Developmental exposure to the brominated flame retardant DE-71 reduces serum thyroid hormones in rats without hypothalamic-pituitary-thyroid axis activation or neurobehavioral changes in offspring

Louise Ramhøj; Terje Svingen; Karen Mandrup; Ulla Hass; Søren Peter Lund; Anne Marie Vinggaard; Karin Sørig Hougaard; Marta Axelstad

doi:10.1371/journal.pone.0271614

Peer Review History

Original SubmissionMarch 9, 2022
1 Jun 2022 Decision Letter - Jamie C. DeWitt, Editor PONE-D-22-07082Developmental exposure to the brominated flame retardant DE-71 reduces serum thyroid hormones in rats without hypothalamic-pituitary-thyroid axis activation or neurobehavioral changes in offspringPLOS ONE Dear Dr. Ramhøj, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jul 16 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Reviewers have made several suggested modifications to the submitted manuscript for clarity and completeness. Please be sure to respond to all reviewer recommendations. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Minor editorial revisions will be needed. 1. Line 116: add St. Louis, MO, USA after Aldrich 2. Line 148: add company name, city, state, country after Tecniplast 3. Line 302: add comma before and after therefore 4. Lines 530-531, 542-543, 557-558, 574-575, 601-602, 615-617, 619-620, 653-654, 682, 686-687: titles should be small capitals 5. Line 535: Vitro should be vitro 6. Line 543 should be fixed appropriately 7. Line 568-569: Journal name should be appropriately abbreviated 8. Line 624: Heal should be Health 9. Line 637: pbfe should be PBDE Reviewer #2: 1. Abstract, overall. The Abstract could be rewritten to use more precise language. As currently written, many parts are very general. For example, lines 38-40 do not indicate if this effect was in dams or offspring. Similarly, lines 40-41 indicate that no effects in brain function were observed by standard behavioral assays, but brain function is challenging to evaluate with just behavioral assays. Therefore, it is recommended that the Abstract be rewritten for precision. 2. Introduction (and in Discussion as well). It is suggested that the authors be more precise in the Introduction with respect to use of the term “marked.” It would improve the Introduction if the authors could include effect sizes/percent differences reported for these changes in levels of T4. By how much DE-71 reduces T4 would help readers understand the biological significance of this reported effect. This is done in line 84 and would be helpful if done throughout the Introduction. 3. Introduction, overall. It is recommended that the authors include a short paragraph in the Introduction about PBDEs in general and about DE-71 specifically. Why should this class of chemicals be studied for its developmental effects? What is its use? What is its environmental fate and transport? How are people exposed? What are human health concerns? This additional context would enhance the Introduction. 4. Methods, general. In the Introduction the authors indicate that one reason for undertaking the studies described in the manuscript was to determine if DE-71 affected the developing TH system similarly to TPO-inhibitors. However, it does not appear as if a TPO-inhibitor positive control was included as an experimental group. It is recommended that the authors provide data from such a group if it exists or include a brief statement of rationale about why such a group is not a necessary part of the experimental design. 5. Methods, line 150. Please indicate at what age were the one male and one female pup/litter weaned for Study 2. 6. Methods, lines 167-169. Please describe blood collections from pups on PD3 and PD8. 7. Methods, histopathology. Please provide more information on histopathological examinations, including whether a veterinary pathologist did the scoring, whether evaluators were blinded to dose groups, etc. 8. Methods, behavioral testing. It would be helpful if authors could include a brief statement of rationale for the behavioral tests chosen for their relevance to assessing impacts of TH on brain development. 9. Results, throughout. It is recommended that the authors provide context for terms such as “reduced,” i.e., by including percent differences or effect sizes. 10. Discussion, Table 1. This will make a nice addition to the literature on developmental neurotoxicity of PBDE. 11. Discussion, overall. The Discussion is very nicely put together. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Taisen Iguchi Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0271614.r001
Revision 1
13 Jun 2022 Author Response Response to Reviewer’s Comments - PONE-D-22-07082 We thank the reviewers for their assessment of our manuscript and their insightful suggestions. We have responded to all comments below and amended the manuscript accordingly. Reviewers' comments: Academic editor: Reviewers have made several suggested modifications to the submitted manuscript for clarity and completeness. Please be sure to respond to all reviewer recommendations. Thank you, we have revised the manuscript and believe that we have addressed all comments as detailed below and in the revised manuscript. Reviewer #1: Minor editorial revisions will be needed. Thank you for pointing out these errors and omissions. They are all now corrected in the revised manuscript. 1. Line 116: add St. Louis, MO, USA after Aldrich 2. Line 148: add company name, city, state, country after Tecniplast 3. Line 302: add comma before and after therefore 4. Lines 530-531, 542-543, 557-558, 574-575, 601-602, 615-617, 619-620, 653-654, 682, 686-687: titles should be small capitals 5. Line 535: Vitro should be vitro 6. Line 543 should be fixed appropriately The citation has been updated and it follows PLOS ONE citation for websites. 7. Line 568-569: Journal name should be appropriately abbreviated 8. Line 624: Heal should be Health Health is abbreviated to Heal in the PLOS ONE citation style. 9. Line 637: pbfe should be PBDE Reviewer #2: 1. Abstract, overall. The Abstract could be rewritten to use more precise language. As currently written, many parts are very general. For example, lines 38-40 do not indicate if this effect was in dams or offspring. Similarly, lines 40-41 indicate that no effects in brain function were observed by standard behavioral assays, but brain function is challenging to evaluate with just behavioral assays. Therefore, it is recommended that the Abstract be rewritten for precision. A1: We have revised the abstract to be more specific including in which animals effects were observed. We have also specified that our study concerns standard behavioral assays and, as the reviewer, we conclude that we lack more sensitive assays: “thus we propose that we lack assays to identify developmental neurotoxicity caused by chemicals disrupting the TH system through various mechanisms”. 2. Introduction (and in Discussion as well). It is suggested that the authors be more precise in the Introduction with respect to use of the term “marked.” It would improve the Introduction if the authors could include effect sizes/percent differences reported for these changes in levels of T4. By how much DE-71 reduces T4 would help readers understand the biological significance of this reported effect. This is done in line 84 and would be helpful if done throughout the Introduction. A2: Thank you for this suggestion, we have specified the language and % reductions in the introduction. 3. Introduction, overall. It is recommended that the authors include a short paragraph in the Introduction about PBDEs in general and about DE-71 specifically. Why should this class of chemicals be studied for its developmental effects? What is its use? What is its environmental fate and transport? How are people exposed? What are human health concerns? This additional context would enhance the Introduction. A3: We have added a paragraph in the introduction on this issue (lines 73-79): “Now banned, PBDEs were for decades used as flame retardants in both industrial and consumer products. Today, human exposure continues due to environmental contamination, persistence and bioaccumulation. Thus, the primary exposure routes are food and house dust [20]. DE-71 is a technical mixture of PBDEs and its congeners are still found in human serum, breast milk and house dust [20,21]. In humans and animals, exposure to PBDEs has been associated with a range of effects including neurotoxicity, disruption of the reproductive and thyroid hormone systems” 4. Methods, general. In the Introduction the authors indicate that one reason for undertaking the studies described in the manuscript was to determine if DE-71 affected the developing TH system similarly to TPO-inhibitors. However, it does not appear as if a TPO-inhibitor positive control was included as an experimental group. It is recommended that the authors provide data from such a group if it exists or include a brief statement of rationale about why such a group is not a necessary part of the experimental design. A4: Thank you, we agree that it was a bit unclear and have added a sentence in the introduction line 105-107 to indicate that TPO-inhibitor induced effects serve as positive control: “The study design and endpoints are aligned with our previous studies of TPO inhibitors propylthiouracil, methimazole and amitrole [6,10] (refer to Table 1 for summary results). Also, the discussion elaborates on this issue. 5. Methods, line 150. Please indicate at what age were the one male and one female pup/litter weaned for Study 2. A5: specified to PD27. 6. Methods, lines 167-169. Please describe blood collections from pups on PD3 and PD8. A6: thank you, lines 174-175 specify that all blood samples are trunk blood. The following description pertains to ages, pooling and sex. We realize that the statement lines 165-166 were unclear and have rewritten the text so that it is now clearly stated that all blood collections are trunk blood. “In both studies all animals, as specified below, were killed by decapitation under CO2/O2 anesthesia and trunk blood collected”. 7. Methods, histopathology. Please provide more information on histopathological examinations, including whether a veterinary pathologist did the scoring, whether evaluators were blinded to dose groups, etc. A7: On lines 215-217 we have added “All histological evaluations were performed by a veterinary pathologist and scoring was done blinded to exposure group.” 8. Methods, behavioral testing. It would be helpful if authors could include a brief statement of rationale for the behavioral tests chosen for their relevance to assessing impacts of TH on brain development. A8: see A4 9. Results, throughout. It is recommended that the authors provide context for terms such as “reduced,” i.e., by including percent differences or effect sizes. A9: Specification of effects sizes have been added to the results on pages 12-13. 10. Discussion, Table 1. This will make a nice addition to the literature on developmental neurotoxicity of PBDE. A10: Thank you for this assessment. 11. Discussion, overall. The Discussion is very nicely put together. A11: We thank the reviewer for the thoughtful comment. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0271614.r002
4 Jul 2022 Decision Letter - Jamie C. DeWitt, Editor Developmental exposure to the brominated flame retardant DE-71 reduces serum thyroid hormones in rats without hypothalamic-pituitary-thyroid axis activation or neurobehavioral changes in offspring PONE-D-22-07082R1 Dear Dr. Ramhøj, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jamie C. DeWitt Academic Editor PLOS ONE Additional Editor Comments (optional): Thank you for thoughtfully responding to reviewer concerns. Reviewers' comments: https://doi.org/10.1371/journal.pone.0271614.r003
Formally Accepted
7 Jul 2022 Acceptance Letter - Jamie C. DeWitt, Editor PONE-D-22-07082R1 Developmental exposure to the brominated flame retardant DE-71 reduces serum thyroid hormones in rats without hypothalamic-pituitary-thyroid axis activation or neurobehavioral changes in offspring Dear Dr. Ramhøj: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Jamie C. DeWitt Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0271614.r004

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