PROOF Correction on Atom Indonesia Name of All Authors : Dwi Ramadhani, Sofiati Purnami

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PROOF Correction on Atom Indonesia

Name of All Authors : Dwi Ramadhani, Sofiati Purnami

^,

Mitsuaki Yoshida

Article Title : Comparison of Radiosensitivity of Human Chromosomes 1, 2 and 4 from One Healthy Donor

E-mail : [email protected]

Line

Number Original Text Correction Note / Change

22 fluorescence in situ hybridization fluorescence in situ hybridization 33-34 prematurechromosome condensation

fluorescence in situhybridization

prematurechromosome condensation- fluorescence in situhybridization

288 Translocation Number at Doses of 1, 3, and 5 Gy

Translocation number at doses of 1, 3, and 5 Gy

316 Total numbert Total number

334-335 Breakpoint positions in chromosome 1 at doses of (a) 1 Gy; (b) 3 Gy; and (c) 5 Gy; chromosome 2 at doses of (d) 1 Gy; (e) 3 Gy; and (f) 5 Gy; and chromosome 4 at doses of (g) 1 Gy;

(h) 3 Gy; and (i) 5 Gy.

Breakpoint positions in

chromosome 1 at doses of (a) 1 Gy; (d) 3 Gy; and (g) 5 Gy;

chromosome 2 at doses of (b) 1 Gy; (e) 3 Gy; and (h) 5 Gy; and chromosome 4 at doses of (c) 1 Gy; (f) 3 Gy; and (i) 5 Gy.

Could it is possible to make the Fig 4 more bigger because the red arrow in 1a cannot be seen

338 ionizing-radiation-induced ionizing radiation induced

442 Anonymous International Atomic Energy

Agency

Please return to Atom Indonesia Editorial Office via e-mail: [email protected] This original sheet should be returned to:

PPIKSN-BATAN, Secretariat of Atom Indonesia, Gedung 71, Lantai 1, Kawasan Puspiptek Serpong, Tangerang, Indonesia 15310

Author Signature(s) 11/5/2016

Dwi Ramadhani

(2)

1

2 3

Comparison of Radiosensitivity of Human

4

Chromosomes 1, 2 and 4 from One Healthy Donor

5 6

D. Ramadhani^*1, S. Purnami¹ and M. Yoshida² 7

1Center forRadiation Safety Technology and Metrology, National Nuclear Energy Agency

8

Jl. Lebak Bulus Raya No. 49, Jakarta 12070, Indonesia

9

2Department of Radiation Biology, Institute of Radiation Emergency Medicine, Hirosaki University, Japan

10 11

12

A R T I C L E I N F O A B S T R A C T

13 14

Article history:

Received 29 September 2015

Received in revised form 29 March 2016 Accepted 31 March 2016

Keywords:

Chromosome FISH

Ionizing radiation Radiosensitivity Translocation

In general, it was assumed that the chromosome aberration induced by ionizing radiation is proportional to the chromosome size. From this viewpoint, the higher chromosome size, the more resistant to radiation. However, different opinions, in which chromosomes are particularly sensitive or resistant to radiation, are also still followed until now. Here in this research, we compared the chromosome sensitivity between chromosomes number 1, 2, and 4 using the FISH (fluorescence in situ hybridization) technique. From this research, we expect that the information obtained could show clearly whether a longer chromosome is more frequently involved in translocations and also more resistant to radiation than a shorter one.

The type of chromosome aberration considered was limited only to translocation and we used one sample donor in order to avoid donor variability. The whole blood from a healthy female was irradiated with γ-rays with doses of 1, 3 and 5 Gy,

respectively. Isolated lymphocytes from the whole blood were then cultured for 48 hours. After the culture process was completed, preparations of harvest and

metaphase chromosomes were carried out. Chromosomes 1, 2, and 4 were stained with different fluorochromes. The translocation of each chromosome at each dose point was subsequently evaluated from 50 images obtained from an automated metaphase finder and capturing system. An additional analysis was performed to identify which chromosome arm was more frequently involved in translocation.

Further analyses were also conducted with the aim of determining which chromosome band had a higher frequency of radiation-induced breakage. The experimental results showed that chromosome number 4 was more frequently involved in translocations compared to chromosomes 1 and 2 at 5 Gy. In contrast, at doses of 1 and 3 Gy translocations involving chromosomes number 1 and 2 were more numerous compared to the ones involving chromosome 4. However, if the number of translocation was accumulated for all the doses applied, the chromosome number 4 was the chromosome most frequently involved in translocations.

Breakpoint analysis revealed that in chromosome 1, chromosome 2, and chromosome 4, the highest chromosome bands as break position were in band q32, p13, and q21, respectively. It can be concluded that chromosome 4 is more sensitive to radiation in all doses point, despite having less DNA content than chromosomes 1 and 2. Thus, it was showed that our research cannot support the general assumption about chromosome aberration induced by radiation being proportional to DNA content.

15

INTRODUCTION^ 16 17

The chromosomal radiosensitivity is 18

representative of the individual radiosensitivity [1].

19

Corresponding author.

E-mail address: [email protected] DOI:

It can be measured using several cytogenetic 20

techniques, and one of the most useful method for 21

assessing it is fluorescence in situ hybridization 22

(FISH). FISH has been shown to be a useful method 23

for assessment of chromosomal radiosensitivity [2].

24

A differential susceptibility of chromosomes for 25

aberration induction will be discernible through a 26

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Summary of comments: Article #428 (Uncorrected Proof).pdf

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Author: windows 8 Subject: Highlight Date: 2016-05-12 18:33:00

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should be italic all the words

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D. Ramadhani et al. / Atom Indonesia Vol. xxx No. xxx (2016) xxx - xxx

systematic and extensive analysis. In general 27

the chromosome aberration induced by radiation 28

will be proportional to deoxyribonucleic acid 29

(DNA) content that is proportional to the 30

chromosome size [3].

31

Pandita et al. [4], using premature 32

chromosome condensation fluorescence in situ 33

hybridization (PCC-FISH) in human lymphocytes at 34

G0 stage, has shown that there is a direct 35

relationship between chromosome size and 36

aberration frequency. Luomahaara et al. [5]

37

examined the distribution of breakpoints that were 38

induced by radiation chromosomes 1, 2, and 4.

39

In their study, they obtained their samples from 40

donors from radiation accident victims in Estonia in 41

1994 and from in vitro irradiated lymphocytes.

42

They showed the localization of the breaks in the 43

chromosome and examined the correlation of the 44

break points with DNA proportion content.

45

They also showed that the yield of chromosome 46

exchanges was equal to DNA content for both the 47

accidental radiation exposure victims, in vivo, and 48

irradiated lymphocytes, in vitro. However, Wojcik 49

and Strefferr [6] showed that, in general, 50

chromosome 1 was more frequently involved in 51

translocations compared to chromosome 2, even 52

though this result was not always reproducible.

53

The translocation is a type of chromosomal 54

aberration in which a large segment of one 55

chromosome breaks off and attaches to a different 56

chromosome. Chromosome translocations are now 57

considered as a valuable biomarker of radiation 58

exposure and cancer risk [7,8]. Several studies 59

showed that chromosome translocations in 60

peripheral blood lymphocytes can be considered as 61

the most reliable biomarker for measurement of 62

low-dose radiation effects and for retrospective 63

biodosimetry [9-12]. Another study also revealed 64

that the analysis results of translocations using FISH 65

after in vitro irradiation correlated with clinical 66

response to radiation in prostate cancer patients.

67

Based on that result, the authors suggested that the 68

cytogenetic assay should be considered as a 69

potential predictor of radiosensitivity [13]. Even 70

now, there are techniques that can be used as 71

radiosensitivity predictors, such as the γ-H2AX 72

assay. Djuzenova et al. showed that the γ-H2AX 73

assay shows the potential for use in screening the 74

individual radiosensitivities of breast cancer 75

patients [14].

76

Until now, there has been no agreement on to 77

which extent the chromosomes are particularly 78

sensitive or resistant to radiation. However, several 79

studies supported the assumption that the higher 80

the chromosome size, the more resistant the 81

chromosome is to radiation. It seems that donor 82

variability is probably a contributing factor to the 83

disagreement, as most studies were performed using 84

lymphocytes of a homogeneous donor [15,16].

85

The works of Wojcik and Streffer [6] and of 86

Sommer et al. [16] seem to support this argument.

87

Their works show that the types of aberration 88

studied affected the radiation sensitivity of 89

chromosomes; for example, human chromosome 90

number 1 was more susceptible to translocations 91

than that of chromosome 2, while Wojcik and 92

Streffer [6] showed that chromosome number 2 was 93

more prone to deletions than that of chromosome 1.

94

The aim of this research was to compare the 95

sensitivities of chromosomes number 1, 2 and 4 96

using FISH technique. The aberration type analyzed 97

was limited only to translocation, and only one 98

sample donor was used in this research to avoid 99

donor variability. In this work, we assumed that the 100

higher the chromosome size is, the more prone the 101

chromosome is to be involved in the translocation 102

and also the more radiation-resistant it is. Additional 103

analyses were also conducted to identify the 104

chromosome arms that more involved in the 105

translocation and to detect the higher frequency 106

bands as breakpoint position induced by radiation.

107 108 109

EXPERIMENTAL METHODS 110

111

Subjects and irradiation 112 113

Eighty milliliters of blood was collected by 114

venipuncture from one 41-year-old healthy female 115

donor without a history of ionizing radiation 116

exposure beyond routine diagnostic exposures.

117

The whole blood was then irradiated with γ-rays 118

from ¹³⁷Cs in doses of 1, 3, and 5 Gy, respectively, 119

with a dose rate of 0.649 Gy/min at the Institute for 120

Environmental Sciences in the Rokkasho village, 121

Aomori prefecture, Japan.

122 123 124

Blood culture 125 126

Lymphocytes to be cultured were isolated 127

from whole blood using Vacutainer CPT Tube (BD 128

Biosciences USA). Cultures were set up in Roswell 129

Park Memorial Institute 1640 (RPMI 1640) culture 130

medium supplemented with 4-(2-hydroxyethyl)-1- 131

piperazineethanesulfonic acid (HEPES) and 132

L-glutamine, 20% fetal bovine serum (FBS), 133

kanamycin, colcemid 0.05 µg/mL, and 134

phytohaemagglutinin (PHA). The culture was 135

maintained in a 5%-humidified CO2 incubator at 136

37°C for 48 hours. Cells were then treated with 137

hypotonic shock (0.075 M KCl) for 15 min at 138

37°C and fixed in acetic acid and methanol 139

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should be italic all the words

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(1:3). Subsequently, 20-25 µL of cell 140

suspension was dropped onto clean slides for 141

metaphase chromosome preparation and allo 142

wed to air-dry. Then, slides were kept overnight 143

at -80°C.

144 145 146

Fluorescence in situ hybridization (FISH) 147 148

The slides that were kept overnight in the 149

previous procedure were hardened for 1 hour at 150

65°C. Commercial chromosome DNA probes 151

(MetaSystems, Altlussheim, Germany) were used 152

to directly label chromosome 1 (Texas Red), 153

2 (fluorescein isothiocyanate/FITC), and 4 154

(FITC/Texas Red) following the manufacturer's 155

recommended protocol. The chromosome 156

DNA probe cocktail was prepared as a premix 157

containing equal amounts of the probe 158

for each chromosome. Seven microliters 159

of the probe mixture were applied on the 160

slide depending on the size of the hybridization 161

area. The area was then covered with 162

15 mm × 40 mm coverslips and sealed with 163

rubber cement. Slides and probes were 164

denatured simultaneously at 70°C for two min.

165

The slides were incubated at 37°C overnight 166

in a humidified atmosphere. The rubber 167

cement was then removed from the slides 168

and they were treated for the post-hybridization 169

step washed with 0.4× saline sodium citrate 170

(SSC) buffer at 73°C for two min. Subsequently, the 171

slides were treated in 0.5× SSC / 0.05% of Tween 172

20 at room temperature for 30 s. Finally, they were 173

rinsed twice briefly in distilled water to prevent salt 174

crystal formation. Air-dried slides were then 175

embedded and counterstained with 15 µL of 4’, 176

6-diamidino-2-phenylindole (DAPI) and mounted 177

with cover glass; nail polish was used to prevent 178

the DAPI from leaving the area in cover slip.

179 180 181

Fluorescence image capture and analysis 182 183

The automatic metaphase finder was 184

performed with a Zeiss Axioplan 2 Imaging 185

epifluorescent microscope connected to a Cool Cube 186

(MetaSystems, Altlussheim, Germany) and 187

the Metafer 4 imaging system (MetaSystems, 188

Altlussheim, Germany). Fluorescent images of 189

metaphase spreads were captured and analyzed with 190

the ISIS software from MetaSystems and ImageJ 191

1.49. The translocation in each chromosome 192

was evaluated separately, and at each dose used, 193

50 metaphases were scored and the yields of the 194

translocations involving chromosomes 1, 2, and 195

4 were recorded.

196

Chromosome arm identification 197 198

The identification of which chromosome arm 199

(p or q arm) that was getting involved in 200

translocation was carried out manually for 201

chromosomes number 2 and 4. Since the 202

chromosomes 2 and 4 were submetacentric, the 203

shortest arm was identified as the p arm.

204

For example, in Fig. 1 it can be seen clearly that the 205

centromere in chromosome 4 was in the dashed line 206

and the shorter arm is defined as the chromosome’s 207

p arm. Therefore, the longer arm was taken as the q 208

arm. For chromosome 1 the arm identification was 209

performed by measuring the length of each arm to 210

define the centromeric ratio. The arm that gives a 211

centromeric ratio in the 0.510-0.520 range, or closer 212

to it, is defined as the q arm based on Morton [17].

213

For instance, in Fig. 1 the length of the upper arm 214

(red arrow) is 27.203 pixels and the length of the 215

bottom arm (orange arrow) is 35.355 pixels.

216

The total length of the upper and bottom arms is 217

62.558 pixels. If the upper arm is assumed as the 218

q arm then the centromeric ratio would be 0.43, 219

while if the bottom arm is considered as the q arm it 220

will give a centromeric ratio of 0.56. Since the 221

bottom arm gives the closer centromeric ratio to 222

0.510 then it was defined as the q arm.

223

The centromeric ratio is defined by equation (1) 224

as follows.

225 226

𝐶𝑅 = _(𝑝+𝑞)^𝑞 (1)

227 228

q : Length of q arm

229

p : Length of p arm

230

CR: Centromeric ratio

231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247

Fig. 1. Chromosomes 1 and 4 arm identification using

248

centromeric ratio (CR) and manual process.

249 250 251

Breakpoint analysis 252 253

The breakpoint analysis was carried out by 254

measuring the loss of chromosome area from the 255

q

p

q

(5)

original chromosome one through observation of the 256

short arm. The area loss percentage was converted 257

to chromosome length, and then it was plotted to 258

chromosome image obtained from International 259

System for Human Cytogenetic Nomenclature 260

(ISCN) 2009 from the Atlas of Genetics and 261

Cytogenetics in Oncology and Haematology website 262

(http://atlasgeneticsoncology.org/index.html) [18].

263

A detailed process of this method is available in 264

additional file of this paper. The method used in this 265

paper was a modified form of Schilling et al. [19]

266 267 268

RESULTS AND DISCUSSION 269 270

Many studies have previously been conducted 271

for many years up to present to identify the 272

chromosomes that are particularly sensitive to 273

ionizing radiation. Here in this research, it was 274

clearly found that for the highest dose used (5 Gy), 275

chromosome number 4 underwent more 276

translocations than did chromosomes 1 and 2, while 277

at 1 and 3 Gy, the number of translocations of 278

chromosomes number 1 and 2 was higher than that 279

of chromosome 4, as shown in Fig. 2. However, for 280

the total number of translocation for all doses, 281

chromosome number 4 showed the most, as shown 282

in Fig. 3. The results of total translocation in 283

chromosomes 1, 2 and 4 from 50 metaphases at 284

doses of 1, 3, and 5 Gy were summarized on 285

Table 1, Fig. 2, and Fig. 3.

286 287

Table 1. Translocation Number at Doses of 1, 3, and 5 Gy

288 289

Radiation Doses (Gy)

Chromosomes Number

Number of Translocation

s

Chromosomes Arm Number Involved in Translocation

1

1 20 11 in p arm and

9 in q arm

10 in q arm

4 11 11 in q arm

3

12 in q arm

2 12 4 in p arm and

8 in q arm

4 24 3 in p arm and

11 in q arm

5

1 19 7 in p arm and

12 in q arm

2 15 5 in p arm and

10 in q arm

4 24 6 in p arm and

18 in q arm

290

Our experimental results showed that the q 291

chromosome arms underwent more translocation 292

that did the p arms. In total, for all doses, q 293

chromosome arms underwent 101 translocations, 294

while p arms underwent 60. Moreover, at 1 Gy, 295

there was no translocation for the p arm of 296

chromosome 4. At 3 and 5 Gy, the q arms of 297

chromosome 4 underwent three times as many 298

translocations as did the p arms (Table 1). Possibly, 299

a factor that causes q arms to get translocated more 300

frequently is the larger size of the q arm compared 301

to the p arm. As can be seen in Table 1 and Fig. 2, 302

for all doses, the q arms of chromosome 2 and 4 303

underwent more translocations than the p arms.

304

The p arms of chromosome 1 also underwent fewer 305

translocations than the q arms for all doses, even 306

though the difference was smaller. It is possible that 307

the difference is smaller because chromosome 1 is 308

metacentric; it has equal-sized p and q arms.

309 310

311

Fig. 2. Number of translocations in chromosomes 1, 2, and 4 at

312

doses of 1, 3, and 5 Gy.

313 314

315 Fig. 3. Total numbert of translocations in chromosomes 1, 2,

316

and 4 for all applied doses.

317 318

In our study, it has been shown that the most 319

frequent break position of chromosome 1 was in 320

band q32. This experimental result is in good 321

agreement with the results of Barrios et al. that 322

examined the lymphocytes from cancer patients 323

after radiotherapy [20]. Barrios et al. argued that the 324

band q32 is a hot point for clastogenic effects 325

(causing chromosomal breakage) of ionizing 326

radiations. For chromosome 2, the break position 327

was depicted in band p13, while for chromosome 4 328

was in band q21. This research only considered the 329

break positions that are located in light bands, as 330

only those bands contain the active genes.

331

0 5 10 15 20 25 30

1 2 4 1 2 4 1 2 4

Number of Translocations

Chromosomes Number

p arm q arm

0 10 20 30 40 50 60 70

1 2 4

Number of Translocations

Chromosomes Number

p arm q arm

1 Gy 3 Gy 5 Gy

Page:4

Translocation number at doses of 1, 3, and 5 Gy

number

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332 333

Fig. 4. Breakpoint positions in chromosome 1 at doses of (a) 1 Gy; (b) 3 Gy; and (c) 5 Gy; chromosome 2 at doses of (d) 1 Gy; (e) 3

334

Gy; and (f) 5 Gy; and chromosome 4 at doses of (g) 1 Gy; (h) 3 Gy; and (i) 5 Gy.

335 336

Our experimental results also showed that the 337

ionizing-radiation-induced breakpoints in the 338

chromosomes were not random. Figure 4 of our 339

experimental data shows that the q21 breakpoint 340

on chromosome 4 seems to have a high breakage 341

frequency for all doses. Breakages were 342

more frequent in light bands compared to dark 343

bands in all chromosome number at all 344

doses. Possible explanation for these finding 345

is that the light bands contain more active genes 346

than do the dark bands. Our findings also support 347

other several studies that found that breakages 348

induced by radiation were more frequent in 349

light bands that are considered as gene-rich 350

regions [21,22].

351

From previous studies it was known that 352

chromosomal radiosensitivity based on the 353

translocation in lymphocytes can be proposed as a 354

predictive assay for detection of radiosensitive 355

individuals [23]. Several studies used chromosome 356

translocations to identify the cancer patients with 357

higher radiosensitivity. A study by Huber et al.

358

showed that translocations can be used as a test to 359

identify breast tumour patients with potentially 360

elevated radiosensitivities [23].

361

In biodosimetry using painting of several 362

chromosomes, there was an assumption that the 363

frequencies of chromosomal aberrations were 364

proportional to their size, which is important 365

because it will extrapolate to the whole 366

genome [24]. In contrast, our experimental 367

research found that the frequencies of chromosome 368

aberrations were not proportional to their 369

size. Based on these findings, for a biodosimetry 370

purpose, it is possible that biodosimetry 371

using painting of all chromosomes (multicolor 372

FISH) is better than with painting only 373

several chromosomes. Multicolor FISH is also 374

considered as the best method for assessment 375

of a chromosome’s structural damage because it 376

allows unstable and stable aberrations to be 377

detected [25].

378

Other methods such as telomeric and 379

pancentromeric probes combined with chromosome 380

paint probes can also be use in order to accurately 381

discriminate between translocations and dicentrics 382

[26]. This technique can also overcome the problem 383

of the high cost of the probes for several 384

laboratories [27]. A study by M’kacher . showed 385

that using the telomere and centromere (TC) 386

staining probes it can provides the most precise 387

cytogenetic biological dosimetry currently available.

388

Another advantage of TC staining method is that it 389

does not require a high level of expertise to identify 390

the chromosome aberrations induced by radiation 391

exposure [28].

392

Could it is possible to make the Fig 4 more bigger because the red arrow in 1a cannot be seen

Breakpoint positions in chromosome 1 at doses of (a) 1 Gy; (d) 3 Gy; and (g) 5 Gy; chromosome 2 at doses of (b) 1 Gy; (e) 3 Gy; and (h) 5 Gy; and chromosome 4 at doses of (c) 1 Gy; (f) 3 Gy; and (i) 5 Gy.

ionizing radiation induced

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CONCLUSION 393 394

Based on the experimental results, it can be 395

concluded that chromosome 4 was more sensitive to 396

radiation in all doses point despite having less DNA 397

content than chromosome 1 and 2. The general 398

assumption about radiation-induced chromosomal 399

aberration being proportional to DNA content 400

cannot be supported by our experimental results.

401

For a biodosimetry purpose it is possible that 402

multicolor FISH will give a better result than 403

painting only several chromosomes. A comparison 404

of the radiation dose estimates using multicolor 405

FISH and three-color FISH should be conducted in 406

the next experiment to validate or invalidate this 407

assumption.

408 409 410

ACKNOWLEDGMENT 411 412

The authors gratefully acknowledge the 413

Institute of Radiation Emergency Medicine at 414

Hirosaki University and National Nuclear Energy 415

Agency of Indonesia (BATAN) via the Center for 416

Technology of Radiation Safety and Metrology 417

(PTKMR) for supporting this research. This 418

research was conducted under the MEXT Program 419

in 2013-2014.

420 421 422

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