JOMPAC

Journal of Medicine and Palliative Care (JOMPAC) is an open access scientific journal with independent, unbiased, and double-blind review under international guidelines. The purpose of JOMPAC is to contribute to the literature by publishing articles on health sciences and medicine.

EndNote Style
Index
Review
Use and comparison of MTT, XTT and iCELLigence methods in the evaluation of drug toxicity
Toxicology tests are one of the fundamental methods used in biological and pharmaceutical research. These tests are used to evaluate the viability, proliferation and toxic responses of cells. The study of biological activities of cells plays a critical role in drug development, cancer research, toxicology and various biotechnological applications. Drug toxicology is an important field of research to determine the harmful effects of drugs on cellular and biological systems. In vitro tests are widely used for accurate evaluation of drugs and their toxic effects. These tests examine the effects of drugs on cell cultures, rapidly revealing their potential harmful effects in terms of time and resources. This review discusses the advantages of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and XTT (sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4 methoxy-6-nitro) benzene-sulfonic acid hydrate) tests, which are widely used as cytotoxicity tests, as well as the newer method iCELLigence. MTT and XTT are widely used and reliable tests that measure cell metabolism; both methods are very effective in assessing cell viability, but provide limited dynamic data. In contrast, iCELLigence is a newer technology and provides more in-depth data by monitoring the real-time responses of cells. iCELLigence continuously monitors the growth rate and morphological changes of cells, allowing for more comprehensive and sensitive results compared to traditional methods. Comparison of these methods allows determining which methodology provides more appropriate results according to different research needs.These tests are also used to define the concentration range over which more comprehensive and detailed in vitro testing can be performed to obtain meaningful data on parameters such as genotoxicity, mutation induction or programmed cell death. This review aims to compare these three methods and discuss their advantages and limitations in the assessment of drug toxicity.


1. Broadhead CL, Combes RD. The current status of food additives toxicity testing and the potential for application of the three Rs.ATLA. 2001; 29(4):471-485. doi:10.1177/026119290102900403
2. Çinarli M, Ataol ÇY, Zeyrek CT, Kiray E, Demirkalp ANC. Synthesis, single crystal investigations, DFT studies, biological activities, DNA cytotoxicity and molecular docking studies of copper (II) complex derived from the new o-vanillin Schiff base ligand.Polyhedron. 2024; 117315:1-15. doi:10.1016/j.poly.2024.117315
3. Gribaldo L, Casati S, Figliuzzi L, Marafante E. In vitro myelotoxicity of environmental contaminants. Environ Toxicol Pharmacol. 1995;6(2): 135-141. doi:10.1016/S1382-6689(98)00029-5
4. Bácskay I, Nemes D, Fenyvesi, F, et al. Role of cytotoxicity experiments in pharmaceutical development.Cytotoxicity. 2018;8:131-146. doi:10.5772/intechopen.72539
5. Gutiérrez AC, Hoyos CG, Cock, JV, et.al. Health and toxicological effects of nanocellulose when used as a food ingredient: a review.Carbohydr Polym. 2024;323:121382. doi:10.1016/j.carbpol.2023.121382
6. Ali A, Banerjee S, Kamaal S, et al. Ligand substituent effect on the cytotoxicity activity of two new copper (ii) complexes bearing 8-hydroxyquinoline derivatives: validated by MTT assay and apoptosis in MCF-7 cancer cell line (human breast cancer).RSC Adv. 2021;11(24): 14362-14373. doi:10.1039/D1RA00172H
7. Terr AI. The cytotoxic test.West J Med. 1983;139(5):702.
8. Fenech M, Crott J, Turner J, Brown S. Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide.Mutagenesis. 1999;14(6):605-612. doi:10.1093/mutage/14.6.605
9. Riss TL, Moravec RA, Niles AL. Cytotoxicity testing: measuring viable cells, dead cells, and detecting mechanism of cell death. In: Stoddart MJ. Eds.Mammalian cell viability: methods and protocols. Humana Press. 2011. doi:10.1007/978-1-61779-108-6_12
10. Wang K, Shindoh H, Inoue T, Horii I. Advantages of in vitro cytotoxicity testing by using primary rat hepatocytes in comparison with established cell lines.J Toxicol Sci. 2002;27(3):229-237. doi:10.2131/jts.27.229
11. Kumar P, Nagarajan A, Uchil PD. Analysis of cell viability by the MTT assay.ColdSpringHarb Protoc. 2018;2018(6):469-471. doi:10.1101/pdb.prot095505
12. Zegers J, Peters M, Albada B. DNA G-quadruplex-stabilizing metal complexes as anticancer drugs. J Biol Inorg Chem. 2023;28(2):117-138. doi:10.1007/s00775-022-01973-0
13. Scudiero DA, Shoemaker RH, Paull KD, et al. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.Cancer Res. 1988;48(17): 4827-4833.
14. Aslantürk ÖS. In vitro cytotoxicity and cell viability assays: principles, advantages, and disadvantages. In: Larramendy ML, Soloneski S. eds.Genotoxicity-A predictable risk to our actual world. 2018. doi:10. 5772/intechopen.71923
15. Ghasemi M, Turnbull T, Sebastian S, Kempson I. The MTT assay: utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis.Int J Mol Sci. 2021;22(23):12827. doi:10.3390/ijms222312827
16. Şener LT, Albeni z G, Dinç B, Albeniz I. iCELLigence real-time cell analysis system for examining the cytotoxicity of drugs to cancer cell lines.Exp Ther Med. 2017;14(3):1866-1870. doi:10.3892/etm.2017.4781
17. Teng Z, Kuang X, Wang J, Zhang X. Real-time cell analysis-a new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity.J VirolMethods. 2013;193(2):364-370. doi:10.1016/j.jviromet.2013.06.034
18. Berg K, Hansen MB, Nielsen SE. A new sensitive bioassay for precise quantification of interferon activity as measured via the mitochondrial dehydrogenase function in cells (MTT-method).Apmis. 1990;98(1-6): 156-162. doi:10.1111/j.1699-0463.1990.tb01016.x
19. Supino R. MTT assays, In: O’Hare S, Atterwil CK eds. In vitro toxicity testing protocols. Methods in Molecular Biology™, Humana Press, New Jersey, 1995.
20. Stockert JC, Blázquez-Castro A, Cañete M, Horobin RW, Villanueva Á. MTT assay for cell viability: intracellular localization of the formazan product is in lipid droplets.Acta Histochem. 2012;114(8):785-796. doi:10. 1016/j.acthis.2012.01.006
21. Huet O, Petit JM, Ratinaud MH, Julien R. NADH-dependent dehydrogenase activity estimation by flow cytometric analysis of 3-(4, 5-dimethylthiazolyl-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction.Cytometry A. 1992;13(5):532-539. doi:10.1002/cyto.990130513
22. Wang X, Ge J, Wang K, Qian J, Zou Y. Evaluation of MTT assay for measurement of emodin-induced cytotoxicity.Assay Drug Dev Technol. 2006;4(2):203-207. doi:10.1089/adt.2006.4.203
23. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.J ImmunolMethods. 1983;65(1-2):55-63. doi:10.1016/0022-1759(83)90303-4
24. Wang S, Yu H, Wickliffe JK. Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO<sub>2</sub>.ToxicolIn Vitro. 2011;25(8):2147-2151. doi:10.1016/j.tiv.2011. 07.007
25. Goodwin CJ, Holt SJ, Downes S, Marshall NJ. Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS.J ImmunolMethods. 1995;179(1):95-103. doi:10.1016/0022-1759 (94)00277-4
26. Zhang W, Zhu M, Wang F, et al. Mono-sulfonated tetrazolium salt based NAD (P) H detection reagents suitable for dehydrogenase and real-time cell viability assays.Anal Biochem. 2016;509:33-40. doi:10.1016/j.ab. 2016.06.026
27. Hawser S. Adhesion of different Candida spp. to plastic: XTT formazan determinations.J Med Vet Mycol. 1996;34(6):407-410. doi:10. 1080/02681219680000721
28. Hawser S. Comparisons of the susceptibilities of planktonic and adherent Candida albicans to antifungal agents: a modified XTT tetrazolium assay using synchronised C. albicans cells.J Med Vet Mycol. 1996;34(2):149-152. doi:10.1080/02681219680000231
29. Skehan P., Storeng R., Scudiero D., et al. New colorimetric cytotoxicity assay for anticancer-drug screening.JNCI:J Natl Cancer Inst. 1990; 82(13):1107-1112. doi:10.1093/jnci/82.13.1107
30. Ke N, Wang X, Xu X, Abassi YA. The xCELLigence system for real-time and label-free monitoring of cell viability. Methods Mol Biol. 2011;740: 33-43. doi:10.1007/978-1-61779-108-6_6
31. Giaever I and Keese CR: A morphological biosensor for mamma&shy;lian cells. Nat. 1993;366(6455):591-592. doi:10.1038/366591a0
32. Lee RC. Cell injury by electric forces.Ann N Y Acad Sci. 2006;1066(1):85-91. doi:10.1196/annals.1363.007
33. Unwin N. The structure of ion channels in membranes of excitable cells.Neuron. 1989;3(6):665-676. doi:10.1016/0896-6273(89)90235-3
34. Wang T, Hu N, Cao J, Wu J, Su K, Wang P. A cardiomyocyte-based biosensor for antiarrhythmic drug evaluation by simultaneously monitoring cell growth and beating.Biosens Bioelectron.2013;49:9-13. doi:10.1016/j.bios.2013.04.039
35. Schr&ouml;terov&aacute; L, Kr&aacute;lov&aacute; V, Vor&aacute;cov&aacute; A, Ha&scaron;kov&aacute; P, Rudolf E, Cervinka, M. Antiproliferative effects of selenium compounds in colon cancer cells: comparison of different cytotoxicity assays.Toxicol in vitro. 2009; 23(7):1406-1411. doi:10.1016/j.tiv.2009.07.013
36. Atmaca H, Bozkurt E, Kısım A, Uslu R. Comparative analysis of XTT assay and xCELLigence system by measuring cytotoxicity of resveratrol in human cancer cell lines.Turk Biyokim Derg. 2016;41(6):413-421. doi: 10.1515/tjb-2016-0128
37. Garcia SN, Gutierrez L, McNulty A. Real-time cellular analysis as a novel approach for in vitro cytotoxicity testing of medical device extracts.J Biomed Mater Res A. 2013;101(7):2097-2106. doi:10.1002/jbm.a.34507
38. T&uuml;rker Şener L, Albeniz G, Din&ccedil; B, Albeniz I. iCELLigence real-time cell analysis system for examining the cytotoxicity of drugs to cancer cell lines.Exp Ther Med. 2017;14(3):1866-1870. doi:10.3892/etm.2017.4781
39. Burton JD. The MTT assay to evaluate chemosensitivity. In: Blumenthal RD. eds.Chemosensitivity: Volume 1 In Vitro Assays. 2005. doi:10. 1385/1-59259-869-2:069
40. Mahto SK, Chandra P, Rhee SW. In vitro models, endpoints and assessment methods for the measurement of cytotoxicity.J Toxicol Environ Health Sci. 2010;2(2):87-93. doi:10.1007/BF03216487
41. Lundstrom K. Cell-impedance-based label-free technology for the identification of new drugs.ExpertOpinDrugDiscov. 2017;12(4):335-343. doi:10.1080/17460441.2017.1297419
Volume 6, Issue 1, 2025
Page : 66-71
_Footer