TRIBUTYLTIN-INDUCED EFFECTS ON MAPK SIGNALING IN ASCIDIAN EMBRYOS

Among the class of organotin compounds, the most well known is tributyltin (TBT). Organotin have many applications, which include use in PVC, as catalyst in chemical reactions, agricultural pesticides and antifungal treatments for textile polymers. In particular TBT is used in marine antifoulant paints to prevent the growth of organisms such as barnacles on the hull of ships. Extensive use in antifouling paints led to the widespread distribution of TBT and its breakdown products in the global marine, sediment and biota. High levels of TBT in the waters were found to have impaired reproduction, by inhibiting embryogenesis and larval development in a variety of marine organisms. Symptoms of the exposure to high levels of TBT in some invertebrates includes the development of male sexual characteristics as a penis and vas deferens by females (imposex). Ascidians are a good model for the study of embryonal development. They are also sensitive bioindicators of habitat degradation. The effects of tributyltin (IV) chloride (TBT chloride) solutions on ascidian embryos of Ciona intestinalis at different stages of development have been described. Previously, we carried out observations with both the light and the electron microscope on Ciona intestinalis embryos and larvae incubated in TBT solutions. This studies showed morphological and ultrastructural modifications of the embryos and larvae after incubation in TBT chloride at different concentrations. To understand molecular effects of TBT-induced on ascidians embryogenesis we have set out to study the effects of TBT at different concentrations, testing the activity of some protein with a basic role in embryonic development. In ascidian embryos, a fibroblast growth factor (FGF)-like signal has been proposed to be involved in induction of notochord and mesoderm formation. A main pathway is a protein kinase transduction pathway, which includes Ras, Raf, mitogenactivated protein (MAP) kinase and extracellular signal-regulated kinase/MAP kinase (ERK). The aim of this work in progress is to understand whether the TBT exposure on ascidian embryos at different stage of development cause alterations in tyrosine phosphorylation pattern and in MAPK activity. Tyrosine phosphorylation promotes cell growth, differentiation and apoptosis, due to activity of receptor tyrosine kinases and furthermore different stressors are known to stimulate tyrosine kinase activity. At first we focused our attention on tyrosine phosphorylation pattern after ascidian embryos to different stage of development TBT treatment. Phosphorylated proteins pattern is evaluated by SDS-PAGE electrophoresis and Western blotting on protein extract of ascidianembryos incubated with TBT, using anti-phosphotyrosine-antibody directed against mammalian phosphotyrosine. Preliminary results showed a different pattern on protein phosphorylation in response to the incubation with TBT in μM range. Since MAPKs play a key role in animal responses to a wide variety of environmental stresses, we have thought to test the role of MAPK pathway proteins such as MAPK p38 (Thr 180 and Tyr 182), p44/42 (Thr 202/Tyr 204) and c-Jun Nterminal kinases (JNK) after TBT treatment.