Protective Effect of Emblica officinalis in Cyprinus carpio against Hepatotoxicity Induced by Malachite Green: Ultrastructural and Molecular Analysis

Round 1

Reviewer 1 Report

The manuscript entitled "Protective effect of Emblica officinalis in Cyprinus carpio against hepatotoxicity induced by Malachite Green: Ultrastructural and molecular analysis" investigated the mitigating effect of fruit extract on the dye toxicity. The authors revealed that the E. officinalis addition reduced oxidative stress and restored histoarchitecture.

Overall, this is a good manuscript presenting novel results. Some minor comments are as below:

Abstract

1. line 15. "GI-control, GII-MG ..." Describe as "group I control, group II MG, ..."

2. lines 16 to 17. Replace "," in line 16 with ":", and replace ";" in line 17 with ",".  

Materials and Methods

3. line 125. Spell out anti-oxidant enzyme names.

4. line 132. RNA integrity could not be judged by a spectrophotometer such as Nano drop. It should be assessed by electrophoresis-based methods, such as Bioanalyzer and TapeStation.

5. line 134. Clarify the amount of total RNA used for cDNA synthesis.

6. line 160 to 163. Clarify which parameters were used for PCA. According to the Discussion part, biomarkers on oxidative stress were used for PCA. However, I wonder why the authors did not include other parameters (e.g., HSP70 and CYP1A) into PCA analysis.

Results

7. line 317. Proximate? maybe "Principle".

Author Response

Authors are thankful to reviewers for providing valuable suggestion for improvising the manuscript. We have tried to inculcate insightful comments given by the reviewers.

Response to reviewer’s comment:

Reviewer 1: (revisions have been highlighted in yellow)

1. line 15. "GI-control, GII-MG ..." Describe as "group I control, group II MG, ..."

Response: Changes have been made in the abstract

2. lines 16 to 17. Replace "," in line 16 with ":", and replace ";" in line 17 with ",".  

     Response: Changes have been made

3. line 125. Spell out anti-oxidant enzyme names.

     Response: Done (line no. 125-126)

4. line 132. RNA integrity could not be judged by a spectrophotometer such as Nano drop. It

    should be assessed by electrophoresis-based methods, such as Bioanalyzer and             

    TapeStation.

    Response: Agreed to the comment by reviewer, integrity is assessed by the Gel-       

    Electrophoresis. Accordingly, statement has been modified (Line 133).

5. line 134. Clarify the amount of total RNA used for cDNA synthesis.

    Response: 2µl of RNA have been added as per the cDNA synthesis kit instruction.   

    It has been added in the manuscript as well (line 136).

6. line 160 to 163. Clarify which parameters were used for PCA. According to the Discussion

     part, biomarkers on oxidative stress were used for PCA. However, I wonder why the

     authors did not include other parameters (e.g., HSP70 and CYP1A) into PCA analysis.

     Response: PCA was carried out to define the most important parameter, which could be

     used as key factor for individual variation in biochemical changes (antioxidants in current   

     study) and histopathological alterations. However, determination of effect on genetic

     expression could not serve any statistical significance. Thus, genetic expression was not

     considered.

7. line 317. Proximate? maybe "Principle".

    Response: Thankful of reviewer, made the corrections.

Reviewer 2 Report

This is very interesting article, well prepared with an extensive discussion. Methods are well described and obtained results are presented in logical way.

35 - please give some examples of using MG as an aquaculture therapeutic

105 - what kind of methods?

127 - 10 000x  - probably you mean 10 000 rpm?

130 - qRT-PCR? - you mean Real Time RT-PCR (qRT-PCR) or reverse transcription (RT-PCR), because description fits to RT-PCR

179 - there is „fE” on the Figure 1C, but Ef in description

Check carrerfully figures description. There are some abbreviation on photos, which are not included in desription.

Author Response

Authors are thankful to reviewers for providing valuable suggestion for improvising the manuscript. We have tried to inculcate insightful comments given by the reviewers.

Response to reviewer’s comment:

Reviewer 2 (modifications are highlighted in green)

1. 35 - please give some examples of using MG as an aquaculture therapeutic

Response: MG is used in Saprolegniasis and Ichthyophthirius multifilis infection. It has been mentioned in the manuscript as well.

2. 105 - what kind of methods?

Response: Following histopathological processing methods of Baker, 1945 and Pearse 1968.

3. 127 - 10 000x  - probably you mean 10 000 rpm?

Response: It is typographical mistake. Yes, it should be 1000 rpm. (Line 128)

4. 130 - qRT-PCR? - you mean Real Time RT-PCR (qRT-PCR) or reverse transcription (RT-PCR), because description fits to RT-PCR

Response: It is Real-Time Reverse Transcription PCR. For this RNA was extracted, cDNA was synthesised, and quantitative estimation of gene expression has been made on Real time PCR.

5. 179 - there is „fE” on the Figure 1C, but Ef in description.

Response: Made the correction.

6. Check carefully figures description. There are some abbreviations on photos, which are not included in description.

Response: Carefully re-revised and re-checked the description and abbreviation for photographic figures.

Reviewer 3 Report

This manuscript represents a significant amount of work.  In many countries, treatment of fish with MG is not legal.  However, the information in this manuscript might be useful for people in countries where the use of MG is legal.

Some of the terminology used here is not consistent to what has been described for fish.  I recommend review of Fish Ecotoxicology pp 141-164 (Architectural pattern, tissue and cellular morphology in livers of fishes: Relationship to experimentally-induced neoplastic responses).  Unlike mammalian livers, fish livers do not have resident perisinusoidal macrophages (Kupffer cells) or central veins (line 166), and some species (like Cyprinus carpio) have intrahepatic exocrine pancreas.

Abstract line 21 – “were registered” may be replaced with “occurred”

Abstract line 22 – Can this be reworded to state the nature of the “significant alterations”; now it simply states that significant alterations occurred, which provides little useful information.

Line 38 – I recommend using the term “lesions” rather than “pathologies.”

Line 63 (Methods) – the methods described in this paper seem very similar to that described in reference 9 (Sinha and Jindal 2019) and the feed looks like it is the same in both experiments.  Whatever is identical does not need to be repeated here, but can be shortened by saying something like, “The feed used for this experiment is from the same batch as previously described (Sinha and Jindal 2019)”.

Line 96 – does the analysis have precision to three decimal places?  If so, report as is.  If not, then report to level of precision verified for the test (e.g., rather than 640.092, report 640.1 or 640).

Line 104 – Please add information about how many scorers were used, whether analysis was conducted while the scorer was blinded to the treatment, and whether scores were validated (e.g., were 10% of the scored sections also scored by a different scorer or by the same scorer at a different time).

Figure 2 – the resolution of the light micrographs available for my review does not allow me to independently confirm many of the described features.  Therefore, I recommend preparing higher-resolution versions of the light micrographs before publication.  As it is, I have the following concerns:

1. the hypertrophic cells (HT in 2A) look like exocrine pancreas,
2. the region of 2A labelled as “congested erythrocytes” would be better labelled as a “congested vessel,”
3. the whorls associated with bile canaliculi in 2C are probably not myelin (here and elsewhere, they might better be identified as something like “membrane whorls”)
4. I am not able to assess the nucleus in F labelled as a Kupffer cell, and
5. the mitochondrion labeled in H does not seem to be very swollen.

General notes for all figure labels:

1. I recommend using a sans serif font (e.g., Arial) for figure labels rather that the serif fonts used here (sans serif are easier to read).
2. For light micrographs, I recommend using scale bars (as with the electron micrographs) rather that what I assume is the magnification of the objective lens that was used for the image. This can be done manually by photographing a stage micrometer using the same lens and pixel resolution setting on the camera.

Lines 172 and 272 – “were observed” may be replaced with “were” [readers understand that reported findings are observed findings]; similar revisions can be applied to “was observed” [line 302 et al.], “was evident” [line 234 et al.], “was seen” [line 240], and “noticed”.  Some of these uses may be replaced with “occurred”.

Figure 3 – the resolution of the light micrographs available for my review does not allow me to independently confirm many of the described features.  Therefore, I recommend preparing higher-resolution versions of the light micrographs before publication.  As it is, I have the following concerns:

1. The first two words of the ‘A’ caption should probably be “Livers have” rather than “Hepatocytes showed”; oedema is the accumulation of interstitial fluid, and fibrosis is the accumulation collagen (which is always interstitial); therefore, by definition, it is not possible to have hepatocyte oedema or fibrosis. Also, scientists might show their posters at a meeting, but the posters simply have figures and text that attendees can read.
2. Necrosis in 3A – I am a diagnostic pathologist, and I cannot recall having necrosis—an acute lesion—in livers like this one that seem to have abundant glycogen. Therefore, the “necrosis” diagnosis here might not be correct.
3. Congested sinusoid in 3F – I am not able to confirm this finding in the image provided for review. Also, what is the difference between the “congested blood sinusoid (cBS)” in 3A and the “congested sinusoid (cS)” in 3F.  If they are the same, I recommend using the same name and abbreviation for both.

Line 240 – My understanding of the methods is that for the cohort that received E. officinalis and MG, they were fed both at the same time for the entire experiment.  Therefore, here and elsewhere it would be better to say that the inclusion of E. officinalis might have “protected liver architecture and maintained glycogen” rather than “reinstated liver architecture and alleviated glycogen.”  Be sure that other references to the role of E. officinalis focus on something like a protective mechanism rather than a restorative mechanism (e.g. line 400).

Figure 4 – the resolution of the light micrographs available for my review does not allow me to independently confirm many of the described features.  Therefore, I recommend preparing higher-resolution versions of the light micrographs before publication.  As it is, I have the following concerns:

1. The colour saturation of 4B is excessive.
2. The caption for I should lead with something like “Protected liver” rather than “Restored liver”,

Figure 5 caption – it is not clear what is meant by “‘a’ & ‘b’ indicates significant (P < 0.05) change with respect to control and MG treatment respectively (n=9).”  The figures list ‘a’ on the MG treatment and ‘b’ on the MG + EO treatment, but in some cases the MG+EO treatment does not seem to be different from the control (e.g., H2O2 at 30 and 60 days).

Figure 6 caption – it is not clear what is meant by “‘a’ & ‘b’ indicates significant (P < 0.05) change with respect to control and MG treatment respectively (n=9).”  In 6A, 60 days, the two bars labelled ‘a’ seem to be significantly different from each other.  In 6B, 60 days, the MG=EO bar does not seem to be different from the control.

Line 302 vs. Table 1. Caption – Does ND designate “not de-tectable” [line 302] or “Not determined” [Table 1 caption line 311]?

Lines 312-313, “‘b’ indicates significant (P<0.05) difference compared to MG treatment”; is the line 309 ‘b’ being compared to MG 30 day and the line 310 ‘b’ being compared to MG 60 day?  Please clarify.

Lines 330 – 332 – The wording here might benefit from revision.  I agree that lysosomes and auto-phagosomes indicate the defence system of hepatocytes, but other cells (identified here as Kupffer cells) are not hepatocytes.

Line 339 – the abbreviation LMG needs to be defined

Line 368 – I infer that the goal is to have this sentence refer to EO+MG having greater SOD and CAT activity than MG alone.  If so, clarification is needed, because the wording could also be interpreted to mean that EO alone increases SOD and CAT activity compared to the control diet, but based on Figure 5 this does not seem to be the case.

Line 382 – the study design does not allow for a strong conclusion related to the mechanism of MG removal; therefore, this line needs revision to something like, “EO supplementation facilitated MG removal from the body, probably by…”

Author Response

Authors are thankful to reviewers for providing valuable suggestion for improvising the manuscript. We have tried to inculcate insightful comments given by the reviewers.

Response to reviewer’s comment:

Reviewer 3: (Revisions have been highlighted in turquoise)

1. Some of the terminology used here is not consistent to what has been described for fish. I recommend review of Fish Ecotoxicology pp 141-164 (Architectural pattern, tissue and cellular morphology in livers of fishes: Relationship to experimentally induced neoplastic responses).  Unlike mammalian livers, fish livers do not have resident perisinusoidal macrophages (Kupffer cells) or central veins (line 166), and some species (like Cyprinus carpio) have intrahepatic exocrine pancreas.

Response: Corrections have been made in the manuscript as per the suggestion.

2. Abstract line 21 – “were registered” may be replaced with “occurred”.

Response: Replacement has been done.

3. Abstract line 22 – Can this be reworded to state the nature of the “significant alterations”; now it simply states that significant alterations occurred, which provides little useful information.

Response: Suggestion has been incorporated as ‘significant change in the expression level’.

4. Line 38 – I recommend using the term “lesions” rather than “pathologies.”

Response: Made the replacement as per suggestion.

5. Line 63 (Methods) – the methods described in this paper seem very similar to that described in reference 9 (Sinha and Jindal 2019) and the feed looks like it is the same in both experiments.  Whatever is identical does not need to be repeated here, but can be shortened by saying something like, “The feed used for this experiment is from the same batch as previously described (Sinha and Jindal 2019)”.

Response:  Reframed the sentence as per suggestion (line 100-101).

6. Line 96 – does the analysis have precision to three decimal places?  If so, report as is.  If not, then report to level of precision verified for the test (e.g., rather than 640.092, report 640.1 or 640).

Response: Values are reported to one decimal point.

7. Line 104 – Please add information about how many scorers were used, whether analysis was conducted while the scorer was blinded to the treatment, and whether scores were validated (e.g., were 10% of the scored sections also scored by a different scorer or by the same scorer at a different time).

Response: Random scoring of 50 sections from each experimental group were made, analysed, and calculated by the author (109-110)

8. Figure 2 – the resolution of the light micrographs available for my review does not allow me to independently confirm many of the described features.  Therefore, I recommend preparing higher-resolution versions of the light micrographs before publication.  As it is, I have the following concerns:

1. the hypertrophic cells (HT in 2A) look like exocrine pancreas,

Response: Agreed, mistakenly false identification was done.

2. the region of 2A labelled as “congested erythrocytes” would be better labelled as a  

   “congested vessel,”

   Response: Labelling is improved as per the suggestion.

3. the whorls associated with bile canaliculi in 2C are probably not myelin (here and elsewhere, they might better be identified as something like “membrane whorls”).

Response: Myelin whorls are replaced by term membrane whorls

4. I am not able to assess the nucleus in F labelled as a Kupffer cell, and

Response: As per the guidance, I am correcting the labelling.

5. the mitochondrion labelled in H does not seem to be very swollen.

Response: It is labelled as mitochondrion only, not to be considered as swollen.

9. General notes for all figure labels:
10. I recommend using a sans serif font (e.g., Arial) for figure labels rather that the serif fonts used here (sans serif are easier to read).

Response: Improvised the labelling with Arial. 

2. For light micrographs, I recommend using scale bars (as with the electron micrographs) rather that what I assume is the magnification of the objective lens that was used for the image. This can be done manually by photographing a stage micrometer using the same lens and pixel resolution setting on the camera.

Response: With due respect, the present experiment and light micrography were performed in 2018, so for now it is not possible to make any change considering this suggestion. Kindly consider the magnification (10X & 40X) for the light microscopy.  

10. Lines 172 and 272 – “were observed” may be replaced with “were” [readers understand that reported findings are observed findings]; similar revisions can be applied to “was observed” [line 302 et al.], “was evident” [line 234 et al.], “was seen” [line 240], and “noticed”.  Some of these uses may be replaced with “occurred”.

             Response: Made the suggested replacements.

11. Figure 3 – the resolution of the light micrographs available for my review does not allow me to independently confirm many of the described features.  Therefore, I recommend preparing higher-resolution versions of the light micrographs before publication.  As it is, I have the following concerns:
12. The first two words of the ‘A’ caption should probably be “Livers have” rather than “Hepatocytes showed”; oedema is the accumulation of interstitial fluid, and fibrosis is the accumulation collagen (which is always interstitial); therefore, by definition, it is not possible to have hepatocyte oedema or fibrosis. Also, scientists might show their posters at a meeting, but the posters simply have figures and text that attendees can read.

Response: Agreed with your explanations, thus corrected the misidentification.

2. Necrosis in 3A – I am a diagnostic pathologist, and I cannot recall having necrosis—an acute lesion—in livers like this one that seem to have abundant glycogen. Therefore, the “necrosis” diagnosis here might not be correct.

Response: I would like to explain that

1. Histopathological and TEM images are not the same section.
2. At places, prominent necrosis was evident.
3. Moreover, the glycogen content recorded was comparatively very low compared to control fish liver.

3. Congested sinusoid in 3F – I am not able to confirm this finding in the image provided for review. Also, what is the difference between the “congested blood sinusoid (cBS)” in 3A and the “congested sinusoid (cS)” in 3F.  If they are the same, I recommend using the same name and abbreviation for both.

Response: It is typographical mistake, it should be erythrocyte infiltration, thus corrected.

12. Line 240 – My understanding of the methods is that for the cohort that received  officinalis and MG, they were fed both at the same time for the entire experiment.  Therefore, here and elsewhere it would be better to say that the inclusion of E. officinalis might have “protected liver architecture and maintained glycogen” rather than “reinstated liver architecture and alleviated glycogen.”  Be sure that other references to the role of E. officinalis focus on something like a protective mechanism rather than a restorative mechanism (e.g. line 400).

             Response: Suggestions has been incorporated at the specified lines ( line 258-259).

13. Figure 4 – the resolution of the light micrographs available for my review does not allow me to independently confirm many of the described features.  Therefore, I recommend preparing higher-resolution versions of the light micrographs before publication.  As it is, I have the following concerns:
14. The colour saturation of 4B is excessive.

Response: Image quality has been improved.

2. The caption for I should lead with something like “Protected liver” rather than “Restored liver”,

Response: Incorporated the replacement.

14. Figure 5 caption – it is not clear what is meant by “‘a’ & ‘b’ indicates significant (P < 0.05) change with respect to control and MG treatment respectively (n=9).”  The figures list ‘a’ on the MG treatment and ‘b’ on the MG + EO treatment, but in some cases the MG+EO treatment does not seem to be different from the control (e.g., H2O2 at 30 and 60 days).

Response: Plausible explanation to the graph has been made (line 286-287), hope it will make it clear.

15. Figure 6 caption – it is not clear what is meant by “‘a’ & ‘b’ indicates significant (P < 0.05) change with respect to control and MG treatment respectively (n=9).”  In 6A, 60 days, the two bars labelled ‘a’ seem to be significantly different from each other.  In 6B, 60 days, the MG=EO bar does not seem to be different from the control.

Response: Graph is to be read as

• ‘a’ indicates significant (P<0.05) change in groups (II, III, IV) compared to control group. While ‘b’ indicates significant change compared to the MG treatment (group II) in the amelioration group (Group IV).
• In case of Fig. 6A, EO showed significant difference, due to typographic error.

While in Fig 6B, if I am able to get the question. MG+EO has been designated with ‘b’, indicating significant change compared to MG treatment.

16. Line 302 vs. Table 1. Caption – Does ND designate “not de-tectable” [line 302] or “Not determined” [Table 1 caption line 311]?

Response: Typographical mistake has been corrected.

17. Lines 312-313, “‘b’ indicates significant (P<0.05) difference compared to MG treatment”; is the line 309 ‘b’ being compared to MG 30 day and the line 310 ‘b’ being compared to MG 60 day?  Please clarify.

Response: Yes, it is the same.

18. Lines 330 – 332 – The wording here might benefit from revision.  I agree that lysosomes and auto-phagosomes indicate the defence system of hepatocytes, but other cells (identified here as Kupffer cells) are not hepatocytes.

            Response: Made the necessary modifications.

19. Line 339 – the abbreviation LMG needs to be defined.

            Response: LMG is leuco -MG, modifications made in the manuscript.

20. Line 368 – I infer that the goal is to have this sentence refer to EO+MG having greater SOD and CAT activity than MG alone.  If so, clarification is needed, because the wording could also be interpreted to mean that EO alone increases SOD and CAT activity compared to the control diet but based on Figure 5 this does not seem to be the case.

Response: Remodelled the sentence to make it more appropriate (line 373-375).

21. Line 382 – the study design does not allow for a strong conclusion related to the mechanism of MG removal; therefore, this line needs revision to something like, “EO supplementation facilitated MG removal from the body, probably by…”

Response: Incorporated the revision (line 388).