Cytoprotective and Antigenotoxic Properties of Organic vs. Conventional Tomato Puree: Evidence in Zebrafish Model

Round 1

Reviewer 1 Report

Title: Cytoprotective and Antigenotoxic Properties of Organic Vs. 2 Conventional Tomato Puree: Evidence in Zebrafish Model authored by Mottola et al.

Manuscript is well written and presented good, however authors need to give justification for the following comments.

What is the fish size used for the experiment?

Is it possible to collect sufficient blood from such a small fish which can meet many assays?

0.5 mg/L tomato puree in the water, what was the volume of water used, authors say after 7 days water was changed, does not the puree degrades?

What is the difference between P1+H2O2 and P2+H2O2

Authors say P1 and P2 are conventional tomato puree (having pesticides in it). When P1 and P2 in combination/co-exposure with H2O2 should have shown more negative effect than H2O2 alone. But the results are contrary. What could be the reasons?

Table 1 : What does the ‘/’ (slash) indicates?

Line 288: Various types of tomato puree? I guess only two different types, not various.

Line 327: grown?

Any data on how long the conventional tomatoes and organic tomatoes were grown?

Author Response

Response to the Reviewers

Reviewer 1

Manuscript is well written and presented good, however authors need to give justification for the following comments.

R: We thank the reviewer for appreciating our manuscript, we have revised it according to the suggestion of Reviewer as follows.

What is the fish size used for the experiment?

R: Zebrafish size was about 3.5 cm in length. This information has been added in the methods (page 3, lines 102-103).

Is it possible to collect sufficient blood from such a small fish which can meet many assays?

R: Yes, we collect at least 25 microliters of nucleated blood cells (erythrocytes) from each fish and this was enough to meet the requirements of each assay (page 4, line 131).  

0.5 mg/L tomato puree in the water, what was the volume of water used, authors say after 7 days water was changed, does not the puree degrade?

R: The volume of water used was 10 L per tank (page 3, lines 103-104). The water in the tank was changed every 7 days, and fish were exposed to fresh tomato puree to avoid degradation (page 3, line 125).

What is the difference between P₁+H₂O₂ and P₂+H₂O₂?

R: P₁ and P₂ are two different brand of conventional tomato puree, tested at the same concentration in combination with H₂O₂. This has been clarified at page 3, lines 106-111.

Authors say P₁ and P₂ are conventional tomato puree (having pesticides in it). When P₁ and P₂ in combination/co-exposure with H₂O₂ should have shown more negative effect than H₂O₂ alone. But the results are contrary. What could be the reasons?

R: We thank the reviewers for this important comment. In P₁ and P₂ tomato groups, produced by conventional farming methods for which the use of pesticides is allowed, the presence of residues can limit the antigenotoxic power of antioxidants naturally present in the tomato. Hence, natural antioxidants can still counteract the effects of H₂O₂ exposure, although to a much lower extent than organic tomato product (page 2, lines 84-89).

Table 1 : What does the ‘/’ (slash) indicates?

R: Slash indicates “no variation of bands”. We have added this information in the Table 1 legend.

Line 288: Various types of tomato puree? I guess only two different types, not various.

R: We rephrased the sentence for clarity as : “The aim of this study was to evaluate the potential antioxidant, cytoprotective and antigenotoxic impact of tomato puree obtained by organic and conventional cultivations” (page 9, lines 314-316).

Line 327: grown?

R: It was revised.

Any data on how long the conventional tomatoes and organic tomatoes were grown?

R: Commercial tomato products P₁ and P₂ were purchased at a local market while organic tomatoes were grown as follows. The tomato seedlings were transplanted on 30 April 2020 at the three-true-leaf stage in a local farm. Plants grew under natural conditions and were cultivated in compliance with the organic farming method. Fruits were harvested 70-80 days after the transplant in July 2020 and processed in puree (page 3, lines 110-115).

Reviewer 2 Report

The manuscript by Mottola et al. “Cytoprotective and Antigenotoxic Properties of Organic vs. 2 Conventional Tomato Puree: Evidence in Zebrafish Model” is an interesting result from a somewhat novel experiment. The results are statistically significant for some treatments and the agrofood industry should take interest in these findings. Before acceptance there needs to be significant revision to the text, especially to the Materials and Methods, and Results sections.

Three flaws in this study are worth noting. First, there is the lack of chemical analysis of the purees to determine what chemicals could be present and responsible for observed effects. The authors rightly acknowledge this in the Discussion.  Second, there are no tank replicates. All fish from each treatment regime were held in the same tank so any tank effect that could be a source of the observed effects is unknown. Location, ambient light, and ambient noise are all known to influence stress levels in fish held in captivity. Third, the dose of exposure is questionable. The authors explain why it was chosen, but is it biologically relevant? Would a human be exposed for 14 days continuously, morning, noon, and night to tomato extracts? A factory worker in a tomato canning factory could be exposed to fumes, but ingestion of significant amounts daily is unlikely.

Most of the grammar is fine but science is a language of precision. Certain words like “specimen” can be replaced by more precise nouns like “cells”, erythrocytes”, or “fish”, or modify “specimen” to include any of the latter. The caption for Fig. 3 suggests as written, that blood cells were exposed to the puree ± H₂O₂ rather than whole fish.

The M&M describing the cell viability is overly brief and vague. How many microscope fields were examined? How many cells were counted? What is the magnification used for viewing the cells? For replication of experiments by other investigators, or to evaluate the protocol properly there is a need for fine detail including manufacture/reference number of equipment or reagents. This is a simple protocol yet there is still a need for some basic information like magnification and number of cells counted. Similarly, in lines 123-125 of the explanation of the design there is information about preparation of blood samples that belongs in the sections further down were blood cells are used in the different assays. The text “…mixed with phosphate-buffered saline…… 1× and subsequently centrifuged at 2000 rpm for 10 minutes…” is part of the other sections of M&M for the cell-based assays.

I also think the Discussion should include some text discussing the significance of the H₂O₂ treatment. In Fig. 2 of the results it appears to be the only active factor used in the trials bringing to question the significance of Commercial puree products (although clearly this is not so), but there is no discussion of this. Additional corrections are listed below.

• The use of the hyphenated “not-exposed” should be replaced by the grammatically correct “unexposed”.
• Correct “trice” on line 186
• Replace “DCF assay” for cellular “ROS assay”. The assay name should reflect what is being measured not the substrate used in the assay.
• If data is presented as a rate there is usually a time component, and there is a measure of change over time, or it is a unit rate and the denominator is 1. Don´t conflate rate with percentage, and label all graphs, captions, and graphic axes accordingly and appropriately.
• Correct line 75, change “case” to “cases”
• Correct line 76, add the article “the”: “; however, the general population…”
• Correct line 199 to read as “…further decreasing to 50% after 14 days”
• Correct lines 178-179 by including the necessary articles so as to read: “First, the volume of blood sample was washed twice with an equal volume of PBS…”
• Correct line 165 by replacing “e” with “and”.
• Include parentheses around “Nikon Eclipse E600”
• Rewrite line 244-245 to read: “…showed a characteristic pattern of polymorphic bands”
• In Figure 5 include all three bars for the “not exposed” control just as was shown for the other graphs.
• The first use of DFI% in the main text of the introduction should provide the full title “DNA Fragmentation Index”. Nowhere in the manuscript is the correct title used.
• Line 339 there is the phrase “determines a recovery” which is semantically unclear. Consider rephrasing this sentence.

Author Response

Reviewer 2

The manuscript by Mottola et al. “Cytoprotective and Antigenotoxic Properties of Organic vs. 2 Conventional Tomato Puree: Evidence in Zebrafish Model” is an interesting result from a somewhat novel experiment. The results are statistically significant for some treatments and the agrofood industry should take interest in these findings. Before acceptance there needs to be significant revision to the text, especially to the Materials and Methods, and Results sections.

R: We thank the reviewer for appreciating our manuscript.

Three flaws in this study are worth noting. First, there is the lack of chemical analysis of the purees to determine what chemicals could be present and responsible for observed effects. The authors rightly acknowledge this in the Discussion.  

R: We agree with the Reviewer that this represents a limitation of our study, as pointed out in the discussion. Although this is the first study investigating the cytoprotective and genoprotective effects of organic and conventional commercial tomato puree in zebrafish, further research is needed in this field.

Second, there are no tank replicates. All fish from each treatment regime were held in the same tank so any tank effect that could be a source of the observed effects is unknown. Location, ambient light, and ambient noise are all known to influence stress levels in fish held in captivity.

R: We fully agree with the reviewer and apologize for this transcription error. We revised section 2.1 to clarify that a total of 240 fish were raised in a room with no ambient noise, in 16 tanks of 10 liters of water volume containing 15 fish each. These replicates allowed to limit the effects of the environment on stress levels in fish kept in captivity (page 3, lines 102-105).

Third, the dose of exposure is questionable. The authors explain why it was chosen, but is it biologically relevant? Would a human be exposed for 14 days continuously, morning, noon, and night to tomato extracts? A factory worker in a tomato canning factory could be exposed to fumes, but ingestion of significant amounts daily is unlikely.

R: This study is based on Zebrafish as animal model and aims to provide insight about the cytoprotective and genoprotective effects of tomatoes obtained by using different cultivation approaches. Although relevant, ours are preliminary results, and it would be premature, and not in the authors’ intentions, to translate them and draw any conclusion for human exposure. As the Reviewer fairly pointed out, it is unlikely for a human to be exposed at the same concentration tested by us for 14 days. Hence, more research is needed to investigate any possible impact of tomato consumption in humans.

Most of the grammar is fine but science is a language of precision. Certain words like “specimen” can be replaced by more precise nouns like “cells”, erythrocytes”, or “fish”, or modify “specimen” to include any of the latter. The caption for Fig. 3 suggests as written, that blood cells were exposed to the puree ± H₂O₂ rather than whole fish.

R: We followed the reviewer's suggestion and replaced the word “specimen” with more specific names such as “zebrafish”, “cell”, and “DNA” throughout the manuscript as well as in the figure 3 caption.

The M&M describing the cell viability is overly brief and vague. How many microscope fields were examined? How many cells were counted? What is the magnification used for viewing the cells? For replication of experiments by other investigators, or to evaluate the protocol properly there is a need for fine detail including manufacture/reference number of equipment or reagents. This is a simple protocol yet there is still a need for some basic information like magnification and number of cells counted.

R: We thank the reviewer for this important comment. We have described the protocol in more detail at page 4, section “Cell viability test”.

Similarly, in lines 123-125 of the explanation of the design there is information about preparation of blood samples that belongs in the sections further down were blood cells are used in the different assays. The text “…mixed with phosphate-buffered saline…… 1× and subsequently centrifuged at 2000 rpm for 10 minutes…” is part of the other sections of M&M for the cell-based assays.

R: According to the suggestion, we moved the sentence at the beginning of section 2.3. (page 4, lines 162-164).

I also think the Discussion should include some text discussing the significance of the H₂O₂ treatment. In Fig. 2 of the results it appears to be the only active factor used in the trials bringing to question the significance of Commercial puree products (although clearly this is not so), but there is no discussion of this.

R: We expanded the discussion on H₂O₂ treatment, further clarifying the reason why it was used to test the cytoprotective and genoprotective tomato powers (page 10, lines 366-376).

Additional corrections are listed below.

• The use of the hyphenated “not-exposed” should be replaced by the grammatically correct “unexposed”.

R: It was corrected

• Correct “trice” on line 186

R: It was corrected

• Replace “DCF assay” for cellular “ROS assay”. The assay name should reflect what is being measured not the substrate used in the assay.

R: It was corrected

• If data is presented as a rate there is usually a time component, and there is a measure of change over time, or it is a unit rate and the denominator is 1. Don´t conflate rate with percentage, and label all graphs, captions, and graphic axes accordingly and appropriately.

R: According to Reviewer’s suggestion, we substituted the term “rate” with a more appropriate “percentage”.

• Correct line 75, change “case” to “cases”

R: It was corrected

• Correct line 76, add the article “the”: “; however, the general population…”

R: It was corrected

• Correct line 199 to read as “…further decreasing to 50% after 14 days”

R: It was corrected

• Correct lines 178-179 by including the necessary articles so as to read: “First, the volume of blood sample was washed twice with an equal volume of PBS…”

R: It was corrected

• Correct line 165 by replacing “e” with “and”.

R: It was corrected

• Include parentheses around “Nikon Eclipse E600”

R: It was included

• Rewrite line 244-245 to read: “…showed a characteristic pattern of polymorphic bands”

R: It was done

• In Figure 5 include all three bars for the “not exposed” control just as was shown for the other graphs.

R: It was revised.

• The first use of DFI% in the main text of the introduction should provide the full title “DNA Fragmentation Index”. Nowhere in the manuscript is the correct title used.

R: It was revised (page 9, line 298).

• Line 339 there is the phrase “determines a recovery” which is semantically unclear. Consider rephrasing this sentence.

R: This sentence has been rephrased as: “The results showed that the co-exposure of the pesticide-free tomato puree with H2O2 resulted in lower DNA fragmentation and induced mutations” (page 10, lines 376-378).

Round 2

Reviewer 2 Report

The work by Mottloa and coauthors has improved somewhat and with only a three additional minor corrections can be accepted for publication. 

1) In section 2.5 put "DNase and RNase free" in parentheses.

2) In section 2.6, line 211, correct "trice" to "thrice", or "twice", whichever is correct for this part of the text. However, the authors are advised to use "triplicate" in such instants rather than "thrice" for scientific works and reserve "thrice" for more prosaic text.

3) Rewrite line 256 (mark-up version) to read as "...in the case of exposure to individual treatments...."

4) All of the graphs are still lacking a label for the X-axis. It shoud be understood that a color key is not the same as an axis label. The axis label shows what is measured and the units. The color key is for differentiation of groups and can be placed aside the graph or below the X-axis label. Please correct all figures accordingly.

Author Response

The work by Mottloa and coauthors has improved somewhat and with only a three additional minor corrections can be accepted for publication. 

R: Authors thank the Reviewer for his/her important feedback and suggestions.

• In section 2.5 put "DNase and RNase free" in parentheses.

R: It was revised.

• In section 2.6, line 211, correct "trice" to "thrice", or "twice", whichever is correct for this part of the text. However, the authors are advised to use "triplicate" in such instants rather than "thrice" for scientific works and reserve "thrice" for more prosaic text.

R: Authors prefer not to use the term “triplicate” because, in section 2.6, the same step (washing) was repeated more than one time for the same sample. We have modified “thrice” and “twice” as “for three and two times”, respectively.

• Rewrite line 256 (mark-up version) to read as "...in the case of exposure to individual treatments...."

R: It was revised.

• All of the graphs are still lacking a label for the X-axis. It shoud be understood that a color key is not the same as an axis label. The axis label shows what is measured and the units. The color key is for differentiation of groups and can be placed aside the graph or below the X-axis label. Please correct all figures accordingly.

R: we have included a label for X-axis in all the graphs [Percentage (%)] as per Reviewer’s suggestion.