Zucker R M, Elstein K H, Easterling R E, Massaro E J
Northrop Services, Inc., Environmental Sciences, Research Triangle Park, NC 27709.
Toxicol Lett. 1988 Oct;43(1-3):201-18. doi: 10.1016/0378-4274(88)90029-x.
Flow cytometric and light/fluorescence microscopic analyses indicate that tributyltin (TBT) alters the plasma membrane/cytoplasm complex of the murine erythroleukemic cell (MELC) in a dose-dependent and time-dependent manner. The flow cytometric parameter axial light loss, a measure of cell volume, decreases in cells exposed to 5 microM TBT relative to control cells or cells exposed to 50 microM TBT. The flow cytometric parameter 90 degrees light scatter, a function of refractive index and a measure of protein content, increases as a function of TBT concentration above 0.5 microM. Following exposure to TBT concentrations greater than 0.5 microM, but less than 50 microM, DNA distribution across the cell cycle cannot be resolved adequately by flow cytometry. Also, the cells become resistant to solubilization of the cell membrane/cytoplasm complex by nonionic detergents. Relative to logarithmically growing cells, MELC in the stationary phase of the growth cycle and butyric acid-differentiated cells exhibit decreased plasma membrane permeability resulting in increased carboxyfluorescein (CF) retention derived from the intracellular hydrolysis of carboxyfluorescein diacetate (CFDA). Similarly, cells exposed to TBT concentrations below 50 microM exhibit increased cellular CF retention. Viability in terms of CFDA hydrolysis/CF retention and propidium iodide (PI) exclusion is not decreased by exposure to TBT concentrations below 1 microM. At doses between 5 and 50 microM, however, cells exhibit both CF and PI fluorescence simultaneously and are programmed for death. At TBT concentrations greater than 1.0 microM, MELC plasma membrane potential, measured with the cyanine dye, 3,3'-dihexyloxacarbocyanine iodide (DiOC6) decreases at the same time that the uptake of PI is observed. In conjunction with other data, the concentration-dependent increase in CF fluorescence, resistance to detergent-mediated solubilization of the plasma membrane/cytoplasm complex, and increase in 90 degrees light scatter suggest fixation (protein denaturation, cross-linking, etc.) as a mechanism of the toxic action of TBT.
流式细胞术以及光/荧光显微镜分析表明，三丁基锡（TBT）以剂量和时间依赖的方式改变小鼠红白血病细胞（MELC）的质膜/细胞质复合体。流式细胞术参数轴向光损失（一种细胞体积的测量指标），在暴露于5微摩尔/升TBT的细胞中相对于对照细胞或暴露于50微摩尔/升TBT的细胞有所降低。流式细胞术参数90度光散射（一种与折射率有关且为蛋白质含量测量指标的函数），在TBT浓度高于0.5微摩尔/升时随浓度增加而升高。在暴露于TBT浓度大于0.5微摩尔/升但小于50微摩尔/升后，流式细胞术无法充分分辨细胞周期中DNA的分布情况。此外，细胞对非离子去污剂溶解质膜/细胞质复合体变得具有抗性。相对于对数生长期的细胞，处于生长周期静止期的MELC以及丁酸分化细胞表现出质膜通透性降低，导致源自羧基荧光素二乙酸酯（CFDA）细胞内水解的羧基荧光素（CF）保留增加。同样，暴露于低于50微摩尔/升TBT浓度的细胞表现出细胞CF保留增加。就CFDA水解/CF保留和碘化丙啶（PI）排斥而言，暴露于低于1微摩尔/升的TBT浓度不会降低细胞活力。然而，在5至50微摩尔/升的剂量下，细胞同时表现出CF和PI荧光，并被编程死亡。在TBT浓度大于1.0微摩尔/升时，用花青染料3,3'-二己基氧杂羰花青碘化物（DiOC6）测量的MELC质膜电位降低，同时观察到PI的摄取。结合其他数据，CF荧光的浓度依赖性增加、对去污剂介导的质膜/细胞质复合体溶解的抗性以及90度光散射的增加表明固定作用（蛋白质变性、交联等）是TBT毒性作用的一种机制。
