Transcriptomes of Wet Skin Biopsies Predict Outcomes after Ionizing Radiation Exposure with Potential Dosimetric Applications in a Mouse Model

Round 1

Reviewer 1 Report

This study performed in mice is aimed at analysing the modulation of gene expression following increasing doses of ionising radiations. Although the transposition from animals to humans is not obvious, such a study may be of interest to manage the problems of exposure to ionising radiations in humans if the design is appropriate.

The study is well performed, although the number of animals per condition (5) might be considered as low. Anyway, the data are of interest to analyse the transcriptomic modulation promoted by ionising radiations.

Major point :

In the discussion, what is lacking is the deduction from the data of some biomarkers (gene modulation) following low doses of irradiation. This would be particularly important (if transposed to humans) for the follow-up of workers exposed to ionising radiations. The discussion is long and might be more synthetic and focused on the most relevant conclusion.

Minor points :

·      Methods, line 138 : replace "d0" by "h0".

·      Methods, lines 138-140 : the biopsies were probably harvested under anaesthesia, so this must be indicated.

·      Methods, line 159 : it is not obvious to grind frozen biopsies with the use of a mortar and a pestle; was a particular equipment used for that ? Please describe this step in more details.

·      Methods, lines 170-171 : a RT-PCR is indicated (which allows the analysis of the modulation of a few genes), whereas the study is performed using a whole genome microarray. So, the term "PCR" is not adequate. Please clarify.

·      In the pdf file, the "Expr Fold change" column of the Table 5 is too narrow, rendering the data difficult to read, so in the final form for publication be sure that the tables will be properly printed and easy to read.

Author Response

Reviewer 1:

We thank the reviewer for the time they invested in the critical review of the manuscript. We have addressed all the points and concerns brought up primarily by adjusting the manuscript. We hope that the reviewer will find our answers and adjustments satisfactory. We are ready to answer any additional questions or inquiries she/he may still have. Below is our point-by-point response to the reviewers’ concerns.

Comments and Suggestions for Authors

This study performed in mice is aimed at analysing the modulation of gene expression following increasing doses of ionising radiations. Although the transposition from animals to humans is not obvious, such a study may be of interest to manage the problems of exposure to ionising radiations in humans if the design is appropriate.

The study is well performed, although the number of animals per condition (5) might be considered as low. Anyway, the data are of interest to analyse the transcriptomic modulation promoted by ionising radiations.

Major point :

In the discussion, what is lacking is the deduction from the data of some biomarkers (gene modulation) following low doses of irradiation. This would be particularly important (if transposed to humans) for the follow-up of workers exposed to ionising radiations. The discussion is long and might be more synthetic and focused on the most relevant conclusion.

• The discussion section was reduced as much as possible without taking out any of the points that summarize the manuscript finds.

Minor points :

• Methods, line 138 : replace "d0" by "h0".
• We thank the reviewer. This was to denote that day 0 (d0) samples were collected at 2 hours post-exposure (h2).
• Methods, lines 138-140 : the biopsies were probably harvested under anaesthesia, so this must be indicated.
• The sentence now reads “Briefly, the animals’ dorsa were shaved using standard veterinary clippers and 1 cm2 biopsy from each animal was collected while the animal is under anesthesia as described above”.
• Methods, line 159 : it is not obvious to grind frozen biopsies with the use of a mortar and a pestle; was a particular equipment used for that? Please describe this step in more details.

- Done. The following sentence was added in the material and methods section “A Porcelain Mortar, Size 0, 50 mL, 70 mm Diameter, Coors, 60310 and a Porcelain Pestle, Size 0, 114 mm Long, Coors, 60311 (Porcelain Mortars and Pestles. CoorsTek, Manufacturer: Coorstek, Family Part #: COORS TSI-603) were used to grind the tissue for RNA extraction”.

• Methods, lines 170-171 : a RT-PCR is indicated (which allows the analysis of the modulation of a few genes), whereas the study is performed using a whole genome microarray. So, the term "PCR" is not adequate. Please clarify.
• We thank the reviewer for this catch. We have removed any reference to RT PCR.
• In the pdf file, the "Expr Fold change" column of Table 5 is too narrow, rendering the data difficult to read, so in the final form for publication be sure that the tables will be properly printed and easy to read.
• We agree with the reviewer. We were surprised to see this in the version downloaded from the journal. It seems this happened during the editing of the manuscript at the journal. We will make sure that all tables and figures are properly illustrated in the final version.

Reviewer 2 Report

The manuscript describes several genes as potential dosimetry markers. The work is interesting and with some useful data, but major changes are required.

1. Figures 3, 5, 6, and 7 are low quality. Due to this inconvenience, the written results cannot be compared with them.

2. Why did the authors use radiation doses 1, 3, 6, and 20 Gy? Did the authors determine LD₅₀ for the mice?

3. How can the findings made in this study be used in clinical practice?

4. The dosimetry has to be fast and precise. Did the study meet these demands?

Author Response

Reviewer 2:

We thank the reviewer for the time invested in the critical review of the manuscript. We have addressed all the points and concerns brought up primarily by adjusting the manuscript. We hope that the reviewer will find our answers and adjustments satisfactory. We are glad to answer any additional questions or inquiries. Below is a point-by-point response to the reviewer’s concerns.

Comments and Suggestions for Authors

The manuscript describes several genes as potential dosimetry markers. The work is interesting and with some useful data, but major changes are required.

1. Figures 3, 5, 6, and 7 are low quality. Due to this inconvenience, the written results cannot be compared with them.

- We were surprised to see the same problem in the version downloaded from the journal. It seems the quality of the indicated figures was reduced during manuscript editing at the journal which would make the comparison a little cumbersome. We will bring this to the attention of the journal editor and make sure that all tables and figures are of the best possible quality at publication.

2. Why did the authors use radiation doses 1, 3, 6, and 20 Gy? 

The doses were selected to include a lethal dose within the duration of the experiment (20Gy is lethal in less than 28 days) and to cover doses that cause severe morbidities a few post-exposure days (6Gy), survivable with or without medical care (1, 3 Gy) in mice. The goal was to assess the potential of genomics in differentiating lethal and nonlethal IR doses in the used mouse population and explore any transcriptomic trends or alterations associated with IR exposures that require urgent versus delayed medical support under mass casualty nuclear event. Similar to other diagnostics development, it is important to note that any newly indicated trends require further assessment and validation using a larger and more diverse population of mice to include different strains, ages, and comorbidities before extending findings to responses in humans.

While confident of the reported findings and potential for diagnostics applications, the authors highlighted that the results are still in the proof-of-principle phase and additional validation is required before consideration in dosimetric radiation diagnostics and countermeasures.

- Did the authors determine LD₅₀ for the mice?

The LD50 of radiation in mice was established in previous work by many other coworkers in the field. We selected the IR doses and study duration based on LD50 described by others and our resource limitations. The survival rates in our results, where all animals that have received 6Gy or less survived the 28 days duration of the study while animals exposed to 20Gy didn’t survive past day 7, suggests strongly that our results fell in the same LD50 range described elsewhere. A precise assessment of the LD50 requires the use of a large number of animals, long durations of animals housing and monitoring, and difficult IACUC approval for already published work that all fell beyond the scoop and goals of this work goals and resources.

3. How can the findings made in this study be used in clinical practice?

- The study findings after validation would guide the manufacturing of a panel of genes (10 – 25) transcription quantification that can be used to identify the absorbed dose of IR and determine the immediate triage and care plan for IR-exposed individuals in a mass or small radiation or nuclear event. Additional validation is necessary to validate the manuscript findings and transcriptomic trends using a mice population with larger age diversity, then confirmation of results in humans.

The authors adhered to report data-supported findings under the study described experimental conditions. The authors acknowledged the limitations of the findings at this stage to avoid any claims that are not supported by data.   

4. The dosimetry has to be fast and precise. Did the study meet these demands?

- We thank the reviewer for bringing up this important point. A downside of this work is that it requires a skin biopsy which might be a deterrent for some patients. Technical difficulties, such as RNA extraction, and assessment of transcription of a short panel of biomarker genes should don’t pose a challenge given recent advances in molecular technology and the urgent need for a device that addresses this medicinal gap in response to radiation threats.

We are currently assessing the trends identified in this work using blood samples for the same animal model using different age populations of mice to simplify the processes of sample collection and mRNA isolation. Alternatively, we are testing immediate sequencing techniques to simplify transcription quantification.

In essence, the authors indicated throughout the discussion that the results are solid but limited by the used experimental conditions and still at the proof-of-concept level. Further advancements are needed before introducing the translational applications of findings. Validation using a wider animal population is underway given the known diverse responses to radiation based on age and comorbidities of mice.   

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors answered all questions with valid points.