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)
Grewal
K K
KK
Department of Pharmacology and Toxicology, Faculty of Medicine, Queen's University, Kingston, Ont., Canada.
Racz
W J
WJ
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