Underexplored Molecular Mechanisms of Toxicity
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments to authors:
The manuscript entitled “Underexplored Molecular Mechanisms of Toxicity” by Arowolo & Suvorov described an interesting phenomenon in a statistical way that molecules and mechanisms in toxicity research may have bias. The approach is acceptable, but the overall conclusion is not convincing (enough). The manuscript is not ready to be published before a serious re-thinking and revision.
My major concern is that the focus on certain genes (mechanisms and pathways) may not be due to social bias, but rather an imbalance in research work distribution on chemicals. The situation might be as follows: a limited number of chemicals, which target specific genes, attract significant attention from toxicology researchers, leading to other genes being overlooked. This is because the chemicals related to these other genes are under-investigated.
Author Response
Reviewer 1: My major concern is that the focus on certain genes (mechanisms and pathways) may not be due to social bias, but rather an imbalance in research work distribution on chemicals. The situation might be as follows: a limited number of chemicals, which target specific genes, attract significant attention from toxicology researchers, leading to other genes being overlooked. This is because the chemicals related to these other genes are under-investigated.
Response: We are thankful to the reviewer for sharing an alternative hypothesis explaining the reason of the bias in research attention to specific genes. We tested and rejected this hypothesis in our previous research (PMID: 33182146). To test this hypothesis, we calculated sensitivity of genes to chemical exposures using data from non-overlapping lists of chemicals. The results demonstrated that as soon as big enough (saturated) list of chemicals is used the resulting sensitivity values per genes, and enriched pathways stay the same. In other words, research bias towards specific genes, and neglect of other genes is not determined by chemicals which are the current research focus. This finding is discussed in the manuscript – lines 87-89.
Reviewer 2 Report
Comments and Suggestions for AuthorsI have gone through the manuscript titled "Underexplored Molecular Mechanisms of Toxicity" bearing number JoX (ISSN 2039-4713). Although the manuscript is good, the scientific rationale sound, and the results timely and important, some more aspects need to be considered like:
The hypothesis of the study of the predominance of certain genes due to a social phenomenon is presented more as an opinion, I consider that more argument is needed. It could be indicated in the discussion section instead of the introduction.
What was the rationale for using only vertebrates in the analysis, or the human genome plus murine models, why, other organisms, such as invertebrates, were not used.
The insensitive not explored genes represent a large percentage, there is no mention or discussion of them.
Although the reference was added, it is better to briefly explain the rationale for cutoff point identification that defines the threshold between underexplored and well-explored genes.
The data shown in Fig 1-A may also be shown in the form of a table. It may be good to highlight the results
Functional cluster analysis, using DAVID (or other) of the underexplored sensitive genes list could be performed. The top 10-20 clusters could be listed.
At least for the most significantly enriched biological categorie (fatty acid catabolism) a map of the pathway by KEGG or reactome could be shown to identify at what level, the most underexplored sensitive genes are located.
It could also be interesting to see general graphs of the cellular component and the molecular functions of the underexplored genes.
An association of the functions of the most underexplored sensitive genes with potential diseases could be shown and discussed.
Author Response
Reviewer 2: I have gone through the manuscript titled "Underexplored Molecular Mechanisms of Toxicity" bearing number JoX (ISSN 2039-4713). Although the manuscript is good, the scientific rationale sound, and the results timely and important, some more aspects need to be considered like:
Response: We are very thankful for the positive review of the manuscript.
Reviewer 2: The hypothesis of the study of the predominance of certain genes due to a social phenomenon is presented more as an opinion, I consider that more argument is needed. It could be indicated in the discussion section instead of the introduction.
Response: The goal of the current study is to identify genes/molecular pathways which respond to chemical exposures but are underexplored by toxicological research. Completely different design of the study is needed to prove that medico-biological research is biased towards a subset of genes due to social reasons. These studies have been conducted by other groups and are discussed in the introduction – lines 26-48. The studies cited in these paragraphs provide an unambiguous proof that social factors are the major source of bias. Given that our study does not contribute to the understanding of the causes of this bias we believe we cannot contribute significantly to the matter in Discussion section.
Reviewer 2: What was the rationale for using only vertebrates in the analysis, or the human genome plus murine models, why, other organisms, such as invertebrates, were not used.
Response: Given that our study is gene-centric and combines data from hundreds of studies we need to ensure that gene IDs are the same across all studies and used databases and that big enough numbers of studies are present to generalize (identification of gene sensitivities to a range of compounds and identification of publications per gene). These constraints were the reason for the selection of humans and major mammalian models (mice and rats) for this study. Additionally, inclusion of distant organisms will complicate the enrichment analysis as among groups of genes (say underexplored sensitive to chemicals) some genes may belong to mammals, some to insects, etc. … We have added this information to the manuscript – lines 96-101. Additionally, we added the discussion of the relevance of our findings for other species – lines 224-237.
Reviewer 2: The insensitive not explored genes represent a large percentage, there is no mention or discussion of them.
Response: We conducted this analysis and have added the relevant text (lines 150-154) and Supplemental Table 2.
Reviewer 2: Although the reference was added, it is better to briefly explain the rationale for cutoff point identification that defines the threshold between underexplored and well-explored genes.
Response: We have added this explanation – lines 111-115.
Reviewer 2: The data shown in Fig 1-A may also be shown in the form of a table. It may be good to highlight the results
Response: Given the size of the table (16,096 genes) we are unable to include it in the text of the manuscript. So, we include this table as a new Supplemental Table 1.
Reviewer 2: Functional cluster analysis, using DAVID (or other) of the underexplored sensitive genes list could be performed. The top 10-20 clusters could be listed.
Response: We added DAVID analysis – see lines 161-172 and Supplemental Table 3.
Reviewer 2: At least for the most significantly enriched biological categorie (fatty acid catabolism) a map of the pathway by KEGG or reactome could be shown to identify at what level, the most underexplored sensitive genes are located.
Response: To illustrate highly enriched pathways relevant to lipid metabolism we added figures S1 and S2 illustrating maps of steroid biosynthesis and peroxisome pathways with highly chemically-sensitive underexplored genes marked.
Reviewer 2: It could also be interesting to see general graphs of the cellular component and the molecular functions of the underexplored genes.
Response: We added enriched molecular function categories to table 1. There were no significantly enriched cellular component categories.
Reviewer 2: An association of the functions of the most underexplored sensitive genes with potential diseases could be shown and discussed.
Response: We added a Metascape analysis with DisGeNET disease categories and identified that underexplored sensitive genes are associated with drug induced liver injury, hepatic steatosis and other pathology of metabolism of lipids and carbohydrates, - see lines 172-175 and Fig. S3. We also added the discussion of the relevance of underexplored genes to human diseases in discussion – lines 194-205.
Reviewer 3 Report
Comments and Suggestions for AuthorsIn this manuscript, Arowolo and Suvorov describe underexplored molecular mechanisms of toxicity based on publicly available databases. Metabolism of fatty acids, amino acids, and glucose were identified as major underexplored toxicity mechanisms. I found this article "clean", interesting, and scientifically relevant. I have just three minor suggestions:
- Line 32: I found super interesting the authors cited “G protein-coupled receptors” at this point. The involvement of CCR5 in non-classical mechanisms of genotoxicity was recently explored in this article (https://doi.org/10.1016/j.fct.2024.114511), which the authors may find interesting. This article suggestion may also be useful for the discussion section.
- 2.1. Sensitivity of genes to chemical exposures: Data from the Comparative Toxicogenomics Database (CTD) are constantly being updated. Please cite the date (or CTD Revision number) when the data was extracted from the CTD. Also, correct to “Toxicogenomics” (line 85) and add a link for the database (maintaining the reference).
- Discussion: It may be interesting to add a paragraph to the discussion section highlighting that these results are important for both human toxicology and ecotoxicology, as many toxicity mechanisms affecting wildlife, for example, are varied and may not correspond to the classical toxicity mechanisms described for humans. The results described in this study contribute to advancing aspects of ecotoxicology too.
Author Response
Reviewer 3: In this manuscript, Arowolo and Suvorov describe underexplored molecular mechanisms of toxicity based on publicly available databases. Metabolism of fatty acids, amino acids, and glucose were identified as major underexplored toxicity mechanisms. I found this article "clean", interesting, and scientifically relevant. I have just three minor suggestions:
Response: We are very thankful for the positive review of the manuscript.
Reviewer 3: - Line 32: I found super interesting the authors cited “G protein-coupled receptors” at this point. The involvement of CCR5 in non-classical mechanisms of genotoxicity was recently explored in this article (https://doi.org/10.1016/j.fct.2024.114511), which the authors may find interesting. This article suggestion may also be useful for the discussion section.
Response: We are thankful for this reference, and we used it in our discussion – lines 199-205.
Reviewer 3: - 2.1. Sensitivity of genes to chemical exposures: Data from the Comparative Toxicogenomics Database (CTD) are constantly being updated. Please cite the date (or CTD Revision number) when the data was extracted from the CTD. Also, correct to “Toxicogenomics” (line 85) and add a link for the database (maintaining the reference).
Response: We made the changes as advised.
Reviewer 3: - Discussion: It may be interesting to add a paragraph to the discussion section highlighting that these results are important for both human toxicology and ecotoxicology, as many toxicity mechanisms affecting wildlife, for example, are varied and may not correspond to the classical toxicity mechanisms described for humans. The results described in this study contribute to advancing aspects of ecotoxicology too.
Response: We are thankful for this advice, and we added a paragraph in the Discussion to cover the relevance of our findings for ecotoxicology – lines 224-237.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThis is a significantly improved version of the manuscript; I thank the authors for clarifying some of the issues. Congratulations to the authors for this exciting work.
I would just like to add that the authors have shown new figures (S1-S3) that highlight the importance of the research. I strongly suggest that they be presented as figures within the article and not as supplementary material.
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Author Response
Comment 1: This is a significantly improved version of the manuscript; I thank the authors for clarifying some of the issues. Congratulations to the authors for this exciting work.
Response 1: We are very thankful for the positive assessment of our effort.
Comment 2: I would just like to add that the authors have shown new figures (S1-S3) that highlight the importance of the research. I strongly suggest that they be presented as figures within the article and not as supplementary material.
Response 2: We moved figures S1-S3 to the main text of the manuscript.
