Effects of 9,10-phenanthrenequione on antioxidant indices and metabolite profiles in Takifugu obscurus plasma

Derived from polycyclic aromatic hydrocarbons (PAHs), oxygenated-PAHs (oxy-PAHs) may pose hazards to aquatic organisms, which remain largely unknown. Takifugu obscurus is an important anadromous fish species of high economic and ecological values. In the present study, T. obscurus was acutely exposed to 44.29 µg l−1 9,10-phenanthrenequione (9,10-PQ) for 96 h. Changes of antioxidant indices and metabolite profiles in plasma were compared between 9,10-PQ treatment and the control. The results showed that 9,10-PQ treatment significantly increased malondialdehyde (MDA) content during 6 to 96 h, increased superoxide dismutase (SOD) and catalase (CAT) activities at 6 h, but decreased them at 96 h. These results indicated that 9,10-PQ induced oxidative stress to fish. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis revealed that four metabolic pathways were influenced in response to treatment with 9,10-PQ, including glycerophospholipid metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, purine metabolism and sulfur metabolism. These pathways are associated with antioxidant mechanisms, biosynthesis of neurotransmitters and innate immune functions. Thus, the as-obtained results confirmed that 9,10-PQ induced oxidative stress and raised concerns of neurotoxicity and immunotoxicity to fish. Overall, the present study posed a high environmental risk of oxy-PAHs to aquatic ecosystems.

3. Some data show plateau such as MDA. Then how this response can be correlated with longtern exposure.
4. MDA (I guess lipid peroxidation) is described as an antioxidant (see 3.4 and 4.1). Figures 1,2 and 3 authors write that significant differences (p < .05) between control and 9,10-PQ treatment are marked by asterisks (Student's t-test). However, nowhere I could find asterisks.

In
Decision letter (RSOS-191356.R0) 06-Apr-2020 Dear Dr Jiang, The editors assigned to your paper ("Effects of 9,10-phenanthrenequione on antioxidant indices and metabolite profiles in Takifugu obscurus plasma") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Kind regards, Lianne Parkhouse Editorial Coordinator Royal Society Open Science openscience@royalsociety.org on behalf of Dr Berat Haznedaroglu (Associate Editor) and Kevin Padian (Subject Editor) openscience@royalsociety.org Subject Editor's comments: Thanks for your submission. The reviewers had some useful comments and I hope you will attend to them specifically in your revised manuscript. In addition, the following points were raised: -The justification for exposing the fish species to 50% of the 96 h LC50 is not provided or unclear. The authors should please supply the information.
-The basis for exposing the animal for a period of 96 h though being acute is not clear. Can nontarget organisms to exposed tot his level of oxygenated PAHs? I hope you find these comments helpful, and best wishes for your revisions.

Reviewers' Comments to Author:
Reviewer: 1 Comments to the Author(s) The research with title: Effects of 9,10-phenanthrenequione on antioxidant indices and metabolite profiles in Takifugu obscurus plasma, was revised. The article focuses on studying the effect of 9,10-PQ in T. obscurus, as well as identify the metabolic pathways was influenced in response, allowing important information to be provided to clarify the effects of these pollutants on aquatic organisms. However, some points need to be improved and clarified in order to be able to consider publishing the work.
In the Introduction section: 1. On line 52, the authors mention the interest in assessing the effects of these pollutants on aquatic organisms. However, in reviewing references 7 and 8, it was found that these articles focus primarily on the effects on human populations, so it is advisable to look for other references focused on aquatic organisms. 2. Check line 53 to 55, the information presented does not match the original reference, since in this study they focused on studying A. brevis. In a part of the article they mention that the effects presented by this species is equivalent to the evaluation criteria of bioassays where Rhepoxinius abronius is used.
In the material and methods section: 1. On line 53 of page 2, justify the reason for using 50% of the LC50 of 9,10-PQ. 2. In line 55 to 56 on page 2 the authors mention that during the exhibition, the 75% of the culture media were renewed daily. The stability kinetic of 9,10-PQ was performed previously, what then is the actual concentration to which the organisms were exposed? Clarify this point. 3. Is the term " cultural media" correct? check the grammar 4. It is important that authors review the title of point 3.4 "Determination of antioxidant indices". Whereas MDA content levels are a biomarker of oxidative damage or index of damage. In contrast, enzyme activity (SOD and CAT) refers to enzymatic antioxidant status. Check the review the definition of antioxidant indices.
In the results section: 1. In section 4.1, it is recommended to indicate the percentages of the increase or decrease with respect to the control in the times where a significant difference occurred, this in order to appreciate the magnitude of the change in the biomarkers of oxidative stress.
In the discussion section: 1. In line 50 on page 5, specify that the levels of LPO that increased in P. vivípara exposed to 200 ug/L phenanthrene were in the muscle. 2. Review the citation on line 59, page 5, [46] the paragraph does not correspond to that reference. 3. Review the numbering of the citations from page 6.
In the reference section: 1. Review reference 41, missing names of authors 2. Review the numbering of references, starting with reference 45 Reviewer: 2 Comments to the Author(s) Please include the life stage of the fish species used. This is because a differential response might be observed across male and female or life stage.

Reviewer: 3 Comments to the Author(s)
Manuscript title: Effects of 9,10-phenanthrenequione on antioxidant indices and metabolite profiles in Takifugu obscurus plasma Manuscript ID: RSOS-191356 Takifugu obscurus is an interesting fish species due to its unique place in genomics and also due to its value in culinary industry. Authors have presented a comprehensive data on this species under exposure to 9,10-phenanthrenequione. Manuscript is written in a good manner. However, there are few comments.
1. Selection on one PAH 9,10-phenanthrenequione needs to be justified. Is this the dominant PAH? What about BaP that is benzo-a-pyrene? 2. Are plasma data sufficient? I mean, some vital tissue such as liver which is critical for metabolism should have been taken.
3. Some data show plateau such as MDA. Then how this response can be correlated with longtern exposure.
4. MDA (I guess lipid peroxidation) is described as an antioxidant (see 3.4 and 4.1). Figures 1,2 and 3 authors write that significant differences (p < .05) between control and 9,10-PQ treatment are marked by asterisks (Student's t-test). However, nowhere I could find asterisks.

Author's Response to Decision Letter for (RSOS-191356.R0)
See Appendix A.

Comments to the Author(s)
When performing the second review of the manuscript, the author responded to the requested recommendations, so he considered the manuscript it's eligible for publication.

Comments to the Author(s)
Thank you for providing satisfactory responses to the review comments and updates in relevant sections of the manuscript.

Recommendation? Accept as is
Comments to the Author(s) RSOS-191356.R1 Effects of 9,10-phenanthrenequione on antioxidant indices and metabolite profiles in Takifugu obscurus plasma Authors have appropriately revised the manuscript and have also provided satisfactory responses to queries/comments.

Decision letter (RSOS-191356.R1)
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Dear Dr Jiang,
It is a pleasure to accept your manuscript entitled "Effects of 9,10-phenanthrenequione on antioxidant indices and metabolite profiles in Takifugu obscurus plasma" in its current form for publication in Royal Society Open Science. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter.
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Please see the Royal Society Publishing guidance on how you may share your accepted author manuscript at https://royalsociety.org/journals/ethics-policies/media-embargo/. Comments to the Author(s) When performing the second review of the manuscript, the author responded to the requested recommendations, so he considered the manuscript it's eligible for publication.

Reviewer: 3
Comments to the Author(s) RSOS-191356.R1 Effects of 9,10-phenanthrenequione on antioxidant indices and metabolite profiles in Takifugu obscurus plasma Authors have appropriately revised the manuscript and have also provided satisfactory responses to queries/comments.

Reviewer: 2
Comments to the Author(s) Thank you for providing satisfactory responses to the review comments and updates in relevant sections of the manuscript.

Subject Editor's comments:
Thanks for your submission. The reviewers had some useful comments and I hope you will attend to them specifically in your revised manuscript. In addition, the following points were raised: -The justification for exposing the fish species to 50% of the 96 h LC50 is not provided or unclear. The authors should please supply the information.
Re: The justification for exposing the fish species to 50% of the 96 h LC 50 is provided in manuscript named "Histological, oxidative and immune changes in response to 9,10phenanthrenequione, retene and phenanthrene in Takifugu obscurus liver" which was accepted by journal "Journal of Environmental Science and Health, Part A" but not published, and that was a more basic research. The justification in that manuscript was detailly illustrated with data and figure. The exposure concentration was set at 50% 96 h-LC50 as high exposure concentration to test the acute toxicity of 9,10-PQ, which could maximize the acute toxic effect as well as not cause death. We add this explanation in point 3.3 Exposure and sampling according to this comment.
-The basis for exposing the animal for a period of 96 h though being acute is not clear.
Can non-target organisms to exposed to this level of oxygenated PAHs?
Re: The duration of 96 h is a frequently-used period for acute toxicity test. In the Appendix A manuscript, we mentioned "Following the Gulf War oil spill event on 1991, PAHs could be detected in local seawater even after three years, with the concentration of total PAHs ranging from 21.14 µg/L to 320.5 µg/L [5]" in the introduction part, which meaning the sustainability of PAH compounds. This level of pollution could inevitably destroy the water ecological environment and influence diverse non-target organisms. In contrast, the duration of 96 h was a quite short period which could test the acute toxicity of contamination. Thus, we adopted it in our research.
I hope you find these comments helpful, and best wishes for your revisions.
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The research with title: Effects of 9,10-phenanthrenequione on antioxidant indices and metabolite profiles in Takifugu obscurus plasma, was revised. The article focuses on studying the effect of 9,10-PQ on T. obscurus, as well as identify the metabolic pathways was influenced in response, allowing important information to be provided to clarify the effects of these pollutants on aquatic organisms. However, some points need to be improved and clarified in order to be able to consider publishing the work.
In the Introduction section: 1. On line 52, the authors mention the interest in assessing the effects of these pollutants on aquatic organisms. However, in reviewing references 7 and 8, it was found that these articles focus primarily on the effects on human populations, so it is advisable to look for other references focused on aquatic organisms.
Re: In original manuscript, we considered the toxic effect of PAHs on human. Later we deleted this part for weak correlation, but forgot to delete the references. References 9 (Perspective on Ecotoxicology of PAHs to Fish) detailly discussed the ecotoxicology of PAHs to Fish, which is enough to support the point of the sentence, hence we delete references 7 and 8.
2. Check line 53 to 55, the information presented does not match the original reference, since in this study they focused on studying A. brevis. In a part of the article they mention that the effects presented by this species is equivalent to the evaluation criteria of bioassays where Rhepoxinius abronius is used.
Re: We deleted the sentence from line 53 to 55 "For instance, acute exposure to benzo(a)pyrene, a typical PAH, induced significant mortality and chronic treatments with benzo(a)pyrene significantly inhibited the growth of Rhepoxinius abronius" and also deleted reference.
In the material and methods section: 1. On line 53 of page 2, justify the reason for using 50% of the LC50 of 9,10-PQ.
Re: The justification for exposing the fish species to 50% of the 96 h LC 50 is provided in manuscript named "Histological, oxidative and immune changes in response to 9,10phenanthrenequione, retene and phenanthrene in Takifugu obscurus liver" which was accepted by journal "Journal of Environmental Science and Health, Part A" but not published, and that was a more basic research. The justification in that manuscript was detailly illustrated with data and figure. The exposure concentration was set at 50% 96 h-LC50 as high exposure concentration to test the acute toxicity of 9,10-PQ, which could maximize the acute toxic effect as well as not cause death. We add this explanation in point 3.3 Exposure and sampling according to this comment.
2. In line 55 to 56 on page 2 the authors mention that during the exhibition, the 75% of the culture media were renewed daily. The stability kinetic of 9,10-PQ was performed previously, what then is the actual concentration to which the organisms were exposed? Clarify this point.
Re: In our experiment, we renewed the 75% of the culture media for two purposes. First, we had to keep the experimental water environment clean so as to avoid the interference of water environment pollution and ensure the reliability of experimental results. Besides, we had to avoid any death which may be caused by water pollution since the concentration utilized in the experiment was high. Moreover, the replacement of the culture media was beneficial to maintain the exposure concentration, which could increase the accuracy of experimental results.
3. Is the term " cultural media" correct? check the grammar.
Re: We have replaced "cultural media" by "culture media" according this comment. Besides, we also changed the title of point 4.1 "Changes of antioxidant indices" to "Changes of oxidative stress biomarkers".
In the results section: 1. In section 4.1, it is recommended to indicate the percentages of the increase or decrease with respect to the control in the times where a significant difference occurred, this in order to appreciate the magnitude of the change in the biomarkers of oxidative stress.