Steroid Hormones Protect against Fluoranthene Ethoxyresorufin-O-Deethylase (EROD) Activity Inhibition
Round 1
Reviewer 1 Report
The following proposal of research work entitled: “Steroid hormones protect fluoranthene induced ethoxyresoru-2 fin-O-deethylase (EROD) activity inhibition in Anguilla anguilla”, addressed the potential protected effect, over phase I biotransformation enzyme (EROD) inhibited activity by a PAH fluoranthene (FL), offered by two steroidal hormones, cortisol and 17β-estradiol. The authors used Anguilla anguilla liver microsomes, which had induced EROD activity due to in vivo exposure to ß-naphthoflavone (BNF). Indeed, the authors found that these hormones were able to recover EROD activity at FL inhibition concentrations and the cortisol was more efficient than 17β-estradiol. Little is known about the direct effect of hormones over the activity of biotransformation enzymes, therefore I see this work as an interest for the scientific community. I only have a few comments that would need addressing, see them below:
The title is somewhat strange, It has opposite words in it: Induced and inhibition, I suggest removing “induced” and it can be something like:
Steroid hormones protect against fluoranthene ethoxyresorufine-O-deethylase (EROD) activity inhibition in Anguilla anguilla
(for example)
Abstract: ok
Introduction
Line 44: “has genotoxic potential the”… ?
M&M: Ok
Results:
Line 162: please avoid starting a sentence with an abbreviation. Please attend to this throughout the text.
Lines 162-164: grammar incorrect
Lines 162-166: repeats the same information. Try to be more optimized.
Line 168: again, you repeat information that is already given in line 165
Lines 171 to 173: despite the increase not being so pronounced, this recovery was also seen for concentration 0.3µM. This should also be pointed out.
Figure 1 - low graphic quality, please improve (make it similar to fig 2)
Line 183: remove “research it” and add “reach its”
Discussion:
Lines 247 to 254: I don’t see why the expression of AhR has to do in this case, with the functional effect of cortisol over EROD activity. Furthermore, this was an in vitro assay of 3 minutes, it is clear that the expression of any gene was not involved here.
Supplementary material:
Table numbers should help to indicate that they belong to this section, for example be Table S1 instead of Table 1.
Table 2 - You can remove “Experimental” here
Comments for author File: 
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Author Response
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Author Response File: 
Author Response.docx
Reviewer 2 Report
Information are lacking about the individuals used for experiment. The statistical analysis is not correctly done.
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Information about individuals used for experiment is lacking, specifically the sex. As steroids effect is central in the work, the sex of individuals is necessary.
Furthermore, The reviewer don't find the information on the number of fish were studied. For each experiment, the origin of liposomes must be linked to a particular fish and the analysis must take into account 1/ the sex of individuals from which the liposomes originate and 2/ the identity of individuals. Then a GLMM model must be used with fixed effect being the experiment and sex and random effect being the identity of individual.
It is not correct to present series of statistical tests which are not independent.
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Author Response
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Author Response File: Author Response.docx
Reviewer 3 Report
General comments:
The study performed by Carla S. S. Ferreira and colleagues is focused on a very interesting topic, aiming to increase the knowledge regarding the effects of steroid hormones on ethoxyresorufin-O-deethylase (EROD) activity inhibited by contaminants. Considering the high value of EROD activity as a biomarker of exposure to organic contaminants like polycyclic aromatic hydrocarbons, the results achieved are very promising. Further studies and tests are certainly required to provide a better understanding on the relation between endocrine regulation, action of pollutants and EROD, as pointed out by the authors of the present article. English use is fluent and easily understandable. Just few minor revisions are needed.
Title:
The title completely reflects the results of performed study.
Keywords:
I suggest not using keywords already present in the title (i.e. “hormones”)
Introduction:
The state of art is well reported and the aims of the study are clearly explained. I suggest consulting also the following reference about the effects of long-term exposure to another class of pollutants with a multibiomarker approach:
- Stara et al. 2021 (https://doi.org/10.1016/j.envpol.2021.117892)
Materials and Methods
This section is clear and well written, showing an appropriate experimental design. It does not require revisions.
Results
In this section the authors support with good references the results achieved. Graphs are clear and well explained. It does not require revisions.
Discussion
The authors well explain the importance of liver EROD activity as a biomarker of exposure to organic contamination in fish, highlighting the lack of knowledge about the potential modulatory effect of steroid hormones such as cortisol and E2 on EROD activity. Just very few revisions are suggested:
Line 207: I suggest “Solea solea” instead of “Solea Solea”
Author Response
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Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
I regret that the authors did not change statistics taking into account individual effet. When I look at the Figure 1, for example for 0.1 FL concentration, the SE is huge in the bar with cortisol. It means that individuals respond differently. It must be taken into account.
Furthermore, it is not a good practice to perform several tests with the same data such as shown in Figure 1 (the different microsomes originate from the same individuals; they are not independent).
I maintain my position: the authors should perform a glmm with FL and presence/absence of cortisol as fixed effect and individual as random effect.
Author Response
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Author Response File: Author Response.docx
