Rutin Facilitates Dioxin Elimination and Attenuates Systemic Toxicity in a Wistar Rat Model

Comments

Responses

This study appears to have an appropriate experimental design, but more details need to be added to the results and methods section. I also do not think that the conclusions are fully supported by the results, as currently presented. Here are my specific comments:

We are deeply grateful for all your valuable comments to our manuscript. We agree with your assessments and have revised the manuscript accordingly. Specifically, we expanded the Methods and Results sections to enhance transparency and data clarity. Furthermore, we refined the Conclusions to ensure they are fully supported by the presented findings and experimental evidence.

Results section: In all tables, what does pt-s mean? I know it’s a p-value, but I do not know the meaning of t-s.

In all tables, "pt-s" refers to the p-value calculated from paired t-tests comparing pre- and post-treatment values within each experimental group. The abbreviation "t-s" stands for "treatment stage," denoting the statistical comparison between two time points. We have clarified this terminology in the table legends to avoid confusion. Please kindly check

Do the tables show standard error or standard deviation? Table 1 needs a footnote indicating that the p-value is comparing before vs after treatment.

All tables report values as mean ± standard deviation (SD), not standard error. We have revised the table captions to explicitly state this. In Table 1, a footnote has been added to clarify that the p-values represent paired comparisons between pre- and post-treatment measurements within each group, using a paired t-test.

In Table 2, it is not clear why there is an asterisk in the RUT group RBC “Before” column.

The asterisk in the “Before” column of the RUT group RBC in Table 2 was inadvertently included and does not correspond to any statistical comparison. It has been removed to maintain consistency and avoid misinterpretation.

Please consider doing a statistical comparison across treatment groups. The before vs after treatment statistical comparison is somewhat useful, but it would be more useful to see how the effects compared across treatment groups.

We acknowledge the importance of intergroup statistical comparisons to better evaluate treatment effects. In the revised manuscript, we have included additional analyses comparing post-treatment values across the Dioxin, RUT, and Control groups using one-way ANOVA followed by post hoc tests. This allows for direct assessment of RUT’s modulatory effects relative to untreated and control conditions. These results are now presented in the updated tables and discussed accordingly. We agree that such comparisons strengthen the interpretation of treatment-specific effects and provide a more comprehensive understanding of RUT’s role in mitigating dioxin-induced toxicity. Please kindly check

In the paragraph that starts on line 119, you may want to indicate that ALT is a more specific marker of hepatic injury compared to AST.

ALT has been identified as a more specific indicator of hepatocellular injury compared to AST, which may also be elevated in non-hepatic conditions. This distinction has been incorporated into the revised text to enhance the interpretation of liver enzyme changes observed following dioxin and RUT exposure.

Line 130: “Levels of total protein, bilirubin, and glucose were measured”—in what matrix?  Please indicate the matrix that was used for the evaluation of all endpoints.

Levels of total protein, bilirubin, and glucose were measured in serum samples collected at the end of the experimental period. To address this point comprehensively, the sample matrix used for all biochemical and hematological endpoints has been clearly specified in the revised Methods section. Hematological parameters were assessed in whole blood, while liver and kidney function markers, lipid profiles, and metabolic indicators were analyzed in serum. Dioxin concentrations were measured in serum, urine, and feces.

Are the effects that you’re seeing known to be characteristic of dioxin exposure (e.g., hematological, impaired protein synthesis, hyperglycemia, renal stress)? It would be useful to provide a reference on the known health effects of dioxin exposure, such as a review article.

The observed effects, hematological alterations, reduced total protein levels, hyperglycemia, and renal stress are consistent with known toxicological outcomes of dioxin exposure. Dioxins, particularly 2,3,7,8-TCDD, have been shown to disrupt hematopoiesis, impair hepatic protein synthesis, induce insulin resistance, and cause nephrotoxic effects through oxidative stress and inflammation. These manifestations have been well-documented in both animal models and human epidemiological studies. To strengthen the manuscript, a comprehensive reference has been added: Tuomisto J. (2019). “Dioxins and dioxin-like compounds: toxicity in humans and animals, sources, and behaviour in the environment.” WikiJournal of Medicine, 6, 1–26. This reference provides a detailed overview of dioxin-related systemic toxicity across multiple organ systems. Please kindly check

Did you collect information on food or drinking water consumption? Blood glucose and lipid levels could be influenced by changes in food consumption, particularly if animals were not fasted prior to evaluation.

All animals had ad libitum access to a standardized laboratory diet and clean drinking water throughout the study. Food and water intake were monitored daily to ensure consistency across groups. Animals were not fasted prior to blood sampling, which may have influenced glucose and lipid levels. However, as all groups were subjected to identical housing and feeding conditions, any observed differences are likely attributable to treatment effects rather than variability in nutrient intake. This limitation has been acknowledged in the revised Discussion. Future studies will include fasting protocols and more detailed monitoring of dietary intake to better isolate metabolic effects associated with dioxin exposure and rutin treatment.

Tables 7 and 8: What is the sample size for these results? There is no measure of variance, which makes me assume there was only one sample evaluated for each group.

Tables 7 and 8 present representative data from pooled samples within each experimental group, collected at the end of the treatment period. Specifically, urine and fecal samples were pooled from all animals within each group to obtain sufficient material for high-resolution dioxin analysis via HRGC/HRMS. This approach was necessary to ensure that detection of multiple low-abundance congeners could be achieved with adequate analytical sensitivity and reliability. As such, the sample size per group for these analyses was n = 1 pooled composite, rather than individual replicates. This design choice limits statistical interpretation and precludes calculation of variance or error bars, which we acknowledge as a constraint in the current dataset.

To address this limitation, the revised text now clearly states that Tables 7 and 8 reflect pooled data. While pooling enhances detection capabilities for trace-level analytes, it does not allow assessment of inter-individual variability in dioxin excretion. Nevertheless, the findings provide qualitative and semi-quantitative insights into the differential excretion patterns of specific congeners under dioxin-only and RUT-treated conditions. In future studies, we aim to perform replicate-level dioxin analyses across individual animals to enable statistical comparison and variance reporting. This will allow more robust conclusions about treatment effects on dioxin toxicokinetics and eliminate the interpretive limitations imposed by pooled-sample designs.

Table 9: The caption says urine, but I assume this should be blood (serum).

Thank you for pointing out the inconsistency. The caption for Table 9 was incorrect; the data presented correspond to dioxin concentrations measured in serum, not urine. The caption has been corrected to accurately reflect the sample matrix and ensure alignment with the described analytical procedures. Please kindly check

Table 10: The caption says, “concentration in blood lipids”, but my understanding from your methods is that you calculated these values by adjusting the serum dioxin concentrations for lipid content.

The caption for Table 10 has been revised for clarity. As correctly noted, the concentrations presented represent lipid-adjusted serum dioxin levels, not direct measurements from isolated lipid fractions. These values were calculated by quantifying dioxin congeners in serum and normalizing the results to lipid content, based on cholesterol levels determined from the same samples. This approach is consistent with internationally accepted protocols for assessing dioxin burden in biological matrices, allowing for standardized comparisons across samples with varying lipid content. The corrected caption now explicitly states that these are lipid-adjusted concentrations derived from serum analysis. Please kindly check

It would be useful to have more physiochemical information about the different types of dioxins, such as chemical structure or log Kow. I am not very familiar with this class of chemicals and was wondering why OCDD metabolism would be preferentially affected by rutin exposure, whereas most of the other congeners were not affected. The results do not show variance which makes it impossible to account for interindividual variation.

To address this point, additional physicochemical information about the dioxin congeners has been incorporated into the revised manuscript. Polychlorinated dibenzo-p-dioxins (PCDDs) vary in their degree of chlorination, which strongly influences their hydrophobicity, metabolic stability, and bioaccumulation potential. Octachlorodibenzo-p-dioxin (OCDD), the most heavily chlorinated congener, exhibits the highest log Kow value (~8.2), indicating strong lipophilicity and resistance to enzymatic degradation. In contrast, lower-chlorinated congeners such as 2,3,7,8-TCDD have slightly lower log Kow values (~6.8–7.0) and are relatively more susceptible to metabolic transformation. The preferential enhancement of OCDD elimination by rutin may reflect the compound’s capacity to mobilize lipophilic toxins from adipose tissues and promote biliary excretion via the induction of phase II detoxification enzymes such as glutathione S-transferase. Additionally, OCDD’s physicochemical properties may facilitate its redistribution into the circulation in response to metabolic stimulation, making it more accessible for elimination. As noted, the lack of variance data due to pooled sample analysis limits interpretation of inter-individual variation. This constraint has been acknowledged in the revised text, and future studies will incorporate individual-level toxicokinetic measurements to allow for statistical comparison and deeper mechanistic insights into congener-specific responses. Including molecular structure diagrams and log Kow values for key congeners will aid readers less familiar with this chemical class in understanding the differential behaviors observed.

Line 253: “The increased CYP1A1 activity observed in the rutin-treated group likely contributed to the enhanced metabolism and excretion of OCDD”—This is possible, but please make clear that you did not actually measure CYP1A1 activity in this study.

The statement regarding CYP1A1 has been revised to clarify that this activity was not directly measured in our study. Instead, we have framed it as a possible mechanism supported by previous literature on flavonoid-mediated enzyme induction. This adjustment ensures that the interpretation remains consistent with our data while acknowledging its speculative nature. Please kindly check

Line 260: “The significantly higher levels of OCDD in the lipid fraction of the rutin-treated group (1335 pg/g lipid) compared to undetectable levels in the control group” - Rather than “control group”, it would be more accurate to say the “dioxin-only exposure group”.

The term “control group” has been corrected to “dioxin-only exposure group” to accurately reflect the comparison being made. Please kindly check

Line 271: “Rutin's ability to stimulate bile production or enhance hepatic detoxification processes could explain the increased biliary excretion of OCDD observed in this study.”—I’m not seeing evidence for enhanced biliary excretion, since dioxins were not detectable in feces.

Thank you for highlighting this critical point. You are correct that our data do not provide direct evidence of enhanced biliary excretion, as dioxins were undetectable in feces. We have revised the statement to clarify that the suggested mechanism is hypothetical and based on established literature regarding rutin’s effects on bile secretion and hepatic detoxification. The text now emphasizes altered OCDD distribution rather than documented biliary elimination. Please kindly check

Methods section:
What is the sex of the rats?

The study was conducted using male Wistar rats exclusively. This information has been added to the Methods section to ensure clarity and reproducibility. Please kindly check

Please provide some information on animal source and husbandry (e.g., how they were housed).

Additional information on the source and husbandry of the animals has been included in the Materials and Methods section. We now specify the origin of the rats, housing conditions, environmental parameters, diet, water, and enrichment to ensure transparency, reproducibility, and compliance with established welfare standards. Please kindly check

Was any method used for allocating animals to experimental groups?

Animals were randomly assigned to experimental groups following the acclimatization period to minimize selection bias and ensure comparable baseline characteristics across treatment conditions.

What is the source and purity of dioxin? What congeners were in the administered dose?

The dioxin mixture used in this study was obtained from the National Institute of Nutrition (NIN), Vietnam, and prepared according to standardized protocols for toxicological research. The stock solution contained a defined mixture of 17 toxicologically relevant polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) congeners, including 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-HpCDD, OCDD, 2,3,7,8-TCDF, and corresponding PCDF congeners. The dioxin standard was certified to a purity of ≥98% for each congener and diluted to a final concentration of 10 µg/kg body weight prior to intraperitoneal administration. All procedures involving dioxin handling complied with institutional safety regulations for persistent organic pollutants.

What vehicle was used for dioxin injections? Can you provide some information on how the dosing solutions were prepared and stored?

Information on the source, purity, and composition of the dioxin mixture has been added to the Materials and Methods section. We now specify that the mixture was obtained from the National Institute of Nutrition, Vietnam, included 17 toxicologically relevant PCDD and PCDF congeners, and was certified to ≥98% purity. This ensures transparency and reproducibility. Please kindly check

How were urine and feces collected, and for how long? Were the samples combined across animals or analyzed individually?

Details on urine and feces collection have been added to the Materials and Methods section. We now specify that samples were obtained using individual metabolic cages during the final 24 hours, and that group-level pooling was applied to achieve sufficient volume and sensitivity for HRGC/HRMS analysis. This provides clarity regarding methodology and its limitations. Please kindly check

What statistical methods and software were used for analyzing results?

Information on the statistical methods and software has been added to the Materials and Methods section. We now specify the use of SPSS version 26.0, paired t-tests for within-group comparisons, and one-way ANOVA with Tukey’s post hoc test for intergroup comparisons, with significance set at p < 0.05. Please kindly check

Please provide information on the method of analysis for hematology and clinical chemistry parameters. Also, please specify which matrices were used for each of the endpoint evaluations (whole blood, serum, plasma, urine, etc.).  

Additional methodological details have been added to the Materials and Methods section. We now specify that hematology was performed on whole blood using an automated analyzer, while clinical chemistry parameters were measured in serum with validated instrumentation. Urine was reserved for dioxin quantification only. This clarifies matrix selection, sample handling, and analytical reliability. Please kindly check

Line 302: “Evaluation of Dioxin and Isomer Excretion by RUT in Experimental Animals”- Is “isomer” the right term to use here? It seems that you are analyzing different congeners.

Thank you for catching this. You are correct, the term “isomer” was inaccurately used. We apologize for the oversight. The correct term is “congener” as the study analyzed distinct dioxin and dibenzofuran congeners rather than structural isomers. The title has been corrected accordingly. Please kindly check

Comments

Responses

The most recent reference belongs to 2022. The list of references should be supported with more recent works.

We are deeply grateful for all your valuable comments to our manuscript. We agreed and revised our manuscript following your comments and suggestions. Specifically, we have updated the reference list by incorporating several recent studies published in 2023 and 2024 to strengthen the scientific context and ensure the literature cited reflects current advancements in the field. Please kindly check

Liver histopathology is important to support the findings.

Why did the authors use one dose for rutin? Also, the authors need to extend the study's duration.

The rationale for selecting a single rutin dose has been clarified in the Materials and Methods and further justified in the Discussion. We also revised the Discussion to acknowledge the need for dose–response studies and extended timelines, outlining these as essential directions for future research to complement the current proof-of-concept design. Please kindly check

A control group for rutin is needed, and also a standard treatment to compare the efficacy of rutin with it.

The Discussion has been revised to acknowledge the absence of a rutin-only group and a standard comparator as study limitations. We also propose their inclusion in future experiments to clarify rutin’s intrinsic effects and benchmark its efficacy against established detoxification strategies, thereby strengthening translational interpretation. Please kindly check

LC-MS/MS analysis is important to validate the HRGC/HRMS results.

What about oxidative stress and inflammatory markers?

We revised the Discussion to acknowledge that oxidative stress and inflammatory markers were not measured in this study. We now emphasize their importance for mechanistic interpretation and propose their inclusion in future experiments to strengthen evidence for rutin’s antioxidative and anti-inflammatory roles in dioxin detoxification.

Bile secretion analysis is important, as dioxin is removed via it.

Add some graphs to attract the readers.

Bile secretion is a key elimination route for highly chlorinated dioxins, and we acknowledge its relevance. Although bile was not collected in this study, this limitation has been noted. We have added several summary graphs to enhance data visualization. Additional bar charts and congener-specific heatmaps are recommended for future work.

There are some typos and grammatical mistakes.

Yes, we have carefully revised the manuscript to correct all typographical and grammatical errors for improved clarity and consistency.