Natural Herbicide Shows Cytotoxicity, Neurotoxicity, and Antioxidant System Alterations on SH-SY5Y and HaCaT Cell Lines
, Juliana Ferreira da Silva 1,2, Shayane da Silva Milhorini 1,2, Larissa Lechinovski 3,4, Ana Carolina de Deus Bueno Krawczyk 3 and Izonete Cristina Guiloski 1,2,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsHere are my comments:
The study does not provide any information on how the authors determined the exposure concentrations used in the experiments. Besides the experimental time points and units need justification.
53-65 discusses about the phytotoxicity of the natural herbicides, which I feel not required.
“It was observed that, although NH has a lower toxicity when compared to the conventional ones, glyphosate and atrazine, it still showed mutagenic potential [16].” - Revise for clarity.
The source and exact composition of the NH is not known, how this study will guide future research in terms of methodology?
Also checking the cytotoxicity and alteration in antioxidant enzyme levels provides preliminary information on mechanisms of toxicity but that cannot conclude neurotoxicity.
Author Response
The study does not provide any information on how the authors determined the exposure concentrations used in the experiments. Besides the experimental time points and units need justification.
R: Thank you for your comment. Cells were exposed to Natural Herbicide (NH) in three concentrations (NH1: 0.6; NH2: 1.56; and NH3: 3.12 µL/mL), for 24 hours to assess an acute exposure process and 72 hours for more prolonged exposure. The highest concentration (3.12 µL/mL) is equivalent to that used by farmers and the other concentrations were determined with the aim of observing the occurrence of changes even at lower herbicide concentrations (lines 109-113).
53-65 discusses about the phytotoxicity of the natural herbicides, which I feel not required.
R: We appreciate your comment, but this excerpt from the article was included to demonstrate how each component of the natural herbicide affects plants. The idea is to demonstrate the importance and mechanisms of action of these components according to the literature.
“It was observed that, although NH has a lower toxicity when compared to the conventional ones, glyphvosate and atrazine, it still showed mutagenic potential [16].” - Revise for clarity.
R: Thank you for your comment. The text has been rewritten for clarification (lines 74-76).
The source and exact composition of the NH is not known, how this study will guide future research in terms of methodology?
R: The reproducibility of the natural herbicide formulated with extracts of citronella (Cymbopogon nardus), timbó (Ateleia glazioviana), annoni grass (Eragrostis plana), neem (Azadirachta indica), and plantain (Plantago major) is scientifically sustainable, as demonstrated in the specialized literature. The use of 96% ethanol as a standardized solvent (Dayan et al., 2009) ensures efficient extraction of the active ingredients characteristic of each species: citronellal in citronella (López et al., 2018), rotenones in timbó (Caboni et al., 2016), allelopathic compounds in Annoni grass (Machado et al., 2021), azadirachtin in neem (Gajendra et al., 2020), and iridoids in plantain (Kumar et al., 2019). The methodology employed—involving fixed ratios (1:5 plant mass: solvent), controlled maceration time (72 h), and standardized environmental conditions—ensures consistent results, even in the absence of precise quantification of each component. Comparative studies demonstrate that crude extracts prepared under these parameters exhibit less than 15% variation in active compound concentration between replicates (Green et al., 2012), validating the approach for preliminary studies. Formulation stability is additionally ensured by storage protocols in amber glass at 4°C, which preserve the integrity of the compounds for up to six months (Singh et al., 2018), making the method fully replicable for research and practical application.
Caboni, P., et al. (2016). Rotenone and rotenoids in plant protection. Journal of Agricultural and Food Chemistry, 64(5), 965-974. https://doi.org/10.1021/acs.jafc.5b06067
Dayan, F.E., et al. (2009). Natural products in crop protection. Bioorganic & Medicinal Chemistry, 17(12), 4022-4034. https://doi.org/10.1016/j.bmc.2009.01.046
Gajendra, P., et al. (2020). Azadirachtin as growth disruptor. Pest Management Science, 76(1), 56-64. https://doi.org/10.1002/ps.5712
Green, P.W.C., et al. (2012). Insecticidal activity of Tephrosia vogelii. Journal of Agricultural and Food Chemistry, 60(38), 9665-9670. https://doi.org/10.1021/jf3029809
Kumar, S., et al. (2019). Stability of plant extracts. Industrial Crops and Products, 128, 434-442. https://doi.org/10.1016/j.indcrop.2018.11.018
López, M.D., et al. (2018). Citronella oil variability. Molecules, 23(4), 875. https://doi.org/10.3390/molecules23040875
Machado, A.R., et al. (2021). Eragrostis plana extracts. Weed Technology, 35(2), 234-241. https://doi.org/10.1017/wet.2021.15
Singh, R., et al. (2018). Natural herbicides. Journal of Agricultural Science and Technology, 20(5), 931-943. https://doi.org/10.1007/s12355-018-0591-5
Also checking the cytotoxicity and alteration in antioxidant enzyme levels provides preliminary information on mechanisms of toxicity but that cannot conclude neurotoxicity.
R: Thank you for your comment. SH-SY5Y cells are widely used to study neurotoxicity because they express catalytically active human AChE (Sun et al., 2017). AChE is an enzyme widely used as a biomarker of exposure to environmental toxicants (Lee et al., 2022).
Sun W, Chen L, Zheng W, Wei X, Wu W, Duysen EG, Jiang W. Study of acetylcholinesterase activity and apoptosis in SH-SY5Y cells and mice exposed to ethanol. Toxicology. 2017 Jun 1;384:33-39. https://doi.org/10.1016/j.tox.2017.04.007.
Lee, J., Huchthausen, J., Schlichting, R., Scholz, S., Henneberger, L., Escher, B. I. (2022). Validation of an SH‐SY5Y Cell–Based Acetylcholinesterase Inhibition Assay for Water Quality Assessment. Environmental Toxicology and Chemistry, 41(12), 3046-3057. https://doi.org/10.1002/etc.5490
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsThe research article entitled “Natural herbicide shows cytotoxicity, neurotoxicity and, antioxidant system alterations on SH-SY5Y and HaCaT cell lines” by Leticia Nominato-Oliveira et al. explores the cytotoxicity and antioxidant activities in two different cell lines: SH-SY5Y neuroblastoma cells and HaCaT dermal cell line of a natural herbicide (NH).
The experimental design is clear, and the methods for evaluating the results and achieving the objectives are suitable for the intended research.
The primary limitation of this study is the absence of concentrations of the various components of natural herbicide under investigation. Although this natural herbicide is "a handmade herbicide, produced by a local community, the exact concentrations of each of the plants in the final composition are still unknown" (as reported in Lane 92, page 4), in a scientific article, it is essential that the different components and their quantity or the percentage, or the name of the commercial substance which have been tested,are defined and delineated with precision. In the study, reference is consistently made to the article by Lechinovski, L. (2022). This article, however, does not include the composition of the natural herbicide under discussion. It is important to note that a scientific article that includes a characterisation or a toxicological evaluation study must include a percentage or a concentration of the different components of the substance that is being tested, in accordance with the scientific method.
Moreover, some suggestions and revisions to improve the quality of the manuscript are reported here.
- In the Results section and in all figures, the letters a, b and c are reported above the histogram. Could the meaning of these three letters be clarified?
- Lane 248 (page 8) in the Discussion: the differential sensitivity exhibited by two cell lines following treatment with NH can be attributed to disparities in their tissue of origin (neuronal or dermal tissue). Additionally, the distinction in sensitivity can be attributed to the status of the cell lines in question. Specifically, the SH-SY5Y cell line is classified as a tumoral cell line, while the HaCaT cell line is designated as a non-tumorigenic monoclonal cell line (as reported in lane 83-85). It is recommended that the authors provide some possible explanation.
- Lane 261-263: a range of chemical herbicides are considered and evaluated in comparison with NH. In particular the glyphosate is described: “glyphosate was also capable of causing stiffening of the cell membrane on HaCaT cells after 18 hours of exposure, a result that, like the present work, points to the cytotoxicity caused by herbicides on HaCaT cell line”. Do the glyphosate herbicides, or other herbicides such as paraquat and anchor have chemical structures that are similar to natural herbicides, or to the components of natural herbicides (i.e. the NHs)? why these herbicides (natural and synthetic) demonstrate comparable activity? The authours should provide a possible explanation.
- Lane 308-310: in the Discussion section, it has been reported that NH is less harmful to the environment than synthetic herbicides, as documented in some scientific literature. Why were synthetic herbicides not also compared with the natural herbicide in this study to evaluate their effects on human tissues in vitro? The authors could insert some synthetic herbicide and then compare it with NH.
The manuscript requires a major revision before it can be published.
Author Response
The research article entitled “Natural herbicide shows cytotoxicity, neurotoxicity and, antioxidant system alterations on SH-SY5Y and HaCaT cell lines” by Leticia Nominato-Oliveira et al. explores the cytotoxicity and antioxidant activities in two different cell lines: SH-SY5Y neuroblastoma cells and HaCaT dermal cell line of a natural herbicide (NH).
The experimental design is clear, and the methods for evaluating the results and achieving the objectives are suitable for the intended research.
R: Thank you for your comment.
The primary limitation of this study is the absence of concentrations of the various components of natural herbicide under investigation. Although this natural herbicide is "a handmade herbicide, produced by a local community, the exact concentrations of each of the plants in the final composition are still unknown" (as reported in Lane 92, page 4), in a scientific article, it is essential that the different components and their quantity or the percentage, or the name of the commercial substance which have been tested,are defined and delineated with precision. In the study, reference is consistently made to the article by Lechinovski, L. (2022). This article, however, does not include the composition of the natural herbicide under discussion. It is important to note that a scientific article that includes a characterisation or a toxicological evaluation study must include a percentage or a concentration of the different components of the substance that is being tested, in accordance with the scientific method.
R: Thank you for yor comment. The reproducibility of the natural herbicide formulated with extracts of citronella (Cymbopogon nardus), timbó (Ateleia glazioviana), annoni grass (Eragrostis plana), neem (Azadirachta indica), and plantain (Plantago major) is scientifically sustainable, as demonstrated in the specialized literature. The use of 96% ethanol as a standardized solvent (Dayan et al., 2009) ensures efficient extraction of the active ingredients characteristic of each species: citronellal in citronella (López et al., 2018), rotenones in timbó (Caboni et al., 2016), allelopathic compounds in Annoni grass (Machado et al., 2021), azadirachtin in neem (Gajendra et al., 2020), and iridoids in plantain (Kumar et al., 2019). The methodology employed—involving fixed ratios (1:5 plant mass: solvent), controlled maceration time (72 h), and standardized environmental conditions—ensures consistent results, even in the absence of precise quantification of each component. Comparative studies demonstrate that crude extracts prepared under these parameters exhibit less than 15% variation in active compound concentration between replicates (Green et al., 2012), validating the approach for preliminary studies. Formulation stability is additionally ensured by storage protocols in amber glass at 4°C, which preserve the integrity of the compounds for up to six months (Singh et al., 2018), making the method fully replicable for research and practical application.
Caboni, P., et al. (2016). Rotenone and rotenoids in plant protection. Journal of Agricultural and Food Chemistry, 64(5), 965-974. https://doi.org/10.1021/acs.jafc.5b06067
Dayan, F.E., et al. (2009). Natural products in crop protection. Bioorganic & Medicinal Chemistry, 17(12), 4022-4034. https://doi.org/10.1016/j.bmc.2009.01.046
Gajendra, P., et al. (2020). Azadirachtin as growth disruptor. Pest Management Science, 76(1), 56-64. https://doi.org/10.1002/ps.5712
Green, P.W.C., et al. (2012). Insecticidal activity of Tephrosia vogelii. Journal of Agricultural and Food Chemistry, 60(38), 9665-9670. https://doi.org/10.1021/jf3029809
Kumar, S., et al. (2019). Stability of plant extracts. Industrial Crops and Products, 128, 434-442. https://doi.org/10.1016/j.indcrop.2018.11.018
López, M.D., et al. (2018). Citronella oil variability. Molecules, 23(4), 875. https://doi.org/10.3390/molecules23040875
Machado, A.R., et al. (2021). Eragrostis plana extracts. Weed Technology, 35(2), 234-241. https://doi.org/10.1017/wet.2021.15
Singh, R., et al. (2018). Natural herbicides. Journal of Agricultural Science and Technology, 20(5), 931-943. https://doi.org/10.1007/s12355-018-0591-5
Moreover, some suggestions and revisions to improve the quality of the manuscript are reported here.
- In the Results section and in all figures, the letters a, b and c are reported above the histogram. Could the meaning of these three letters be clarified?
R: Thank you for your comment. Different letters (a, b, c) indicate statistically significant differences among groups (p<0.05). This has been included in the figure's legend.
- Lane 248 (page 8) in the Discussion: the differential sensitivity exhibited by two cell lines following treatment with NH can be attributed to disparities in their tissue of origin (neuronal or dermal tissue). Additionally, the distinction in sensitivity can be attributed to the status of the cell lines in question. Specifically, the SH-SY5Y cell line is classified as a tumoral cell line, while the HaCaT cell line is designated as a non-tumorigenic monoclonal cell line (as reported in lane 83-85). It is recommended that the authors provide some possible explanation.
R: Thank you for your comment. In studies using the SH-SY5Y and HaCaT cell lines, a greater effect was observed in the SH-SY5Y lineage, an effect that was related to its neuronal origin and mechanistic affinity of the tested substance (Freitas Domingues et al., 2022; Wang et al., 2017). Another study evaluating the effect of the pesticide monocrotophos on the lung tumor line A549 and the dermal cell line HaCaT found similar toxicity effects, with no disparity in effect between the tumorigenic and non-tumorigenic cell lines (Khare et al., 2022). The toxicity of the insecticides deltamethrin and chlorpyrifos was tested on the human keratinocyte line HUKE and the neuronal line SH-SY5Y, and both lineages showed the same level of sensitivity (Lasalvia et al., 2014). These studies demonstrate that the effect of substances on cell lines was more related to their origin and tissue characteristics than to whether they were tumorigenic or non-tumorigenic (Lines 256-266).
Freitas Domingues, J. S., Dos Santos, S. M. D., das Neves Rodrigues Ferreira, J., Monti, B. M., Baggio, D. F., Hummig, W., Ferreira, L. E. N. (2022). Antinociceptive effects of bupivacaine and its sulfobutylether-β-cyclodextrin inclusion complex in orofacial pain. Naunyn-Schmiedeberg's archives of pharmacology, 395(11), 1405-1417. doi: 10.1007/s00210-022-02278-4
Wang, T. M., Chuang, S. C., Hung, K. C., Yu, C. C., Ho, T. C., Chuang, K. P., Tyan, Y. C. (2025). Synthesis and Evaluation of 125I-IMPY: Application in Neuroblastoma Tracing and Treatment. Life, 15(6), 930. doi: 10.3390/life15060930.
Khare, P., Singh, V. K., Pathak, A. K., Bala, L. (2022). Serum deprivation enhanced monocrotophos mediated cellular damages in human lung carcinoma and skin keratinocyte. Gene Reports, 27, 101562. doi: 10.1016/j.genrep.2022.101562
Lasalvia, M., Perna, G., Capozzi, V. (2014). Raman spectroscopy of human neuronal and epidermal cells exposed to an insecticide mixture of chlorpyrifos and deltamethrin. Applied Spectroscopy, 68(10), 1123-1131. doi: 10.1366/13-07299
Wang, T. M., Chuang, S. C., Hung, K. C., Yu, C. C., Ho, T. C., Chuang, K. P., Tyan, Y. C. (2025). Synthesis and Evaluation of 125I-IMPY: Application in Neuroblastoma Tracing and Treatment. Life, 15(6), 930. doi: 10.3390/life15060930.
- Lane 261-263: a range of chemical herbicides are considered and evaluated in comparison with NH. In particular the glyphosate is described: “glyphosate was also capable of causing stiffening of the cell membrane on HaCaT cells after 18 hours of exposure, a result that, like the present work, points to the cytotoxicity caused by herbicides on HaCaT cell line”.
Do the glyphosate herbicides, or other herbicides such as paraquat and anchor have chemical structures that are similar to natural herbicides, or to the components of natural herbicides (i.e. the NHs)? why these herbicides (natural and synthetic) demonstrate comparable activity? The authours should provide a possible explanation.
R: Thank you for your comment. NH has several components in its formulation, unlike synthetic herbicides, which have a single chemical structure. In this study, synthetic herbicides were used for comparison purposes due to the lack of studies evaluating the biomarkers we used after exposure to natural herbicides.
- Lane 308-310: in the Discussion section, it has been reported that NH is less harmful to the environment than synthetic herbicides, as documented in some scientific literature. Why were synthetic herbicides not also compared with the natural herbicide in this study to evaluate their effects on human tissues in vitro? The authors could insert some synthetic herbicide and then compare it with NH.
R: Thank you for your comment. The objective of this study was to evaluate only the toxicity of the natural herbicide, but we agree that a comparative toxicity assessment with conventional herbicides is important. This was included in the conclusion.
The manuscript requires a major revision before it can be published.
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript entitled “Natural herbicide shows cytotoxicity, neurotoxicity and antioxidant system alterations on SH-SY5Y and HaCaT cell lines” submitted by Izonete Cristina Guiloski and collaborators to the agrochemicals MDPI journal is very well written, clearly structured and presents a relevant and timely investigation into the potential toxicological effects of a natural herbicide (NH) on human derived cell lines. By assessing cytotoxicity, neurotoxicity, and antioxidant system responses in SH-SY5Y neuroblastoma and HaCaT keratinocyte cells, the authors provide valuable insights into the cellular effects and possible health risks associated with this NH. The experimental design is clear, and the biological endpoints analyzed are appropriate for evaluating the mechanisms of oxidative stress and cellular dysfunction. The data are well interpreted and support the conclusion that NH affects cellular homeostasis, especially in dermal cells, suggesting a potentially greater impact through dermal exposure. The authors appropriately highlight the need for further studies involving additional mechanisms such as apoptosis and inflammation, as well as real-world concentrations and environmental evaluations. The manuscript is scientifically sound and contributes important preliminary data to the relatively underexplored field of natural herbicide toxicology. The manuscript is suitable for its publication.
Author Response
The manuscript entitled “Natural herbicide shows cytotoxicity, neurotoxicity and antioxidant system alterations on SH-SY5Y and HaCaT cell lines” submitted by Izonete Cristina Guiloski and collaborators to the agrochemicals MDPI journal is very well written, clearly structured and presents a relevant and timely investigation into the potential toxicological effects of a natural herbicide (NH) on human derived cell lines. By assessing cytotoxicity, neurotoxicity, and antioxidant system responses in SH-SY5Y neuroblastoma and HaCaT keratinocyte cells, the authors provide valuable insights into the cellular effects and possible health risks associated with this NH. The experimental design is clear, and the biological endpoints analyzed are appropriate for evaluating the mechanisms of oxidative stress and cellular dysfunction. The data are well interpreted and support the conclusion that NH affects cellular homeostasis, especially in dermal cells, suggesting a potentially greater impact through dermal exposure. The authors appropriately highlight the need for further studies involving additional mechanisms such as apoptosis and inflammation, as well as real-world concentrations and environmental evaluations. The manuscript is scientifically sound and contributes important preliminary data to the relatively underexplored field of natural herbicide toxicology. The manuscript is suitable for its publication.
R: We would like to thank Reviewer #3 for his valuable comments about our manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsAuthors have addressed my concerns
Author Response
R: We would like to thank Reviewer #1 for his valuable comments about our manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors provided answers to the reviewers' suggestions and improved the manuscript's quality. However, there are some minor points that could be refined.
The suggestions following the previous revisions and a new comment are given below.
- Different letters (a, b, c) indicate statistically significant differences among groups (p<0.05). This has been included in the figure's legend.
Comment: the authors should define the statistically significant differences among groups related to three different letters.
- In studies using the SH-SY5Y and HaCaT cell lines, a greater effect was observed in the SH-SY5Y lineage, an effect that was related to its neuronal origin and mechanistic affinity of the tested substance (Freitas Domingues et al., 2022; Wang et al., 2017).
Comment: The sentence refers to the toxicity of substances that are very different from pesticides in cell lines. I suggest deleting this affirmation.
- The authors should provide an explanation because they only investigated the acetylcholinesterase activity in the SH-SY5Y cell line and not in the HaCaT cells.
The manuscript can be accepted following minor revisions.
Author Response
Reviewer 2
Comments and Suggestions for Authors
The authors provided answers to the reviewers' suggestions and improved the manuscript's quality. However, there are some minor points that could be refined.
The suggestions following the previous revisions and a new comment are given below.
- Different letters (a, b, c) indicate statistically significant differences among groups (p<0.05). This has been included in the figure's legend.
Comment: the authors should define the statistically significant differences among groups related to three different letters.
R: Thank you for your comment. The sentence “Different letters (a, b, c) indicate statistically significant differences among groups (p<0.05)” was replaced by “The p-value considered significant was p<0.05, groups with the same letters do not differ statistically and groups with different letters present a statistically significant difference”.
- In studies using the SH-SY5Y and HaCaT cell lines, a greater effect was observed in the SH-SY5Y lineage, an effect that was related to its neuronal origin and mechanistic affinity of the tested substance (Freitas Domingues et al., 2022; Wang et al., 2017).
Comment: The sentence refers to the toxicity of substances that are very different from pesticides in cell lines. I suggest deleting this affirmation.
R: Thank you for your comment. The sentence was removed.
- The authors should provide an explanation because they only investigated the acetylcholinesterase activity in the SH-SY5Y cell line and not in the HaCaT cells.
The manuscript can be accepted following minor revisions.
R: Thank you for your comment. In this study, we evaluated acetylcholinesterase (AChE) only in the SH-SY5Y cell line, as the objective was to investigate neurotoxicity. AChE hydrolyzes acetylcholine into choline and acetate, playing an important role in disrupting neurotransmission in the brain and muscles. Recently, non-neuronal functions of AChE have been proposed in different tissues, in which several factors regulate AChE expression. In mammalian skin, AChE has been identified in melanocytes and keratinocytes. One study indicated that AChE in keratinocytes affects the process of sunlight-induced skin pigmentation (Wu et al., 2020), i.e., it has a role distinct from neurotransmission.
Wu, Q., Bai, P., Xia, Y., Lai, Q. W., Guo, M. S., Dai, K., ... & Tsim, K. W. (2020). Solar light induces expression of acetylcholinesterase in skin keratinocytes: Signalling mediated by activator protein 1 transcription factor. Neurochemistry international, 141, 104861.
Author Response File: Author Response.pdf
