Nakajima-Shimada J, Hirota Y, Aoki T
Department of Parasitology, Juntendo University School of Medicine, Tokyo, Japan.
Antimicrob Agents Chemother. 1996 Nov;40(11):2455-8. doi: 10.1128/AAC.40.11.2455.
Trypanosoma cruzi, the causative agent of Chagas' disease, exhibits two different developmental stages in mammals, the amastigote, an intracellular form that proliferates in the cytoplasm of host cells, and the trypomastigote, an extracellular form that circulates in the bloodstream. We have already established an in vitro culture system using mammalian host cells (HeLa) infected with T. cruzi in which the time course of parasite growth is determined quantitatively. We adopted this system for the screening of anti-T. cruzi agents that would ideally prove to be effective against trypanosomes with no toxicity to the host cell. Of the purine analogs tested, allopurinol markedly inhibited the growth of amastigotes in a dose-dependent manner, with no lethal effect on trypomastigotes. 3'-Deoxyinosine and 3'-deoxyadenosine also suppressed T. cruzi growth inside the host cell, with the concentrations causing 50% growth inhibition being 10 and 5 microM, respectively, in contrast to a concentration causing 50% growth inhibition of 3 microM for allopurinol. Among the pyrimidine analogs examined, 3'-azido-3'-deoxythymidine (zidovudine) significantly reduced the growth of the parasite at concentrations as low as 1 microM. The anti-human immunodeficiency virus agents 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine caused a decrease in amastigote growth, while 2',3'-dideoxycytidine and 2',3'-dideoxyuridine had no inhibitory effect. When Swiss 3T3 fibroblasts were used as host cells, allopurinol, 3'-deoxyinosine, 3'-deoxyadenosine, and 3'-azid-3'-deoxythymidine also markedly inhibited T. cruzi proliferation. These results indicate that our culture system is useful as a primary screening method for candidate compounds against T. cruzi on the basis of two criteria, namely, intracellular replication by the parasite and host-cell infection rate.
克氏锥虫是恰加斯病的病原体，在哺乳动物体内呈现两种不同的发育阶段：无鞭毛体，一种在宿主细胞质中增殖的细胞内形式；以及锥鞭毛体，一种在血液中循环的细胞外形式。我们已经建立了一种体外培养系统，该系统使用感染了克氏锥虫的哺乳动物宿主细胞（HeLa细胞），在这个系统中可以定量确定寄生虫生长的时间进程。我们采用这个系统来筛选抗克氏锥虫药物，理想情况下，这些药物应证明对锥虫有效且对宿主细胞无毒。在所测试的嘌呤类似物中，别嘌呤醇以剂量依赖的方式显著抑制无鞭毛体的生长，对锥鞭毛体无致死作用。3'-脱氧肌苷和3'-脱氧腺苷也抑制宿主细胞内克氏锥虫的生长，导致50%生长抑制的浓度分别为10微摩尔和5微摩尔，相比之下，别嘌呤醇导致50%生长抑制的浓度为3微摩尔。在所检测的嘧啶类似物中，3'-叠氮-3'-脱氧胸苷（齐多夫定）在低至1微摩尔的浓度下就能显著降低寄生虫的生长。抗人类免疫缺陷病毒药物2',3'-双脱氧肌苷和2',3'-双脱氧腺苷导致无鞭毛体生长减少，而2',3'-双脱氧胞苷和2',3'-双脱氧尿苷没有抑制作用。当使用瑞士3T3成纤维细胞作为宿主细胞时，别嘌呤醇、3'-脱氧肌苷、3'-脱氧腺苷和3'-叠氮-3'-脱氧胸苷也显著抑制克氏锥虫的增殖。这些结果表明，我们的培养系统作为一种基于两个标准（即寄生虫的细胞内复制和宿主细胞感染率）的抗克氏锥虫候选化合物的初步筛选方法是有用的。
