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
[email protected]
Toxicology 2014, Chicago, 20-22 Oct.
1-2 Gy gamma Radiation
Incubation
for repair
Detection of oxidized
DNA base in serum
1 Gy
–20-40 DSB
–~1000 SSB
–~2000 base damages (500-700 8-oxo-dG)
–~10000 ionizations in a cell
8-Oxo-dG repair pathways
NER, up to 28 bp
C
G
A
T
T
A
C
G
NIR, damaged base with sugar and phosphate
BER, damaged base
(hOGG1)
(Michaels, 1991, BM Ames 1991)
Repaired 8-oxo-dG is released to urine via blood serum
where it can be detected.
Extracellular 8-oxo-dG as a sensitive marker
for oxidative stress in vivo and in vitro
Extracellular 8-oxo-dG
250
220
8-oxo-dG (%)
• 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.
190
160
130
Expected
100
• DNA is not the main source
for extracellular 8-oxo-dG
70
0
0,5
(n=4 and 3 exp)
Haghdoost S. et al. Free Radic. Res. 2005
1
1,5
Dose (Gy)
(In vitro study, HPLC)
2
2,5
Chromatin structure
Number of bases
3 x 109
Chromosome nr 1
8.9 cm
DNA/cell
100 cm
10 m
Protective effect of chromatin structure
Ljungman, M. et al., 1991
Histone free
Svoboda and Harms-Ringdahl,
2005
8-oxo-dG
DNA single strand breaks
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
(dGTP+ ROS
(dATP+ ROS
8-oxo-dGTP)
8-oxo-dATP/2-OH-dATP)
8-oxo-dGTP
8-oxo-dATP
hMTH1
8-oxo-dGMP +2p
8-oxo-dG+p
(NUDT5)
Urine, Serum, Medium
(Haghdoost, et al., 2005, 2006)
T, Tajiri, et al., 1995
Nucleotide pool cleaning up system
hMTH1
8-oxo-dGTP
8-oxo-dG+p
8-oxo-dGMP +2p
(NUDT5)
Urine, Serum, Medium
Nucleotide
pool
(2005)
Polymerase
DNA synthesis
8-oxo-G--C
hOGG1
G—C
8-oxo-G
8-oxo-G--A
T--A
hMYH1 (mut y)
IN DNA
8-oxo-G—C +A
(1990)
hOGG1
G—C
8-oxo-G
2-OH-dA: AT-GC
8-oxo-dG: TA-GC
G--C
Pool size and extracellular 8-oxo-dG
Extracellular 8-oxo-dG (pmol/10 6 cells)
0,6
0,5
V79/p
0,4
VH10
0,3
0,2
0,1
Leukocytes
0
0
5
10
dGTP (pmol/106 cells) in nucleotide pool
Haghdoost S. et al, Free rad. Bio. Med. 2006
15
dNTP sanitization
B
dGTP
8-oxo-dGTP
hMTH1 hMTH1 shRNA
8-oxo-dGmp +pp
?
Extracellular 8-oxo-dG
Extracellular levels of 8-oxo-dG in VH10 cells KD in MTH1
8-oxo-dG (%)
300
250
200
150
100
50
0
0,5
1
Dose (Gy)
1,5
2
Haghdoost S. et al, Free rad. Bio. Med. 2006
UVA and oxidative stress
Ozon and
atmosphere
Ozon and
atmosphere
5%
95%
www.greengoods.com
Clonogenic survival of TK6 cells KD in MTH1
N-Tr
Tr
100
Survival (%)
Actin
hMTH1
10
1
0
50
100
Dose (kJ/m2)
Fotouhi, et. al. Mut. Res. 2011
150
TK6 cells KD in MTH1
8-oxo-dGTP in cytoplasm
Intracellular 8-oxo-dG (%)
400
*
300
MTH1 ND
200
100
0
0
18
29
Dose (kJ/m2)
dG (TP, MP, DP) in cytoplasm
dG (N-Tr)
dG (Tr)
100
80
60
40
20
0
0
18
Dose (kJ/m2)
29
160
*
Extracellular 8-oxo-dG (%).
Intracelular dG (%)
120
8-oxo-dG in medium
120
80
40
0
0
Fotouhi, et. al. Mut. Res. 2011
18
Dose (kJ/m2)
29
Mutation frequency induced by UVA in
transfected and non-transfected TK6 cells
A. Fotouhi et al. Mut. Res. 2011
Mutation frequency
1.5
*
hMTH transfected
1
*
0.5
0
0
18
29
Dose (kJ/m2)
Equal to 1h sunshine on the French riviera at noon
Point mutations induced by UVA
8-oxo-dATP
Fotouhi, A. et al, Mut. Res. 2013
Mutation rate: the role of MTH1
UVB
UVC
LD50 dose
MTH1 har minor
role in UVC mutagenecity
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
• Exposure to Gamma radiation?
Gamma radiation
Shakeri-Manesh, S. et. al. Rad. Env. Biophys. 2014
Effect of MTH1 on micronuclei induction: UVA, B, C and gamma radiation
LD50
MTH1 +
MTH1 -
Final summary
dNTP (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
Radiobiology groups at SU