TBT-induced apoptosis in Tunicate haemocytes

Early events in apoptosis include chromatincondensation followed by DNA fragmentation as well as translocation of phosphatidylserine (PS) in the outer plasma membrane. Organotin compounds increase intracellular Ca2+ levels and cause apoptosis in mammalian cells. In investigating whether TBT may also induce apoptosis in haemocytes of the ascidian Botryllus schlosseri, we exposed haemocytes to this xenobiotic at the sublethal dose of 10 µM, causing cell shrinkage and inhibition of phagocytosis and respiratory burst. Apoptosis was revealed as (i) chromatin condensation, with Acridine Orange nuclear staining; (ii) DNA fragmentation, with the TUNEL reaction; (iii) PS translocation, with the annexin-V assay; and (iv) loss of membrane permeability with the Trypan Blue diffusion assay. After 1 h of exposure, nuclear changes, i.e. significant collapse and cleavage of chromatin, were observed and cytoplasm blebbing occurred, together with surface alterations triggered by PS exposure. Haemocyte mortality increased significantly only after 2 h. All these apoptotic events may be closely related to a TBT-induced cytosolic calcium increase resulting in activation of endonucleases.