Tetrazolium-Based Cytotoxicity Tests May Not Always Reflect Accurate Results

Abstract

Background: Cell viability and/or cytotoxicity analysis is one of the most important tools used for biological evaluation in vitro studies. The selection of the right cytotoxicity tests is critical to form the basis for in vivo and preclinical studies, specifically for cancer research. In the present study, we aimed to investigate the cytotoxic effects of bromelain, a widely-used phytochemical product in the medical field, and idarubicin, an anthracycline antibiotic used in the treatment of cancer, in normal lymphocytes and a promyelocytic leukemia cell line (HL-60) with MTT, WST-1, and luminescent ATP assays and to compare the results of these tests..
Materials and Methods: We obtained peripheral blood lymphocytes from healthy, young, non-smoker male volunteers and obtained the HL-60 cell line from the American Type Culture Collection (ATCC). Bromelain and idarubicin were added in increasing concentrations to both cell lines. Cells were incubated at 37°C in a carbon dioxide incubator for 24 h. After incubation, cytotoxicity levels were determined by MTT, WST-1, and ATP assays, and morphological evaluations were performed by fluorescent staining.
Results: The MTT and WST-1 assays demonstrated that cell viability/formazan formation increased with bromelain concentration; however, the luminescent ATP assay demonstrated that cell viability decreased with increasing concentrations of bromelain. Whereas fluorescent staining methods confirmed the ATP assay results, the MTT and WST-1 assays contradicted the ATP assay results. The cytotoxic effects of idarubicin were similar in the two cell lines according to the three different measurement methods and were positively correlated with the results of the fluorescent staining methods.
Conclusion: The detection of cell viability and cytotoxicity by bromelain with the MTT and WST-1 assays in lymphocytes and HL-60 cells is limited. To obtain accurate and reliable results from cytotoxicity studies, a measurement method should be carefully selected by considering that the phytochemicals to be tested could interfere with the results, and the results should be verified by other methods.

Keywords

• Bromelain,idarubicin,cytotoxicity tests,interference,tetrazolium salts

Tetrazolium Bazlı Sitotoksisite Testleri Her Zaman Doğru Sonuçları Yansıtmayabilir

Abstract

Amaç: Hücre canlılığı ve/veya sitotoksisite analizleri in vitro çalışmalarda biyolojik değerlendirme için kullanılan en önemli göstergelerdendir. Özellikle kanser araştırmalarında, in vivo ve preklinik çalışmalara kaynaklık etmesi bakımından sitotoksisite testlerinin seçimi kritik öneme sahiptir. Bu çalışmada medikal kullanımı yaygın fitokimyasal bir ürün olan bromelain ve kanser tedavisinde kullanılan antrasiklin antibiyotik bir ilaç olan idarubisin’in MTT, WST-1 ve Luminesan ATP yöntemleriyle sitotoksik etkilerinin, normal lenfosit hücre kültürü ve HL-60 promyelositik lösemi hücre hattında araştırılması ve testler arasındaki ilişkinin incelenmesi amaçlandı.
Materyal ve Metod: Periferal kan lenfositleri sigara içmeyen, sağlıklı, gönüllü, genç erkek bireyden, HL-60 promyelosik lösemi hücre hattı ise ATCC firmasından sağlandı. Bromelain ve idarubisinin artan konsantrasyonları her iki hücre hattına eklendi. Hücreler, 24 saat 37 °C’de karbondioksit inkübatöründe inkübe edildi. İnkübasyon sonrası MTT, WST-1 ve ATP analizleriyle sitotoksisite düzeyleri araştırılırken, floresan boyama yöntemleriyle de morfolojik incelemeler yapıldı.
Bulgular: MTT ve WST-1 analizlerinde, bromelain konsantrasyon artışına paralel olarak hücre canlılığının/formazan oluşumunun arttığı, ATP testinde ise konsantrasyon artışıyla hücre canlılığının azaldığı bulundu. Floresan boyama yöntemleri ile ATP analiz sonuçları doğrulanırken, MTT ve WST-1 negatif ilişkiliydi. İdarubisin’in sitotoksik etkisinin her iki hücre hattında 3 ölçüm yöntemi ile benzer olduğu, floresan boyama yöntemleri ile de pozitif ilişkili olduğu bulundu
Sonuç: Bromelain’in lenfosit ve HL-60 hücre dizileri üzerinde MTT ve WST-1 analiz yöntemleri ile hücre canlılığı/sitotoksisite tespitinde bazı ölçüm sınırlamalarına neden olduğu bulunmuştur. Sitotoksisite çalışmalarında doğru ve güvenilir sonuçlar elde etmek için analiz edilecek fitokimyasalların interferenslere sebep olabileceği göz önüne alınarak yöntem seçimi dikkatlice yapılmalı ve başka metodlarla sonuçlar doğrulanmalıdır.

Keywords

• Bromelain,idarubisin,sitotoksisite testleri,interferans,tetrazolyum

References

1. 1. Estanqueiro M, Amaral MH, Conceição J, Lobo JM. Nanotechnological carriers for cancer chemotherapy: the state of the art. Colloids Surf B. 2015; 126: 631–48.
2. 2. Tokur O, Aksoy A. İn Vitro Sitotoksisite Testleri. Harran Üniversitesi Veteriner Fakültesi Dergisi. 2017; 6(1): 112-118.
3. 3. Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, et all. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ. 2009; 16: 1093-1107.
4. 4. Riss TL, Moravec RA, O’brien M A, Hawkins EM, Niles A. Homogeneous multiwell assays for measuring cell viability, cytotoxicity, and apoptosis. In “Handbook Of Assay Development In Drug Discovery”, Ed; Minor LK, CRC Press. 2006; 385-405.
5. 5. Crouch SPM, Kozlowski R, Slater KJ, Fletcher J. The use of ATP bioluminescence as a measure cell proliferation and cytotoxicity. J Immunol Methods. 1993; 160: 81-88.
6. 6. Fan F, Wood KV. Bioluminescent assays for highthroughput screening. Assay Drug Dev Techn. 2007; 5(1): 127-136.
7. 7. Longo-Sorbello GSA, Saydam G, Banerjee D, Bertino JR. Cytotoxicity and cell growth assays. In “Cell Biology: A Laboratory Handbook”, Ed; Celis JE, Elseiver Academic Press, Burlington. 2006: 315-325.
8. 8. Mossman T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65: 55-63.

9. 9. Riss TL, Moravec RA. Cell proliferation assays: improved homogeneous methods used to measure the number of cells in culture. In “Cell Biology”, Ed; Celis JE, Elseiver Academic Press, Burlington. 2006: 25-31.
10. 10. Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev. 2005; 11: 127–152.
11. 11. Riss TL, Moravec RA, Niles AL, Duellmann S, Benink HA, Worzella TJ, Minor L. Assay guidance manual: Cell viability assays. 2016 Available from: https://www.ncbi.nlm.nih.gov/books/NBK144065/
12. 12. Stepanenko AA, Dmitrenko VV. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability. Gene. 2015; 574(2): 193-203.
13. 13. Aluko BT, Oloyede OI, Afolayan AJ. Polyphenolic contents and free radical scavenging potential of extracts from leaves of Ocimum americanum L. Pak J Biol Sci. 2013; 16: 22–30.
14. 14. Majo DD, Giammanco M, Guardia ML, Tripoli E, Giammanco S, Finotti E. Flavanones in citrus fruit: structure–antioxidant activity relationships. Food Res Int. 2005; 38: 1161–1166.
15. 15. Kocyigit A, Koyuncu I, Taskin A, Dikilitas M, Bahadori F, Turkkan B. Antigenotoxic and antioxidant potentials of newly derivatized compound naringenin-oxime relative to naringenin on human mononuclear cells. Drug Chem Toxico. 2016; 39: 66-73.
16. 16. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods. 1983; 65(1‑2): 55‑63.
17. 17. Liu K, Liu PC, Liu R, Wu X. Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry. Med Sci Monit Basic Res. 2015; 21: 15-20.
18. 18. Rahman A, Hussain A. Anticancer activity and apoptosis inducing effect of methanolic extract of Cordia dichotoma against human cancer cell line. Bangladesh J Pharmacol 2015; 10: 27-34.
19. 19. Adan A, Kiraz Y, Baran Y. Cell Proliferation and Cytotoxicity Assays. Curr Pharm Biotechnol. 2016; 17(14): 1213-1221.
20. 20. Chrzanowska C, Hunt SM, Mohammed R, Tilling PJ. The use of cytotoxicity assays for the assessment of toxicity. In: EHT 9329, Final Report to the Department of the Environment. 1990.
21. 21. Riss TL, Moravec RA. Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev Techn. 2004, 2: 51-62.
22. 22. Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Okhura Y, Ueno KA. Combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biological & Pharmaceutical Bulletin. 1996; 19(11): 1518-1520
23. 23. Özlem Sultan Aslantürk. In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages, Genotoxicity - A Predictable Risk to Our Actual World, IntechOpen 2017:1-17. DOI: 10.5772/intechopen.71923
24. 24. Sliwka L, Wiktorska K, Suchocki P, Milczarek M, Mielczarek S, Lubelska K, et all. The comparison of MTT and CVS assays for the assessment of anticancer agent interactions. PLoS One. 2016; 11(5): e0155722. DOI: 10.1371/journal.pone.0155772.
25. 25. Weyermann J, Lochmann D, Zimmer AA. Practical note on the use of cytotoxicity assays. Int. J. Pharm. 2005; 288: 369–376.
26. 26. van Tonder A, Joubert AM, Cromarty AD. Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Res Notes. 2015; 20; 8:47.
27. 27. Karakas D, Ari F, Ulukaya E. The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant extracts. Turk J Biol. 2017; 18;41(6):919-925.
28. 28. Ulukaya E, Colakogullari M, Wood EJ. Interference by anti-cancer chemotherapeutic agents in the MTT-tumor chemosensitivity assay. Chemotherapy. 2004; 50: 43-50.
29. 29. Peng L, Wang B, Ren P. Reduction of MTT by flavonoids in the absence of cells. Colloid Surface B 2005; 45: 108-111.
30. 30. Ganapathy-Kanniappan S, Morgan RH. The pyruvic acid analogue 3-bromopyruvate interferes with the tetrazolium reagent MTS in the evaluation of cytotoxicity. Assay Drug Dev Techn. 2010; 8(2): 258–62.
31. 31. Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nogziger TH, et all. Evaluation of a soluble tetrazoliun/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research. 1988; 48: 4827-4833.
32. 32. "Water Soluble Tetrazolium Salts (WSTs)" interchim.com. Interchim Retrieved 2013, Available from: https://www.interchim.fr/ft/F/F98881.pdf
33. 33. Wang YJ, Zhou SM, Xu G, Gao YQ. Interference of Phenylethanoid Glycosides from Cistanche tubulosa with the MTT Assay. Molecules. 2015; 20(5): 8060-8071.
34. 34. Bruggisser R, von Daeniken K, Jundt G, Schaffner W, Tullberg-Reinert H. Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay. Planta Med. 2002; 68(5): 445-448.
35. 35. Ulukaya E, Ozdikicioglu F, Oral AY, Demirci M. The MTT assay yields a relatively lower result of growth inhibition than the ATP assay depending on the chemotherapeutic drugs tested. Toxicol In Vitro. 2008; 22(1): 232-239.
36. 36. Braun K, Stürzel CM, Biskupek J, Kaiser U, Kirchhoff F, Lindén M. Comparison of different cytotoxicity assays for in vitro evaluation of mesoporous silica nanoparticles. Toxicol In Vitro. 2018; 52: 214-221.
37. 37. Neufeld BH, Tapia JB, Lutzke A, Reynolds MM. Small Molecule Interferences in Resazurin and MTT-Based Metabolic Assays in the Absence of Cells. Anal Chem. 2018; 90(11): 6867-6876.