ALTERATIONS OF HEPATOCYTE CA2+ HOMEOSTASIS BY TRIETHYLATED LEAD (ET(3)PB(+)) - ARE THEY CORRELATED WITH CYTOTOXICITY

Isolated rat hepatocytes were used to investigate the biochemical mechanisms of toxicity of triethyllead (Et(3)Pb(+)), a highly neurotoxic degradation product of the antiknocking petrol additive tetraethyllead. As early as 5 min from the addition of 50 mu M Et(3)Pb(+) to hepatocyte suspensions a decrease of mitochondrial membrane potential and of the capacity of mitochondria and microsomes to retain Ca2+ occurred. A dose-dependent release of mitochondrial Ca2+ as well as an inhibition of microsomal Ca2+-ATPase activity were also evident when Et(3)Pb(+) (from 2.5 mu M up to 50 mu M) was added to, respectively, isolated liver mitochondria and microsomes. Further experiments using hepatocytes loaded with the Ca2+ indicator Fura-2AM demonstrate that 1 min from addition of Et(3)Pb(+) the cytosolic free Ca2+ levels increased by about 3-fold. High affinity plasma membrane Ca2+-ATPase activity was also significantly inhibited in hepatocytes treated with Et(3)Pb(+), suggesting that an impairement of the mechanisms controlling the efflux of extracellular Ca2+ was concomitantly involved in the rise in cytosolic Ca2+ concentration. The increase in the cytosolic Ca2+ levels caused by Et(3)Pb(+) was followed by a rapid decline of cell viability. However, the addition of EGTA or of the intracellular Ca2+ chelator BAPTA/AM did not affect either the time-course or the extent of cytotoxicity. Conversely, fructose, a glycolytic substrate that was able to support ATP production, prevented hepatocyte death. Thus, the depletion of cellular energy stores rather than the increase in cytosolic Ca2+ appears to be the mechanism by which Et(3)Pb(+) causes irreversible injury in isolated hepatocytes.