1. 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.

2. 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

3. (hOGG1) Repaired 8-oxo-dG is released to urine via blood serum where it can be detected. (Michaels, 1991, BM Ames 1991) 8-Oxo-dG repair pathways

4. 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 (n=4 and 3 exp) Extracellular 8-oxo-dG Expected (In vitro study, HPLC)

5. Number of bases 3 x 10 9 Chromosome nr 1 8.9 cm DNA/cell 100 cm 10 m Chromatin structure

6. Histone free 8-oxo-dG DNA single strand breaks Svoboda and Harms-Ringdahl, 2005 Ljungman, M. et al., 1991 Protective effect of chromatin structure

7. 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

8. hMTH1 8-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-dGTP 8-oxo-dATP Nucleotide pool cleaning up system T, Tajiri, et al., 1995 (Haghdoost, et al., 2005, 2006)

9. 8-oxo-dGTP hMTH1 8-oxo-dGMP +2p Polymerase DNA synthesis 8-oxo-G--C 8-oxo-G--A hOGG1 G—C 8-oxo-G hMYH1 (mut y) 8-oxo-G—C +A hOGG1 G—C 8-oxo-G T--A G--C (NUDT5) 8-oxo-dG+p Urine, Serum, Medium IN DNA Nucleotide pool (1990) (2005) 2-OH-dA: AT-GC 8-oxo-dG: TA-GC

10. Haghdoost S. et al, Free rad. Bio. Med. 2006 Pool size and extracellular 8-oxo-dG

11. dGTP 8-oxo-dGTP 8-oxo-dGmp +pp hMTH1 dNTP sanitization Extracellular 8-oxo-dG ? hMTH1 shRNA B

12. Haghdoost S. et al, Free rad. Bio. Med. 2006 Extracellular levels of 8-oxo-dG in VH10 cells KD in MTH1

13. UVA and oxidative stress 5% 95% Ozon and atmosphere Ozon and atmosphere www.greengoods.com

14. Fotouhi, et. al. Mut. Res. 2011 Clonogenic survival of TK6 cells KD in MTH1

15. 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

16. Mutation frequency induced by UVA in transfected and non-transfected TK6 cells hMTH transfected A. Fotouhi et al. Mut. Res. 2011 Equal to 1h sunshine on the French riviera at noon

17. 8-oxo-dATP Fotouhi, A. et al, Mut. Res. 2013 Point mutations induced by UVA

18. LD50 dose UVB UVC Mutation rate: the role of MTH1 MTH1 har minor role in UVC mutagenecity

19. 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

20. Exposure to Gamma radiation?

21. Gamma radiation Shakeri-Manesh, S. et. al. Rad. Env. Biophys. 2014

22. MTH1 + MTH1 - Effect of MTH1 on micronuclei induction: UVA, B, C and gamma radiation LD50

23. 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

24. Radiobiology groups at SU