ATOM INDONESIA JOURNAL
Referee’s Report
Article No. : # 428
Title of Paper : Comparison of Human Chromosomes 1, 2 and 4 Radiosensitivities From One Healthy Donor
Referee Name :
Comment on Descriptions 1. Title
[ ] Appropriate [√] Should be changed 2. Abstract
Yes[ ] No[√] Is the length reasonable?
Yes[√] No[ ] Is it an appropriate summary of the content?
3. Main Text
Yes[ ] No[√] Is there anything new in this work?
Yes[ ] No[√] Is the relation to previous studies adequately stated?
Yes[√] No[ ] Are the assumption(s) and/or method(s) described comprehensively?
Yes[ ] No[√] Are the new results adequately emphasized?
Line # Referee’s Comments
1-2 Title: Comparison of Radiosensitivity of Human Chromosomes 1, 2 and 4 Radiosensitivities From from One Healthy Donor
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In general, it was assumed that the chromosome aberration induced by ionizing radiation was is proportional to the chromosome size. From this viewpoint, These mean a the higher chromosome size, was the more resistant the chromosome to radiation. However, Unfortunately conflicted different opinions, in about which chromosomes were are particularly sensitive or resistant to radiation, still exist until now. Here in this research, we compared the chromosomes chromosome sensitivity between chromosomes number 1, 2 and 4 using a FISH (fluorescence in situ hybridization) (FISH) technique. From this research, we expect that the information obtained could show clearly It expected that from this research we can add the information to clearly whether the higher chromosome size means is more prone apt to be involved in translocation and also more resistant to radiation. The type of chromosome aberration chromosome aberration type was limited only to translocation and we used one sample donor in this research in order could to avoid donor variability. The whole bloods blood from a healthy female were was irradiated with γ-rays of 1, 3 and 5 Gy, respectively of γ-rays. Isolated lymphocytes from the whole blood then were cultured for forty-eight hours. After cultured process completed, preparations of harvest and metaphase chromosomes preparations were carried out. conducted after cultured process was done.
Chromosomes 1, 2 and 4 then were stained with different fluorochromes. Translocation of in each chromosome at each dose point then was subsequently evaluated from fifty images obtained using from automated metaphase finder and capturing system. An additional analysis was performed to identify which chromosome arm that more involved in translocation was performed. Further analysis was also conducted with the aim of detecting which chromosome band that has a higher frequency as a breakpoint position induced by radiation also conducted in this research. The experimental results showed that chromosome number 4 was more involved in the translocation compared to chromosome 1 and 2 at dose 5 Gy. In contrast, at In doses 1 and 3 Gy translocation involving chromosomes number 1 and 2 was higher compared to the chromosome 4. But However, if the number of translocation was accumulated in all doses, the chromosome number 4 then the was the highest chromosome number involved in the translocation was chromosome number 4.
Breakpoint analysis revealed that in chromosome 1, chromosome 2, and chromosome 4, the highest chromosome bands as break position was were in bands q32, p13, and q21, respectively. In chromosome 2 the highest chromosome band as break position was in band p13. While in chromosome 4 highest chromosome band as break position was in band q21. Overall It can be concluded that chromosome 4 was is more sensitive to radiation in all doses point. Even though the DNA content of chromosome 4 is lesser compared to chromosome 1 and 2, it was showed that our research cannot support the general assumption about chromosome aberration induced by radiation
being proportional to DNA content. cannot be sup ed by our research.
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It Chromosomal radiosensitivity can be measured measure using several cytogenetic cytogenetics techniques and one of the most useful method for assessing it is Among all the techniques the fluorescence in situ hybridization (FISH) method has been shown to be a assessment of chromosomal radiosensitivity [2].
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A systematic and extensive analysis about chromosomal radiosensitivity reveal that there was A differential susceptibility of chromosomes for aberration induction will be discernible through a
systematic and extensive analysis. It was assuming In general, that the chromosome aberration induced by radiation will be being proportional to deoxyribonucleic acid (DNA) content that DNA content in general is also proportional to the chromosome size [3].
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Several studies support these assumption, for example study performed by Pandita et al. [4] using premature chromosome condensation fluorescence in situ hybridization (PCC-FISH) in human lymphocytes at G0 stage has shown that there is a direct relationship between chromosome size was directly related to and aberration frequency. Luomahaara et al. [5] examined the break points distribution in chromosomes 1, 2, and 4 induced by radiation. In this study, they used both samples of donors from the radiation accident victims in Estonia in 1994 and in vitro irradiated lymphocytes. They could show the localization of the breaks in the chromosome and examined the correlation of the break points to with DNA proportion content. The radiation accident victims in Estonia in 1994 were used in Luomahaara et al. study as a sample donors and in vitro irradiated lymphocytes [5]. It was also shown was showed that the yield of chromosomes exchanges was equal to DNA content both in vivo at accidental radiation exposure victims and in vitro in irradiated lymphocytes. However, in general, Study by Wojcik and Streffer [6] showed that in general the chromosome 1 was more frequent involved in translocations compare to the chromosome 2, even though the result was not always reproducible.
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Even though until now there was no agreement to which extent the chromosomes are
particularly sensitive or resistant to radiation, several studies supported support the assumption that the more higher the chromosome size, means the more resistant the chromosome to the radiation until now there was no agreement exists as to which chromosomes are particularly sensitive or resistant to radiation. It also seems that the donor variability could be a contributory contributing factor to the disagreement that problem, as most studies were performed using with lymphocytes of a homogenous single donor [7]. Works of Results studies by Wojcik and Streffer [6], and also Sommer et al. [7] seem seems to support this view argument. Their works show that the types of aberration studied affected also influenced the radiation sensitivity of chromosomes, for example, human chromosome number 1 was more susceptible to
translocations as compared to than that of chromosome 2, while Wojcik and Streffer [6] showed that chromosome number 2 was more prone to deletions compare to than that of chromosome 1 as showed in studies by Wojcik and Streffer [6].
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The aim of this research was to compare the chromosome chromosomes sensitivity between chromosomes, number 1, 2 and 4, using a FISH technique. The analysis of aberration type was limited only to translocation that analyze and to avoid donor variability only one sample donor was used in this research to avoid donor variability. In this work, we assumed that From this research authors expect it can clearly the assumption was a the higher chromosome size, means the more prone the chromosome involved in the translocation and also more resistant to
radiation. In this research an Additional analysis was also conducted to identify which the chromosome arms that more involved in the translocation and to Further analysis also done in order to detected the band that has a higher frequency detect the higher frequency bands as breakpoint position induced by radiation.
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Eighty millilitres mL of whole blood were collected by venipuncture from one female healthy donor with age 41 years without a history of ionizing radiation exposure beyond routine
diagnostic exposures. The whole blood then was were irradiated with 137Cs γ-rays at the dose 1, 3 and 5 Gy, respectively, with a dose rate of 0.649 Gy/min at The Institute for Environmental Sciences in Rokkasho Village, Aomori Prefecture, Japan.
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Lymphocytes were isolated from whole blood before culture was performed using Vacutainer CPT Tube (BD Biosciences USA). Cultures were obtained from were set up in of Roswell Park Memorial Institute (RPMI 1640). Culture medium was supplemented with HEPES and L- Glutamine, 20% Fetal Bovine Serum (FBS), Kanamycin, Colcemid 0.05 μg/mL and
Phytohaemagglutinin (PHA). The culture was maintained in a 5% humidified CO2 incubator at 37°C for 48 hours. Cells were then treated with by hypotonic shock (0.075 M KCl) for 15 min at 37°C and fixed in acetic acid and methanol (1:3). Subsequently, 20-25 μL of cell suspension was dropped onto clean slides for metaphase chromosome preparation and allowed to air dry. Then,
slides then were kept overnight in ultra low. (Please define, what kind of ultra low in this case?)
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The slides that were kept overnight in the previous procedure were then hardening hardened for 1 hour at 65°C. A Commercial directly labeled whole chromosome DNA probes (MetaSystems, Altlussheim, Germany) were used to label directly chromosome 1 (Texas Red), 2 (FITC) and 4 (FITC/Texas Red) following the manufacturer's recommended protocol.
128 Seven microliters of the probe mixture were was applied on the slide depending…………
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The area is then covered with coverslips cover slips 15 × 40 mm and sealed with rubber cement.
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The slides were then incubated at 37°C overnight in a humidified atmosphere. Rubber cement then was removed from the slides and slides they were treated for the post-hybridization step that washed were washes in with 0.4× saline sodium citrate (SSC) buffer at 73°C for 2 min.
Subsequently, slides then were treated in 0.5× SSC / 0.05 % of Tween 20 at room temperature for 30 sec. Finally, slides they were rinse shortly twice in distilled water to prevent salt crystal formation. Air-dried slides were then embedded and counterstained with 15 μL of 4’,6-
diamidino-2-phenylindole (DAPI) and mounted mount with cover glass and nail polish was used to prevent the DAPI out from the area in cover glass
149 The automatically metaphase metaphases finder was performed ………….
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The translocation in each chromosome was evaluated separately and at each of dose point 50 metaphases were scored and the yield of the translocation involving chromosomes 1, 2 and 4 was were recorded.
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Identification of which chromosome arm (p or q arm) that getting involved in translocation was carried out done manually for chromosomes number 2 and 4. Since the chromosomes 2 and 4 were belong to submetacentric sub metacentric chromosome type, then the shortest arm was identified as p arm. Therefore In contrast, the longest arm was considered as q arm. For
chromosomes 1 the arm identification was performed done by measuring measured the length of each arm to define the centromeric ratio value. The arm that gave a centromeric ratio value closer or in range 0.510 to 0.520 defined as q arm based on data published Morton [8] (For more detailed explanation, see Figure 1 for more detail).
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Please define clearly and briefly the title of Figure1.
The long statement you attached on the Figure 1 is not a title of figure. If you think the statement is very important for explaining Figure 1, it is better you put it in the text body
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Breakpoint analysis was carried out done by measuring measure the percentage loss of
chromosome area from the original chromosome one through observation of as viewed from the short arm. The area loss percentage percentage loss area was then converted to chromosome length, and then it was plotted to chromosome image obtained from ISCN (International System for Human Cytogenetic Nomenclature) 2009 that can be downloaded from the website of Atlas of Genetics and Cytogenetics in Oncology and Haematology
(http://atlasgeneticsoncology.org/index.html) [9]. A detailed detail process of this method is available can be found in the additional file of this paper. The method used in this paper was a modified form of Schilling et al. [10]
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It was clearly depicted that in the highest dose (5 Gy), the chromosome number 4 underwent involved more translocation than that of compared to chromosomes 1 and 2 (Figure Fig.2).
While doses of 1 and 3 Gy, translocation of involving chromosomes number 1 and 2 was higher than that of compared to chromosome 4. However, But if the total number of translocation was accumulated in for all doses, then it can be shown that the highest chromosome number involved in translocation was 4 showed the highest (Figure Fig.).
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Our experimental results research also showed the q chromosome arms underwent was higher having a translocation that was higher than that of p arms. (Please clarify your statement why q arms are higher than p arms) Moreover Even at the dose of 1 Gy, there was completely no translocation involved in the p arms of chromosome number 4. At the dose of 5 Gy on the chromosome 4, it was also found out seen that the translocation of q arms involved in translocation compared to is higher than that of p arms (Figure 2). (Please also clarify this statement)
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Breakpoint analysis resulted in revealed that in chromosome 1 the highest chromosome band of chromosome 1 defined as a break position was shown in band q32 (red arrow). For In
chromosome 2, the highest chromosome band as the break position was depicted in band p13 (red arrow), while for in chromosome 4 was in band q21 (red arrow) (Figure 4). The break position considered in this research was only the break position direct to the light bands that considered, since only due to this area the genes were active that is only containing the active genes.
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Many studies have been already conducted for many years until now to identify which chromosomes that particularly sensitive to ionizing radiation. In our this study, it has been shown showed that chromosome 1 the highest chromosome band as has a break position at band q32 that is the highest chromosome. This experimental result is Our finding was in a good agreement with the result of research by Barrios et al. that examined the lymphocytes from cancer patients after radiotherapy [11]. Barrios et al. argued They proposed that the band q32 as is a ‘hot point’ for a clastogenic effect of ionizing radiation. (Please clarify, what is clastogenic).
In our research, it appears also seen that q arms were was more affected by radiation compared to p arms. (Please discuss this experimental result based on the literatures or on your scientific opinion, why the q arms are more affected than the p arms).
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Our experimental results also showed that the break point in the chromosomes induced by ionizing radiation was not random. (Please clarify this statement). Figure 4 of our experimental data shows that It was also clearly seen in the breakpoint position on chromosome 4 which was is the q21 band that occurs in a higher frequency in the light bands rather than the dark bands in all doses and all chromosome numbers. (Please clarify this experimental result based on the literatures or your scientific opinion) Band q21 in chromosome 4 in our research seen have a high frequent as a breakpoint in all doses. It also showed that the breakpoints were frequent in light bands compared to dark bands in all chromosomes number at all doses
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These paragraphs are not relevant for this Discussion Section due to no relationship in supporting the discussion of your experimental results. It is better you remove the paragraphs from this section and move them to the introduction section.
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Please revise this paragraph!!!
You reported that your experimental data concerning “the frequencies of chromosome
aberrations were not proportional to their size”, but you mentioned based on literatures (?) that “ the frequencies of chromosome aberrations were proportional to their size was important in biodosimetry using FISH”. Please clarify your last statement of “it is possible if biodosimetry using painting all chromosomes (multicolor FISH) was better compare with painting only several chromosomes” with two contradictory previous statements.
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Please revise your conclusion!!
It is normally in the conclusion section; first we conclude our experimental results that have a close relationship with (a). the title of our research or paper and (b). the problems associated with title that we would like to solve them, then finally we make suggestion or advice regarding with the experimental results we obtained.
Final comments and recommendations:
Most experimental results in the Tables and the Figures are only reported, not discussed in the Discussion Section. Many statements in the Discussion Section are confused and it looks the
conclusion in the Conclusion Section is not based on the discussion results in the Discussion Section.
Some important terminologies associated with the research, for example translocation, etc., need to be defined before we describe their roles in the experiment.
This paper is recommended to be [ ] Accepted without further revision [ ] Accepted with minor revision [√] Major Revision is required [ ] Rejected
