Comparative Toxicity of Vegan Red, E124, and E120 Food Dyes on Three Rapidly Proliferating Model Systems

Round 1

Reviewer 1 Report

General comment: The manuscript brings useful information about the toxic effects of three common red food dyes,  (i) a vegan red (VEG) preparation, (ii) the cochineal E120, and (iii) the artificial E124 on three rapidly proliferating models (i.e., PNT1A cells, Artemia salina nauplii, and Cucumis sativus seedlings). The authors found that vegan red impairs cell growth, delays naupliar hatching and early growth in Artemia, and reduces shoot/root biomass in Cucumis. Their obtained findings demonstrate that the three dyes affect development and that the vegan substitute is as unsafe as the E124 and E120. The reviewer believed that the present study is interesting and potentially could contribute to the research field, however, there are some concerns and questions that require be addressed to clarity and improve the present version.

Specific comments:

Title

The title is informative and relevant to the major findings.

Abstract

In the abstract, the aim of the study is clearly mentioned and major results are also properly presented. However, information on treatment doses (concentrations) and treatment duration are missing in the abstract.

Introduction

The introduction is one of the main sections of a research article. Unfortunately, the introduction section of the current manuscript does not provide sufficient background for the study. The research gap/question is not clearly outlined. The authors should briefly discuss the toxicity mechanisms of common red food dyes in various cells, Artemia salina nauplii, and Cucumis sativus seedlings.

Materials and Method

In general, well described. No major concern.

Results

The results section is not well explained. Please compare your results with other previously published reports. Please give an explanation,

-Why was the cell viability increased in vegan red-treated cells?

Discussion

The discussion section could be more focused. The discussion doesn’t properly reflect the results obtained in this study. Authors can give more emphasis to discussing their findings from multiple angles in this section.

-Please explain in detail the statement in lines 312-314.

- Conclusion could be a separate section that would answer the aims of the study and should discuss major limitations of the present study and opportunities to inform future research.

Additional comments:

-Please consider removing repeated “* Correspondence: *Corresponding:” in line 11.

 

-Font style and size should be checked in lines 298 and 300.

Author Response

We thank this Reviewer to his/her precious comments in improving our manuscript. We have strictly followed the Reviewer recommendation in amending the criticisms. We have highlighted in the text the changes by word tracking system. Below are reported the point-to point response to Reviewer comments (R= reviewer comment; A= Author response)

R :  English language and style, fine/minor spell check required

A: We have revised and improved it

ABSTRACT

R: Abstract information on treatment doses (concentrations) and treatment duration are missing. 

A: Thank you for the comment , we added the informations required 

INTRODUCTION

R:- The introduction section of the current manuscript does not provide sufficient background for the study.

A:  Following the suggestion by both reviewers, we have amended this point. In the revised version we have reported background information on different species, including humans.

R: -Authors should briefly discuss the toxicity mechanisms of common red food dyes in various cells, Artemia salina nauplii, and Cucumis sativus seedlings.

A: As far as we were able to find, in the literature there are no data on the toxicity mechanisms of E124 or E120 or vegan red preparation in brine shrimp or Cucumis. The only paper available has been published by our group and demonstrates interference with the oxidative state (ref 9) with consequences on growth (ref 8). On lines 52-53 a sentence referring to the two published papers has been added. 

R: -The research gap/question is not clearly outlined.

A: We revised lines 79-89 so to better clarify the aim of the research and the rationale of the investigations carried out.

RESULTS

R : The results section is not well explained. Please compare your results with other previously published reports. Please give an explanation.

A: We have improved it. In lines 326 and 354 we compared previously published results for E124. For E120 there are no data to compare since, apart from mortality (added on line 358), no other parallel data are available. The same is for the vegan pigment that was absent in the previous study (Motta et al 2019). In addition, as stated above, no data on red dye toxicity could be found in our models.

R : Why was the cell viability increased in vegan red-treated cells?

A: The question is already addressed in lines 297-306.

DISCUSSION

R : The discussion section could be more focused. The discussion doesn’t properly reflect the results obtained in this study. Authors can give more emphasis to discussing their findings from multiple angles in this section.

A : We improved the discussion in some points in order to clarify important aspects see lines  328-344 and lines 372-383 . We hope this new version of discussion may fulfil the reviewer expectation. 

R: Please explain in detail the statement in lines 312-314.

A: Sentence amended, now lines 333-336

R: Conclusion could be a separate section that would answer the aims of the study, and should discuss major limitations of the present study and opportunities to inform future research.

A:  A  separate section was made, discussion of  major limitations  was add on lines 386-391, and opportunities to inform future research were add  on lines 393-395.

Additional comments:

R: Please consider removing repeated “* Correspondence: *Corresponding:” in line 11.

A: Done

R: Font style and size should be checked in lines 298 and 300.

 A: We have checked and corrected

Reviewer 2 Report

The submitted research manuscript by Avallone et al., is organized, well executed and falls within the scope of the journal. However, below are the points which are needed to clear for improving the quality of the presentation.

1.      Abstract section should be polished for the better clarity of the work aimed study.

2.      Author should also discuss in brief 2-3 lines about the food dyes and related their harm to human health.

3.      Why MTT assay indicated that after 24 hours of exposure, vegan red induced a significant increase and E124 a significant decrease in cell viability?

4.      Please check the scale bar in Figure 7.

 

Author Response

We are grateful to this reviewer for his/her valuable comments. We have improved the manuscript following the Reviewer instruction and recommendations. We have highlighted in the text of the manuscript all the changes in tracking word system. Below we have reported the responses to each question (R= reviewer comment; A= Author response).

R: English language and style, fine/minor spell check required

A: Revised

R1: Abstract section should be polished for the better clarity of the work aimed study.

A1: The aim has been explained more clearly in lines 19-25

R2: Author should also discuss in brief 2-3 lines about the food dyes and related their harm to human health.

A2: Thanks for the comment, we added required explanation in:

line 48-53, more info on E124 effect on children, plants, and animal models

lines 56-58, more info on allergic responses to E120 reported

lines 70-75. more detail on vegan red effects

Reference numbering was changed accordingly in the text and bibliography

R3: Why MTT assay indicated that after 24 hours of exposure, vegan red induced a significant increase and E124 a significant decrease in cell viability?

A3: For the vegan red preparation, the explanation may reside in the antioxidant effects and/or presence of antifungal proteins in the extract (lines 298-303). For E124 the effect may depend on cytotoxicity (demonstrated on allium cepa meristematic cells, a line of fast proliferating cells as the cells used in this study; Santana, G. M.; do Anjos Sousa, J. J.; Peron, A.P. Action of Ponceau 4R (E-124) food dye on root meristematic cells of Allium cepa. L Acta Sci Biol Sci 2015, 37(1),101-106. DOI: 10.4025/actascibiolsci.v37i1.23119.

R4: Please check the scale bar in Figure 7.

A4: The figure was checked and replaced in the new version of the manoscript

Round 2

Reviewer 1 Report

OK