Cytoskeleton alterations by tributyltin (TBT) in tunicate phagocytes

The severe and continuous contamination of coastal sea waters by organotin compounds (OTC) used as biocides in antifouling paints has led in the last decade to the worldwide rising of several studies about the effects induced by TBT and its degradation products on various marine organisms. As a part of an environmental biomonitoring program our studies on tunicates aim to evaluate the real impact of OTC on the benthonic fauna in the Venice Lagoon ecosystem. Previously, we demonstrated the in vitro immunotoxicity of OTC at low concentrations in the blood cells of the colonial ascidian Botryllus schlosseri. In this species, this effect of OTC does not cause any cytolysis and/or mortality of the hemocytes, although irreversible and significant decreases in yeast-phagocytosis, respiratory burst, and calmodulin-Ca2+-ATPase activity occur. However, this enzyme activity is recoverable in the presence of a calmodulin excess in the medium. In those experiments we always observed a considerable change in the shape of the hemocytes, which turned spherical, amoebocytes withdrawing their long pseudopodia. The number of cultured hemocytes never decreased showing that probable alteration of cell components by OTC did not interfere with the ability of the cells to attach to substrate through the adhesion plaques. In this research the toxic effects on cytoskeleton were evaluated immunocytochemically using anti-actin and anti-tubulin monoclonal antibodies on cultured hemocytes previously treated with 10 µM TBT for 60 min at 25 °C. The samples were specifically prepared for observations under light, confocal fluorescence, and electron transmission microscopes, according to the standard protocols. Results obtained give for the first time demonstration that TBT is able to strongly interact with the polymerization and the organization of microfilaments and microtubules. The decrease in yeast-phagocytosis may be related to a direct mechanism of action of TBT on cytoskeleton through an alteration of calcium homeostasis. This effect would induce an excess of intracellular Ca2+ accumulation which may cause an internal disorganization of the cytoskeleton proteins, externally appearing as a shape change of the hemocytes. Calmodulin can partially restore the cytoskeleton architecture. Indeed, these severe alterations inhibit phagocytosis at a very early stage, i.e., from the stage of particle adhesion, whereas the toxic effects on the oxidative phosphorylation of mitochondria, i.e., on the respiratory burst of phagocytes, take place later.