Loss of Calcium Homeostasis Leads to Progressive Phase of Chlordecone-Potentiated Carbon Tetrachloride Hepatotoxicity

Abstract

Earlier work has shown increased hepatocellular free Ca2+ levels in rats receiving a single subtoxic dose of CCl4 after dietary pretreatment with nontoxic (10 ppm, 15 days) levels of chlordecone (CD), indicating a significant perturbation of Ca2+ homeostasis in the interactive toxicity of CD + CCl4 combination treatment. In the present study, the mitochondrial and microsomal ability to sequester Ca2+ as well as plasma membrane translocase activity was investigated, since it is known that cells maintain normal Ca2+ homeostasis by these mechanisms. Hepatic plasma membrane Ca2+-ATPase (high and low affinity components) as well as 45Ca uptake by mitochondria and microsomes was measured using a range of calcium concentrations in Ca2+-EGTA-buffered medium at different time points after a single ip administration of CCl4 (100 μl/kg). Male Sprague-Dawley rats were maintained for 15 days either on a normal diet or on a diet containing 10 ppm CD prior to CCl4 injection. Hepatic plasma membranes, devoid of microsomal and mitochondrial contamination, were prepared using polyethyleneimme-coated beads. CD treatment alone did not significantly decrease the plasma membrane Ca2+ATPase activity. Similarly, CCl4 treatment alone did not alter Ca2+-ATPase in hepatic plasma membranes at any concentration of free Ca2+ in assay medium employed in this study. The interactive combination treatment, however, resulted in significant, irreversible, and specific inhibition of the high affinity component of the hepatic plasma membrane Ca2+-ATPase at early time points. Low affinity Ca2+-ATPase was not affected with any treatment protocol. CD pretreatment alone significantly inhibited 45Ca uptake by mitochondria and microsomes when incubated at 10 μM and higher, concentrations much higher than normal cytosolic levels, but not at lower concentrations of Ca2+. CCl4 administration to both normal and CD-pretreated rats resulted in significant inhibition of microsomal and mitochondrial 45Ca uptake as early as 1 hr at all concentrations of free calcium. While the extent of inhibition was greater and irreversible after CD + CCl4 treatment, it was reversible after normal diet + CCl4 treatment. Phosphorylation of proteins was determined in order to investigate if the inhibition of microsomal 45Ca uptake during CD + CCl4 toxicity might be correlated to decreased phosphorylation of any particular protein involved in Ca2+ transport. SDS-polyacrylamide gel electrophoresis of microsomal protein revealed at least 30 Comassie blue stainable bands. Of these, 6 bands were phosphorylated when microsomes were incubated with [32P]ATP. After CCl4 treatment, the band corresponding to ∼20 kDa protein in liver microsomes decreased in its intensity in both normal and CD rats, suggesting that CCl4-induced inhibition of microsomal 45Ca uptake was due to decreased phosphorylation of ∼20 kDa protein. The decrease was significantly greater after CD + CCl4 combination treatment. These results suggest that an irreversible perturbation of Ca2+ homeostasis is a critical event in the liver injury associated with the hepatotoxicity of CD + CCl4 combination treatment.