The microculture tetrazolium assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was used to estimate the 50% inhibitory concentration of chloroquine, quinine, artemisinin, and atovaquone using a Plasmodium falciparum in vitro culture system. The MTT assay was compared to the standard tritiated hypoxan-thine assay and to a previously described method, the 2,2′-di-p-nitrophenyl-5,5′-diphenyl-3,3′-[3,3′-dimethoxy-4,4′-diphenylenel-ditetrazolium chloride (NBT) assay. In general, the results show that the three assays generate comparative results.
The results of this study suggest that the MTT method is able to give a profile of cytotoxic dose response effects over a wide range of concentrations of a drug. The method may be used in work that does not require extreme pre-cision and sensitivity, for instance, as a portable rapid screen to assay natural products for in vitro cytotoxic ac-tivity against Plasmodium falciparum.
References
1.
Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D. (1979). Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother16:710.
2.
Maker, M.T., Ries, J.M., Williams, J.A., Bancroft, J.E., Piper, R.C., Gibbins, B.L., Hinrichs, D.J. (1993). Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am. J. Trop. Med. Hyg.48:739.
3.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immun. Meth. 65:55.
4.
Druilhe, P., Gentilini, M. (1982). Culture in vitro de Plasmodium falciparum: Int6ret et limites-m6thodologie. Medicine Tropicale42(4):437.
5.
Lambros, C., Vanderberg, J.P. (1979). Synchronization of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol. 65:418.
6.
Geary, T., Divo, A., Jensen, J. (1989). Stage specific actions of antimalarial drugs on Plasmodium falciparum in culture. Am. J. Trop. Med. Hyg.40:240.
7.
Mirovsky, P., Gay, F., Bustos, D., Mazier, D., Gentilini, M. (1989). Cloning of a fresh isolate of Plasmodium falciparum and drug sensitivity of the clones. Trans. R. Soc. Trop. Med. Hyg.84:511.
8.
Maker, M.T., Hinrichs, D.J. (1993). Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessement of parasitemia. Am. J. Trop. Med. Hyg.48:205.
9.
Sladowski, D., Steer, S.J., Clothier, R.H., Balls, M. (1993). An improved MTT assay. J. Immun. Meth.157:203.
10.
Beutler, E., Baluda, M.C. (1963). Methemoglobin reduction: Studies of the interaction between cell populations and of the role of methylene blue. Blood22(3):329.
11.
Fairbanks, V.F., Lampe, L.T. (1968). A tetrazolium-linked cytochemical method for estimation of glucose-6-phosphate dehydrogenase activity in individual erythrocytes: Applications in the study of heterozygotes for glucose-6-phosphate dehydrogenase deficiency. Blood31(5):589.
12.
Theakston, R.D.G., Fletcher, K.A. (1973). A technique for the cytochemical demonstration in the electron microscope of glucose-6-phosphate dehydrogenase activity in erythrocytes of malaria-infected animals. J. Microscopy97:315.
13.
Gay, F. (1992). Chemoresistance of Plasmodium falciparum: Studies on human infected populations and malaria parasite populations. Doctoral dissertation, University of Montpellier, Montpellier, VT.
14.
Bustos, M.D.G., Gay, F., Diquet, B. (1994). In-vitro tests on Philippine isolates of Plasmodium falciparum against four standard antimalarials and four quinghaosu derivatives. Bull. WHO72(5):729.
15.
Basco, L.K., Marquet, F., Makler, M.M., Le Bras, J. (1995). Plasmodium falciparum and Plasmodium vivax: Lactate dehydrogenase activity and its application for in vitro drug susceptibility assay. Exp. Parasitol.80:260.
16.
Nargi, F.E., Yang, T.J. (1993). Optimization of the L-M cell bioassay for quantitating tumor necrosis factor a in serum and plasma. J. Immun. Meth.159:81.
17.
Sherman, I.W. (1979). Biochemistry of Plasmodium (malaria parasites). Microb. Rev.43(4):453-495.
18.
Tilton, W.M., Seaman, C., Carriero, D., Piomelli, S. (1991). Regulation of glycolysis in the erythrocyte: Role of the lactate/pyruvate and NAD/NADH ratios. RBC Glycolysis118:146.
19.
Trager, W., Jensen, J.B. (1976). Human malaria parasites in continuous culture. Science193:673.
