Ionic Liquid 1-Octyl-3-Methylimidazolium (M8OI) Is Mono-Oxygenated by CYP3A4 and CYP3A5 in Adult Human Liver

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is mainly edited correctly and contains important information from a scientific and social point of view.
Requires slight adjustments:
1. Figure 1c is illegible,
2. The description of statistical methods should be included as a subsection in the Materials and Methods chapter.

Author Response

Thank you for your email of the 15^th June 2024.  We would like to thank the reviewers for their helpful and constructive comments.  We have uploaded a revised manuscript as a track changes document so that changes can be identified as required.  In response to the comments made by the reviewers, please see responses below:

Reviewer #1

Requires slight adjustments:
1. Figure 1c is illegible,
2. The description of statistical methods should be included as a subsection in the Materials and Methods chapter.

 

We thank the reviewer for their advice.  We have:

1. Increased the size of the Fig1c so that it is more legible.
2. The statistical methods were already described in the Materials and Methods section 2.10 of the manuscript. On advice of other reviewers, this remains there and legends are shortened.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors of the work jox-2992090, I make some constructive observations on your work.

Introduction

-I suggest that you please include the objective of the work

-I suggest that information on the chemical structures of the products M80I and COOH7IM be included, it is not enough to cite other works.

Section 2.2

-Line 101, please indicate if the procedure is similar or with changes according to the bibliographic citation (11).

-Line 102, please include the approval number, as well as the name of the institution where the Local Ethics Committee belongs.

-Line 165, TRIzol is the generic name of: TRI Reagent®, please clearly indicate which product you used, as well as the name of the manufacturer.

-Line 167, please briefly describe what you did, it is not enough to include a bibliographical citation.

-Line 210, please briefly describe what you did, it is not enough to include a bibliographic citation}

Results

-Table 1. The information in the table should be described in the “Introduction” section

-Line 233, what do the bibliographic citations (15,16) mean? the data are concordant or discordant, or are results of (15,16).

-Figure 1. Lines 248, 250, what does the bibliographic citation (10) mean?

-Line 267, Figure 1, has sections A, B, C; Please include which section to go to.

-Figure 2, please omit non-relevant information, for example, the statistical test used, they already said that in the “methods” section, it is enough to describe the significance value.

-Lines 322-332, The text seems more like a discussion than a presentation of their results, please limit yourself to describing what you found.

-Lines 352-360, The text seems more like a discussion than a presentation of their results, please limit yourself to describing what you found.

-Discussion, it seems to me that the discussion is sufficient but not good.

Author Response

Reviewer #2

Dear authors of the work jox-2992090, I make some constructive observations on your work.

Introduction

-I suggest that you please include the objective of the work

We have modified the abstract to more explicitly indicate the objective of this study: “The objective of this study was to examine the metabolism of M8OI in humans in more detail”.  This is now also re-iterated in the introduction.

-I suggest that information on the chemical structures of the products M80I and COOH7IM be included, it is not enough to cite other works.

The structures were already essentially present in Fig1a.  However, we have clarified with additional details in the legend.

Section 2.2

-Line 101, please indicate if the procedure is similar or with changes according to the bibliographic citation (11).

Now indicated

-Line 102, please include the approval number, as well as the name of the institution where the Local Ethics Committee belongs.

It stated in the manuscript Institutional Review Board Statement: “Human hepatocytes were isolated from organs after undergoing normothermic machine perfu-sion as part of an existing research project (IRAS number 179433, NHS Blood and Transplant liv-er study 52, NuTH R&D study 7483) essentially as previously outlined [10].  After transfer to the Newcastle Transplant Tissue Biobank [Newcastle and North Tyneside Research Ethics Commit-tee 1 (5979, REC:17/NE/0022)].”.  That this is the case is now indicated.

-Line 165, TRIzol is the generic name of: TRI Reagent®, please clearly indicate which product you used, as well as the name of the manufacturer.

This is now done.

-Line 167, please briefly describe what you did, it is not enough to include a bibliographical citation.

This is now done.

-Line 210, please briefly describe what you did, it is not enough to include a bibliographic citation}

This is now done.

Results

-Table 1. The information in the table should be described in the “Introduction” section

Table 1 moved to introduction section

-Line 233, what do the bibliographic citations (15,16) mean? the data are concordant or discordant, or are results of (15,16).

Line 233 states “Fig. 1c demonstrates that HO8IM was barely detectable in hepatocyte cultures except in the presence of the CYP2E1 and ADH inhibitor pyrazole [15,16]”.  Refs 15 and 16 report that pyrazole inhibits the enzymes CYP2E1 and ADH.  I have modified the sentence to clarify to thus:

Fig. 1c demonstrates that HO8IM was barely detectable in hepatocyte cultures, except in the presence of pyrazole, which is a CYP2E1 and ADH inhibitor [15,16]

-Figure 1. Lines 248, 250, what does the bibliographic citation (10) mean?

Reference 10 is providing a citation that demonstrates that these non-specific inhibitors suggests that a CYP(s) an ADH(s) and an AcDH(s) are likely involved in the metabolism of M8OI.

-Line 267, Figure 1, has sections A, B, C; Please include which section to go to.

This now done

-Figure 2, please omit non-relevant information, for example, the statistical test used, they already said that in the “methods” section, it is enough to describe the significance value.

This now done, and for all the other figure legend statements.

-Lines 322-332, The text seems more like a discussion than a presentation of their results, please limit yourself to describing what you found.

The text has been modified and discussion text moved to discussion section as suggested.

-Lines 352-360, The text seems more like a discussion than a presentation of their results, please limit yourself to describing what you found.

The text has been modified and discussion text moved to discussion section as suggested.

-Discussion, it seems to me that the discussion is sufficient but not good.

The discussion has been modified.

Reviewer 3 Report

Comments and Suggestions for Authors

About "The ionic liquid 1-octyl-3-methylimidazolium (M8OI) is mono- 2 oxygenated by CYP3A4 and CYP3A5 in adult human liver". This paper has poor logic. The manuscript is not suitable for publication in this journal.Here are my comments:

(1) Comparisons of gene expression or any other biomarker between the sexes based solely on hepatocyte samples from three individuals (only one of whom was female) cannot draw generality conclusions, such as CYP3A4 and CYP3A5 subtypes are more expressed in women than in men;

(2) In this paper, only cell experiments were used for verification, and neither in vitro nor in vivo verification was carried out;

(3) The discussion part of this paper is not logical.

Author Response

Reviewer #3

1) Comparisons of gene expression or any other biomarker between the sexes based solely on hepatocyte samples from three individuals (only one of whom was female) cannot draw generality conclusions, such as CYP3A4 and CYP3A5 subtypes are more expressed in women than in men;

The reviewer is correct and in fact we already stated this in our discussion.  We accept the limitations inherent in using human hepatocytes.  However, as a qualitative study it clearly identifies for the first time the isoforms of CYP the mediate the metabolism of M8OI and that in the absence of CYP3A4, in occasional individuals, there is negligible metabolism.  This is novel new data. 

It should be appreciated that the use of primary human hepatocytes is challenging but can be highly informative for the general population.  A search for papers in the Journal of Xenobiotics on PubMed shows that 162 papers have been published since 2015.  In that time, no papers have been published using human hepatocytes. This is because isolating viable primary human hepatocytes is challenging in terms of access to tissue, cost (The purchase of just 5 million cryopreserved human hepatocytes for the final part of this study, in total was around $3500 and these are substandard compared to fresh cells!) and limited expertise.  Our procedure is unique in that it uses a re-conditioning technique that involves whole organ perfusion with human blood and technologies employed in transplantation.  The work presented in this paper therefore represents a body of work that could not be performed by any other lab in the UK, and possibly few in the EU.

(2) In this paper, only cell experiments were used for verification, and neither in vitro nor in vivo verification was carried out;

The paper is an in vitro study using human hepatocytes and supersome preparations.  Therefore we do not understand the suggestion by the reviewer that “neither....in vitro .... verification was carried out”!  I am uncertain what the reviewer means by in vivo verification, since this would mean administering M8OI to human participants, which is unethical.

(3) The discussion part of this paper is not logical.

We have re-written the discussion and thank the reviewer for their advice.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors of the work JoX-2992090, it seems to me that you resolved all the observations I made about your work in a good way. I extend congratulations for the improvements to your work.

Author Response

Response: We note this reviewer deems our manuscript acceptable.  We thank the reviewer for helping us to improve our manuscript. 

Reviewer 3 Report

Comments and Suggestions for Authors

About The article "The ionic liquid 1-octyl-3-methylimidazolium (M8OI) is mono-oxygenated by CYP3A4 and CYP3A5 in adult human liver", My suggestions are as follows:

1. I suggest the author increase the number of samples, as three samples are not enough to support the conclusion;

2. Comparisons of gene expression or any other biomarker between the sexes based solely on liver cell samples from three individuals, only one of whom was female, cannot draw generalizations; 

3. It is suggested that the author increase the conditions for sample selection.

Author Response

This report demonstrates for the first time that, as stated in the title: “The ionic liquid 1-octyl-3-methylimidazolium (M8OI) is mono-oxygenated by CYP3A4 and CYP3A5 in adult human liver”.  It is therefore a qualitative study and examination of more human donors would not change this conclusion.  It is NOT a quantitative study that claims to identify the proportion of the human population (or any differences between that proportion between males and females) that metabolise M8OI.  In case the reviewer is unclear about what this means in terms of our study, a qualitative study indicates how M8OI is metabolised (i.e. via CYP3A4 and 5) and for example, why ketoconazole enhances the toxicity of M8OI in human hepatocytes (because it inhibits M8OI metabolism by the above CYPs).  To draw reliable conclusions quantitatively on this issue would require at least several tens of donors which is not feasible (it would probably take 10-20 years based on past experience).  Indeed, the conclusion we make in our study might be drawn from the supersome data alone and therefore the human hepatocyte data could be considered superfluous.  The added value of the human hepatocyte data is:

1. We demonstrate metabolism in human hepatocytes.
2. We demonstrate for the first time that an individual with negligible CYP3A4 also has negligible metabolism of M8OI. This is somewhat of a chance finding and it is unlikely such an individual will be found any time in the near future (let alone another 2 donors, to satisfy this reviewer). The point is, that this individual uniquely supports - in the most biologically realistic human liver system possible - that CYP3A4 is a major player in M8OI metabolism.
3. We demonstrate that the metabolism of M8OI to the hydroxylated and carboxylated metabolites is a detoxification pathway (using 5 pooled donors).

This reviewer does not seem to appreciate that the use of fresh primary human hepatocytes in research is rarely performed because it is so challenging in terms of resources and difficulties with respect to tissue access.  Most tissue is reserved for transplantation, which understandably takes priority.  This is probably why not a single paper has been published in your Journal with human hepatocytes.  We re-iterate what we said previously:

A search for papers in the Journal of Xenobiotics on PubMed shows that 162 papers have been published since 2015.  In that time, no papers have been published using human hepatocytes.

We hope the reviewer can appreciate our response.  In actual fact, the requests are likely not possible to fulfil since the time and resources required far exceed what is available from funding authorities in terms of support.  It should be appreciated that to perform these studies has required surgical research fellows travelling around the UK to harvest organs from donors; rapid transport to Newcastle, re-conditioning (up to 24 hours warm perfusion with human blood with constant monitoring) followed by a separate collagenase perfusion to isolate hepatocytes.  A key problem still impacting this procedure is the availability of human blood, which has been either unavailable or limited since the COVID pandemic.

If this reviewer’s view prevails (1 of 3 reviews in total, the 2 other reviewers are content), it will be unlikely that this Journal will ever publish data using human hepatocytes.  Given that the Journal’s focus is on xenobiotics and the liver is the major determinant in xenobiotic metabolism and toxicity, it seems the Journal will be limiting its impact in terms of the papers it will publish if it does not understand the qualitative nature of our manuscript and the issues surrounding the use of human hepatocytes in research.