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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,

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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. 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

introduction

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
introduction1

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

introduction2

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

introduction3

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.

introduction4

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.

introduction5

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

introduction6

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.
introduction7

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

introduction8

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.

methodology

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
methodology1

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

methodology2

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
methodology3

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

methodology4

24th International Conference on Nuclear Tracks in Solids

Bologna, 1-5 September 2008

Methodology
  • Alpha-particle irradiation

16 μmPADC film

fluid

-particles

methodology5

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
methodology6

24th International Conference on Nuclear Tracks in Solids

Bologna, 1-5 September 2008

Methodology

16 μmPADC films glued to holes

2mm diameter holes

methodology8

24th International Conference on Nuclear Tracks in Solids

Bologna, 1-5 September 2008

Methodology

Agarose

Shallow regions

methodology9

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
methodology10

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

methodology11

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
methodology12

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)
methodology13

24th International Conference on Nuclear Tracks in Solids

Bologna, 1-5 September 2008

Sharing same medium

Sharing same medium

Irradiation experiment

Control experiment

Methodology
methodology14

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
methodology15

24th International Conference on Nuclear Tracks in Solids

Bologna, 1-5 September 2008

Methodology

Signal of apoptotic cell

methodology16

24th International Conference on Nuclear Tracks in Solids

Bologna, 1-5 September 2008

Methodology
  • Determining the absorbed dose
methodology17

24th International Conference on Nuclear Tracks in Solids

Bologna, 1-5 September 2008

Methodology
  • Determining the absorbed dose
methodology18

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
methodology19

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
results and discussion

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

results and discussion1

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

conclusions

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.