The Therapeutic Potential of Oxyhydrogen Gas in Oncology: A Study on Epstein–Barr Virus-Immortalised B-Lymphoblastoid (TK6) Cells

Round 1

Reviewer 1 Report

Thanks for your review manuscript entitled" The Therapeutic Potential of Oxyhydrogen Gas in Oncology: A  Study on Epstein-Barr Virus-immortalised B-lymphoblastoid  (TK6) Cells'' I read this manuscript with great interest and felt it needs some modifications to improve your manuscript for considering in this journal. I am sharing my comments and suggestions details below.

Comments and suggestions

1. In the abstract,  the abstract starts with a general statement about B-cells and their relevance to the immune system, but it could benefit from a brief introductory sentence explaining why studying them in this context is important.

2.  Methods: While the methods are mentioned, it would be helpful to briefly outline the experimental setup or approach used in the study. What are the key variables and controls? Please check cell name, materials, reagent, and company name has been written correctly.

3. Please check Figure 1 & 2 for retention of H₂ and O₂ plotted corrected. Also, check the figure resolution.

4. Figure 3 (A & B) is not clear. Please increase figure resolution as per journal guidelines for visualization.

5. In Figure 5, Ogyhydrogen 24 hours G2(10%; S/P: 19%), similarly after 48 hours (G2(10%; S/P: 19%)) is correct?

6. Summary is necessary as a separate section? Or it can be added end of the discussion section.

7.  Please check reference formatting as per journal guidelines.

8. Is it necessary to put citations in the conclusion sections?

9. Please check all typos, syntax, and grammatical errors throughout the manuscript.

 Moderate editing of English language required

Author Response

Reviewer 1

Thank you for your valuable contribution. To address your comments I have altered the manuscript as follows:

I have added a short sentence and amended the abstract to reflect the wider aspects of this research

I have updated the methods section to include the sterilisation and infusion protocols. All names, materials etc. are clear within the text and researchers can access reagents, suppliers etc. with the information available.

All figures have been updated; this should solve the resolution issues. The data and plotting in original figures 1 and 2 (now 3 and 4) are correct. Figure 5 has been updated with precise (not rounded) data for clarity – thank you for pointing this out it is greatly appreciated.

I would like to keep the summary section separate from the discussion as I am aware many researchers like to skim-read articles and this will allow them to extract the key points readily.

References have been updated

Citations in the conclusion have been removed

The document has been through an AI check for punctuation, grammar and syntax – English spellings have been changed to American English.

Reviewer 2 Report

 I thought overall the manuscript was very good.
I did think there needed to be a better rational for why oxyhydrogen was being utilized rather than simply hydrogen. Is it thought to be more effective?

In much of the citing literature, simply hydrogen is the substance described rather than oxyhydrogen.w

In the graphical abstract, I didn’t understand what was being illustrated in line 2. An explanation would be helpful, although I wasn’t able to see what the grey block was illustrating.

In citing literature, oxyhydrogen was utilized.

Are they treated as equivalent, or are there differences?

Was this a total replacement for air as a breathing mixture, or was it given as an additive to atmospheric air, such as liters per minute via nasal cannula.

Is there a concern about oxyhydrogen being an explosive mixture?

I can’t think of a more explosive mixture than 30%O2/70%H2.

In Fig.5, SubG is a heading, however in the text, Sub G1 is reported. Are these the same?

I should be corrected.

Does oxyhydrogen actually kill the malignant cells, or is it simply inhibitory?
Can you make such a statement or is it speculative?

Author Response

Thank you kindly for your valuable comments. To address your findings I have altered the manuscript as follows:

I have added a short section to the rationale explaining why oxyhydrogen was utilised – this may also address the comment that oxyhydrogen is cited in the references. Unfortunately, to date I am not aware of any direct comparative studies and any differences between H2 and oxyhydrogen have yet to be investigated. Comparative analysis will be a very exciting development in the field.

The graphical abstract has been updated

The gas was infused into the cell growth media, described in the methods section

All figures have been updated to resolve resolution issues (Sub G – Sub G1 also amended)

At this juncture, it would be pure speculation as to whether oxyhydrogen kills or inhibits growth as much more research in this area is required.

Ignition of oxyhydrogen gas is a concern; however, this has been well addressed in the current literature. I have added a very short section highlighting the risks of oxyhydrogen.

Reviewer 3 Report

This study investigated the effects of dissolved oxyhydrogen on p53-positive immortalised B-cells (TK6 cells). They found that oxyhydrogen may inhibit the proliferation of TK6 cells by promoting cell apoptosis, suggesting a potential inhibitory effect of oxyhydrogen treatment on EB virus infection-related tumors. In summary, this study provides some reference value for the application of oxyhydrogen in anti-tumor therapy. However, the study also has some issues as follows:

1. The introduction section mentions that oxyhydrogen is produced by the electrolysis of water in the presence of catalysts such as potassium hydroxide. In fact, it can also be produced by directly electrolyzing pure water without the need for potassium hydroxide.

2. In the materials and methods section, it is mentioned that hydrogen concentration was measured every 5 minutes using the H2Blue™ titration method, but data for the 20th and 25th minutes are missing in Figure 1. Similarly, the monitoring of oxygen concentration in Figure 2 also lacks data for these two time points.

3. Figure 3B is not clear, especially the asterisk is partially obscured.

4. It would be beneficial to include a control group treated with pure hydrogen in the study to compare the differences between oxyhydrogen treatment and pure hydrogen treatment. This would help determine if the effects on TK6 cells are due to hydrogen.

5. The study focused on cell lines infected with EB virus, so it's important to investigate whether oxyhydrogen has any impact on the EB virus itself and if its effects are related to influencing the EB virus.

Author Response

Thank you kindly for your valuable comments. To address your findings I have altered the manuscript as follows:

It is my understanding that most commercial devices utilise a catalyst for water electrolysis, however, the word ‘typically’ has been added for clarity

I have amended the methods section to clearly explain when the measurements were taken – thank you for pointing out this discrepancy, it is greatly appreciated

All figures have been updated as there were resolution issues

I am not aware of any direct comparative studies and any differences between H2 and oxyhydrogen have yet to be made available. Comparative analysis will be a very exciting development in the field, as would the direct effects of H2/oxyhydrogen on virus replication, however, with regret, this is beyond the scope of this study.

Round 2

Reviewer 1 Report

Thanks for improving your manuscript.