Glifosatın glukoz-6-fosfat dehidrogenaz enzim aktivitesi üzerindeki etkisinin in vitro koşullarda değerlendirilmesi

Year 2022, Volume: 47 Issue: 1, 143 - 151, 31.03.2022

Kezban Kartlaşmış , Nurten Dikmen

https://doi.org/10.17826/cumj.996838

Cited By: 1

Abstract

Amaç: Bu çalışmada, substrat Glukoz-6-fosfat (G6P) ile moleküler yapı benzerliği ve enzimin kofaktör olarak Mg+2 kullanması nedeniyle, eritrosit G6PD enzimi üzerindeki in vitro etkisi incelendi.
Gereç ve Yöntem: Çalışmada sağlıklı ve G6PD enzim eksikliği olan erkek bireylerin kan örneklerinden yararlanıldı. Hazırlanan hemolizatların enzim aktiviteleri modifiye Beutler yöntemi ile ölçüldükten sonra deney ortamına farklı derişimlerde (5.3x10-3, 5.3x10-4, 5.3x10-5, 5.3x10-6 mmol/mL) glifosat eklenerek aktivite üzerindeki etkisi değerlendirildi. Daha sonra antioksidan N-asetilsisteinin normal ve mutant enzimler üzerindeki etkisi in vitro deney koşullarında glifosatlı ve glifosatsız ortamda karşılaştırıldı.
Bulgular: Sağlıklı örnekte eritrosit G6PD aktivitesi 12, enzim eksikliği bulunan olguda ise 2.5 U/gr Hb olarak saptandı. Deney ortamına glifosat eklenmesinden sonra en yüksek aktivite kaybı 60. dakikada saptandı. En yüksek inhibisyon oranı ise 5.3x10-3 mmol/mL glifosatın etkilediği çalışma setinde gözlendi. 4.7x10-7 mmol/mL N-Asetilsistein, normal aktivitedeki G6PD enziminde glifosatın oluşturduğu inhibisyonu azalttı ancak mutant G6PD enzim aktivitesi üzerinde herhangi bir değişikliğe neden olmadı.
Sonuç: Bu çalışmayla glifosatın insanlarda in vitro olarak eritrosit G6PD enzimini inhibe ettiği gösterildi. İlaveten laboratuvar ortamında G6PD enzim aktivitesinin ölçüldüğü ve/veya kullanıldığı deneysel süreçte, negatif interferans ajanı olarak davrandığı gösterildi. Ayrıca bulaşı durumlarında glifosatın insanlarda zararlı etkiye neden olmayacak limit sınırları ile ilgili çalışmalar yapılmalıdır.

Keywords

Glifosat, G6PD enzimi, inhibisyon, N-asetilsistein

Evaluation of the effect of glyphosate on glucose-6-phosphate dehydrogenase enzyme activity in vitro conditions

Abstract

Purpose: The aim of this study was to investigate in vitro effect of glyphosate on Glucose 6-phosphate dehydrogenase (G6PD) enzyme activity.
Materials and Methods: In terms of G6PD enzyme deficiency, samples taken from healthy and enzyme deficient male individuals were studied. After the hemolysates were prepared from blood sample, G6PD enyzme activities were determined by the modified Beutler method. Then, the effects of different concentrations (5.3x10-3, 5.3x10-4, 5.3x10-5, 5.3x10-6 mmol/mL) of glyphosate on G6PD activity were evaluated in normal and mutant enzymes. In addition, the in vitro effect of the antioxidant N-acetylcysteine (NAC) on the enzyme was investigated in the presence of glyphosate and without glyphosate.
Results: While the result of normal erythrocyte G6PD activity was 12U/g for the individual, the result for the individual with enzyme deficiency was 2.5U/g Hb. The glyphosate’s maximum activity loss in the G6PD enzyme was observed in the 60th minute incubation. The highest inhibition was observed at 5.3x10-3 mmol/mL glyphosate. 4.7x10-7 mmol/mL N-Acetylcysteine partially increased the inhibition of glyphosate in the G6PD enzyme in healthy individuals, but had no effect on mutant G6PD.
Conclusion: In humans, it is predicted that glyphosate affects G6PD enzyme activity in vitro and is an interference agent in the experimental process. In case of contamination, studies on limits of glyphosate that will not cause harmful effects in humans should be continued.

Keywords

Glyphosate, G6PD enzyme, inhibition, N-acetylcysteine.

References

• 1. Grube A, Donaldson D, Kiely T, Wu L. Pesticides Industry Sales and Usage. Washington, DC , EPA, 2011.
• 2. Székács A, Darvas B. Forty years with glyphosate. In Herbicides-Properties, Synthesis and Control of Weeds (Ed MNA El-Ghany Hasaneen):247-84. London, InTech, 2012.
• 3. Krebs C. Farmers look to broader strategies to battle weeds. AG J. March. 2011;11.
• 4. Antier C, Kudsk P, Reboud X, Ulber L, Baret PV, Messéan A. Glyphosate use in the European agricultural sector and a framework for its further monitoring. Sustainability. 2020;12:5682.
• 5. Knuuttila P, Knuuttila H. The crystal and molecular structure of n-(phosphonomethyl) glycine (glyphosate). Acta Chem Scand B. 1979;3:623-626.
• 6. Silva V, Montanarella L, Jones A, Fernández-Ugalde O, Mol HG, Ritsema CJ et al. Distribution of glyphosate and aminomethylphosphonic acid (ampa) in agricultural topsoils of the European Union. Sci Total Environ. 2018;621:1352-1359.
• 7. Lemke N, Murawski A, Schmied-Tobies MI, Rucic E, Hoppe H-W, Conrad A et al. Glyphosate and aminomethylphosphonic acid (ampa) in urine of children and adolescents in germany–human biomonitoring results of the german environmental survey 2014–2017 (geres v). Environ Int. 2021;156:106769.
• 8. Kostopoulou S, Ntatsi G, Arapis G, Aliferis KA. Assessment of the effects of metribuzin, glyphosate, and their mixtures on the metabolism of the model plant lemna minor l. Applying metabolomics. Chemosphere. 2020;239:124582.
• 9. Mennan H, Kaya-Altop E, Belvaux X, Brants I, Zandstra BH, Jabran K et al. Investigating glyphosate resistance in amaranthus palmeri biotypes from Turkey. Phytoparasitica. 2021:1-10.
• 10. Ozbay B, Akyol NH, Akyol G, Ozbay I. Sorption and desorption behaviours of 2, 4‐d and glyphosate in calcareous soil from Antalya, Turkey. Water Environ J. 2018;32:141-8.
• 11. Inci D, Galvin L, Al-Khatib K, Uludağ A. Sumatran fleabane (Conyza sumatrensis) resistance to glyphosate in peach orchards in Turkey. HortScience. 2019;54:873-9.
• 12. Cap MC, Eşen D. Effects of application date and rate of foliar-applied glyphosate on pine seedlings in Turkey. J For Res (Harbin). 2018;29:583-91.
• 13. Steinrücken H, Amrhein N. The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phosphate synthase. Biochem Biophys Res Commun. 1980;94:1207-12.
• 14. Kim H-W, Perez JA, Ferguson SJ, Campbell ID. The specific incorporation of labelled aromatic amino acids into proteins through growth of bacteria in the presence of glyphosate: Application to fluorotryptophan labelling to the H+-ATPase of Escherichia coli and nmr studies. FEBS Lett. 1990;272:34-6.
• 15. Barrett K, McBride M. Oxidative degradation of glyphosate and aminomethylphosphonate by manganese oxide. Environ Sci Technol. 2005;39:9223-8.
• 16. Mertens M, Höss S, Neumann G, Afzal J, Reichenbecher W. Glyphosate, a chelating agent—relevant for ecological risk assessment? Environ Sci Pollut Res Int. 2018;25:5298-317.
• 17. Grandcoin A, Piel S, Baures E. Aminomethylphosphonic acid (AMPA) in natural waters: Its sources, behavior and environmental fate. Water Res. 2017;117:187-97.
• 18. Heap I, Duke SO. Overview of glyphosate‐resistant weeds worldwide. Pest Manag Sci. 2018;74:1040-9.
• 19. Konak Ş, Polat M. Glukoz 6 fosfat dehidrogenaz enzim eksikliği; tanı ve tedavi. Mehmet Akif Ersoy University Journal of Health Sciences Institute. 2015;3:77-83.
• 20. Sasmaz I. Glukoz-6-fosfat dehidrogenaz enzim eksikligi/glucose-6-phosphate dehydrogenase deficiency. Turk Pediatr Arch. 2009:35-39.
• 21. Gómez-Manzo S, Marcial-Quino J, Vanoye-Carlo A, Serrano-Posada H, Ortega-Cuellar D, González-Valdez A et al. Glucose-6-phosphate dehydrogenase: Update and analysis of new mutations around the world. Int J Mol Sci. 2016;17:2069.
• 22. Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiency. Lancet. 2008;371:64-74.
• 23. Beutler E. G6PD deficiency. Blood. 1994;84:3613-36.
• 24. Yüregir G, Varinli İ, Donma O. Glucose 6-phosphate dehydrogenase deficiency both in red blood cells and lenses of the normal and cataractous native population of Çukurova, the southern part of Turkey. Ophthalmic Res. 1989;21:158-61.
• 25. Jain SK. Glutathione and glucose-6-phosphate dehydrogenase deficiency can increase protein glycosylation. Free Radic Biol Med. 1998;24:197-201.
• 26. Aksu TA, Esen F, Dolunay MS, Alıcıgüzel Y, Yücel G, Çalı Ş et al. Erythrocyte glucose-6-phosphate dehydrogenase (1.1. 1.49) deficiency in Antalya province, Turkey: An epidemiologic and biochemical study. Am J Epidemiol. 1990;131:1094-7.
• 27. İsbir T, Yüregir G. Çukurova'da HbS ve G6PD enzim eksikliği ve aralarındaki İlişki. Doğa Bilim Dergisi. 1984:232-44.
• 28. Luzzatto L, Battistuzzi G. Glucose-6-phosphate dehydrogenase. Adv Hum Genet. 14. 1985:217-329.
• 29. Aldini G, Altomare A, Baron G, Vistoli G, Carini M, Borsani L et al. N-acetylcysteine as an antioxidant and disulphide breaking agent: The reasons why. Free Radic Res. 2018;52:751-62.
• 30. Daruich J, Zirulnik F, Gimenez MaSa. Effect of the herbicide glyphosate on enzymatic activity in pregnant rats and their fetuses. Environ Res. 2001;85:226-231.
• 31. Glusczak L, dos Santos Miron D, Moraes BS, Simões RR, Schetinger MRC, Morsch VM et al. Acute effects of glyphosate herbicide on metabolic and enzymatic parameters of silver catfish (rhamdia quelen). Comp Biochem Physiol C Toxicol Pharmacol. 2007;146:519-24.
• 32. Cherni AE, Trabelsi D, Chebil S, Barhoumi F, Rodríguez-Llorente ID, Zribi K. Effect of glyphosate on enzymatic activities, rhizobiaceae and total bacterial communities in an agricultural tunisian soil. Water Air Soil Pollut. 2015;226:1-11.
• 33. Agostini LP, Dettogni RS, Dos Reis RS, Stur E, Dos Santos EV, Ventorim DP et al. Effects of glyphosate exposure on human health: Insights from epidemiological and in vitro studies. Sci Total Environ. 2020;705:135808.
• 34. Turkmen R, Birdane YO, Demirel HH, Yavuz H, Kabu M, Ince S. Antioxidant and cytoprotective effects of n-acetylcysteine against subchronic oral glyphosate-based herbicide-induced oxidative stress in rats. Environ Sci Pollut Res. 2019;26:11427-37.
• 35. Beutler E, Blume K, Kaplan J, Löhr G, Ramot B, Valentine W. International committee for standardization in haematology: Recommended screening test for glucose‐6‐phosphate dehydrogenase (g‐6‐pd) deficiency. Br J Haematol. 1979;43:465-467.
• 36. Bilen E, Çiçekli Ö, Aksoy U, Altindisli A. Dünya ve Türkiyede Organik Tarım. Organik Tarım İmak Ofset; 2012:8-37.
• 37. Özkan E, Aydın B. Türkiyenin farklı bölgelerindeki çeşitli tarımsal ürün maliyetlerinin ve etkileyen unsurların karşılaştırmalı değerlendirmesi. International Journal of Agricultural and Natural Sciences. 2012;5:134-138.
• 38. Madsen HL, Christensen H, Gottlieb-Petersen C. Stability constants of copper (u), zinc, manganese (ii), calcium, and magnesium complexes of n-(phosphonomethyl) glycine (glyphosate). Acta Chem Scand A. 1978;32:79-83.
• 39. Fendoğlu BY, Koçer-Gümüşel B, Erkekoğlu P. Endokrin bozucu kimyasal maddelere ve etki mekanizmalarına genel bir bakış. Hacettepe University Journal of the Faculty of Pharmacy. 2019;39:30-43.
• 40. Trasande L, Aldana SI, Trachtman H, Kannan K, Morrison D, Christakis DA et al. Glyphosate exposures and kidney injury biomarkers in infants and young children. Environ Pollut. 2020;256:113334.
• 41. Dai P, Hu P, Tang J, Li Y, Li C. Effect of glyphosate on reproductive organs in male rat. Acta Histochem. 2016;118:519-26.
• 42. Laguarda-Miro N, Ferreira FW, García-Breijo E, Ibáñez-Civera J, Gil-Sánchez L, Garrigues-Baixauli J. Glyphosate detection by voltammetric techniques. A comparison between statistical methods and an artificial neural network. Sens Actuators B Chem 2012;171:528-36.
• 43. Tarazona JV, Tiramani M, Reich H, Pfeil R, Istace F, Crivellente F. Glyphosate toxicity and carcinogenicity: A review of the scientific basis of the european union assessment and its differences with iarc. Arch Toxicol. 2017;91:2723-43.
• 44. Bancone G, Chu CS. G6pd variants and haemolytic sensitivity to primaquine and other drugs. Front Pharmacol. 2021;12:283.
• 45. Altay Ç, Gümrük F. Red cell glucose-6-phosphate dehydrogenase deficiency in Turkey. Turk J Haematol. 2008;25:1-7.
• 46. Akoğlu T, Çığ S, Kümı M, Erdoğan A, Anıl H. Glucose—6—phosphate dehydrogenase deficiency in çukurova province, Turkey. Int J Epidemiol. 1981;10:27-9.
• 47. Pochron S, Simon L, Mirza A, Littleton A, Sahebzada F, Yudell M. Glyphosate but not roundup® harms earthworms (Eisenia fetida). Chemosphere. 2020;241:125017.
• 48. Yang H-C, Wu Y-H, Liu H-Y, Stern A, Chiu DT-Y. What has passed is prolog: New cellular and physiological roles of g6pd. Free Radic Res. 2016;50:1047-64.