Assessment of the In Vitro Phosphatidylinositol Glycan Class A (PIG-A) Gene Mutation Assay Using Human TK6 and Mouse Hepa1c1c7 Cell Lines
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript entitled “Assessment of the In-Vitro Pig-A Gene Mutation Assay Using Human TK6 and Mouse Hepa1c1c7 Cell Lines” written by Zhang et al presents a subject of interest to the readers of the journal, the development and optimization of mouse Hepa1c1c7 cell line as model for in vitro Pig-A gene mutation assay. The manuscript is overall well written, the introduction section provides sufficient background information to support the experimental part and the methods are properly chosen to prove the aim of the study. Despite the fact that the obtained results are convincing in terms of applying this model for the evaluation of mutagenic potential of different xenobiotics that require prior activation via metabolization, I have major concerns regarding the description and the quality of results and discussions section presentation that should be addressed prior to publication.
Major concerns:
1. The authors should decide whether they organize their manuscript with a common Results and discussions section or different sections. If they choose different sections, then the present form of the Results section should be modified, since this part includes multiple paragraphs that should be moved to Discussions section, for example: lines 250 – 259; lines 283-287; lines 321-328, etc.
2. The authors present statistical significance within the figures and tables, but there are not mentioned the statistical tests used. A subheading “Statistical analysis” should be included in the “Materials and methods” section.
3. The authors should explain the selection of the concentrations for EMS and PAHs, in particular the use of different concentrations of EMS for different cell lines (800, 600 and 300 µM for TK6 cells and 16000 µM for Hepa1c1c7). Moreover, a concentration of 12000 µM EMS was mentioned in the Methods (line 213), but was not found into the Results section.
4. Figure 2, lines 247-248: “OD is supposed to be linear related to cell number, however, the linear relationship in Hepa1c1c7 cell line, especially with high cell density, is not as strong as in TK6cell line.” should be removed from the legend of Figure 2 and added to the correspondent results.
5. Figures 3 and 4: The notes from the upper side of the figures should be deleted or replaced with more comprehensible notations.
6. Figure 6, lines 312-216:” B[a]P exposure did 312not show cytotoxicity effect on TK6cell line, however, the MTT assay exhibited an extremely high live cell percentage after B[a]P exposure. Significant difference between methods after B[a]P exposure can be found in both TK6and Hepa1c1c7 cell lines. A possible reason might be high hydrophobicity of B[a]P.” should be removed from the legend of Figure 6 and added to the correspondent results. The statistical tests applied must be mentioned in the Figure 6 legend.
7. Table 3 and table 4 are the same; the authors should correct the error.
8. Table 3 and figure 7 present the same data; the authors should decide how they want to present their data: as a table or as a graph; it is unacceptable to keep both. The data from the table could be inserted in the text to the correspondent results.
9. Similar suggestion for table for and figure 8, table 5 and figure 11, and table 7 and figure 12.
10. Legends of the figures 8, 10 and 11 must be rewritten as recommended at point 3 and 5.
11. The equations for: RICC calculation, Mutation Frequency, RPD calculation, and cell cycle number should be moved to the Materials and methods section as separate subheadings or to the correspondent methods described.
12. The Discussions section should be reorganized to include the paragraphs that were removed from the Results sections.
Minor concerns:
1. A grammar and spelling check is recommended since several grammar errors were identified.
2. All the abbreviations should be explained at first use, for example, EPA, GPI, PM, PI, PNH etc.
3. The correct forms of “in-vitro” and “in-vivo” are “in vitro” and “in vivo”. The authors should make the correction within the manuscript.
4. Introduction: The objectives/aims of the study are described in the final paragraph of the introduction, so I would recommend the relocation of the lines 72-84 to the end of introduction section.
5. Materials and methods section: The lines 126-133 should be moved to Discussions section.
6. In all the figures/tables that present statistical significance must be mentioned the statistical test used and the level of significance, p value.
7. The following reference https://doi.org/10.1093/mutage/gew059 could be also consulted for the improvement of the Discussions section.
Comments on the Quality of English Language
Minor editing is recommended since grammar and spelling errors were identified within the manuscript.
Author Response
Comment 1: The authors should decide whether they organize their manuscript with a common Results and discussions section or different sections. If they choose different sections, then the present form of the Results section should be modified, since this part includes multiple paragraphs that should be moved to Discussions section, for example: lines 250 – 259; lines 283-287; lines 321-328, etc.
Response 1:
Thank you for your suggestion regarding the organization of the manuscript. I have moved the specified sections to the appropriate parts as follows:
- Lines 250-259 have been moved to the Discussion section (lines 505-513).
- Lines 283-287 have been moved to the Discussion section (lines 499-504).
- Lines 321-328 have been moved to the Introduction section (lines 112-123).
Please let me know if these adjustments are satisfactory.
Comment 2: The authors present statistical significance within the figures and tables, but there are not mentioned the statistical tests used. A subheading “Statistical analysis” should be included in the “Materials and methods” section.
Response 2: A "Statistical Analysis" section has been added to the "Materials and Methods" section on lines 274-277, clearly outlining the statistical tests used for data analysis. All relevant figures and tables have been updated to include the specific statistical tests applied, ensuring that the results are accurately interpreted. These changes have been highlighted in red for easy reference.
Comment 3: The authors should explain the selection of the concentrations for EMS and PAHs, in particular the use of different concentrations of EMS for different cell lines (800, 600 and 300 µM for TK6 cells and 16000 µM for Hepa1c1c7). Moreover, a concentration of 12000 µM EMS was mentioned in the Methods (line 213), but was not found into the Results section.
Response 3: The mention of 12,000 µM EMS in the Methods section was a typographical error and has been corrected to 16,000 µM. The EMS concentrations of 800, 600, and 300 µM for the TK6 cell line were selected based on a combination of literature recommendations and our pilot studies, aiming for a 10% survival rate to ensure adequate cytotoxic impact for further mutagenicity testing. The PAH concentrations used for the TK6 cells were matched with those used for the Hepa1c1c7 cell line to maintain consistency across experiments. These corrections and clarifications have been made in lines 218-221 and 229-235 of the manuscript.
Comment 4: Figure 2, lines 247-248: “OD is supposed to be linear related to cell number, however, the linear relationship in Hepa1c1c7 cell line, especially with high cell density, is not as strong as in TK6cell line.” should be removed from the legend of Figure 2 and added to the correspondent results.
Response: The observation about the linear relationship between OD and cell number has been removed from the legend of Figure 2 and integrated into the corresponding results section for clarity.
Comment 5: Figures 3 and 4: The notes from the upper side of the figures should be deleted or replaced with more comprehensible notations.
Repsonse: The notes from the upper side of Figures 3 and 4 have been removed to improve clarity.
Comment 6: Figure 6, lines 312-216:” B[a]P exposure did 312not show cytotoxicity effect on TK6cell line, however, the MTT assay exhibited an extremely high live cell percentage after B[a]P exposure. Significant difference between methods after B[a]P exposure can be found in both TK6and Hepa1c1c7 cell lines. A possible reason might be high hydrophobicity of B[a]P.” should be removed from the legend of Figure 6 and added to the correspondent results. The statistical tests applied must be mentioned in the Figure 6 legend.
Response 6: The information regarding the lack of cytotoxicity observed with B[a]P exposure and the significant differences between methods in TK6 and Hepa1c1c7 cell lines has been removed from the legend of Figure 6 and incorporated into the corresponding results section. Additionally, the statistical tests applied have been included in the Figure 6 legend and highlighted in red.
Comment 7: Table 3 and table 4 are the same; the authors should correct the error.
Response 7: The issue has been resolved. Table 4 has been updated to present the live cell numbers for Hepa1c1c7 cells, correcting the previous error where Tables 3 and 4 were identical.
Comment 8: Table 3 and figure 7 present the same data; the authors should decide how they want to present their data: as a table or as a graph; it is unacceptable to keep both. The data from the table could be inserted in the text to the correspondent results.
Response 8: The intention behind including both tables and figures was to provide detailed numerical data and highlight trends visually. However, to avoid redundancy, I have updated Figure 7 to include only the RICC data for both cell lines, and removed the duplicated information from Table 3. This ensures that the data are not repeated and are presented in a more streamlined manner.
Comment 9: Similar suggestion for table for and figure 8, table 5 and figure 11, and table 7 and figure 12.
Response 9: I have removed the duplicate information from Table 4 and Figure 8, Table 5 and Figure 11, and Table 7 and Figure 12. The tables have been deleted to avoid redundancy, retaining only the figures to highlight the trends.
Comment 10: Legends of the figures 8, 10 and 11 must be rewritten as recommended at point 3 and 5.
Response 10: Figure 8 has been removed, and Figures 10 and 11 have been renumbered to Figures 9 and 10, respectively. The legends for Figures 9 and 10 have been revised in accordance with the recommendations provided in points 3 and 5.
Comment 11: The equations for: RICC calculation, Mutation Frequency, RPD calculation, and cell cycle number should be moved to the Materials and methods section as separate subheadings or to the correspondent methods described.
Response 11: The equations for RICC calculation, Mutation Frequency, RPD calculation, and cell cycle number have been moved to the Materials and Methods section under separate subheadings or incorporated into the corresponding methods descriptions. Please refer to lines 244-249 and 254-273 for the updated information.
Comment 12: The Discussions section should be reorganized to include the paragraphs that were removed from the Results sections.
Response 12: Sure! The Discussions section has been reorganized to include the paragraphs that were previously removed from the Results section.
Comment 13: A grammar and spelling check is recommended since several grammar errors were identified.
Response 13: All identified grammar and spelling errors have been corrected.
Comment 14: All the abbreviations should be explained at first use, for example, EPA, GPI, PM, PI, PNH etc.
Response 14: All abbreviations, including EPA, GPI, PM, PI, PNH, and others, have been defined upon their first use.
Comment 15: The correct forms of “in-vitro” and “in-vivo” are “in vitro” and “in vivo”. The authors should make the correction within the manuscript.
Repsonse 15: All instances of “in-vitro” and “in-vivo” have been corrected to the proper forms, “in vitro” and “in vivo,” respectively.
Comment 16: Introduction: The objectives/aims of the study are described in the final paragraph of the introduction, so I would recommend the relocation of the lines 72-84 to the end of introduction section.
Repsonse 16: The objectives and aims of the study, originally described in lines 72-84, have been relocated to the end of the Introduction section, now appearing in lines 112-123.
Comment 17: Materials and methods section: The lines 126-133 should be moved to Discussions section.
Response 17: The content from lines 126-133 has been moved to the Discussions section, now appearing in lines 489-496.
Comment 18: In all the figures/tables that present statistical significance must be mentioned the statistical test used and the level of significance, p value.
Response 18: The statistical tests used and the levels of significance (p-values) have been added to all figures and tables that present statistical significance.
Comment 19: The following reference https://doi.org/10.1093/mutage/gew059 could be also consulted for the improvement of the Discussions section.
Response 19: Thank you for suggesting the reference. It is indeed a valuable article. Although I have not yet had the opportunity to fully review it, I will consider it for further enhancement of the Discussions section. I would appreciate the opportunity to discuss my thoughts and findings regarding this reference with you once I have finished reading it.
Reviewer 2 Report
Comments and Suggestions for AuthorsIn this manuscript, Wenhao Zhang et al. described the evaluation of the in vitro Pig-A gene mutation assay using human TK6 and mouse Hepa1c1c7 cell lines.
The authors did not pass the affiliation, but only the email addresses.
The manuscript needs some revisions before it can be considered for publication.
The purpose of these tests needs to be understood. Some cytotoxicity techniques were compared on two cell lines, but what was the objective, and what was the result of these studies?
The material and methods chapter should be improved. For example, for cell cultures, it is incomplete, and for cell viability tests, the description of the method is unclear.
Author Response
Comment 1: The authors did not pass the affiliation, but only the email addresses.
Response 1: The affiliation information has been added to the manuscript between lines 5-12.
Comment 2: The manuscript needs some revisions before it can be considered for publication
Response: The requested revisions have been made, and the changes are highlighted in red throughout the manuscript for easy reference.
Comment 3: The purpose of these tests needs to be understood. Some cytotoxicity techniques were compared on two cell lines, but what was the objective, and what was the result of these studies?
Response 3: The objective of comparing different cytotoxicity techniques on the two cell lines (TK6 and Hepa1c1c7) was to determine the most reliable method for assessing cell viability and cytotoxicity during mutagenicity testing. These tests were crucial to evaluate how different cytotoxicity methods, including the MTT assay and propidium iodide (PI) staining, performed under various conditions and exposure levels. The results of this comparison showed that PI staining combined with flow cytometry provided a more consistent and reliable measure of cytotoxicity, particularly when used to assess cell viability in adherent and suspension cells. This allowed us to better understand how cytotoxicity influenced mutagenicity and led to the decision to use PI staining for further studies.
Comment 4: The material and methods chapter should be improved. For example, for cell cultures, it is incomplete, and for cell viability tests, the description of the method is unclear.
Response4: The Materials and Methods section has been thoroughly revised to provide more clarity and detail. The cell culture procedure has been expanded to include specific conditions, such as temperature, media components, and passage protocols. Additionally, the description of the cell viability tests has been improved to ensure that the method is clear, including details about reagents, incubation times, and analytical techniques. All updates are highlighted in red in the manuscript for easier reference.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe study has potential and is addressing the most current concern regarding pollution of the air, water, and foodstuff with PAHs. The authors aimed to develop and present new in vitro model for testing mutagenicity in mammalian cells. The target gene for evaluating mutagenic potential should be the one encoding for the protein PIG-A. Though, the experimental design is deficient. The authors tested PAHs in two cell lines. The TK& cell line without endogenous metabolic system and Hepa1c1c7 which are capable to metabolically transform substances such as PAH and turn them from non-mutagenic to mutagenic. However, cultures were not treated with same PAHs except for B[a]P. Even in treatment with it different final concentrations were used for cell lines that they used in “parallel” treatments. Thus, it is not possible to compare the mutagenic potential of PAHs used for the treatment between metabolically inactive and active cell line and properly evaluate them as possible models for testing mutagenicity in vitro. I would recommend filling these gaps by additionally perform the treatment of TK6 with methyl chrysene or chrysene at the same concentrations as those used in treatment of Hepa1c1c7.
In Abstract and throughout entire manuscript: in vitro and other latin names and terms should be written by letters in italic.
Introduction
Line 38: in allignment to the concurrent OECD test gudeline Ames test should include Escherichia coli unable to synthesize tryptophan.
Line 40: “…caused many…” should read “…caused by many…”.
Lines 42-44: Please rephrase the sentence. False negative results of the Ames test and issues of extrapolation of its results to humans is not due to “provides quick results at low cost”. It might be because bacteria are prokaryotes and humans eukaryotes or just due to limitation of the test itself, but similar limitations and caution in extrapolation of the results to humans are present in other tests either, even when mammalian cells are used as a model.
Lines 49-51: TK and HPRT (in vitro mammalian mutation assays) do not detect reversible mutations, which is the case in the Ames test. They detect direct mutation of the genes for proteins TK and HPRT.
6-thioguanine is a selective agent for detection of cells with mutated HPRT but not TK. To detect mutants in TK assay 3-fluothymidine. Please, do correct manuscript in accordance to those two remarks.
Line 62: In the sentence’s part “…is catalyzed by P450…” P450 has no meaning. Please correct it.
Line 79: Please correct saying “model alkylated agent” to “model alkylation agent”. EMS alkylates DNA bases.
Line 81: Please provide a literature source which corroborates that EMS forms DNA adducts.
Line 88: The sentence is senseless. By translation synthesized peptide cannot be transferred into protein precursor. The saying used in the following sentence is, using the verb attached, is more adequate.
Please provide full words for the acronyms PM, PNH…
Line 91: It is not clear what is undergoing remodeling – protein precursors, PMI-A, PMI-AP? Please, rephrase this entire sentence.
Line 102: Please, provide a short explanation the role of phycoerythrin-conjugate (PE) in detection of PGI-A mutation.
Methodology
What was the temperature applied in cell cultivation?
Line 153, 180: After detaching the cells with trypsin, how was the proteolytic activity of trypsin halted?
Line 156: instead saying that plates were incubated do write that cells were cultivated.
Line 200-216: Why TK6 cells were not treated with methyl chrysene or chrysene? If they were treated, what were the final concentrations of these PAHs?
Why only 1 concentration of PAHs was tested?
On what grounds were concentrations used in the treatment chosen?
Why different concentration of EMS were applied in TK6 and Hepa1c1c7 cells treatment?
Results
How were results presented in figures 4 and 5 obtained? What was the agent the cells were treated with and what was the final concentration of it?
Figure 7 and 8: RICC for vehicle control should be presented either.
Line 361: Please provide the year for the quotation Kruger et al. All citations in the text of the manuscript should be aligned with the JoX instructions for references formatting.
Lines 405-408: The authors stated that EMS at 300 mM did not induce mutations in TK6 cells. Then, they referred to Yu et al. also observed mutation at 200 mM of EMS. The authors should decide whether Yu et al. did detect mutations or not. If not, then it should be stated that they lacked to detect them.
Comments on the Quality of English LanguageA moderate editing is needed.
Author Response
Comment 1: In Abstract and throughout entire manuscript: in vitro and other latin names and terms should be written by letters in italic.
Repsonse 1: All instances of "in vitro" and other Latin names and terms have been revised to be in italics throughout the Abstract and the entire manuscript.
Comment 2: In allignment to the concurrent OECD test gudeline Ames test should include Escherichia coli unable to synthesize tryptophan.
Response 2: Thank you for pointing this out. The revised version is located at line 43-45
Comment 3: “…caused many…” should read “…caused by many…”.
Response 3: Revised.
Comment 4: Please rephrase the sentence. False negative results of the Ames test and issues of extrapolation of its results to humans is not due to “provides quick results at low cost”. It might be because bacteria are prokaryotes and humans eukaryotes or just due to limitation of the test itself, but similar limitations and caution in extrapolation of the results to humans are present in other tests either, even when mammalian cells are used as a model.
Response 4: The sentence has been revised to address your concerns. The revised text is now located at lines 48-50.
Comment 5: TK and HPRT (in vitro mammalian mutation assays) do not detect reversible mutations, which is the case in the Ames test. They detect direct mutation of the genes for proteins TK and HPRT.
6-thioguanine is a selective agent for detection of cells with mutated HPRT but not TK. To detect mutants in TK assay 3-fluothymidine. Please, do correct manuscript in accordance to those two remarks.
Response 5:
Thank you for your observations. The manuscript has been revised to reflect that TK and HPRT in vitro mammalian mutation assays do not detect reversible mutations, unlike the Ames test. Instead, they detect direct mutations in the genes encoding the proteins TK and HPRT. Specifically, 6-thioguanine is used as a selective agent to detect cells with mutated HPRT, while 3-fluorothymidine is used to identify mutants in the TK assay. The revisions addressing these points can be found in lines 56-58 and 62-65.
Comment 6: In the sentence’s part “…is catalyzed by P450…” P450 has no meaning. Please correct it.
Response 6: The term “P450” has been revised to “CYP450” for clarity.
Comment 7: Please correct saying “model alkylated agent” to “model alkylation agent”. EMS alkylates DNA bases.
Response 7: The term “model alkylated agent” has been corrected to “model alkylation agent,” as EMS alkylates DNA bases.
Comment 8: Please provide a literature source which corroborates that EMS forms DNA adducts.
Response 8: The information regarding EMS forming DNA adducts has been moved to line 122, and relevant literature has been added to support this statement.
Comment 9: The sentence is senseless. By translation synthesized peptide cannot be transferred into protein precursor. The saying used in the following sentence is, using the verb attached, is more adequate.
Response 9: The sentence has been revised for clarity and accuracy, and the revised version has been moved to lines 93-96. The term "attached" has been used appropriately in the updated text.
Comment 10: Please provide full words for the acronyms PM, PNH…
Response 10: Full words for the acronyms PM and PNH have been provided in the manuscript at line 99 and 103.
Comment 11: It is not clear what is undergoing remodeling – protein precursors, PMI-A, PMI-AP? Please, rephrase this entire sentence.
Response 11: The sentence has been rewritten for clarity to specify what is undergoing remodeling. The revised text can be found in lines 96-99.
Comment 12: Please, provide a short explanation the role of phycoerythrin-conjugate (PE) in detection of PGI-A mutation.
Response 12: A short explanation of the role of phycoerythrin-conjugate (PE) in the detection of PGI-A mutations has been added. Please refer to lines 109-111 for this information.
Comment 13: What was the temperature applied in cell cultivation?
Response 13: The temperature applied in cell cultivation was 37°C.
Comment 14: Line 153, 180: After detaching the cells with trypsin, how was the proteolytic activity of trypsin halted?
Response 14: The proteolytic activity of trypsin was halted by adding a fivefold volume of complete medium. The cells were then resuspended and repetitively pipetted to disaggregate any cell clusters. This information has been added to lines 165-167.
Comment 15: instead saying that plates were incubated do write that cells were cultivated.
Response 15: Revised
Comment 16: Why TK6 cells were not treated with methyl chrysene or chrysene? If they were treated, what were the final concentrations of these PAHs?
Response 16: TK6 cells were not treated with 5-MC (methyl chrysene) or chrysene because they lack the CYP450 enzyme family, which is necessary for the metabolism of PAHs. Therefore, B[a]P (benzo[a]pyrene) was chosen as a representative PAH for these experiments.
Comment 17: Why only 1 concentration of PAHs was tested?
Response 17: We selected a single concentration of PAHs based on our pilot study, aiming for a 10% survival rate at 48 hours after a 4-hour exposure. Due to the high lipid solubility of PAHs, 10 µM was the maximum concentration achievable in the cytotoxicity experiments. Higher concentrations, such as 15 µM, resulted in insoluble crystals in the 96-well plates.
Comment 18: On what grounds were concentrations used in the treatment chosen?
Response 18: The treatment concentrations were determined based on pilot studies and online literature. For the TK6 cell line, EMS concentrations were chosen based on literature recommendations and our pilot study aimed at achieving a 10% survival rate. The PAH concentrations for TK6 cells were aligned with those used for the Hepa1c1c7 cell line to maintain consistency.
Comment 19: Why different concentration of EMS were applied in TK6 and Hepa1c1c7 cells treatment?
Response 19: Different concentrations of EMS were used for TK6 and Hepa1c1c7 cells due to their varying sensitivities to EMS. Our pilot study indicated that Hepa1c1c7 cells are less sensitive to EMS compared to TK6 cells. Consequently, a significantly higher concentration (16000 µM) was required for Hepa1c1c7 cells to achieve a 10% survival rate after 48 hours of exposure.
Comment 20: How were results presented in figures 4 and 5 obtained? What was the agent the cells were treated with and what was the final concentration of it?
Response 20: The results presented in Figures 4 and 5 were obtained using flow cytometry after staining the cells with propidium iodide (PI). A 100 µg/mL PI stock solution was diluted 1:100 in the experimental wells, resulting in a final concentration of approximately 1.48 µM PI.
Comment 21: Figure 7 and 8: RICC for vehicle control should be presented either.
Response 21: RICC (Relative Index of Cell Culture) for vehicle control groups is typically set to 1, as it serves as a baseline for comparing proliferation between exposure and control groups. Since the RICC values for control groups are consistently 1, presenting these values separately is generally unnecessary.
Comment 22: Please provide the year for the quotation Kruger et al. All citations in the text of the manuscript should be aligned with the JoX instructions for references formatting.
Response 22: The year for the quotation by Kruger et al. has been provided, and all citations in the manuscript have been revised to align with the JoX reference formatting instructions.
Comment 23: The authors stated that EMS at 300 mM did not induce mutations in TK6 cells. Then, they referred to Yu et al. also observed mutation at 200 mM of EMS. The authors should decide whether Yu et al. did detect mutations or not. If not, then it should be stated that they lacked to detect them.
Response 23: We acknowledge the inconsistency between our results and those of Yu et al. (2021) and Kruger et al. (2015). In our study, exposure to 300 µM EMS did not result in a significant increase in GPI(-) cells in the TK6 cell line, while both Yu et al. and Kruger et al. observed genotoxic effects at lower concentrations (200 µM). This discrepancy may be due to differences in experimental conditions, such as cell density, exposure duration, or variations in assay sensitivity. We have added this clarification to the manuscript.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThis resubmitted form of the manuscript is much improved.
The authors added essential elements that clarify the purpose and direction of this study.
The discussions were much improved which led to a better understanding of the results obtained.
Overall, I consider the article could be a useful contribution to the journal. I recommend the manuscript for publication.
Reviewer 3 Report
Comments and Suggestions for AuthorsThank you for correcting the manuscript and elaborating your position regarding the remarks that are not corrected.
