Intracellular calcium disruption as a secondary event in acetaminophen-induced hepatotoxicity.
To examine a role for disturbances in intracellular calcium homeostasis in acetaminophen-induced hepatotoxicity, freshly isolated mouse hepatocytes were incubated with 1.0 mM acetaminophen for 1.5 h to allow for covalent binding and initiation of cell damage. The hepatocytes were then washed and the cells incubated in fresh medium containing either 2.0 mM N-acetylcysteine or 1.5 mM dithiothreitol for the duration of a 4-h incubation period. These agents were used as tools in the elucidation of the biochemical events responsible for acetaminophen-induced cell necrosis. The reduced protein sulfhydryl content, cytosolic [Ca2+], and plasma membrane integrity were quantitated. Acetaminophen produced protein sulfhydryl depletion, an increased cytosolic [Ca2+], and cell injury; however, cytotoxicity preceded the increase in [Ca2+]. Both N-acetylcysteine and dithiothreitol restored the acetaminophen-induced protein sulfhydryl loss. Dithiothreitol prevented both further cell injury and an increase in the cytosolic [Ca2+]. However, cell death and a subsequent increase in cytosolic [Ca2+] proceeded unabated following N-acetylcysteine addition. Although both agents restored protein sulfhydryl content, in view of their contrasting ultimate effects on cell viability the role of reduced protein sulfhydryl depletion in acetaminophen-induced hepatic injury requires further investigation. The increase in cytosolic [Ca2+] with acetaminophen alone and with subsequent N-acetylcysteine addition was determined to be a secondary event in cell injury because cytotoxicity occurred by 1.5 h; however, the increase in cytosolic [Ca2+] was not observed until 2.5 h. Additional evidence for changes in cytosolic [Ca2+] as a secondary event was obtained by incubating the hepatocytes with acetaminophen in the presence of fura 2.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pharmacology and Toxicology, Faculty of Medicine, Queen's University, Kingston, Ont., Canada.
K K
KK
Grewal
W J
WJ
Racz
D000082:Acetaminophen / Q000633:toxicity*; D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D056486:Chemical and Drug Induced Liver Injury / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D016257:Fura-2 / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D013438:Sulfhydryl Compounds / Q000378:metabolism
D002614:Chelating Agents; D013438:Sulfhydryl Compounds; D000082:Acetaminophen; D002118:Calcium; D004229:Dithiothreitol; D016257:Fura-2; D000111:Acetylcysteine