Bone Marrow Oxidative Stress and Acquired Lineage-Specific Genotoxicity in Hematopoietic Stem/Progenitor Cells Exposed to 1,4-Benzoquinone^†

Round 1

Reviewer 1 Report

In this study, Dewi and colleagues exposed bone marrow cells ex vivo to a physiologically-relevant metabolite of benzene; 1,4-benzoquinone. The authors assessed several relevant endpoints in different types of bone marrow cells to a range of physiologically-relevant concentrations. The study was well-designed and the results are convincing.

Comments:

1. The abstract contains a lot of acronyms which are not used subsequently in the abstract (CFC, GSH, SOD, MDA, PC). Please use only the long form.
2. Please provide more information on the strain of mice used and the rationale behind it.
3. Was any sample eliminated from analysis or is in fact N=3 for every group of every endpoint? The phrasing "Each experiment was repeated at least three times" (line 210) is misleading. Please replace with "Each experiment was conducted in triplicate" if appropriate.
4. In the result section, please include a brief description of the purpose/goal of the assays performed at the beginning of each subsection.
5. Figure 1B: This is a duplicate of figure 2A (MDA). Please include the correct figure.
6. Figure 3 is mislabeled as figure 5 in section 3.3.
7. Information in sections 3.2, 3.3 and 3.4 is not clearly distributed. The subheadings are extremely similar and confusing. As table 1 is supporting the data displayed in figures 3 and 4, section 3.2 should be eliminated and table 1 referenced alongside figures 3 and 4 in sections 3.3 and 3.4.
8. Figure 3 should be incorporated as the 2 first panels of figure 4, making comparisons easier. 
9. Please correct the fonts and placement of asterisk signs in figure 4.
10. Please verify the concentrations listed on lines 329 and 330.
11. On the English language and style，it seemed that there were minor mistakes throughout the manuscript (e.g. lines 45, 70, 84, 240, 358, 376, 377, 386, 409).

Author Response

Author's Reply to the Review Report (Reviewer 1)

Comments:

1. The abstract contains a lot of acronyms which are not used subsequently in the abstract (CFC, GSH, SOD, MDA, PC). Please use only the long form.

We have revised the terms by using only the long form in the abstract.

1. Please provide more information on the strain of mice used and the rationale behind it.

We thank you for this useful suggestion. The information has been added into the manuscript under section 2.2. Isolation of mouse bone marrow cells and cell culture condition (highlighted in yellow; page 3, line 133).

1. Was any sample eliminated from analysis or is in fact N=3 for every group of every endpoint? The phrasing "Each experiment was repeated at least three times" (line 210) is misleading. Please replace with "Each experiment was conducted in triplicate" if appropriate.

We have revised the description to "Each experiment was conducted in triplicate"(line 212, page 5)

1. In the result section, please include a brief description of the purpose/goal of the assays performed at the beginning of each subsection.

We thank you for this useful suggestion. The information have been added into the manuscript under section 3.1 (page 5),  3.2 (page 7) and 3.3 (page 8) . The new sentences are highlighted in yellow.

Figure 1B: This is a duplicate of figure 2A (MDA). Please include the correct figure.

We have revised and changes has been made. The new figure is presented as Figure Figure 3a (page 7)

1. Figure 3 is mislabeled as figure 5 in section 3.3.

We have revised and changes has been made. Figure 3 is corrected in Section 3.3 (page 8)

1. Information in sections 3.2, 3.3 and 3.4 is not clearly distributed. The subheadings are extremely similar and confusing. As table 1 is supporting the data displayed in figures 3 and 4, section 3.2 should be eliminated and table 1 referenced alongside figures 3 and 4 in sections 3.3 and 3.4.

We thank you for this useful suggestion. Previous section 3.2 is eliminated. The new sections are 3.2 and 3.3  that separate findings for bone marrow cells from  lineage-committed progenitors . Table 1 is rephrased as Figure 4 and referenced alongside figures 5 and 6 in respective subsections.

1. Figure 3 should be incorporated as the 2 first panels of figure 4, making comparisons easier. 

We thank you for this useful suggestion. However, we would like to maintain the figures as it is to avoid confusion in data presentation and discussion. We hope this request can be considered.

1. Please correct the fonts and placement of asterisk signs in figure 4.

We have revised the fonts and placement of asterisk signs in Figure 5 on page 10 (new figure numbering after manuscript revision).

1. Please verify the concentrations listed on lines 329 and 330.

Thank you for the suggestion and the concentrations has been listed as highlighted in yellow (Line 375, page 12)

1. On the English language and style，it seemed that there were minor mistakes throughout the manuscript (e.g. lines 45, 70, 84, 240, 358, 376, 377, 386, 409).

            We have revised the use of languages and writing style throughout the manuscript.             Some of the sentences highlighted here by reviewer are no longer available following        revision of manuscript which include deletion of sentences, mainly in discussion       section.

Reviewer 2 Report

Major comments

In this study, the authors investigate the effect of benzene and benzene derivatives on hematopoietic and stem cell progenitors. The experiments are sound but somewhat preliminary. The major issue is that DNA damage is monitored only with a comet assay. No effort is being made to look at other forms of DNA damage, e.g. DSBs, H2Ax phosphorylation, status of repair and recombination genes, etc. Therefore, at best this study is preliminary and it only says that when cell are treated with benzene they respond by producing oxidative species and appear to have their DNA damaged.  

This reviewer does not necessarily believe that the study is not worthy of dissemination but only that the writing should be much more condensed. Mainly, the discussion is too long. There is no need to have three pages of discussion on three small findings. Please rewrite the discussion to fit one page maximum. Reintroducing statements in the discussion which were already stated in the introduction only makes the paper more tedious to read.

This sentence: “We found crucial differences in genotoxic susceptibility based on percentages 34 of DNA in tail between myeloid and pre-B lymphoid progenitors that appear to acquire significant 35 DNA damage starting from low concentration 1,4-BQ exposure (2.5 µM) as compared to the 36 erythroid progenitor which the significant damage notable starting at 5 µM 1,4-BQ” may be key to this study. The data that support this sentence appear to be in Figure 5. However, the way Figure 5 is organized it is hard to tell. Please reorganize this figure to graph myeloid and pre-myeloid side by side. Is there really a difference between a and c at concentration 12? Is this difference statistically significant? Did you run a statistical test between these two values? Note that the control at 0 concentration is higher for a than b. If you adjust for this, will there still be a difference? The same goes for e and f when compared with the other panels.

Minor comments

Line 51. …benzene is able to act.. OR …benzene acts

Line 60. No need to say ..lipid peroxidation in vivo…It is clear that it is in vivo, otherwise why study it.

Line 74. Among the most common…

Line 78…comprised of HSCs…

Line 87 linking…toward benzene toxicity. Sounds awkward. Consider rephrasing. Maybe say linking…with benzene toxicity.

Line 98. This sentence is redundant with the end of the previous paragraph. Consider removing.

Figure 1 is awkwardly stretched out. Check the shape of your mouse! Consider resizing.

For figures 1 and 2 please indicate in the legend at what hour the measurements are taken. I know it is in the methods but a short statement in the legend would help the reader.

Why are there paragraph signs on figure 3?

Line 228. …with the concentration of 100nM was used…

Please convert Table 1 into a figure. Also organize your panels so that they are symmetrical.

Line 319. Benzene metabolites are able.

The stars indicating significance on figure 5 are of different sizes. Also the text. Also if you say “percentage…” no need to include the sign (%). Either include % or remove “percentage.”

Author Response

Author's Reply to the Review Report (Reviewer 2)

Major comments

In this study, the authors investigate the effect of benzene and benzene derivatives on hematopoietic and stem cell progenitors. The experiments are sound but somewhat preliminary. The major issue is that DNA damage is monitored only with a comet assay. No effort is being made to look at other forms of DNA damage, e.g. DSBs, H2Ax phosphorylation, status of repair and recombination genes, etc. Therefore, at best this study is preliminary and it only says that when cell are treated with benzene they respond by producing oxidative species and appear to have their DNA damaged. 

• We thank you for this useful suggestion. Regarding reporting of DNA damage, we do agree that the current data is preliminary in the context of lineage-specific responses involving hematopoietic progenitors.
• We are conducting the study of DNA damage by staging. Phase 1 is analysis of DNA damage using comet assay focusing on 4 different types of cell populations comprise of bone marrow cells, and lineage-committed hematopoietic progenitors for myeloid, Pre-B lymphoid and erythroid lineages. Such comparative study to address benzene genotoxic effects has never been reported previously and crucial as these cells are vital for maintenance of hematopoietic system.
• Thus we believe this finding can be considered as one of major study to initiate further assessment in term of benzene’s genotoxic effects targeting hematopoietic stem cells niche.
• Moreover, we are currently conducting Phase 2 of the study focusing on DNA repair mechanisms involving these population of cells.

This reviewer does not necessarily believe that the study is not worthy of dissemination but only that the writing should be much more condensed. Mainly, the discussion is too long. There is no need to have three pages of discussion on three small findings. Please rewrite the discussion to fit one page maximum. Reintroducing statements in the discussion which were already stated in the introduction only makes the paper more tedious to read.

Thank you for highlighting this concern. Discussion part has been simplified as per suggestion by emphasizing on the main points only.

This sentence: “We found crucial differences in genotoxic susceptibility based on percentages 34 of DNA in tail between myeloid and pre-B lymphoid progenitors that appear to acquire significant 35 DNA damage starting from low concentration 1,4-BQ exposure (2.5 µM) as compared to the 36 erythroid progenitor which the significant damage notable starting at 5 µM 1,4-BQ” may be key to this study. The data that support this sentence appear to be in Figure 5. However, the way Figure 5 is organized it is hard to tell. Please reorganize this figure to graph myeloid and pre-myeloid side by side. Is there really a difference between a and c at concentration 12? Is this difference statistically significant? Did you run a statistical test between these two values? Note that the control at 0 concentration is higher for a than b. If you adjust for this, will there still be a difference? The same goes for e and f when compared with the other panels.

After considering reviewer comment, we agree to change the way the data is presented. New Figures which display comparison of DNA damage status between progenitors are enclosed in the revised manuscript (page 10 and 11)

Minor comments

Line 51. …benzene is able to act.. OR …benzene acts

Correction has been made and highlighted in yellow (Line 52)

Line 60. No need to say ..lipid peroxidation in vivo…It is clear that it is in vivo, otherwise why study it.

Correction has been made (Line 61)

Line 74. Among the most common…

Correction has been made and highlighted in yellow (Line 75)

Line 78…comprised of HSCs…

Correction has been made and highlighted in yellow (Line 78)

Line 87 linking…toward benzene toxicity. Sounds awkward. Consider rephrasing. Maybe say linking…with benzene toxicity.

Correction has been made and highlighted in yellow (Line 87)

Line 98. This sentence is redundant with the end of the previous paragraph. Consider removing.

Correction has been made and the sentences has been removed (Line 98)

Figure 1 is awkwardly stretched out. Check the shape of your mouse! Consider resizing.

Correction has been made (Page 3)

For figures 1 and 2 please indicate in the legend at what hour the measurements are taken. I know it is in the methods but a short statement in the legend would help the reader.

The information have been added into the manuscript (Line 242, Figure 2 ,page 6 , and Line 247, Figure 3, page 7)

Why are there paragraph signs on figure 3?

Paragraph signs have been removed and new figure 3 has been included in page 7

Line 228. …with the concentration of 100nM was used…

Correction has been made and highlighted in yellow (Line 254 page 7)

Please convert Table 1 into a figure. Also organize your panels so that they are symmetrical.

We thank you for the suggestions and changes has been made.

Line 319. Benzene metabolites are able.

Correction has been made and highlighted in yellow (Line 364)

The stars indicating significance on figure 5 are of different sizes. Also the text. Also if you say “percentage…” no need to include the sign (%). Either include % or remove “percentage.”

We thank you for the suggestions and correction has been made (page 9 and 10)

Round 2

Reviewer 2 Report

The authors have made significant changes to this version of the paper. This reviewer is satisfied. 

The only one comment I have is about figure 4 micrograph panels. They are not cropped to the exact same size. I suggest that an effort be made to crop them so that they are the same size. It would be more visually appealing. As I recall MDPI journals copy editors may make grammatical textual changes but do not adjust figures.