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Mechanism of non-DNA targeted mutagenesis: the role of intra cellular nucleotide pool
Siamak Haghdoost, PhD
Department of Molecular Bioscience, Wenner-Gren Institute
Stockholm University
Stockholm, Sweden
Toxicology 2014, Chicago, 20-22 Oct.
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1-2 Gy gamma Radiation
Incubation for repair
Detection of oxidizedDNA base in serum
1 Gy
–20-40 DSB–~1000 SSB–~2000 base damages (500-700 8-oxo-dG)–~10000 ionizations in a cell
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C
T
C
T
G
A
G
A
NER, up to 28 bp
BER, damaged base
NIR, damaged base with sugar and phosphate
(hOGG1)
Repaired 8-oxo-dG is released to urine via blood serumwhere it can be detected.
(Michaels, 1991, BM Ames 1991)
8-Oxo-dG repair pathways
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Extracellular 8-oxo-dG as a sensitive marker for oxidative stress in vivo and in vitro
• Amount of 8-oxo-dG excreted by leukocytes, exposed to 1 Gy, is 35 times higher than what is expected to be formed in DNA.
• DNA is not the main source for extracellular 8-oxo-dG
Haghdoost S. et al. Free Radic. Res. 2005
70
100
130
160
190
220
250
0 0,5 1 1,5 2 2,5
Dose (Gy)
8-ox
o-dG
(%
)
(n=4 and 3 exp)
Extracellular 8-oxo-dG
Expected
(In vitro study, HPLC)
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Number of bases 3 x 109
Chromosome nr 1 8.9 cm
DNA/cell 100 cm
10 m
Chromatin structure
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Histone free
8-oxo-dGDNA single strand breaks
Svoboda and Harms-Ringdahl, 2005
Ljungman, M. et al., 1991
Protective effect of chromatin structure
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Summary
• Increase in extracellular 8-oxo-dG observed after in vitro irradiation of whole blood
• This yield• saturates above a dose of 1 Gy• is signifcantly different between individuals.
• Indications for a radiation induced stress response that would work primarily on the dNTP pool
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hMTH18-oxo-dGMP +2p
(NUDT5)8-oxo-dG+p
Urine, Serum, Medium
(dGTP+ ROS 8-oxo-dGTP)(dATP+ ROS 8-oxo-dATP/2-OH-dATP)
8-oxo-dGTP8-oxo-dATP
Nucleotide pool cleaning up system
T, Tajiri, et al., 1995(Haghdoost, et al., 2005, 2006)
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8-oxo-dGTPhMTH1
8-oxo-dGMP +2p
PolymeraseDNA synthesis
8-oxo-G--C 8-oxo-G--A
hOGG1
G—C 8-oxo-G
hMYH1 (mut y)
8-oxo-G—C +AhOGG1
G—C8-oxo-G
T--A
G--C
(NUDT5)8-oxo-dG+p
Urine, Serum, Medium
IN DNA
Nucleotide pool
(1990)
(2005)
2-OH-dA: AT-GC8-oxo-dG: TA-GC
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0
0,1
0,2
0,3
0,4
0,5
0,6
0 5 10 15
dGTP (pmol/106 cells) in nucleotide pool
Ext
race
llula
r 8-
oxo-
dG (
pmol
/10
6 c
ells
)
V79/p
VH10
Leukocytes
Haghdoost S. et al, Free rad. Bio. Med. 2006
Pool size and extracellular 8-oxo-dG
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dGTP
8-oxo-dGTP
8-oxo-dGmp +pp
hMTH1
dNTP sanitization
Extracellular 8-oxo-dG
?
hMTH1 shRNA
B
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50
100
150
200
250
300
0 0,5 1 1,5 2Dose (Gy)
8-ox
o-dG
(%
)
Haghdoost S. et al, Free rad. Bio. Med. 2006
Extracellular levels of 8-oxo-dG in VH10 cells KD in MTH1
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UVA and oxidative stress
5%
95%
Ozon and atmosphere
Ozon and atmosphere
www.greengoods.com
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N-Tr Tr
Actin
hMTH1
N-Tr Tr
Actin
hMTH11
10
100
0 50 100 150
Dose (kJ/m2)
Su
rviv
al (
%)
Fotouhi, et. al. Mut. Res. 2011
Clonogenic survival of TK6 cells KD in MTH1
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0
20
40
60
80
100
120
0 18 29
Dose (kJ/m2)
Intra
celu
lar d
G (%
)
dG (N-Tr)
dG (Tr)
0
100
200
300
400
0 18 29
Dose (kJ/m2)
Intra
cellu
lar 8
-oxo
-dG
(%)
*
0
40
80
120
160
0 18 29
Dose (kJ/m2)
Ext
race
llula
r 8
-oxo
-dG
(%
).
*
Fotouhi, et. al. Mut. Res. 2011
dG (TP, MP, DP) in cytoplasm
8-oxo-dGTP in cytoplasm
8-oxo-dG in medium
TK6 cells KD in MTH1
MTH1 ND
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Mutation frequency induced by UVA in transfected and non-transfected TK6 cells
0
0.5
1
1.5
0 18 29
Dose (kJ/m2)
Mu
tatio
n fr
eq
ue
ncy
*
*hMTH transfected
A. Fotouhi et al. Mut. Res. 2011
Equal to 1h sunshine on the French riviera at noon
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8-oxo-dATPFotouhi, A. et al, Mut. Res. 2013
Point mutations induced by UVA
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LD50 doseUVB
UVC
Mutation rate: the role of MTH1
MTH1 har minorrole in UVC mutagenecity
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Exposure of the MTH1-transfected cells to UVA:
•MTH1 has no effect on suvival (UVA, B, C) •High 8-oxo-dGTP in cytoplasm, •Low 8-oxo-dG in the medium•High mutation rate
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• Exposure to Gamma radiation?
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Gamma radiation
Shakeri-Manesh, S. et. al. Rad. Env. Biophys. 2014
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MTH1 +
MTH1 -
Effect of MTH1 on micronuclei induction: UVA, B, C and gamma radiation
LD50
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Final summarydNTP (NTP?) is a significant mutagenic target for free radicals particularly for UVA.
MTH1 does not protect cells from radiation induced chromosomal damages
MTH1 does not influense survival of the cells exposed to UV and Gamma radiation
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Radiobiology groups at SU