Intracellular calcium and pH alterations induced by tri-n-butyltin chloride in isolated rainbow trout hepatocytes: a flow cytometric analysis

The effects of tri-n-butyltin chloride (TBT) on ionic homeostasis on isolated trout hepatocytes were investigated by flow cytometry (FCM), using the Ca(2+)-sensitive and pH-sensitive fluorescent probes Indo-1 and SNARF-1, respectively. Cell viability was monitored concurrently. Treatment of hepatocytes with 1 and 5 microM TBT caused a rapid and sustained elevation of cytosolic free Ca2+ concentration [Ca2+]i and an important cytoplasmic acidification. These changes were dependent upon TBT concentration and were maintained over 60 min, the maximum exposure period investigated. At 0.5 microM TBT, there was a slight but not significant increase in [Ca2+]i and a significant reduction in intracellular pH (pHi) only after 60 min of exposure. A rise in [Ca2+]i and cytoplasmic acidification were observed before loss of viability was detectable. Experiments carried out in Ca(2+)-free medium suggest that TBT mainly mobilizes Ca2+ from intracellular stores in trout hepatocytes. The cytoplasmic acidification following TBT exposure seems to be caused by the combination of intracellular Ca2+ mobilization and by direct action of TBT. The present results suggest that ionic homeostasis perturbations could be early events in the mechanism of cell injury by TBT.