İNSAN AKCİĞER HÜCRELERİNDE ROUNDUP® TOKSİSİTESİNİN DEĞERLENDİRİLMESİ

Öz

Amaç: Bu çalışmada, en yaygın olarak kullanılan glifosat bazlı herbisitlerden (GBH) biri olan Roundup’ın, insan bronşiyal epitel hücrelerine (BEAS-2B) toksik etkileri değerlendirilmiştir.

Gereç ve Yöntem: Hücre canlılığının 24 ve 48 saatte değerlendirilmesi amacıyla 3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazolyum bromür ve nötral red alım testleri uygulanmıştır. Apoptoz tespiti için Muse analiz cihazı; apoptotik nükleer değişikliklerin değerlendirilmesi için Hoechst boyama tekniği kullanılmıştır. Ayrıca reaktif oksijen türleri (ROT) oluşumunun değerlendirilmesi için diklorofloresin diasetat yöntemi uygulanmıştır.

Sonuç ve Tartışma: Her iki sitotoksisite testinden benzer yarı maksimal inhibisyon konsantrasyonları elde edilmiştir. 24 saatte 200 µM, 48 saatte ise 50 µM maruziyetten sonra BEAS-2B hücrelerinin canlılığında önemli ölçüde azalma olduğu tespit edilmiştir. Her iki zamanda da Roundup maruziyetinin doza bağlı olarak apoptoza neden olduğu ve apoptozun maruz kalma süresinin uzamasıyla arttığı bulunmuştur. Ayrıca, Roundup maruziyeti sonrasında hücresel ROT oluşumunun indüklendiği gözlenmiştir. Bu bulgular GBH’lerin sağlıklı insan akciğer hücrelerinde ROT üretimini ve apoptozu arttırdığını göstermektedir. GBH’lere başlıca maruziyet yollarından birinin inhalasyon olduğu göz önüne alındığında bu çalışmanın sonuçları önem arz etmektedir.

Anahtar Kelimeler

• Apoptosis
• glyphosate
• lung cells
• reactive oxygen species
• toxicity

Proje Numarası

THD-2018-8670; THD-2018-8671

EVALUATION OF ROUNDUP® TOXICITY IN HUMAN LUNG CELLS

Öz

Objective: In this study, toxic effects of Roundup, one of the most common glyphosate-based herbicides (GBHs), were assessed on human bronchial epithelial cells (BEAS-2B).

Material and Method: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and neutral red uptake assays were implemented for evaluation of cell viability at 24 and 48 h. Apoptosis detection was made by Muse analyzer while Hoechst staining was employed to detect apoptotic nuclear changes. In addition, dichlorofluorescein diacetate assay was used for the assessment of reactive oxygen species (ROS) formation.

Result and Discussion: Similar half maximal inhibitory concentrations were obtained from cytotoxicity assays. Results showed that significant reduction in the viability of BEAS-2B cells started to occur from 200 µM at 24 h and 50 µM at 48 h treatment times. Roundup treatments were found to cause apoptosis in a dose-dependent manner in both time points and apoptosis increased with increasing exposure time. Moreover, it was observed that cellular ROS formation was induced following Roundup exposure. These findings suggest that GBHs can stimulate ROS production, as well as apoptosis on healthy human lung cells which is important considering inhalation is one of the primary exposure routes to this group of chemicals.  

Anahtar Kelimeler

• Apoptosis
• glyphosate
• lung cells
• reactive oxygen species
• toxicity

Destekleyen Kurum

Erciyes University

Proje Numarası

THD-2018-8670; THD-2018-8671

Kaynakça

1. 1. Boocock, M.R., Coggins, J.R. (1983). Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate. FEBS Letters, 154(1), 127-133. [CrossRef]
2. 2. Duke, S.O., Powles, S.B. (2008). Glyphosate: A once‐in‐a‐century herbicide. Pest Management Science, 64(4), 319-325. [CrossRef]
3. 3. Williams, A.L., Watson, R.E. DeSesso, J.M. (2012). Developmental and reproductive outcomes in humans and animals after glyphosate exposure: A critical analysis. Journal of Toxicology and Environmental Health, Part B, 15(1), 39-96. [CrossRef]
4. 4. Bai, S.H., Ogbourne, S.M. (2016). Glyphosate: environmental contamination, toxicity and potential risks to human health via food contamination. Environmental Science and Pollution Research, 23(19), 18988-19001. [CrossRef]
5. 5. Chang, F.C., Simcik, M.F., Capel, P.D. (2011). Occurrence and fate of the herbicide glyphosate and its degradate aminomethylphosphonic acid in the atmosphere. Environmental Toxicology and Chemistry, 30(3), 548-555. [CrossRef]
6. 6. Soares, D., Silva, L., Duarte, S., Pena, A., Pereira, A. (2021). Glyphosate use, toxicity and occurrence in food. Foods, 10(11), 2785. [CrossRef]
7. 7. Agency for Toxic Substances and Disease Registry (ATSDR). Toxicological Profile for Glyphosate. (2020). From https://wwwn.cdc.gov/ Accessed date: 29 September 2022.
8. 8. Agostini, L.P., Dettogni, R.S., Dos Reis, R.S., Stur, E., Dos Santos, E.V., Ventorim, D.P., Garcia, F.M., Cardoso, R.C., Graceli, J.B., Louro, I.D. (2020). Effects of glyphosate exposure on human health: Insights from epidemiological and in vitro studies. Science of the Total Environment, 705, 135808. [CrossRef]

9. 9. Dill, G.M., Sammons, R.D., Feng, P.C., Kohn, F., Kretzmer, K., Mehrsheikh, A., Bleeke, M., Honegger, J.L., Farmer, D., Wright, D., Haupfea,r E.A. (2010). Glyphosate: discovery, development, applications, and properties. In: V.K. Nandula (Ed.) Glyphosate Resistance in Crops and Weeds: History, Development, and Management, (pp. 1-33). John Wiley & Sons, Inc.
10. 10. Wang, X., Lu, Q., Guo, J., Ares, I., Martínez, M., Martínez-Larrañaga, M.R., Wang, X., Anadón, A., Martínez, M.A. (2022). Oxidative stress and metabolism: A mechanistic insight for glyphosate toxicology. Annual Review of Pharmacology and Toxicology, 62, 617-639. [CrossRef]
11. 11. Mesnage, R., Bernay, B., Seralini, G.E. (2013). Ethoxylated adjuvants of glyphosate-based herbicides are active principles of human cell toxicity. Toxicology, 313(2-3), 122-128. [CrossRef]
12. 12. Mesnage, R., Defarge, N., Spiroux de Vendomois, J., Seralini, G.E. (2015). Potential toxic effects of glyphosate and its commercial formulations below regulatory limits. Food and Chemical Toxicology, 84, 133-153. [CrossRef]
13. 13. Martins-Gomes, C., Silva, T.L., Andreani, T., Silva, A.M. (2022). Glyphosate vs. glyphosate-based herbicides exposure: A review on their toxicity. Journal of Xenobiotics, 12(1), 21-40. [CrossRef]
14. 14. International Agency for Research on Cancer (IARC). Monographs Volume 112: evaluation of five organophosphate insecticides and herbicides. (2015). From https://www.iarc.who.int/. Accessed date: 20 September 2022.
15. 15. United States Environmental Protection Agency (USEPA). Revised glyphosate issue paper: Evaluation of carcinogenic potential. (2018). From https://www.regulations.gov/. Accessed date: 20 September 2022.
16. 16. European Chemical Agency (ECHA). Glyphosate: No change proposed to hazard classification. (2022). From https://echa.europa.eu/. Accessed date: 3 October 2022.
17. 17. Bradberry, S.M., Proudfoot, A.T., Vale, J.A. (2004). Glyphosate poisoning. Toxicological Reviews, 23(3), 159-167. [CrossRef]
18. 18. Sidthilaw, S., Sapbamrer, R., Pothirat, C., Wunnapuk, K., Khacha-Ananda, S. (2022). Effects of exposure to glyphosate on oxidative stress, inflammation, and lung function in maize farmers, Northern Thailand. BMC Public Health, 22(1), 1-10. [CrossRef]
19. 19. Sies, H. (1997). Oxidative stress: oxidants and antioxidants. Experimental Physiology, 82(2), 291-295. [CrossRef]
20. 20. Hengartner, M.O. (2000). The biochemistry of apoptosis. Nature, 407(6805), 770-776. [CrossRef]
21. 21. Chaufan, G., Coalova, I., Molina M.D.C.R.D. (2014). Glyphosate commercial formulation causes cytotoxicity, oxidative effects, and apoptosis on human cells: differences with its active ingredient. International Journal of Toxicology, 33(1), 29-38. [CrossRef]
22. 22. Hao, Y., Chen, H., Xu, W., Gao, J., Yang, Y., Zhang, Y., Tao, L. (2019). Roundup confers cytotoxicity through DNA damage and Mitochondria-Associated apoptosis induction. Environmental Pollution, 252 (Pt A), 917-923. [CrossRef]
23. 23. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65(1-2), 55-63. [CrossRef]
24. 24. Borenfreund, E., Puerner, J.A. (1985). Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicology Letters, 24(2-3), 119-124. [CrossRef]
25. 25. Wang, H., Joseph, J.A. (1999). Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radical Biology and Medicine, 27(5-6), 612-616. [CrossRef]
26. 26. Hao, Y., Zhang, Y., Ni, H., Gao, J., Yang, Y., Xu, W., Tao, L. (2019). Evaluation of the cytotoxic effects of glyphosate herbicides in human liver, lung, and nerve. Journal of Environmental Science and Health, Part B, 54(9), 737-744. [CrossRef]
27. 27. Gasnier, C., Dumont, C., Benachour, N., Clair, E., Chagnon, M.C., Seralini, G.E. (2009). Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Toxicology, 262(3), 184-191. [CrossRef]
28. 28. Ergun, H., Cayir, A. (2021). Exposure to glyphosate and tetrachlorvinphos induces cytotoxicity and global DNA methylation in human cells. Toxicology and Industrial Health, 37(10), 610-618. [CrossRef]
29. 29. Fotakis, G., Timbrell, J.A. (2006). In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters, 160(2), 171-177. [CrossRef]
30. 30. Lopes, F.M., Sandrini, J.Z., Souza, M.M. (2018). Toxicity induced by glyphosate and glyphosate-based herbicides in the zebrafish hepatocyte cell line (ZF-L). Ecotoxicology and Environmental Safety, 162, 201-207. [CrossRef]
31. 31. Goulart, T.L., Boyle, R.T., Souza, M.M. (2015). Cytotoxicity of the association of pesticides Roundup Transorb(R) and Furadan 350 SC(R) on the zebrafish cell line, ZF-L. Toxicology in Vitro, 29(7), 1377-1384. [CrossRef]
32. 32. Conte, F.M., Cestonaro, L.V., Piton, Y.V., Guimarães, N., Garcia, S.C., da Silva, D.D., Arbo, M.D. (2022). Toxicity of pesticides widely applied on soybean cultivation: Synergistic effects of fipronil, glyphosate and imidacloprid in HepG2 cells. Toxicology in Vitro, 84, 105446. [CrossRef]
33. 33. Kwiatkowska, M., Michałowicz, J., Jarosiewicz, P., Pingot, D., Sicińska, P., Huras, B., Zakrzewski, J., Jarosiewicz, M., Bukowska, B. (2020). Evaluation of apoptotic potential of glyphosate metabolites and impurities in human peripheral blood mononuclear cells (in vitro study). Food and Chemical Toxicology, 135, 110888. [CrossRef]
34. 34. Martini, C.N., Gabrielli, M., Vila Mdel, C. (2012). A commercial formulation of glyphosate inhibits proliferation and differentiation to adipocytes and induces apoptosis in 3T3-L1 fibroblasts. Toxicology in Vitro, 26(6), 1007-1013. [CrossRef]
35. 35. Redza-Dutordoir, M., Averill-Bates, D.A. (2016). Activation of apoptosis signalling pathways by reactive oxygen species. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1863(12), 2977-2992. [CrossRef]
36. 36. Hao, Y., Zhang, Y., Cheng, J., Xu, W., Xu, Z., Gao, J., Tao, L. (2020). Adjuvant contributes Roundup's unexpected effects on A549 cells. Environmental Research, 184, 109306. [CrossRef]
37. 37. Bai, G., Zhou, R., Jiang, X., Zou, Y., Shi, B. (2022). Glyphosate‐based herbicides induces autophagy in IPEC‐J2 cells and the intervention of N‐acetylcysteine. Environmental Toxicology, 37(8):1878-1890. [CrossRef]
38. 38. Silva, A.M., Martins-Gomes, C., Ferreira, S.S., Souto, E.B., Andreani, T. (2022). Molecular Physicochemical Properties of Selected Pesticides as Predictive Factors for Oxidative Stress and Apoptosis-Dependent Cell Death in Caco-2 and HepG2 Cells. International Journal of Molecular Sciences, 23(15), 8107. [CrossRef]