1. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Study of influence of catechins on bystander responses in alpha- particle radiobiological experiments using thin PADC films Y.L. Law and K.N. Yu Department of Physics and Materials Science, City University of Hong Kong

2. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Objectives 1.To investigate the relationship between the radiation dose and induction of DNA damages in unirradiated cells 2. To investigate the scavenging effect of catechins on reactive oxygen species in unirradiated cells.

3. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Introduction Radiobiological effects of ionizing radiation Alpha particle Interacts with DNA  Double-strand breaks (DSBs) Free radicals  DSBs DSBs Most lethal Cell death, chromosome aberration, gene mutation…

4. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Introduction Catechins Polyphenolic compounds in green tea EGCG (epigallocatechin gallate) – most abundant Cancer protection Antioxidant effects Scavengers of reactive oxygen species (ROS)

5. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Introduction Radiation-induced bystander effect  Induction of biological effects in cells that are not directly traversed by an ionizing radiation Underlying mechanisms: 1.Gap junction-mediated intercellular communication 2.Soluble factor such as ROS released by irradiated cells

6. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Introduction Polyallyldiglycol carbonate (PADC) films -Most commonly used solid-state nuclear track detectors (SSNTDs) -Transparent -Relatively biocompatible -Does not dissolve in alcohol during sterilization Commercially available 100 µm thick PADC film

7. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Methodology 22 µm PADC film base cell dish + 1000 µm PADC films = Set-up: PADC film cell dish

8. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Methodology Set-up: Alpha-particle irradiation Cell type : Chinese hamster ovary (CHO) Cell culture substrate : 22 & 1000 µm PADC films Alpha source : 241 Am (activity = 5 µCi and main alpha energy = 5.49 MeV under vacuum )  -particle beam nutritive medium 22 µm PADC film 1000 µm PADC film

9. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Methodology 21 34  -particle irradiation add EGCG (in DMSO) BY cells only Trypsinize the cells Pelleted by centrifugation for TUNEL assay single cell BY cells only

10. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Methodology TUNEL assay T erminal deoxynucleotidyl transferase d U TP transferase-mediated n ick e nd- l abeling Common method for detecting DNA fragmentation results from apoptosis Identified by enzyme TdT dUTPs – marker

11. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Methodology: TUNEL assay Fixation Permeabilisation Labeling reaction with TUNEL reaction mixture Flow Cytometry 2% paraformaldehyde in PBS – 1h in RT To stabilize the cell – reaction stopped

12. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Fixation Permeabilisation Labeling reaction with TUNEL reaction mixture Flow Cytometry Methodology: TUNEL assay 0.1% Triton X-100 and 0.1% sodium citrate -10 min on ice Dissolve the cell membranes – allow larger dye molecules access into the cell

13. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Fixation Permeabilisation Labeling reaction with TUNEL reaction mixture Flow Cytometry Methodology: TUNEL assay 90% Label solution and 10% TUNEL enzyme - 37 ℃ in dark for 1 h Labels DNA strand breaks generated during apoptosis

14. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Fixation Permeabilisation Labeling reaction with TUNEL reaction mixture Flow Cytometry Methodology: TUNEL assay Detect and quantify fluorescein labels using laser beam Counting of 10000 cells

15. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Methodology 21 34 Fixation Capture image Etching 37 ℃ 14 N KOH solution Capture image of tracks and cells IR cells

16. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Results and Discussions Number of cell hits After fixation (CHO cells only) After etching (CHO cells and tracks) Alpha-particle track CHO cells

17. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Results and Discussions As low as 1% of the cell nuclei were actually traversed by alpha particles in all the samples Bystander damages occur even at very low doses DNA damages in bystander cells seems to be independent of the number of irradiated cells Number of cell hits

18. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Results and Discussions 0 μM1μM10 μM20 μM ** ** *P < 0.05, **P < 0.005 compared to the corresponding control (0 μM) Results of TUNEL assay For example: Bystander sample = 33.75% Unirradiated control = 17.22% Result: 16.33% of TUNEL positive signal

19. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Results and Discussions 0 μM1μM10 μM20 μM ** ** *P < 0.05, **P < 0.005 compared to the corresponding control (0 μM) Without any treatment Results of TUNEL assay Signal transduction from irradiated cells to bystander cells occurred within a short time

20. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Results and Discussions 0 μM1μM10 μM20 μM ** ** *P < 0.05, **P < 0.005 compared to the corresponding control (0 μM) Results of TUNEL assay

21. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Results and Discussions Results of TUNEL assay Scavenging effect of EGCG in 1 μM on bystander cells is the most significant Previous study showed EGCG significantly reduced the amount of DNA strand breaks in 1-2 μM concentrations (Anderson et al., 2001) ROS might be involved in the induction of DNA breaks Anderson, R.F., Fisher, L.J., Hara, Y., Harris, T., Mak, W.B., Melton, L.D., Packer, J.E., Anderson, R.F., Fisher, L.J., Hara, Y., Harris, T., Mak, W.B., Melton, L.D., Packer, J.E., Anderson, R.F., Fisher, L.J., Hara, Y., Harris, T., Mak, W.B., Melton, L.D., Packer, J.E., Anderson, R.F., Fisher, L.J., Hara, Y., Harris, T., Mak, W.B., Melton, L.D., Packer, J.E., 2001. Green tea catechins partially protect DNA from OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals. Carcinogenesis. 22(8), 1189-93.

22. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Conclusions Bystander damages occur at very low doses The signal transduction from irradiated cells to unirradiated cells occur within a short time after irradiation EGCG scavenges ROS at a significant level in the bystander cells produced by alpha-particles irradiation Further study is needed to find out which types of ROS are involved in the medium-mediated bystander effects.

23. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Study of influence of catechins on bystander responses in alpha-particle radiobiological experiments using thin PADC films Y.L. Law and K.N. Yu Thank you!