Are tributyltin-induced cytoskeletal alterations mediated by interaction with calmodulin?

Tributyltin chloride (TBTC), a powerful antifouling biocide, acts as immunosuppressant in Vertebrates, since it causes lymphocyte depletion, impairs phagocytosis, decreases the activity and the number of polymorphonuclear leukocytes. In the colonial ascidian Botryllus schlosseri we observed irreversible and significant decrease in yeast phagocytosis at sublethal dose (10 µM) associated with considerable changes in the shape of phagocytes which withdraw their pseudopodia and become spherical in relation to structural damage to cytoskeletal components. Subsequent and prolonged washes in sea water never succeed in restoring the amoeboid shape, suggesting an irreversible interaction between TBTC and cytoskeletal components. However, phagocytes do not change their morphology when 80 µg/ml calmodulin (CaM) are added tgether with 10 µM TBTC. In these cytoskeletal alterations the F-actin undergoes an extensive depolymerization resulting in the absence of fluorescence labelling by phalloidin-FITC. Analogously, microtubules are not recognizable as single filaments with anti-alpha-tubulin immunofluorescence although the centrosome is not affected. In the presence of increasing CaM concentrations in the incubation medium we observed that microtubules form an aster beginning from the concntration of 20 µg/ml CaM. At 80 µg/ml thin microtubules are rebuilt in pseudopodia and the amoeboid shape of the phagocytes is restored. At the same concentration only scattered spots of F-actin are observed corresponding with the adhesion plaques. Microfilaments appear only beginning from 120 µg/ml CaM and therefore they are more vulnerable than microtubules. Experiments with isodynamic mixtures of TBTC and specific CaM inhibitor, i.e. chlorpromazine (CPZ) and N-(6-aminohexyl)-5-chloronaphtalene-1-sulfonamide (W7), reveal a combined effect of antagonism. On the other hand, similar experiments with thapsigargin, a specific inhibitor of CaM-dependent Ca2+-ATPase show an effect of potentiation. We think that TBTC can interact on the same CaM receptor sites of CPZ and W7 in a Ca2+-CaM hydrophobic region. This interaction may induce a conformational change which prevents the regulative activity of CaM on Ca2+-ATPase. Consequently, the cell Ca2+ homeostasis is deeply altered. An excess of intracellular Ca2+ accumulate which, together with the inhibition of CaM-dependent kinases, produce an extensive cytoskeletal disaggregation. (This work was supported by grants of the italian C.N.R. and M.U.R.S.T. "Sistema Lagunare Veneziano")