Tributyltin triggers apoptosis in trout hepatocytes: the role of Ca2+, protein kinase C and proteases.
The purpose of the present study was to study the mechanisms involved in the induction of apoptosis and by tributyltin (TBT) in rainbow trout hepatocytes, and to examine the role of intracellular Ca2+, protein kinase C (PKC) and proteases in the apoptotic process. The intracellular Ca2+ chelator BAPTA-AM has a suppressive effect on TBT-mediated apoptosis. However, exposure to the ionophore A23187 is not sufficient to induce apoptosis in trout hepatocytes. The results obtained also show that TBT stimulates PKC gamma and delta translocation from cytosol to the plasma membrane in trout hepatocytes after 30 min of exposure. However, PKC gamma translocation is down-regulated after 90 min of treatment. The addition of protein kinase inhibitors (staurosporine and H-7) not only fails to inhibit apoptosis induced by TBT, but also leads to enhancement of DNA fragmentation. These inhibitors also afford a remarkable protection against the loss of plasma membrane integrity caused by TBT exposure. PMA, a direct activator of PKC, fails to stimulate DNA fragmentation. In addition, Z-VAD.FMK is an extremely potent inhibitor of TBT-induced apoptosis in trout hepatocytes, indicating that the activation of ICE-like proteases is a key event in this process. The cysteine protease inhibitor N-ethylmaleimide also prevented TBT-induced DNA fragmentation. Taken together, these data allow for the first time to suggest a mechanistic model of TBT-induced apoptosis. We propose that TBT could trigger apoptosis through a step involving Ca2+ efflux from the endoplasmic reticulum or other intracellular pools and by mechanisms involving cysteine proteases, such as calpains, as well as the phosphorylation status of apoptotic proteins such as Bcl-2 homologues.
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Université du Québec, Montréal, Département de Chimie, Canada.
Moutardier
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Denizeau
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D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D017686:Oncorhynchus mykiss; D011480:Protease Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D051745:Protein Kinase C-delta; D014220:Trialkyltin Compounds / Q000633:toxicity*; D014874:Water Pollutants, Chemical / Q000633:toxicity