Dose–Effect Relationship of the Immunotoxicity, Neurotoxicity, Gastrointestinal Toxicity, and Hepatotoxicity of the Maillard Reaction Product 2-Acetylfuran
, Qingxue Chen 4, Shubo Luo 2, Dongying Cui 2, Sinan Mu 2, Jufang Li 2, Qinggang Xie 2 and Yajun Xu 1,3,5,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have comprehensively examined the toxicity of 2-acetylfuran, which can form in food during heating, using zebrafish embryos and larvae. Given the current need to avoid mammalian experiments whenever possible, this is a paper worthy of consideration. However, there are numerous unclear points regarding the experimental methods and results, necessitating substantial revisions.
General comments：
1) Is this research ultimately focused on fish, or are mammals such as humans also being considered? If the latter, then Reviewer believes the specific concentration-dependent numerical values for fish—particularly during early developmental stages—are not very significant; what matters is the presence of concentration dependence itself.
2) The authors should include an introduction or discussion on basic toxicity data, such as acute toxicity and genotoxicity in mammals. If this information is unknown or varies between testing facilities, summarizing it accordingly is acceptable. Additionally, any available information on non-mammals, including fish, would be valuable. Is there any fragmentary knowledge regarding developmental toxicity? These may be included in the references, but they should also be addressed in the main text.
3) The zebrafish larvae only begin to develop their liver around 72 hpf, and it takes additional time to reach adequate metabolic enzyme activity. This is likely quite different from adult mammals, both in terms of species differences and developmental stages. What are the authors’ thoughts on this point?
4) In the introduction or discussion section, please present the known sources of 2-acetylfuran contamination and exposure conditions. If these are unknown, please note it like this in the text.
5) Overall, only fluorescence microscope images are presented, making it unclear which specific area is being shown. To enable identification, brightfield images should be added. If brightfield images were not taken, at the very least, the area depicted in the figure must be clearly indicated. Most images are simply too dark. They're extremely hard to see, so adjustments are necessary. In some cases, this might make body contours visible.
Specific comments：
1) A chapter from L101 (2.2. Determination of MNLC and LC₁₀ for 2-Acetylfuran): A 1% DMSO solution was used as the solvent control, but this concentration is too high. Lower concentrations of DMSO have been reported to affect expressions of many genes in developing zebrafish. Does the solution containing 2-Acetylfuran also contain 1% DMSO? Also, under what conditions was the compound exposure performed? Was it in a 96-well plastic plate for cell culture? The original manuscript does not rule out the possibility of a flow-through system.
2) A chapter from L111 (2.3. Immunotoxicity Assessment): The source of the TG fish is unclear. Reviewer believes this is the lineage used in many papers. However, since Readers cannot confirm the extent to which EGFP expression corresponds with immune cells. Please add source of the TG fish or provide references describing its properties.
3) L112: The authors should add the method for obtaining 2-acetylfuran.
4) A chapter from L124 (2.4. Behavioral Analysis): The authors appear to have used equipment from a specific manufacturer for the behavioral experiments; please add a note regarding this point.
5) L181: Is the 4% tissue fixative referring to 4% paraformaldehyde? Please specify which fixative was used.
6) L223: Does MNLC stand for Maximum Non-Lethal Concentration? Please add the full name on first mention. First of all, what method did the authors use to draw the best-fit line and calculate each value of MNLC? This should be added to the methods section.
7) "3.4. Neurotoxicity of 2-Acetylfuran" and "Figure 4": The two sentences from line 256 to line 259 are not needed because this is too long as an opening discussion, and very common. The second sentence lacks a period. The images in sections A and B of Figure 4 are too small and unclear. The reviewer cannot follow the authors' descriptions in the text. Does A indicate cell death in the brain, and B in the spinal cord? Does “neuronal” in the upper section of C refer to the brain? In any case, it is difficult to agree that the graph shown in C accurately represents images.
8) Figure 5: Reviewers cannot clearly identify the intestinal tract from the diagram. A clearer diagram should be used, and guide lines should be added. Moreover, is it even possible to reliably identify the wild-type intestinal tract under a standard microscope?
9) A sentence from L299 to L301: This sentence should be moved to the discussion section.
10) Figure 5: No bright-field whole-body images of zebrafish larvae are provided, but is delayed absorption of the yolk sac extension distinct from overall growth inhibition? In the high‑concentration treatment group, the swim bladders did not inflate. This was considered evidence of growth retardation. In fact, the authors themselves mention that 2-acetylfuran causes developmental delays (L397-398).
11) L309: What do the authors mean by "fluorescent brightness of the liver"? It doesn't look different at all.
12) Figure 6, (C): The H&E microscopic images are out of focus.
Author Response
Responds to reviewer 1
The authors have comprehensively examined the toxicity of 2-acetylfuran, which can form in food during heating, using zebrafish embryos and larvae. Given the current need to avoid mammalian experiments whenever possible, this is a paper worthy of consideration. However, there are numerous unclear points regarding the experimental methods and results, necessitating substantial revisions.
General comments：
1) Is this research ultimately focused on fish, or are mammals such as humans also being considered? If the latter, then Reviewer believes the specific concentration-dependent numerical values for fish—particularly during early developmental stages—are not very significant; what matters is the presence of concentration dependence itself.
Reply: Thank you for your constructive suggestion. The initial aim of this study was to comprehensively clarify the effects of 2-Acetylfuran on the major organs of the host. Naturally, our greatest hope is that this finding will have guiding significance for fish, mammals, and even humans. However, it is inevitable that concentrations across different species are not entirely equivalent. Therefore, the concentration results in this study may most directly reflect the toxicity of 2-Acetylfuran in fish. It may have some guiding significance for mammals.
At the outset of our experimental design, a literature search revealed no reports of specific toxic concentrations of 2-Acetylfuran. Therefore, we selected a concentration range (0.062, 0.125, 0.250, 0.500, and 1.00 µL/mL), a relatively large range that can well reflect the lethality of 2-Acetylfuran in zebrafish. Subsequently, based on concentration and lethality, we screened several concentrations that were significant in previous studies (including 1/9 MNLC, 1/3 MNLC, MNLC, and LC₁₀) to specifically assess the effects of 2-Acetylfuran in different organs. A core objective of this study is to establish an understanding of the toxicity of different concentrations of 2-Acetylfuran. This also provides valuable insights for future research on Maillard reaction products (especially 2-Acetylfuran).
2) The authors should include an introduction or discussion on basic toxicity data, such as acute toxicity and genotoxicity in mammals. If this information is unknown or varies between testing facilities, summarizing it accordingly is acceptable. Additionally, any available information on non-mammals, including fish, would be valuable. Is there any fragmentary knowledge regarding developmental toxicity? These may be included in the references, but they should also be addressed in the main text.
Reply: Thank you for your constructive suggestion. We have supplemented previous reports on the safety (or toxicity) assessment of 2-Acetylfuran in mammalian and fish studies, and supplemented the relevant references. The supplementary content is as follows:
“A 90 days evaluation in rats confirmed that 22.6 mg/kg body weight daily for 2-acetylfuran is a safe dose [9]. Furthermore, the EFSA Animal Feed Additives and Products or Substances Group has reported safe doses of 2-acetylfuran in several spe-cies, including cats (2 mg/kg body weight), dogs (11.9 mg/kg body weight), and orna-mental fish (44.2 mg/kg body weight) [10]. Based on these findings, the group states that the use of 2-acetylfuran in animal feed at doses ≤0.5 mg/kg is safe in all animal diets. These findings provide a safe range for 2-acetylfuran exposure, but toxicity from high-concentration exposures has not yet been reported. In particular, there is a lack of dose-response data on the effects of 2-acetylfuran on specific organs, such as the im-mune system, nervous system, gastrointestinal tract, and liver.” (Lines 68-77 of the revised manuscript)
3) The zebrafish larvae only begin to develop their liver around 72 hpf, and it takes additional time to reach adequate metabolic enzyme activity. This is likely quite different from adult mammals, both in terms of species differences and developmental stages. What are the authors’ thoughts on this point?
Reply: Thank you for your constructive suggestion. We strongly agree with your point. This is a point we previously overlooked. This study chose zebrafish as the evaluation model for several reasons. First, zebrafish have excellent visibility, allowing for direct observation of the morphology of specific cells or organs in vivo. This facilitates the most direct and accurate toxicity results. Third, avoiding the use of mammals to assess substance toxicity is beneficial to animal welfare. However, we overlooked the differences in liver development between zebrafish and mammals. This suggests that we need to consider this aspect when assessing the toxicity of 2-acetylfuran in other species in the future. Thank you again for your valuable feedback.
Furthermore, although the liver development of zebrafish differs significantly from that of mammals, the liver is still a major detoxification organ in zebrafish. Therefore, the results can also partially reflect the toxicity of 2-acetylfuran in other species. In addition, the results of this study can directly reflect the hepatotoxicity risk of 2-acetylfuran in fish.
4) In the introduction or discussion section, please present the known sources of 2-acetylfuran contamination and exposure conditions. If these are unknown, please note it like this in the text.
Reply: Thank you for your constructive suggestion. We have supplemented the discussion section with relevant information on 2-acetylfuran sources and exposure conditions, and added corresponding references. The supplementary information is as follows:
“2-acetylfuran is produced during the Maillard reaction, a process involving the dehydration, rearrangement, and cleavage of reducing sugars and amino acids. This process primarily occurs during the processing of protein- and carbohydrate-rich foods or feeds [5, 16, 17]. Furthermore, 2-acetylfuran is a commonly used flavoring additive in feeds, thereby exposing mammals or fish to its diet[10]. In recent years, the toxicity risks of Maillard reaction products such as 2-acetylfuran have received increasing attention.” (Lines 356-361 of the revised manuscript)
5) Overall, only fluorescence microscope images are presented, making it unclear which specific area is being shown. To enable identification, brightfield images should be added. If brightfield images were not taken, at the very least, the area depicted in the figure must be clearly indicated. Most images are simply too dark. They're extremely hard to see, so adjustments are necessary. In some cases, this might make body contours visible.
Reply: Thank you for your constructive suggestion. We have provided example images of the complete zebrafish for all fluorescence images and other images, in which we have marked the analyzed areas with lines of a specific color. We have adjusted some of the darker images and rearranged all images, enlarging the size of individual sub-images. The modified figures include Figure 2 (A-C), 4 (A and B), 5 (A and B), and 6 (A and C).
Specific comments：
1) A chapter from L101 (2.2. Determination of MNLC and LC₁₀ for 2-Acetylfuran): A 1% DMSO solution was used as the solvent control, but this concentration is too high. Lower concentrations of DMSO have been reported to affect expressions of many genes in developing zebrafish. Does the solution containing 2-Acetylfuran also contain 1% DMSO? Also, under what conditions was the compound exposure performed? Was it in a 96-well plastic plate for cell culture? The original manuscript does not rule out the possibility of a flow-through system.
Reply: Thank you for your constructive suggestion. 2-Acetylfuran was also diluted with 1% DMSO; therefore, the solution containing 2-Acetylfuran also contains 1% DMSO. 1% DMSO is an appropriate dose, which has been commonly used in previous studies. In addition, we set up a solvent control group with 1% DMSO and a blank control group (NC) without treatment. In acute toxicity and other studies, 1% DMSO did not show toxicity compared to the NC group. We have added solvent-related information to the manuscript.
2-Acetylfuran was used to expose zebrafish to the aquatic environment in which they lived. Specifically, zebrafish were housed in 6-well plates, with 30 zebrafish per well. 3 mL of an aqueous solution containing or without 2-acetylfuran (dissolved in 1% DMSO) was added to each well.
The revised section 2.2 is as follows:
“The acute lethality of 2-acetylfuran (Purity is 99%, Merck KGaA, Darmstadt, Germany) was first assessed in 2-day-post-fertilization (dpf) wild-type AB zebrafish larvae. Larvae were exposed for 3 days to a series of nominal 2-acetylfuran concentra-tions (0.062, 0.125, 0.250, 0.500, and 1.00 µL/mL). A normal control (NC) and a vehicle control containing 1% DMSO were included in parallel. Simultaneously, all concentra-tions of 2-acetylfuran were dissolved in 1% DMSO. Zebrafish were reared in 6-well plates with 30 zebrafish per well. 3 mL of aqueous solution containing or without 2-Acetylfuran (dissolved in 1% DMSO) was added to each well. Mortality in each group was recorded daily, and dead larvae were promptly removed. Origin 8.0 software was used to fit the concentration-mortality ratio of 2-acetylfuran, and a nonlinear fitting was employed to generate the optimal concentration-mortality effect curve for 2-acetylfuran. The MNLC and LC₁₀ of 2-acetylfuran were recorded using the 1% and 10% mortality rates from the curve, respectively. These values were then used to define a subtoxic exposure concentration set (1/9 MNLC, 1/3 MNLC, MNLC, and LC₁₀) for subsequent toxicity assessments.” (Lines 115-118 of the revised manuscript)
2) A chapter from L111 (2.3. Immunotoxicity Assessment): The source of the TG fish is unclear. Reviewer believes this is the lineage used in many papers. However, since Readers cannot confirm the extent to which EGFP expression corresponds with immune cells. Please add source of the TG fish or provide references describing its properties.
Reply: Thank you for your constructive suggestion. The transgenic zebrafish used in this study were provided by Hunter Biotechnology Inc. (Hangzhou, China). Imaging of the three strains corresponds to three different immune cell types. We have incorporated this information into the manuscript. The revised manuscript's statement regarding the zebrafish type and origin is as follows:
“Immunotoxic effects of 2-acetylfuran were examined using three transgenic lines that label distinct immune cell populations: Tg (mpx: EGFP) for neutrophils, Tg (mpeg1: EGFP) for macrophages, and Tg (rag2: DsRed) for T cells in the thymus. The three transgenic zebrafish strains were provided by Hunter Biotechnology Inc. (Hangzhou, China).” (Lines 128-129 of the revised manuscript)
3) L112: The authors should add the method for obtaining 2-acetylfuran.
Reply: Thank you for your constructive suggestion. 2-acetylfuran was purchased from Merck KGaA (Darmstadt, Germany) with a purity of 99%. We have added this source information to the manuscript. (Lines 111-112 of the revised manuscript)
4) A chapter from L124 (2.4. Behavioral Analysis): The authors appear to have used equipment from a specific manufacturer for the behavioral experiments; please add a note regarding this point.
Reply: Thank you for your constructive suggestion. We used a behavior analysis system (V3.11, ViewPoint Life Sciences, France) to collect data on zebrafish locomotor abilities. We have added information about this instrument to the manuscript. (Lines 144-145 of the revised manuscript)
5) L181: Is the 4% tissue fixative referring to 4% paraformaldehyde? Please specify which fixative was used.
Reply: Thank you for your constructive suggestion. 4% tissue fixative is 4% paraformaldehyde. We corrected this error in the manuscript. The corrected sentence is:
“Zebrafish were fixed in 4% paraformaldehyde and processed using a standard histo-logical workflow, including graded dehydration, paraffin embedding, sectioning, and H&E staining.” (Lines 195-196 of the revised manuscript)
6) L223: Does MNLC stand for Maximum Non-Lethal Concentration? Please add the full name on first mention. First of all, what method did the authors use to draw the best-fit line and calculate each value of MNLC? This should be added to the methods section.
Reply: Thank you for your constructive suggestion. We have added the full name of MNLC to the manuscript. Furthermore, we have added the fitting method for the concentration-mortality curve of 2-acetylfuran and the method for calculating MNLC and LC10 values. The additions are as follows:
“Origin 8.0 software was used to fit the concentration-mortality ratio of 2-acetylfuran, and a nonlinear fitting was employed to generate the optimal concentration-mortality effect curve for 2-acetylfuran. The MNLC and LC₁₀ of 2-acetylfuran were recorded us-ing the 1% and 10% mortality rates from the curve, respectively. These values were then used to define a subtoxic exposure concentration set (1/9 MNLC, 1/3 MNLC, MNLC, and LC₁₀) for subsequent toxicity assessments.”c
7) "3.4. Neurotoxicity of 2-Acetylfuran" and "Figure 4": The two sentences from line 256 to line 259 are not needed because this is too long as an opening discussion, and very common. The second sentence lacks a period. The images in sections A and B of Figure 4 are too small and unclear. The reviewer cannot follow the authors' descriptions in the text. Does A indicate cell death in the brain, and B in the spinal cord? Does “neuronal” in the upper section of C refer to the brain? In any case, it is difficult to agree that the graph shown in C accurately represents images.
Reply: Thank you for your constructive suggestion. We agree with your point that excessive description or discussion in the results section is unreasonable. Therefore, we have deleted lines 256-259. We have also adjusted the size of Figures 4A and 4B for easier reading. Figure 4A refers to apoptotic cells of the central nervous system; Figure 4B refers to the length of peripheral motor neurons. The bar chart in Figure 4C is the quantitative fluorescence result of the images in Figures A and B. We have adjusted the layout of Figure 4 to combine these two bar charts with Figures 4A or 4B respectively. Furthermore, in Figure 4C, "neuronal" refers to the central nervous system. "Neuronal" might be an inaccurate expression, and we have corrected "neuronal" in Figure 4C to "central nervous system."
8) Figure 5: Reviewers cannot clearly identify the intestinal tract from the diagram. A clearer diagram should be used, and guide lines should be added. Moreover, is it even possible to reliably identify the wild-type intestinal tract under a standard microscope?
Reply: Thank you for your constructive suggestion. We adjusted the layout of Figure 5 to make the visual and fluorescent images of the gastrointestinal tract clearer. In Figure 5A, we added an example image of the complete gastrointestinal tract of a zebrafish, and marked the gastrointestinal tract region with a red curve. Furthermore, the zebrafish gastrointestinal tract was photographed using a dissecting microscope (SZX7, OLYMPUS, Japan), which is capable of identifying the zebrafish's gastrointestinal tract.
9) A sentence from L299 to L301: This sentence should be moved to the discussion section.
Reply: Thank you for your constructive suggestion. We have already moved this sentence to the discussion section.
10) Figure 5: No bright-field whole-body images of zebrafish larvae are provided, but is delayed absorption of the yolk sac extension distinct from overall growth inhibition? In the high‑concentration treatment group, the swim bladders did not inflate. This was considered evidence of growth retardation. In fact, the authors themselves mention that 2-acetylfuran causes developmental delays (L397-398).
Reply: Thank you for your constructive suggestion. Figure 6A shows the location and changes of both the liver and yolk sac. The yolk sac is marked with a blue curve. The yolk sac provides energy and nutrients during early zebrafish development. An increased area of delayed absorption in the yolk sac can partially reflect developmental delay in zebrafish. Furthermore, the area of delayed absorption in the yolk sac can also reflect delayed liver development and lipid metabolism disorders.
11) L309: What do the authors mean by "fluorescent brightness of the liver"? It doesn't look different at all.
Reply: Thank you for your constructive suggestion. "liver fluorescent brightness" is a misspelling; the word "fluorescent" was mistakenly entered. It refers to liver brightness.
12) Figure 6, (C): The H&E microscopic images are out of focus.
Reply: Thank you for your constructive suggestion. We replaced the H&E image in Figure 6C with a clearer image and enlarged it for easier reading.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study reports that 2-acetylfuran exposure impairs zebrafish immunity, behavior, neural development, gastrointestinal growth, and liver function. The effects were dose-dependent and became pronounced near sublethal concentrations. Notably, immune cell populations and hepatotoxic endpoints were affected even at lower doses, highlighting potential health risks associated with this Maillard reaction product. While the central proposition is excellent and innovative, and the experimental design is relevant, the manuscript requires minor clarifications and targeted revisions on several aspects, as detailed below.
1) The abstract is informative and clear. However, I suggest briefly contextualizing its relevance to food processing and dietary exposure at the beginning to enhance its impact for the journal’s audience.
2) The manuscript should specify the supplier, purity, and preparation of 2-acetylfuran, as these details are essential for reproducibility in toxicology studies.
3) The concentrations of 2-acetylfuran are expressed as µL/mL (v/v), but the rationale for using volumetric units is not explained. Please clarify stock preparation, solubility, the solvent used, and why molarity or mass-based units were not adopted.
4) Figures 2, 4, 5, and 6 are well-designed, rich in detail, and effectively illustrate the experimental findings. However, in the current layout they appear relatively small, which limits visualization of zebrafish morphological features and histological sections. I recommend increasing their display size to improve clarity and reader interpretation.
5) The statistical analysis states that multiple two-group comparisons were performed using unpaired t-tests. Given that the experimental design includes multiple dose levels, a one-way ANOVA followed by an appropriate post-hoc test (e.g., Dunnett or Tukey) would be more suitable to control Type I error and improve inference reliability.
6) The manuscript explains the meaning of statistical symbols (*, **, ***) in the Methods, but this information should also appear in the figure legends for clarity. Additionally, the figures do not specify which reference group was used for statistical comparison (e.g., NC or vehicle). Explicitly stating the comparison group in each legend would improve interpretability.
7) It would be helpful to contextualize the tested concentrations with reported levels of 2-acetylfuran in heat-processed foods, to assess whether the observed toxicity occurs within realistic human exposure ranges.
8) Although the phenotypic and histological endpoints are robust, the absence of biochemical or oxidative stress markers limits mechanistic interpretation. Even basic assays (e.g., ROS, MDA, antioxidant enzymes, caspase activity) would strengthen the causal framework and facilitate comparison with other furan derivatives.
Author Response
Responds to reviewer 2
This study reports that 2-acetylfuran exposure impairs zebrafish immunity, behavior, neural development, gastrointestinal growth, and liver function. The effects were dose-dependent and became pronounced near sublethal concentrations. Notably, immune cell populations and hepatotoxic endpoints were affected even at lower doses, highlighting potential health risks associated with this Maillard reaction product. While the central proposition is excellent and innovative, and the experimental design is relevant, the manuscript requires minor clarifications and targeted revisions on several aspects, as detailed below.
1) The abstract is informative and clear. However, I suggest briefly contextualizing its relevance to food processing and dietary exposure at the beginning to enhance its impact for the journal’s audience.
Reply: Thank you for your constructive suggestion. We have supplemented the abstract with information on the relevance of 2-Acetylfuran to food processing and dietary intake. The supplementary information is as follows:
“2-Acetylfuran is a product of the Maillard reaction and is widely found, especially in heat-processed foods such as grain products, baked goods, and dairy products. Alt-hough 2-Acetylfuran contributes to flavor, high concentrations may be toxic.” (Lines 17-19 of the revised manuscript)
2) The manuscript should specify the supplier, purity, and preparation of 2-acetylfuran, as these details are essential for reproducibility in toxicology studies.
Reply: Thank you for your constructive suggestion. 2-acetylfuran was purchased from Merck KGaA (Darmstadt, Germany) with a purity of 99%. We have added this source information to the manuscript. (Lines 111-112 of the revised manuscript)
3) The concentrations of 2-acetylfuran are expressed as µL/mL (v/v), but the rationale for using volumetric units is not explained. Please clarify stock preparation, solubility, the solvent used, and why molarity or mass-based units were not adopted.
Reply: Thank you for your constructive suggestion. The 2-acetylfuran we used is a colorless, transparent liquid. Therefore, the stock solution was prepared according to volume fraction. Its purity is 99%, so it was prepared as a pure substance. The solvent was 1% DMSO.
4) Figures 2, 4, 5, and 6 are well-designed, rich in detail, and effectively illustrate the experimental findings. However, in the current layout they appear relatively small, which limits visualization of zebrafish morphological features and histological sections. I recommend increasing their display size to improve clarity and reader interpretation.
Reply: Thank you for your constructive suggestion. We have reformatted the fluorescence images and other subfigures in Figures 2, 4, 5, and 6, resulting in significantly larger magnifications. All figures are now easier to read and understand.
5) The statistical analysis states that multiple two-group comparisons were performed using unpaired t-tests. Given that the experimental design includes multiple dose levels, a one-way ANOVA followed by an appropriate post-hoc test (e.g., Dunnett or Tukey) would be more suitable to control Type I error and improve inference reliability.
Reply: Thank you for your constructive suggestion. We have reanalyzed all the data in the manuscript. The current analysis uses one-way ANOVA to compare differences between groups, followed by Tukey's post-hoc tests. We have also revised the representation of differences in all figures. Furthermore, we have revised the description of statistical analysis in the Methods section.
The revised text is as follows:
“2.11. Statistical Analysis
All quantitative data are presented as mean ± standard deviation (SD), with n = 3–10 per group depending on the specific assay. One-way analysis of variance (ANOVA) was used to compare differences among groups, followed by Tukey’s post hoc test for pairwise comparisons. Data processing was performed using SPSS version 26.0 (IBM SPSS Inc., New York, NY, USA). p values < 0.05 were considered statistically significant, and differences are indicated by different letters. Furthermore, all fluorescence-related images acquired in this study were not edited or manipulated outside of standard processing procedures. Because the images were acquired while the zebrafish were alive, all images were region-specific, and complete images were not collected.” (Lines 202-209 of the revised manuscript)
6) The manuscript explains the meaning of statistical symbols (*, **, ***) in the Methods, but this information should also appear in the figure legends for clarity. Additionally, the figures do not specify which reference group was used for statistical comparison (e.g., NC or vehicle). Explicitly stating the comparison group in each legend would improve interpretability.
Reply: Thank you for your constructive suggestion. Because the statistical analysis has been modified to one-way ANOVA, differences are represented using different letters. We have added annotations to all figures to explain the interpretation of differences.
7) It would be helpful to contextualize the tested concentrations with reported levels of 2-acetylfuran in heat-processed foods, to assess whether the observed toxicity occurs within realistic human exposure ranges.
Reply: Thank you for your constructive suggestion. We reviewed previous literature on 2-acetylfuran concentrations in foods and performed simple conversions between this information and the concentrations used in this study. Generally, 2-acetylfuran levels in properly processed and stored foods are lower than the concentrations at which toxicity was observed in this study. However, improperly processed products (such as oxidized fish oil) showed significantly elevated 2-acetylfuran concentrations, reaching levels considered toxic. We have added this information to the discussion section of the manuscript. The additions are as follows:
“One study reported 2-acetylfuran levels in 24 fruit- and meat-based canned infant food samples, all <6 ng/g. This concentration is less than 1/9 MNLC in this study, reflecting that conventionally processed and normally stored products are generally not toxic with 2-acetylfuran. Another study found 2-acetylfuran concentrations as high as 359.49 ng/g in oxidized fish oil. This concentration is higher than 1/3 MNLC in this study. This suggests that the product may pose a risk of 2-acetylfuran toxicity under conditions of overprocessing or improper storage. In conclusion, these results indicate that the con-centration tested in this study is of practical significance.” (Lines 369-376 of the revised manuscript)
8) Although the phenotypic and histological endpoints are robust, the absence of biochemical or oxidative stress markers limits mechanistic interpretation. Even basic assays (e.g., ROS, MDA, antioxidant enzymes, caspase activity) would strengthen the causal framework and facilitate comparison with other furan derivatives.
Reply: Thank you for your constructive suggestion. We strongly agree with your point. However, in our initial experimental design, we neglected the effects of 2-acetylfuran on oxidative stress. This is because our primary focus was on macroscopic toxicity from the organ or immune perspective. Seeing your comments, we also feel that adding biochemical or oxidative stress biomarker analysis, and even related signaling pathway changes, would be very valuable. This would greatly enhance the mechanistic aspects of the manuscript. Unfortunately, the samples from previous experiments have been exhausted, preventing us from directly detecting these indicators. Furthermore, zebrafish rearing was outsourced to a third-party company, and conducting experiments again may take a very long time. Your comments have been very helpful, reminding us to consider these issues in advance in future research.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsNo comments.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe revised manuscript provides a clear investigation into the toxicological effects of 2-acetylfuran in zebrafish. In summary, exposure disrupts immunity, behavior, neural development, gastrointestinal growth, and liver function in a dose-dependent manner, with pronounced impairments near sublethal concentrations and notable immune and hepatic alterations at low doses. The authors have satisfactorily addressed all previous concerns, and in my opinion the manuscript is now suitable for publication in its current form.
