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  • Carlos Fernandes 1,*,†,
  • Afonso J. C. Videira 1,† and
  • Filomena S. G. Silva 1,*
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous

Round 1

Reviewer 1 Report

The article describes the cytotoxicity properties of new mitochondriotropic antioxidants. They are compared with those described earlier. Based on the results obtained, two compounds were selected for further, possibly preclinical studies. It is proposed to use the compounds for the development of therapy for neurodegenerative diseases and liver diseases. The material is presented in great detail, perhaps even unnecessarily.  

Comments on the text of the manuscript:

  1. Experiments were carried out on cell cultures. However, the text does not explain in detail why these particular cell cultures were chosen. It is necessary to supplement the justification and description of the used cell models. This is important because Hep G2 is not only a hepatic, but a cancer cell line.
  2. The inscriptions and numbers in the figures are small and difficult to read. This is especially true for Figures 5, 6 and 7.
  3. Figures 6 and 7, S5 and S6 should be turned over and the panels on them should be enlarged. It is difficult to read the text upside down.

Author Response

October 19th, 2021

Re: Cytotoxicity and mitochondrial effects of phenolic and quinone-based mitochondria-targeted and untargeted antioxidants on human neuronal and hepatic cell lines: a comparative analysis by Fernandes et al.

 

Dear Reviewer,

Please find enclosed the authors' answers on the above-referenced manuscript. The authors are grateful for the comments suggested by the reviewer and agree that these led to a considerable improvement in the quality of the manuscript, enhancing the visibility of this scientific work. Taking this opportunity, we also performed another revision of the English language in the manuscript. Alterations in the manuscript in response to the reviewer are included in blue.

The authors' response (in italic format) follows the reviewers' respective comments (in bold format).

  1. Experiments were carried out on cell cultures. However, the text does not explain in detail why these particular cell cultures were chosen. It is necessary to supplement the justification and description of the used cell models. This is important because Hep G2 is not only a hepatic, but a cancer cell line.

The authors insert the followed paragraph:

"Pag 4, line 109: Although HepG2 is a human hepatoma cell line, it is one of the most used cell models in cytotoxicity screening of different compounds due to having a stable phenotype, high availability, easy handling and phase I and phase II metabolizing activities (PMID: 26272135)."

  1. The inscriptions and numbers in the figures are small and difficult to read. This is especially true for Figures 5, 6 and 7.

The size of the inscriptions and numbers inside the figures were increased for Figures 2-7 as suggested by the reviewer. We acknowledge that they were too small and also increased them for Figure S1-4. The authors also corrected the number of independent experiments in the legends because in the assays in figures 4, S3 and S8, only 3 independent experiments were performed

  1. Figures 6 and 7, S5 and S6 should be turned over and the panels on them should be enlarged. It is difficult to read the text upside down.

The authors performed the modifications on Figures 6, 7, S5, and S6, as suggested by the reviewer.

  1. Please revise your reference formatting according to MDPI's layout guide

The authors performed the revision of the manuscript to attempt the MDPI's layout guide

 

Reviewer 2 Report

The manuscript by Fernandes et al., reported the effects of phenolic- and quinone-based mitochondria targeted antioxidants including synthesized mitoCIN library compounds as well as MitoQ/SkQ1 on cytotoxicity and mitochondria, and compared these effects with those of mitochondria non-targeting antioxidants like resveratrol/CoQ10. This is an interesting comparison of novel mitochondria targeting compounds with those of existing mitochondria targeting and non-targeting antioxidants, which could have potential therapeutic effects in several disorders related to mitochondrial dysfunction. Here, the authors used several cell-based in vitro assays to measure cytotoxicity, metabolic and functional effects on mitochondria using two different cellular models (HepG2 and SH-SY5Y cell lines), and demonstrated that mitoCINs MC4 and MC7.2 with shorter alkyl chain length and apyrogallol group have significantly lower (100-1000 fold) cytotoxicity compared to MitoQ/SkQ1 with optimal mitochondrial functional effects, suggesting a potential therapeutic application of these compounds in disorders related to mitochondrial dysfunction. However, there are few minor concerns that need to be addressed.

Results: Why do the authors chose to present the figures as two different comparisons: one with mitoCINs and the other with MitoQ/SkQ1/resveratrol/CoQ10? Does this mean both the mitoCINs and MitoQ/SkQ1/resveratrol/CoQ10 experiments were performed separately? Please provide clarity on this and include it in the methods/results section.

In some places, figures were not referred in the description of results. Please include referred figures in the description of results (in pages 11, 12 and 18).

Given that there are several comparisons in this study, it would be easier for readers if the authors can summarize the results of different assays and the key findings in these assays in a tabular format (Columns: Assay, key finding, Potency/Effect size - like MC4<MC7.2<...).

Author Response

October 19th, 2021

Re: Cytotoxicity and mitochondrial effects of phenolic and quinone-based mitochondria-targeted and untargeted antioxidants on human neuronal and hepatic cell lines: a comparative analysis by Fernandes et al.

 

Dear Reviewer,

Please find enclosed the authors' answers on the above-referenced manuscript. The authors are grateful for the comments suggested by the reviewer and agree that these led to a considerable improvement in the quality of the manuscript, enhancing the visibility of this scientific work. Taking this opportunity, we also performed another revision of the English language in the manuscript. Alterations in the manuscript in response to the reviewer are included in blue.

 

The authors' response (in italic format) follows the reviewers' respective comments (in bold format).

 

  1. Results: Why do the authors chose to present the figures as two different comparisons: one with mitoCINs and the other with MitoQ/SkQ1/resveratrol/CoQ10? Does this mean both the mitoCINs and MitoQ/SkQ1/resveratrol/CoQ10 experiments were performed separately? Please provide clarity on this and include it in the methods/results section.

In order to clarify this question made by the reviewer, the authors added the following sentence to the manuscript:

"Pag 5, line 188.189: "The experiments with MitoCIN compounds, mitochondrial-targeted antioxidants, non-targeted antioxidants, MitoCIN parentals and alkyl-TPP chains were performed in parallel under the same experimental conditions. We selected to initially present the figures with two different comparisons only for the sake of clarity. We have now incorporated a new set of supplementary figures which remove this separation and describe relevant comparisons that were made in the text (see below)"

  1. In some places, figures were not referred in the description of results. Please include referred figures in the description of results (in pages 11, 12 and 18).

We acknowledge the reviewer for this comment. The authors added several figures (Figures S4, S5, S6, S7, S9, S10, S13, S14, S15 and S16) related to the comparisons referred by the reviewer. A careful revision related to the numbering of the figures was performed

  1. Given that there are several comparisons in this study, it would be easier for readers if the authors can summarize the results of different assays and the key findings in these assays in a tabular format (Columns: Assay, key finding, Potency/Effect size - like MC4<MC7.2<...).

We acknowledge the reviewer for this valuable suggestion. The authors incorporated this comparison in a tabular format (Table I,page 33).

Pag 32, "as observed individually for each assay in Table 1"

Table 1 Comparison of cytotoxicity and mitochondrial effects of MitoCIN compounds in HepG2 and differentiated SH-SY5Y cells

Assay

Key finding

Potency/Effect size

Sulforhodamine B

Cell mass/ Cytotoxicity

HepG2: MC4<MC7.2<MC3<MC6.2

SH-SY5Y: MC4≈MC7.2<MC3<MC6.2

Resazurin

Cell metabolic activity/ Cytotoxicity

HepG2: MC4<MC7.2<MC3<MC6.2

SH-SY5Y: MC4≈MC7.2<MC3<MC6.2

Intracellular ATP levels

Cytotoxicity

HepG2: MC4=MC7.2<MC3≈MC6.2

SH-SY5Y: MC4<MC7.2≈MC3<MC6.2

TMRM

 

Mitochondrial polarization

HepG2: MC4<MC6.2≈MC3<MC7.2

SH-SY5Y: MC4≈MC7.2<MC3<MC6.2

Cellular oxygen consumption and extracellular acidification

Mitochondrial respiration and indirect measurement of cellular glycolytic activity

HepG2: MC4≈MC7.2≈MC3≈MC6.2

SH-SY5Y: MC4<MC7.2<MC3<MC6.2

 

  1. Please revise your reference formatting according to MDPI's layout guide

The authors performed the revision of the manuscript to attempt the MDPI's layout guide