Articles Organotin-Induced Caspase Activation and Apoptosis in Human Peripheral Blood Lymphocytes He ´le `ne Stridh,* ,†,‡ Ian Cotgreave, † Malin Mu ¨ ller, †,‡ Sten Orrenius, † and Dulceaydee Gigliotti § Institute of Environmental Medicine, Department of Medicine, Division of Respiratory Medicine, Microbiology and Tumorbiology Centre and Department of Microbiology, Pathology and Immunology, Division of Biomedical Laboratory Technology, Karolinska Institutet, Stockholm, Sweden Received July 19, 2000 In the present study, we show that the immunotoxicant, tributyltin (TBT), induces a dose- dependent activation of caspases followed by typical apoptotic morphology in resting human peripheral blood lymphocytes. TBT also caused an early loss of mitochondrial membrane potential (Δ Ψm ) and release of cytochrome c, suggesting that apoptosis was triggered by the mitochondrial pathway. When CD4+ T-cells were sorted from peripheral blood and exposed to TBT for 30 min, caspase activation and apoptosis were induced. Interestingly, in the sorted CD8+ T-cell population, caspase activation was not observed until 2 h of TBT exposure, suggesting that these cells were more resistant toward TBT. Moreover, a time-dependent induction of caspase activity was also detected in CD3-stimulated peripheral blood lymphocytes. This caspase activation was not associated with cytochrome c release or loss of mitochondrial Δ Ψ and did not lead to apoptotic morphology, although it did lead to both PARP and DFF cleavage. We also noticed a concomitant induction of Hsp27, and it awaits to be seen if this chaperone may interfere with the processing of nuclear protein substrates downstream from these primary caspase-3 substrates. Moreover, no increase in caspase activation or induction of apoptosis was observed after TBT treatment in these cells. Instead, the cells were directed toward necrotic deletion. Taken together, these data suggest that TBT-induced deletion of peripheral lymphocytes is likely to be a component in the overall risk for immunotoxic responses in exposed humans. Introduction Organotin compounds are important organometallic chemicals, which are used in a variety of technical applications. Of the trialkyltins, particularly tributyltin (TBT) 1 is applied as an active component in wood preservatives, disinfectants, and antifoulant paints (1, 2). Consequently, these compounds are now present in the environment, exerting cytotoxic effects to a broad range of species including mammals (3, 4). Several in vivo studies have shown that low doses of TBT target the mammalian immune system, thereby causing immuno- suppression by an elimination of cortical thymocytes (5-9). The toxicity of the organotins has been previously studied in experimental animal systems. The di- and the tributyltin compounds have been shown to cause atrophy both of the thymus and of the thymus-dependent areas of the spleen and lymph nodes in rats. Thus, the weight and overall morphology of rat lymphoid tissues, as well as peripheral lymphocyte counts and total serum immu- noglobulin concentrations, have all been shown to be greatly affected by oral exposure to TBT (8-10). The TBT-induced atrophy of the rat thymus was associated with a selective elimination of cortical thymocytes, resulting in a marked depletion of small-sized, nonpro- liferating cells (11). Signs of extensive cell destruction within the tissue were not observed. Consequently, the T-cell-dependent immune functions in rats fed with TBT were suppressed. The mechanisms of the immunotoxicity of TBT are still not clear. Some studies suggest that it is the major metabolite of TBT, dibutyltin (DBT), 1 which is respon- sible for the thymic toxicity. The primary effect of DBT is thought to interrupt the maturation of the developing * To whom correspondence and should be addressed. Phone: +46 8 5177 6183. Fax: +46 8 5177 54 51. E-mail: helene.stridh@ks.se. † Institute of Environmental Medicine. ‡ Department of Medicine, Division of Respiratory Medicine. § Microbiology and Tumorbiology Centre and Department of Micro- biology, Pathology and Immunology, Division of Biomedical Laboratory Technology. 1 Abbreviations: huPBL, human peripheral blood lymphocytes; TBT, tributyltin; TPT, triphenyltin; DBT, dibutyltin; DEVD-AMC, Ac-Asp- Glu-Val-Asp-(7-amino-4-methylcoumarin); MPT, mitochondrial perme- ability transition; (ΔΨm) mitochondrial membrane potential; NP-40, octylphenoxy polyethoxy ethanol; CHAPS, (3-cholamidopropyldimethy- lammonio)-1-propane sulfonate; TMRE, tetramethylrhodamine ethy- lester; FACS, fluorescence activated cell sorter; G3PDH; glyceraldehyde- 3-phosphate -dehydrogenase; PARP, poly (ADP-ribose)polymerase; DFF/ICAD, DNA fragmentation factor/inhibitor of caspase-activated inhibitor of caspase-activated DNase. 791 Chem. Res. Toxicol. 2001, 14, 791-798 10.1021/tx000156c CCC: $20.00 © 2001 American Chemical Society Published on Web 06/26/2001