RADIATION DAMAGE TO DNA : FROM NUCLEOSIDES TO THE CELL

1
RADIATION DAMAGE TO DNA FROM NUCLEOSIDES TO THE
CELL

• Jean Cadet, Thierry Douki, Didier Gasparutto
• Jean-Luc Ravanat
• Département de Recherche Fondamentale sur la
  Matière Condensée, SCIB/Laboratoire Lésions des
  Acides Nucléiques, CEA/Grenoble, Grenoble,
  France.

2
OXIDATIVE DAMAGE TO DNA
PHOTOSENSITIZATION IONIZING RADIATION
OXIDATIVE METABOLISM UV LASER PULSES
XENOBIOTICS
1O2 - e- .OH H2O2 O2-.
- Modified bases - Abasic sites - Single
double strand breaks - DNA-protein cross-links -
Aldehyde adducts to aminobases
LETHALITY MUTAGENESIS
CARCINOGENESIS AGING
3
Effects of photons of the electromagnetic range
on DNA
Visible
3 eV
Photosensitization one-electron oxidation, 1O2

Near-UV
4 eV
Excitation of the bases dimerization, adducts
Far-UV
5.5 eV
Vacuum UV
Photoionization bases, 2-deoxyribose
25 keV
Soft X-ray
Atom photoexcitation Auger effect (P 2.153
keV)
5 keV
X-rays Gamma rays
DNA ionization direct effect (radical
cations) Water radiolysis indirect effect (.OH
radical, e-aq)
4 MeV
Heavy ions
Multiple events clustered damage
4
Radiation-induced damage to DNA
Indirect effect radiolysis of water
X
ADN
X
X
X
X
X
e-aq, H, HO
Base
Base
H
H
e-
O
Direct effect ionization of DNA bases and
2-deoxyribose
5
Scale of radiation events in the cell
Time Events
Experimental
approaches
10-18 sec 10-12 sec 10-6 sec 100
sec 106 sec
Excitations ionizations Intratrack reactions
Radiation physics Track simulation
Indirect effects of water radicals .OH, e-aq, H.
Pulse radiolysis
Target radical reduction/oxidation
Rapid mix techniques
Enzymic removal of O2.-, H2O2
Enzymic repair of damaged molecules
Repair deficient cells
Puck plating techniques
Cell proliferation
Late tissue effects
Animal models
6
EFFECTS OF IONIZING RADIATION ON NUCLEIC ACIDS

• - SINGLE LESIONS
• due to single events such as .OH, ionization,
  secondary electrons, Auger effect .
• - MULTIPLE LESIONS
• tandem lesions due to either a single event
  (.OH or ionization )
• clustered lesions due to multionization
  processes involving several events and/or
  reactive species ionization of DNA, water
  radiolysis species secondary electron

7
OXIDATIVE DNA DAMAGE

• - OLIGONUCLEOTIDE STRAND BREAKS
• (hydrogen abstraction at 2', 4' and 5'
  carbons)
• - ABASIC SITES
• hydrolysis of the N-glycosidic bond
  (modified bases)
• oxidation at C1 (2-deoxyribonolactone)
• BASE LESIONS (about 70 modifications identified)
• DNA-PROTEIN CROSSLINKS
• ALDEHYDE ADDUCTS TO AMINOBASES
• (breakdown products of LOOH and oxidation
  products of 2-deoxyribose)
• ALKALI-LABILE SITES
• (abasic sites and a few oxidized bases including
  thymine glycols, 5-formyluracil, hydantoins )

8
OXIDATION OF NUCLEIC ACIDS (general objectives)
- Model compounds Nucleosides and
oligonucleotides for structural and mechanistic
studies
- Isolated DNA Search for oxidative lesions,
requiring the development of assays based on
the chemical and spectroscopic features of the
targeted lesions
- DNA in cells and tissues Need of sensitive
assays aimed at singling out targeted lesions (at
least 1 modification per 106 to 107 normal
bases)
9
DNA lesions(model compounds)
Structural and mechanistic studies (NMR, MS,
X-rays) Theoretical aspects
Synthesis
Damage measurement
Modified oligonucleotides
HPLC (EC, MS/MS) Comet assay Immunoassays
Reactivity
.
Biological role (mutagenesis, translesional
synthesis)
Human Animals
Isolated cells Tissues
Biological fluids
DNA
Repair
Biomarkers
10
RADIATION-INDUCED DAMAGE TO ISOLATED AND CELLULAR
DNA (outline)

• Mechanistic studies on isolated DNA and models
• - .OH radical degradation of the thymine base
• - reactions of the guanine radical cation
• - Radiation-induced damage to cellular DNA
• - HPLC-MS/MS measurement
• - Modified comet assay

11
OXIDATIVE BASE DAMAGE TO DNA (current situation)

• - THYMINE almost complete available information
  for isolated DNA and model compounds
• - CYTOSINE comprehensive mechanism for radical
  oxidation of dCyd. Paucity of information for
  isolated DNA
• - GUANINE complex reactions with still a strong
  need of further investigations on both dGuo and
  isolated DNA
• - ADENINE apparent lack of information (need to
  be further checked)

12
Reactions of .OH radical with thymine
13
OXIDATIVE DAMAGE TO GUANINE (mechanistic
aspects)
- Single damage (one-electron oxidation)
- Modulating effect of 8-oxo-7,8-dihydroguanine
(one-electron oxidation)
- Tandem lesions (.OH radical, one-electron
oxidation)

- Charge transfer reaction within DNA
14
Main chemical reactions of the guanine radical
cation
Kasai et al, JACS, 1992 Cadet et al, JACS
1994 Gasparutto et al, JACS 1998 Ravanat et al,
JACS 2003
15
RADICAL OXIDATION OF GUANINE AND ADENINE IN
ISOLATED DNA(similarities and differences)

• Similar degradation products from
  8-hydroxy-7,8-dihydro
• purinyl radicals 8-oxo-7,8-dihydro- and Fapy
  purine derivatives
• - 2-Hydroxyadenine barely detectable with
  yield much lower than that of FapyAde
• On the overall degradation of guanine 10-fold
  less efficient than that
• of adenine in isolated DNA but not in
  nucleosides.
• - Unknown reasons of the apparent lower
  susceptibility of
• adenine to both .OH radical and one-electron
  oxidants.

16
Modulating effect of 8-oxodGuo on one-electron
oxidative damage to DNA

• - Ionization potential of 8-oxodGuo is lower than
  that of other DNA nucleosides including dGuo
  (Prat et al, J. Am. Chem. Soc., 120, 845-846,
  1998)
• - Rate constant for one-electron oxidation of
  8-oxodGuo is about 2 orders of magnitude higher
  with respect to that of dGuo (Steenken et al, J.
  Am. Chem. Soc., 122, 2373-2374, 2000)
• - 8-OxodGuo could be the ultimate sink of
  one-electron oxidation process within DNA

17
Comparative and competitive susceptibility of
dGuo and 8-oxodGuo to one-electron oxidant
(photoexcited riboflavin)
(Ravanat et al, J. Am. Chem. Soc., 125, 2030,
2003)
18
Radical oxidation reactions of isolated
2-deoxyguanosine
19
8-OXO-7,8-DIHYDROGUANINE(available information
on its formation)

• - Ubiquitous DNA oxidation product
• singlet oxygen (1O2)
• one-electron oxidation
• .OH radical
• peroxynitrite (ONOO-)
• - Present in tandem base modifications that
  involve initial oxidation reactions of thymine or
  cytosine
• one-electron oxidation
• .OH radical

20
Oxidative DNA damage tandem lesions (one initial
radical hit)
N
N
H
O
H
O
H
O
(5R)-5,8-cyclodGuo
(5S)-5',8-cyclodAdo
Fo/8-oxodGuo
8-oxodGuo/Fo
21
Mechanism of formation of 8-oxodGuo/formamido
tandem lesions (Douki et al, Chem. Res.
Toxicol., 2002)
22
CHARGE MIGRATION WITHIN DOUBLE STRANDED DNA

• - radical cations may be produced within
  nucleobases and 2-deoxyribose moieties
  (ionization)
• ionizing radiation
• bi-photonic excitation (high intensity laser
  pulses)
• - positive hole migration was found to occur
  toward guanine and also adenine (lesser
  extent) through
• hopping
• phonon-assisted polaron-like hopping
• super-exchange

23
Biphotonic products oxidized nucleosides
T
Tn
Ionization energy
hn
S1
ISC
T1
hn
S0
Singlet state
Triplet state
Monophotonic products pyrimidine dimers
24
Effects of the intensity on the quantum yield of
formation of oxidized nucleosides upon exposure
of DNA to UV laser pulses.
25
2
c,s TltgtT
t,s TltgtT
ThdGly
8-oxodGuo
1.8
8-oxodAdo
1.6
1.4
1.2
quantum yield (x1000)
1
0.8
0.6
0.4
0.2
0
denatured (heated)
control
spermidine 0.1 mM
Effects of denaturation and addition of polyamine
on the quantum yield of formation of modified
bases upon exposure of DNA to UV laser pulses
(intensity 347 mJ/cm2).
26
Effects of the addition of TRIS on the quantum
yield of formation of oxidized bases upon
exposure of DNA to UV laser pulses (intensity
347 mJ/cm2).
27
OXIDATIVE BASE DAMAGE TO CELLULAR DNA(current
situation)

• Isolated DNA and model compounds
• More than 70 lesions have been identified as
  oxidative degradation products of thymine,
  cytosine, adenine, guanine and 5-methylcytosine
• Cellular DNA only 11 base lesions have been
  accurately measured
• Adenine (2)
• Cytosine (1)
• Guanine (2),
• Thymine (6)

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MEASUREMENT OF OXIDATIVE BASE DAMAGE TO DNA

• - As DNA fragments (bases, nucleosides or
  nucleotides)
• - In whole DNA
• - In intact cells

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MEASUREMENT OF OXIDATIVE BASE DAMAGE TO CELLULAR
DNA(individual measurement)
- Chromatographic methods HPLC-MS/MS
Optimization of DNA extraction conditions
- Applications Effects of ?-rays and heavy
ions on human monocytes
30
DNA extraction for chromatographic assays

• Still a critical step
• Various methods have been proposed with or
  without phenol
• The background level is within the range of
  one up to several 8-oxodGuo residue per 106 bp
• Chaotropic method associated with a metal
  chelator (desferioxamine)
• The background level is lower than one 8-oxodGuo
  residue per 106 bp (Helbock et al, PNAS, 95,
  288-93, 1998 Ravanat et al, Carcinogenesis, 23,
  1911-8, 2002)

31
OXIDATIVE DNA BASE DAMAGEHPLC-MS/MS
(electrospray ionization mode) measurements

• - Most recent method (tandem mass spectrometers)
• - More sensitive than any other chromatographic
  methods by about a factor of 10 (this depends on
  the targeted lesion)
• - More straightforward than GC-MS (no
  derivatization) and versatile than HPLC-ECD
  (almost all compounds can be detected)
• - Extension to more sensitive analytical methods
  (micro-HPLC, capillary electrophoresis)

32
Principle of the HPLC-MS/MS assay for the
measurement of oxidized nucleosides
dN
OxdN
dN
dN
dN
dN
DNA digestion (dN nucleosides OxdNs oxidized
nucleosides)
33
HPLCMS/MS of oxidized nucleosides (separation -
detection quantitation)
Asa.
Integration
Ais
MS/MS
Hydrolyzed DNA sample Q1 is
M1
F1
C18 / 2.1mm
Mn
Fn
Gradient Formate / MeCN
34
Gamma ray-mediated formation of DNA damage in
isolated calf thymus DNA in aerated aqueous
solutions (lesions /106 DNA bases/ Gy)
11.4 ? 1.2
5.3 ? 1.4
15.3 ? 0.1
7.6 ? 4.4
Measurement within the dose rang 0 100 Gy
35
(No Transcript)
36
Yields of formation of 11 base modifications in
the DNA of monocytes upon exposure to 60Co ?-rays
(LET 0.2 keV/µm) and 12C6 ions (LET 25 keV/µm)
DNA base modifications
?-rays 12C6 (per 109 bases and per
Gy) 5,6-Dihydroxy-5,6-dihydrothymidine
97 62 (4 diastereomers) 5-(Hydrox
ymethyl)-2-deoxyuridine
29 12 5-Formyl-2-deoxyuridine
22 11 5-Hydroxy-2-deoxyuridine ?
0.2 lt 0.2 8-Oxo-7,8-dihydro-2-deoxyadenosin
e 3 3 Adenine formamidopyrimidine 5
1 8-Oxo-7,8-dihydro-2-deoxyguanosine
20 10 Guanine formamidopyrimidine
39 22
37
RADIATION-INDUCED DAMAGE TO CELLULAR
DNA(conclusions)

• - Thymine is a better substrate than guanine
• (guanine is a better target than adenine)
• - Occurrence of reduction processes for purine
  base damage
• Fapypurines gt 8-oxo-7,8-dihydropurine
• - Relatively low yields of base damage and
  strand breaks
• (with respect to steady-level of oxidative
  DNA damage)
• - Major role played by clustered damage
• DNA double strand breaks
• base lesion single strand break (or
  another base damage)

38
MEASUREMENT OF OXIDATIVE BASE DAMAGE TO CELLULAR
DNA

• Isolated cells
• Modified comet assay (use of DNA repair
  enzymes to convert base damage into strand
  breaks)
• More sensitive but less specific than
  HPLC-MS/MS

- Applications Effects of ionizing
radiation
39
The Comet Assay
60Co
Agarose gel
Cells
Microscope slide
Untreated cells
Alkaline cell lysis
Electrophoresis (pH13)
DNA migration
Cells
untreated
Detection of
í
CSB, CDB SAL
modified
Irradiated cell
Fluorescence labeling of DNA prior to analysis
40
Cleavage of DNA by formamidopyrimidine
glycosylase (Fpg) at a 8-oxo-7,8-dihydroguanine
site

Fpg
Fpg
Single strand break and 8-oxoGua release
41
Comet assay associated with DNA glycosylases (Fpg)
Control cells treated wiht Fpg
Control cells
8 Gy irradiated treated with Fpg
8 Gy irradiated cell
42
Detection of radiation-induced DNA damage using
an optimized version of the comet assay
SSB strand breaks ALS alkali-labile
sites Endo III sites oxidized pyrimidines Fpg
sites oxidized purines
Cultured human monocytes exposed to ?-rays
43
Formation of 8-oxodGuo, SSB DSB ALS and DNA
repair glycosylases (Fpg and endo III) sensitive
sites in ?irradiated THP-1 tumoral monocytes
SSB DSB ALS Fpg sites
endo III sites Background level/109 bases
130 90 90
Background level/cell
910 630 630 Yield /109
bases/Gy 62 22
25 Yield/cell/Gy 430 151
171
44
Damage in a mammalian cell nucleus (1 Gy of
low-LET radiation)
- Initial physical damage Ionizations in cell
nucleus 100 000 Ionizations directly
in DNA 2 000 Excitations
directly in DNA 2 000 - - -
Selected biochemical damage (Ward 1988) DNA
strand breaks DNA 1 000
8-Hydroxyadenine 700 Diol de
thymine 200 DNA
double-strand breaks 40
DNA-proteins crosslinks 150
Selected cellular effect Lethal events
0.2-0.8 D.T. Goodhead, IJRB,
65, 7-17 1994
45
RADIATON DAMAGE TO DNA (future work)
- Clustered damage - DNA-protein crosslinks -
DNA-aldehyde adducts (LOOH) - Bystander
effects - Effects of electrons of low energy
46
OXIDATIVE DAMAGE TO CELLULAR DNA(conclusion)

• - The steady-state level of the main oxidized
  bases is within the range of 1 lesion per 106 -
  107 normal bases.
• - The level of radical induced 8-oxoGua is lower
  than that of FapyGua and thymine glycol.
• - The level of 8-oxoAde and FapyAde is about
  10-fold lower than that of 8-oxoGua.
• - 2-Hydroxyadenine is not detectable.

47
OXIDATIVE DAMAGE TO CELLULAR DNA(ESCODD)

• European Standards Committee on Oxidative DNA
  Damage set in 1997 with EC funding over the
  period 2000-2003 it has involved 25 member
  laboratories in Europe and one in Japan
• Objectives to standardize and validate
  procedures for measuring 8-oxodGuo as a biomarker
  of DNA oxidation
• Levels of 8-oxodGuo (0.5) and Fpg-sensitive sites
  (0.1) per 106 bases in the DNA of human
  lymphocytes by HPLC and enzymic methods
• It will be necessary to re-examine anti-oxidant
  studies that are based on claims of 8-oxodGuo
  higher values than 1 per 106 bases
• (Collins et al, Arch. Biochem. Biophys., 423,
  57-65, 2004)