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

Isolated rat hepatocytes were used to investigate the biochemical mech anisms of toxicity of triethyllead (Et(3)Pb(+)), a highly neurotoxic d egradation product of the antiknocking petrol additive tetraethyllead. As early as 5 min from the addition of 50 mu M Et(3)Pb(+) to hepatocy te suspensions a decrease of mitochondrial membrane potential and of t he 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 live r mitochondria and microsomes. Further experiments using hepatocytes l oaded with the Ca2+ indicator Fura-2AM demonstrate that 1 min from add ition of Et(3)Pb(+) the cytosolic free Ca2+ levels increased by about 3-fold. High affinity plasma membrane Ca2+-ATPase activity was also si gnificantly inhibited in hepatocytes treated with Et(3)Pb(+), suggesti ng that an impairement of the mechanisms controlling the efflux of ext racellular Ca2+ was concomitantly involved in the rise in cytosolic Ca 2+ 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 d id not affect either the time-course or the extent of cytotoxicity. Co nversely, fructose, a glycolytic substrate that was able to support AT P production, prevented hepatocyte death. Thus, the depletion of cellu lar energy stores rather than the increase in cytosolic Ca2+ appears t o be the mechanism by which Et(3)Pb(+) causes irreversible injury in i solated hepatocytes.