TRIBUTYLTIN-INDUCED EFFECTS ON MAPK SIGNALING IN ASCIDIAN EMBRYOS

Among the class of organotin compounds, the most well known istributyltin (TBT). Organotin have many applications, which includeuse in PVC, as catalyst in chemical reactions, agricultural pesticidesand antifungal treatments for textile polymers. In particular TBT isused in marine antifoulant paints to prevent the growth of organismssuch as barnacles on the hull of ships. Extensive use in antifoulingpaints led to the widespread distribution of TBT and its breakdownproducts in the global marine, sediment and biota. High levels of TBTin the waters were found to have impaired reproduction, by inhibitingembryogenesis and larval development in a variety of marineorganisms. Symptoms of the exposure to high levels of TBT in someinvertebrates includes the development of male sexual characteristicsas a penis and vas deferens by females (imposex). Ascidians are a goodmodel for the study of embryonal development. They are also sensitivebioindicators of habitat degradation. The effects of tributyltin (IV)chloride (TBT chloride) solutions on ascidian embryos of Cionaintestinalis at different stages of development have been described.Previously, we carried out observations with both the light and theelectron microscope on Ciona intestinalis embryos and larvae incubatedin TBT solutions. This studies showed morphological andultrastructural modifications of the embryos and larvae afterincubation in TBT chloride at different concentrations. Tounderstand molecular effects of TBT-induced on ascidiansembryogenesis we have set out to study the effects of TBT at differentconcentrations, testing the activity of some protein with a basic role inembryonic development. In ascidian embryos, a fibroblast growthfactor (FGF)-like signal has been proposed to be involved in inductionof notochord and mesoderm formation. A main pathway is a proteinkinase transduction pathway, which includes Ras, Raf, mitogenactivatedprotein (MAP) kinase and extracellular signal-regulatedkinase/MAP kinase (ERK). The aim of this work in progress is tounderstand whether the TBT exposure on ascidian embryos atdifferent stage of development cause alterations in tyrosinephosphorylation pattern and in MAPK activity. Tyrosinephosphorylation promotes cell growth, differentiation and apoptosis,due to activity of receptor tyrosine kinases and furthermore differentstressors are known to stimulate tyrosine kinase activity. At first wefocused our attention on tyrosine phosphorylation pattern afterascidian embryos to different stage of development TBT treatment.Phosphorylated proteins pattern is evaluated by SDS-PAGEelectrophoresis and Western blotting on protein extract of ascidianembryos incubated with TBT, using anti-phosphotyrosine-antibodydirected against mammalian phosphotyrosine. Preliminary resultsshowed a different pattern on protein phosphorylation in response tothe incubation with TBT in μM range. Since MAPKs play a key role inanimal responses to a wide variety of environmental stresses, we havethought 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 Nterminalkinases (JNK) after TBT treatment.