1 / 38

Alpha-particle-induced bystander effects between zebrafish embryos in vivo

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

oke
Download Presentation

Alpha-particle-induced bystander effects between zebrafish embryos in vivo

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Alpha-particle-induced bystander effects between zebrafish embryos in vivo E.H.W. Yum1, V.W.Y. Choi1, V.W.T. Li2, S.H. Cheng2, and K.N. Yu1 1Department of Physics and Materials Science, City University of Hong Kong 2Department of Chemistry and Biology, City University of Hong Kong

  2. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 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

  3. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Introduction • 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 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

  4. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Ionizing radiation Cell A Cell B Introduction • Bystander Effect Responds as it has been irradiated • When they are put in contact or they share the same medium 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

  5. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Ionizing radiation Yes, there have been recent researches on bystander effect in vivo in mice and in fish Mouse A Mouse B Introduction • Bystander effect in vivo are always questioned Responds as it also receive radiation????? 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.

  6. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 Introduction • Recent researches on bystander effects in vivo 0.5 Gy X-ray This communication signals involved secretion of a chemical messenger into the water 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.

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

  8. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 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.

  9. 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 = Introduction αparticles DNA damage Apoptosis  

  10. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 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.

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

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

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

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

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

  16. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 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

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

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

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

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

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

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

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

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

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

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

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

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

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

  30. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 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

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

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

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

  34. 24th International Conference on Nuclear Tracks in Solids Bologna, 1-5 September 2008 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.

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

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

More Related