Review Reports - The Use of Nutritional Interventions to Enhance Genomic Stability in Mice and Delay Aging

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The ms. "The use of nutritional interventions to enhance genomic stability in mice and delay aging", by van Galen et al., is an interesting review of what is reported on the possible effects of diet styles and nutrients on DNA preservation and repair and on the consquence this may have on aging rate.

The ms. is well written, the review of the literature is accurate. Information is well presented and sound interpatation f the wealth of studies are presente.

I would suggest the authors to report at least a summary of the search criteria ( in particular keywords) in Materials and Methods as this is essential to understand the results obtained.

Author Response

Reviewer 1

The ms. is well written, the review of the literature is accurate. Information is well presented and sound interpatation f the wealth of studies are presente.

I would suggest the authors to report at least a summary of the search criteria ( in particular keywords) in Materials and Methods as this is essential to understand the results obtained.

We thank the reviewer for the positive comments. We have expanded the methods section and added overall search terms. The full details of our search can be found in Supplemental file 1 as indicated. See addition Material and Methods line 126-128: “Search queries included at least one primary keyword from the subject of DNA damage and repair, one secondary keyword related to aging and health, one tertiary keyword specific for the type of nutritional intervention and all restricted to Mus Musculus (supplemental file 1).”

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a topic of clear relevance by examining a broad range of nutritional interventions and their potential impact on genomic stability. However, for the following reasons, it does not currently meet the standards required for publication:

• Interpretations of subtle DNA damage markers (γH2AX, 8-OHdG, etc.) are inconsistent, and tissue-specific differences are not adequately discussed.
• The conclusion that antioxidant or metabolic regulation leads to reduced DNA damage is overly generalized, and the causal pathways are not clearly articulated.
• Evidence supporting several dietary interventions—particularly plant derivatives and synthetic drugs—relies heavily on single studies, limiting generalizability.
• Comparative criteria between the robust effects of CR/DR and the partial effects of other interventions are unclear, and the evidence hierarchy is not established.
• The mechanistic connection between Figures 2–3 and the corresponding narrative in the text is insufficiently explained, resulting in breaks in logical flow.
• Findings from natural aging models and those from disease or genetically modified models are discussed on the same conceptual level, blurring translational relevance.

Author Response

Reviewer 2

Interpretations of subtle DNA damage markers (γH2AX, 8-OHdG, etc.) are inconsistent, and tissue-specific differences are not adequately discussed.

We thank the reviewer for this question and agree that not all identified publications show equal changes in DNA damage markers. To help the reader we have added classification systems in Table 1 to both Biomarkers and Detection methods, with explanatory footnote on line 111-114: “$DNA damage biomarkers which have a broad specificity or can be secondary to other lesions. 1-3Degree of accuracy/reliability of the detection method (1 high; 2 intermediate or depending on antibody specificity, and 3 lower/variable).”.

Moreover, we acknowledge this in our discussion as limitation of the study and included references specifically focusing on tissue-, strain- or sex-specific differences. New included Line 654-666: “Limitations of this study, are the lack of a formal quality assessment, as usually indicated in systematic reviews. Instead, our semi-systematic search did result into a broad overview of literature, where we do acknowledge potential bias and for several compounds or interventions yet lack of reproducibility. Such interventions are therefore also more depicted on the left side of Figure 3. Concomitantly, also tissue specific effects, strain variability or sex differences are not discussed or only marginally indicated, in line with the limited studies identified for several dietary interventions, particularly plant derivatives and synthetic drugs, or assessed only a single or less specific biomarker, limiting generalizability. However, we do refer to such reviews with a focus on the most robust health and lifespan extending intervention of restricting calories (34, 44, 48). Moreover, this review was intended to reveal interventions that could enhance genomic stability and to serve as guide for future longevity research. As such we did reveal many connections of nutritional based interventions affecting genome integrity for further exploration.”

The conclusion that antioxidant or metabolic regulation leads to reduced DNA damage is overly generalized, and the causal pathways are not clearly articulated.

We have revised our wording and changed sections related to this e.g., 3.4 Micronutrients line 399-407: “When present at adequate levels, micronutrients appear to delay age-associated metabolic changes and reduce genomic instability. However, to what extent these effects are attributable to antioxidant activity or metabolic regulation, rather than alternative mechanisms, is currently largely unknown (130). Beyond direct redox actions, micro-nutrients may indirectly influence genome integrity by modulating cellular stress re-sponses, metabolic homeostasis, or damage-tolerance pathways in a compound-specific manner. Such effects could alter either the burden of DNA damage or its downstream consequences, highlighting the need for further research in this area.”

Also, we down-tone the conclusions on antioxidants in our discussion section line 726-742: “One key area of debate in this context is the role of oxidative stress and antioxidants in genomic stability. While many studies focus on the antioxidative properties of a sub-stance, evidence that ROS at physiological levels causes genomic damage (259–261), or that antioxidants are beneficial protectors of the genome (262–264), is heavily debated. Moreover, studies in humans have often yielded negative results, with various meta-analyses even suggesting a likely increase in disease incidence (265,266). While the exact reasons for this are unknown, dosage and context might be an important factor, highlighting the limited ability of in-vitro antioxidant assays to predict in-vivo efficacy (267). For example, while physiological levels of vitamin C behave largely as an anti-oxidant in-vivo, pharmacological dosing can shift toward pro-oxidant activity via Fenton chemistry. And, although the increase in oxidative DD and engagement of DNA repair pathways can in some situations be beneficial (e.g. cancer treatment), it does show the importance of situational awareness and balanced application of these and similar types of compounds (268,269). Additionally, the genome protective effects of many so-called "antioxidative" compounds extend beyond simple ROS scavenging, as they often also modulate stress resilience pathways and energy metabolism to improve cellular function, longevity, and genomic maintenance (270–272).”

Evidence supporting several dietary interventions—particularly plant derivatives and synthetic drugs—relies heavily on single studies, limiting generalizability.

We agree with the reviewer and have, as indicated above, included this in a new specific section addressing our study limitations (Line 654-666). This aspect was included in our Figure 3 but was lacking in explanation. Now we added Discussion Line 632-644: “In this review, we highlighted and assessed the evidence of known nutritional interventions that improve genomic stability and healthy aging in mice. Many interventions discussed here, particularly CR/DR and their mimetics, act on more than one mechanistic layer (e.g. different DNA repair processes, metabolism and/or antioxidant systems; also see Figure 2), complicating causal interpretation when relying solely on DD markers or lifespan readouts. Moreover, while some dietary interventions have been studied for decades, others are supported by only a limited number of small studies, and relatively few directly assess DD or DNA repair. In figure 3 we provide a qualitative overview of the discussed literature. Interventions were positioned based on the amount and consistency of available data, and how the reported outcomes relate to genomic stability (DD markers and/or repair readouts). As such, figure 3 should be viewed as a guide to where evidence is stronger and where important gaps remain.”

Comparative criteria between the robust effects of CR/DR and the partial effects of other interventions are unclear, and the evidence hierarchy is not established.

We likely were unclear in the formulation of the type of review we have generated. To make readers aware that this is not a systematic review we have more explicitly stated this in our Material and Methods section lines 121-122 “search queries using a semi-systematic approach without formal quality assessment” and 133-137 “This approach was chosen to maximize the global overview and identification of potentially relevant literature as many studies likely contain or assessed only one type of nutritional intervention or DNA damage detection method or looked at specific mouse models or tissue types. Although no formal quality assessment was performed, yielding a potential bias,…”.

Information related to specificity of the damage biomarkers and detection methods evaluated in the various studies following different interventions has been added to Table 1, and the various types of interventions graded based on available evidence into Figure 3 (horizontal positioning) and related sections, as indicated above.

The mechanistic connection between Figures 2–3 and the corresponding narrative in the text is insufficiently explained, resulting in breaks in logical flow.

We thank the reviewer for this comment and have expanded on the mechanistic explanation for Figure 2 in lines 305-316: “Integrating findings from the broader literature, we propose a (simplified) scenario for the effects of various forms of dietary interventions on lifespan and health, at least in part by influencing DD and DNA repair mechanisms as presented in figure 2, while recognizing interactive mechanisms, such as hormesis-induced resilience, likely modulate these processes both directly and indirectly. Various of the above indicated diets, overall reduces the proliferation rate of healthy body cells, which distinctively affects replication dependent DNA repair processes like HR and XLR, as well as replication independent DNA repair processes such as TCR and NHEJ. Combined with the sup-pression of metabolic activity and ROS levels this results in a global reduction of genomic instability lowering the incidence of cells undergoing apoptosis or entering a state of cellular senescence, preventing systemic inflammaging and functional decline of tissues (25-27, 35, 41, 45, 46, 49, 51, 52, 56, 61, 64, 69, 71-76) (Figure 2). Furthermore,..”, and for both Figure 2 and 3 in the beginning of our Discussion (lines 631-666).

Findings from natural aging models and those from disease or genetically modified models are discussed on the same conceptual level, blurring translational relevance.

We highly appreciate pointing out this issue. Throughout the entire results section we have now more explicitly stated when studies assessed wild-type aging models or specific mutant mice to avoid confusion. Please see our version included with all changes highlighted in red.

Reviewer 3 Report

Comments and Suggestions for Authors

This critical review evaluates the article titled “The use of nutritional interventions to enhance genomic stability in mice and delay aging.” The manuscript covers a broad spectrum of dietary and oral interventions and their potential roles in modulating genomic stability. While comprehensive, the review exhibits methodological and interpretive weaknesses that merit detailed analysis. The article successfully integrates metabolism, nutrition, and DNA damage into a coherent biological rationale. However, it conflates reduced DNA damage with improved stress resilience or altered metabolic rate—mechanisms that are distinct and should be treated separately. Additionally, causality is often implied from correlational data, particularly when relying on biomarkers such as γH2AX, which lack high specificity.The authors gathered a wide array of mouse-based studies but did not implement any formal evidence grading. The heavy reliance on progeroid or DNA-repair–deficient models limits generalizability to natural aging. Furthermore, several cited interventions are supported only by one or two studies, often with methodological limitations such as small sample sizes or non-physiological exposure levels.

With regards to analysis of specific nutritional interventions caloric and dietary restriction (CR/DR) are appropriately identified as the most robust interventions, with consistent effects on genomic stability. In contrast, micronutrient and antioxidant sections lack critical appraisal, especially given contradictory findings in humans. NAD+ precursors are promising but require clearer differentiation between effects on DNA repair versus mitochondrial or inflammatory pathways.

In addition, the review briefly acknowledges translational challenges but does not deeply analyze differences in metabolic rate, lifespan, or environmental exposure between mice and humans. The weak clinical outcomes of antioxidant therapies and practical barriers to long-term caloric restriction warrant further exploration.

However, there are some limitations: limited critical evaluation of study quality, overreliance on single biomarkers, excessive focus on mouse models with poor generalizability and insufficient discussion of dosage, hormesis, and nutrient interactions.

Some recommendations for improvement.

Implementing an evidence-grading system.
Distinguishing mechanistic pathways affecting genomic stability.
Reducing emphasis on single-study findings.
Discussing sex differences, strain variability, and dose–response effects.
Adding a detailed analysis of human translational challenges.
Clarifying the limitations of DNA damage biomarkers.

In conclusion, this review recognizes the scientific importance of examining nutritional influences on genomic stability. Although the article provides an extensive compilation of findings, its conclusions would be strengthened by a more rigorous methodology, clearer mechanistic distinctions, and a more realistic appraisal of translational applicability. With improved evidence grading and critical analysis, the review could serve as a strong reference in nutrition and aging research.

Author Response

Reviewer 3

Some recommendations for improvement.

Implementing an evidence-grading system.

We thank the reviewer for the positive notes above. We likely were unclear in the formulation of the type of review we have generated. To make readers aware that this is not a systematic review we have more explicitly stated this in our Material and Methods section lines 121-122 “search queries using a semi-systematic approach without formal quality assessment” and 133-137 “This approach was chosen to maximize the global overview and identification of potentially relevant literature as many studies likely contain or assessed only one type of nutritional intervention or DNA damage detection method or looked at specific mouse models or tissue types. Although no formal quality assessment was performed, yielding a potential bias,…”.

In terms of grading the specificity of the damage biomarkers and detection methods evaluated in the various studies following different interventions we have added classification systems in Table 1 to both Biomarkers and Detection methods, with explanatory footnote on line 111-114: “$DNA damage biomarkers which have a broad specificity or can be secondary to other lesions. 1-3Degree of accuracy/reliability of the detection method (1 high; 2 intermediate or depending on antibody specificity, and 3 lower/variable).”.

The various types of interventions were graded based on available evidence into Figure 3 (horizontal positioning), but this aspect was lacking any explanation. Now we added Discussion Line 632-644: “In this review, we highlighted and assessed the evidence of known nutritional interventions that improve genomic stability and healthy aging in mice. Many interventions discussed here, particularly CR/DR and their mimetics, act on more than one mechanistic layer (e.g. different DNA repair processes, metabolism and/or antioxidant systems; also see Figure 2), complicating causal interpretation when relying solely on DD markers or lifespan readouts. Moreover, while some dietary interventions have been studied for decades, others are supported by only a limited number of small studies, and relatively few directly assess DD or DNA repair. In figure 3 we provide a qualitative overview of the discussed literature. Interventions were positioned based on the amount and consistency of available data, and how the reported outcomes relate to genomic stability (DD markers and/or repair readouts). As such, figure 3 should be viewed as a guide to where evidence is stronger and where important gaps remain.”

Distinguishing mechanistic pathways affecting genomic stability.

We have highlighted the various genomic instability pathways in the introduction, section lines 60-68, and the type of lesion they resolve in Table 1. Specific references that in great detailed describe these processes (including References 11 and 13) are highlighted.

We additionally expanded the introduction by adding lines 89-94: “Nutritional components can either provoke or protect against DD through multiple, partially overlapping mechanisms such as; (i) direct effects on DD formation or repair capacity, (ii) changes in metabolic rate and energy flux that alter the production of endogenous genotoxic stress, and (iii) enhanced cellular and organismal resilience that improves tolerance to damage without necessarily reducing lesion burden.”, and the results section related to Figure 2 by adding lines 305-316: “Integrating findings from the broader literature, we propose a (simplified) scenario for the effects of various forms of dietary interventions on lifespan and health, at least in part by influencing DD and DNA repair mechanisms as presented in figure 2, while recognizing interactive mechanisms, such as hormesis-induced resilience, likely modulate these processes both directly and indirectly. Various of the above indicated diets, overall reduces the proliferation rate of healthy body cells, which distinctively affects replication dependent DNA repair processes like HR and XLR, as well as replication independent DNA repair processes such as TCR and NHEJ. Combined with the sup-pression of metabolic activity and ROS levels this results in a global reduction of genomic instability lowering the incidence of cells undergoing apoptosis or entering a state of cellular senescence, preventing systemic inflammaging and functional decline of tissues (25-27, 35, 41, 45, 46, 49, 51, 52, 56, 61, 64, 69, 71-76) (Figure 2). Furthermore,..”

Specifically related to NAD in relation to genomic instability pathways (section 3.7) we have added lines 471-474: “Importantly, reported health benefits of NAD+ precursors can reflect both NAD+-dependent DD signaling and/or repair and indirect effects via improved mitochondrial function, redox homeostasis, and inflammatory signaling that may reduce the upstream burden of DD.”, lines 491-493: “NR significantly abrogated γH2AX foci formation and related senescence markers, an effect that may reflect reduced damage accumulation and/or altered damage signaling”, lines 496-498: “despite their inherently lowered DNA repair capacity, suggesting that these health benefits likely arise from mechanisms distinct from direct enhancement of repair.”, and lines 510-514: “While direct stimulation of NAD⁺-dependent DNA repair pathways may contribute, reductions in DD markers are likely mediated indirectly through improved mitochondrial function and the consequent reduction in endogenous damage burden. Distinguishing direct repair enhancement from secondary protective effects in-vivo remains an important objective for future research.”, and related discussion on lines 687-691: NAD⁺ precursors such as NMN and NR, for example, activate sirtuins and improve mitochondrial function, recapitulating some CR/DR-associated benefits; however, current evidence suggests that their effects on genomic stability are largely indirect, arising from improved metabolic homeostasis, reduced inflammation, and enhanced cellular stress resilience rather than from direct stimulation of DNA repair.”.

The generalizability of the model systems highlighted has been further clarified in the introduction lines 74-78: “Notably, progeroid DNA repair–deficient mouse models such as Xpg-/- and Ercc1Δ/- mice in which DD persists accelerated resulting in shortened lifespan, exhibit epigenetic altera-tions, transcriptional changes, systemic biomarker shifts, and multi-organ functional decline that closely mirror those observed during natural aging, supporting their use as mechanistic models of physiological aging (12,13).”.

Throughout the entire results section we have now also more explicitly stated when studies assessed such specific mutant models or wild-type aging mice to avoid confusion. Please see our version included with all changes highlighted in red.

Reducing emphasis on single-study findings.

We agree with the reviewer and have, as indicated above, included this in a new specific section addressing our study limitations, lines 654-658: “Limitations of this study, are the lack of a formal quality assessment, as usually indicated in systematic reviews. Instead, our semi-systematic search did result into a broad overview of literature, where we do acknowledge potential bias and for several compounds or interventions yet lack of reproducibility. Such interventions are therefore also more depicted on the left side of Figure 3.”, and related discussion section lines 637-644: “Moreover, while some dietary interventions have been studied for decades, others are supported by only a limited number of small studies, and relatively few directly assess DD or DNA repair. In figure 3 we provide a qualitative overview of the discussed lit-erature. Interventions were positioned based on the amount and consistency of available data, and how the reported outcomes relate to genomic stability (DD markers and/or repair readouts). As such, figure 3 should be viewed as a guide to where evidence is stronger and where important gaps remain.”.

Discussing sex differences, strain variability, and dose–response effects.

Along the same lines, this is included in the above indicated Limitations section, lines 658-666: “Concomitantly, also tissue specific effects, strain variability or sex differences are not discussed or only marginally indicated, in line with the limited studies identified for several dietary interventions, particularly plant derivatives and synthetic drugs, or assessed only a single or less specific biomarker, limiting generalizability. However, we do refer to such reviews with a focus on the most robust health and lifespan extending intervention of restricting calories (34, 44, 48). Moreover, this review was intended to reveal interventions that could enhance genomic stability and to serve as guide for future longevity research. As such we did reveal many connections of nutritional based interventions affecting genome integrity for further exploration.”, with specific references added to reviews on these topics.

Adding a detailed analysis of human translational challenges.

The connection of mouse findings to potential human relevance is slightly strengthened in the discussion section, on lines 678-684: “However, efficacy in humans remains inconclusive, potentially reflecting adherence challenges, mismatches with standard-of-care regimens, or biological differences (245–249). Species-specific physiological features, such as higher mass-specific metabolic rates, shorter lifespans, and accelerated molecular turnover in mice, are likely to shape both damage accumulation and responsiveness to intervention. These differences suggest that dietary interventions that robustly extend lifespan or reduce DD in mice may require careful adjustment in timing, duration, or intensity to achieve benefits in humans.” In addition to the already discussed translational challenges.

Clarifying the limitations of DNA damage biomarkers.

We thank the reviewer for this comment and have added “However, accurately measuring physiological (i.e., low) levels of DD and reliably assessing DNA repair activity remains technically challenging (Table 1).” on lines 78-80 and included a grading system for DNA damage biomarkers and detection systems within Table 1.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

While the manuscript provides a comprehensive overview of dietary interventions affecting genomic stability, the complexity and breadth of the topic make it challenging for readers to readily grasp the overall conceptual structure and relative strength of evidence across interventions. In particular, the distinction between the robust effects of caloric/dietary restriction and the more context-dependent or partial effects of other interventions, as well as the interpretative limitations of commonly used DNA damage markers, are not immediately intuitive from the text alone.

The addition of a unifying main conceptual figure summarizing (i) the relative hierarchy of evidence across dietary interventions, (ii) the distinction between reduced DNA damage, enhanced DNA repair, and improved damage tolerance or cellular resilience, and (iii) the specificity and limitations of key DNA damage biomarkers (e.g., γH2AX, 8-OHdG) would substantially enhance clarity and accessibility for the reader. Such a figure would also help to more explicitly connect Figures 2–3 with the accompanying narrative and reinforce the intended interpretative framework of the review.

Overall, this type of integrative visual summary would strengthen the manuscript by clearly conveying that the review aims to organize and contextualize existing evidence, rather than to draw definitive causal conclusions for individual interventions.

Author Response

We thank the reviewer for this comment and have updated both Figure 2 and 3 of which the latter has also been added as graphical abstract.

Figure 2 now shows dietary interventions which less robust effects in lighter tones, as also further explained in its figure legend (see new changes indicated in blue on lines 327 “As caloric/dietary restriction yields the most robust effects other diets are depicted in lighter tones” and 332 “Green and red outlines indicate positive and negative processes respectively”) and corresponding main text on line 306 “particularly CR/DR”.

Figure 3 has two extra panels incorporated to show:

(a) the relationships between DNA damage burden, DNA repair, DNA damage response signaling, and cellular health;

(b) the detection fidelity and biomarker specificity of commonly used DNA damage markers, as in our previous revision (see changes indicated in red) added to Table 1 and corresponding introduction lines 78-80: “However, accurately measuring physiological (i.e., low) levels of DD and reliably assessing DNA repair activity remains technically challenging (Table 1)” and now further expanded (see changes indicated in red and blue) lines 89-93: “Nutritional components can either provoke or protect against DD through multiple, partially overlapping mechanisms such as; (i) direct effects on DD formation or repair capacity, (ii) changes in metabolic rate and energy flux that alter the production of endogenous genotoxic stress, and (iii) enhanced cellular and organismal resilience which could indicate for example improved tolerance to damage without necessarily reducing lesion burden or enhanced DD response signaling”;

in addition to now (c) the grading of evidence by positioning of interventions. The corresponding figure legend (lines 656-667) and discussion (lines 634-649) has been updated accordingly.