Abstract
AbstractTributyltin (TBT), widely used as an antifouling biocide, is the most abundant pesticide in coastal environments. One of its main toxic effects is immunosuppression in both vertebrates and invertebrates. At sublethal doses of TBT, phagocytes lose their ability to move towards and ingest foreign particles. For short‐term cultures of haemocytes (60 min at 25 °C) of a marine invertebrate, the colonial ascidian Botryllus schlosseri, exposed to 10−5 M TBT, we previously reported dose‐ and time‐dependent impairment of yeast phagocytosis and changes in cell morphology related to cytoskeleton disorganization. These effects are Ca2+‐dependent, since inactivation of Ca2+‐adenosine triphosphatase and a sustained increase in cytosolic Ca2+ occurred. As TBT can antagonize the effect of chlorpromazine, a specific calmodulin (CaM) inhibitor, and the co‐presence of exogenous CaM and TBT in the incubation medium resulted in the absence of effects, we hypothesized an interaction between TBT and CaM. TBT may remove endogenous CaM from cell proteins, thus inactivating them and causing alteration of Ca2+ homeostasis. With the aim of confirming the hypothesis of a direct TBT–CaM interaction, we first studied the effects of co‐incubation of TBT with other exogenous proteins on restoring the ability of phagocyte morphology. Although bovine serum albumin was never able to restore cell morphology, the effect of human spectrin was similar to that described for CaM, suggesting a common non‐specific mechanism of action based on the interaction of TBT with exposed hydrophobic pouches. We also analysed the conformational changes of pure CaM (10−5 M) in the presence of various concentrations (10−4 to 10−3 M) of TBT and its degradation products, dibutyltin (DBT) and monobutyltin (MBT), by circular dichroism. Results indicate the dose‐ and time‐dependent interaction of TBT with CaM. This interaction is a non‐covalent interaction, probably hydrophobic in nature, between the aliphatic chains of TBT and the hydrophobic regions of Ca2+‐activated CaM. DBT and MBT turned out to be less active in inducing CaM conformational changes, without any significant differences between the two compounds. Copyright © 2002 John Wiley & Sons, Ltd.
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