Development of an Electron Microbeam for Cell Culture Studies
Radiation Chemistry; Effects of Radiation On DNA and ... · DNA is a Primary Target • Microbeam...
Transcript of Radiation Chemistry; Effects of Radiation On DNA and ... · DNA is a Primary Target • Microbeam...
1
Radiation Chemistry
Effects of Radiation On DNA and
Chromosomes
Kathryn D Held PhD
Massachusetts General Hospital
Harvard Medical School
2
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull 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
ndash 10-6
s Chemical Events
Reactions of radicals
Minutes to hours BiochemicalCellular 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
bull All biological effects produced by ionizing
radiation result from the chemical events that
occur shortly after the initial deposition of
radiation energy
bull Absorption of IR by matter produces ions and
excited molecules
A A+ + e-
A A
bull The number of species produced is proportional
to dose
5
Ionizing Radiation
bull Free radicals - atoms or molecules that
have one or more unpaired electron
ndash designated by ldquobullrdquo
ndash may be formed by division of a covalent
bond
RS Rbull + Sbull
ndash may be charged or neutral
ndash 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
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
8
Water Radiolysis Summary
H2O bullOH bullH e-aq H2 H2O2
bullOH 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
+ + bullOH
e- + H2O bullH + OH-
H2O bullH + bullOH
Electron hydration
e- + (H2O)n e-aq
Spur reactionsbullH + bullH H2
bullOH + bullOH H2O2
bullOH + bullH H2O
Diffusion
10
G Values (Yields) of Primary Radiolysis
Species in Neutral Water
-H2O bullbullbullbullOH
bullbullbullbullH e
-aq H2 H2O2
γγγγ-rays and
electrons of
01 ndash 20 MeV
043 028 006 028 005 007
32 MeV αααα-
particles031 009 004 008 010 010
(G-value = moles of material formed or changed by an energy absorption of 1 J)
Note
bull For low LET radiation the most common radiolysis species are OH and
e-aq
bull With higher LET radiation yields of radical species decrease and
molecular species increase
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
2
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull 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
ndash 10-6
s Chemical Events
Reactions of radicals
Minutes to hours BiochemicalCellular 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
bull All biological effects produced by ionizing
radiation result from the chemical events that
occur shortly after the initial deposition of
radiation energy
bull Absorption of IR by matter produces ions and
excited molecules
A A+ + e-
A A
bull The number of species produced is proportional
to dose
5
Ionizing Radiation
bull Free radicals - atoms or molecules that
have one or more unpaired electron
ndash designated by ldquobullrdquo
ndash may be formed by division of a covalent
bond
RS Rbull + Sbull
ndash may be charged or neutral
ndash 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
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
8
Water Radiolysis Summary
H2O bullOH bullH e-aq H2 H2O2
bullOH 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
+ + bullOH
e- + H2O bullH + OH-
H2O bullH + bullOH
Electron hydration
e- + (H2O)n e-aq
Spur reactionsbullH + bullH H2
bullOH + bullOH H2O2
bullOH + bullH H2O
Diffusion
10
G Values (Yields) of Primary Radiolysis
Species in Neutral Water
-H2O bullbullbullbullOH
bullbullbullbullH e
-aq H2 H2O2
γγγγ-rays and
electrons of
01 ndash 20 MeV
043 028 006 028 005 007
32 MeV αααα-
particles031 009 004 008 010 010
(G-value = moles of material formed or changed by an energy absorption of 1 J)
Note
bull For low LET radiation the most common radiolysis species are OH and
e-aq
bull With higher LET radiation yields of radical species decrease and
molecular species increase
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
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
ndash 10-6
s Chemical Events
Reactions of radicals
Minutes to hours BiochemicalCellular 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
bull All biological effects produced by ionizing
radiation result from the chemical events that
occur shortly after the initial deposition of
radiation energy
bull Absorption of IR by matter produces ions and
excited molecules
A A+ + e-
A A
bull The number of species produced is proportional
to dose
5
Ionizing Radiation
bull Free radicals - atoms or molecules that
have one or more unpaired electron
ndash designated by ldquobullrdquo
ndash may be formed by division of a covalent
bond
RS Rbull + Sbull
ndash may be charged or neutral
ndash 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
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
8
Water Radiolysis Summary
H2O bullOH bullH e-aq H2 H2O2
bullOH 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
+ + bullOH
e- + H2O bullH + OH-
H2O bullH + bullOH
Electron hydration
e- + (H2O)n e-aq
Spur reactionsbullH + bullH H2
bullOH + bullOH H2O2
bullOH + bullH H2O
Diffusion
10
G Values (Yields) of Primary Radiolysis
Species in Neutral Water
-H2O bullbullbullbullOH
bullbullbullbullH e
-aq H2 H2O2
γγγγ-rays and
electrons of
01 ndash 20 MeV
043 028 006 028 005 007
32 MeV αααα-
particles031 009 004 008 010 010
(G-value = moles of material formed or changed by an energy absorption of 1 J)
Note
bull For low LET radiation the most common radiolysis species are OH and
e-aq
bull With higher LET radiation yields of radical species decrease and
molecular species increase
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
4
Ionizing Radiation
bull All biological effects produced by ionizing
radiation result from the chemical events that
occur shortly after the initial deposition of
radiation energy
bull Absorption of IR by matter produces ions and
excited molecules
A A+ + e-
A A
bull The number of species produced is proportional
to dose
5
Ionizing Radiation
bull Free radicals - atoms or molecules that
have one or more unpaired electron
ndash designated by ldquobullrdquo
ndash may be formed by division of a covalent
bond
RS Rbull + Sbull
ndash may be charged or neutral
ndash 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
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
8
Water Radiolysis Summary
H2O bullOH bullH e-aq H2 H2O2
bullOH 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
+ + bullOH
e- + H2O bullH + OH-
H2O bullH + bullOH
Electron hydration
e- + (H2O)n e-aq
Spur reactionsbullH + bullH H2
bullOH + bullOH H2O2
bullOH + bullH H2O
Diffusion
10
G Values (Yields) of Primary Radiolysis
Species in Neutral Water
-H2O bullbullbullbullOH
bullbullbullbullH e
-aq H2 H2O2
γγγγ-rays and
electrons of
01 ndash 20 MeV
043 028 006 028 005 007
32 MeV αααα-
particles031 009 004 008 010 010
(G-value = moles of material formed or changed by an energy absorption of 1 J)
Note
bull For low LET radiation the most common radiolysis species are OH and
e-aq
bull With higher LET radiation yields of radical species decrease and
molecular species increase
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
5
Ionizing Radiation
bull Free radicals - atoms or molecules that
have one or more unpaired electron
ndash designated by ldquobullrdquo
ndash may be formed by division of a covalent
bond
RS Rbull + Sbull
ndash may be charged or neutral
ndash 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
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
8
Water Radiolysis Summary
H2O bullOH bullH e-aq H2 H2O2
bullOH 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
+ + bullOH
e- + H2O bullH + OH-
H2O bullH + bullOH
Electron hydration
e- + (H2O)n e-aq
Spur reactionsbullH + bullH H2
bullOH + bullOH H2O2
bullOH + bullH H2O
Diffusion
10
G Values (Yields) of Primary Radiolysis
Species in Neutral Water
-H2O bullbullbullbullOH
bullbullbullbullH e
-aq H2 H2O2
γγγγ-rays and
electrons of
01 ndash 20 MeV
043 028 006 028 005 007
32 MeV αααα-
particles031 009 004 008 010 010
(G-value = moles of material formed or changed by an energy absorption of 1 J)
Note
bull For low LET radiation the most common radiolysis species are OH and
e-aq
bull With higher LET radiation yields of radical species decrease and
molecular species increase
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
6
Direct and Indirect Actions of
Ionizing Radiation (from Hall 1994)
Low LET
70
30
7
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
8
Water Radiolysis Summary
H2O bullOH bullH e-aq H2 H2O2
bullOH 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
+ + bullOH
e- + H2O bullH + OH-
H2O bullH + bullOH
Electron hydration
e- + (H2O)n e-aq
Spur reactionsbullH + bullH H2
bullOH + bullOH H2O2
bullOH + bullH H2O
Diffusion
10
G Values (Yields) of Primary Radiolysis
Species in Neutral Water
-H2O bullbullbullbullOH
bullbullbullbullH e
-aq H2 H2O2
γγγγ-rays and
electrons of
01 ndash 20 MeV
043 028 006 028 005 007
32 MeV αααα-
particles031 009 004 008 010 010
(G-value = moles of material formed or changed by an energy absorption of 1 J)
Note
bull For low LET radiation the most common radiolysis species are OH and
e-aq
bull With higher LET radiation yields of radical species decrease and
molecular species increase
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
7
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
8
Water Radiolysis Summary
H2O bullOH bullH e-aq H2 H2O2
bullOH 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
+ + bullOH
e- + H2O bullH + OH-
H2O bullH + bullOH
Electron hydration
e- + (H2O)n e-aq
Spur reactionsbullH + bullH H2
bullOH + bullOH H2O2
bullOH + bullH H2O
Diffusion
10
G Values (Yields) of Primary Radiolysis
Species in Neutral Water
-H2O bullbullbullbullOH
bullbullbullbullH e
-aq H2 H2O2
γγγγ-rays and
electrons of
01 ndash 20 MeV
043 028 006 028 005 007
32 MeV αααα-
particles031 009 004 008 010 010
(G-value = moles of material formed or changed by an energy absorption of 1 J)
Note
bull For low LET radiation the most common radiolysis species are OH and
e-aq
bull With higher LET radiation yields of radical species decrease and
molecular species increase
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
8
Water Radiolysis Summary
H2O bullOH bullH e-aq H2 H2O2
bullOH 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
+ + bullOH
e- + H2O bullH + OH-
H2O bullH + bullOH
Electron hydration
e- + (H2O)n e-aq
Spur reactionsbullH + bullH H2
bullOH + bullOH H2O2
bullOH + bullH H2O
Diffusion
10
G Values (Yields) of Primary Radiolysis
Species in Neutral Water
-H2O bullbullbullbullOH
bullbullbullbullH e
-aq H2 H2O2
γγγγ-rays and
electrons of
01 ndash 20 MeV
043 028 006 028 005 007
32 MeV αααα-
particles031 009 004 008 010 010
(G-value = moles of material formed or changed by an energy absorption of 1 J)
Note
bull For low LET radiation the most common radiolysis species are OH and
e-aq
bull With higher LET radiation yields of radical species decrease and
molecular species increase
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
9
Mechanisms of Water Radiolysis
Ionization and Excitation
H2O H2O+ + e-
H2O H2O
Ion-molecule interaction and dissociation
H2O+ + H2O H3O
+ + bullOH
e- + H2O bullH + OH-
H2O bullH + bullOH
Electron hydration
e- + (H2O)n e-aq
Spur reactionsbullH + bullH H2
bullOH + bullOH H2O2
bullOH + bullH H2O
Diffusion
10
G Values (Yields) of Primary Radiolysis
Species in Neutral Water
-H2O bullbullbullbullOH
bullbullbullbullH e
-aq H2 H2O2
γγγγ-rays and
electrons of
01 ndash 20 MeV
043 028 006 028 005 007
32 MeV αααα-
particles031 009 004 008 010 010
(G-value = moles of material formed or changed by an energy absorption of 1 J)
Note
bull For low LET radiation the most common radiolysis species are OH and
e-aq
bull With higher LET radiation yields of radical species decrease and
molecular species increase
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
10
G Values (Yields) of Primary Radiolysis
Species in Neutral Water
-H2O bullbullbullbullOH
bullbullbullbullH e
-aq H2 H2O2
γγγγ-rays and
electrons of
01 ndash 20 MeV
043 028 006 028 005 007
32 MeV αααα-
particles031 009 004 008 010 010
(G-value = moles of material formed or changed by an energy absorption of 1 J)
Note
bull For low LET radiation the most common radiolysis species are OH and
e-aq
bull With higher LET radiation yields of radical species decrease and
molecular species increase
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
11
Reactions of bullOH
bull Oxidation of inorganic compoundsbullOH + Mn OH- + Mn+1
bull Addition to free radicals and unsaturated
organicsbullOH + CH2=CH2
bullCH2-CH2OH
bull Abstraction from saturated organicsbullOH + CH3COCH3
bullCH2COCH3 + H2O
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
12
e-aq and bullH
bull Reducing species
bull Frequently undergo diffusion-controlled
reactions
bull Reactions do not seem to be biologically
damaging
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
13
Reactions of Primary Radicals
with Oxygen
bull Formation of perhydroxyl and
superoxide radicals
O2 + bullH HO2bull
O2 + e-aq O2
-bull
O2-bull + H+ HO2
bull pK = 488
bull Reactions with organic radicals
O2 + Rbull RO2bull
Note These reactions are thought to be responsible for
the Oxygen Effect
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
14
Radical Scavenging
bull Use of a compound that selectively reacts
with certain free radicals
bull Simplifies more complex radiation
chemistry
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
15
Radical Scavengers
Additive Reaction Active
Species
Remaining
N2O N 2O + e-
aq + H 2O
rarrrarrrarrrarr bullbullbullbullOH + OH
- + N 2
bullbullbullbullOH (H
bullbullbullbull)
bullbullbullbullOH
scavengers
RH + bullbullbullbullOH rarrrarrrarrrarr R
bullbullbullbull +
H 2O e
-
aq (Hbullbullbullbull)
oxygen O 2 + e-
aq rarrrarrrarrrarr O 2
-bullbullbullbull
O 2 + bullbullbullbullH rarrrarrrarrrarr HO 2
bullbullbullbull
bullbullbullbullOH O 2
bullbullbullbull-
HO 2bullbullbullbull
acid e-
aq + H+ rarrrarrrarrrarr Hbullbullbullbull
Hbullbullbullbull
bullbullbullbullOH
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
16
bullOH Scavengers Decrease Radiation-
induced DNA Damage (from Roots and Okada 1972)
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
17
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
18
DNA is a Primary Target
bull Microbeam experiments show cell nucleus to be more sensitive than cytoplasm
bull Halogenated base analogues sensitize cells and DNA
bull Radioisotopes in DNA are more lethal than when in RNA or protein
bull DNA repair deficient cells are radiation sensitive drugs that inhibit DNA repair usually are radiosensitizers
bull Oxygen and LET modify survival cytogeneticdamage and biological activity of DNA in similar manner
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
19
Reactions of bullOH with DNA Bases and Sugar
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
20
Reactions of bullOH with DNA Bases and
Sugar
bull Subsequent to radical production in
DNA a multitude of products can be
formed
bull Eg 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
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
21
Types of DNA Lesions from IR
From McMillan
and Steel 1993
Breaks
bull SSB
bull DSB
Base damages
bull Change
bull Loss (abasic sites)
Crosslinks
bull DNA-DNA
bull DNA-protein
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
22
Measurement of DNA Damages
bull Base damages
ndash Enzyme sensitivity
ndash HPLC GC-MS GC-EC
ndash Immunological probes
bull Strand breaks
ndash Gel electrophoresis (alkaline for SSB
neutral for DSB)
ndash Comet assay (alkaline for SSB neutral for
DSB)
ndash Foci of DNA repair-related proteins(eg γγγγ-H2AX)
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
23
Pulsed Field Gel Electrophoresis
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
24
Comet Assay (Single Cell Gel Electrophoresis)
bull Embed cells in gel and lyse
bull Electrophorese
bull Quantify amount of DNA in ldquotailrdquo (damaged) versus ldquoheadrdquo
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
25
Comparison of Assays for Breaks
(from Olive 1992)
Note
bull More SSBs
than DSBs
bull Breaks usually
linear with
dose
bull Killing usually
shouldered
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
26
Foci of DNA Repair-Related Proteins (eg
γγγγ-H2AX) as Measure of DNA DSBs
(from Bonner 2003)
γγγγ-H2AX ndash phosphorylated histone H2A variant X
Foci ndash fluorescent ldquoblobsrdquo representing aggregates of protein
recognized by antibodies
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
27
γγγγ-H2AX Foci as a Measure of DSBs
(from Rothkamm and Loumlbrich 2003)
(o = focicell ∆ = PFGE)
slope = 35 DSBcellGyNote
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
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
28
Foci as Measure of DSBs
bull Caution γγγγ-H2AX foci have been seen
after treatments that do not directly
cause DSBs eg hypoxia hydrogen
peroxide
bull Other DNA repair-related proteins also
form foci and are being used as
surrogates for DSBs eg 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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
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
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
30
Energy Deposition Events (spurs
blobs short tracks)
100 to 500 eV
lt 100 eV lt5000 eV
Primary
gt5000 eV
Branch TracksDelta rays
Blobs
Short Tracks
Spurs
(from Mozunder and Magee 1966)
bullbull bullbullbullbullbullbull
bull bullbullbullbullbullbullbull
bullbullbull
bull
bullbullbull
bullbull bullbull
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
31
Energy Deposition Events for Low
LET Radiation
ENTITY
ENERGY
DEPOSITED SIZE
NUMBER OF
WATER
MOLECULES
PER EVENT
ENERGY
()
EVENTS
()
Spur lt100 eV 4 nm (diam) 1100 ~80 95
Blob lt500 eV 7 nm (diam) 6000 ~20 5
Short track 500-5000 eV
DNA 2 nm (diam)
Nucleosome
disc
Thickness 57 nm
Radius of 55 nm
Electrons with sufficient energy to form short tracks will also produce spurs and blobs (from
Ward 1988)
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
32
Clustered Lesions (Multiply
Damaged Sites) (from Steel 1993)
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
33
Biological Consequences of
Clustered Lesions (MDS)
bull Harder to repair accurately than single
lesions
bull Unrepairedndash Block DNA replication
ndash Loss of genetic integrity
bull Mispaired
ndash May lead to DSBs
ndash Deletions could be produced
bull Repair could be completed accurately
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
34
Measured Clustered Lesions
Relative Cluster Frequencies
in Human Cells
DSB 1
Oxidized purines 1
Oxidized pyrimidines 09
Abasic sites 075
(from Sutherland et al 2002)
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
35
Chromatin Structure is Important in Radiation Damage to DNA
bull Presence of histones
chromatin
condensation
bull Regionally multiply
damaged sites
bull Actively transcribing
vs non-transcribing
DNA
bull Nuclear matrix
attachment sites
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
36
Importance of HistonesChromatin
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
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
37
Which DNA lesion is most important
biologically
Good correlation
between DNA DSBs
and cell killing
(from Radford 1985)
38
Most Important Lesion
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
38
Most Important Lesion
bull To date most data suggest DSBs
bull Most assays for DSBs will include
Clustered Lesions
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
39
Biological Consequences
DNA damage
Accurate repair Misrepair No repair
Mutations
Chromosome aberrations
Genomic instability
Neoplastic transformation
Cell deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
40
Effects of Radiation On DNA and
Chromosomes
bull Introduction to Radiation Chemistry
bull Water Radiolysis
bull DNA Damage
bull Chromosome Aberrations
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
41
Chromosome Aberrations
bull Reflect
ndash initial DNA damage
ndash its repair (or nonmisrepair)
bull Two general types
ndash Chromosome aberrations
bull G1 irradiation
bull Both sister chromatids involved
ndash Chromatid aberrations
bull S or G2 irradiation
bull 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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
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
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
43
Examples of Chromosome Aberrations
dicentrics
tricentric
fragment
ring
44
Examples of Chromatid Aberrations
quadra-radials
complex
exchange
45
Chromosome Aberrations
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
44
Examples of Chromatid Aberrations
quadra-radials
complex
exchange
45
Chromosome Aberrations
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
45
Chromosome Aberrations
bull Principal aberrations
ndash Dicentrics
ndash Rings
ndash Acentric fragments
ndash Translocations
ndash Anaphase bridges
bull Exchange-type aberrations can be symmetric or asymmetric
bull Aberrations can be stable or unstable
bull Dicentrics (eg 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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
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
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
47
Techniques Used in Chromosome
Analysis
bull Premature Chromosome Condensation (PCC)
bull Fluorescence in Situ Hybridization (FISH chromosome ldquopaintingrdquo)
Use of FISH-based techniques has made it clear that
bull radiation-induced chromosome aberrations are more complex than previously realized
bull 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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
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 deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
51
Biological Consequences
DNA damage
Accurate repair Misrepair No repair
Mutations
Chromosome aberrations
Genomic instability
Neoplastic transformation
Cell deathinactivation
Mitotic
Apoptotic
Long-term arrest
Survival
no mutations
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
52
Take Home Messages - 1
bull Indirect action produces most damage from low LET radiation bullOH is the most critical water radiolysis species
bull A plethora of DNA damages are produced by IR
ndash Numerous techniques can be used to measure the damage
ndash Using foci of DNA repair-related proteins to measure DSBs is currently of great interest
bull IR produces clustered lesions (multiply damaged sites) that are probably most important biologically
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward
53
Take Home Messages - 2
bull Chromatin structure is important for radiation
damage to DNA and its repair
bull The biological consequences of misrepair or no
repair include mutations aberrations genomic
instability cell deathinactivation
bull It is assumed that most cell death results from
DNA damage relationships between loss of
clonogenicity apoptosis or long-term arrest are
not straightforward