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Calcium‐dependent DNA damage and adenosine 3′,5′‐cyclic monophosphate‐ independent glycogen phosphorylase activation in an in vitro model of acetaminophen‐induced liver injury
Author(s) -
Salas V M,
Corcoran G B
Publication year - 1997
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.510250621
Subject(s) - acetaminophen , dna damage , glycogen phosphorylase , chemistry , biochemistry , hepatocyte , calcium , lactate dehydrogenase , microbiology and biotechnology , glycogen , biology , dna , in vitro , enzyme , organic chemistry
Abstract Acetaminophen (N‐acetyl‐p‐aminophenol [APAP]) hepatotoxicity is a process characterized by Ca 2+ deregulation. Cellular functions utilizing Ca 2+ as a second messenger molecule affect both cytosolic and nuclear signal transduction. Many studies have independently shown Ca 2+ ‐ related effects on target molecules in response to toxic doses of APAP; however, the primary Ca 2+ target resulting in liver necrosis has not been determined. We hypothesize that Ca 2+ ‐dependent DNA damage is a critical event in liver necrosis caused by alkylating hepatotoxins. In this study, Ca 2+ ‐dependent endonuclease activity was determined from DNA single‐strand lesions measured by fluorometric analysis of DNA unwinding. The status of cytosolic Ca 2+ was determined by measuring Ca 2+ ‐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 Ca 2+ ‐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 Ca 2+ 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 Ca 2+ chelator, ethylene glycol‐bis (β‐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 Ca 2+ availability plays a major role in the progression of APAP‐dependent cellular necrosis, and that the nucleus is a critical target for APAP hepatotoxicity.