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Detection of Biochemical, Cytotoxic, and Genotoxic Damage Caused by Glufosinate-Ammonium on the Zebrafish Cell Line

Year 2025, Volume: 46 Issue: 2, 240 - 249, 30.06.2025

Harika Eylül Esmer Duruel , Figen Esin Kayhan

https://doi.org/10.17776/csj.1595139

Abstract

Pesticides widely used in agriculture can enter aquatic ecosystems and threaten non-target organisms in the environment. In this study, sublethal toxic effects of glufosinate-ammonium on Zebrafish (Danio rerio) liver fibroblast cell line (ZFL) were investigated. Cytotoxicity and genotoxicity tests; MTT, neutral red uptake, lactate dehydrogenase, trypan blue tests were observed at 24 hours, 48 hours, 72 hours and 96 hours; apoptosis and necrosis detection at 48 and 96 hours, cell proliferation detection at 72 hours, micronucleus test at 96 hours. A sublethal dose of 2000 mgL-1 was determined as the initial concentration and there are dilution differences between the tests. As a result of the tests; Decreases were observed in all applications compared to the negative control in MTT, neutral red uptake, trypan blue, % necrosis and cell proliferation detection tests; in lactate dehydrogenase and % apoptosis tests, an increase were observed in all applications compared to the negative control. In the micronucleus test, it was determined that glufosinate-ammonium application stimulated micronucleus formation compared to the negative control. Biochemical tests were performed in ZFL cell lines with 96-hour application. Selected groups; control group, 250 mgL-1, 500 mgL-1 and 1000 mgL-1 glufosinate-ammonium doses. Evaluated biochemical parameters; lipid peroxidation determination, reduced glutathione determination, catalase enzyme activity determination, acetylcholinesterase enzyme activity determination and total protein determination. As a result of biochemical experiments; lipid peroxidation level at 1000 mgL-1; catalase enzyme activity 250 mgL-1; total protein levels in all concentrations increased compared to the control. lipid peroxidation level at 250 mgL-1 and 500 mgL-1; catalase enzyme activity 500 mgL-1 and 1000 mg/L; GSH level and AChE enzyme activity decreased at all glufosinate-ammonium dose applications. It is thought that the obtained results will provide important contributions to the literature and shed light on further research.

Keywords

Pesticide Zebrafish Oxidative stress Cytotoxicity Genotoxicity

Project Number

FEN-C-DRP-230119-0010

References

  • [1] Saeed T., Sawaya W.N., Ahmad N., Rajagopal S., Al-Omair A., Organophosphorus pesticide residues in the total diet of Kuwait. Arab J Sci Eng, 30 (2005) 17–28.
  • [2] Kumar V., Kumar P., Pesticides in agriculture and environment: Impacts on human health, In Contaminants in Agriculture and Environment: Health Risks and Remediation, 1 (2019) 76–95.
  • [3] Bonifacio A.F., Hued A.C., Single and joint effects of chronic exposure to chlorpyrifos and glyphosate based pesticides on structural biomarkers in Cnesterodon decemmaculatus. Chemosphere, 236 (2019) 124311.
  • [4] Mellanby K., Pesticides and pollution. Pesticides and Pollution, 1967.
  • [5] Hashmi T.A., Qureshi R., Tipre D., Menon S., Investigation of pesticide residues in water, sediments and fish samples from Tapi River, India as a case study and its forensic significance. Environ Forensics, 21 (2020) 1–10.
  • [6] Rahman M.Z., Hossain Z., Mollah M.F.A., Ahmed G.U., Effect of Diazinon 60 EC on Anabas testudineus, Channa punctatus and Barbodes gonionotus, Naga, The ICLARM Quarterly, 25 (2) (2002).
  • [7] Natesan V., Kim S.J., Lipid metabolism, disorders and therapeutic drugs–review. Biomol Ther (Seoul), 29 (2021) 596.
  • [8] Kayhan F., Esmer Duruel H.E., Kızılkaya Ş., Dinç S., Kaymak G., Akbulut C, Yön Ertuğ N.D., Toxic effects of herbicide tribenuron-methyl on liver tissue of zebrafish (Danio rerio). Fresenius Environ Bull, 29 (12A) (2020) 11175-11179.
  • [9] Villeneuve D.L., Larkin P., Knoebl I., Miracle A.L., Kahl M.D., Jensen K.M., et al., A graphical systems model to facilitate hypothesis-driven ecotoxicogenomics research on the teleost brain− pituitary− gonadal axis. Environ Sci Technol, 41 (2007) 321–30.
  • [10] Halim N., Kuntom A., Determination of glufosinate ammonium in crude palm oil: use of the modified quechers method and LC-MS/MS detection. J Oil Palm Res, 25 (2013) 84–91.
  • [11] Dayan F.E., Barker A., Bough R., Ortiz M., Takano H., Duke S.O., Herbicide mechanisms of action and resistance, In Comprehensive Biotechnology, 3rd Ed. (2019) 36–48.
  • [12] Kwok M.L., Hu X.L., Meng Q., Chan K.M., Whole-transcriptome sequencing (RNA-seq) analyses of the zebrafish liver cell line, ZFL, after acute exposure to Cu2+ ions. Metallomics, 12 (2020) 732–51.
  • [13] Bols N.C., Dayeh V.R., Lee L.E.J., Schirmer K., Use of fish cell lines in the toxicology and ecotoxicology of fish. Piscine cell lines in environmental toxicology. Biochemistry and Molecular Biology of Fishes, 6 (2005) 43–84.
  • [14] Žegura B., Filipič M., The application of the Comet assay in fish cell lines. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 842 (2019) 72–84.
  • [15] Mosmann T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 65 (1983) 55–63.
  • [16] Krone P.H., Blechinger S.R., Evans T.G., Ryan J.A., Noonan E.J., Hightower L.E., Use of fish liver PLHC-1 cells and zebrafish embryos in cytotoxicity assays. Methods, 35 (2005) 176–87.
  • [17] Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P., Molecular Biology of the Cell, Garland Science Textbooks 2007.
  • [18] Strober W., Trypan blue exclusion test of cell viability. Curr Protoc Immunol, 111 (2015) A3-B.
  • [19] Parrilla I., Vazquez J.M., Cuello C., Gil M.A., Roca J., Di Berardino D., et al., Hoechst 33342 stain and uv laser exposure do not induce genotoxic effects in flow-sorted boar spermatozoa. Reproduction, 128 (2004) 615–21.
  • [20] Şekeroğlu V., Atlı-Şekeroğlu Z., Genotoksik hasarın belirlenmesinde mikronükleus testi. Türk Hijyen ve Deneysel Biyoloji Dergisi, 68 (2011) 241–52.
  • [21] Wang Y., Tang M., Review of in vitro toxicological research of quantum dot and potentially involved mechanisms. Science of the Total Environment, 625 (2018) 940–62.
  • [22] Ledwożyw A., Michalak J., Stȩpień A., Ka̧dziołka A., The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clinica Chimica Acta, 155 (1986) 275–83.
  • [23] Beutler E., Glutathione in red blood cell metabolism. A Manuel of Biochemical Methods, (1975).
  • [24] Bradford M.M., A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Chem, 72 (1973) 249–54.
  • [25] Aebi H., Catalase in vitro Methods of Enzymatic Analysis. 2nd Ed, FL 121 (1974).
  • [26] Ellman G.L, Courtney KD, Andres Jr V, Featherstone RM., A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol, 7 (1961) 88–95.
  • [27] Benachour N., Sipahutar H., Moslemi S., Gasnier C., Travert C., Seralini G.E., Time-and dose-dependent effects of roundup on human embryonic and placental cells. Arch Environ Contam Toxicol, 53 (2007) 126–33.
  • [28] Mesnage R., Defarge N., Spiroux de Vendômois J., Séralini G.E., Major pesticides are more toxic to human cells than their declared active principles. Biomed Res Int, 26 (2014) 179691.
  • [29] Nenni M., Toksikolojide in vitro Hücre Temelli Sitotoksisite Çalışmaları. Ege Üniversitesi Sağlık Bilimleri Enstitüsü Yüksek Lisans tezi (2019).
  • [30] Bonomo M.M., Fernandes J.B., Carlos R.M., Fernandes M.N., Mitochondrial and lysosomal dysfunction induced by the novel metal-insecticide [Mg (hesp) 2 (phen)] in the zebrafish (Danio rerio) hepatocyte cell line (ZF-L). Chem Biol Interact, 307 (2019) 147–53.
  • [31] Goulart T.L.S., Boyle R.T., Souza M.M., Cytotoxicity of the association of pesticides Roundup Transorb® and Furadan 350 SC® on the zebrafish cell line, ZF-L. Toxicology in Vitro, 29 (2015) 1377–84.
  • [32] Kanat Ö.N., Selmanoğlu G., Neurotoxic effect of fipronil in neuroblastoma SH-SY5Y cell line. Neurotox Res 2020;37:30–40.
  • [33] Karacaoğlu E., Flusilazole-induced damage to SerW3 cells via cytotoxicity, oxidative stress and lipid metabolism: An in vitro study. Pestic Biochem Physiol, 180 (2022) 104998.
  • [34] Orta Yılmaz B., Sodyum florürün leydig hücrelerinde steroidogenik yolak üzerinde in vitro etkileri, İstanbul Üniversitesi, Fen Bilimleri Enstitüsü, Doktora tezi, (2017).
  • [35] Bonomo M.M., Fernandes J.B., Carlos R.M., Fernandes M.N., Biochemical and genotoxic biomarkers and cell cycle assessment in the zebrafish liver (ZF-L) cell line exposed to the novel metal-insecticide magnesium-hespiridin complex. Chemosphere, 250 (2020) 126416.
  • [36] Repetto M., Semprine J., Boveris A., Lipid peroxidation: chemical mechanism, biological implications and analytical determination. Lipid Peroxidation, 1 (2012) 3–30.
  • [37] Gasnier C., Benachour N., Clair E., Travert C., Langlois F., Laurant C., et al., Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines. Journal of Occupational Medicine and Toxicology, 5 (2010) 1–13.
  • [38] Larsen K., Najle R., Lifschitz A., Virkel G., Effects of sub-lethal exposure of rats to the herbicide glyphosate in drinking water: glutathione transferase enzyme activities, levels of reduced glutathione and lipid peroxidation in liver, kidneys and small intestine. Environ Toxicol Pharmacol, 34 (2012) 811–8.
  • [39] Van der Oost R., Beyer J., Vermeulen N.P.E., Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ Toxicol Pharmacol, 13 (2003) 57–149.

Zebra Balığı Hücre Hattında Glufosinat-Amonyumun Neden Olduğu Biyokimyasal, Sitotoksik ve Genotoksik Hasarın Tespiti

Year 2025, Volume: 46 Issue: 2, 240 - 249, 30.06.2025

Harika Eylül Esmer Duruel , Figen Esin Kayhan

https://doi.org/10.17776/csj.1595139

Abstract

Tarımda yaygın olarak kullanılan pestisitler, sucul ekosistemlere girebilir ve çevredeki hedef dışı organizmaları tehdit edebilir. Bu çalışmada, glufosinat-amonyumun Zebra balığı (Danio rerio) karaciğer fibroblast hücre hattı (ZFL) üzerindeki subletal toksik etkileri incelenmiştir. Hücre hatlarının sudaki kirleticilerin toksisitesini test etmede kullanılması, hedef dışı organizmaların toksisitesini test etme yöntemlerine alternatiftir. ZFL hücre hatlarında biyokimyasal incelemeler 96 saatlik uygulama ile gerçekleştirilmiştir. Seçilen gruplar; Kontrol grubu 250 mgL-1, 500 mgL-1 ve 1000 mgL-1 glufosinat-amonyum dozlarından oluşmaktadır. Değerlendirilen biyokimyasal parametreler; lipid peroksidasyon tayini, redükte glutatyon tayini, katalaz enzim aktivitesi tayini, asetilkolinesteraz enzim aktivitesi tayini ve toplam protein tayinidir. Uygulamalar sonucunda kontrole karşı yapılan değerlendirmede değişiklik olduğu belirlenmiştir. Sitotoksisite ve genotoksisite testleri olarak; MTT, Nötral kırmızı alımı, Laktat dehidrogenaz, Hücre çoğalmasının belirlenmesi, Trypan mavisi, Apoptozis ve nekroz tespiti, Mikronükleus testi uygulandı. Tüm değişiklikler doza ve süreye bağlı olarak tespit edildi. Glufosinat-amonyum uygulamasını takiben oksidatif stresin indüklenmesi ve bunun sonucunda hücrelerde oluşan hasar değerlendirildi. Elde edilen sonuçların literatüre önemli katkılar sağlayarak daha ileri araştırmalara ışık tutacağı düşünülmektedir.

Keywords

pestisit zebra balığı oksidatif stres sitotoksisite genotoksisite

Project Number

FEN-C-DRP-230119-0010

References

  • [1] Saeed T., Sawaya W.N., Ahmad N., Rajagopal S., Al-Omair A., Organophosphorus pesticide residues in the total diet of Kuwait. Arab J Sci Eng, 30 (2005) 17–28.
  • [2] Kumar V., Kumar P., Pesticides in agriculture and environment: Impacts on human health, In Contaminants in Agriculture and Environment: Health Risks and Remediation, 1 (2019) 76–95.
  • [3] Bonifacio A.F., Hued A.C., Single and joint effects of chronic exposure to chlorpyrifos and glyphosate based pesticides on structural biomarkers in Cnesterodon decemmaculatus. Chemosphere, 236 (2019) 124311.
  • [4] Mellanby K., Pesticides and pollution. Pesticides and Pollution, 1967.
  • [5] Hashmi T.A., Qureshi R., Tipre D., Menon S., Investigation of pesticide residues in water, sediments and fish samples from Tapi River, India as a case study and its forensic significance. Environ Forensics, 21 (2020) 1–10.
  • [6] Rahman M.Z., Hossain Z., Mollah M.F.A., Ahmed G.U., Effect of Diazinon 60 EC on Anabas testudineus, Channa punctatus and Barbodes gonionotus, Naga, The ICLARM Quarterly, 25 (2) (2002).
  • [7] Natesan V., Kim S.J., Lipid metabolism, disorders and therapeutic drugs–review. Biomol Ther (Seoul), 29 (2021) 596.
  • [8] Kayhan F., Esmer Duruel H.E., Kızılkaya Ş., Dinç S., Kaymak G., Akbulut C, Yön Ertuğ N.D., Toxic effects of herbicide tribenuron-methyl on liver tissue of zebrafish (Danio rerio). Fresenius Environ Bull, 29 (12A) (2020) 11175-11179.
  • [9] Villeneuve D.L., Larkin P., Knoebl I., Miracle A.L., Kahl M.D., Jensen K.M., et al., A graphical systems model to facilitate hypothesis-driven ecotoxicogenomics research on the teleost brain− pituitary− gonadal axis. Environ Sci Technol, 41 (2007) 321–30.
  • [10] Halim N., Kuntom A., Determination of glufosinate ammonium in crude palm oil: use of the modified quechers method and LC-MS/MS detection. J Oil Palm Res, 25 (2013) 84–91.
  • [11] Dayan F.E., Barker A., Bough R., Ortiz M., Takano H., Duke S.O., Herbicide mechanisms of action and resistance, In Comprehensive Biotechnology, 3rd Ed. (2019) 36–48.
  • [12] Kwok M.L., Hu X.L., Meng Q., Chan K.M., Whole-transcriptome sequencing (RNA-seq) analyses of the zebrafish liver cell line, ZFL, after acute exposure to Cu2+ ions. Metallomics, 12 (2020) 732–51.
  • [13] Bols N.C., Dayeh V.R., Lee L.E.J., Schirmer K., Use of fish cell lines in the toxicology and ecotoxicology of fish. Piscine cell lines in environmental toxicology. Biochemistry and Molecular Biology of Fishes, 6 (2005) 43–84.
  • [14] Žegura B., Filipič M., The application of the Comet assay in fish cell lines. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 842 (2019) 72–84.
  • [15] Mosmann T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 65 (1983) 55–63.
  • [16] Krone P.H., Blechinger S.R., Evans T.G., Ryan J.A., Noonan E.J., Hightower L.E., Use of fish liver PLHC-1 cells and zebrafish embryos in cytotoxicity assays. Methods, 35 (2005) 176–87.
  • [17] Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P., Molecular Biology of the Cell, Garland Science Textbooks 2007.
  • [18] Strober W., Trypan blue exclusion test of cell viability. Curr Protoc Immunol, 111 (2015) A3-B.
  • [19] Parrilla I., Vazquez J.M., Cuello C., Gil M.A., Roca J., Di Berardino D., et al., Hoechst 33342 stain and uv laser exposure do not induce genotoxic effects in flow-sorted boar spermatozoa. Reproduction, 128 (2004) 615–21.
  • [20] Şekeroğlu V., Atlı-Şekeroğlu Z., Genotoksik hasarın belirlenmesinde mikronükleus testi. Türk Hijyen ve Deneysel Biyoloji Dergisi, 68 (2011) 241–52.
  • [21] Wang Y., Tang M., Review of in vitro toxicological research of quantum dot and potentially involved mechanisms. Science of the Total Environment, 625 (2018) 940–62.
  • [22] Ledwożyw A., Michalak J., Stȩpień A., Ka̧dziołka A., The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clinica Chimica Acta, 155 (1986) 275–83.
  • [23] Beutler E., Glutathione in red blood cell metabolism. A Manuel of Biochemical Methods, (1975).
  • [24] Bradford M.M., A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Chem, 72 (1973) 249–54.
  • [25] Aebi H., Catalase in vitro Methods of Enzymatic Analysis. 2nd Ed, FL 121 (1974).
  • [26] Ellman G.L, Courtney KD, Andres Jr V, Featherstone RM., A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol, 7 (1961) 88–95.
  • [27] Benachour N., Sipahutar H., Moslemi S., Gasnier C., Travert C., Seralini G.E., Time-and dose-dependent effects of roundup on human embryonic and placental cells. Arch Environ Contam Toxicol, 53 (2007) 126–33.
  • [28] Mesnage R., Defarge N., Spiroux de Vendômois J., Séralini G.E., Major pesticides are more toxic to human cells than their declared active principles. Biomed Res Int, 26 (2014) 179691.
  • [29] Nenni M., Toksikolojide in vitro Hücre Temelli Sitotoksisite Çalışmaları. Ege Üniversitesi Sağlık Bilimleri Enstitüsü Yüksek Lisans tezi (2019).
  • [30] Bonomo M.M., Fernandes J.B., Carlos R.M., Fernandes M.N., Mitochondrial and lysosomal dysfunction induced by the novel metal-insecticide [Mg (hesp) 2 (phen)] in the zebrafish (Danio rerio) hepatocyte cell line (ZF-L). Chem Biol Interact, 307 (2019) 147–53.
  • [31] Goulart T.L.S., Boyle R.T., Souza M.M., Cytotoxicity of the association of pesticides Roundup Transorb® and Furadan 350 SC® on the zebrafish cell line, ZF-L. Toxicology in Vitro, 29 (2015) 1377–84.
  • [32] Kanat Ö.N., Selmanoğlu G., Neurotoxic effect of fipronil in neuroblastoma SH-SY5Y cell line. Neurotox Res 2020;37:30–40.
  • [33] Karacaoğlu E., Flusilazole-induced damage to SerW3 cells via cytotoxicity, oxidative stress and lipid metabolism: An in vitro study. Pestic Biochem Physiol, 180 (2022) 104998.
  • [34] Orta Yılmaz B., Sodyum florürün leydig hücrelerinde steroidogenik yolak üzerinde in vitro etkileri, İstanbul Üniversitesi, Fen Bilimleri Enstitüsü, Doktora tezi, (2017).
  • [35] Bonomo M.M., Fernandes J.B., Carlos R.M., Fernandes M.N., Biochemical and genotoxic biomarkers and cell cycle assessment in the zebrafish liver (ZF-L) cell line exposed to the novel metal-insecticide magnesium-hespiridin complex. Chemosphere, 250 (2020) 126416.
  • [36] Repetto M., Semprine J., Boveris A., Lipid peroxidation: chemical mechanism, biological implications and analytical determination. Lipid Peroxidation, 1 (2012) 3–30.
  • [37] Gasnier C., Benachour N., Clair E., Travert C., Langlois F., Laurant C., et al., Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines. Journal of Occupational Medicine and Toxicology, 5 (2010) 1–13.
  • [38] Larsen K., Najle R., Lifschitz A., Virkel G., Effects of sub-lethal exposure of rats to the herbicide glyphosate in drinking water: glutathione transferase enzyme activities, levels of reduced glutathione and lipid peroxidation in liver, kidneys and small intestine. Environ Toxicol Pharmacol, 34 (2012) 811–8.
  • [39] Van der Oost R., Beyer J., Vermeulen N.P.E., Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ Toxicol Pharmacol, 13 (2003) 57–149.
There are 39 citations in total.

Details

Primary Language English
Subjects Genotoxicity and Cytotoxicity, Ecotoxicology
Journal Section Natural Sciences
Authors

Harika Eylül Esmer Duruel 0000-0002-0792-2062

Figen Esin Kayhan 0000-0001-7754-1356

Project Number FEN-C-DRP-230119-0010
Publication Date June 30, 2025
Submission Date December 3, 2024
Acceptance Date May 3, 2025
Published in Issue Year 2025Volume: 46 Issue: 2

Cite

APA Esmer Duruel, H. E., & Kayhan, F. E. (2025). Detection of Biochemical, Cytotoxic, and Genotoxic Damage Caused by Glufosinate-Ammonium on the Zebrafish Cell Line. Cumhuriyet Science Journal, 46(2), 240-249. https://doi.org/10.17776/csj.1595139