LET

70%

30%

7

Effects of Radiation On DNA and

Chromosomes

• Introduction to Radiation Chemistry

• Water Radiolysis

• DNA Damage

• Chromosome Aberrations

8

Water Radiolysis Summary

H2O •OH, •H, e-aq, H2, H2O2

•OH is the most important biologically.

9

Mechanisms of Water Radiolysis

Ionization and Excitation

H2O H2O+ + e-

H2O H2O*

Ion-molecule interaction and dissociation

H2O+ + H2O H3O

+ + •OH

e- + H2O •H + OH-

H2O* •H + •OH

Electron hydration

e- + (H2O)n e-aq

Spur reactions•H + •H H2•OH + •OH H2O2•OH + •H H2O

Diffusion

10

G Values (Yields) of Primary Radiolysis

Species in Neutral Water

-H2O••••OH

••••H e

-aq H2 H2O2

γγγγ-rays and

electrons of

0.1 – 20 MeV

0.43 0.28 0.06 0.28 0.05 0.07

32 MeV αααα-

particles0.31 0.09 0.04 0.08 0.10 0.10

(G-value = moles of material formed or changed by an energy absorption of 1 J)

Note:

• For low LET radiation, the most common radiolysis species are ·OH and

e-aq.

• With higher LET radiation, yields of radical species decrease and

molecular species increase.

11

Reactions of •OH

• Oxidation of inorganic compounds•OH + Mn OH- + Mn+1

• Addition to free radicals and unsaturated

organics•OH + CH2=CH2

•CH2-CH2OH

• Abstraction from saturated organics•OH + CH3COCH3

•CH2COCH3 + H2O

12

e-aq and •H

• Reducing species

• Frequently undergo diffusion-controlled

reactions

• Reactions do not seem to be biologically

damaging

13

Reactions of Primary Radicals

with Oxygen

• Formation of perhydroxyl and

superoxide radicals

O2 + •H HO2

•

O2 + e-aq O2

-•

O2-• + H+ HO2

• pK = 4.88

• Reactions with organic radicals

O2 + R• RO2

•

Note: These reactions are thought to be responsible for

the Oxygen Effect.

14

Radical Scavenging

• Use of a compound that selectively reacts

with certain free radicals

• Simplifies more complex radiation

chemistry

15

Radical Scavengers

Additive Reaction Active

Species

Remaining

N2O N 2O + e-

aq + H 2O

→→→→ ••••OH + OH

- + N 2

••••OH (H

••••)

••••OH

scavengers

RH + ••••OH →→→→ R

•••• +

H 2O e

-

aq (H••••)

oxygen O 2 + e-

aq →→→→ O 2-••••

O 2 + ••••H →→→→ HO 2

••••

••••OH, O 2

••••-,

HO 2••••

acid e-

aq + H+ →→→→ H••••

H••••,

••••OH

16

•OH Scavengers Decrease Radiation-

induced DNA Damage (from Roots and Okada 1972)

17

Effects of Radiation On DNA and

Chromosomes

• Introduction to Radiation Chemistry

• Water Radiolysis

• DNA Damage

• Chromosome Aberrations

18

DNA is a Primary Target

• Microbeam experiments show cell nucleus to be more sensitive than cytoplasm.

• Halogenated base analogues sensitize cells and DNA.

• Radioisotopes in DNA are more lethal than when in RNA or protein.

• DNA repair deficient cells are radiation sensitive; drugs that inhibit DNA repair usually are radiosensitizers.

• Oxygen and LET modify survival, cytogeneticdamage and biological activity of DNA in similar manner.

19

Reactions of •OH with DNA Bases and Sugar

20

Reactions of •OH with DNA Bases and

Sugar

• Subsequent to radical production in

DNA, a multitude of products can be

formed.

• E.g., from thymine alone, more than 30

radiolysis products have been identified,

with quite different yields

21

Types of DNA Lesions from IR

From McMillan

and Steel 1993

Breaks

• SSB

• DSB

Base damages

• Change

• Loss (abasic sites)

Crosslinks

• DNA-DNA

• DNA-protein

22

Measurement of DNA Damages

• Base damages

– Enzyme sensitivity

– HPLC, GC-MS, GC-EC

– Immunological probes

• Strand breaks

– Gel electrophoresis (alkaline for SSB; neutral for DSB)

– Comet assay (alkaline for SSB; neutral for DSB)

– Foci of DNA repair-related proteins(e.g., γγγγ-H2AX)

23

Pulsed Field Gel Electrophoresis

24

Comet Assay (Single Cell Gel Electrophoresis)

• Embed cells in gel and lyse

• Electrophorese

• Quantify amount of DNA in “tail” (damaged) versus “head”

25

Comparison of Assays for Breaks

(from Olive 1992)

Note:

• More SSBs

than DSBs

• Breaks usually

linear with

dose

• Killing usually

shouldered

26

Foci of DNA Repair-Related Proteins (e.g.,

γγγγ-H2AX) as Measure of DNA DSBs

(from Bonner 2003)

γγγγ-H2AX – phosphorylated histone H2A variant X

Foci – fluorescent “blobs” representing aggregates of protein

recognized by antibodies

27

γγγγ-H2AX Foci as a Measure of DSBs

(from Rothkamm and Löbrich 2003)

(o = foci/cell; ∆ = PFGE)

slope = 35 DSB/cell/GyNote:

Number of foci increases linearly with dose, with same slope

as DSBs measured by PFGE.

Even after very low doses, some foci remain at 24 h.

28

Foci as Measure of DSBs

• Caution: γγγγ-H2AX foci have been seen

after treatments that do not directly

cause DSBs, e.g., hypoxia, hydrogen

peroxide

• Other DNA repair-related proteins also

form foci and are being used as

surrogates for DSBs, e.g., 53BP1,

RAD51

29

Number of Radiation-Induced Lesions

Type of Lesion Number per Gray

Double strand breaks 40

Single strand breaks 1000

Base damages 1000-2000

Sugar damages 800-1000

DNA-DNA crosslinks 30

DNA-protein crosslinks 150

Alkali-labile sites 200-300

Number of Clustered Lesions not yet quantified.

30

Energy Deposition Events (spurs,

blobs, short tracks)

100 to 500 eV

< 100 eV 5000 eV

Branch TracksDelta rays

Blobs

Short Tracks

Spurs

(from Mozunder and Magee 1966)

•• ••••••

• •••••••

•••

•

•••

•• ••

31

Energy Deposition Events for Low

LET Radiation

ENTITYENERGY

DEPOSITED SIZE

NUMBER OF

WATER

MOLECULES

PER EVENT

ENERGY

(%)

EVENTS

(%)

Spur

32

Clustered Lesions (Multiply

Damaged Sites) (from Steel 1993)

33

Biological Consequences of

Clustered Lesions (MDS)

• Harder to repair accurately than single

lesions

• Unrepaired– Block DNA replication

– Loss of genetic integrity

• Mispaired

– May lead to DSBs

– Deletions could be produced

• Repair could be completed accurately

34

Measured Clustered Lesions

Relative Cluster Frequencies

in Human Cells

DSB 1

Oxidized purines 1

Oxidized pyrimidines 0.9

Abasic sites 0.75

(from Sutherland et al. 2002)

35

Chromatin Structure is Important in Radiation Damage to DNA

• Presence of histones/

chromatin

condensation

• Regionally multiply

damaged sites

• Actively transcribing

vs non-transcribing

DNA

• Nuclear matrix

attachment sites

36

Importance of Histones/Chromatin

Condensation

Recent studies suggest histone deacetylase (HDAC)

inhibitors may be radiation sensitizers.

(from Campausen et al. 2004a) (from Campausen et al. 2004b)

37

Which DNA lesion is most important

biologically?

Good correlation

between DNA DSBs

and cell killing

(from Radford 1985)

38

Most Important Lesion?

• To date, most data suggest DSBs

• Most assays for DSBs will include

Clustered Lesions

• Clustered Lesions may be most

important for cell killing

39

Biological Consequences

DNA damage

Accurate repair Misrepair No repair

Mutations

Chromosome aberrations

Genomic instability

Neoplastic transformation

Cell death/inactivation

Mitotic

Apoptotic

Long-term arrest

Survival;

no mutations

40

Effects of Radiation On DNA and

Chromosomes

• Introduction to Radiation Chemistry

• Water Radiolysis

• DNA Damage

• Chromosome Aberrations

41

Chromosome Aberrations

• Reflect

– initial DNA damage

– its repair (or non/misrepair)

• Two general types

– Chromosome aberrations

• G1 irradiation

• Both sister chromatids involved

– Chromatid aberrations

• S or G2 irradiation

• Usually only one chromatid involved

42

Chromosome versus Chromatid Aberrations (from McMillan and Steel 1993)

43

Examples of Chromosome Aberrations

dicentrics

tricentric

fragment

ring

44

Examples of Chromatid Aberrations

quadra-radials

complex

exchange

45

Chromosome Aberrations

• Principal aberrations:

– Dicentrics

– Rings

– Acentric fragments

– Translocations

– Anaphase bridges

• Exchange-type aberrations can be symmetric or asymmetric.

• Aberrations can be stable or unstable.

• Dicentrics (e.g., in lymphocytes) are a good biomarker of radiation exposure.

46

Micronuclei formation is sometimes used as

a surrogate for chromosome aberrations

Micronuclei can

result from

chromosome

deletions or

fragments

47

Techniques Used in Chromosome

Analysis

• Premature Chromosome Condensation (PCC)

• Fluorescence in Situ Hybridization (FISH; chromosome “painting”)

Use of FISH-based techniques has made it clear that:

• radiation-induced chromosome aberrations are more complex than previously realized.

• complexity of aberrations increases with LET.

48

Example of mFISH

Metaphase chromosomes

from lymphocytes of

plutonium-exposed

individual showing

complex rearrangements (from Anderson et al. 2005)

49

Dose Response Curve for Chromosome

Aberrations is Linear-Quadratic

(From Hall 2000)

50

Good Correlation Between Chromosome Aberrations and Loss of Clonogenicity

(from Hall

2000)

51

Biological Consequences

DNA damage

Accurate repair Misrepair No repair

Mutations

Chromosome aberrations

Genomic instability

Neoplastic transformation

Cell death/inactivation

Mitotic

Apoptotic

Long-term arrest

Survival;

no mutations

52

Take Home Messages - 1

• Indirect action produces most damage from low LET radiation; •OH is the most critical water radiolysis species.

• A plethora of DNA damages are produced by IR.

– Numerous techniques can be used to measure the damage.

– Using foci of DNA repair-related proteins to measure DSBs is currently of great interest.

• IR produces clustered lesions (multiply damaged sites) that are probably most important biologically.

53

Take Home Messages - 2

• Chromatin structure is important for radiation

damage to DNA and its repair.

• The biological consequences of misrepair or no

repair include mutations, aberrations, genomic

instability, cell death/inactivation.

• It is assumed that most cell death results from

DNA damage; relationships between loss of

clonogenicity, apoptosis or long-term arrest are

not straightforward.