Loss of calcium homeostasis leads to progressive phase of chlordecone-potentiated carbon tetrachloride hepatotoxicity.
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 Ca(2+)-ATPase (high and low affinity components) as well as 45Ca uptake by mitochondria and microsomes was measured using a range of calcium concentrations in Ca(2+)-EGTA-buffered medium at different time points after a single ip administration of CCl4 (100 microliters/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 polyethyleneimine-coated beads. CD treatment alone did not significantly decrease the plasma membrane Ca(2+)-ATPase activity. Similarly, CCl4 treatment alone did not alter Ca(2+)-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 Ca(2+)-ATPase at early time points. Low affinity Ca(2+)-ATPase was not affected with any treatment protocol. CD pretreatment alone significantly inhibited 45Ca uptake by mitochondria and microsomes when incubated at 10 microM 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 Coomassie blue stainable bands. Of these, 6 bands were phosphorylated when microsomes were incubated with [32P]ATP.(ABSTRACT TRUNCATED AT 400 WORDS)
Kodavanti
P R
PR
Division of Pharmacology and Toxicology, College of Pharmacy and Health Sciences, Northeast Louisiana University, Monroe 71209-0470.
Rao
V C
VC
Mehendale
H M
HM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002251:Carbon Tetrachloride / Q000008:administration & dosage / Q000633:toxicity*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D007631:Chlordecone / Q000633:toxicity*; D004032:Diet; D004591:Electrophoresis, Polyacrylamide Gel; D006706:Homeostasis; D007274:Injections, Intraperitoneal; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D008855:Microscopy, Electron, Scanning; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D011506:Proteins; D002251:Carbon Tetrachloride; D000252:Calcium-Transporting ATPases; D007631:Chlordecone; D002118:Calcium