title: Calcium-dependent DNA damage and adenosine 3',5'-cyclic monophosphate-independent glycogen phosphorylase activation in an in vitro model of acetaminophen-induced liver injury.
abstract: Acetaminophen (N-acetyl-p-aminophenol [APAP]) hepatotoxicity is a process characterized by Ca2+ deregulation. Cellular functions utilizing Ca2+ as a second messenger molecule affect both cytosolic and nuclear signal transduction. Many studies have independently shown Ca2+-related effects on target molecules in response to toxic doses of APAP; however, the primary Ca2+ target resulting in liver necrosis has not been determined. We hypothesize that Ca2+-dependent DNA damage is a critical event in liver necrosis caused by alkylating hepatotoxins. In this study, Ca2+-dependent endonuclease activity was determined from DNA single-strand lesions measured by fluorometric analysis of DNA unwinding. The status of cytosolic Ca2+ was determined by measuring Ca2+-dependent activation of glycogen phosphorylase a. Primary cultures of mouse hepatocytes exposed to a toxic concentration of APAP showed twofold and greater increases in glycogen phosphorylase a stimulation at 6 hours, which was reversible with Ca2+-chelating agents. Cell death was preceded by a large decline in intact, double-stranded DNA. Following toxic administration of APAP, the percentage of total double-stranded DNA was significantly reduced by 2 hours. At 6 and 24 hours, genomic integrity was compromised by 26% and 37%, respectively, compared with untreated controls. Hepatotoxic effects of APAP-mediated Ca2+ deregulation were confirmed in both primary mouse hepatocytes and the human hepatoblastoma HepG2 cell line by lactate dehydrogenase (LDH) release and tetrazolium reduction using the 3-4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide thiazol blue(MTT) assay. The Ca2+ chelator, ethylene glycol-bis (beta-aminoethyl ether) N',N',N', N'-tetraacetic acid (EGTA), blocked APAP-induced phosphorylase a activation and necrotic cell death, but failed to inhibit phosphorylase a activation by the adenosine 3',5'-cyclic monophosphate (cAMP) analogue, dibutyryl cAMP, indicating little or no contribution of the cAMP pathway to phosphorylase a stimulation during APAP-induced necrotic death. Results with these in vitro models of liver injury are interpreted as supporting the hypothesis that increased Ca2+ availability plays a major role in the progression of APAP-dependent cellular necrosis, and that the nucleus is a critical target for APAP hepatotoxicity.
authors.affiliation: Toxicology Program, University of New Mexico College of Pharmacy, Albuquerque 87131-1066, USA.
authors.forename: V M
authors.initials: VM
authors.lastname: Salas
authors.forename: G B
authors.initials: GB
authors.lastname: Corcoran
mesh_terms: D000082:Acetaminophen / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D016923:Cell Death; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury; D000242:Cyclic AMP / Q000502:physiology*; D004249:DNA Damage; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D008297:Male; D051379:Mice; D009336:Necrosis; D010762:Phosphorylase a / Q000378:metabolism; D006005:Phosphorylases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000082:Acetaminophen; D000242:Cyclic AMP; D010762:Phosphorylase a; D006005:Phosphorylases; D002118:Calcium