Effect of 2-acetylaminofluorene on intracellular free Ca2+ in isolated rat hepatocytes.

The effect of 2-acetylaminofluorene (2-AAF) on the intracellular free Ca2+ ([Ca2+]i) and viability of isolated rat hepatocytes has been investigated using the fluorescent probes quin 2 and propidium iodide respectively. At the highest concentration tested (224 microM), 2-AAF produces an elevation of [Ca2+]i which shows a biphasic profile. A small initial increase is observed during the first 5 min; this is followed by a considerable rise which reaches up to 2.5 times the control value at 15 min. These changes in intracellular calcium are not accompanied by detectable alterations in cell viability. In order to determine the mechanisms by which this effect of 2-AAF takes place, three calcium antagonists, namely verapamil, TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) and ruthenium red (RuR), have been used. The results suggest that the first phase is dependent upon internal Ca2+ store mobilization, while the second phase seems to be related to Ca2+ entry from the extracellular space. The data obtained with RuR further indicate that mitochondria may be involved in the perturbation of calcium homeostasis caused by 2-AAF. In addition, in the experiments involving antagonists, no consistent pattern emerges that suggests a close relationship between intracellular Ca2+ levels and cell viability. The present study provides further information on the mechanisms by which these well-known hepatotoxin 2-AAF may interact with liver cells. It also shows that when these cells are exposed to a toxin, short-term changes in [Ca2+]i may not be accompanied by loss of cell viability, and conversely, that changes in cell viability may occur without alterations in [Ca2+]i.