Triphenyltin pesticides in sea water as immunotoxins for tunicates

Among the biocidal triorganotin compounds, triphenyltin derivatives (TPTs, Fentin), i.e. acetate (TPTA), hydroxide (TPTH), and chloride (TPTC) are used world wide as fungicide – mainly against Cercospora beticola -, herbicides, antifeedings, and molluscicides. They are of environmental concern owing to direct introduction into the marine water and sediments by leaching from employment in agriculture with particular contamination of coastal areas as in the Venice Lagoon. As lipophilic compounds, TPTs accumulate by oral and via gill intake especially in the fatty compartments of the teleostean fishes. In the latters the resulted bioconcentration is responsible of mortality after continue exposure, together with the decreased resistance to infections. Such effect appears in accord with the highly selective toxicity on the immune system in mammals. Our previous studies showed that organotin compounds, such as butyltin derivatives mainly employed in antifouling paints, are immunotoxic in tunicates causing inhibitory effects on yeast phagocytosis by hemocytes of the colonial ascidian Botryllus schlosseri, which is spread in Venice Lagoon. Therefore we have extended the study to TPTs with the aim of investigating whether hemocytes of this species were still sensitive biosensors with the same mechanism. TPTs were first dissolved in DMSO and then diluted and always employed at concentrations of 0.1, 1, 10 µM in sea water. After 1 hr exposure, viability of hemocytes was not affected up to 100 µM for TPTA and TPTC, and 1 mM for TPTH, indicating and about tenfold lesser sensitivity than mammal thymocytes. Only at 100 µM TPTs caused some morphological changes without cytolysis, suggesting an interaction with cytoskeletal constituents. Analogously to butyltin, TPTs exerted significant inhibitory effects on yeast phagocytosis – TPTC > TPTA > TPTH – in a concentration-dependent manner. The same order of inhibition was observed for Ca2+-ATPase activity, suggesting that the inhibition of this enzyme and of phagocytosis might be strictly linked, as we demonstrated with butyltin. So, in general, organotins may influence cell calcium homeostasis. As for alkyl trorganortins – TBT > TET > TMT -, addition of 20 µg/ml calmodulin (CaM) was able to reverse the inhibition of Ca2+-ATPase activity by TPTs, but not restoring the phagocytosis index. These results supported the hypothesis that the mitochondrial oxidative phosphorylation might also be involved. In fact, O2°- production was inhibited – TPTA > TPTC > TPTH – in a concentration-dependent manner, but not restored by CaM. So CaM does not seem to be involved in the respiratory burst of mitochondria. In conclusion, TPTs inhibit the mitochondrial oxidative phosphorylation in tunicates as shown in teleosts, but impair the cell-mediated immunity compromising the phagocyte system, differently from mammals where e.g. TPTH is not immunotoxic. Therefore tunicates appear effective candidates for sentinel organisms of organotin pollutants.