Alphaparticleinduced bystander effects between zebrafish embryos in vivo

1
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
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
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
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
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
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., ONeill, 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
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/F
ish/fish_stage.html
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.

9
Introduction
a particles
DNA damage
Apoptosis
?
?
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
• Bladens 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.
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Methodology

• Dechorionation of zebrafish embryos
• Alpha-particle irradiation
• Bystander signal exposure
• Apoptotic cell staining
• Determining the absorbed dose

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Methodology

• Dechorionation of zebrafish embryos
• Range of 5.5 MeV a-particles in water 42 µm
• The chorion will absorb all the alpha-particle
  energies

220um
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Methodology
Water
Agarose Layer
Zebrafish embryos with chorions

• The chorions were removed by hands with forceps

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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

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Methodology

• Alpha-particle irradiation

16 µm PADC film
fluid
?-particles
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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

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Methodology
16 µm PADC films glued to holes
2mm diameter holes
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Methodology
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Methodology
Agarose
Shallow regions
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Methodology

• Bystander effect exposure (Part 1)
• Aim to prove the existence of alpha-particle-induc
  ed bystander effects

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Methodology

• Bystander effect exposure (Part 1)
• Aim at proving the existence of
  alpha-particle-induced bystander effects

Control experiment
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Methodology

• Bystander effect exposure (Part 2)
• Aim at identifying the relationship between the
  alpha-particle absorbed dose and the bystander
  effect

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Methodology

• Bystander effect exposure (Part 2)

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Methodology
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Methodology

• Apoptotic cell staining
• At 24 hours post fertilization
• Using acridine orange
• Photos taking
• No. of apoptotic cells were counted

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Methodology
Signal of apoptotic cell
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Methodology

• Determining the absorbed dose

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Methodology

• Determining the absorbed dose

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Methodology

• Determining the absorbed dose

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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

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Results and Discussion
Average no. of apoptotic cells naive gt control
naive
N Average number of apoptotic cells n sample
size P p values obtained
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Results and Discussion
The p values lt0.05
N Average number of apoptotic cells n sample
size P p values obtained
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Results and Discussion
y ABx
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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.

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Thank You!
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