Shaked-Mishan P, Ulrich N, Ephros M, Zilberstein D
Departments of Biology and Pediatrics, Carmel Medical Center and the Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 32000, Israel.
J Biol Chem. 2001 Feb 9;276(6):3971-6. doi: 10.1074/jbc.M005423200. Epub 2000 Nov 10.
The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (Sb(V)). Its mechanism of action is unknown because of the limited information available about intracellular antimony metabolism and about the genes that regulate these processes. Herein, flow injection-inductively coupled plasma mass spectrometry (ICP-MS), flow injection hydride generation ICP-MS, and ion chromatography ICP-MS were used to measure antimony accumulation and intracellular metabolism in the human protozoan parasite Leishmania donovani. Amastigotes (the intracellular form) and promastigotes (the extracellular form) accumulate Sb(V) and Sb(III) via separate transport systems. Stage-specific intracellular Sb(V) reducing activity was apparent in amastigotes, which reduced the negligibly toxic Sb(V) to highly toxic Sb(III). This amastigote-specific reducing activity was deficient in the Pentostam-resistant mutant L. donovani Ld1S.20. These data indicate that parasite susceptibility to Sb(V) correlates with its level of Sb(V) reducing activity. Also, in promastigotes of both wild-type L. donovani and the Pentostam-resistant mutant L. donovani Ld1S.20, Sb(V) inhibited the toxicity of Sb(III) but not of As(III). Both Sb(V) and Sb(III) were toxic to wild-type amastigotes. However, as observed in promastigotes, in mutant amastigotes Sb(V) inhibits Sb(III) but not As(III) activity. Anion exchange chromatography showed that intracellular antimony metabolism occurred in both promastigotes and amastigotes. These data demonstrate that the interaction between the two antimony oxidation states occurs intracellularly, within the parasite. The results also indicate that Sb(V) anti-leishmanial activity is dependent on its reduction to Sb(III). The mechanism of this novel intracellular Sb(V) reduction has yet to be identified, and it may or may not be enzymatic. This is the first description of intracellular Sb(V) reducing activity in Leishmania as well as in any prokaryotic or eukaryotic cell.
人类内脏利什曼病的标准治疗方法是使用五价锑（Sb(V)）。由于关于细胞内锑代谢以及调节这些过程的基因的可用信息有限，其作用机制尚不清楚。在此，采用流动注射-电感耦合等离子体质谱法（ICP-MS）、流动注射氢化物发生ICP-MS和离子色谱ICP-MS来测量人体原生动物寄生虫杜氏利什曼原虫中的锑积累和细胞内代谢。无鞭毛体（细胞内形式）和前鞭毛体（细胞外形式）通过不同的转运系统积累Sb(V)和Sb(III)。阶段特异性的细胞内Sb(V)还原活性在无鞭毛体中很明显，它将毒性可忽略不计的Sb(V)还原为剧毒的Sb(III)。这种无鞭毛体特异性的还原活性在对喷他脒耐药的杜氏利什曼原虫突变体Ld1S.20中缺乏。这些数据表明寄生虫对Sb(V)的敏感性与其Sb(V)还原活性水平相关。此外，在野生型杜氏利什曼原虫和对喷他脒耐药的杜氏利什曼原虫突变体Ld1S.20的前鞭毛体中，Sb(V)抑制Sb(III)的毒性，但不抑制As(III)的毒性。Sb(V)和Sb(III)对野生型无鞭毛体均有毒性。然而，如在前鞭毛体中观察到的那样，在突变体无鞭毛体中Sb(V)抑制Sb(III)但不抑制As(III)的活性。阴离子交换色谱显示细胞内锑代谢在前鞭毛体和无鞭毛体中均有发生。这些数据表明两种锑氧化态之间的相互作用发生在寄生虫细胞内。结果还表明Sb(V)的抗利什曼活性取决于其还原为Sb(III)。这种新的细胞内Sb(V)还原机制尚未确定，它可能是酶促的，也可能不是。这是首次描述利什曼原虫以及任何原核或真核细胞中的细胞内Sb(V)还原活性。
