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

Siamak.Haghdoost@su.se

Toxicology 2014, Chicago, 20-22 Oct.

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

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

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)

Number of bases 3 x 109

Chromosome nr 1 8.9 cm

DNA/cell 100 cm

10 m

Chromatin structure

Histone free

8-oxo-dGDNA single strand breaks

Svoboda and Harms-Ringdahl, 2005

Ljungman, M. et al., 1991

Protective effect of chromatin structure

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

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)

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

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

dGTP

8-oxo-dGTP

8-oxo-dGmp +pp

hMTH1

dNTP sanitization

Extracellular 8-oxo-dG

?

hMTH1 shRNA

B

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

UVA and oxidative stress

5%

95%

Ozon and atmosphere

Ozon and atmosphere

www.greengoods.com

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

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

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

8-oxo-dATPFotouhi, A. et al, Mut. Res. 2013

Point mutations induced by UVA

LD50 doseUVB

UVC

Mutation rate: the role of MTH1

MTH1 har minorrole 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

MTH1 +

MTH1 -

Effect of MTH1 on micronuclei induction: UVA, B, C and gamma radiation

LD50

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

Radiobiology groups at SU