Yarlett N, Goldberg B, Nathan H C, Garofalo J, Bacchi C J
Haskins Laboratories, New York, New York 10038.
Exp Parasitol. 1991 Feb;72(2):205-15. doi: 10.1016/0014-4894(91)90138-m.
Clinical isolates of Trypanosoma brucei rhodesiense, which were resistant to arsenical drugs in murine infections, were examined for resistance in vitro. A rapid lysis assay was developed which was able to predict in vivo sensitivity to melarsoprol (Mel B, Arsobal) and melarsen oxide. The assay was based on the finding that long slender bloodforms of drug-sensitive isolates would lyse in the presence of arsenicals upon incubation in heat-inactivated fetal bovine serum. On the basis of plots of decrease in the absorbance of trypanosome suspensions vs time of incubation with drug, L50 values, reflecting the drug concentration necessary for lysis of 50% of the cells within 30 min. were calculated for five strains. These values ranged from less than 30 microM for arsenical-sensitive strains to greater than 75 microM in proven arsenic refractory isolates. Calcium was essential for lysis, and the presence of the Ca2+ chelator EGTA (10 mM) in serum delayed lysis of sensitive strains. Ca2+ channel antagonists (Verapamil, Diltiazem), however, did not enhance lysis of refractory isolates when used at 20 to 30 microM. Intracellular concentrations of reduced trypanothione, the apparent target of arsenicals, were similar for all isolates, approximately 1.02 +/- 0.28 nmol/10(8) cells, as detected by monobromobimane derivitization and HPLC analysis. Uptake of melarsen oxide was found to be reduced in arsenical refractory strains. Uptake was judged by reduction of free reduced trypanothione as a result of formation of the trypanothione-arsenic complex Mel T. Little change was found in arsenical-resistant strains, but sensitive strains had 50 to 70% reductions in trypanothione levels after incubation with a low (1 microM) level of melarsen oxide.
对布氏罗得西亚锥虫的临床分离株进行了体外耐药性检测，这些分离株在鼠类感染中对砷剂耐药。开发了一种快速裂解试验，该试验能够预测体内对美拉胂醇（美拉B，砷苯甘胺）和氧化美拉胂的敏感性。该试验基于以下发现：药物敏感分离株的长而细的血鞭毛体在热灭活胎牛血清中孵育时，在砷剂存在下会裂解。根据锥虫悬浮液吸光度的下降与药物孵育时间的关系图，计算了五个菌株的L50值，该值反映了在30分钟内使50%的细胞裂解所需的药物浓度。这些值范围从砷剂敏感菌株的小于30微摩尔/升到经证实的砷剂难治性分离株的大于75微摩尔/升。钙对于裂解至关重要，血清中Ca2+螯合剂EGTA（10毫摩尔）的存在会延迟敏感菌株的裂解。然而，Ca2+通道拮抗剂（维拉帕米、地尔硫䓬）在20至30微摩尔使用时，并不会增强难治性分离株的裂解。通过单溴联苯胺衍生化和高效液相色谱分析检测到，所有分离株中砷剂的明显靶标——还原型锥虫硫醇的细胞内浓度相似，约为1.02±0.28纳摩尔/10^8个细胞。发现砷剂难治性菌株中氧化美拉胂的摄取减少。通过还原型锥虫硫醇的减少来判断摄取情况，这是由于形成了锥虫硫醇 - 砷复合物美拉T。在砷剂耐药菌株中未发现明显变化，但敏感菌株在与低水平（1微摩尔）氧化美拉胂孵育后，锥虫硫醇水平降低了50%至70%。
