Melatonin and DNA Integrity: The Impact of Exogenous Administration in Exercise-Induced Oxidative Stress—A Systematic Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The systematic review titled, “Melatonin and DNA Repair: The Impact of Exogenous Administration in Exercise-Induced Oxidative Stress” submitted by Vanessa Bertolucci et al., focusing on the role of exogenous melatonin supplementation in mitigating oxidative stress and supporting DNA repair in healthy physically active individuals undergoing intense exercise. The review is well-structured, methodologically sound, the synthesis of existing clinical trials is clear, and the discussion offers mechanistic insights that add value to the interpretation of findings. This manuscript makes a valuable contribution by synthesizing clinical evidence on melatonin’s role in reducing exercise-induced oxidative stress and potential DNA protection. Mainly emphasizing the limitations regarding DNA repair data and improving methodological appraisal, it will offer a strong, impactful resource for researchers and clinicians in exercise physiology and sports medicine. I accept this review subject to the following improvements.

1. The Introduction section could briefly mention potential confounding factors such as diet or sleep quality, which may influence oxidative stress and melatonin’s efficacy.
2. Figure 1 and 2 are not cited in the text. Kindly cite these figures.
3. Only one included study directly measured DNA damage via comet assay, with others relying mainly on oxidative stress markers. This limits strong conclusions regarding melatonin’s impact on DNA repair mechanisms. It is suggested that emphasize this limitation more explicitly and recommend that future clinical studies incorporate direct DNA damage/repair biomarkers, including enzyme activity assays such as OGG1, APE1 and advanced genomic integrity tests.
4. It was noticed that all reported studies involved exclusively male participants, mainly adolescents and young adults. This limits generalizability across genders and ages. I would suggest to highlight this demographic limitation and suggest expanding future research to include female and older populations to assess potential sex-specific and age-related effects.
5. Considerable variability exists in dosing (5-100 mg), timing (pre-vs-post-exercise), and supplementation duration (single vs multi-day). This heterogeneity complicates direct comparisons and dose-response interpretations. Recommend more standardized dosing protocols in future trials, possibly stratifying by exercise type and intensity.
6. Some figures (e.g., Figure 3 circle plot) are informative but could be better described in the main text to improve accessibility for readers less familiar with biomarker overlap visualizations.
7. The manuscript summaries the absence of formal Cochrane RoB 2 or CONSORT-based risk of bias assessment for discussed RCTs. Consider adding at least a qualitative or semi-quantitative risk of bias evaluation to strengthen the critical appraisal and contextualize findings.
8. It is suggested to expand the discussion on melatonin’s pharmacokinetics, especially regarding immediate vs extended-release formulations and their practical relevance for athletes.
9. The manuscript is generally well-written, but minor typographical errors, inconsistent abbreviation use, and occasional sentence complexity may reduce readability. It is suggested to conduct a thorough editorial proofreading to improve clarity, ensure consistent use of abbreviations, and simplify complex sentences where possible.

Author Response

Reviewer 1:
Comments and Suggestions for Authors
The systematic review titled, “Melatonin and DNA Repair: The Impact of Exogenous Administration in Exercise-Induced Oxidative Stress” submitted by Vanessa Bertolucci et al., focusing on the role of exogenous melatonin supplementation in mitigating oxidative stress and supporting DNA repair in healthy physically active individuals undergoing intense exercise. The review is well-structured, methodologically sound, the synthesis of existing clinical trials is clear, and the discussion offers mechanistic insights that add value to the interpretation of findings. This manuscript makes a valuable contribution by synthesizing clinical evidence on melatonin’s role in reducing exercise-induced oxidative stress and potential DNA protection. Mainly emphasizing the limitations regarding DNA repair data and improving methodological appraisal, it will offer a strong, impactful resource for researchers and clinicians in exercise physiology and sports medicine. I accept this review subject to the following improvements.

1.    The Introduction section could briefly mention potential confounding factors such as diet or sleep quality, which may influence oxidative stress and melatonin’s efficacy.

Response: We thank the reviewer for this insightful comment. We appreciate the recognition of the review’s structure, methodological approach, and relevance to the field. 
In response, we have revised the Introduction to briefly address potential confounding factors such as dietary antioxidant intake and sleep quality, both of which may modulate oxidative stress responses and influence the physiological and pharmacological actions of melatonin:
line 409-4013: “Emerging evidence suggests that dietary antioxidants (e.g., polyphenols, vitamins C and E) can independently mitigate exercise-induced oxidative stress and may interact synergistically or antagonistically with melatonin supplementation (Beck et al., 2021). Likewise, sleep duration and quality are closely linked to endogenous melatonin secretion and circadian regulation, thereby influencing baseline antioxidant capacity and responsiveness to exogenous melatonin (Reiter et al., 2007)”

 

2.    Figure 1 and 2 are not cited in the text. Kindly cite these figures.

Response: We thank the reviewer for noticing this. We have checked and the figures are cited in the text as follows: figure1, line: 140  and figure 2, line:632.

 

3.    Only one included study directly measured DNA damage via comet assay, with others relying mainly on oxidative stress markers. This limits strong conclusions regarding melatonin’s impact on DNA repair mechanisms. It is suggested that emphasize this limitation more explicitly and recommend that future clinical studies incorporate direct DNA damage/repair biomarkers, including enzyme activity assays such as OGG1, APE1 and advanced genomic integrity tests.

Response: We appreciate the reviewer's comment and welcome the opportunity to adjust this point. We have already acknowledged this limitation in our original discussion section. However, in response to the reviewer's suggestion, we have now strengthened and expanded the Conclusions of this item to emphasize the gap in the current evidence base. The section: "Current evidence suggests exogenous MLT may protect against exercise-induced oxidative stress and potentially attenuate DNA damage. However, further research directly assessing DNA damage and repair biomarkers is essential to confirm these effects and elucidate the underlying mechanisms in physically active individuals and athletes.”; has been amended to:
Line: 1144 “Current evidence suggests that exogenous melatonin may protect against exercise-induced oxidative stress and potentially attenuate DNA damage. However, most of the included studies assessed only indirect oxidative stress markers, with limited direct assessment of DNA damage or repair. To confirm these effects and better understand the underlying mechanisms, future clinical trials should incorporate validated biomarkers of DNA integrity and repair (e.g., comet assay, enzyme activity assays such as OGG1, APE1), as well as advanced genomic stability tests.”

 

4.    It was noticed that all reported studies involved exclusively male participants, mainly adolescents and young adults. This limits generalizability across genders and ages. I would suggest to highlight this demographic limitation and suggest expanding future research to include female and older populations to assess potential sex-specific and age-related effects.

Response: We thank the reviewer for this important observation. We agree that the demographic homogeneity across the included studies -predominantly involving young male participants- represents a key limitation for the generalizability of findings. As noted, this issue was already addressed in the Discussion section of our manuscript, where we emphasized the predominance of male cohorts and the underlying factors contributing to the underrepresentation of females, such as hormonal variability and challenges with experimental standardization. In response to the reviewer’s valuable suggestion, we have now reinforced the wording of this section to explicitly highlight the implications for both sex- and age-related generalizability, and to more clearly recommend that future research efforts include female and older populations to explore potential sex-specific and age-dependent responses to exercise-induced oxidative stress and melatonin supplementation.

We believe these additions improve the clarity and translational relevance of the discussion. The section: “Current evidence in physical activity research is predominantly derived from studies involving exclusively male cohorts, underscoring the necessity to expand investigations into female populations. This disparity may stem from multifactorial challenges, including the hormonal variability inherent to female physiology and the potential confounding effects of oral contraceptive use, which complicate experimental standardization [128]. Additionally, enrollment biases persist, as males exhibit higher voluntary participation rates in exercise-related trials [124]. Such limitations emphasize critical knowledge gaps regarding sex-specific physiological responses to physical exertion. Future studies must prioritize elucidating the impact of biological dimorphism (e.g., hormonal fluctuations, metabolic differences) on exercise outcomes to ensure translational relevance across diverse populations. Addressing these methodological shortcomings will enhance the generalizability of findings and inform tailored interventions in public health and clinical practice.”; has been amended to:

Line:1122 “Current evidence in physical activity research is predominantly derived from studies involving exclusively male cohorts, primarily adolescents and young adults. This demographic homogeneity limits the generalizability of findings across sexes and age groups. The underrepresentation of female participants may stem from multifactorial challenges, including the hormonal variability inherent to female physiology and the potential confounding effects of oral contraceptive use, which complicate experimental standardization [128]. Moreover, males tend to exhibit higher voluntary participation rates in exercise-related trials, contributing to enrollment bias [124]. Similarly, older adults are rarely included in such studies, despite age-related differences in oxidative stress responses, hormonal regulation, and melatonin metabolism. These limitations underscore critical knowledge gaps regarding sex- and age-specific physiological responses to physical exertion and melatonin supplementation. Future studies must prioritize the inclusion of female and older populations to elucidate the impact of biological dimorphism and age-associated changes on exercise-induced oxidative stress and DNA repair mechanisms. Addressing these methodological shortcomings will enhance the translational applicability of findings and support the development of tailored strategies in sports medicine and public health.”

 

5.    Considerable variability exists in dosing (5-100 mg), timing (pre-vs-post-exercise), and supplementation duration (single vs multi-day). This heterogeneity complicates direct comparisons and dose-response interpretations. Recommend more standardized dosing protocols in future trials, possibly stratifying by exercise type and intensity.

Response: We thank the reviewer for this important observation. In fact, we had already acknowledged this point in the Discussion. However, in response to the reviewer’s suggestion, we have reinforced and clarified this aspect to more explicitly emphasize the need for standardization in future studies and the potential benefit of stratifying protocols by exercise modality and intensity.

Revised paragraph in the Discussion (modified for clarity and alignment with the reviewer’s comment):

Line 994: “The variability in supplementation protocols—particularly regarding dosage (ranging from 5 to 100 mg), timing of administration (pre- vs. post-exercise), and duration (single vs. multi-day)—significantly limits direct comparisons and complicates dose-response interpretations across studies.”

 

6.    Some figures (e.g., Figure 3 circle plot) are informative but could be better described in the main text to improve accessibility for readers less familiar with biomarker overlap visualizations.

Response: We appreciate the reviewer’s thoughtful comment. We would like to clarify that Figure 3 is not a biomarker-specific plot, but rather a visual representation of the overlap and diversity in biomarker selection across the included studies. Its purpose is to highlight the heterogeneity in oxidative stress biomarkers assessed rather than to present detailed quantitative data. For readers interested in comprehensive and study-specific information on biomarkers, methodologies, and outcomes, this information is systematically detailed in Table 2. However, figure 2 was enhanced and incorporated in a new format.

7.    The manuscript summaries the absence of formal Cochrane RoB 2 or CONSORT-based risk of bias assessment for discussed RCTs. Consider adding at least a qualitative or semi-quantitative risk of bias evaluation to strengthen the critical appraisal and contextualize findings.

We thank the reviewer for highlighting the importance of a formal risk of bias assessment. Although the original studies did not report standardized quality evaluations, we addressed this by conducting a conceptual appraisal based on the Cochrane RoB 2 tool, as described in Section 3.4. We have now clarified this approach in the Methods section 2.7.

Line :593 “2.7. Risk of Bias
Since none of the included studies reported a self-assessment of risk of bias, we conducted an independent evaluation using the Risk of Bias 2 (RoB 2) tool developed by the Cochrane Collaboration, which is widely recognized as the methodological gold standard for assessing risk of bias in randomized controlled trials (RCTs). The RoB 2 tool evaluates five distinct domains of potential bias: the randomization process, deviations from intended interventions, missing outcome data, measurement of the outcome, and selection of the reported result. For each included study, two reviewers independently applied the structured signaling questions provided by RoB 2 to each relevant domain, ensuring a comprehensive and systematic assessment. Any discrepancies between reviewers were resolved through discussion, and when consensus could not be reached, a third reviewer was consulted. The risk of bias was categorized as low, some concerns, or high for each outcome, in accordance with the RoB 2 guidance. This rigorous approach, including outcome-specific assessments and transparent documentation of judgments, enhances the reliability and reproducibility of our findings and aligns with current best practices as outlined in the Cochrane Handbook for Systematic Reviews of Interventions and the PRISMA 2020 guidelines. Details in Supplementary Table S6. Risk of Bias Assessment.”

8.    It is suggested to expand the discussion on melatonin’s pharmacokinetics, especially regarding immediate vs extended-release formulations and their practical relevance for athletes.

We appreciate the reviewer’s suggestion and have accordingly expanded and clarified the discussion on melatonin pharmacokinetics, including the differences between immediate- and extended-release formulations.

Line 1013: “Timing and formulation choice are therefore critical, since ER MLT can maintain protective plasma concentrations overnight or across multi-day recovery phases, potentially enhancing resistance to exercise-induced oxidative stress, particularly following late or nocturnal training sessions(Ishihara et al., 2021). Nonetheless, further research is needed to establish optimal dosing strategies tailored to exercise type and intensity.” has been incorporated in the discussion section. 

 

9.    The manuscript is generally well-written, but minor typographical errors, inconsistent abbreviation use, and occasional sentence complexity may reduce readability. It is suggested to conduct a thorough editorial proofreading to improve clarity, ensure consistent use of abbreviations, and simplify complex sentences where possible.

Response: We thank the reviewer for noting these important editorial aspects. Following the suggestion, we have thoroughly proofread the manuscript to correct typographical errors, ensure consistent use of abbreviations, and improve sentence clarity and readability.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled "Melatonin and DNA Repair: The Impact of Exogenous Administration in Exercise-Induced Oxidative Stress" is a well-written systematic review that explores the potential effects of melatonin on exercise-induced oxidative stress. The authors have addressed the topic clearly in both the introduction and discussion sections, effectively supported by informative figures and tables.

However, I have a significant concern regarding the literature search methodology. The review appears to rely solely on the PubMed database. For a comprehensive systematic review, it is essential to include additional databases such as MEDLINE, Google Scholar, Scopus and Embase. This broader search strategy would ensure a more thorough and unbiased analysis of the available literature.

Author Response

Reviewer 2:

Comments and Suggestions for Authors
The manuscript entitled "Melatonin and DNA Repair: The Impact of Exogenous Administration in Exercise-Induced Oxidative Stress" is a well-written systematic review that explores the potential effects of melatonin on exercise-induced oxidative stress. The authors have addressed the topic clearly in both the introduction and discussion sections, effectively supported by informative figures and tables.
However, I have a significant concern regarding the literature search methodology. The review appears to rely solely on the PubMed database. For a comprehensive systematic review, it is essential to include additional databases such as MEDLINE, Google Scholar, Scopus and Embase. This broader search strategy would ensure a more thorough and unbiased analysis of the available literature.
Response: We thank the reviewer for this valuable observation regarding our search strategy. In response, we expanded our literature search to include PubMed, MEDLINE, and Scopus, which we considered to be the most robust and relevant databases for the topic. This updated strategy is now reflected in the revised PRISMA flow diagram. The additional sources confirmed the consistency of our initial results and enhanced the comprehensiveness and reliability of the review. Please find the details in section 2.2. Search Strategy, Line: 448.

Reviewer 3 Report

Comments and Suggestions for Authors

Melatonin (MLT) is a hormone with antioxidant and anti-inflammatory properties. The submitted work by Bertolucci et al., "Melatonin and DNA repair. The impact of Exogenous administration in exercise-induced oxidative stress" discusses the effect of MLT on oxidative stress induced by extreme exercise in athletes. The work would be significant, since extreme exercise by top athletes causes oxidative damage to the athletes' muscles, which has a major negative impact on the athletes' health. However, the work requires several corrections, clarifications and additions.

Main recommendations and comments:

1. The title of the work does not reflect the authors' intention. It does not follow from the text of the manuscript that the authors of the evaluated publications monitored DNA repair in the only evaluated work. The information provided shows that the mentioned work monitored DNA damage via 8-OHdG.
2. The methodology has several shortcomings and limitations for achieving a serious result and conclusion. From a selection of 65 publications, the authors selected 6 publications and only one work was evaluated. Other criteria also show a significant degree of heterogeneity - a small number of athletes included in the studies, the type of athletic activity is not stated, the daily or continuous dose of MLT administered, the method of administration, the length of the administration period.

Minor comments:

3. It is necessary to explain the abbreviations mentioned for the first time in the text (e.g. OGGI, BER, SSB, on p. 4) and all those mentioned below.
4. The equations and markers of DNA damage are incorrect: The Fenton reaction is not explained correctly. As stated in the work, it is incorrect. Fe2+ does not exist in the biological system, but is bound to some biological chelator. Therefore, "Fenton reaction" should be stated instead of "Fenton type reaction". In the biological system Haber-Weiss reaction during oxidative stress is more common: H2O2 + O2.- ---> ^.OH + ^-OH +O2
5. The products of oxidative DNA damage are not correctly listed: 8-oxo-7,8-dihydroguanine is not 8-OHdG, the letter "d" is not from "dihydro", but is the symbol for the deoxynucleoside 8-OH-deoxyguanosine = 8-OHdG (sometimes dGuo is also used). For 8-oxo-7,8-dihydroguanine, the symbol is 8-OHGua = 8-oxo-dG. The tautomeric form of these oxidatively damaged nitrogenous bases / nucleosides is 8-oxo-G = 8-oxo-Gua / 8-oxo-dG=8-oxo-dGuo.
6. Figure 1 - explain in the text under Figure PPAR, Reaction catalyzed by GPx: not quantitatively correct - what is OOH ??

Author Response

Reviewer 3

Comments and Suggestions for Authors
Melatonin (MLT) is a hormone with antioxidant and anti-inflammatory properties. The submitted work by Bertolucci et al., "Melatonin and DNA repair. The impact of Exogenous administration in exercise-induced Oxidative Stress discusses the effect of MLT on oxidative stress induced by extreme exercise in athletes. The work would be significant, since extreme exercise by top athletes causes oxidative damage to the athletes' muscles, which has a major negative impact on the athletes' health. However, the work requires several corrections, clarifications and additions.
Main recommendations and comments:

1.    The title of the work does not reflect the authors' intention. It does not follow from the text of the manuscript that the authors of the evaluated publications monitored DNA repair in the only evaluated work. The information provided shows that the mentioned work monitored DNA damage via 8-OHdG.

Response: We appreciate the reviewer’s observation. We acknowledge that the studies included in this review did not directly assess DNA repair mechanisms, but rather focused on markers of DNA oxidative damage. However, we consider these findings as indirect evidence of potential effects on DNA integrity, which may reflect upstream modulation of repair processes. Nevertheless, based on our theoretical framework and understanding of the oxidative stress and DNA damage-repair continuum, we consider that the findings reported by the included clinical trials in humans—despite being indirect—remain scientifically valid. The measurement of markers such as 8-OHdG in the context of exercise-induced oxidative stress offers relevant insights into the potential protective effects of melatonin on genomic integrity, which may plausibly involve modulation of repair processes upstream.  In response to this, we have revised the title of the manuscript from:
“MLT and DNA Repair: The Impact of Exogenous Administration in Exercise-Induced Oxidative Stress “
to:
“MLT and DNA Integrity: The Impact of Exogenous Administration in Exercise-Induced Oxidative Stress — A Systematic Review”
This revised title better aligns with the content of the manuscript and the nature of the evidence synthesized, which focuses on indirect markers of oxidative stress and DNA damage, rather than direct measurement of DNA repair mechanisms.
To clarify this point, we have revised the title and strengthened the conclusions of the manuscript. In particular, Section 5. “Conclusions
Current evidence suggests that exogenous melatonin may protect against exercise-induced oxidative stress and potentially attenuate DNA damage. However, most of the included studies assessed only indirect oxidative stress markers, with limited direct assessment of DNA damage or repair. To confirm these effects and better understand the underlying mechanisms, future clinical trials should incorporate validated biomarkers of DNA integrity and repair (e.g., comet assay, enzyme activity assays such as OGG1, APE1), as well as advanced genomic stability tests.”

2.    The methodology has several shortcomings and limitations for achieving a serious result and conclusion. From a selection of 84 publications, the authors selected 6 publications and only one work was evaluated. Other criteria also show a significant degree of heterogeneity - a small number of athletes included in the studies, the type of athletic activity is not stated, the daily or continuous dose of MLT administered, the method of administration, the length of the administration period.

Response: We thank the reviewer for this important observation. We would like to clarify that the methodological approach of this systematic review strictly followed the PRISMA 2020 guidelines, including a comprehensive database search, predefined eligibility criteria, independent screening by multiple reviewers, and critical appraisal of the included studies. The final selection of six clinical trials reflects the current state of published evidence that meets the inclusion criteria focused on human intervention studies assessing the effects of exogenous melatonin on oxidative stress and DNA integrity in the context of physical exercise.
We fully agree with the reviewer that the available studies present important limitations, including small sample sizes, variability in melatonin dosing regimens, heterogeneous administration protocols, and often limited reporting on exercise characteristics. These issues are thoroughly acknowledged and discussed in detail in the "Limitations" section of the manuscript, as well as in Supplementary Table S3, which outlines methodological heterogeneity and risk of bias using the Cochrane RoB 2 tool. Highlighting these gaps is, in fact, one of the core contributions of our review, reinforcing the need for more rigorous and standardized clinical research in this field.
Importantly, despite these differences, we observed relatively consistent findings across the included studies regarding the potential protective role of melatonin against exercise-induced oxidative stress. This convergence of evidence, although preliminary and based on indirect indicators, supports the plausibility of melatonin's beneficial effects and underscores the need for further well-designed clinical trials incorporating standardized protocols and direct biomarkers of DNA integrity and repair.

 

Minor comments

3.    It is necessary to explain the abbreviations mentioned for the first time in the text (e.g. OGGI, BER, on p. 4) and all those mentioned below.

Response: Thank you for noting this. We have carefully reviewed the manuscript and ensured that all abbreviations, including OGG1 and BER,  are properly defined upon their first appearance in the text. We also verified the consistency of abbreviation use throughout the manuscript.

 

4.    The equations and markers of DNA damage are incorrect: The Fenton reaction is not explained correctly. As stated in the work, it is incorrect. Fe2+ does not exist in the biological system, but is bound to some biological chelator. Therefore, "Fenton reaction" should be stated instead of "Fenton type reaction". In the biological system Haber-Weiss reaction during oxidative stress is more common: H2O2 + O2.- ---> .OH + -OH +O2
Response: We are grateful for the reviewer’s insight; the sentence has been modified as shown below (lines 106-114):
In biological systems, H₂O₂ participates in the iron-catalyzed Haber-Weiss reaction, the physiologically relevant pathway for hydroxyl radical generation during exercise. Unlike classical Fenton chemistry, intracellular iron exists bound to proteins or as chelated complexes in the labile iron pool. This iron catalyzes the two-step process: superoxide reduces Fe³⁺ to Fe²⁺, followed by Fe²⁺ reacting with H₂O₂ in a Fenton-type reaction. 
O₂⁻· + H₂O₂ → ·OH + OH⁻ + O₂
This net reaction represents the major pathway for hydroxyl radical production in exercising muscle, particularly in microdomains where exercise-induced metabolic changes may transiently increase iron availability from storage proteins.

5.    The products of oxidative DNA damage are not correctly listed: 8-oxo-7,8-dihydroguanine is not 8-OHdG, the letter "d" is not from "dihydro", but is the symbol for the deoxynucleoside 8-OH-deoxyguanosine = 8-OHdG (sometimes dGuo is also used). For 8-oxo-7,8-dihydroguanine, the symbol is 8-OHGua = 8-oxo-dG. The tautomeric form of these oxidatively damaged nitrogenous bases / nucleosides is 8-oxo-G = 8-oxo-Gua / 8-oxo-dG=8-oxo-dGuo.
Response: We sincerely thank the reviewer for this valuable correction and for pointing out the precise biochemical nomenclature regarding oxidatively damaged DNA products. You are absolutely right in stating that:
●    8-oxo-7,8-dihydroguanine refers to the oxidized nitrogenous base and should be abbreviated as 8-OHGua. We confirm that the term “8-oxo-7,8-dihydroguanine” does not appear in the current revised version of our manuscript.

●    8-OHdG stands for 8-hydroxy-2'-deoxyguanosine, the oxidized deoxynucleoside form, where the “d” correctly denotes the deoxyribose moiety, not “dihydro”. As indicated on line 133 of our manuscript

●    The term 8-oxo-G is a tautomeric representation used more broadly and can refer to either the base (8-oxo-Gua) or the nucleoside (8-oxo-dG/8-oxo-dGuo), depending on context
We acknowledge the confusion caused by our initial phrasing and have revised the manuscript to ensure accurate terminology is used throughout. 

 

6.    Figure 1 - explain in the text under Figure PPAR, Reaction catalyzed by GPx: not quantitatively correct - what is OOH ??
Response: Thank you for your valuable feedback regarding the representation of the glutathione peroxidase (GPx) reaction and the notation "OOH" in Figure 1. We appreciate the opportunity to clarify and improve the accuracy of our figure. In the original figure, "OOH" was used as a generic abbreviation for hydroperoxides, which may include hydrogen peroxide (H₂O₂) and organic hydroperoxides. However, this notation lacks specificity and may cause confusion regarding the precise substrates of GPx enzymes. The more accurate and widely accepted notation is "R-OOH," where "R" denotes an organic moiety, representing lipid or other organic hydroperoxides (e.g., lipid hydroperoxides formed during oxidative stress) (Pei et al., 2023).    

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript “Melatonin and DNA Repair: The Impact of Exogenous Administration in Exercise-Induced Oxidative Stress” by Bertolucci et al is a systematic review about the role of melatonin in attenuating exercise-induced oxidative stress. The authors also note that they are considering the role of melatonin in protecting DNA integrity and modulating DNA repair, but DNA repair is not studied in this review. Thus the name of the paper and the possibility of publishing in this journal DNA (MDPI) are questionable. I leave this for the Editor to consider.

After use of the search protocol only six clinical trials were included to the Results. The purpose of such a strict selection of literature is not entirely clear and in my opinion do not correspond to the fact that the authors consider their review as a step to design the studies with standardized protocols to confirm melatonin genoprotective potential in sports medicine and “aimed to synthesize clinical evidence on the impact of exogenous MLT supplementation in attenuating exercise-induced oxidative stress, protecting DNA integrity, and modulating DNA repair in healthy, physically active individuals”. Perhaps the authors should more focus not on the fact that they prove the effectiveness of melatonin in DNA repair, but on the fact that there is really little data that fits their strict criteria.

How correct is to discard the old papers? It could be better to discuss them. Perhaps there was no data suitable for the PICO criteria, but not reviewing them at all is strange.

 

Line 161: to 𝐻2𝑂2 e 𝑂2 – and?

Line 190: synthesized in numerous other tissues, including mitochondria themselves – mitochondria are not tissues.

Line 216: thereby protecting DNA [5,8,51] – there is nothing about DNA in ref [51].

Line 271: The objective of this comprehensive review is to critically examine the current scientific literature surrounding MLT’s role in mitigating exercise-induced oxidative stress and DNA damage – Perhaps it would be better to really examine the current scientific literature surrounding MLT’s role in DNA damage, thus it could be closer to the topic of the journal.

Table 1 and Figure 4 are not cited in the text. The authors should check it for other tables, figures and supplementary materials.

S2 table is not entirely in English. Tables 1 and 2 also contain words not in English.

Line 485: A time analysis of the publications in this literature review shows a range between 2017 and 2023 – put the year in the Table.

Line 523: Two studies in the text, but there are three references 76, 77, 78.

Fig. 3: There are 5 papers in the picture, where is the sixth (Farjallah et al.)?

Line 665: glucose was also explored in several studies – reference is needed.

Figure 4: There is nothing about OGG1 increase in the text.

Author Response

Reviewer 4:

Comments and Suggestions for Authors
The manuscript “Melatonin and DNA Repair: The Impact of Exogenous Administration in Exercise-Induced Oxidative Stress” by Bertolucci et al is a systematic review about the role of melatonin in attenuating exercise-induced oxidative stress. The authors also note that they are considering the role of melatonin in protecting DNA integrity and modulating DNA repair, but DNA repair is not studied in this review. Thus the name of the paper and the possibility of publishing in this journal DNA (MDPI) are questionable. I leave this for the Editor to consider.

1.    After use of the search protocol only six clinical trials were included to the Results. The purpose of such a strict selection of literature is not entirely clear and in my opinion do not correspond to the fact that the authors consider their review as a step to design the studies with standardized protocols to confirm melatonin genoprotective potential in sports medicine and “aimed to synthesize clinical evidence on the impact of exogenous MLT supplementation in attenuating exercise-induced oxidative stress, protecting DNA integrity, and modulating DNA repair in healthy, physically active individuals”. Perhaps the authors should focus more not on the fact that they prove the effectiveness of melatonin in DNA repair, but on the fact that there is really little data that fits their strict criteria.
Response: We sincerely thank the reviewer for their thoughtful comments and critical appraisal. We fully agree that the number of clinical trials meeting our eligibility criteria is limited. However, our intention was not to exclude relevant insights arbitrarily, but rather to apply a rigorous and transparent methodological framework based on the PRISMA 2020 guidelines and the PICO strategy, which we believe are essential to ensure the reproducibility and reliability of systematic reviews.
Our aim was to synthesize the available clinical evidence on its potential impact in the context of exercise-induced oxidative stress and DNA integrity. Indeed, one of our key findings—and a central message of the manuscript—is the recognition of how scarce and heterogeneous the current literature is. We emphasize this gap throughout the discussion and in the conclusion, highlighting the urgent need for more robust, standardized, and targeted clinical trials in this field. For greater transparency, we have clarified this point in the manuscript and provided a PRISMA flowchart detailing the reasons for exclusion.
Ultimately, we view this review not as definitive proof of melatonin’s genoprotective efficacy, but as a critical starting point for identifying existing limitations and informing future, well-designed clinical studies in sports medicine and exercise physiology.
Additionally, in response to the reviewer’s concern, we have revised the language in the conclusion section to more accurately reflect the preliminary nature of current evidence. The revised version underscores the limitations in existing studies and frames the findings as suggestive rather than confirmatory regarding melatonin’s genoprotective potential. We hope this adjustment addresses the concern about overstatement and aligns the conclusions with the quality and quantity of available data.

 

2.    How correct is to discard the old papers? It could be better to discuss them. Perhaps there was no data suitable for the PICO criteria, but not reviewing them at all is strange.
Response: Thank you for your observation. As indicated in the Methods section, our systematic search was limited to the last 10 years in order to capture the most recent and relevant evidence. Despite this time restriction, all the studies that met our predefined eligibility criteria and were ultimately included in the review were published between 2017 and 2023. Furthermore, we confirm that all included studies involved exclusively human participants, in alignment with our inclusion criteria.

 

3.    Line 161: to ?2?2 e ?2 – and?
Response: We appreciate the reviewer’s comment, and have revised the sentence accordingly (lines 102-104):
[...] The superoxide radical from these multiple sources undergoes dismutation-catalyzed by superoxide dismutase (SOD) enzymes-forming the less reactive but more diffusible H₂O₂:
[...]

 

4.    Line 190: synthesized in numerous other tissues, including mitochondria themselves – mitochondria are not tissues.
Response: We are grateful for the reviewer’s attentive comment, and we have accordingly revised the sentence as follows (lines 336-339):
[...]. Although primarily synthesized by the pineal gland following a circadian rhythm, melatonin is also produced in various peripheral cells and organs, with mitochondria themselves acting as important intracellular sites of its synthesis, [...]

 

5.    Line 216: thereby protecting DNA [5,8,51] – there is nothing about DNA in ref [51].
Response: We thank the reviewer for this careful observation. We agree that reference [51] does not support the statement regarding DNA protection. This phrase was inadvertently retained in an earlier version of the manuscript. We confirm that, once the modifications suggested by the editor and other reviewers were incorporated, this sentence was removed from the text, and reference [51] no longer appears in this context in the current revised version.

 

6.    Line 271: The objective of this comprehensive review is to critically examine the current scientific literature surrounding MLT’s role in mitigating exercise-induced oxidative stress and DNA damage – Perhaps it would be better to really examine the current scientific literature surrounding MLT’s role in DNA damage, thus it could be closer to the topic of the journal.
Response: We thank the reviewer for this insightful suggestion. We have carefully considered focusing the objective solely on DNA damage; however, we chose to retain the broader phrasing—“mitigating exercise-induced oxidative stress and DNA damage”—because it more accurately reflects the dual antioxidant and genome-protective actions of melatonin that we survey. Indeed, while melatonin’s general antioxidant properties have been extensively characterized across numerous physiological and pathological contexts, its specific capacity to safeguard DNA integrity and influence repair pathways after intense exercise remains remarkably underexplored. By maintaining this dual emphasis, our review not only highlights a critical gap in the literature but also underscores the urgent need for rigorously designed clinical trials in human athletes to dissect melatonin’s role in post-exercise DNA repair. We believe this approach best positions our manuscript to illuminate an emerging research frontier and to catalyze future studies in humans.

 

7.    Table 1 and Figure 4 are not cited in the text. The authors should check it for other tables, figures and supplementary materials.
Response: We thank the reviewer for this valuable observation. We carefully verified the current version of the manuscript to ensure that Table 1, Figure 4, as well as all other tables, figures, and supplementary materials, are properly cited and integrated into the main text.
We appreciate the reviewer’s attention to detail, which helped us improve the coherence and completeness of the manuscript.

 

8.    S2 table is not entirely in English. Tables 1 and 2 also contain words not in English.
Response: Thank you for noticing this detail. We have carefully reviewed the content of Supplementary Table S2, as well as Tables 1 and 2, and we confirm that any remaining terms not in English have now been corrected to ensure full consistency with the language of the manuscript.

 

9.    Line 485: A time analysis of the publications in this literature review shows a range between 2017 and 2023 – put the year in the Table.
Response: We thank the reviewer for this observation. As noted, our systematic review was conducted with a 10-year publication window, and all articles selected for inclusion were indeed published between 2017 and 2023. We confirm that the publication year has now been added to the summary table to provide a clearer overview of the temporal distribution of the included studies.

 

10.    Line 523: Two studies in the text, but there are three references 76, 77, 78.
Response: Thank you for your careful observation. We confirm that this issue has been corrected in the current version of the manuscript. The citation of the studies by Farjallah et al. and the numerical discrepancy in line 680 have both been revised to accurately reflect the correct number of studies and corresponding references. We appreciate the reviewer’s attention to detail. In line 680: “ Farjallah et al. conducted three separate studies [7,39,65].”

 

11.    Fig. 3: There are 5 papers in the picture, where is the sixth (Farjallah et al.)?
Response: We thank the reviewer for bringing this to our attention. We acknowledge that certain information was inadvertently omitted from the original version of the figure due to a formatting oversight. Upon review, we identified the missing data and have now thoroughly revised the figure to ensure that all relevant elements are correctly and clearly presented. We appreciate the reviewer’s careful observation, which allowed us to correct this issue and improve the quality and completeness of the manuscript.

 

12.    Line 665: glucose was also explored in several studies – reference is needed.
Response: Thank you for your observation. We appreciate your suggestion and have now incorporated the appropriate references to support the statement regarding glucose as a biomarker evaluated in several studies.
The revised sentence now reads:
Line 938: “In addition to these biomarkers, glucose was also explored in several studies (Ghosal et al., 2013; Nordlie et al., 1999) . Glucose plays a central role in energy production, and during aerobic activity, it is primarily disposed of in skeletal muscle.”

 

13.    Figure 4: There is nothing about OGG1 increase in the text.
Response: We thank the reviewer for this observation. We would like to clarify that the reference to OGG1 activity is indeed included in the current version of the manuscript.
Specifically, OGG1 is mentioned in the Introduction, line 134, in the context of DNA repair biomarkers:
“Finally, DNA repair biomarkers—including direct measures such as the comet assay […] and the activity of repair enzymes like OGG1 and APE1—capture the genotoxic consequences of exercise-induced ROS and the cellular capacity for genomic maintenance and repair [25–27].”
Additionally, we discuss the role of OGG1 within the section starting at line 388, highlighting its mechanistic relevance:
“DNA Repair Biomarkers: Most intriguingly, MLT appears to influence DNA integrity through multiple complementary mechanisms. […] Additionally, and perhaps more importantly, MLT activates the Base Excision Repair (BER) pathway through enhanced OGG1 and APE1 activity [47,63,64], as shown in Figure 5.”
We have double-checked that this is now correctly aligned with Figure 5, where OGG1 is depicted as part of the BER pathway influenced by MLT.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors, 

Thank you for taking into consideration my suggestion. Your manuscript is now suitable for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

Authors accepted reviewer recommendations

Reviewer 4 Report

Comments and Suggestions for Authors

Accept in present form