Ascorbic Acid Protects Bone Marrow from Oxidative Stress and Transient Elevation of Corticosterone Caused by X-ray Exposure in Akr1a-Knockout Mice
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Bo and co-workers studied the effect of ascorbic acid on blood cell count recovery in low dose irradiated mice. They conclude that ascorbic acid is reducing irradiation-derived damages in the bone marrow compartment. They suggest it is caused by the antioxidative properties of ascorbic acid.
The work is interesting, the authors have used a sophisticated animal model, and several in vitro methods to prove their hypothesis.
Did authors separately count the number of granulocytes after irradiation? Granulocyte count reflects better the condition in the bone marrow because of their short life span.
The aim of the irradiation is to make space for hematopoietic stem cell (HSC) transplantation. If ascorbic acid protects bone marrow cells from irradiation damage, would it be feasible to initiate ascorbic acid dosing 1-2 days after the irradiation, when also the “cytokine storm” is at its highest level? Can you speculate how ascorbic acid influence to the survival of HSCs?
The authors should discuss the effect of ascorbic acid on HSCs and on the effiency of HCS homing and engraftment. Please look at the previously published paper PMID: 19179476.
Author Response
Thank you very much for taking time to review our manuscript and favorable comments on our manuscript. We have included our responses after the reviewer's comments.
Reviewer 1
Bo and co-workers studied the effect of ascorbic acid on blood cell count recovery in low dose irradiated mice. They conclude that ascorbic acid is reducing irradiation-derived damages in the bone marrow compartment. They suggest it is caused by the antioxidative properties of ascorbic acid.
The work is interesting, the authors have used a sophisticated animal model, and several in vitro methods to prove their hypothesis.
- Did authors separately count the number of granulocytes after irradiation? Granulocyte count reflects better the condition in the bone marrow because of their short life span.
Our responses: Thank you for kind advice. We have also counted granulocytes separately. Since the population of granulocytes was small, at approximately 10% of white blood cells, and no significant differences were observed among the experimental groups of mice, so the data were not shown. Accordingly, we wish to provide the data as the new supplementary figures (new Supplementary Figure S1) rather than as main data.
- The aim of the irradiation is to make space for hematopoietic stem cell (HSC) transplantation. If ascorbic acid protects bone marrow cells from irradiation damage, would it be feasible to initiate ascorbic acid dosing 1-2 days after the irradiation, when also the “cytokine storm” is at its highest level? Can you speculate how ascorbic acid influence to the survival of HSCs?
Our responses: Thank you for asking the critical issues. It is well known that dividing cells, where DNA replication occurs, are highly susceptible to oxidative damage caused by radicals and other ROS produced by ionizing radiation such as X-ray. Among pleiotropic functions of Asc, it acts as a strong antioxidant by donating electrons and eliminates oxygen radicals [see ref.7 and 8]. Asc may also stimulate hematopoietic process by supporting enzymatic reactions, such as prolyl hydroxylase, or by epigenetically regulating gene expression as a cofactor [new ref 51]. We have added a brief statement concerning these issues to Discussion (page 10, Line 396-399).
- The authors should discuss the effect of ascorbic acid on HSCs and on the effiency of HCS homing and engraftment. Please look at the previously published paper PMID: 19179476.
Our responses: Thank you for kindly informing us the reference. In this study, we did not perform implantation, so we did not mention this issue. According to the reviewer’s advice, now we have briefly discussed the issue by quoting references (new ref.37, 38, page 9, Line 339-348).
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors,
please find my comments in the attached file.
Best regards
Comments for author File: 
Comments.pdf
Dear authors,
the English of your manuscript is well understandable.
Best regards
Author Response
Thank you very much for taking time to review our manuscript and favorable comments on our manuscript. We have included our responses after the reviewer's comments.
Reviewer 2
Title: Ascorbic Acid Protects the Bone Marrow from the Oxidative Stress and Transient Elevation of Corticosterone Caused by X-ray Exposure in Akr1a-Knockout Mice
In this study, the efficacy of ascorbic acid (Asc) to mitigate the effects of X-ray exposure was tested in mice. As rodents can synthesize ascorbic acid, a knock-down mouse without this ability was used (Akr1a-KO).
Mice were exposed to 2 Gy X-ray and white blood cell (WBC) counts, Asc and ACTH and cortisone levels were followed. ACTH and corticosterone were increased after exposure. Asc alleviated the effects on WBC counts and hormone levels. An in vitro experiment revealed that Asc reduces the effects of X-ray on viability of bone marrow cells.
The manuscript reveals an important connection between different organs (pituary gland, liver, adrenal gland and bone marrow) after whole body exposure of mice to X-rays. The results are important and relevant. The major weakness of the manuscript is the incomplete description of the methods, see below. Furthermore, only female mice were used, although the use of both sexes is highly recommended. This might be attributed to the higher aggressiveness of the male Akr1a-KO mice.
Our responses: Gender affects various physiological functions, so there may be gender differences in the finding in this study too, as pointed out by the reviewer. In case of plasma corticosterone, however, we have actually reported its increase in both male and female mice [see ref 20, 21]. Although there may be differences in their extent between gender, we consider that the similar trends could also occur in male mice regarding concept of the findings. However, since this study did not aim to examine gender differences, we do not see the significance of examining male mice.
Methods:
- Picolab 5053: Asc content of this diet?
Our responses: Since Asc is decomposed during autoclave sterilization, Asc is not included in Picolab 5053. We have briefly explained this issue in Material Methods 2.1.
- WT mice drank water and Akr1a-KO mice water containing 1.5 mg/ml Asc before the start of the experiment. Did they have comparable Asc levels in blood and tissues?
Our responses: Thank you for asking. In case of the experiment in Fig. 1, we used WT and Akr1a-KO mice supplemented with or without 1.5 mg/mL Asc (WT ± Asc and Akr1a-KO ± Asc). As indicated at 0 h in Figure 2, plasma and tissue levels of Asc were comparable between WT mouse and Akr1a-KO + Asc mouse.
- mouse blood was collected => How much blood was collected and where?
Our responses: We collected 100 µL of mouse blood from submandibular vein using 5 mm Goldenrod Animal Lancet (MEDIpoint, New York, USA). We have added this issue to Material Methods 2.3.
- “In order to isolate the BMCs, the femurs were removed from both WT and Akr1a-KO mice.” => I suppose the mice were euthanized before this procedure, so please describe how and at what timepoint after irradiation.
Our responses: We euthanized WT and Akr1a-KO mice by cervical dislocation at indicated time following exposure to X-rays. We have added this statement to Material Methods 2.4.
- “All bone marrow was flushed out” => using a solution such as PBS?
Our responses: As you considered, we flushed out with PBS. We added this issue to Material Methods 2.4.
- Cell Strainer => The company is missing
Our responses: Thank you for pointing out. We have added the company information to the text.
- RBC lysis buffer => The company and the volume that was used per sampler are missing
Our responses: Thank you for pointing out. RBC lysis buffer was prepared by mixing reagents by us, as mentioned in the text. We have added the volume 100 µL to the text.
- “BMCs were frozen with liquid nitrogen until use”: Please indicate the cell number and the freezing medium. How long was the maximal storage time?
Our responses: Thank you for asking. After centrifugation, we aspirated the supernatant and froze the remaining cell pellet without counting the cell number. When the frozen samples were used in the experiment, the protein concentration was measured before use. The samples were used within one month. We have mentioned these in the text.
- “To measure Asc levels, plasma, liver, adrenal glands and BMCs were collected and 106 used for the experiments following exposure to X-rays.” => please indicate how much (weight or volume) of each entity was used, and also the volume of the lysis buffer.
Our responses: We have added the weight of the samples and the volume of the lysis buffer to Material Methods 2.5.
- “Samples were incubated with Naph-DiPy” => please indicate the concentration and the supplier.
Our responses: We have added the concentration and the supplier of Naph-Dipy to Material Methods 2.5.
- 2.5 “The results were corrected per unit of protein.” => please indicate how the protein content was determined.
Our responses: The protein concentration of the samples was measured by Bio-Rad Protein Assay Dye Reagent. We have added the information to Material Methods 2.5.
- “Blood was collected from the mice on the first day following exposure to X-rays and again 3 days later.” => How much blood was collected and where?
Our responses: We collected 100 µL of mouse blood from submandibular vein by 5 mm Goldenrod Animal Lancet. We have added this information to Material Methods 2.3.
- “centrifugation at 800 × g for 5 min” => at room temperature?
Our responses: We have added the temperature to Material Methods 2.6.
- 2.7 “The results were corrected per unit of protein.” => please indicate how the protein content was determined.
Our responses: The protein concentration of the samples was measured by Bio-Rad Protein Assay Dye Reagent. We have added this information to Material Methods 2.7.
- “specific antibodies” => information on these antibodies is missing (which antibody, supplier, dilution)
- “HRP-conjugated secondary antibodies” => information on these antibodies is missing (which antibody, supplier, dilution)
Our responses: We have added the antibodies information together with a reference [new ref 33] to Material Methods 2.8.
- “RNA from mouse livers and BMCs was purified using ISOGEN II” => please indicate the approximate weight or volume, respectively, of these samples.
We have respectively added the weight of the samples to Material Methods 2.9.
- “cDNA was prepared using a primescript cDNA synthesis kit” => How much RNA did you use in the reverse transcription? How did you quantify the RNA? How did you check RNA integrity?
Our responses: The concentration and integrity of RNA were measured, using a spectrophotometer, and 500 ng of RNA were used for cDNA synthesis. We have added the information to Material Methods 2.9.
- RT-qPCR => a description how gene expression was calculated is missing. SVCT1 and SVCT2 mRNA levels are shown, so how did you calculate these levels from the qPCR data? To which sample did you normalize the mRNA levels? Did you use a reference gene?
Our responses: Thank you for pointing out. The relative mRNA level of SVCT1 and SVCT2 was normalized to that of HPRT1, which was used as the internal control. We have added the method to Material Methods 2.9.
- “RPMI1640 culture media containing 10% FBS and 50 U/ml each of penicillin/streptomycin” => suppliers are missing
Our responses: Thank you for pointing out. We have added the supplier information to Material Methods 2.11.
- “50 μM Asc or 200 nM dexamethasone” => suppliers are missing
Our responses: We have added the supplier information of Asc and dexamethasone to Material Methods 2.1 and 2.11, respectively.
- Fig. 1 => It would be helpful to explain the abbreviations in the figure legend.
Our responses: Thank you for kind advice. Although full spellings are provided in the text, we have added full spellings regarding WBC and WT ADX to the Fig. 1 legend again for readers’ convenience.
Discussion
- Nrf2 is an important player in the antioxidant response. What about NF-κB? It can be activated by ionizing radiation. It could also be involved in the response described in this manuscript.
Our responses:
Main function of NRF2 is induction of genes responsible for anti-oxidation, which includes glutathione-related redox ant detoxification system. Accordingly, inhibition of NRF-2 by glucocorticoid increases oxidative damage as described in Discussion. Upon activation, however, NF-κB mainly induces pro-inflammatory genes, although it is also involved in induction of anti-oxidative genes to some extent, such as manganese-superoxide dismutase. Hence, suppression of inflammatory reaction through inhibition of NF-κB is a dominant action of glucocorticoid. Thus, the two transcriptional regulators work in nearly opposite ways. We have added brief statement concerning this issue by quoting a reference on NF-κB [new ref 44]. (new ref.37, 38, page 9, Line 383-385)
Minor Comments
Our responses: Thank you very much for pointing out. We have corrected all of the issues pointed out below. We also have replaced “μl” and ”ml” to “μL” and ”mL”, respectively, throughout the manuscript including figures.
Line 64 Cells uptake Asc => Cells take up Asc
Line 76 (2Gy) => (2 Gy)
M is not a SI unit, use mol/L
mM is not a SI unit, use mmol/L
Line 147 Quantitative RT-PCR => please explain the full abbreviation
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors,
most of the points were addressed in this revision.
A few points need to be improved:
- “cDNA was prepared using a primescript cDNA synthesis kit” => How much RNA did you use in the reverse transcription? How did you quantify the RNA? How did you check RNA integrity?
Our responses: The concentration and integrity of RNA were measured, using a spectrophotometer, and 500 ng of RNA were used for cDNA synthesis. We have added the information to Material Methods 2.9.
To my understanding, a spectrometer can be used to determine concentration and purity of RNA, but not the integrity. (Micro-)gel electrophoresis is required to determine RNA integrity.
- RT-qPCR => a description how gene expression was calculated is missing. SVCT1 and SVCT2 mRNA levels are shown, so how did you calculate these levels from the qPCR data? To which sample did you normalize the mRNA levels? Did you use a reference gene?
Our responses: Thank you for pointing out. The relative mRNA level of SVCT1 and SVCT2 was normalized to that of HPRT1, which was used as the internal control. We have added the method to Material Methods 2.9.
In Supplementary Table S1, a gene ID (e.g. from NCBI) is missing. Also, the resulting length of the amplicon should be mentioned.
The calculation of the relative mRNA level based on Ct values is still missing. As no PCR efficiency determination was used, I guess that the delta-delta-Ct method was used. Please explain.
Suppl. Fig. 1 B y-axis legend & depiction: “neutrophil count” => “Neutrophil count”
Author Response
Thank you gain for taking time to review our manuscript. We have included our responses after your comments.
most of the points were addressed in this revision.
A few points need to be improved:
- “cDNA was prepared using a primescript cDNA synthesis kit” => How much RNA did you use in the reverse transcription? How did you quantify the RNA? How did you check RNA integrity?
Our responses: The concentration and integrity of RNA were measured, using a spectrophotometer, and 500 ng of RNA were used for cDNA synthesis. We have added the information to Material Methods 2.9.
To my understanding, a spectrometer can be used to determine concentration and purity of RNA, but not the integrity. (Micro-)gel electrophoresis is required to determine RNA integrity.
Our responses: Thank you for your comments. As you thought, we determined concentrations of RNA by a spectrometer. Although we did perform gel electrophoresis, the quality of the RNA was considered to be assured since no significant difference was observed in the amplification level of the reference gene, HPRT1.
- RT-qPCR => a description how gene expression was calculated is missing. SVCT1 and SVCT2 mRNA levels are shown, so how did you calculate these levels from the qPCR data? To which sample did you normalize the mRNA levels? Did you use a reference gene?
Our responses: Thank you for pointing out. The relative mRNA level of SVCT1 and SVCT2 was normalized to that of HPRT1, which was used as the internal control. We have added the method to Material Methods 2.9.
-In Supplementary Table S1, a gene ID (e.g. from NCBI) is missing. Also, the resulting length of the amplicon should be mentioned.
Our responses: Thank you for your comment. We have added the gene ID and product length to Supplementary Table S1.
-The calculation of the relative mRNA level based on Ct values is still missing. As no PCR efficiency determination was used, I guess that the delta-delta-Ct method was used. Please explain.
Our responses: Thank you for pointing out. As you mentioned, we used delta-delta-Ct method. We have added the method to Material Methods 2.9.
Suppl. Fig. 1 B y-axis legend & depiction: “neutrophil count” => “Neutrophil count”
Our responses: Thank you for pointing out. We corrected the notation in Supplementary Figure 1B.
