Stokkermans T J, Schwartzman J D, Keenan K, Morrissette N S, Tilney L G, Roos D S
Department of Pathology, Dartmouth Medical School, Hanover, New Hampshire 03756, USA.
Exp Parasitol. 1996 Dec;84(3):355-70. doi: 10.1006/expr.1996.0124.
Submicromolar concentrations of several dinitroaniline herbicides have been found to specifically inhibit intracellular replication of the protozoan parasite Toxoplasma gondii. IC50 concentrations for T. gondii survival were approximately 100 nM for ethalfluralin and oryzalin and approximately 300 nM for trifluralin. Primary human fibroblasts employed as host cells for parasite culture were unaffected at > 100-fold higher concentrations. Extracellular parasites were unaffected by these drugs, but within 8 hr after treatment of infected cell cultures, intracellular tachyzoites formed large amorphous bodies containing distorted nuclei. Parasite cytokinesis was completely blocked by drug treatment; nucleic acid synthesis, however, continued at near-normal levels for several days in the continuous presence of drug. All dinitroanilines appear to block nuclear division by inhibition of intranuclear spindle formation, but other cytoskeletal components were differentially affected by the various drugs tested. Subpellicular microtubules were absent in oryzalin-treated parasites, and large fragments of the inner membrane complex were observed throughout the parasite cytoplasm. In contrast, subpellicular microtubules and the inner membrane complex remained intact in ethalfluralin-treated parasites, but the endoplasmic reticulum and nuclear envelope were highly distended. Cytoskeletal elements associated with the conoid were not affected by any of the dinitroanilines tested, and treatment with the Ca2+ ionophore A23187 failed to trigger release of drug-treated parasites from infected cells. Mutant parasites resistant to oryzalin, ethalfluralin, or trifluralin were selected by chemical mutagenesis and examined for cross-resistance. An ethalfluralin-resistant mutant displayed cross-resistance to both oryzalin and trifluralin, while a trifluralin-resistant mutant was sensitive to oryzalin and only partially resistant ethalfluralin; an oryzalin-resistant mutant exhibited higher resistance to ethalfluralin and trifluralin than to oryzalin itself. Similarities between Apicomplexan and plant tubulin are discussed.
已发现几种亚微摩尔浓度的二硝基苯胺类除草剂能特异性抑制原生动物寄生虫刚地弓形虫的细胞内复制。乙丁氟灵和安磺灵对刚地弓形虫存活的IC50浓度约为100 nM，氟乐灵约为300 nM。用作寄生虫培养宿主细胞的原代人成纤维细胞在高于100倍的浓度下不受影响。细胞外寄生虫不受这些药物影响，但在处理感染细胞培养物8小时后，细胞内速殖子形成了含有扭曲细胞核的大无定形物体。药物处理完全阻断了寄生虫的胞质分裂；然而，在持续存在药物的情况下，核酸合成在数天内仍维持在接近正常的水平。所有二硝基苯胺似乎都通过抑制核内纺锤体形成来阻断核分裂，但其他细胞骨架成分受到所测试的各种药物的不同影响。在经安磺灵处理的寄生虫中，表膜下微管缺失，在整个寄生虫细胞质中观察到内膜复合体的大片段。相比之下，在经乙丁氟灵处理的寄生虫中，表膜下微管和内膜复合体保持完整，但内质网和核膜高度扩张。与锥体相关的细胞骨架成分不受所测试的任何二硝基苯胺影响，用Ca2+离子载体A23187处理未能促使经药物处理的寄生虫从感染细胞中释放。通过化学诱变筛选出对安磺灵、乙丁氟灵或氟乐灵耐药的突变寄生虫，并检测其交叉耐药性。一株对乙丁氟灵耐药的突变体对安磺灵和氟乐灵均表现出交叉耐药性，而一株对氟乐灵耐药的突变体对安磺灵敏感，对乙丁氟灵仅部分耐药；一株对安磺灵耐药的突变体对乙丁氟灵和氟乐灵的耐药性高于对安磺灵本身。文中讨论了顶复门寄生虫与植物微管蛋白之间的相似性。
