24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

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 • 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: • Gap junction-mediated intercellular communication • 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 usedsolid-state nuclear track detectors (SSNTDs) -Transparent -Relatively biocompatible -Does not dissolve in alcohol during sterilization Commercially available 100 µm thick PADC film

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

8. 1000 µm PADC film nutritive medium 22 µm PADC film -particle beam 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 µmPADC films Alpha source : 241Am (activity = 5 µCiandmain alpha energy = 5.49 MeV under vacuum)

9. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Methodology 1 2 -particle irradiation BY cells only add EGCG (in DMSO) 3 4 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 • Terminal deoxynucleotidyl transferase dUTP • transferase-mediated nick end-labeling • 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 TUNELreaction 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 Methodology: TUNEL assay Fixation Permeabilisation Labeling reaction with TUNELreaction mixture Flow Cytometry • 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 Methodology: TUNEL assay Fixation Permeabilisation Labeling reaction with TUNELreaction mixture Flow Cytometry 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 Methodology: TUNEL assay Fixation Permeabilisation Labeling reaction with TUNELreaction mixture Flow Cytometry 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 1 2 Fixation IR cells 3 4 Etching 37℃ Capture image of tracks and cells Capture image 14 N KOH solution

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

17. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Results and Discussions Number of cell hits • 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

18. 0 μM 1μM 10 μM 20 μM 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Results and Discussions Results of TUNEL assay * * For example: Bystander sample = 33.75% Unirradiated control = 17.22% Result: 16.33% of TUNEL positive signal ** *P < 0.05, **P < 0.005 compared to the corresponding control (0 μM)

19. * * ** 0 μM 1μM 10 μM 20 μM Without any treatment *P < 0.05, **P < 0.005 compared to the corresponding control (0 μM) 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Results and Discussions Results of TUNEL assay Signal transduction from irradiated cells to bystander cells occurred within a short time

20. 0 μM 1μM 10 μM 20 μM 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Results and Discussions Results of TUNEL assay * * ** *P < 0.05, **P < 0.005 compared to the corresponding control (0 μM)

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.,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!