Flow cytometric analysis of the cellular toxicity of tributyltin

Abstract

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 μM TBT relative to control cells or cells exposed to 50 μm TBT. The flow cytometric parameter 90° light scatter, a function of refractive index and a measure of protein content, increases as a function of TBT concentration above 0.5 μM. Following exposure to TBT concentrations greater than 0.5 μM, but less than 50 μm, 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 carboxyfruoreseein diacetate (CFDA). Similarly, cells exposed to TBT concentrations below 50 μM 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 μM. At doses between 5 and 50 μM, however, cells exhibit both CF and PI fluorescence simultaneously and are programmed for death. At TBT concentrations greater than 1.0 μM, 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° light scatter suggest fixation (protein denaturation, cross-linking, etc.) as a mechanism of the toxic action of TBT.