Effects of tributyltin on biomembranes: Alteration of flow cytometric parameters and inhibition of Na +, K +-ATPase two-dimensional crystallization

Abstract

Carboxyfluorescein diacetate (CFDA) is a lipophilic nonfluorescent molecule that readily crosses the cell membrane. In the cytoplasm, it is hydrolyzed by nonspecific esterases to carboxyfluorescein (CF), a negatively charged fluorescent molecule, which is retained incompletely by cells with an intact plasma membrane. Exposure (4 hr) of the murine erythroleukemic cell (MELC) to micromolar quantities (0.1 to 5.0 μ m) of tributyltin (TBT) results in increased cellular CF fluorescence. The increase occurs within a range below a critical value of the product (CPV) of the concentration ( C) of TBT × duration ( T) of exposure to TBT. Fluorescence increase is a sensitive indicator of the interaction of TBT with the cell: it is observed following exposure to 0.1 μ m TBT for 4 hr at 37°C. In the range above the CPV, cellular CF fluorescence is reduced apparently resulting from perturbation of membrane structure. For example, exposure of MELC to 2.5 μ m TBT for 4 hr at 37°C produces resistance to detergent-mediated cytolysis and inhibition of vanadate-mediated two-dimensional crystallization of Na +, K +-ATPase molecules in porcine renal microsomal membrane preparations, a process requiring molecular mobility within the membrane. Taken together, the increased cellular CF fluorescence and resistance of the MELC to cytolysis along with the inhibition of Na +, K +-ATPase crystallization in the microsomal membrane preparations suggest fixation (protein denaturation, cross-linking, etc.) at the level of the plasma membrane as a mode of toxic action of TBT.