Alterations of hepatocyte Ca²⁺ homeostasis by triethylated lead (Et₃Pb⁺): are they correlated with cytotoxicity?

Isolated rat hepatocytes were used to investigate the biochemical mechanisms of toxicity of triethyllead (Et₃Pb⁺), a highly neurotoxic degradation product of the antiknocking petrol additive tetraethyllead. As early as 5 min from the addition of 50 μM Et₃Pb⁺ 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 Et₃Pb⁺ (from 2.5 μM up to 50 μM) 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 Et₃Pb⁺ 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 Et₃Pb⁺, 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 Et₃Pb⁺ 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 Et₃Pb⁺ causes irreversible injury in isolated hepatocytes.