Tempol Attenuates Methotrexate-Induced Osteotoxicity via Antioxidant Mechanisms: Impairment of Protection by GPX4 Inhibition Through ML210

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the protective effects of Tempol against Methotrexate (MTX)-induced osteotoxicity in MLO-Y4 osteocyte-like cells and evaluates the impact of GPX4 inhibition via ML210 on this protection. The topic is timely and relevant, particularly considering the increasing interest in oxidative stress, ferroptosis, and redox-modulating therapies in chemotherapy-induced toxicity.

The study is well-structured and presents a coherent mechanistic framework linking oxidative stress, apoptosis, MAPK activation, and GPX4-dependent redox regulation. The experimental design is straightforward and logically organized. The findings are generally consistent and biologically plausible.

However, several conceptual and methodological issues should be addressed before the manuscript can be considered for publication.

Major Comments

-Although the manuscript strongly emphasizes GPX4 inhibition and ferroptosis-related mechanisms, no direct ferroptosis markers were evaluated.

• No lipid peroxidation assay (e.g., MDA, 4-HNE, BODIPY-C11 staining) was performed.
• No ferroptosis-specific markers (ACSL4, SLC7A11, FTH1, etc.) were analyzed.
• No ferroptosis inhibitors (e.g., ferrostatin-1 or liproxstatin-1) were used to validate ferroptotic involvement.

-The conclusion that ML210 enhances ferroptosis remains indirect. The authors should either:

• Include direct ferroptosis measurements, or
• Moderate their claims and limit conclusions to GPX4-dependent oxidative stress mechanisms rather than ferroptosis per se.

-The central mechanistic claim of the study is that Tempol protection depends on GPX4 activity. However:

• GPX4 protein expression or activity was not directly quantified.
• Total GPx activity was measured, but this does not specifically reflect GPX4.

Given that ML210 is a GPX4 inhibitor, direct assessment of GPX4 protein levels or activity would significantly strengthen the manuscript.

-The study primarily evaluates caspase-3, bax, and bcl-2, which are classical apoptotic markers.

However, ferroptosis is typically caspase-independent. The manuscript should clarify:

• Whether the observed cell death is predominantly apoptotic,
• Or whether ferroptotic and apoptotic pathways coexist.

Currently, the mechanistic boundaries between apoptosis and ferroptosis remain unclear.

-The concentrations used:

• MTX (10⁻⁵ M),
• Tempol (100 µM),
• ML210 (1 µM),

should be justified with references or preliminary dose–response data.

 

Author Response

-Although the manuscript strongly emphasizes GPX4 inhibition and ferroptosis-related mechanisms, no direct ferroptosis markers were evaluated.

• No lipid peroxidation assay (e.g., MDA, 4-HNE, BODIPY-C11 staining) was performed.
• No ferroptosis-specific markers (ACSL4, SLC7A11, FTH1, etc.) were analyzed.
• No ferroptosis inhibitors (e.g., ferrostatin-1 or liproxstatin-1) were used to validate ferroptotic involvement.
• Lipid peroxidation assays (MDA and 4‑HNE and/or BODIPY‑C11), measurements of ACSL4, SLC7A11 and FTH1, and ferrostatin‑1/liproxstatin‑1 experiments were not performed, but the claim of “ferroptosis” was moderated and the findings were presented as GPX4‑dependent oxidative stress mechanisms.

-The conclusion that ML210 enhances ferroptosis remains indirect. The authors should either:

• Include direct ferroptosis measurements, or
• Moderate their claims and limit conclusions to GPX4-dependent oxidative stress mechanisms rather than ferroptosis per se.
• the claim of “ferroptosis” was moderated and the findings were presented as GPX4‑dependent oxidative stress mechanisms.

-The central mechanistic claim of the study is that Tempol protection depends on GPX4 activity. However:

• GPX4 protein expression or activity was not directly quantified.
• Total GPx activity was measured, but this does not specifically reflect GPX4.

Given that ML210 is a GPX4 inhibitor, direct assessment of GPX4 protein levels or activity would significantly strengthen the manuscript.

GPX4-specific activity or protein expression measurements were not performed; only total GPx activity was reported. This limitation was explicitly stated in the Results and Discussion, and claims that ML210 acted via GPX4 were softened as indirect (functional) evidence. It was noted that direct GPX4 protein/activity measurements will be performed in future studies.

-The study primarily evaluates caspase-3, bax, and bcl-2, which are classical apoptotic markers.

However, ferroptosis is typically caspase-independent. The manuscript should clarify:

• Whether the observed cell death is predominantly apoptotic,
• Or whether ferroptotic and apoptotic pathways coexist.

Currently, the mechanistic boundaries between apoptosis and ferroptosis remain unclear.

Additional experiments to determine whether cell death was apoptotic or involved concurrent ferroptotic pathways were not performed The available data indicated a potential overlap between apoptosis and GPX4‑dependent oxidative damage.

 

-The concentrations used:

• MTX (10⁻⁵ M),
• Tempol (100 µM),
• ML210 (1 µM),

should be justified with references or preliminary dose–response data.

A reference to the doses used in our previous studies was added.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates the protective effects of Tempol against Methotrexate-induced osteotoxicity and explores the mechanistic role of GPX4 inhibition using ML210. The topic is timely and clinically relevant, as osteotoxicity remains an important complication of chemotherapy, and antioxidant-based strategies are gaining increasing interest. The study is well-organized and provides mechanistic insights linking oxidative stress, apoptosis, MAPK signaling, and ferroptosis-related pathways. However, several conceptual, methodological, and presentation issues should be addressed to improve the scientific rigor and clarity of the manuscript.

The manuscript addresses an interesting hypothesis regarding the dependency of Tempol’s protective effects on GPX4 activity. However, the novelty should be better emphasized. The authors should clearly state what distinguishes this work from previous studies on antioxidant protection against Methotrexate-induced bone toxicity. More discussion is needed regarding the emerging role of ferroptosis in bone pathology and how this study advances the current understanding.

While the study links GPX4 inhibition to reduced Tempol efficacy, the mechanistic evidence remains indirect. Direct assessment of lipid peroxidation (e.g., MDA, 4-HNE, or BODIPY-C11 staining) is recommended to confirm ferroptotic involvement. Measurement of ferroptosis-related markers such as ACSL4, SLC7A11, or FSP1 would strengthen the conclusions. The authors should also clarify whether the observed effects are due to ferroptosis, apoptosis, or a combination of both.

The choice of drug concentrations and treatment duration should be better justified. The rationale for selecting MTX (10⁻⁵ M), Tempol (100 μM), and ML210 (1 μM) should be supported by literature or dose–response pilot experiments. The absence of dose–response or time-course analyses limits the translational relevance of the findings.

The manuscript lacks essential functional endpoints: Cell viability (e.g., MTT, CCK-8, or LDH release) should be included. Bone-related functional markers such as ALP activity, osteogenic gene expression, or mineralization would increase the relevance to osteotoxicity.

In methodology, provide catalog numbers for all key reagents. Clarify whether ELISA or Western blot was used for protein detection. The homogenization protocol should specify buffer composition and protein normalization procedures.

The statistical methods are insufficiently described. The authors should specify the statistical tests used, number of replicates, and whether normality and variance assumptions were tested. Details regarding post hoc analysis and significance thresholds are required.

In the results, figures and tables should include sample size and statistical annotations. Some results are repetitive and should be summarized more concisely. The authors should avoid overstating conclusions based on correlative findings.

The discussion section should compare the findings with recent studies on antioxidant and ferroptosis-targeted therapies in bone toxicity. Address the dual antioxidant/pro-oxidant nature of Tempol in more depth. Explore potential clinical implications and limitations.

The English could be improved to more clearly express the research. Grammatical and typographical errors are present and should be corrected.

Standardization of abbreviations is recommended (e.g., TPL vs Tempol).

Ensure consistent formatting of units and symbols.

Author Response

This manuscript investigates the protective effects of Tempol against Methotrexate-induced osteotoxicity and explores the mechanistic role of GPX4 inhibition using ML210. The topic is timely and clinically relevant, as osteotoxicity remains an important complication of chemotherapy, and antioxidant-based strategies are gaining increasing interest. The study is well-organized and provides mechanistic insights linking oxidative stress, apoptosis, MAPK signaling, and ferroptosis-related pathways. However, several conceptual, methodological, and presentation issues should be addressed to improve the scientific rigor and clarity of the manuscript.

The manuscript addresses an interesting hypothesis regarding the dependency of Tempol’s protective effects on GPX4 activity. However, the novelty should be better emphasized. The authors should clearly state what distinguishes this work from previous studies on antioxidant protection against Methotrexate-induced bone toxicity. More discussion is needed regarding the emerging role of ferroptosis in bone pathology and how this study advances the current understanding.

Novelty was emphasized: It was stated that Tempol’s GPX4 dependency was functionally tested using ML210. The role of ferroptosis in bone pathology and the study’s contribution were briefly expanded in one paragraph; relevant references were added.

While the study links GPX4 inhibition to reduced Tempol efficacy, the mechanistic evidence remains indirect. Direct assessment of lipid peroxidation (e.g., MDA, 4-HNE, or BODIPY-C11 staining) is recommended to confirm ferroptotic involvement. Measurement of ferroptosis-related markers such as ACSL4, SLC7A11, or FSP1 would strengthen the conclusions. The authors should also clarify whether the observed effects are due to ferroptosis, apoptosis, or a combination of both.

Lipid peroxidation assays (MDA, 4‑HNE and/or BODIPY‑C11) and analyses of ACSL4, SLC7A11, and FSP1 were not performed and this limitation was stated in the manuscript. Additional experiments to distinguish apoptosis vs. ferroptosis (caspase and ferroptosis inhibitors) were recommended; if not performed, results were rephrased to indicate a possible overlap between apoptosis and GPX4‑dependent oxidative damage.

 

The choice of drug concentrations and treatment duration should be better justified. The rationale for selecting MTX (10⁻⁵ M), Tempol (100 μM), and ML210 (1 μM) should be supported by literature or dose–response pilot experiments. The absence of dose–response or time-course analyses limits the translational relevance of the findings.

Selections of MTX (10⁻⁵ M), Tempol (100 µM), and ML210 (1 µM) were supported by references to doses used in our previous studies and/or preliminary dose–response pilot analyses.
The choice of treatment duration was justified with literature references.
The absence of dose–response and time‑course analyses was acknowledged as a limitation in the Discussion and stated to be planned for future studies.

The manuscript lacks essential functional endpoints: Cell viability (e.g., MTT, CCK-8, or LDH release) should be included. Bone-related functional markers such as ALP activity, osteogenic gene expression, or mineralization would increase the relevance to osteotoxicity.

Cell viability assays (MTT/CCK‑8 and LDH release)bone‑related functional markers was noted as a limitation and inclusion was planned for follow‑up studies.

In methodology, provide catalog numbers for all key reagents. Clarify whether ELISA or Western blot was used for protein detection. The homogenization protocol should specify buffer composition and protein normalization procedures.

Catalog numbers and supplier information for all key reagents were provided.
Which protein assays were performed by ELISA and which by Western blot were specified.
The homogenization protocol was detailed: buffer composition (e.g., RIPA: 50 mM Tris‑HCl pH 7.4, 150 mM NaCl, 1% NP‑40, 0.5% sodium deoxycholate, 0.1% SDS) and use of protease/phosphatase inhibitors were stated.
Protein quantification and normalization methods were reported

The statistical methods are insufficiently described. The authors should specify the statistical tests used, number of replicates, and whether normality and variance assumptions were tested. Details regarding post hoc analysis and significance thresholds are required.

The statistical methods were updated.

In the results, figures and tables should include sample size and statistical annotations. Some results are repetitive and should be summarized more concisely. The authors should avoid overstating conclusions based on correlative findings.

The mentioned corrections were made.

 

The discussion section should compare the findings with recent studies on antioxidant and ferroptosis-targeted therapies in bone toxicity. Address the dual antioxidant/pro-oxidant nature of Tempol in more depth. Explore potential clinical implications and limitations.

Findings were compared with recent studies on antioxidant and ferroptosis‑targeted therapies in bone toxicity.
Tempol’s dual antioxidant and pro‑oxidant properties were discussed in greater detail.
Potential clinical implications and study limitations were addressed.

The English could be improved to more clearly express the research. Grammatical and typographical errors are present and should be corrected.

Standardization of abbreviations is recommended (e.g., TPL vs Tempol).

Ensure consistent formatting of units and symbols.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed the reviewer’s comments point by point and have adequately revised the manuscript. Therefore, I recommend the manuscript for acceptance.

Reviewer 2 Report

Comments and Suggestions for Authors

Accepted in the present form