Title:Inhibitory effects of astaxanthin on azoxymethane-induced colonic preneoplastic lesions in C57/BL/KsJ-db/db mice

Transcription

1 Author's response to reviews Title:Inhibitory effects of astaxanthin on azoxymethane-induced colonic preneoplastic lesions in C57/BL/KsJ-db/db mice Authors: Takahiro Kochi Masahito Shimizu Takafumi Sumi Masaya Kubota Yohei Shirakami Takuji Tanaka Histaka Moriwaki Version:3Date:14 October 2014 Author's response to reviews: see over

2 October 14, 2014 Professor Andrzej Wincewicz Associate Editor, BMC Gastroenterology Re: Revision of BMC Gastroenterology: MS: Title: Inhibitory effects of astaxanthin on azoxymethane-induced colonic preneoplastic lesions in C57/BL/KsJ-db/db mice Dear Professor Wincewicz, Thank you for your letter dated August 21, 2014, regarding the above manuscript and for the reviewers comments. Enclosed please find a revised manuscript including new data (new Table 3 and Figures 1, 2, and 3), which we believe addresses all of the concerns raised by the three reviewers, and a point-by-point list of our responses to the criticisms. Revised parts are highlighted in red. We thank you and the reviewers for your thoughtful suggestions and insights, which have enriched the manuscript and produced a more balanced and better account of the research. We hope that the revised manuscript is now suitable for publication in your journal BMC Gastroenterology. Respectfully yours, Masahito Shimizu, M.D., Ph.D. Department of Gastroenterology/Internal Medicine Gifu University Graduate School of Medicine

3 1-1 Yanagido, Gifu, Japan Phone: Fax:

4 Responses to Reviewer #1 We are pleased that, in the overall comments, this reviewer feels that our manuscript is well-designed study. We found that the reviewers comments most helpful and have revised the manuscript accordingly. We responded to the concerns raised by Reviewer #1 as follows. 1. Please mention the duration (8-weeks) of astaxanthin intervention in the abstract. Following this suggestion, we added the duration of astaxanthin intervention in the Abstract section (Page 2, line 11). 2. Page 6: What is the composition of the basal diet CRF-1 (the formula of Charles River)? Is this different from the conventional AIN diet formulations? At the least, please provide a reference to the CRF-1 diet. According to this comment, we described the composition of the CRF-1 diet (Page 6, lines 18 to 20). 3. Page 7 (ACF): Please cross-refer the paper by Bird RP for ACF identification, it is okay to use your own reference as a modification. According to this comment, we cited the suggested paper by Bird RP (new Reference #29). 4. Page 7 (ACF-method), Page 10 (ACF-results and Figure 1B = the ACF per unit area (cm2) can be confusing. Please provide the total mean number of ACF/group, mean number of large ACF, and ACF distribution in colon = please see papers from Tanaka T group or Rao CV group. This may give additional information supporting the chemorpeventive properties of Astaxanthin. Please modify results and discussion accordingly.

5 Following this suggestion, we changed the manner of ACF counting and modified the results accordingly (Page 10, lines 15 to 17 and new Table 3). Unfortunately we did not evaluate ACF distribution in the present study. We deeply appreciate your valuable suggestion. 5. The "no change" results of serum glucose, insulin and adiponectin upon astaxanthin exposure was surprising; the supporting statement in the discussion is weak (Page 16, 2nd para). Please include a couple of references with stronger statement to support this "no change". According to this comment, we revised the Discussion section by citing new reference (Page 16, line 20 to Page 17, line 6 and new Reference #54). important comment that gave us a better perspective of our discussion. We thank your 6. Figures (Legends) and Tables: Please change the headings from Group 1, Group 2, Group 3 and Group 4 to None, Astaxanthin, AOM, AOM+Astaxanthin, respectively. And in results and discussion, please use the same. The use of Group 1, 2... is very confusing to the reader. According to this suggestion, we revised the text and the headings of Figures and Tables. Responses to Reviewer #2 We are very pleased to learn the favorable comments of this reviewer. Responses to Reviewer #3 We appreciate your favorable evaluation of this manuscript. The concerns raised by this reviewer have become great help for us to improve the manuscript. We responded to the concerns raised by Reviewer #3 as follows.

6 Materials and methods a) Page no 6 line no 17: treatment with astaxanthin is mentioned as 8 weeks but in abstract it is mentioned as 12 weeks. Please clarify. Treatment period with astaxanthin in abstract was incorrect, so we have corrected the text (Page 2, line 11). Thank you very much for letting me know. b) PCR conditions for real time RT-PCR should be mentioned 8, lines 11 to 12). According to this suggestion, we mentioned the PCR cycling conditions (Page c) Page no 8 line no 22: replace with 8-hydroxy-2-deoxyguanosine We are afraid that there is character corruption in the text, so we could not found real intention of reviewer. As suggested by this reviewer, we believe 8-hydroxy-2 -deoxyguanosine is standard nomenclature (Page 9, line 5). Results Western blotting for SOD, GPx, IL-1b, IL-6, and TNF-a should be included. Based on this comment, we tried to perform Western blotting; however, specimen of colonic mucosa was very limited and there were not sufficient samples for this experiment. So, we performed additional real time RT-PCR to reinforce our result. Inflammatory mediators, including F4/80 and chemokine (C-X-C motif) ligand (CXCL)2, and catalase, an antioxidant enzyme, were assessed and text was modified accordingly (Page 11, lines 7 to 11, Page 11, line21 to Page 12, line 3, new Figure 2C, new Figure 3B, and new Reference #34). suggestion. We deeply appreciate your valuable Discussion Page no 14 line no 6: replace with TNF-a

7 We are afraid that there is character corruption in the text, so we could not found real intention of reviewer. We consider that suggested word is TNF-α and this is standard nomenclature. List of abbreviations Line no 1: replace with 8-hydroxy-2-deoxyguanosine We are afraid that there is character corruption in the text, so we could not found real intention of reviewer. As suggested by this reviewer, we believe 8-hydroxy-2 -deoxyguanosine is standard nomenclature. References The reference list has to be updated. There are several papers on the subject published in 2013 and Of relevance to this manuscript are recent papers by Hong et al (2013), Tuominen et al (2013), Khan et al (2013), Kavitha et al (2013) and Kochi et al (2014). The authors should discuss their data in the light of these findings. Following this suggestion, we revised the text by citing some of suggested papers published in 2013 and 2014 (Page 4, line 15, Page 4, line19 to 21, Page 15, lines 4 to 5 and new Refs #10, #13, and #41). Figures Figure 1: representative photomicrographs for ACF and BCAC for all groups have to be included. According to this comment, we revised the figure (new Figure 1). We showed representative photomicrographs of ACF and BCAC in AOM-treated groups because these lesions developed only in mice treated with this carcinogen Format Tables have to be presented after figures as per the journal format

8 Based on this comment, we have rearranged the Tables and Figures.