1. Alpha-particle-induced bystander effects between zebrafish embryos in vivo E.H.W. Yum 1, V.W.Y. Choi 1, V.W.T. Li 2, S.H. Cheng 2, and K.N. Yu 1 1 Department of Physics and Materials Science, City University of Hong Kong 2 Department of Chemistry and Biology, City University of Hong Kong 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

2. Introduction Objectives:  To study whether alpha-particles can induce in vivo bystander effects  Identifying the relationship between the alpha-particle absorbed dose and the bystander effect  Zebrafish embryos as the model 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

3. Radiation-induced Bystander Effect At cell level: Unirradiated cells respond as if they have been irradiated  When contact with irradiated cells  Or in medium previously holding irradiated cells Introduction Yang, H., Asaad, N., Held, K.D., 2005. Medium-mediated intercellular communication is involved in bystander responses of X-ray- irradiated normal human fibroblasts. Oncogene. 24, 2096-2103 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

4. Introduction Bystander Effect Responds as it has been irradiated Ionizing radiation Cell ACell B Yang, H., Asaad, N., Held, K.D., 2005. Medium-mediated intercellular communication is involved in bystander responses of X-ray- irradiated normal human fibroblasts. Oncogene. 24, 2096-2103  When they are put in contact or they share the same medium 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

5. Introduction Bystander effect in vivo are always questioned Responds as it also receive radiation????? Ionizing radiation Mouse AMouse B Morgan, W.F., 2003. Non-targeted and delayed effects of exposure to ionizing radiation: II. Radiation-induced genomic instability and bystander effect in Vivo, clastogenic factors and transgenerational effects. Radiation research 159, 581-595. Yes, there have been recent researches on bystander effect in vivo in mice and in fish 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

6. Introduction Recent researches on bystander effects in vivo 0.5 Gy X-ray Mothersill, C., Bucking, C., Smith, R.W., Agnihotri, N., O’Neill, A., Kilemade, M., Seymour, C.B., 2006. Communication of Radiation-induced Stress or Bystander Signals between Fish in Vivo. Environ. Sci. Technol. 40, 6859-6864. Mothersill, C., Smith, R.W., Agnihotri, N., Seymour, C.B., 2007. Characterization of a radiation-induced stress response communicated in vivo between zebrafish. Environ. Sci. Technol. 41, 3382-3387. This communication signals involved secretion of a chemical messenger into the water 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

7. Introduction Zebrafish development stages: http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage. html 1.5 hours after fertilization 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 4 hours after fertilization Embryo cell Chorion 16 hours after fertilization 24 hours after fertilization

8. Introduction Why study alpha particles ?  An ionizing radiation with high linear energy transfer (LET)  Furthermore, alpha-particle emitters can be easily found in our environment. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

9. Introduction α particlesDNA damageApoptosis  24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Different in the number of apoptotic signals between control and irradiation experiment DNA damage due to alpha- particle irradiation =

10. Introduction In this project: Irradiation : 1.5 hours after fertilization  No repair mechanism at this stage  can magnify our results for investigation Apoptotic cell staining : 24 hours after fertilization  Bladen’s group found it the best time to investigate the no. of apoptotic cell after trying different time stages Walker, C., Streisinger, G., 1983. Induction of mutations by gamma rays in pregonial germ cells of zebrafish embryos. Genetics. 103, 125-136 Bladen, C.L., Lam, W.K., Dynan, W.S., Kozlowski, D.J., 2005. DNA damage response and Ku80 function in the vertebrate embryo. Nucleic Acids Research 33, 3002-3010. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

11. Methodology 1.Dechorionation of zebrafish embryos 2.Alpha-particle irradiation 3.Bystander signal exposure 4.Apoptotic cell staining 5.Determining the absorbed dose 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

12. Methodology Dechorionation of zebrafish embryos  Range of 5.5 MeV α-particles in water = 42 µ m  The chorion will absorb all the alpha-particle energies 220um 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

13. Methodology Agarose Layer Water Zebrafish embryos with chorions  The chorions were removed by hands with forceps 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

14. Methodology Alpha-particle irradiation Alpha particles ( passing through a large and variable thickness of fluid)  To control the incident alpha-particle energy  alpha particles should pass through the substrate which was in contact with the embryo cells water 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

15. Methodology Alpha-particle irradiation fluid 16 μm PADC film  -particles 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

16. Methodology PADC films of 16 μm were used  polyallyldiglycol-carbonate  Repeating unit of PADC: Most importantly:  It is biocompatible and can record the number of alpha particles hitting the embryo cells 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

17. Methodology 16 μm PADC films glued to holes 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 2mm diameter holes

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

19. Methodology Agarose Shallow regions 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

20. Methodology Bystander effect exposure (Part 1)  Aim to prove the existence of alpha-particle-induced bystander effects Irradiated embryos Agarose Layer (Unirradiated) Naïve embryos 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Irradiation experiment

21. Methodology Bystander effect exposure (Part 1)  Aim at proving the existence of alpha-particle-induced bystander effects Unirradiated embryos Agarose Layer Control Naïve embryos 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Control experiment

22. Methodology Bystander effect exposure (Part 2)  Aim at identifying the relationship between the alpha-particle absorbed dose and the bystander effect Irradiated embryos Agarose Layer Na ï ve embryos 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Irradiation experiment

23. Methodology Bystander effect exposure (Part 2) Agarose layer Water Irradiated zebrafish embryo Naïve zebrafish embryos 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

24. Methodology 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Irradiated Embryos Naïve Embryos Control Embryos Control Naïve Embryos Sharing same medium Irradiation experiment Control experiment

25. Methodology Apoptotic cell staining:  At 24 hours post fertilization  Using acridine orange  Photos taking  No. of apoptotic cells were counted 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

26. Methodology Signal of apoptotic cell 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

27. Methodology Determining the absorbed dose 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

28. Methodology Determining the absorbed dose 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

29. Methodology Determining the absorbed dose Bottom layer of embryo cells Cell area in contact with PADC film 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

30. Methodology Why counting? It is tedious and time consuming.  The size and shape of the cells varied significantly  The contact area of the cell with the PADC film varied significantly  Impractical to estimate the no. of alpha-particles striking the embryos cells using track density by assumed geometry 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

31. Results and Discussion Part 1 experiment N =Average number of apoptotic cells n = sample size P = p values obtained 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Average no. of apoptotic cells: naive > control naive

32. Results and Discussion Part 1 experiment N =Average number of apoptotic cells n = sample size P = p values obtained 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 The p values <0.05

33. Results and Discussion 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Part 2 experiment y = A+Bx

34. Conclusions Our results gave strong support to the existence of alpha-particle-induced bystander effects between zebrafish embryos in vivo A general positive correlation between the apoptotic signals in the naive embryos and the alpha-particle dose absorbed by the irradiated embryos. These supported the bystander factors involved chemical messengers secreted into the water medium. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

35. Thank You! 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008

36. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008