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

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

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



Radiation-induced Bystander Effect

At cell level:Unirradiated cells respond as if they have been irradiated When contact with irradiated cellsOr 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



IntroductionBystander Effect

Responds as it has been irradiated

Ionizing radiation

Cell A Cell 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



IntroductionBystander effect in vivo are always questioned

Responds as it also receive radiation?????

Ionizing radiation

Mouse A Mouse 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



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



Introduction Zebrafish development stages:

http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html

1.5 hours after fertilization



4 hours after fertilization

Embryo cell

Chorion



IntroductionWhy study alpha particles ?

An ionizing radiation with high linear energy transfer (LET)

Furthermore, alpha-particle emitters can be easily found in our environment.



Introduction

α particles DNA damage Apoptosis



Different in the number of apoptotic signals between control and irradiation experiment

DNA damage due to alpha-particle irradiation=

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

1. Dechorionation of zebrafish embryos

2. Alpha-particle irradiation

3. Bystander signal exposure

4. Apoptotic cell staining

5. Determining the absorbed dose



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



Methodology

Agarose Layer

Water

Zebrafish embryos with chorions

The chorions were removed by hands with forceps



MethodologyAlpha-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



MethodologyAlpha-particle irradiation

fluid16 μm PADC film -particles



MethodologyPADC films of 16 μm were used polyallyldiglycol-carbonate Repeating unit of PADC:

H2C O C

O

O CH2 CH2 O CH2 CH2 O C O

O

CH2

CH CH2 n

CHH2Cn

Most importantly:It is biocompatible and can record the number of alpha particles hitting the embryo cells



Methodology

16 μm PADC films glued to holes



2mm diameter holes

Methodology



Methodology

Agarose

Shallow regions



MethodologyBystander effect exposure (Part 1) Aim to prove the existence of alpha-particle-induced bystander effects

Irradiated embryos

Agarose Layer (Unirradiated)

Naïve embryos



Irradiation experiment

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

Unirradiated embryos

Agarose LayerControl Naïve embryos



Control experiment

MethodologyBystander effect exposure (Part 2) Aim at identifying the relationship between the alpha-particle absorbed

dose and the bystander effect

Irradiated embryos

Agarose Layer Naïve

embryos




MethodologyBystander effect exposure (Part 2)

Agarose layer

Water

Irradiated zebrafish embryo

Naïve zebrafish embryos



Methodology



Irradiated Embryos

Naïve

Embryos

Control Embryos

Control Naïve Embryos

Sharing same medium

Sharing same medium


Control experiment

MethodologyApoptotic cell staining:At 24 hours post fertilizationUsing acridine orangePhotos takingNo. of apoptotic cells were counted



Methodology

Signal of apoptotic cell



MethodologyDetermining the absorbed dose







Bottom layer of embryo cells

Cell area in contact with PADC film



MethodologyWhy counting? It is tedious and time consuming.

The size and shape of the cells varied significantlyThe 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 DiscussionPart 1 experiment

N =Average number of apoptotic cells

n = sample size

P = p values obtained



Average no. of apoptotic cells:

naive > control naive

Results and DiscussionPart 1 experiment

N =Average number of apoptotic cells

n = sample size

P = p values obtained



The p values <0.05

Results and Discussion

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.00

50

100

150

200

250

300

A = 37.0±8.37 (p<0.00043)B = 28700±10700 (p<0.0015)

Ap

op

totic

Sig

na

l

Absorbed Dose (mGy)



Part 2 experiment

y = A+Bx

ConclusionsOur results gave strong support to the existence of alpha-particle-induced bystander effects between zebrafish embryos in vivoA 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.



Thank You!





Documents

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