Dear Dr. Merrick,

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.

In the submitted manuscript, authors have aimed to evaluate the translocation of Benzo(a)pyrene (BaP) reactive metabolites across human mammary epithelial cell membranes and the findings of this manuscript suggest that BaP reactive metabolites like BPDE (BaP-diol-epoxide) can easily translocate across cell membranes despite robust conjugation systems and ready supplies of essential co-substrates for sulfate or GSH conjugations. However, the submitted manuscript needs major revision and cannot be recommended in its current form for publication in esteemed journal Plos One. It is requested that authors must revise the manuscript according to learned reviewers’ suggestions. The corrections made in the manuscript should be highlighted. So, it would be easier to identify the modified content from the original submitted manuscript. 

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: N/A

Reviewer #2: I Don't Know

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Breast cancer is the most common type of cancer in women, and the global incidence of this disease continues to rise.

Despite advancements in early detection and therapeutic strategies, breast cancer remains a remarkable health challenge because of its complex pathogenesis and diverse clinical manifestations.

Like all kinds of cancer, the etiology of breast cancer involves a variety of factors, including genetics, diet, lifestyle, and environmental factors. Nonetheless, germline mutations predisposing to breast cancer account for 10% of the cases, and the mutated genes encode for proteins involved in the DNA-damage repair, cell-cycle control, and chromatin remodeling processes. On the other hand, somatic mutations are the most frequent. A significant contribution to somatic mutations is due to environmental exposure. Within this framework, a considerable contribution to the mutation burden is due to polycyclic aromatic hydrocarbons (PAHs), of which benzo (a) pyrene (BaP) is a representative. PHAs' exposure occurs through multiple sources, including natural (e.g., volcanic eruption), industrial (e.g., air pollution due to industrial emissions and vehicle exhausts), and lifestyle choices (e.g., tobacco smoke). BaP is a potent DNA mutagenic molecule, thus tightly associated with carcinogenesis. However, cells counteract such "xenobiotic" stress caused by BaP exposure by creating less toxic BaP adducts, also thanks to the intervention of glutathione (GSH). Based on their previous inquiries in the manuscript titled "Translocation of Benzo(a)pyrene Reactive Metabolites Across Human Mammary Epithelial Cell Membranes", Merrick A.B. and colleagues tested the hypothesis of a potential inverse distribution of cellular DNA/protein adducts and extracellular protein BaP upon GSH depletion or augmentation in T47D cells. Overall, the manuscript presents some interesting hints; nonetheless, before publication amendments are required.

* Given that there is no mention of any form of a specific enzyme kinetic rate in the entire article, I would exercise caution when using the phrase "…specific activity…" as it could be misunderstood or cause confusion for the readers. The rate at which a substrate is converted into a particular product by unit time is sometimes referred to as specific activity.

* More solid evidence regarding the greater quantity of extracellular protein adducts (such as BSA and alpha 1 anti-trypsin) in comparison to intracellular protein is requested from the authors. Since BSA is the main protein component and a primary part of FBS, I must admit that I am not startled if we consider that cells are cultivated in the presence of FBS. What happens if cells are starved? Or reducing the amount of serum? Does a dosage dependency exist? BSA depleted FBS?

* Robust mechanistic data supporting the translocation of BaP reactive metabolites across a phospholipid bilayer are missing. I mean, while BaP entering cells is easy due to the very hydrophobic features of the molecules, once the molecule has been hydroxylated, its features change. Indeed, hydroxylated forms are more polar, thus cannot freely diffuse through the plasma membrane as effectively as the parent compound. A possibility is that both adducts and BaP-free hydroxylated forms are secreted via the Endoplasmic Reticulum (ER). Consistently, CYP proteins, which are mostly localized within the ER, and GSH coordinate detoxification through sequential action where first CYP activate, or functionalize the BaP, making it more water soluble, and then GSH can conjugate with these modified substrates, rendering them less toxic and facilitating their secretion. The authors should clarify this issue. Did they ever check ER? To gain insight into the issue, this route should be considered.

* Ultimately, the CYP issue needs to be strengthened. The authors refer only to the transcript amount, and even from cells cultured in different conditions. As such, this part is a little bit too speculative. I would advise either strengthening it by providing protein data (e.g., Western Blot) or omitting this last part.

Minor concerns

* Acronyms and abbreviations when cited for the first time should be spelled out (e.g., line 126: CNDB; line 547: FCS; etc.)

* A few typos seem scattered through the main text (e.g., line 111, etc.).

Reviewer #2: This is a thoughtful, well designed study on an important biological question. The manuscript is well written and easy to understand even for people outside the field. The basic scientific question asked is simple, which I consider a plus. The work does not provide answers to how these adducts affect human health, but the information provided here will be important for future studies.

Reviewer #3: The authors investigated the dynamics of BaP adduct formation and distribution in T47D mammary cells in connection with the role of BaP and PAH in tumor formation and involvement in cancer malignancies. The authors showed the kinetics of the distribution of BaP adducts by using BSO and Bap treatments/pre-treatments to alter GSH levels in the cells. The authors also mined the available datasets to demonstrate the expression levels of the enzymes and transcription factors, relevant for Bap conjugation and bioactivation.

The data is sound, generally well interpreted. Methods are presented with appropriate references where applicable. Legends contain necessary details.

Figure 1 and 2 can be merged into 2 panel figure. Same for Figure 3 and 4 – 4 panel single figure.

Lines 332 -334 “…….but only decreased nuclear protein adducts by 11% from control”. Table 1 however indicates an increase in nuclear protein adducts by 11%. The authors should correct and elaborate.

While the authors provide plausible explanation for the observed kinetics differences in Figure 3, how would the authors explain the difference in BSO curve relative to control in cellular vs extracellular adduct formation? Is it expected that GSH activity induced by BSO treatment would lead to lower escape to the media? The trend in total BaP adduct in Fig 4 goes more in line with the cellular BaP adduct kinetics of Fig 3. Please, include a sentence either in results or discussion.

For RNA-seq analysis the authors should cite packages used for processing appropriately and move the dataset accession numbers from Methods to Data Availability section.

The authors should give the article additional round of proofreading and address these minor corrections.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Translocation of benzo(a)pyrene reactive metabolites across human mammary epithelial cell membranes

PONE-D-25-43097R1

Dear Dr. Merrick,

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.

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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,

Pankaj Singh, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: I Don't Know

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: The authors have directly addressed my concern that while their work is important, well written and technically sound, it does not answer the question of how these adducts affect carcinogenesis and human health; This concern has been adequately addressed by the authors in the Conclusion. I want to reiterate, however, that I have no doubt that BaP adducts cause cancer, but the cell and molecular mechanisms for this carcinogenesis remains unknown. I hope the authors continue this important work.

Reviewer #3: Line 377 the word "reactive" is double typed, please check. The rest of the concerns are corrected and addressed.

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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 #2: No

Reviewer #3: No

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