Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The study selected eight human proteins as targets, including hormone receptors (estrogen receptor, androgen receptor, thyroid hormone receptor, and vitamin D receptor), a metabolic enzyme (CYP1A1), a nervous system-related enzyme (acetylcholinesterase), and blood transport proteins (human serum albumin and hemoglobin alpha chain). Although these proteins represent various physiological systems, the manuscript does not provide a systematic rationale for selecting these particular eight proteins. Furthermore, the biological relevance between these targets and diseases associated with aviation pollutant exposure is not sufficiently discussed, making the reasoning appear somewhat abrupt.
2. AutoDock Vina is a semi-empirical method that uses a scoring function, which often favors compounds with strong hydrophobicity and high molecular weight (such as high-ring PAHs). Have you considered using free energy calculations (such as MM/PBSA) to further validate high-affinity complexes?
3. Is it possible to increase the standard deviation and bias by only listing the average binding energy in the table? These pieces of information help to assess the stability and credibility of the docking results.
4. The effects of the combined action of multiple pollutants (dual ligand docking) deserve attention, yet so far only one example involving HSA has been reported. It is recommended to expand this research to other proteins or additional combinations, and to integrate existing studies to analyze synergistic or antagonistic mechanisms.
5. The author mentioned in the conclusion section that it can be used to develop exposure limits or SAF optimization directions. Could a preliminary "pollutant priority list" or "structure-toxicity reference model" also be proposed? This would further enhance the article's guiding significance for environmental regulatory policies

Author Response

Reviewer 1’s Comment

Comment 1

The study selected eight human proteins as targets, including hormone receptors (estrogen receptor, androgen receptor, thyroid hormone receptor, and vitamin D receptor), a metabolic enzyme (CYP1A1), a nervous system-related enzyme (acetylcholinesterase), and blood transport proteins (human serum albumin and hemoglobin alpha chain). Although these proteins represent various physiological systems, the manuscript does not provide a systematic rationale for selecting these particular eight proteins. Furthermore, the biological relevance between these targets and diseases associated with aviation pollutant exposure is not sufficiently discussed, making the reasoning appear somewhat abrupt.

Response 1:

We thank the reviewer for the constructive Comment. We performed a systematic analysis of the list of proteins reported in the literature to interact with or be affected by air pollutants such as VOCs, PAHs, particulate matter and organophosphate esters. We compiled >40 candidates from peer‑reviewed toxicology literature and databases linked to combustion‑derived VOCs, PAHs, or organophosphate esters. We selected a subset of proteins from this list to identify proteins that perform discrete critical cellular and physiological functions, disruption of which is associated with disease states such as cancer, endocrine, cardiopulmonary, neurotoxicity related to air pollution. Based on these criteria, we selected the final set of eight target proteins reported in this study. We have expanded Table 1 to specify health effects associated with changes in the structure or effects of pollutant interactions on the selected proteins. In addition, we have included additional text in the Results (section 3.1) to explain the biological relevance between the selected target proteins and diseases associated with combustion-derived air pollutants.

 

Comment 2

AutoDock Vina is a semi-empirical method that uses a scoring function, which often favors compounds with strong hydrophobicity and high molecular weight (such as high-ring PAHs). Have you considered using free energy calculations (such as MM/PBSA) to further validate high-affinity complexes?

Response 2:

We thank the reviewer for the suggestion. In this manuscript, we have included a table in the Supporting Information (Table S3) compiling the molecular docking scores of pollutant-protein complexes obtained from the scientific literature and compared this with the results from this work. We show that the binding scores obtained in this work are consistent with the scores obtained in previous studies for interactions of human serum albumin with benzene, toluene, p-xylene, acetylcholinesterase complexed with TCP and TPHP. This consistency across independent simulation results strengthens the credibility of our results and suggests that these results represent physiologically plausible interactions.

We agree with the reviewer that free energy calculations such as MM/PBSA offer a more rigorous thermodynamic estimate of the binding and are useful for validating the high affinity complexes. We have already initiated a follow-up research study where we perform molecular dynamics simulations of pollutant-protein complexes to characterize the interactions and free-energy calculations. This work will be complemented by in vitro toxicological assays to experimentally assess the biological relevance of these interactions.

 

Comment 3

Is it possible to increase the standard deviation and bias by only listing the average binding energy in the table? These pieces of information help to assess the stability and credibility of the docking results.

Response 3:

We thank the reviewer for this comment. We present the standard deviations for all protein–ligand complexes based on triplicate docking simulations in Table S2 in the Supporting Information. The average standard deviation across all complexes is less than 0.3 kcal/mol, indicating strong convergence and reproducibility of the docking results for the majority of the systems studied. We also identified a few complexes, primarily those involving CYP1A1 with larger PAHs that exhibited standard deviations exceeding 1 kcal/mol. To address this, we increased the exhaustiveness parameter to 64. This refinement led to improved convergence in several of these cases, reducing the variability in binding energy estimates while some complexes such as dibenzo-ah-anthracene and Fluorene. We intend to conduct a more detailed follow-up study using molecular dynamics simulations and free energy calculations to better characterize these specific complexes. This future work will further clarify the nature and stability of the interactions identified here.

 

Comment 4

The effects of the combined action of multiple pollutants (dual ligand docking) warrant attention; however, so far, only one example involving HSA has been reported. It is recommended to expand this research to other proteins or additional combinations, and to integrate existing studies to analyze synergistic or antagonistic mechanisms.

Response 4:

We thank the reviewer for the suggestion. We have performed further analysis to characterize the effect of the simultaneous binding of multiple pollutants. We assessed the simultaneous binding of VOC-PAH, PAH-OPE (organophosphate ester) and VOC-OPE pollutant category pairs on representative proteins that display high binding affinities to the individual ligands. We used o-xylene (VOC), benzo-a-pyrene (PAH) and tricresyl phosphate (OPE) to represent the three pollutant categories. The target proteins tested comprised of the androgen receptor, human serum albumin and acetylcholinesterase. Our results show the significantly larger binding affinities of pollutant pairs relative to the affinities determined for individual ligand binding, highlighting the potential detrimental effects of these interactions on protein structure and function. These results are presented in Table 4 and the associated text copied below.  Further experimental analysis of the toxicological effects is necessary for regulators and policymakers to make data-driven informed decisions to establish regulations for these pollutant classes.

“Our docking results demonstrate the strong binding characteristics of pollutants commonly released during aviation fuel combustion with proteins involved in critical functions in humans. In the ambient environment, the presence of a mixture of pollutants released in the emissions may influence the binding of more than one ligand to these critical proteins 37. To assess the effect of the simultaneous binding of multiple pollutants to the target proteins, we performed molecular docking simulations of pairs of ligands, representing VOC-PAH, PAH-OPE, and VOC-OPE interactions, with three proteins – androgen receptor, acetylcholinesterase and human serum albumin. The ligands representing the three pollutant categories are p-xylene (VOC), benzo-a-pyrene (PAH) and tricresyl phosphate (OPE). Analysis of the binding revealed stronger binding of the pair of ligands represented by the larger calculated binding affinities for all three pollutant pairs relative to the individual pollutant binding scores (Table 4). The binding affinity of the two-ligand binding shows significantly higher binding affinity (for example, -18 kcal/mol for androgen binding to p-xylene and benzo-a-pyrene) relative to the individual pollutant docking (-6.7 and -13.3 kcal/mol for p-xylene and benzo-a-pyrene, respectively). These observations illustrate the potential health effects of exposure to mixtures of these pollutants. These results further highlight the need to perform toxicological studies to characterize the effect of multi-pollutant interactions. By correlating in-silico binding affinities with in vivo toxicity profiles, regulatory authorities and public health practitioners can better identify which pollutants present the greatest risk and develop targeted mitigation or monitoring strategies to protect airport ground crews and nearby populations.”

 

Comment 5

The author mentioned in the conclusion section that it can be used to develop exposure limits or SAF optimization directions. Could a preliminary "pollutant priority list" or "structure-toxicity reference model" also be proposed? This would further enhance the article's guiding significance for environmental regulatory policies

Response 5:

We thank the reviewer for this suggestion. We have updated the conclusions section to include a list of priority pollutants that may serve as a guide for regulatory authorities to mitigate exposure of airport personnel and nearby communities to these pollutants. The updated text we added to the manuscript is copied below:

‘In summary, the findings from this work suggest that emission standards should be stratified by pollutant class, particularly targeting high-affinity binders such as benzo-a-pyrene and PAHs with a larger number of aromatic rings such as fluoranthene, pyrene, cyclopenta-cd-pyrene, benzo-a-pyrene, benzo-g-chrysene, dibenz-ah-anthracene, dibenzo-ae-pyrene, and benzo-ghi-perylene, and organophosphate esters such as TCP and TPHP. Occupational exposure limits for ground personnel must be tightened using real-time air monitoring of these priority compounds. As the aviation sector pivots towards sustainable aviation fuels (SAFs), our affinity data can provide a strategy for selecting feedstocks and SAF production pathways that minimize aromatic hydrocarbon pollutants. Finally, this work underscores the urgent need for targeted toxicological and epidemiological studies to quantify the real-world risk of exposure to toxic and carcinogenic compounds in aircraft engine exhaust and to develop effective interventions to mitigate the health risks associated with chronic exposure to these hazardous pollutants.’

Reviewer 2 Report

Comments and Suggestions for Authors

The reviewed article is consistent with the journal's profile. It is a very interesting contribution to the field of air transport's impact on the natural environment, and in particular on living organisms, including humans. However, the results and the form of their presentation should be refined before final publication in the journal.
1.    The abstract is more of a description of the work than material that will encourage the reader to read it. It should be supplemented with numerical values and the most important conclusion.
2.    The work uses many abbreviations and symbols; an appropriate list would improve the readability of the study. 
3.    The review section lacks sufficient information about the identified “research gap.” 
4.    The purpose of the research is explained in a rather vague manner.
5.    There is no information on how the model can be validated and implemented in further work.

Author Response

Reviewer 2’s Comment

Comment 1

The reviewed article is consistent with the journal's profile. It is a very interesting contribution to the field of air transport's impact on the natural environment, and in particular on living organisms, including humans. However, the results and the form of their presentation should be refined before final publication in the journal.

 

The abstract is more of a description of the work than material that will encourage the reader to read it. It should be supplemented with numerical values and the most important conclusion.

Response 1:

We thank the reviewer for the suggestion. We have modified the abstract of the manuscript to include specific details and numerical values, highlighting the contributions of this work and clearly stating the importance and application of its results.

 

Comment 2

The work uses many abbreviations and symbols; an appropriate list would improve the readability of the study. 

Response 2:

We have included a list of abbreviations at the start of the manuscript to improve readability. It is copied here for the reviewer’s reference:

Abbreviations: AR Androgen Receptor; COPD Chronic Obstructive Pulmonary Disease; CO Carbon Monoxide; CO₂ Carbon Dioxide; CYP1A1 Cytochrome P450 isoform 1A1; ER Estrogen Receptor alpha; HSA Human Serum Albumin; NOₓ Nitrogen Oxides; PAHs Polycyclic (polyaromatic) Aromatic Hydrocarbons; PLIP Protein-Ligand Interaction Profiler; PM Particulate Matter; RMSD Root-Mean-Square Deviation; SAF Sustainable Aviation Fuel; SOₓ Sulfur Oxides; TCP Tricresyl phosphate; TDCIPP Tris(1,3-dichloro-2-propyl)-phosphate; TPHP Triphenyl Phosphate; VOCs Volatile Organic Compounds

 

Comment 3

The review section lacks sufficient information about the identified “research gap.” 

Response 3:

We have updated the introduction to clearly highlight the research gaps, the rationale for this research, and how it addresses the identified knowledge gaps. We have also updated Table 1 to provide the biological relevance and disease effects associated with the choice of the eight proteins selected for this work.

 

Comment 4

The purpose of the research is explained in a rather vague manner.

Response 4:

We have updated the Introduction to clearly specify the research gaps and the purpose of this research (also specified in response to comment 3).

 

Comment 5

There is no information on how the model can be validated and implemented in further work.

Response 5:

We thank the reviewer for this comment.

In this manuscript, we have included a table in the Supporting Information (Table S3) compiling the molecular docking scores of pollutant-protein complexes obtained from the scientific literature and compared this with the results from this work. We show that the binding scores obtained in this work are consistent with the scores obtained in previous studies for interactions of human serum albumin with benzene, toluene, p-xylene, acetylcholinesterase complexed with TCP and TPHP. This consistency across independent simulation results strengthens the credibility of our results and suggests that these results represent physiologically plausible interactions. We also present the standard deviations for all protein–ligand complexes based on triplicate docking simulations in Table S2 in the Supporting Information. The average standard deviation across all complexes is less than 0.3 kcal/mol, indicating strong convergence and reproducibility of the docking results for the majority of the systems studied. In addition, we have initiated a follow-up research study where we perform molecular dynamics simulations of pollutant-protein complexes to characterize the interactions and free-energy calculations. This work will be complemented by in vitro toxicological assays to experimentally assess the biological relevance of these interactions.

Reviewer 3 Report

Comments and Suggestions for Authors

The in-silico characterization of molecular interactions of aviation-derived pollutants with human proteins was presented in this paper.
The paper presents an in-silico characterization of molecular interactions between eight human proteins, which span a broad range of physiological functions, and over thirty aviation-derived pollutants categorized into three chemical groups: volatile organic compounds, polyaromatic hydrocarbons, and organophosphate esters. The results included in this work indicate that polyaromatic hydrocarbons and organophosphate esters exhibit strong binding affinity, correlating positively with aromatic ring count, with these interactions stabilized by hydrophobic, π-π stacking, and hydrogen bonding interactions.
The work notes that the molecular mechanisms of the interactions of pollutants with proteins can interfere with hormonal signaling, xenobiotic metabolism, oxygen transport, and neurotransmission.

In fact, the combustion of aviation jet fuel emits a complex mixture of pollutants linked to adverse health outcomes among airport personnel and nearby communities. Therefore, it is justified to address this topic. However, it should also be noted that replacing combustion with alternative solutions in the aviation sector is an extremely challenging task.

The methodology employed in this study is appropriate for the research objectives and is described in sufficient detail.
The study itself is well-structured and clearly presented.
Experimental results are communicated in a coherent and accessible manner. Furthermore, the selection of references is relevant and effectively supports the context and findings of the research.
The added value of the work lies in the Supporting Information.

Questions and Suggestions:
- In my opinion, the sentence "Our results indicate the harmful effect of the pollutants released from aviation jet fuel emissions and underscore the need for targeted toxicological studies to quantify the real-world risk of exposure to toxic and carcinogenic compounds in aircraft engine exhaust" from the Abstract should be revised, as it includes general knowledge about the impact of aviation fuel combustion products on health, which is not solely derived from the study itself.
- I believe it would be preferable to use the phrase "This study showed that..." instead of "Our research..." throughout the text.
Were other research methods also considered? If yes or no, it would be worth mentioning this and explaining why. This would add value to the paper.
- Were any difficulties encountered during topic analysis, measurement, or data analysis? If so, they should also be discussed.
- It should also be emphasized that replacing combustion in aviation is an extremely complex challenge. This justifies the relevance of addressing emission issues in this work.
- The Conclusions section should be rewritten. It would be beneficial to highlight the potential implications of the results for future research and the possible benefits arising from the acquired knowledge. Additionally, the study’s contribution to the development of research in this area should be emphasized.
- I suggest that the Conclusions chapter be shortened to include only key findings. The more descriptive content could be moved to the Results and Discussion section. This would help readers extract the most essential information while allowing those interested in a deeper analysis to explore the full content.

Author Response

Reviewer 3’s Comment

Comment 1

The in-silico characterization of molecular interactions of aviation-derived pollutants with human proteins was presented in this paper. The paper presents an in-silico characterization of molecular interactions between eight human proteins, which span a broad range of physiological functions, and over thirty aviation-derived pollutants categorized into three chemical groups: volatile organic compounds, polyaromatic hydrocarbons, and organophosphate esters. The results included in this work indicate that polyaromatic hydrocarbons and organophosphate esters exhibit strong binding affinity, correlating positively with aromatic ring count, with these interactions stabilized by hydrophobic, π-π stacking, and hydrogen bonding interactions. The work notes that the molecular mechanisms of the interactions of pollutants with proteins can interfere with hormonal signaling, xenobiotic metabolism, oxygen transport, and neurotransmission.

In fact, the combustion of aviation jet fuel emits a complex mixture of pollutants linked to adverse health outcomes among airport personnel and nearby communities. Therefore, it is justified to address this topic. However, it should also be noted that replacing combustion with alternative solutions in the aviation sector is an extremely challenging task.

The methodology employed in this study is appropriate for the research objectives and is described in sufficient detail. The study itself is well-structured and clearly presented. Experimental results are communicated in a coherent and accessible manner. Furthermore, the selection of references is relevant and effectively supports the context and findings of the research. The added value of the work lies in Supporting Information.

 

Response 1:

We thank the reviewer for the favorable comments and critical pointers to improve the clarity of the article. We agree with the reviewer that, at present, deploying alternative solutions to replace fossil jet fuel combustion is challenging. The emitted pollutants are relevant to fossil fuel combustion. We would like to highlight that this work characterizes the harmful effects of aviation-derived pollutants on proteins that perform key functions in cells. In addition to providing valuable new information on the interaction of selected aircraft exhaust pollutants and human proteins, this study aims to inform the formulation and selection of sustainable aviation fuels (SAFs), which is already underway and rapidly intensifying in several jurisdictions worldwide. It is expected that SAF use at some airports will be significant in the next few years to a decade or two, impacting workers and local communities in those specific locations. We intend for this work to inform the choice of biomass and selection of sustainable aviation fuel production pathways that will minimize the production of combustion-derived pollutants released from the replacement of conventional jet fuel with SAF that will be significant at a number of airports in certain jurisdictions.

 

Comment 2

Questions and Suggestions:

In my opinion, the sentence "Our results indicate the harmful effect of the pollutants released from aviation jet fuel emissions and underscore the need for targeted toxicological studies to quantify the real-world risk of exposure to toxic and carcinogenic compounds in aircraft engine exhaust" from the Abstract should be revised, as it includes general knowledge about the impact of aviation fuel combustion products on health, which is not solely derived from the study itself.

Response 2:

We have updated the Abstract based on this and other reviewers’ comments. The updated abstract includes specific details and numerical values to highlight the contributions of this work and clearly states the importance and application of the results of this work.

 

Comment 3

I believe it would be preferable to use the phrase "This study showed that..." instead of "Our research..." throughout the text.

Response 3:

We have replaced the text where appropriate with the reviewer’s suggestion.

 

Comment 4

Were other research methods also considered? If yes or no, it would be worth mentioning this and explaining why. This would add value to the paper.

Response 4:

This work focused on molecular docking simulations to characterize the interaction strength between aviation-derived air pollutants and a set of target proteins to identify and screen the most potent pollutants for more in-depth analysis. We already initiated a follow-up to this research to integrate molecular dynamics simulations and in-vitro toxicological studies to gain further insights into the molecular mechanisms of these interactions and their biological relevance to pollution-related health effects. We specify this in the conclusions with emphasis on the need to perform toxicological studies to quantify real-world risk-exposure analyses.

 

Comment 5

Were any difficulties encountered during topic analysis, measurement, or data analysis? If so, they should also be discussed.

Response 5:

One of the difficulties encountered was ensuring that the docking simulations results were converged and the results reproducible. In this study, we performed triplicate molecular docking simulations to quantitate the quality of docking. Docking simulations of larger PAHs showed some differences in the poses for some ligands in the triplicate simulation runs. This required testing and identification of the exhaustiveness parameter that accounted for the large size of these PAHs. For the larger PAHs, increasing the exhaustiveness to 64 provided convergent docking simulation results for the triplicate runs. The average standard deviation across all complexes is less than 0.3 kcal/mol, indicating strong convergence and reproducibility of the docking results for the majority of the systems studied. We also identified a few complexes, primarily those involving CYP1A1 with larger PAHs that exhibited standard deviations exceeding 1 kcal/mol. To address this, we increased the exhaustiveness parameter to 64. This refinement led to improved convergence in several of these cases, reducing the variability in binding energy estimates while some complexes such as dibenzo-ah-anthracene and Fluorene. We attribute the larger values to the exploration of all potential binding sites in the protein. We intend to conduct a more detailed follow-up study using molecular dynamics simulations and free energy calculations to better characterize these specific complexes. This future work will further clarify the nature and stability of the interactions identified here.

 

Comment 6

It should also be emphasized that replacing combustion in aviation is an extremely complex challenge. This justifies the relevance of addressing emission issues in this work.

Response 6:

We thank the reviewer for identifying the relevance of addressing the issues of jet fuel emissions in this work. With regards to the replacement of combustion, we agree that this is indeed very challenging. We clarify the aim and scope of this work as a response to comment 1 above.

 

Comment 7

The Conclusions section should be rewritten. It would be beneficial to highlight the potential implications of the results for future research and the possible benefits arising from the acquired knowledge. Additionally, the study’s contribution to the development of research in this area should be emphasized.

Response 7:

We thank the reviewer for the pointers about modifying the conclusions section. We have updated the Conclusions and present a list of priority pollutants that regulators may use to establish targeted exposure limits through real-time monitoring of these pollutants. We further emphasize the use of this list to identify sustainable aviation fuel production pathways that minimize the production of these combustion products.

 

Comment 8

I suggest that the Conclusions chapter be shortened to include only key findings. The more descriptive content could be moved to the Results and Discussion section. This would help readers extract the most essential information while allowing those interested in a deeper analysis to explore the full content.

Response 8:

We thank the reviewer for the suggestion. We have updated the Conclusions to reduce the text and to emphasize the main results and highlight the application of the knowledge acquired in this work to inform regulators and SAF industry to make informed decisions on the selection of biomass and SAF-production pathways to minimize production of the combustion products identified in this work to have the largest effect on target proteins tested.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Have co factors been considered during the molecular docking of acetylcholinesterase and P450 enzymes? This is the first very important question. Because cofactors play an important role in enzyme catalysis. 

Author Response

Comment 1

Have co factors been considered during the molecular docking of acetylcholinesterase and P450 enzymes? This is the first very important question. Because cofactors play an important role in enzyme catalysis.

Response 1:

We thank the reviewer for specifying the cofactors. In our docking simulations, the heme prosthetic group of CYP1A1 and hemoglobin was retained and treated as part of the receptor. Acetylcholinesterase does not require a non‑protein cofactor. The grid box dimensions for CYP1A1 with prosthetic group were set to 40 Å around the binding site. This information is clarified in the Methods section and is copied below for the reviewer’s reference.

Reviewer 3 Report

Comments and Suggestions for Authors

The paper has been significantly revised. Furthermore, all inquiries and suggestions have been addressed.

Overall, the work has been adequately revised.

However, it would be beneficial to further condense the Conclusions chapter. This approach could facilitate the reader's understanding of the research topic and potentially increase the number of citations. 

Author Response

Comment 1

The paper has been significantly revised. Furthermore, all inquiries and suggestions have been addressed.

Overall, the work has been adequately revised.

However, it would be beneficial to further condense the Conclusions chapter. This approach could facilitate the reader's understanding of the research topic and potentially increase the number of citations.

Response 1:

We thank the reviewer for the suggestion. We have made the conclusion more concise while providing a clear summary of this work, its applications and next steps.