CITY UNIVERSITY OF HONG KONG DEPARTMENT OF PHYSICS …lbms03.cityu.edu.hk/oaps/ap2008-4209-cwy200.pdf · Figure 2 Geometry of track development with the incident angle normal to the

CITY UNIVERSITY OF HONG KONG

DEPARTMENT OF

PHYSICS AND MATERIALS SCIENCE

BACHELOR OF SCIENCE (HONS) IN APPLIED PHYSICS 2007-2008

PROJECT REPORT

Studying the effects of alpha particles on zebrafish embryos with the help of CR-39

nuclear track detectors

by

Choi Wing Yan

March 2008

Studying the effects of alpha particles on zebrafish embryos with the help of CR-39

nuclear track detectors

by

Choi Wing Yan

Submitted in partial fulfilment of the

requirements for the degree of

BACHELOR OF SCIENCE (HONS)

IN

APPLIED PHYSICS

from

City University of Hong Kong

March 2008

Project Supervisor : Prof. Peter K.N. Yu

Table of Content

A. List of Figures i

B. List of Tables ii

C. Acknowledgments iv

D. Abstract v

1. Introduction and Objectives 1

2. Literature Review 3

2.1 Solid state nuclear track detectors (SSNTDs) 3

2.1.1 Formation of latent tracks 3

2.1.2 Formation of etched track 5

2.1.3 Shape of the alpha track 6

2.2 Zebrafish embryo 7

2.3 Logistic regression 8

3. Mehtodology 9

3.1 Preparation of CR-39 detector 9

3.2 Preparation of zebra fish embryos 9

3.2.1 Soften the chorions of the embryos 10

3.2.2 Dechorionation 10

3.3 Irradiation of the embryos by alpha particles 10

3.4 Recording the positions of embryos and alpha-particle hits 12

3.5 Locating the effective irradiated area of the embryo 13

3.6 Calculation of the absorbed dose 15

3.6.1 Calculation of the average energy absorbed by the cells 15

3.6.2 Calculation of the mass of cells 17

3.6.3 Counting the alpha-particle tracks 18

4. Results 20

4.1 Experiment with 2-min irradiation 20




4.5 Results from the logistic regression model 28

5. Discussion 32

5.1 Characteristics of morphologic abnormalities 32

5.2 Relationship between time of irradiation and malformation 33

occurrence

5.3 Relationship between absorbed dose group and malformation occurrence

33

5.4 Relationship between absorbed dose and probability of malformation occurrence

35

5.5 Irradiation at 4 hpf 36

5.6 Counting the numbers of alpha-particle tracks on the detectors 37

5.7 Errors and possible improvements 38

5.7.1 Damages of the embryos during dechorionation 38

5.7.2 Finding the effective irradiated area 38

5.7.3 Determination of mutation occurrence due to radiation 39

6. Conclusions 40

7. References 41

8. Appendix 43

i

A. List of figures

Figure 1 Input for finding the range of He in water 4

Figure 2 Geometry of track development with the incident angle

normal to the detector surface

5

Figure 3 Variation of alpha particle track structure with dip angle 7

Figure 4 Plastic tray with holes on the top for holding the embryos 9

Figure 5 A top view of four embryos inside a hole and placed on top

of the CR-39 detector

11

Figure 6 A desired orientation of an embryo 11

Figure 7a 16 photos combined into one photo with visible alpha-

particle tracks

13

Figure 7b Superimposed photos of the hole with embryos and the same

hole with alpha-particle tracks

13

Figure 8 Variables describing the embryo used in calculations 14

Figure 9 Only circular tracks (corresponding to normal incidence of

alpha particles) are counted

19

Figure 10 Images of zebrafish embryos that show (from left to right)

curvature of spine, shortening of body length, pericardial

edema and micro-ophthalmia

32

Figure 11 Number of normal (blue) and malformed (red) zebrafish

embryos in different absorbed-dose groups

34

Figure 12 Effective irradiated areas of the embryos 38

ii

B. List of Tables

Table 1 Energy E (MeV) of alpha particles after travelling

different distances in water

17

Table 2 Images of zebrafish embryos at 48 hpf with 2-min

irradiation

20

Table 3 The determined number of alpha-particle tracks, absorbed

dose and equivalent dose in zebrafish embryos with 2-

min irradiation

21


irradiation

22



min irradiation

22

Table 6 Images of the zebrafish embryos at 48 hpf with 6-min

irradiation

23



min irradiation

24


irradiation (set A)

24



min irradiation (set A)

26


irradiation (set B)

27



min irradiation (set B)

28

Table 12 Grouping of absorbed doses 28

Table 13 Input of the logistic regression model, with 0 representing

no malformation and 1 representing the presence of

malformation

29

iii

Table 14 Percentage of malformation occurrence in different

ranges of absorbed doses from the 5 sets of experiments

33

Table 15 Absorbed doses for all the malformed zebrafish for

different irradiation time and their corresponding

probability of malformation occurrence calculated from

Eq. (13)

35

Table 16 Probability of malformation occurrence determined from

Eq. (13)

36

iv

C. Acknowledgement:

I would like to take this opportunity to express my deepest gratitude to my supervisor,

Professor Peter K.N. Yu for his patient guidance and support. He always gives me

valuable suggestion and comment on doing the project.

Thanks also to my tutor Miss E.H.W. Yum, she teaches me the technique on doing the

experiment and gives me lots of advice throughout this period.

I would like to thank Mr. K.S. Ng for giving me advice and sharing his opinion on

doing the logistic regression model.

Last but not least, thanks to my family and friends for giving me support.

v

Abstract

Alpha particles from inhaled radon progeny are the most common source of

irradiation of the human respiratory tract. Not only the human respiratory tract may be

affected, radon may dissolve into the blood and enter other organ through the blood

circulatory system. Radon progeny in the blood of a pregnant woman may affect the

embryo through the exchange of nutrient at the placenta. It is therefore important to

estimate the effect caused by alpha particles to the embryo by using zebrafish embryo

as a vertebrate model.

Study has been shown that alpha particle would cause morphologic abnormalities in

48 hpf zebrafish embryo. In this project, we want to further study the relationship

between occurrence of malformation and the absorbed dose of the zebrafish embryo.

CR-39 solid-state nuclear track detectors with a thickness of 16 μm are used as

support substrates for holding the zebrafish embryos during irradiation. It can also

help to record the alpha particle incident position so that the number of alpha particle

actually going into the embryo cells can be found, thus, it can enable the calculation

of absorbed dose of the embryo cells.

In this study, planar Am241 source with an activity of 0.1151 Ciμ and main alpha

energy equal to 5.49 MeV was employed. Five sets of experiments with irradiation

times of 2, 4, 6 and 8 min were conducted. Observations were made when the

irradiated embryos were developed to 48 hpf to determine whether malformation had

occured or not.

Among 112 irradiated zebrafish embryos, 21 of them showed morphologic

abnormalities. The smallest value of absorbed dose that caused malformation in this

study was 0.51 mGy and the largest value was 2.2 mGy. These correspond to 5.17 and

22 mSv equivalent doses in human, respectively.

1

1. Introduction and objectives:

Radon is an element that comes from geological materials such as rocks and soil. It is

present in air and is radioactive. A number of research works have been conducted to

investigate the biological effects of radon on human [1]. It shows that radon present

in air can cause health hazard to human, especially lung cancer [2].

In fact, radon gas itself does not adversely affect humans; most of the radon gas

inhaled would be exhaled directly through the respiratory system. The main cause of

biological effect is radon progeny, which is the daughter of radon due to a chain of

decay. Some radon progeny are alpha emitters. Since alpha particles have strong

ionizing power, they have larger ability to cause double strand breaks of DNA when

compared with beta particles and gamma rays, so alpha particles have more important

radiological consequence [3]. Radon progeny that breathed into the respiratory track

can diffuse into the blood stream and irradiate the organs in the body [4]. The main

objective of this project is to study effects on a human embryo if a pregnant woman

breathes in air that contains radon and if the human embryo is irradiated by alpha

particles emitted from radon progeny.

By the use of zebrafish, Danio rerio, a small vertebrate species from Southeast Asia,

we can study the biological effects caused by alpha particles on the human embryo

with different absorbed doses. Since the zebrafish has a relatively close genetic

relationship with human [5], studying the effects on the zebrafish can enable us to

assess the effects of alpha particles on the human embryo.

Previous study has shown that alpha-particle irradiation at 4 hours post fertilization

(hpf) of zebrafish embryos with absorbed dose ranged from 0.41 to 2.3mGy would

2

cause morphologic abnormalities’ development when examined at 48 hpf [6]. We

want to further investigate the relationship between the absorbed dose and the

malformation of zebrafish embryos by increasing the time of irradiation to the

embryos and also the probability of malformation occurrence.

CR-39 films with a thickness of 16 μm were prepared by chemical etching. During

alpha particle irradiation of the zebrafish embryos, the amount of absorbed dose will

be controlled by the time of irradiation. CR-39 detector will be used as support

substrates for holding the embryos. Invisible alpha particles passing through the

CR-39 detector would leave some tracks on the detector. Through a chemical etching

process, the tracks are enlarged, so we can directly record the position of the cell

being hit by alpha particles and the dose absorbed by the embryo cells can be

calculated.

3

2. Literature Review:

2.1 Solid state nuclear track detectors (SSNTDs)

Solid state nuclear track detectors (SSNTDs) were first discovered by D.A.Young in

the year 1985. A numbers of etch pits, called “tracks”, were accidentally found

when a LiF crystal was placed in contact with a uranium foil and irradiated with slow

neutrons followed by a treatment with chemical reagent [7]. This finding begins the

investigation of solid state nuclear track detectors.

Recently, the most commonly used SSNTDs are CR-39 (poly allyl diglycol carbonate)

and LR 115 (cellulose nitrate) detectors [7]. Both types of detectors are important for

recording alpha-particle tracks. In this project, CR-39 detector will be used. When an

alpha particle irradiates a SSNTD, it leaves a submicroscopic damage along the

alpha-particle trajectory; this damaged zone is called the latent track, which is too

small to be observed. In order to record the position of the track under the optical

microscope, an etching process is needed to fix and enlarge the damaged zone [8].

2.1.1 Formation of latent tracks

Alpha particle is a heavy charged particle. When it passes through a medium, it will

cause extensive ionization of the material. The initial charged particle loses its energy

through many small interactions with the electrons in the medium due to the Coulomb

force. Since a heavy charged particle is much heavier than the electrons, during the

interaction, the direction of the particle does not change a lot; as a result the path of

the alpha particle is effectively a straight line.

There is a physical quantity that describes the slowing down of the charged particles

in matter, called the stopping power –dE/dx, where dE is the energy lost in a distance

4

dx. When we take the quantum effects and the relativistic effects into account, the

equation is called Bethe-Bloch expression and is given below:

⎥⎥⎦

⎤

⎢⎢⎣

⎡−−−

−=− U

I

WvmN

vmeZ

dxdE δβ

βπε2

22max

20

20

20

42

2)1(

2ln

4-------------------------------(1)

where Z is the charge of the incident particle, v is velocity of the incident particle,

β=v/c in which c is speed of light, 0m is the rest mass of the electron, N is the number

of electrons per unit volume, I is the average excitation potential of electrons in the

stopping material, maxW is the maximal value of transferred energy of electron, δ is

the correction for polarization of the material and U is non-participation of inner

electrons in the collision [8].

To make the calculations of stopping powers and ranges of charged particles simple,

the computer software called SRIM (Stopping and Range of Ions in Matter) is

developed [9]. In this program, the properties of incident ions and targeted medium

can be input (see Figure 1), and the stopping powers at different energies are output.

Figure 1: Input for finding the range of He in water.

5

2.1.2 Formation of etched track

After a SSNTD is irradiated by an alpha particle, a latent track will be formed as

explained in the above section. The detector will then be exposed to a chemically

aggressive solution called etchant. Aqueous solutions of NaOH or KOH are the most

frequently used etchants [8]. The track development is shown in Figure 2.

Figure 2: Geometry of track development with the incident angle normal to the

detector surface.

In Figure 2, I is the initial detector surface, I’ is the surface after etching, tV is the

etch rate along the particle trajectory which is called the track etch rate, bV is the etch

rate of the undamaged regions of the detector called the bulk etch rate, O represents

the entrance point of the ion and E is the end point of the ion in the detector material,

and the length of OE represents the range of the ion in the detector.

Generally, the bulk etch rate bV only depends on the detector’s material for a given

etchant applied under a specific set of etching conditions, so bV is a constant. The

6

track etch rate tV will depend on the amount of damage located in the track core

region in addition to the etching condition and the materials, so it is in general a

variable in the real case. The track development is governed by the V function which

is the ratio of tV and bV . To form a track, the track etch velocity must be greater than

the bulk etch velocity (i.e., V>1).

2.1.3 Shape of the alpha track

The shape of the alpha track varies with the alpha particle incident angle with respect

to the detector surface, which is called the dip angle. In the above case for normal

incidence, the dip angle is 90 degrees; the alpha particle track opening should be a

circle when viewed from the top. Figure 3 shows the structure of alpha particle track

at different dip angles at the vertical section containing the major axis (i.e., the track

profile) as well as at the detector surface when viewed from above (i.e., the track

opening). These structures were determined using the TRACK_TEST software

available on the website: http://www.cityu.edu.hk/ap/nru/vision.htm

7

Figure 3: Variation of alpha particle track structure with dip angle.

From Figure 3, the track opening becomes elliptical when the dip angle is not o90 . At

smaller dip angles, the track opening becomes shorter than before and becomes a

tear-drop appearance [10].

2.2 Zebrafish embryo

The zebrafish, Danio rerio, has recently become a powerful model to study human

diseases. It is a small vertebrate species from Southeast Asia, however, unlike other

vertebrate species, zebrafish is rapidly and prolifically bred. It is easily maintained in

the laboratory and the unique property of its optically transparent embryos can

facilitate direct observation of the effect of ionizing radiation to the cells [11]. Most

importantly, human and zebrafish genomes share considerable homology including

8

conservation of most DNA repair related genes. This is the main reason why zebrafish

has become a popular animal model system to study gene functions during the

embryonic development [12].

2.3 Logistic regression

Logistic regression is a statistical data analysis method which can be used to study the

relationship between the response variable and one or more explanatory variables. It

can be a biologically suitable model to describe the relationship between the outcome

variable and independent variable. The logistic regression model is different from a

linear regression model in which the outcome variable in the logistic regression model

is binary or dichotomous [13]. The outcome variable is dichotomous means that it has

only two values.

In this project, we want to estimate the relationship between the probability of

mutation occurrence and the absorbed dose. Therefore, the mutation occurrence

probability is the dependent variable while the absorbed dose is the independent

variable. For one independent variable, the probability of an event can be written as

[14]

Prob (event) = ( )XBBe 1011

+−+ ------------------------------------------------(2)

Where 0B and 1B are the estimated regression coefficients. By using the statistical

software called SPSS for Windows, Rel. 15.0.0. 2006 Chicago: SPSS Inc., we can

estimate the regression coefficients and calculate the probability of mutation

occurrence.

9

3. Methodology:

3.1 Preparation of CR-39 detector

The original thickness of CR-39 detectors obtained from the manufacture, Pershore,

was around 100 μm and the dimensions were 30×47cm. They were first cut into a

size of 28.18.1 cm× and then etched to 16 μm thick by using 0.25 M sodium

hydroxide in ethanol. During the etching process, the CR-39 detectors were rinsed by

distilled water once every two hours.

After the etching, the thickness of the CR-39 detectors was measured using a

micrometer with an accuracy of mμ1± . These were glued by epoxy onto the bottom

of a rectangular plastic tray with 8×6 holes on it (see Figure 4).

Figure 4: Plastic tray with holes on the top for holding the embryos.

The CR-39 detectors were attached onto the bottom of the holes, and were used as a

base for holding the zebrafish embryos. Five embryos were placed on top of the

CR-39 detector inside each hole for irradiation.

3.2 Preparation of zebra fish embryos

About 100 newly born zebrafish embryos were obtained. There is a thick chorion

outside the embryo to protect it from damages, which will absorb a significant

fraction of the alpha-particle energy. In order to avoid the loss of energy of alpha

10

particles during their passage through the chorion, we need to remove the chorion

before irradiation.

3.2.1 Soften the chorions of the embryos

The zebrafish embryos were dried by removing all the water with a dropper. A

mixture of 2400 lμ E3 (5mM NaCl, 0.17mM KCl, 0.33mM CaC 2l , 0.33mM 4MgSO ,

0.1% Methylene blue) and 100 lμ enzyme called pronase were added to the dried

embryos and left for about two minutes. After two minutes, they were placed under an

optical microscope to see whether the enzyme had sufficiently digested the embryo. If

so, they were transferred into a beaker and washed by water for at least 3 times.

3.2.2 Dechorionation

After the chorions were softened by the enzyme and washed thoroughly, the embryos

were transferred to a petri dish, which had a layer of agar gel on top of it, and some

E3 was added into the dish. A pair of sharp forceps was used to remove the chorion of

the embryo under the optical microscope. During the dechorionation, we should avoid

touching the embryo by the forceps since this would damage the cells of the embryo.

About 100 dechorioned zebrafish embryos were placed into an incubator to allow

development to 4 hours post fertilization (hpf).

3.3 Irradiation of the embryos by alpha particles

The 4 hpf zebrafish embryos were spherical in shape. They were transferred into the

plastic tray, as shown in Figure 4. Around 40 embryos were put into 10 holes, each

containing 4 embryos. Twenty of these embryos were used as control (i.e., without

irradiation). Figure 5 shows 4 embryos in a hole on top of the CR-39 detector.

11

Figure 5: A top view of four embryos inside a hole and placed on top of the

CR-39 detector.

The embryos were moved slightly so that the cells of the embryo were oriented

towards the lower part of the hole. After that, 1.5% methyl cellulose was added into

each hole to prevent the embryos from changing to another orientation. Figure 6

illustrates a desired the orientation of the embryo.

Figure 6: A desired orientation of an embryo.

The embryos were irradiated by an alpha source at the bottom for 2 minutes. The

alpha source employed in this study was a planar Am241 source with an activity of

0.1151 Ciμ (main alpha energy = 5.49 MeV under vacuum). The alpha particles

passed through the detectors and the fluid around the embryo before reaching the cells,

and will thus lose their energies to the detector and the fluid. The energy loss can be

calculated by using a program called Stopping and Range of Ions in Matter (SRIM).

12

Calculations of the energy loss will be shown in section 3.6.1.

3.4 Recording the positions of embryos and alpha-particle hits

Photos of the irradiated embryos were taken immediately after irradiation to record

the positions of cells of the embryos. The diameter (in mm) of the embryo was found

using the software called Spot Advanced Version 4.6, Diagnostic Instruments Inc. The

unit for the diameter will be transformed to pixels by using a program called Image J

obtained from the website http://rsb.info.nih.gov/ij/download.html, so that we can find

the effective area of the cells being actually irradiated. This will be shown in section

3.5.

The embryos were then labeled and transferred separately to another container which

was placed into the incubator until the embryos developed into 48 hpf. The 48 hpf

zebrafish embryos were observed under the microscope, and photos were taken to

determine whether any malformations had occurred in the irradiated embryos.

The detectors which had been irradiated by the alpha particles were etched in 6.25 M

sodium hydroxide in water at 70 °C for 3 h. After the etching process, tracks visible

under the microscope were formed. A total of 16 photos were taken for each hole at

different parts with a magnification 500x. At this magnification, the shape of the

visible tracks can be observed clearly. The 16 photos were then combined into one

photo as shown in Figure 8a below. The photo of the hole with tracks (Figure 7a) was

combined with the corresponding photos of the hole with embryos (Figure 5) to form

Figure 7b. The position of the cells being hit by alpha particles can be located in this

way.

13

Figure 7a: 16 photos combined into Figure 7b: Superimposed photos of the

one photo with visible alpha-particles hole with embryos and the same hole

tracks. with alpha-particle tracks.

3.5 Locating the effective irradiated area of the embryo

Before the alpha particles reach the cell, they needed to pass through the detector and

the fluid. During this travelling, the alpha particles lose energy continuously, therefore

not all the alpha particles can reach the cell. We defined an effective irradiated area as

the region of which that alpha particle can reach the cells. We assume the embryo is a

sphere, therefore the effective projected area is a circular area. Outside this region, all

energies of the incident alpha particles were lost to the fluid. The radius of this

circular area can be calculated by Eq. (3):

22max )( wRRr −−= -----------------(3)

In this equation, maxr represents the radius of the effective area; R is the radius of the

embryo; w is the range of an 5.49 MeV alpha particle in water after passing through

the detector which is equals to 21.66 mμ as calculated in section 3.6.1. Figure 8

defines the variables used in the calculations, where y is the vertical distance from the

equator of the embryo to the boundary between the cells and the yolk which can be

expressed as

14

y= 22cellRR − --------------------------------------(4)

The red line indicates the boundary between the cells and the yolk.

Figure 8: Variables describing the embryo used in calculations.

After calculating maxr , the superimposed photos were opened in Image J, and an

outline of the embryo was drawn to find out the radius of embryo in pixel. At this

stage, the difference between maxr and radius of embryo could be calculated. Then, the

effective area was plotted by entering a negative value of the difference in the

“enlarge” function in Image J. By doing so, the effective irradiated area and the

zebrafish embryo was concentric.

For the case where all the embryo cells could be oriented towards the bottom as

shown in Figure 6, the alpha-particle tracks appearing in the effective region was

counted according to the scheme described in section 3.6.3. However, in real

situations, it was difficult to orient all embryo cells towards the bottom. In these cases,

the effective irradiated region is no longer a circular area.

15

3.6 Calculation of the absorbed dose

Since the absorbed dose =dmdE , where dE is the absorbed energy in a mass dm, we

need to determine the average energy absorbed by the cells and also the mass of the

cells.

3.6.1 Calculation of the average energy absorbed by the cells

The SRIM program was used to determine the ranges and energies of alpha particles

in air, in the detector as well as in water. The SRIM outputs for the detector and water

are given in Appendix 1 and Appendix 2 respectively.

Calculation of the range of alpha particles with E= 5.49 MeV

We define detx is the range of an alpha particle in the detector with energy 5.49 MeV.

Using the SRIM outputs and interpolation, we have

)23.33()49.55.5(

)77.2823.33()55.5(

detx−−

=−−

3.725-0.112 detx =0.01

detx =33.17 mμ

Therefore, an alpha particle with an energy of 5.49 MeV can travel 33.17 mμ in the

detector.Range of alpha particle after passing through a 16 mμ detector: 33.17-16 =

17.17 mμ

Calculation of energy of an alpha particle that has a range of 17.17 mμ in the

detector

We define detE as the energy of alpha particle with a range in the detector as

17.17 mμ , which is obtained by

)17.1723.17()5.3(

)58.1523.17()25.35.3( det

−−

=−− E

06.05.3

65.125.0 detE−

=

detE =3.49Mev

16

Therefore, after traveling through the detector, the energy of the alpha particle

becomes 3.49 MeV.

Range of alpha particles with energy 3.49 MeV in water

We define w as the range in water of alpha particles after passing through the detector

(i.e., with an energy of 3.49 MeV), which is determined through

)74.21()49.35.3(

)65.1974.21()25.35.3(

w−−

=−−

w−=

74.2101.0

09.225.0

w =21.66 mμ

Therefore, the range in water of the alpha particles after traveling through the detector

was 21.66 mμ .

Calculation of average energy of alpha particles incident on the cells:

The energy of alpha particles incident on the cells varied due to the difference in the

height of water column they traveled. In the extreme case where an alpha particle

immediately reaches the cells after passing through the detector, the energy absorbed

by the cells was 3.49 MeV. In another extreme case where an alpha particle reaches

the rim of the effective irradiated area, the energy absorbed by the cells was zero by

definition. To take into account the variations of the absorbed energy at different

positions, an average absorbed energy is needed for convenience. The average energy

was calculated as shown in Eq. (5):

aE = 2max

0

/2max

rrdrEr

ππ⎥⎥⎦

⎤

⎢⎢⎣

⎡∫ --------(5)

where E is the residual energy of an alpha particle after passing through a water

column at a radial distance r from the contact point between the embryo and the

detector. The values of E of alpha particles after travelling different water distances

are shown in Table 1.

17

Table 1: Energy E (MeV) of alpha particles after travelling different distances in

water.

Distance of the alpha particles travelled in the water column (μm)

Energy E (MeV) remaining after travelling the corresponding distance

1.0 3.373.0 3.135.0 2.877.0 2.599.0 2.3011 1.9913 1.6415 1.2517 0.8119 0.3521 0.043

3.6.2 Calculation of the mass of cells

Since the size of each zebrafish embryo was not exactly the same, the mass of the

embryo cells was also not the same. To find the mass of the cells, we needed to first

determine the volume of the cells. The variables used in the calculations have been

described in Figure 8.

Assuming that the embryo is a sphere, then volume of cells is calculated through the

volume of the hemisphere which can be expressed as shown in Eqs. (6) to (8):

Volume of hemisphere: R

rrR0

32

3 ⎥⎦

⎤⎢⎣

⎡−π = 3

33

32

3RRR ππ =⎥

⎦

⎤⎢⎣

⎡− ------------------------(6)

Volume of the shaded region: y

rrR0

32

3 ⎥⎦

⎤⎢⎣

⎡−π = ⎥

⎦

⎤⎢⎣

⎡−

3

32 yyRπ ------------------------(7)

18

Volume of the cells = Volume of the hemisphere – volume of the shaded region

= ⎥⎦

⎤⎢⎣

⎡+−

332 3

23 yyRRπ --------------------------------------(8)

The mass of cells being irradiated= Volume of cells × density

= ⎥⎦

⎤⎢⎣

⎡+−

332 3

23 yyRRπ ρ ---------------------------(9)

Now that we have the average absorbed energy aE and the mass of cells being

irradiated, we can calculate the absorbed dose as

Absorbed dose = cellsofmasstracksofnoEa

.×

-------------------------------(10)

By calculating the absorbed dose, equivalent dose in zebrafish can be calculated by

Eq. (11) as

Equivalent dose = absorbed dose radiation factor --------------------------(11)

Since human has a 2-fold larger genome than zebrafish [14], equivalent dose in

human can be calculated Eq. (12).

Equivalent dose in human = 2

zebrafishindoseEquivalent -------------------------(12)

3.6.3 Counting the alpha-particle tracks

The number of alpha particles incident onto the cells was found by counting the

number of alpha-particle tracks within the cell region from the superimposed photo as

shown in Fig 7(b). It is remarked that only tracks with circular openings (those

corresponding to normal incidence of alpha particles) were counted.

19

Fig. 9: Only circular tracks (corresponding to normal incidence of alpha

particles) are counted.

20

4. Results:

The experiments had been repeated for 4 different irradiation periods, which were 2, 4,

6, and 8 min. For the experiment with 8-min irradiation, 2 sets of experiments had

been performed. Therefore, a total of 5 sets of results were obtained, with 112

embryos irradiated. The absorbed dose for 61 of those embryos could be obtained.

4.1 Experiment with 2-min irradiation

Table 2: Images of zebrafish embryos at 48 hpf with 2-min irradiation

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

21

The highlighted are the malformed zebrafish embryos.

Table 3: The determined number of alpha-particle tracks, absorbed dose and

equivalent dose in zebrafish embryos with 2-min irradiation.

Labeled number

Number of ion tracks

Absorbed dose(mGy)

Equivalent dose in zebrafish(mSv)

16 75 0.540 10.8 17 82 0.640 12.8 18 40 0.317 6.34 23 143 1.77 35.4 24 152 1.60 32.0 25 59 0.691 13.8 26 108 0.587 11.7 27 156 1.36 27.2

19 20 21 22 23 24

25 26 27

22


Table 4: Images of zebrafish embryos at 48 hpf with 4-min irradiation.

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16



Labeled zebrafish number


Absorbed dose(mGy)


2 234 1.38 27.6 3 293 1.84 36.0 4 80 0.729 14.6 5 92 0.480 9.60

23

6 46 0.419 8.38 7 240 0.366 7.32 8 77 0.456 9.12 9 55 0.517 10.3 10 70 0.646 12.9 11 77 0.697 13.9


Table 6: Images of the zebrafish embryos at 48 hpf with 6-min irradiation

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

24

19 20





Absorbed dose(mGy)


15 353 1.71 34.0 16 128 0.940 18.8 17 126 1.35 27.0 18 67 0.518 10.4 19 349 2.15 43.0


Table 8: Images of zebrafish embryos at 48 hpf with 8-min irradiation (set A)

1 2 3 4 5 6

25

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 23 24

25 26 27 28

26


equivalent dose in zebrafish embryos with 8-min irradiation (set A).



Absorbed dose(mGy)


1 112 0.890 17.8 2 197 1.90 38.0 3 211 1.40 28.0 4 73 0.870 17.4 5 226 1.63 32.6 6 42 0.560 11.2 7 132 1.24 24.8 8 147 1.76 35.2 13 97 1.03 20.6 14 24 0.230 4.60 15 43 0.250 5.00 16 131 1.11 22.2 17 132 1.67 33.4 18 175 1.40 28.0 19 37 0.420 8.40 20 132 0.840 16.8 21 172 1.60 32.0 22 123 2.20 44.0 23 218 1.57 31.4 24 176 1.90 38.0 25 95 0.920 18.4 26 167 1.59 31.8 27 172 1.23 24.6 28 107 0.85 17.0

27

Table 10: Images of zebrafish embryos at 48 hpf with 8-min irradiation (set B).

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21

28


equivalent dose in zebrafish embryos with 8-min irradiation (set B).



Absorbed dose(mGy)


1 80 0.770 15.4 2 74 1.30 26.0 3 56 0.480 9.60 7 32 0.240 4.80 8 72 0.490 9.80 13 115 2.0 40.0 14 84 0.770 15.4 15 108 1.78 35.6 16 38 0.580 11.6 17 101 1.23 24.6 18 117 0.646 12.9 19 46 0.250 5.08 20 222 1.56 31.2 21 113 0.586 11.7

4.5 Results from the logistic regression model

From the absorbed dose values calculated above, we can study the relationship

between the absorbed dose and the probability of mutation occurrence by using the

logistic regression model. Before the analysis, the absorbed doses were arranged into

different absorbed dose groups as shown in Table 12.

Table 12: Grouping of absorbed doses.

absorbed dose group 1: 0 to 0.5 mGy absorbed dose group 2: >0.5 to 1.0 mGy absorbed dose group3:>1.0 to 1.5 mGy absorbed dose group 4: >1.5 to 2.0 mGy absorbed dose group 5: >2.0 to 2.5 mGy

During the analysis, the absorbed dose groups were used instead of the actual

29

absorbed doses. Table 13 shows the experimental data expressed in absorbed dose

groups, with 0 representing no malformation of the zebrafish embryos and 1

representing the presence of malformation. The data in Table 13 were input into the

logistic regression model using the SPSS 15.0 software. The results of the logistic

regression model are shown in Appendix 3.

Table 13: Input of the logistic regression model, with 0 representing no

malformation and 1 representing the presence of malformation

Identifier variable

Absorbed dose Group Absorbed dose ( mGy )

malformation

1 1 0.240 0 2 1 0.250 0 3 1 0.317 0 4 1 0.366 0 5 1 0.419 0 6 1 0.456 0 7 1 0.480 0 8 1 0.480 0 9 1 0.490 0

10 1 0.230 0 11 1 0.250 0 12 1 0.420 0 13 2 0.518 0 14 2 0.540 0 15 2 0.580 0 16 2 0.586 0 17 2 0.587 0 18 2 0.697 0 19 2 0.770 0 20 2 0.770 0 21 2 0.940 0 22 2 0.517 1 23 2 0.640 1 24 2 0.646 1 25 2 0.646 1

30

26 2 0.691 1 27 2 0.729 1 28 2 0.560 1 29 2 0.840 0 30 2 0.850 0 31 2 0.870 0 32 2 0.890 0 33 2 0.920 0 34 3 1.23 0 35 3 1.30 0 36 3 1.35 0 37 3 1.36 1 38 3 1.38 1 39 3 1.11 1 40 3 1.03 0 41 3 1.23 0 42 3 1.24 0 43 3 1.40 0 44 3 1.40 0 45 4 1.60 0 46 4 1.77 0 47 4 1.78 0 48 4 1.84 0 49 4 1.56 1 50 4 1.71 1 51 4 1.57 0 52 4 1.59 0 53 4 1.60 0 54 4 1.63 1 55 4 1.67 0 56 4 1.76 0 57 4 1.90 0 58 4 1.90 0 59 5 2.00 1 60 5 2.15 1 61 5 2.20 1

31

From the results of the analysis, the regression coefficients were found as = -2.506

and = 0.534.

As described in Eq. (2), the probability of an event can be written as

Prob (event) = ( )XBBe 1011

+−+ ----------------------------------(2)

The probability of mutation as derived from the present experiments can therefore be

written as

Prob (mutation) = ( )De 534.0506.211

+−−+ ---------------------------------(13)

where D represents the absorbed dose.

With Eq. (13), we can calculate the absorbed dose that is expected to cause mutation

in 50% of the zebrafish embryos, i.e.,

)534.0506.2(115.0 De +−−+

=

D = 4.69 mGy.

32

5. Discussion:

5.1 Characteristics of morphologic abnormalities

Five sets of experiments have been performed to investigate the relationship between

the absorbed dose and the probability of mutation occurrence. In each set of

experiments, at least 2 zebrafish embryos showed morphologic abnormalities. Those

morphologic abnormalities were characterized by any one or combinations of the

curvature of the spine, shortening of the overall length of the body, pericardial edema,

micro-ophthalmia and microcephaly. Images of such abnormalities are shown in Fig

10. The morphologic abnormalities observed in our experiments using alpha particles

as a source of radiation agreed with those researches by using other ionizing radiation

as a source of radiation, e.g., X ray [11] and gamma ray [12].

Fig. 10. Images of zebrafish embryos that show (from left to right) curvature of spine, shortening of body length, pericardial edema and micro-ophthalmia.

These morphologic abnormalities are likely the results from DNA double strand

breaks. When an ionizing radiation passes through a cell, the radiation or the reactive

free radicals formed by the radiation can break the strands of the DNA. If the

damaged DNA cannot undergo successful repairing, e.g., through homologous

recombination or non-homologous end joining, cell death and mutation can occur,

with mutation possibly leading to morphologic changes.

33

5.2 Relationship between time of irradiation and malformation occurrence

In this study, we use the time of irradiation to control the amount of absorbed dose in

the zebrafish embryos. From the ranges of absorbed doses for different irradiation

time as shown in Table 14, the maximum absorbed dose increases with the irradiation

time as expected. However, the individual values of absorbed dose within each set of

experiment still had a large range, which means that the absorbed dose in each

embryo was not the same even for a specific irradiation time. In other words,

irradiation time can only control the maximum value of the absorbed dose.

Table 14: Percentage of malformation occurrence in different ranges of absorbed

doses from the 5 sets of experiments.

Time of irradiation (min)

Number of malformed embryos

Percentage of malformed embryos (%)

Range of absorbed doses (mGy)

2 5 in 27 embryos 18.5 0.32-1.36 4 4 in 16 embryos 25.0 0.37-1.84 6 2 in 20 embryos 10.0 0.52-2.0 8 (set A) 4 in 28 embryos 14.5 0.23-2.20 8 (set B) 6 in 21 embryos 28.6 0.24-2.15

Table 14 also shows the percentage of malformed embryos for different irradiation

time. No relationship could be drawn for the percentage and the irradiation time. This

is likely due to varying absorbed dose in the zebrafish embryos even for the same

specific irradiation time.

5.3. Relationship between absorbed dose group and malformation occurrence

The absorbed doses from the five sets of experiments were arranged into 5

absorbed-dose groups. Fig. 11 shows a bar chart with the blue data representing the

number of normal zebrafish embryos while the red data representing the number of

34

malformed zebrafish embryos.

It was interesting to observe no malformed embryos for absorbed-dose group one. In

other words, all zebrafish embryos that received less than 0.5 mGy absorbed dose

appeared to be normal at 48 hpf. In contrast, for the absorbed-dose group 5, all the 3

zebrafish embryos showed abnormal appearance.

Figure 11: Number of normal (blue) and malformed (red) zebrafish embryos in

different absorbed-dose groups.

35

5.4 Relationship between absorbed dose and probability of malformation

occurrence

Table 15: Absorbed doses for all the malformed zebrafish for different

irradiation time and their corresponding probability of malformation occurrence

calculated from Eq. (13)

Irradiation time Labeled zebrafish number

Absorbed dose (mGy)

Probability of malformation occurrence

17 0.640 0.10325 0.691 0.106

2

27 1.36 0.1442 1.38 0.1464 0.729 0.1079 0.510 0.097

4

10 0.646 0.10315 1.71 0.1696 19 2.0 0.1925 1.63 0.1636 0.560 0.09916 1.11 0.129

8(set A)

22 2.20 0.20913 2.15 0.20518 0.646 0.103

8 (set B)

20 1.56 0.158

Table 15 shows the absorbed dose for all the malformed zebrafish embryos. The

smallest absorbed dose that caused mutation was 0.51 mGy while the largest value

was 2.2 mGy. The forth column of Table 15 shows the probability of malformation

occurrence, which is calculated from Eq. (13). The probability of malformation

occurrence ranges from 0.097 to 0.209. To further investigate the relationship between

the absorbed dose and the probability of malformation occurrence, we calculated the

36

absorbed dose for different probabilities (see Table 16) by Eq. (13). In particular, the

probability of 50% malformation occurrence corresponds to an absorbed dose of 4.69

mGy, i.e., half of the zebrafish embryos receiving such an absorbed dose are expected

to have malformations. Similarly, if the absorbed dose is increased to 8.81 mGy, we

expect 90% of the zebrafish embryos receiving such an absorbed dose to have

malformations.

Table 16: Probability of malformation occurrence determined from Eq. (13)

Probability of malformation occur absorbed dose (mGy) 0.1 0.5800.2 2.100.3 3.110.4 3.930.5 4.690.6 5.450.7 6.280.8 7.290.9 8.81

5.5 Irradiation at 4 hpf

In the present experiments, 4 hpf zebrafish embryos were irradiated by alpha particles

and they were then placed into the incubator until they developed into 48 hpf. At 48

hpf, the zebrafish embryos were observed under an optical microscope to determine

whether they had malformed. The embryos were irradiated at 4 hpf because at this

time they had already developed into a spherical shape, so that we could conveniently

control the orientation of the embryos during irradiation. It was important to rotate the

embryos to orient the cells towards the detector in such a way that the incoming alpha

particles would strike the cells instead of the yolk (see Fig. 6). This can help control

the absorbed doses for the embryos. As mentioned before, increasing the irradiation

37

time can increase the maximum value of the absorbed doses.

It was also important to have all zebrafish embryos irradiated at the same

developmental stage. McAleer et al. [11] studied the lethal effect of radiation in

zebrafish embryos using X-ray, and found that gastrula stage embryos (1-2 hpf)

showed greater sensitivity to low doses of radiation than blastula stage embryos (4

hpf). In other words, the effect of radiation was dependent on the embryonic stage

during irradiation. If some embryos were irradiated at 1-2 hpf and some were

irradiated at 4 hpf, the results of the experiments might not be consistent.

5.6 Counting the numbers of alpha-particle tracks on the detectors

The number of alpha particles irradiating an embryo was the crucial factor affecting

the absorbed dose. A CR-39 detector was used to record the number of alpha particles

incident onto the embryo cells. When alpha particles pass through the detector, latent

tracks are formed, and the enlarged tracks after etching could be observed under the

optical microscope. Not only the quantity of the alpha particles could be recorded, but

also the positions of the incident particles can be located.

To get a precise number of alpha particles that have entered the cells, we need to

differentiate among alpha particles crossing the detector at different dip angles, which

can be achieved through the shape of the track openings. If the track opening was

circular, the incident alpha particle was normally incident onto the cells; in this case,

the track was counted. However if the track opening had a tear drop structure, the

incident angle was smaller than 50 degrees, and the alpha particle would leave the

detector without entering the embryo cells; in this case, the track was not counted.

38

5.7 Errors and possible improvements

5.7.1 Damages of the embryos during dechorionation

To avoid alpha-particle energy loss in the chorion, the zebrafish embryos should be

dechorionated before irradiation. However, the dechorionation may damage the

embryo cells. In this study, protease was used to soften the chorions of the embryos. If

the time of protease treatment was not controlled well, protease might diffuse into the

embryos and cause damages.

From the control experiments, we can see that some of the embryos had malformed

even under no irradiation of alpha particles. This may be due to damages of the cells

during dechorionation. A possible improvement is to remove the chorion directly by

sharp forceps without adding protease.

5.7.2 Finding the effective irradiated area

Before counting the tracks, we needed to locate the effective irradiated area within the

embryo. If not all the embryo cells were oriented towards the detector, we needed to

locate the cells. However, we could only locate the boundary of the cells from the

superimposed photos, and small deviations in the location might lead to a different

effective area and hence a different number of alpha-particle tracks (see Fig.12).

Fig. 12. Effective irradiated areas of the embryos.

39

5.7.3 Determination of mutation occurrence due to radiation

In this study, we determined whether mutation had occurred or not in the zebrafish

embryos by observing the morphologic abnormalities. However, mutation was not

only shown in the morphologic development, but also shown in the internal organs

which could not be observed directly from the microscope. Research has shown that

ionizing radiation would affect the brain development, leading to an increased cell

death in the brain of zebrafish embryos [11]. If radiation did affect the heart

development, the heart beat rate of the embryos would be changed. Therefore, focus

only on the malformation occurrence in the embryos inevitably had some limitations.

In future studies, we can further try to observe the DNA double strand breaks caused

by ionizing radiation to identify the effects on zebrafish embryos. For example, DNA

strand breaks can be detected by using the TdT-mediated dUTP Nick-end Labeling

(TUNEL) fluorescence method, where the DNA strand breaks would be stained with

colour. With increasing DNA strand breaks caused by ionizing radiation, the signal

obtained from the TUNEL method would increase.

40

6. Conclusion:

In this project, 112 zebrafish embryos were irradiated by alpha particles, 21 of which

showed morphologic abnormalities. The morphologic abnormalities were

characterized by curvature of the spine, shortening of the overall length of the body,

pericardial edema, micro-ophthalmia and microcephaly. The smallest value of

absorbed dose that caused malformation in this study was 0.51 mGy and the largest

value was 2.2 mGy. These values correspond to 5.17 and 22 mSv equivalent dose in

human. This smallest equivalent dose in human was numerically about 2 times larger

than the annual background radiation effective dose which is 2 to 3 mSv/yr.

Furthermore, an equation describing the probability of malformation occurrence can

be obtained from the logistic regression model, which is given by Prob (mutation)

= ( )De 534.0506.211

+−−+ . There was an increasing probability of malformation for

increasing absorbed dose.

41

References

[1] K.N. Yu, B.M.F. Lau, D. Nikezic, 2006. "Assessment of Environmental Radon

Hazard Using Human Respiratory Tract Models". Journal of Hazardous Materials,

132, 98-110.

[2] C. G. Collier, J. C. Strong, J. A. Humphreys, N. Timpson, S. T. Baker, T. Eldred,

L. Cobb, D. Papworth & R. Haylock, 2005. "Carcinogenicity of radon/radon

decay product inhalation in rats – effect of dose, dose rate and unattached

fraction". Int. F. Radiat. Biol., Col. 81, No. 9, pp. 631-647.

[3] K. N. Yu, “Natural radiation and radon,” AP4271/AP8271 Environmental

Radiation.

[4] F.E. Alexander, P.A. McKinney and R.A. Cartwright, 1990. "Radon and

leukaemia". The Lancet, 335: 1336-7

[5] W. B. Barbazuk, I. Korf, C. Kadavi, J. Heyen, S. Tate, E. Wun, J. A.

Bedell, J. D. McPherson, and S. L. Johnson, 2000. “The Syntenic Relationship of

the Zebrafish and Human Genomes”, Genome Research. Vol. 10, Issue 9,

1351-1358.

[6] E.H.W. Yum, C.K.M. Ng, A.C.C. Lin, S.H. Cheng, K.N. Yu, 2007. "Experimental

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Techniques for Environmental Radiation.

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[8] D. Nikezic, K.N. Yu, 2004. "Formation and Growth of Tracks in Nuclear Track

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[9] J.F. Ziegler, 2006. SRIM-2006, http://www.srim.org/

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[11] M.F. McAleer, C. Davidson, W.R. Davidson, B. Yentzer, S.A.Farber, U. Rodeck,

A.P. Dicker, 2005. “Novel use of zebrafish as a vertebrate model to

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61(2005) 10-13.

[12] G.A. Geiger, S.E. Parker, A.P. Beothy, J.A. Tucker, M.C. Mullins,G.D. Kao,

2006. Zebrafish as a ‘‘Biosensor’’? Effects of Ionizing Radiation and Amifostine

on Embryonic Viability and Development. Cancer Res. 66 (2006) 8172-8181.

[13] D.W. Hosmer & S. Lemeshow, 2000. Applied logistic regression. 2nd ed., New

York : Wiley, pp.1-30.

[14] B. G. Tabachnick, & L. S. Fidell, 2007. Using Multivariate Statistics (5th edition).

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[15] C.L. Bladen, W.K. Lam, W.S. Dynan, D.J. Kozlowski, 2005. DNA damage

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(2005) 3002-3010.

43

Appendix: 1. Range of an alpha particle in CR39 calculated from SRIM 2006 ============================================================= Calculation using SRIM-2006 SRIM version ---> SRIM-2006.02 Calc. date ---> March 24, 2008 ============================================================ Disk File Name = alpha particle in cr39 Ion = Helium [2] , Mass = 4.003 amu Target Density = 1.3200E+00 g/cm3 = 1.0724E+23 atoms/cm3 ======= Target Composition ======== Atom Atom Atomic Mass Name Numb Percent Percent ---- ---- ------- ------- C 6 032.43 052.55 H 1 048.65 006.62 O 8 018.92 040.84 ==================================== Bragg Correction = 0.00% Stopping Units = keV / micron See bottom of Table for other Stopping units Ion dE/dx dE/dx Projected Longitudinal Lateral Energy Elec. Nuclear Range Straggling Straggling ----------- ---------- ---------- ---------- ---------- ---------- 10.00 keV 5.366E+01 1.074E+01 1380 A 472 A 425 A 11.00 keV 5.631E+01 1.015E+01 1507 A 498 A 454 A 12.00 keV 5.885E+01 9.640E+00 1631 A 521 A 481 A 13.00 keV 6.130E+01 9.182E+00 1753 A 544 A 507 A 14.00 keV 6.365E+01 8.773E+00 1872 A 564 A 532 A 15.00 keV 6.592E+01 8.403E+00 1990 A 583 A 556 A 16.00 keV 6.812E+01 8.067E+00 2105 A 601 A 578 A 17.00 keV 7.025E+01 7.762E+00 2218 A 618 A 600 A 18.00 keV 7.232E+01 7.481E+00 2329 A 634 A 620 A 20.00 keV 7.629E+01 6.985E+00 2547 A 664 A 659 A

44

22.50 keV 8.096E+01 6.460E+00 2809 A 697 A 703 A 25.00 keV 8.535E+01 6.018E+00 3062 A 726 A 744 A 27.50 keV 8.949E+01 5.640E+00 3306 A 752 A 781 A 30.00 keV 9.341E+01 5.311E+00 3543 A 775 A 815 A 32.50 keV 9.712E+01 5.023E+00 3773 A 797 A 847 A 35.00 keV 1.006E+02 4.769E+00 3997 A 816 A 877 A 37.50 keV 1.040E+02 4.541E+00 4215 A 834 A 904 A 40.00 keV 1.072E+02 4.337E+00 4428 A 851 A 931 A 45.00 keV 1.131E+02 3.985E+00 4840 A 881 A 979 A 50.00 keV 1.185E+02 3.691E+00 5236 A 908 A 1022 A 55.00 keV 1.236E+02 3.442E+00 5619 A 931 A 1061 A 60.00 keV 1.284E+02 3.227E+00 5988 A 952 A 1097 A 65.00 keV 1.330E+02 3.041E+00 6347 A 971 A 1131 A 70.00 keV 1.374E+02 2.876E+00 6696 A 989 A 1162 A 80.00 keV 1.458E+02 2.600E+00 7365 A 1021 A 1217 A 90.00 keV 1.538E+02 2.377E+00 8003 A 1049 A 1266 A 100.00 keV 1.614E+02 2.192E+00 8612 A 1073 A 1310 A 110.00 keV 1.686E+02 2.037E+00 9196 A 1094 A 1349 A 120.00 keV 1.755E+02 1.903E+00 9759 A 1112 A 1384 A 130.00 keV 1.820E+02 1.788E+00 1.03 um 1129 A 1417 A 140.00 keV 1.883E+02 1.687E+00 1.08 um 1144 A 1446 A 150.00 keV 1.942E+02 1.598E+00 1.13 um 1158 A 1474 A 160.00 keV 1.998E+02 1.518E+00 1.18 um 1171 A 1500 A 170.00 keV 2.051E+02 1.447E+00 1.23 um 1183 A 1524 A 180.00 keV 2.102E+02 1.382E+00 1.28 um 1193 A 1546 A 200.00 keV 2.197E+02 1.271E+00 1.37 um 1216 A 1587 A 225.00 keV 2.303E+02 1.156E+00 1.48 um 1243 A 1632 A 250.00 keV 2.396E+02 1.062E+00 1.58 um 1267 A 1673 A 275.00 keV 2.478E+02 9.825E-01 1.69 um 1288 A 1709 A 300.00 keV 2.550E+02 9.153E-01 1.78 um 1307 A 1742 A 325.00 keV 2.613E+02 8.573E-01 1.88 um 1324 A 1772 A 350.00 keV 2.668E+02 8.067E-01 1.97 um 1340 A 1800 A 375.00 keV 2.716E+02 7.622E-01 2.07 um 1355 A 1827 A 400.00 keV 2.756E+02 7.227E-01 2.16 um 1370 A 1851 A 450.00 keV 2.819E+02 6.556E-01 2.33 um 1407 A 1897 A 500.00 keV 2.860E+02 6.006E-01 2.51 um 1442 A 1939 A 550.00 keV 2.885E+02 5.548E-01 2.68 um 1474 A 1977 A 600.00 keV 2.895E+02 5.158E-01 2.85 um 1505 A 2014 A

45

650.00 keV 2.894E+02 4.824E-01 3.03 um 1535 A 2048 A 700.00 keV 2.884E+02 4.532E-01 3.20 um 1564 A 2081 A 800.00 keV 2.843E+02 4.050E-01 3.55 um 1657 A 2145 A 900.00 keV 2.785E+02 3.666E-01 3.90 um 1748 A 2205 A 1.00 MeV 2.717E+02 3.352E-01 4.26 um 1837 A 2264 A 1.10 MeV 2.644E+02 3.091E-01 4.63 um 1926 A 2322 A 1.20 MeV 2.570E+02 2.870E-01 5.02 um 2015 A 2379 A 1.30 MeV 2.497E+02 2.680E-01 5.41 um 2106 A 2437 A 1.40 MeV 2.425E+02 2.515E-01 5.82 um 2197 A 2496 A 1.50 MeV 2.356E+02 2.370E-01 6.23 um 2289 A 2555 A 1.60 MeV 2.290E+02 2.242E-01 6.66 um 2383 A 2615 A 1.70 MeV 2.227E+02 2.128E-01 7.11 um 2479 A 2676 A 1.80 MeV 2.166E+02 2.026E-01 7.56 um 2575 A 2739 A 2.00 MeV 2.054E+02 1.850E-01 8.51 um 2930 A 2868 A 2.25 MeV 1.928E+02 1.671E-01 9.76 um 3459 A 3038 A 2.50 MeV 1.816E+02 1.525E-01 11.10 um 3972 A 3219 A 2.75 MeV 1.717E+02 1.403E-01 12.51 um 4480 A 3410 A 3.00 MeV 1.628E+02 1.301E-01 14.00 um 4986 A 3613 A 3.25 MeV 1.548E+02 1.213E-01 15.58 um 5493 A 3828 A 3.50 MeV 1.476E+02 1.137E-01 17.23 um 6004 A 4054 A 3.75 MeV 1.411E+02 1.071E-01 18.96 um 6519 A 4292 A 4.00 MeV 1.351E+02 1.012E-01 20.77 um 7040 A 4542 A 4.50 MeV 1.247E+02 9.124E-02 24.62 um 8965 A 5076 A 5.00 MeV 1.159E+02 8.317E-02 28.77 um 1.08 um 5657 A 5.50 MeV 1.083E+02 7.647E-02 33.23 um 1.26 um 6282 A 6.00 MeV 1.017E+02 7.082E-02 37.99 um 1.43 um 6952 A 6.50 MeV 9.594E+01 6.599E-02 43.05 um 1.61 um 7665 A 7.00 MeV 9.084E+01 6.181E-02 48.40 um 1.79 um 8420 A 8.00 MeV 8.220E+01 5.491E-02 59.96 um 2.44 um 1.01 um 9.00 MeV 7.636E+01 4.946E-02 72.58 um 3.03 um 1.18 um 10.00 MeV 7.059E+01 4.504E-02 86.19 um 3.61 um 1.37 um ----------------------------------------------------------- Multiply Stopping by for Stopping Units ------------------- ------------------ 1.0000E-01 eV / Angstrom 1.0000E+00 keV / micron 1.0000E+00 MeV / mm 7.5760E-03 keV / (ug/cm2)

46

7.5760E-03 MeV / (mg/cm2) 7.5760E+00 keV / (mg/cm2) 9.3251E-02 eV / (1E15 atoms/cm2) 8.0481E-03 L.S.S. reduced units ================================================================== (C) 1984,1989,1992,1998,2006 by J.P. Biersack and J.F. Ziegler

47

2. Range of an alpha particle in water calculated from SRIM 2006

====================================================== Calculation using SRIM-2006 SRIM version ---> SRIM-2006.02 Calc. date ---> March 24, 2008 ============================================================ Disk File Name = range of alpha in water Ion = Helium [2] , Mass = 4.003 amu Target Density = 1.0000E+00 g/cm3 = 1.0029E+23 atoms/cm3 ======= Target Composition ======== Atom Atom Atomic Mass Name Numb Percent Percent ---- ---- ------- ------- H 1 066.67 011.19 O 8 033.33 088.81 ==================================== Bragg Correction = 2.00% Stopping Units = keV / micron See bottom of Table for other Stopping units Ion dE/dx dE/dx Projected Longitudinal Lateral Energy Elec. Nuclear Range Straggling Straggling ----------- ---------- ---------- ---------- ---------- ---------- 10.00 keV 3.498E+01 9.046E+00 1895 A 675 A 602 A 11.00 keV 3.674E+01 8.553E+00 2077 A 716 A 647 A 12.00 keV 3.842E+01 8.119E+00 2255 A 754 A 689 A 13.00 keV 4.004E+01 7.734E+00 2431 A 790 A 730 A 14.00 keV 4.161E+01 7.388E+00 2604 A 823 A 769 A 15.00 keV 4.312E+01 7.076E+00 2774 A 855 A 806 A 16.00 keV 4.458E+01 6.794E+00 2942 A 884 A 842 A 17.00 keV 4.601E+01 6.536E+00 3107 A 912 A 876 A 18.00 keV 4.739E+01 6.300E+00 3269 A 938 A 909 A 20.00 keV 5.006E+01 5.881E+00 3586 A 988 A 970 A 22.50 keV 5.323E+01 5.440E+00 3970 A 1042 A 1041 A 25.00 keV 5.624E+01 5.067E+00 4341 A 1091 A 1107 A 27.50 keV 5.912E+01 4.749E+00 4700 A 1135 A 1167 A 30.00 keV 6.188E+01 4.472E+00 5047 A 1174 A 1222 A 32.50 keV 6.454E+01 4.230E+00 5385 A 1210 A 1274 A 35.00 keV 6.710E+01 4.015E+00 5712 A 1243 A 1322 A

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37.50 keV 6.959E+01 3.824E+00 6031 A 1273 A 1367 A 40.00 keV 7.200E+01 3.652E+00 6342 A 1300 A 1409 A 45.00 keV 7.662E+01 3.355E+00 6941 A 1351 A 1487 A 50.00 keV 8.101E+01 3.108E+00 7513 A 1394 A 1556 A 55.00 keV 8.520E+01 2.898E+00 8062 A 1433 A 1619 A 60.00 keV 8.922E+01 2.718E+00 8589 A 1467 A 1676 A 65.00 keV 9.309E+01 2.560E+00 9097 A 1497 A 1728 A 70.00 keV 9.682E+01 2.422E+00 9588 A 1524 A 1776 A 80.00 keV 1.039E+02 2.190E+00 1.05 um 1574 A 1862 A 90.00 keV 1.106E+02 2.002E+00 1.14 um 1616 A 1936 A 100.00 keV 1.169E+02 1.846E+00 1.22 um 1652 A 2002 A 110.00 keV 1.229E+02 1.715E+00 1.30 um 1683 A 2060 A 120.00 keV 1.287E+02 1.603E+00 1.38 um 1710 A 2113 A 130.00 keV 1.341E+02 1.506E+00 1.45 um 1735 A 2160 A 140.00 keV 1.394E+02 1.421E+00 1.53 um 1756 A 2204 A 150.00 keV 1.444E+02 1.346E+00 1.59 um 1776 A 2244 A 160.00 keV 1.492E+02 1.279E+00 1.66 um 1794 A 2280 A 170.00 keV 1.538E+02 1.219E+00 1.72 um 1810 A 2315 A 180.00 keV 1.582E+02 1.164E+00 1.79 um 1825 A 2346 A 200.00 keV 1.666E+02 1.070E+00 1.91 um 1856 A 2404 A 225.00 keV 1.761E+02 9.737E-01 2.05 um 1891 A 2467 A 250.00 keV 1.847E+02 8.942E-01 2.19 um 1921 A 2522 A 275.00 keV 1.924E+02 8.277E-01 2.32 um 1948 A 2571 A 300.00 keV 1.994E+02 7.710E-01 2.44 um 1971 A 2615 A 325.00 keV 2.056E+02 7.222E-01 2.57 um 1993 A 2655 A 350.00 keV 2.111E+02 6.796E-01 2.68 um 2012 A 2692 A 375.00 keV 2.159E+02 6.421E-01 2.80 um 2030 A 2726 A 400.00 keV 2.201E+02 6.088E-01 2.91 um 2047 A 2758 A 450.00 keV 2.269E+02 5.523E-01 3.14 um 2089 A 2816 A 500.00 keV 2.317E+02 5.060E-01 3.35 um 2128 A 2868 A 550.00 keV 2.347E+02 4.674E-01 3.57 um 2164 A 2915 A 600.00 keV 2.364E+02 4.346E-01 3.78 um 2198 A 2960 A 650.00 keV 2.369E+02 4.064E-01 3.99 um 2230 A 3002 A 700.00 keV 2.365E+02 3.819E-01 4.20 um 2262 A 3042 A 800.00 keV 2.335E+02 3.412E-01 4.62 um 2362 A 3117 A 900.00 keV 2.286E+02 3.089E-01 5.05 um 2459 A 3189 A 1.00 MeV 2.227E+02 2.824E-01 5.49 um 2556 A 3258 A 1.10 MeV 2.163E+02 2.604E-01 5.95 um 2654 A 3326 A

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1.20 MeV 2.096E+02 2.418E-01 6.42 um 2753 A 3394 A 1.30 MeV 2.031E+02 2.258E-01 6.90 um 2855 A 3462 A 1.40 MeV 1.967E+02 2.119E-01 7.40 um 2958 A 3531 A 1.50 MeV 1.906E+02 1.997E-01 7.91 um 3065 A 3601 A 1.60 MeV 1.847E+02 1.890E-01 8.45 um 3174 A 3673 A 1.70 MeV 1.792E+02 1.794E-01 8.99 um 3285 A 3745 A 1.80 MeV 1.739E+02 1.707E-01 9.56 um 3400 A 3820 A 2.00 MeV 1.643E+02 1.559E-01 10.74 um 3823 A 3975 A 2.25 MeV 1.536E+02 1.408E-01 12.31 um 4464 A 4181 A 2.50 MeV 1.442E+02 1.285E-01 13.99 um 5097 A 4401 A 2.75 MeV 1.360E+02 1.183E-01 15.77 um 5728 A 4637 A 3.00 MeV 1.287E+02 1.096E-01 17.66 um 6362 A 4889 A 3.25 MeV 1.223E+02 1.022E-01 19.65 um 7001 A 5157 A 3.50 MeV 1.164E+02 9.583E-02 21.74 um 7646 A 5441 A 3.75 MeV 1.112E+02 9.022E-02 23.94 um 8298 A 5742 A 4.00 MeV 1.065E+02 8.526E-02 26.23 um 8959 A 6059 A 4.50 MeV 9.824E+01 7.689E-02 31.11 um 1.14 um 6742 A 5.00 MeV 9.130E+01 7.009E-02 36.39 um 1.37 um 7487 A 5.50 MeV 8.536E+01 6.445E-02 42.05 um 1.60 um 8294 A 6.00 MeV 8.023E+01 5.969E-02 48.08 um 1.82 um 9160 A 6.50 MeV 7.573E+01 5.562E-02 54.49 um 2.04 um 1.01 um 7.00 MeV 7.176E+01 5.209E-02 61.27 um 2.27 um 1.11 um 8.00 MeV 6.505E+01 4.628E-02 75.89 um 3.09 um 1.32 um 9.00 MeV 6.079E+01 4.169E-02 91.78 um 3.84 um 1.55 um 10.00 MeV 5.641E+01 3.796E-02 108.85 um 4.55 um 1.80 um ----------------------------------------------------------- Multiply Stopping by for Stopping Units ------------------- ------------------ 1.0000E-01 eV / Angstrom 1.0000E+00 keV / micron 1.0000E+00 MeV / mm 1.0000E-02 keV / (ug/cm2) 1.0000E-02 MeV / (mg/cm2) 1.0000E+01 keV / (mg/cm2) 9.9709E-02 eV / (1E15 atoms/cm2) 8.6356E-03 L.S.S. reduced units ============================================================= (C) 1984,1989,1992,1998,2006 by J.P. Biersack and J.F. Ziegler

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3. Logistic regression output from SPSS for Windows (15.0) Logistic Regression [DataSet0]

Case Processing Summary

Unweighted Cases(a) N Percent

Included in Analysis 61 100.0

Missing Cases 0 .0

Selected Cases

Total 61 100.0

Unselected Cases 0 .0

Total 61 100.0

a If weight is in effect, see classification table for the total number of cases.

Dependent Variable Encoding

Original Value Internal Value

not mutated 0

mutated 1

Block 0: Beginning Block Classification Table(a,b)

Predicted

mutation

Percentage

Correct

Observed not mutated mutated not mutated

not mutated 45 0 100.0 mutation

mutated 16 0 .0

Step 0

Overall Percentage 73.8

a Constant is included in the model.

b The cut value is .500

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Variables in the Equation

B S.E. Wald df Sig. Exp(B)

Step 0 Constant -1.034 .291 12.621 1 .000 .356

Variables not in the Equation

Score df Sig.

Variables DoseGp 4.465 1 .035 Step 0

Overall Statistics 4.465 1 .035

Block 1: Method = Enter Omnibus Tests of Model Coefficients

Chi-square df Sig.

Step 4.483 1 .034

Block 4.483 1 .034

Step 1

Model 4.483 1 .034

Classification Table(a)

Predicted

mutation

Percentage

Correct

Observed not mutated mutated not mutated

not mutated 45 0 100.0 mutation

mutated 13 3 18.8

Step 1

Overall Percentage 78.7

a The cut value is .500

Variables in the Equation

B S.E. Wald df Sig. Exp(B)

DoseGp .534 .261 4.182 1 .041 1.705 Step

1(a) Constant -2.506 .818 9.382 1 .002 .082

a Variable(s) entered on step 1: DoseGp.

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