Abstract
A quantitative colorimetric assay using the oxidation–reduction indicator resazurin was developed to measure cytotoxicity of compounds against the protozoan parasite Trypanosoma cruzi. This method is based on the detection of colorimetric changes caused by the oxidation (blue) and reduction (pink) capabilities of resazurin dye, an indicator for metabolic cell function. To validate the assay, the experimental conditions were adjusted, such as number of parasites, dye concentration, and time of incubation, with respect to linearity and lower limit of detection. We found that absorbances increased linearly, with the plating density of parasites as low as 5–100×104/well (r=0.99; p<0.001) when they were incubated for 5 h at 28°C in the presence of 10% resazurin solution (3 mM). When the cytotoxicity of the reference drugs nifurtimox and benznidazole was measured with this assay and compared to the microscopic counting method, the same range was obtained, demonstrating that the resazurin microtiter assay is valid for the screening of new trypanocidal compounds. This test is very simple, fast, sensitive, and cheap.
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Batchelor RH, Zhou M (2004) Use of cellular glucose-6-phosphate dehydrogenase for cell quantitation: applications in cytotoxicity and apoptosis assays. Anal Biochem 329(1):35–42
Benavides T, Mitjans M, Martinez V, Clapes P, Infante MR, Clothier RH, Vinardell MP (2004) Assessment of primary eye and skin irritants by in vitro cytotoxicity and phototoxicity models: an in vitro approach of new arginine-based surfactant-induced irritation. Toxicology 197(3):229–237
Castro JA, Diaz de Toranzo EG (1988) Toxic effects of nifurtimox and benznidazole, two drugs used against American trypanosomiasis (Chagas’ disease). Biomed Environ Sci 1:19–33
Erb RE, Ehlers MH (1950) Resazurin reducing time as an indicator of bovine semen capacity. J Dairy Sci 33:853–864
Evans SM, Casartelli A, Herreros E, Minnick DT, Day C, George E, Westmoreland C (2001) Development of a high throughput in vitro toxicity screen predictive of high acute in vivo toxic potential. Toxicol In Vitro 15(4–5):579–584
Gabrielson J, Hart M, Jarelov A, Kuhn I, McKenzie D, Mollby R (2002) Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates. J Microbiol Methods 50(1):63–73
Gómez-Barrio A, Martínez-Díaz RA, Atienza J, Escario JA, Diego C, Avendaño C (1997) New derivatives of gentian violet as trypanocides: in vitro and in vivo assays on Trypanosoma cruzi. Res Rev Parasitol 57:25–31
Guerin TF, Mondido M, McClenn B, Peasley B (2001) Application of resazurin for estimating abundance of contaminant-degrading micro-organisms. Lett Appl Microbiol 32(5):340–345
Liu D (1981) A rapid biochemical test for measuring chemical toxicity. Bull Environ Contam Toxicol 26(2):145–149
Mann CM, Markham JL (1998) A new method for determining the minimum inhibitory concentration of essential oils. J Appl Microbiol 84(4):538–544
Martin A, Camacho M, Portaels F, Palomino JC (2003) Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: rapid, simple, and inexpensive method. Antimicrob Agents Chemother 47(11):3616–3619
Mikus J, Steverding D (2000) A simple colorimetric method to screen drug cytotoxicity against Leishmania using dye Alamar Blue. Parasitol Int 48:259–265
Muelas-Serrano S, Nogal-Ruiz JJ, Gomez-Barrio A (2000) Setting of a colorimetric method to determine de viability of Trypanosoma cruzi epimastigotes. Parasitol Res 86:999–1002
O’Brien J, Wilson I, Orton T, Pognan F (2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267(17):5421–5426
Pagé B, Pagé M, Noël C (1993) A new fluorometric assay for cytotoxicity measurements in vitro. Int J Oncol 3:473–476
Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F (2002) Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 46(8):2720–2722
Perrot S, Dutertre-Catella H, Martin C, Rat P, Warnet JM (2003) Resazurin metabolism assay is a new sensitive alternative test in isolated pig cornea. Toxicol Sci 72(1):122–129
Putnam KP, Bombick DW, Doolittle DJ (2002) Evaluation of eight in vitro assays for assessing the cytotoxicity of cigarette smoke condensate. Toxicol In Vitro 16(5):599–607
Rassmusen EV (1999) Use of fluorescent redox indicator to evaluate cell proliferation and viability. In Vitro Mol Toxicol 12:47–58
Räz B, Iten Y, Grether Bühler R, Brun R (1997) The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro. Acta Tropica 68:139–147
Silva LH, Nussensweig V (1953) Sobre uma cepa Trypanosoma cruzi virulenta para o camundongo branco. Folia Clin Biol 20:191–207
Vega C, Rolón M, Martínez-Fernández AR, Escario JA, Gómez-Barrio A (2005) A new pharmacological screening assay with Trypanosoma cruzi epimastigotes expressing beta-galactosidase. Parasitol Res 95(4):296–298
Wang S, Holyoak GR, Panter KE, Liu Y, Evans RC, Bunch TD (1998) Resazurin reduction assay for ram sperm metabolic activity measured by spectrophotometry. Proc Soc Exp Biol Med 217(2):197–202
World Health Organization (1991) Control of Chagas’ disease. WHO Tech Rep Ser 811:1–93
Zhang HX, Du GH, Zhang JT (2004) Assay of mitochondrial functions by resazurin in vitro. Acta Pharmacol Sin 25(3):385–389
Zrimsek P, Kunc J, Kosec M, Mrkun J (2004) Spectrophotometric application of resazurin reduction assay to evaluate boar semen quality. Int J Androl 27(1):57–62
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Rolón, M., Vega, C., Escario, J.A. et al. Development of resazurin microtiter assay for drug sensibility testing of Trypanosoma cruzi epimastigotes. Parasitol Res 99, 103–107 (2006). https://doi.org/10.1007/s00436-006-0126-y
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DOI: https://doi.org/10.1007/s00436-006-0126-y