Reader S, Marion M, Denizeau F
Université du Québec à Montréal, Canada.
Toxicology. 1993 Jun 11;80(2-3):117-29. doi: 10.1016/0300-483x(93)90175-r.
The toxic effects of tri-n-butyltin chloride (TBT) were investigated on isolated trout hepatocytes by flow cytometry (FCM). We developed a procedure permitting the study of cytosolic free calcium in these cells using the new fluorescent probe Fura Red. In parallel, changes in thiol levels upon exposure to TBT were also followed by FCM with the probe 5-chloromethylfluorescein-diacetate. Cell viability was monitored through FCM analysis using propidium iodide. Treatment of hepatocytes with TBT caused a time- and concentration-dependent loss of viability. The results show that TBT induced a sustained elevation of cytosolic free calcium in isolated trout hepatocytes before loss of viability was detectable. Data for the viable cells remaining after incubation with TBT were selected after appropriate gating with the flow cytometer. When this was performed, the data revealed that changes in cytosolic free calcium were not dependent upon TBT concentration and duration of exposure. Moreover, TBT induced a rapid and important depletion of thiols in cells which survived TBT exposure. The present results suggest that alterations in calcium homeostasis and intracellular thiols are involved in the mechanism of trout hepatocyte injury by TBT. FCM is a powerful tool to study metabolic disturbances caused by toxic agents on cells at an individual level.
通过流式细胞术（FCM）研究了三丁基氯化锡（TBT）对分离的鳟鱼肝细胞的毒性作用。我们开发了一种程序，可使用新型荧光探针Fura Red研究这些细胞中的胞质游离钙。同时，还通过用探针5-氯甲基荧光素二乙酸酯的流式细胞术跟踪暴露于TBT后硫醇水平的变化。使用碘化丙啶通过流式细胞术分析监测细胞活力。用TBT处理肝细胞会导致细胞活力随时间和浓度依赖性丧失。结果表明，在可检测到活力丧失之前，TBT诱导分离的鳟鱼肝细胞中胞质游离钙持续升高。在用流式细胞仪进行适当的门控后，选择了与TBT孵育后剩余的活细胞的数据。当进行此操作时，数据显示胞质游离钙的变化不依赖于TBT浓度和暴露持续时间。此外，TBT诱导了在TBT暴露后存活的细胞中硫醇的快速且重要的消耗。目前的结果表明，钙稳态和细胞内硫醇的改变参与了TBT对鳟鱼肝细胞损伤的机制。流式细胞术是在个体水平上研究有毒物质对细胞引起的代谢紊乱的有力工具。
