Influence of TBT on the activity of the detoxicant enzymes, GST and GPX, from haemocytes of the colonial ascidian Botryllus schlosseri

In the last decade, some authors have hypothesised that a considerable part of the immunotoxic effects described for tributyltin (TBT) in mammals was due to Ca2+-independent mechanisms involving changes in sulphydryl groups of both proteins and reduced glutathione (GSH). Among the detoxicant enzymes, which play a multiple role in the metabolism of many xenobiotics of invertebrates and vertebrates, there are glutathione-transferase (GST) and glutathione-peroxidase (GPX). GST catalyses the conjugation reactions of GSH with electrophilic xenobiotics or their derivatives. It has been described in the antioxidant defence systems mainly of the gill of bivalves and as a mediator of xenobiotic detoxication in the liver and kidney of marine fish. In mammals, the activation of GST has been proposed to occur after TBT treatment and the presence of mercapturic acid supports this hypothesis. However, this derivative was never found in molluscs, suggesting that invertebrates possess a different detoxicant system of organotins. GPX is an antioxidant enzyme, which protects from the effects of reactive oxygen species production, requiring GSH consumption. In marine fish, since the inhibition of GPX by various organotin compounds had been recently described, this enzyme can be used as an effective biomarker of these pollutants. High concentrations of GPX were found in the blood of many invertebrates. The GPX activity detected inside the gill of bivalves is associated with GST and is catalysed by this enzyme. In cultured haemocytes of the colonial ascidian Botryllus schlosseri, we have observed by immunocytochemistry that positivity to both GST and Se-dependent GPX antibodies disappeared after incubation, for 60 min at 25 °C, in the presence of 0.1 µM and 1 µM TBT, respectively. This suggests that conformational changes occur probably due to direct interaction of the xenobiotic with these antioxidant enzymes. Moreover, the spectrophotometric assays on the haemocytic lysate have shown that TBT was able to significantly inhibit the GST activity (µmoles/min/mg protein) 2.6 times at 0.01 µM and 4.8 times at 0.1 µM, whereas the Se-dependent GPX activity was inhibited 1.5 times at 0.05 µM TBT. Our data suggest that even in the ascidian blood cells, enzymes involved in detoxication metabolic processes are present and that GST is the most important and sensitive among them.