Effects of coenzyme Q10 and N-acetylcysteine on experimental poisoning by paracetamol in Wistar rats

Rayanne Henrique Santana da Silva; Mariana de Moura; Larissa de Paula; Kelly Carolina Arantes; Marina da Silva; Jaqueline de Amorim; Marina Pacheco Miguel; Danieli Brolo Martins; Daniela de Melo e Silva; Marília Martins Melo; Ana Flávia Machado Botelho

doi:10.1371/journal.pone.0290268

Peer Review History

Original SubmissionFebruary 14, 2023
27 Apr 2023 Decision Letter - Jianhong Zhou, Editor PONE-D-23-03966Effects of coenzyme Q10 and N-acetylcysteine on experimental poisoning by paracetamol in Wistar ratsPLOS ONE Dear Dr. BOTELHO, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jun 10 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 4. Please include a separate caption for each figure in your manuscript. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 6. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. 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Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No Reviewer #3: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: • The idea of the manuscript is important because it discusses the effect of one of the most usable medications for analgesics that we used daily to relieve the pain resulting from headaches or fever without the supervision of a physician, which leads to liver and kidney diseases later. • The manuscript is well written, and the introduction section is focusing on the theme of the manuscript. • The statistical analysis has been performed appropriately and rigorously. • Please refer to the following comments in the reviewed attached manuscript file. 1- Include the page number for the manuscript. 2- Include the “line numbers” to allow reviewers to easily mention the comments. 3- In the results, the authors should consider the ordering in writing the text to be aligned with the figures and the graphs name, as in “Hematological and serum biochemistry evaluation “an increase in erythrocytes, hematocrit, and hemoglobin in the CON+ group and evidenced thrombocytopenia in the group of animals intoxicated with PAR and treated with NAC (Figure 1).”, while in the figure1 they wrote as follows: (A) Mean and standard deviation of hematocrit (HCT), (B) erythrocytes and (C) hemoglobin. 4- The authors should organize each section in the manuscript by adding numbers to the section and subtitles as follows: 1. Introduction. 2. Materials and Methods. 2.1. Experimental Design. 5- In the discussion section, the fourth paragraph “Although PAR overdose produces ROS and changes DNA, and lipid peroxidation [8,23]. in the present study, no differences were observed between the groups”. The highlighted dot should be corrected with coma. 6- Table 2: please can you rewrite it in the proper way. Reviewer #2: - Why were 31 rats divided into five groups? - It is only necessary to use the full spelling of IP once, followed by the abbreviation. - Suggest to standardise the term use for PAR - Paracetamol or acetaminophen Or Tylenol? - The author stated in the statistical analysis that the experimental design is completely randomised. How is the experimental design performed randomisation? Aren't the groups assigned to certain treatments? RESULTS: - In haematological and serum biochemistry evaluation results, how do ALT and AST levels found represent liver morphological status? - Please correct the statement where it said 'it was possible'. The observation has been made, so the results should be definite, not 'possible'. E.g.: 1. It was possible to observe a significant increase of creatinine in the NAC group compared to NAC+CoQ10 group (Figure 2). 2. The results obtained by the analysis demonstrate the mean ± SD (standard deviation) of the groups, where it was possible to observe that there was no statistical difference between them. - The scale bar in fig. 4-5 is small. It is suggested that a clearer scale bar be provided. - Figure 6: Add an arrow as an indicator to the results, such as intact mitochondria/normal nuclei/fat droplets, etc. Other comments: The study is descriptive rather than comprehensive. It would be ideal if some parameters, such as ROS levels, were added to confirm the observation (since PAR overdose did not show any differences between the treatment and control groups.); as well as antioxidant activities (glutathione levels etc). A mechanism study would be preferable to corroborate the findings, particularly on the mechanism of hepatoprotection action of CoQ10 by measuring NRF2, proinflammatory cytokines or mitochondrial enzymes as postulated in the discussion. Several arguments are speculative, with no experimental evidence to back them up. Reviewer #3: The study was well conducted by the authors;however,there are some concerns to revise that are described below For Absrtct; It is better to add one or two as introduction before the aim of study Keywords ubiquinol ; ubiquinone - to anyone that is not familiar with the subject these keywords are difficult - I don’t expect you to go deep in the chemistry of CoQ10 , but the reader should be able to understand all words For Introduction; The introduction section resumes the existing knowledge regarding antioxidant action of NAC and CoQ10 . However, as the importance of the topic, it is recommended to expand and update the literature. For Method ; The method section appears well organized with the relevant paper, but Comet assay was elaborated extensively and it is recommended to be reduced to some extent. Minor comments ; Discussion In the fifth paragraph No significant alteration were observed in GGT,FA and ALT What does FA mean? In the eighth paragraph Nrf2 expression Abbreviation should be explained to readers. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Plos One Comments.docx https://doi.org/10.1371/journal.pone.0290268.r001
Revision 1
2 Jun 2023 Author Response Authors are grateful for all suggestions and have addressed all reviewers’ comments: Reviewer #1: 1- Include the page number for the manuscript We appreciate the suggestion and have included the number of pages in the article. 2- Include the “line numbers” to allow reviewers to easily mention the comments. Thanks for the suggestion and we included the lines in the article. 3- In the results, the authors should consider the ordering in writing the text to be aligned with the figures and the graphs name, as in “Hematological and serum biochemistry evaluation “an increase in erythrocytes, hematocrit, and hemoglobin in the CON+ group and evidenced thrombocytopenia in the group of animals intoxicated with PAR and treated with NAC (Figure 1).”, while in the figure1 they wrote as follows: (A) Mean and standard deviation of hematocrit (HCT), (B) erythrocytes and (C) hemoglobin. We thank you for your comments and have already made the suggested changes. Line 176: We changed the sentence “an increase in erythrocytes, hematocrit, and hemoglobin in the CON+ group and evidenced thrombocytopenia in the group of animals intoxicated with PAR and treated with NAC (Figure 1).” to “an increase in hematocrit, erythrocytes, and hemoglobin in the CON+ group and evidenced thrombocytopenia in the group of animals intoxicated with PAR and treated with NAC (Figure 1).” 4- The authors should organize each section in the manuscript by adding numbers to the section and subtitles as follows: 1. Introduction. 2. Materials and Methods. 2.1. Experimental Design. Thanks for the suggestion and the sections of the manuscript have been organized by adding numbers and subheadings. 5- In the discussion section, the fourth paragraph “Although PAR overdose produces ROS and changes DNA, and lipid peroxidation [8,23]. in the present study, no differences were observed between the groups”. The highlighted dot should be corrected with coma We thank you for your comments and have already made the suggested changes. Line 331: We changed the sentence “Although PAR overdose produces ROS and changes DNA, and lipid peroxidation [8,23]. in the present study, no differences were observed between the groups” to “Although PAR overdose produces ROS and changes DNA, and lipid peroxidation [8,23], in the present study, no differences were observed between the groups”. 6- Table 2: please can you rewrite it in the proper way. Table was rewritten according to suggestions from reviewer Reviewer #2: Why were 31 rats divided into five groups? We had planned to use 35 animals distributed into the five groups, however the university couldn’t provide the necessary number of animals at the time, considering the pandemic situation. We also had two deaths occur due to a gavage mistake, these animals did not participate in the study. - It is only necessary to use the full spelling of IP once, followed by the abbreviation. Thanks for the suggestion. All incorrect uses have been reviewed. - Suggest to standardize the term use for PAR - Paracetamol or acetaminophen Or Tylenol? Thanks for the suggestion. Sentence was reviewed accordingly. - The author stated in the statistical analysis that the experimental design is completely randomized. How is the experimental design performed at randomisation? Aren't the groups assigned to certain treatments? We had previously defined the treatments that we would perform in the project (CON-, CON+, NAC, CoQ10, NAC+CoQ10) and divided the animals into the identified boxes. The choice of animals in each box was performed at random. The experimental design used was completely randomized and the data obtained are presented as mean and standard deviation. The Kolmogorov-Smirnov test was performed to define normality. Those classified as parametric were evaluated by ANOVA and Tukey's post-test. In the case of non-parametric variables, the Kruskal-Wallis test and Dunn's post test of multiple comparisons were used. Spearman's correlation test was used to assess the relationship between serum biomarker levels and histological lesion scores. In all analyses, differences were considered significant when p<0.05. Data were evaluated using GraphPad Prism 7. RESULTS: - In haematological and serum biochemistry evaluation results, how do ALT and AST levels found represent liver morphological status? Hepatocyte damage can be verified by measuring ALT and AST in blood serum. ALT is a cytoplasmic enzyme that catalyzes the pyruvate-forming reaction of gluconeogenesis and is involved in amino acid metabolism. Its half-life in rats ranges from three to four hours, and normal values for this species comprise 35.1 ± 13.3 U/L. Increases in the levels of this liver enzyme reflect cellular damage to hepatocytes and may be linked to drug intoxication, including PAR. In PAR intoxication, there is an increase in the production of the NAPQI, which is positively correlated with the administered amount of the drug. The accumulation of this metabolite contributes to the formation of peroxynitrite with consequent peroxidation, destruction of proteins and damage to hepatocytes. In the present study, ALT did not differ between groups, although it showed a mean similar to AST. It is possible to observe from the scatter plot that some animals, possible outliers, significantly increase the mean and standard deviation of these groups. This finding is associated with and justified by the susceptibility of each individual to PAR intoxication, since the great variability of individual response has been described through genetic and hereditary factors. The observation of the ALT parameter also revealed a significant increase in the NAC group in relation to CON-, which suggests that its protective action was not effective enough to heal or mitigate liver damage. AST, in turn, is a mitochondrial and cytoplasmic enzyme, present in the deamination reaction of aspartate into oxaloacetate, and exhibits significant concentrations in the heart, liver, kidney and skeletal muscle. The normal values of this enzyme, for the studied species, comprise 42.9 ± 10.1 U/L and the elevation of this parameter may be related to damage to hepatocytes. Macroscopy showed congestion and evidence of a multifocal to diffuse lobular pattern in the liver of animals in the CON+ and NAC groups, compatible with what is described in the literature for PAR intoxication without further changes in other organs. - Please correct the statement where it said 'it was possible'. The observation has been made, so the results should be definite, not 'possible'. E.g.: 1. It was possible to observe a significant increase of creatinine in the NAC group compared to NAC+CoQ10 group (Figure 2). 2. The results obtained by the analysis demonstrate the mean ± SD (standard deviation) of the groups, where it was possible to observe that there was no statistical difference between them. Thanks for the suggestions. All have been reviewed accordingly. - The scale bar in fig. 4-5 is small. It is suggested that a clearer scale bar be provided. Thanks a lot for the suggestion. Scales have been modified for better visualization. - Figure 6: Add an arrow as an indicator to the results, such as intact mitochondria/normal nuclei/fat droplets, etc. Thanks a lot for the suggestion. Figure 6 has been revised. Other comments: The study is descriptive rather than comprehensive. It would be ideal if some parameters, such as ROS levels, were added to confirm the observation (since PAR overdose did not show any differences between the treatment and control groups.); as well as antioxidant activities (glutathione levels etc). A mechanism study would be preferable to corroborate the findings, particularly on the mechanism of hepatoprotection action of CoQ10 by measuring NRF2, proinflammatory cytokines or mitochondrial enzymes as postulated in the discussion. Several arguments are speculative, with no experimental evidence to back them up. We unfortunately don’t have the funds to further investigate the mechanisms. We are applying in our country for new funding in order to continue the research and provide such answers in the future. Reviewer #3: The study was well conducted by the authors; however, there are some concerns to revise that are described below For Abstract; It is better to add one or two as introduction before the aim of study Thanks a lot for the suggestion. A paragraph has been added to the abstract. Line 21: “Paracetamol (PAR) is a drug widely used in human and veterinary medicine as an analgesic and antipyretic, often involved in cases of intoxication. The most common clinical signs result from damage to red blood cells and hepatocytes, and this intoxication is considered a model for the induction of acute liver failure”. Keywords: ubiquinol ; ubiquinone To anyone that is not familiar with the subject these keywords are difficult - I don’t expect you to go deep in the chemistry of CoQ10, but the reader should be able to understand all words We are grateful for the suggestion. Keywords were revised. Antioxidant; hepatotoxicity; hepatoprotective; oxidative damage; paracetamol; rats For Introduction; The introduction section resumes the existing knowledge regarding antioxidant action of NAC and CoQ10. However, as the importance of the topic, it is recommended to expand and update the literature. Thanks for the suggestion. The introduction has been modified with updated articles. For Method ; The method section appears well organized with the relevant paper, but Comet assay was elaborated extensively and it is recommended to be reduced to some extent. Minor comments Thanks a lot for the suggestion. The text about comet assay has been shortened. Discussion In the fifth paragraph No significant alteration were observed in GGT, FA and ALT What does FA mean? Thanks a lot for the suggestion. Line 123, 337: FA has been replaced by ALP Line 363 was added Nrf2 means nuclear factor erythroid 2-related factor 2 After the first round of revisions, the following requirements were answered. Please let us know if they are sufficient. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Manuscript was revised accordingly. 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. We wish to acknowledge "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior"—Finance Code 001" for the fellowship to Rayanne Henrique Santana da Silva. Also, we acknowledge the financial support for publication from FAPEG (Research Support Foundation of the State of Goiás), grant 06/2018. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. The graphs for globulin, bilirubin and ALT were added to the supporting information and all “data not shown” were excluded. 4. 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Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. We don’t have a data repository subscription. But all data used can be made available as supportive material in Excel spreadsheets. Thank you for your consideration. We look forward to hearing from you. Sincerely, Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0290268.r002
21 Jul 2023 Decision Letter - Yasmina Abd‐Elhakim, Editor PONE-D-23-03966R1Effects of coenzyme Q10 and N-acetylcysteine on experimental poisoning by paracetamol in Wistar ratsPLOS ONE Dear Dr. BOTELHO, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Sep 04 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Yasmina Abd‐Elhakim Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The study was well conducted by the authors, and the authors have adequately addressed the comments raised in a previous round of review. So I suggest that the manuscript is now acceptable for publication. Reviewer #2: I believe that it is worth mentioning that 35 animals were distributed into the five groups, and later only 31 mice survived. Liver Morphology is often examined through the analysis of the liver's histological features. The authors are looking at the levels of liver enzymes, which are indications of liver function. Please ensure that the terminology you're using in line197 is accurate. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0290268.r003
Revision 2
4 Aug 2023 Author Response Authors are grateful for all suggestions and have addressed all reviewers’ comments: Reviewer #1: 1- The study was well conducted by the authors, and the authors have adequately addressed the comments raised in a previous round of review. So I suggest that the manuscript is now acceptable for publication. Thank you very much for your considerations. Reviewer #2: 1- I believe that it is worth mentioning that 35 animals were distributed into the five groups, and later only 31 mice survived. Thank you for the suggestion. A sentence has been added to the abstract. Line 29: “Thirty-five adult Wistar rats (Rattus novergicus albinus) were randomly assigned to five groups, and thirty-one of these survived the treatments.” Line 83: “Thirty-five adult male Wistar rats (Rattus novergicus albinus) were used, weighing on average 200-300g, distributed in five groups, and thirty-one of these survived the treatments. Animals were acquired at the Federal University of Goiás Bioterium.” 2-Liver Morphology is often examined through the analysis of the liver's histological features. The authors are looking at the levels of liver enzymes, which are indications of liver function. Please ensure that the terminology you're using in line197 is accurate. Thanks for the suggestion, we changed the terminology presented in line 196. Line 196: “To evaluate the liver function ALT and AST were measured.” Attachments Attachment Submitted filename: Response to Reviewers (1).docx https://doi.org/10.1371/journal.pone.0290268.r004
7 Aug 2023 Decision Letter - Yasmina Abd‐Elhakim, Editor Effects of coenzyme Q10 and N-acetylcysteine on experimental poisoning by paracetamol in Wistar rats PONE-D-23-03966R2 Dear Dr. BOTELHO, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Yasmina Abd‐Elhakim Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0290268.r005
Formally Accepted
10 Aug 2023 Acceptance Letter - Yasmina Abd‐Elhakim, Editor PONE-D-23-03966R2 Effects of coenzyme Q10 and N-acetylcysteine on experimental poisoning by paracetamol in Wistar rats Dear Dr. Botelho: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Dr. Yasmina Abd‐Elhakim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0290268.r006

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