Hexavalent Chromium Inhibited Zebrafish Embryo Development by Altering Apoptosis- and Antioxidant-Related Genes

Round 1

Reviewer 1 Report

1 The paper entitled “Hexavalent Chromium Inhibited Zebrafish Embryo Development by Altering Apoptosis- and Antioxidant-Related Genes” is an interesting study and the authors have collected a unique dataset using methodological rigor. The paper is generally well written and structured and it is clear that it is a continuation of the research group's. The introduction describes in a clear manner the principal bases of the study and the materials and methods are well done.

However, in my opinion, the paper should be revised only into few points. 

I attached a copy of work with the line for my comments.

a1. Abstract: Con riferimento a “The chromium-exposed embryos showed attenuated expression of apoptosis-related transcripts such as caspase 3, bcl2, and bax. Moreover, Western blot analysis also demonstrated a downregulation of these apoptosis-related proteins”, in the discussion you say “Real-time RT-PCR and Western blot analyses demonstrated that chromium exposure induced an upregulation of caspase 3 and bax, and the downregulation of bcl2.” So in this way is not clear that chromium treatment is related with an increase in apoptosis pathway.

 

2 2. Line 9: Rephrase this sentence “Zebrafish embryos ex- zebrafish as a model…”

 

3.3.       Line 11: Replace “accompanies” with “are associated”

 

4.4       Line 30: The number of live embryos “was” determined…

 

5.5    Line 33: *Treatment groups?

 

6.6      Line 34: Which lethality indicators have you considered to assess the survival rates?

 

7.7       Line 71: I suggest to use the same format to express the dilutions. You used “1/5000” and “1:10,000”

 

8.8       Line 73: Clarify this sentence because it seems that you used only one secondary antibody

 

9.9       Figure 2: Is missing the photo of 25 µg/L at Day 3

 

110.   Line 131: Maybe is “Figure 4A”?

 

111.   Line 136-137: You already wrote it in lines 132-133

 

112.   Line 194: Rewrite this sentence because it is not clear at all. Perhaps you meant "the creation of ROS by healthy cells”?

Comments for author File: Comments.pdf

Author Response

Dear Prof. Dr. Madhav Bhatia, Editorial Boards and Reviewers,

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

1. Abstract: Con riferimento a “The chromium-exposed embryos showed attenuated expression of apoptosis-related transcripts such as caspase 3, bcl2, and bax. Moreover, Western blot analysis also demonstrated a downregulation of these apoptosis-related proteins”, in the discussion you say “Real-time RT-PCR and Western blot analyses demonstrated that chromium exposure induced an upregulation of caspase 3 and bax, and the downregulation of bcl2.” So in this way is not clear that chromium treatment is related with an increase in apoptosis pathway.

Authors’ response: the characterization of capase 3, bcl2, and bax expression was corrected in the abstract.

2. Line 9: Rephrase this sentence “Zebrafish embryos ex- zebrafish as a model…”

 Author’s response: We have rewritten the sentence.

3. Line 11: Replace “accompanies” with “are associated”

 Author’s response: We have replaced this word.

4. Line 30: The number of live embryos “was” determined…

 Author’s response: We have rewritten the passage.

5. Line 33: *Treatment groups?

 Author’s response: We have rewritten the passage.

6. Line 34: Which lethality indicators have you considered to assess the survival rates?

Author’s response: The lethality indicators, the lethal dose (LD50) value at day 3 and day 7, was added to the Result.

7. Line 71: I suggest to use the same format to express the dilutions. You used “1/5000” and “1:10,000”

 Author’s response: The format to express the dilutions was corrected to “1:5,000” and “1:10,000”.

8. Line 73: Clarify this sentence because it seems that you used only one secondary antibody

Author’s response: We have already covered manipulations with primary antibodies in line 363: “The membrane was incubated with primary antibodies in a blocking buffer overnight at 4°C”.

9       Figure 2: Is missing the photo of 25 µg/L at Day 3

 Author’s response: the zebrafish embryos from 25 µg/L was delayed hatching at day 3, thus we could not get the zebrafish larvae and measure the body length at this time.

10. Line 131: Maybe is “Figure 4A”?

 Author’s response: We have corrected this mistake.

11. Line 136-137: You already wrote it in lines 132-133

Author’s response: The repetition in description of the developmental blocks in embryo treated with 50 100 µg/L and 100 µg/L chromium has been corrected.

12. Line 194: Rewrite this sentence because it is not clear at all. Perhaps you meant "the creation of ROS by healthy cells”?

       Author’s response: We have corrected this sentence.

We hope that our corrections could meet your requirements,

Thank you so much.

 

 

Reviewer 2 Report

The article from Dang et al. reports the effects of hexavalent chromium on zebrafish development with a focus on characterizing changes in cell cycle gene expression, apoptosis and enzymes which are responsible for mitigating reactive oxygen species. Overall, this is a nice report with information that is useful for the toxicology field and expands our knowledge about the developmental consequences of chromium exposure. I have several questions and suggestions for the authors.

 

Major comments

1. Abstract and throughout the manuscript: Zebrafish gene names should be italicized.

2. Section 2.1: the animal approval/institutional oversight for vertebrate animal research/IACUC statement(s) are missing.

 

3. Figure 2: The live images of the chromium treated zebrafish are not consistent with a decrease in tip to tail length compared to the wild-type embryo images. Please clarify.

 

4. Figure 5: How did the authors quantify protein expression based on the Western blots? In some cases, the blots are clearly consistent with the trends reported in the graphs, such as day 3 caspase 3 and Bax data. However, it is more difficult to appreciate the observed trend in Bcl-2 for the day 3 data, just as one example. The methods are not clear in how the numbers for the graphs in A and B were obtained, which must be described within the study to substantiate the graphs.

The manuscript is well written and organized.

Author Response

Dear Prof. Dr. Madhav Bhatia, Editorial Boards and Reviewers,

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

Major comments

1. Abstract and throughout the manuscript: Zebrafish gene names should be italicized.

 

Author’s response: We have corrected all the gene names.

 

2. Section 2.1: the animal approval/institutional oversight for vertebrate animal research/IACUC statement(s) are missing.

 

Author’s response: We have added the information of animal approval in “Institutional Review Board Statement”:

 

3. Figure 2: The live images of the chromium treated zebrafish are not consistent with a decrease in tip to tail length compared to the wild-type embryo images. Please clarify.

 

Author’s response: Figure 2 was corrected and the scale bar was added to this figure.

 

4. Figure 5: How did the authors quantify protein expression based on the Western blots? In some cases, the blots are clearly consistent with the trends reported in the graphs, such as day 3 caspase 3 and Bax data. However, it is more difficult to appreciate the observed trend in Bcl-2 for the day 3 data, just as one example. The methods are not clear in how the numbers for the graphs in A and B were obtained, which must be described within the study to substantiate the graphs.

 

Author’s response: We have added the method for the semiquantitation of exposed bands in western blot in Materials and Methods. This experiment was technically replicated in three times. To quantify protein bands of caspase 3, Bax, and Bcl-2, we used the rectangle to choose the bands at the predicted molecular weight (following the manual of manufacture). The expression of these proteins was normalized to Gapdh.

We hope that our corrections could meet your requirements,

Thank you so much.

 

Reviewer 3 Report

This manuscript utilizes the zebrafish animal model to investigate the toxicity of a heavy metal, hexavalent chromium, with a goal of beginning to understand possible mechanisms for the developmental toxicity of hexavalent chromium.  While new information regarding possible mechanisms of chromium toxicity are provided, it is unclear how much the current results will advance this understanding.  Contradictory data, and the poor quality of some of the supplemental figures, raises questions regarding the reliability of the conclusions described in the manuscript.

 

Major concerns:

 

1.     The authors provide morphological data pertaining to embryo body length, but were other morphological abnormalities observed or characterized?  It appears microphthalmia may occur in some embryos.  As well as cardiac edema.  With the marked effects of chromium on survival, what might presume to observe significant dysmorphologies.

 

2.     There is significant variability in the RT-PCR data, and some of the observed changes, or lack of change, are difficult to put into context vis a vis morphological/survival changes.  The authors proposed molecular model for chromium toxicity is that the chromium decreases Cdks and increases Sod, which leads to increased caspase 3 and Bax, promoting apoptosis, and decreased Bcl2, losing the anti-apoptotic regulation.  But with the lowest chromium dose, that has no effects on morphology, survival, or heart rate, one observes at day 1 no change in Cdk mRNA, a decrease in Sod mRNAs, an increase in caspase3 but not Bax mRNA.  And at day 3 this lowest dose is manifesting changes in mRNAs that would be consistent with cell death.  These data would suggest a toxicity with even the lowest chromium dose, that is not observed in the morphological studies.

 

3.     This same lack of correlation between analyzed genes is observed with the protein expression in Fig 5.  Again, the authors’ present data showing the lowest chromium dose is increasing caspase 3 protein at day 1, but not Bax, while Bcl2 protein is also unchanged.  And Cdk protein is unchanged.  So how would this lowest chromium dose be eliciting these disparate changes in cell death pathway mRNA and proteins, as well as cell cycle markers, but not be impacting observable aspects of zebrafish development?  What is a reasonable mechanistic explanation for these data?

 

4.     The supplemental data blots are poor quality.  Is there poor specificity for the selected antibodies, or pronounced protein degradation?  If the latter this is especially problematic, as variability in protein degradation will lead to inaccurate quantitation of the analyzed protein levels.

 

5.     Other measures of apoptosis should be employed by the authors, to confirm that changes in apoptotic pathway genes/proteins are markedly increasing cell death.  Examination of specific organs is of special interest, such as heart, brain, etc.

 

 

Minor concerns:

 

1.      Statistical analyses are only provided for Figures 4 and 5.

 

2.     Was heart rate assessed at day 3 for embryos treated with 50 µg/L of chromium?

 

3.     The rationale for conducting RT-PCR and western blots for day 1 embryos is not clear, since only survival rate was assessed at this age.  A clear correlation between these possible molecular changes and significant effects on zebrafish development cannot be made.

 

4.     How is the protein expression represented in Fig. 5?  Is it a ratio of the analyzed protein to GADPH protein?

Author Response

 

Dear Prof. Dr. Madhav Bhatia, Editorial Boards and Reviewers,

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

Major concerns:

 

1. The authors provide morphological data pertaining to embryo body length, but were other morphological abnormalities observed or characterized?  It appears microphthalmia may occur in some embryos.  As well as cardiac edema.  With the marked effects of chromium on survival, what might presume to observe significant dysmorphologies.

 

Author’s response: heavy metal showed multiple morphological abnormalities  on zebrafish embryo development. In this study, we focus on the three parameters of zebrafish embryo concluding survival rate, body length, and heart rate. We have found that chromium induced changes in these properties of zebrafish. In further research, we will assess more morphological abnormalities as you mention in above comment.

 

2. There is significant variability in the RT-PCR data, and some of the observed changes, or lack of change, are difficult to put into context vis a vis morphological/survival changes.  The authors proposed molecular model for chromium toxicity is that the chromium decreases Cdks and increases Sod, which leads to increased caspase 3 and Bax, promoting apoptosis, and decreased Bcl2, losing the anti-apoptotic regulation.  But with the lowest chromium dose, that has no effects on morphology, survival, or heart rate, one observes at day 1 no change in Cdk mRNA, a decrease in Sod mRNAs, an increase in caspase3 but not Bax mRNA.  And at day 3 this lowest dose is manifesting changes in mRNAs that would be consistent with cell death.  These data would suggest a toxicity with even the lowest chromium dose, that is not observed in the morphological studies.
3. This same lack of correlation between analyzed genes is observed with the protein expression in Fig 5.  Again, the authors’ present data showing the lowest chromium dose is increasing caspase 3 protein at day 1, but not Bax, while Bcl2 protein is also unchanged.  And Cdk protein is unchanged.  So how would this lowest chromium dose be eliciting these disparate changes in cell death pathway mRNA and proteins, as well as cell cycle markers, but not be impacting observable aspects of zebrafish development?  What is a reasonable mechanistic explanation for these data?

 

Author’s response for comments 2 and 3: In this study we found that at the lowest concentrations of chormium, there was no difference in the survival rate of fish embryos, although the expression of some genes decreased at day 1 and day 3. However, in At higher chromium concentrations, changes in the expression of these genes were more pronounced, and were associated with fish embryo survival. However, the survival rate of zebrafish embryos is associated with many other genes, so we will evaluate these for the next study.

 

 

4. The supplemental data blots are poor quality.  Is there poor specificity for the selected antibodies, or pronounced protein degradation?  If the latter this is especially problematic, as variability in protein degradation will lead to inaccurate quantitation of the analyzed protein levels.

 

Author’s response: In this study, we selected the band size according to the manufacturer's instructions to evaluate the expression level of protein. The evaluation procedure has been added in the research materials and methods section. The intensity of these bands was determined and the expression of these proteins was normalized to Gapdh.

 

5. Other measures of apoptosis should be employed by the authors, to confirm that changes in apoptotic pathway genes/proteins are markedly increasing cell death.  Examination of specific organs is of special interest, such as heart, brain, etc.

 

Author’s response: Thank you so much for your comments, these are very kind guidance to clearly estimate changes in apoptosis of zebrafish embryos. Unfortunately, we could not order and import reagents and kits for apoptosis evaluation in this time, thus we will carry out these experiments in the further research.

 

Minor concerns:

 

1. Statistical analyses are only provided for Figures 4 and 5.

 

Author’s response: the information of statistical analyses was concluded for these Figures.

 

2. Was heart rate assessed at day 3 for embryos treated with 50 µg/L of chromium?

 

Author’s response:  the heart rate was not assessed at day 3 for embryos treated with 50 µg/L of chromium, because zebrafish embryos stopped developing in early stage.

 

3. The rationale for conducting RT-PCR and western blots for day 1 embryos is not clear, since only survival rate was assessed at this age.  A clear correlation between these possible molecular changes and significant effects on zebrafish development cannot be made.

 

Author’s response:  In this study, we assess the changes in the expression of apoptosis- and antioxidant-related genes in zebrafish embryos under the chormium treatment. Although there was not much difference in survival, it is necessary to estimate the changes in the expression of these genes when zebrafish embryos were exposed to chromium. In the further research, we would like to perform more experiment the assess the changes of zebrafish embryo morphology during early developmental stage, especially the immunostaining.

 

 

4. How is the protein expression represented in Fig. 5?  Is it a ratio of the analyzed protein to GADPH protein?

 

Author’s response: We have added the method (using Image J) for the semiquantitation of exposed bands in western blot in Materials and Methods. To quantify protein bands of caspase 3, Bax, and Bcl-2, we used the rectangle to choose the bands at the predicted molecular weight (following the manual of manufacture). The intensity of these bands was determined and the expression of these proteins was normalized to Gapdh.

We hope that our corrections could meet your requirements,

Thank you so much.

Round 2

Reviewer 3 Report

The authors have addressed some of the major concerns, and provided sufficient explanation for some of the other concerns that could not be addressed.  Statistical analysis of all figures is now included.

The authors' explanation for their analysis of protein expression using Western blots does not allay concerns about the quality of the blots.  If the lower MW proteins are degradation products of the specific proteins being analyzed, then just analyzing the band with the manufacturer's recommended MW will not capture all of the protein in the analysis.  And variable degradation under different chromium concentrations could skew the analysis and conclusions.

Author Response

Dear Prof. Dr. Madhav Bhatia, Editorial Boards and Reviewers,

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

Comment: The authors' explanation for their analysis of protein expression using Western blots does not allay concerns about the quality of the blots.  If the lower MW proteins are degradation products of the specific proteins being analyzed, then just analyzing the band with the manufacturer's recommended MW will not capture all of the protein in the analysis.  And variable degradation under different chromium concentrations could skew the analysis and conclusions.

Author’s response: Thank you so much for your comment. In this study, the primary antibodies (to caspase 3, bax, and bcl2) are polyclonal, they may recognize multiple lower molecular weight bands due to their ability to recognize multiple epitopes. This could result in the appearance of other bands in the blot film that are not the target proteins. Thus, the western blot bands we used for analysis are specific bands as the guidance on manufacture.

We hope that our explanation could meet your requirements,

Thank you so much.