Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via interaction with calmodulin

In the colonial ascidian Botryllus schlosseri, tributyltin (TBT), a powerfu l antifouling biocide, acts as immunotoxic xenobiotic since, at a sublethal concentration (10 mu M), it causes an irreversible and significant decreas e in in vitro yeast phagocytosis; associated with considerable changes in t he shape of phagocytes, which withdraw their pseudopodia and become spheric al, due to structural damage of cytoskeletal components. The addition of TB T to the culture medium causes a significant decrease in the amoebocytic in dex, i.e. the percentage of amoeboid-shaped haemocytes, and prolonged washi ng in sea water never succeeds in restoring amoeboid shape. In these cytosk eletal alterations, F-actin undergoes extensive depolymerisation, resulting in the absence of FITC-phalloidin fluorescence. Microtubules are not recog nisable as single filaments with anti-alpha-tubulin immunofluorescence, alt hough the centrosome is not affected. The addition of increasing exogenous calmodulin (CaM) concentrations (from 20 to 120 mu M) after incubation in T BT determines a significant increase in the amoebocytic index, although it is not able to bring it to that of controls, suggesting that CaM in the med ium in any case externally exerts an influence on haemocytes pretreated wit h TBT. The copresence of TBT and exogenous CaM at concentrations higher tha n 80 mu g/ml restores the amoebocytic index and cytoskeletal morphology. Th e latter appears complete for microtubules and partial for microfilaments. Experiments with isodynamic mixtures of TBT and specific CaM inhibitors, i. e. chlorpromazine (CPZ) and N-(6-aminohexyl)-5-chloronaphtalene-1-sulfonami de (W-7), reveal the synergistic effect of antagonism, indicating competiti on for the same site - a Ca2+-CaM hydrophobic region - by both interacting substances and, therefore, the formation of a TBT-CaM complex. Instead, iso dynamic mixtures with thapsigargin, an inhibitor of Ca2+-ATPase of the endo plasmic reticulum, have an effect of potentiation, suggesting that TBT indi rectly interacts with this Ca2+-ATPase activity. We hypothesise that the ma in mechanism of action of TBT in B. schlosseri phagocytes is alteration of Ca2+ homeostasis by means of direct interaction with endogenous CaM, which induces a conformational change preventing the regulative activity of CaM o n Ca2+-ATPase. Consequently, an excess of cytosolic Ca2+ accumulates which, together with the inhibition of CaM-dependent kinases and Ca2+-regulated p roteins. produces extensive cytoskeletal disorganisation. (C) 2000 Elsevier Science B.V. All rights reserved.