TBT-induced apoptosis in tunicate haemocytes

Early events in apoptosis include chromatin condensation followed by DNA fr agmentation as well as translocation of phosphatidylserine (PS) in the oute r plasma membrane. Organotin compounds increase intracellular Ca2+ levels a nd cause apoptosis in mammalian cells. In investigating whether TBT may als o induce apoptosis in haemocytes of the ascidian Botryllus schlosseri, we e xposed haemocytes to this xenobiotic at the sublethal dose of 10 mu M, caus ing cell shrinkage and inhibition of phagocytosis and respiratory burst. Ap optosis was revealed as (i) chromatin condensation, with Acridine Orange nu clear staining; (ii) DNA fragmentation, with the TUNEL reaction; (iii) PS t ranslocation, with the annexin-V assay; and (iv) loss of membrane permeabil ity with the Trypan Blue diffusion assay. After 1 h of exposure, nuclear ch anges, i.e, significant collapse and cleavage of chromatin, were observed a nd 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 cytoso lic calcium increase resulting in activation of endonucleases. Copyright (C ) 1999 John Wiley & Sons, Ltd.