Toxicological and Nutraceutical Screening Assays of Some Artificial Sweeteners

Round 1

Reviewer 1 Report

Dear author,

Thank you for your valuable manuscript. I read it very interestingly. In the future, I think it will be an opportunity to further develop research in this field.

Itemize what you notice

1. Lines 44-46 are "actually", but please refer to them.

2. How did you determine the concentrations of hydrogen peroxide, ACK, hokaASP, and CYC?

3. Tell us a little more about how ASP and CYC reduce the toxicity of hydrogen peroxide. What is the difference between these and the biochemical mechanism of ACK?

4. There is an error in Table 3. It may be a mistake in the editorial department, but it may not meet the posting criteria. Please check.

5. The content of the abstract and the "conclusion" are different. You should unify.

Author Response

Authors thank all the comments and suggestions of reviewers. Thanks of these, we consider that the manuscript has improved its quality. Below we indicate the modifications asked for.

1. Dear author,

Thank you for your valuable manuscript. I read it very interestingly. In the future, I think it will be an opportunity to further develop research in this field.

Itemize what you notice

Lines 44-46 are "actually", but please refer to them.

Answer: Thank you very much for the comment. We did miss the references. So, two references have been included at the end of the sentence [2,3].

2. How did you determine the concentrations of hydrogen peroxide, ACK, hokaASP, and CYC?

Answer: The working concentration of hydrogen peroxide were stablished by our research group (Romero-Jiménez, et al. 2005) in order to find an assay concentration both for Drosophila ranging 50% toxicity (as we routinely use it as a positive control for toxicity/antitoxicity trials) and, at the same time, that is mutagenic (as we use it as a positive control in genotoxicity/antigenotoxicity trials). Thanks for the comment, authors have added the reference [28] in the 2.4.1 section.

Furthermore, the concentration range of sweeteners was stablished in order to cover the values by the most popular soft drink traders. For this, we have used the concentration of the respective contents by the worldwide distributed cola beverages already determined. For this, two references have been added in the 2.4.1 section ([27] and [8]).

Text was modified as follow:

"The concentrations used in ASP and ACK were 0.0063 mM, 0.025 mM, 0.05 mM, 0.2 mM and 0.815 mM; and 0.0125 mM, 0.05 mM, 0.1 mM, 0.4 mM and 1.6 mM in the CYC assays, according to previous analyses carried out by Lehkorizova, et al. [27] and Cillo [8]. Negative (H2O) and positive (0.15 M H2O2) toxicant concurrent controls were also assayed, according to previous ranging established by Romero-Jiménez, et al. [28]"

3. Tell us a little more about how ASP and CYC reduce the toxicity of hydrogen peroxide. What is the difference between these and the biochemical mechanism of ACK?

Answer: authors thanks the comment of the referee as the paper has improved. Following his/her advice we have included related available information on the different mechanisms in the Discussion section:

“Antioxidant compounds act providing cells with mechanisms to defence themselves from ROS-induced damage. Glutathione, ascorbate, superoxide dismutase, catalase and glutathione-dependent antioxidant enzymes are compounds which exert antioxidant defence [80]. It is demonstrated that after 15 days of ASP consumption (40 mg/kg b.w), rats exerted an increase in superoxide dismutase, glutathione peroxidase and catalase and a reduction of glutathione which means an increase of antioxidant level from free radical production. However, when time exposure to ASP was increased, the free radical production over helmed the antioxidant defence producing a decrease in antioxidant molecules. Therefore, ASP provokes a dose dependent oxidant/antioxidant imbalance in rats [81]. In addition, ASP was found to be antigenotoxic reverting the damage caused by the mycotoxin ochratoxin A feeding rats with 25 mg/kg [82]. Conversely, Abhilash, et al. [83] concluded that ASP was able to modify the antioxidant defence status of rats fed with 1000 mg/kg ASP and be the responsible for oxidative stress [84,85].

With this regard, ACK also produced oxidative stress in Cyprinus carpio modifying the activity of superoxide dismutase and catalase [86] and the pancreas of diabetic rats showed an improvement in antioxidant conditions when this model organism was fed with CYC and saccharin producing glutathione reductase and superoxide dismutase [87].

With this in mind, we hypothesize that the activation of the antioxidant mechanisms depends on the dose of administration showing a dose dependent influence and also depends on the model organism which it is used. Therefore, when cells are treated at low concentrations the antioxidant mechanisms are increased but the oxidant/antioxidant imbalance occurs when the concentration exceeds the threshold allowed by the kind of cell and the model organism and then, the ROS production is higher than the antioxidant activity.” 

4. There is an error in Table 3. It may be a mistake in the editorial department, but it may not meet the posting criteria. Please check.

Answer: Thanks again for the comment. Posting criteria have been checked and table has been modified taking them into account.

5. The content of the abstract and the "conclusion" are different. You should unify.

Answer: Abstract and Conclusion sections have been rewritten to unify them. Changes could be seen in change form in the manuscript.

Reviewer 2 Report

The article describes a very important and up-to-date topic. There is a need of providing more this kind of reports so the final consumers would not be so disoriented and confused with the choice when doing shopping. The article is generally well written, the research well planned, the presentation of results is clear and they are exhaustively described. The discussion provides a deeper insight into results as well as highlight the importance of the research.

I have only minor comments on the English language and length of sentences in the Introduction and Abstract part. Some sentences are a bit confusing.

Authors should also pay attention to the presentation of values in the tables – the decimal numbers should be consistent.

Author Response

Authors thank the comments. Following the suggestions of reviewer 2, English language and length of sentences have been revised and modified as can be seen in the changes form.

Moreover, we are grateful for your editing comments: tables have been checked and presentation of values modified.