Glutathione as a natural TBT scavenger in immunosuppressive effect in ascidians

Tributyltin (TBT) is a widespread biocide belonging to organotin compounds, a group of important industrial chemicals which resulted severe environmental toxicants known to have detrimental effects on immune system. We have previously reported that incubation of haemocytes of the colonial ascidian Botryllus schlosseri to 10 µM TBT caused significant impairment in yeast phagocytosis, disassembly of cytoskeletal components and induction of apoptosis as a consequence of direct interaction on calmodulin affecting Ca2+ homeostasis. However, in the presence of exogenous calmodulin, although Ca2+-ATPase activity and amoeboid morphology of phagocytes were restored, phagocytosis index, i.e., the percentage of haemocytes containing yeast cells, was still inhibited, indicating that, as suggested for rat thymocytes, the potent immunotoxic effect of TBT occurs with both Ca2+-dependent and Ca2+-independent mechanism. With the aim to study the capability of TBT to oxidise intracellular thiols, we first assessed the fraction of phagocytes stained by mercury orange - a specific dye for total thiols - and chlorobimane - a fluorescent dye for reduced glutathione - after exposition to 0.1, 1, 10 µM TBT for 1 h. In both cases, we observed a significant decrease in thiol content already after exposition to 0.1 µM TBT. Evaluation of amoebocytic index, i.e., the percentage of haemocytes with amoeboid shape, after co-incubation with 1 µM TBT and increasing concentrations (10, 20, 50 mM) of exogenous sulphydryl compounds revealed the ability of the latter compounds to restore cell morphology beginning from 20 mM for L-cysteine, reduced glutathione, and the glutathione fragment L-cysteinyl-glutamic acid (-Glu-Cys), whereas L-cystine, N-acetylcysteine, oxidised glutathione, and the glutathione fragment L-cysteinyl-glycine (Cys-Gly, probably resulting in 2,5 diketopiperazine derivative) had no restoring effect. Parallel experiments to evaluate the percentage of phagocytes containing tin, revealed with cacotheline dye, showed a perfectly symmetrical trend after co-incubation of TBT and sulphydryl compounds, indicating a scavenger ability of the natural thiol-compounds bearing free amino and carboxyl groups. As regards glutathione content, isodynamic mixtures of TBT and carmustine (BCNU, a specific inhibitor of glutathione-reductase) showed indifferent behaviour, while those of TBT and N-ethylmaleimide (NEM, an alkylating agent which specifically binds to thiol groups) showed an antagonistic interaction. Moreover, positivity to both glutathione-transferase (GST) and glutathione-peroxidase (GPX) antibody, revealed through immunocytochemical localisation, disappeared after incubation of 1 µM TBT, suggesting conformational changes probably due to direct interaction of the xenobiotic with these antioxidant enzymes involved in detoxification metabolic processes. In conclusion, our results suggest that i) oxidation of thiol groups, a process known to affect cytoskeletal proteins, may be involved in TBT immunotoxicity together with calmodulin interaction, ii) TBT causes depletion in glutathione content through direct interaction with both reduced glutathione and glutathione-dependent enzymes as GST and GPX, iii) although TBT does not interact directly with glutathione-reductase, the activity of this enzyme is inadequate to restore the reduced state of glutathione: the presence of high amounts of oxidised glutathione causes stimulation of Ca2+-releasing property of the inositol-1,4,5,-trisphosphate receptor and both the resulting calcium mobilisation and inhibition of calmodulin-dependent Ca2+-ATPase, increase the intracellular calcium content, and trigger a cascade of secondary events which lead to cell death.