Parasitol Res (2000) 86: 999±1002
Ó Springer-Verlag 2000
ORIGINAL PAPER
S. Muelas-Serrano á J. J. Nogal-Ruiz á A. GoÂmez-Barrio
Setting of a colorimetric method to determine the viability of Trypanosoma cruzi epimastigotes
Received: 8 February 2000 / Accepted: 5 June 2000
Abstract The method most commonly used in screening of drugs for the treatment of Chagas' disease, microscopic counting of viable trypanosomes, is time-consuming, labor-intensive, and dependent on the observer. Although the tetrazolium dye [MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is comparatively quick and accurate, it requires careful attention in design as well as in interpretation of the results. Therefore, we examined under various conditions the sensitivity and speci®city of the MTT assay versus microscopic counting for determination of the viability of Trypanosoma cruzi for drug-screening purposes. We tested dierent concentrations of MTT in phenazine methosulfate (PMS) against T. cruzi epimastigotes of the Y strain in dierent stages of logarithmic growth. In our model, in tests of benznidazole and nifurtimox the optimal concentration of MTT was 2.5 mg/ml of PMS and the optimal incubation period was 75 min. This method detected parasite concentrations of approx. 500,000 epimastigotes/ml (P < 0.01), and the linear correlation between absorbance values and numbers of epimastigotes per milliliter was very strong (approx. R 0.99). The present MTT assay results in faster determination of the activity of compounds, is more objective, and enables testing of several drugs simultaneously. Trypanosoma cruzi causes American trypanosomiasis or Chagas' disease. This illness continues to be a major public health problem in Latin America (Kirchho 1994). The WHO has estimated that approximately 16± 18 million people harbor T. cruzi and that more than 50,000 people die each year of Chagas' disease (WHO
S. Muelas-Serrano á J. J. Nogal-Ruiz á A. GoÂmez-Barrio (&) Departamento de ParasitologõÂ a, Facultad de Farmacia, Universidad Complutense de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain e-mail:
[email protected] Fax: +34-1-3941815
1991). The chemotherapy of Chagas' disease remains an unsolved problem, and the search for alternative drug treatments continues (De Castro 1993). Nifurtimox and benznidazole, the only two drugs currently available for treatment against T. cruzi, are eective in reducing the severity of acute and congenital Chagas' disease but have no role in the treatment of chronic infections (Kirchho 1994). Screening of new trypanocidal compounds has primarily involved microscopic counting, a process that has several drawbacks: it is labor- and time-intensive and highly dependent on the observer. Another method, the MTT assay, which is based on the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan, is widely used for in vitro measurement of the metabolic activity or viability of cell cultures (Sieuwerts et al. 1995). This colorimetric assay has successfully been used with mammalian cells and with some protozoa, such as Leishmania promastigotes (Berg et al. 1994) and amastigotes (Sereno and Lemesre 1997), T. brucei trypomastigotes (Ellis et al. 1993), Entamoeba histolytica (Cedillo-Rivera et al. 1992), Giardia duodenalis trophozoites (Ponce-Macotela et al. 1994), and Tetrahymena pyriformis (Hegyesi and Csaba 1997). The MTT assay is a quick and accurate method. However, attention should be paid to the design of the experiment and the interpretation of the results; the optimal MTT concentration and incubation period should be established (Sieuwerts et al. 1995). Phenazine methosulfate (PMS, 0.22 mg/ml of MTT solution) has been used as an intermediate electron carrier for enhancement of the formazan yield and, hence, reduction of the period of incubation (Cedillo-Rivera et al. 1992; Ponce-Macotela et al. 1994). In this study, T. cruzi epimastigotes of the Y strain were used in all experiments. They were grown axenically at 28 °C in liver-infusion tryptose (LIT) medium supplemented with 10% heat-inactivated fetal calf serum (FCS). For determination of the eect of the MTT concentration used, ®ve solutions of MTT-PMS were prepared at concentrations ranging from 1 to 10 mg
1000
MTT/ml. Of each of these solutions, 100 ll was added to the 24 wells of 5 dierent plates. These plates were seeded at 900 ll/well with a log-phase culture of T. cruzi epimastigotes. We carried out this assay twice, using three dierent concentrations of epimastigotes (six wells per concentration of epimastigotes) and negative controls each time. The concentration of parasites ranged from 0 to 6.34 ´ 106 epimastigotes/ml. After 45 min of incubation at 28 °C, 1 ml of a 10% sodium dodecyl sulfate (SDS)-0.01 N HCl solution was added to dissolve the formazan crystals that had formed. Plates were further incubated for 30 min at room temperature. The absorbance values were read on a scanning multiwell spectrophotometer at a test wavelength of 595 nm (A595). To choose the optimal duration of incubation for cultures in the presence of MTT-PMS, we incubated ®ve plates for periods ranging from 30 to 90 min, after which the experiments proceeded as described above. For determination of the eect of the growth phase, one culture in the stationary phase and two cultures in dierent stages of the log phase were chosen. Multiple dilutions of these were made for establishment of (a) the linear correlation between absorbance values and numbers of epimastigotes per milliliter and (b) the capability of this method to distinguish between two dierent concentrations of epimastigotes. To evaluate the possibility of using the MTT assay as a substitute for microscopic counting in drug screening, we assayed eight dierent concentrations of benznidazole and nine of nifurtimox against epimastigotes of T. cruzi, using both methods to determine their viability. The in vitro activity of these two products was determined as previously described (GoÂmez-Barrio et al. 1997). In brief, cell-culture plates consisting of 24 wells were ®lled at 2 ml/well with T. cruzi epimastigote culture during its exponential growth in LIT medium. Dierent concentrations of benznidazole (range 1±100 lg/ml) and of nifurtimox (range 0.5±100 lg/ml) dissolved in dimethylsulfoxide (DMSO) were added and maintained for 3 days, after which parasites were counted in a hemocytometer for determination of the cytotoxicity percentage (%C) as an ecacy parameter. When the MTT assay was used, 900 ll of culture was harvested into wells; solutions of the compounds were added; the cultures were seeded for 96 h at 28 °C; and the percentage of cytotoxicity was determined; i.e., 100 ll of a 2.5-mg/ml MTT-PMS solution was added to each well, plates were incubated for 75 min, formazan crystals were dissolved with 1 ml of the SDS solution, and plates were read at 595 nm. Each concentration was assayed three times and six growth controls were used in each test. When MTT was used the assays were performed twice. The cytotoxicity percentage was calculated as follows: %C
Gc
Gp =Gc 100
%C
Ac
Am
Ap
Apm =
Ac
Am 100 ;
where Gc represents the mean number of parasites noted per milliliter in control wells; Gp represents the mean number of parasites detected per milliliter according to the dierent doses of product assayed; Ac represents the mean A595 value recorded for control wells; Am represents the mean A595 value recorded for medium/control wells; Ap represents the mean A595 value recorded for culture containing dierent doses of product; and Apm represents the mean A595 value recorded for dierent doses of product in medium. The formazan production was found to depend on the MTT concentration in the culture medium, and it also varies in a cell-speci®c manner (Sieuwerts et al. 1995). The commonly used concentration of MTT solution has been 5 mg/ml (Cedillo-Rivera et al. 1992; Ellis et al. 1993; Hegyesi and Csaba 1997). However, we established the optimal concentration to be 2.5 mg/ml. The absorbance values recorded for each concentration of epimastigotes were very similar at MTT concentrations ranging from 2.5 to 10 mg/ml but exceeded those obtained using the 1-mg/ml MTT concentration. The formazan production was also found to be timedependent up to a maximal period from which a prolongation would result in a decrease in measured absorbance, particularly at higher cell densities (Sieuwerts et al. 1995). The optimal MTT-incubation period in our model was 75 min. The A595 value increased proportionally to the duration of incubation for up to 75 min, after which it remained constant or decreased for a given concentration of epimastigotes. Due to the high concentration of nutrients in fresh culture medium, epimastigotes had greater metabolic activity during the early log phase of growth versus a later phase. Consequently, the production of formazan per cell was also greater. The MTT reduction was found to be proportional to the metabolic activity of epimastigotes. Therefore, the absorbance value recorded for one concentration of epimastigotes varied according to the growth phase in which the parasite is situated; thus, the slope of the line obtained for a culture in the early log phase is greater than that recorded for a culture in the late log phase. This means that the absorbance value is not ®xed for a speci®c concentration of epimastigotes, as it varies with the metabolic activity of the cells in the culture (Fig. 1). The linear correlation between absorbance values and numbers of epimastigotes per milliliter was very high (approx. R 0.99). Moreover, this assay was capable of discerning parasite concentrations of approx. 500,000 epimastigotes/ml (P < 0.01). This correlation, however, disappeared during the stationary growth phase. When the MTT-assay is applied to pharmacological screening, its planning should be carefully done. Some of the test compounds are colored (dyes) and may have reducing activity, which could translate into an increase in A595. To overcome this drawback, medium and compound controls should be prepared. Furthermore, an incubation period of 96 h versus 72 h is required for greater sensitivity, given that the observed dierence in
1001
Fig. 2 Validation of the use of the MTT assay for determination of the viability of epimastigotes of Trypanosoma cruzi in an in vitro test with benznidazole. Dark gray bars represent screening by microscopic counting. Gray bars and white bars express the cytotoxicity percentage values determined in two pharmacological screenings using MTT
Fig. 1a±c Correlation between absorbance values and numbers of epimastigotes per milliliter. Evaluation of the eect of the growth phase in which a parasite is situated on its capacity to reduce MTT to formazan. a Graph presenting the data recorded for an early-logphase culture (R 0.99). b Graph depicting the data recorded for a late-log-phase culture. Note that the line plotted for absorbance values versus numbers of epimastigotes per milliliter has a greater slope (m) in a than in b (R 0.99). c Graph showing that a correlation between A595 and the concentration of epimastigotes does not exist during the stationary growth phase
A595 between growth controls and active products is greater. The performance of this method decreases neither the time required to set up the culture nor the incubation period. It does, however, reduce the time needed to determine the activity of the compounds. In addition, it is more objective and more comfortable for the researcher than is microscopic counting, and more products can be assayed at once. The cytotoxicity percentage values determined for benznidazole by microscopic counting versus the MTT method are shown in Fig. 2, and those recorded for nifurtimox are shown in Fig. 3. These ®ndings are expressed as mean values SD. As the results are quite similar, these two techniques can be said to have the same sensitivity and speci®city for determination of the viability of epimastigotes in vitro. In conclusion, this report demonstrates that it is now possible to perform a rapid, simple, and reliable colorimetric assay for evaluation of trypanocidal activity
Fig. 3 Validation of the use of the MTT assay for determination of the viability of epimastigotes of T. cruzi in an in vitro test with nifurtimox. Dark gray bars represent screening by microscopic counting. Gray bars and white bars express the cytotoxicity percentage values determined in two pharmacological screenings using MTT
against epimastigotes of T. cruzi once the most suitable conditions have been set (concentration of MTT solution, 2.5 mg/ml; incubation period, 75 min). This in vitro model may therefore prove to be a useful tool for investigations of the pharmacological eects of new, potentially antitrypanosomal agents. Acknowledgements The authors are grateful to T. McDougall for carefully reviewing the English version of the manuscript. We declare that all experiments performed comply with current Spanish laws.
References Berg K, Zhai L, Chen M, Kharazmi A, Owen TC (1994) The use of a water-soluble formazan complex to quantitate the cell number and mitochondrial function of Leishmania major promastigotes. Parasitol Res 80: 235±239 Cedillo-Rivera R, Ramirez A, MunÄoz O (1992) A rapid colorimetric assay with the tetrazolium salt MTT and phenazine methosulfate (PMS) for viability of Entamoeba histolytica. Arch Med Res 23: 59±61 De Castro SL (1993) The challenge of Chagas' disease chemotherapy: an update of drugs assayed against Trypanosoma cruzi. Acta Trop (Basel) 53: 83±98
1002 Ellis JA, Fish WR, Sileghem M, Mcodimba F (1993) A colorimetric assay for trypanosome viability and metabolic function. Vet Parasitol 50: 143±149 GoÂmez Barrio A, Martinez Diaz RA, Atienza J, Escario JA, De Diego C, AvendanÄo C (1997) New derivatives of gentian violet as trypanocides: in vitro and in vivo assays on Trypanosoma cruzi. Res Rev Parasitol 57: 25±31 Hegyesi H, Csaba G (1997) Time and concentration dependence of the growth-promoting activity of insulin and histamine in Tetrahymena. Application of the MTT method for the determination of cell proliferation in a protozoan model. Cell Biol Int 21: 289±293 Kirchho LV (1994) American trypanosomiasis (Chagas' disease) and African trypanosomiasis (sleeping sickness). Curr Opin Infect Dis 7: 542±546
Ponce-Macotela M, Navarro-AlegrõÂ a I, MartõÂ nez-Gordillo MN, AÂlvarez-ChacoÂn R (1994) Efecto antigiardiaÂsico in vitro de 14 extractos de plantas. Rev Invest Clin 46: 343±347 Sereno D, Lemesre JL (1997) Use of an enzymatic micromethod to quantify amastigote stages of Leishmania amazonensis in vitro. Parasitol Res 83: 401±403 Sieuwerts AM, Klijn JGM, Peters HA, Foekens JA (1995) The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for assessment of growth characteristics, IC50-values and cell survival. Eur J Clin Chem Clin Biochem 33: 813±823 World Health Organization (WHO) (1991) Control of Chagas' disease. (WHO Tech Rep Ser 811) WHO, Geneva
