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Mechanism of non-DNA targeted mutagenesis: the role of intra cellular nucleotide pool

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.

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Mechanism of non-DNA targeted mutagenesis: the role of intra cellular nucleotide pool

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

  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 Extracellular 8-oxo-dG Expected (n=4 and 3 exp) (In vitrostudy, HPLC) Haghdoost S. et al. Free Radic. Res. 2005

  5. Chromatin structure Number of bases 3 x 109 Chromosome nr 1 8.9 cm DNA/cell 100 cm 10 m

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

  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. (dGTP+ ROS 8-oxo-dGTP) (dATP+ ROS 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

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

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

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

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

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

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

  15. TK6 cells KD in MTH1 8-oxo-dGTP in cytoplasm MTH1 ND dG (TP, MP, DP) in cytoplasm 8-oxo-dG in medium Fotouhi, et. al. Mut. Res. 2011

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

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

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

  19. Exposure of the MTH1-transfected cells to UVA: • MTH1 has no effect on suvival (UVA, B, C) • High8-oxo-dGTP in cytoplasm, • Low8-oxo-dG in the medium • Highmutation rate

  20. Exposure to Gamma radiation?

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

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

  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

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