CALCIUM-DEPENDENT DNA-DAMAGE AND ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE-INDEPENDENT GLYCOGEN-PHOSPHORYLASE ACTIVATION IN AN IN-VITRO MODEL OFACETAMINOPHEN-INDUCED LIVER-INJURY

Acetaminophen (N-acetyl-p-aminophenol [APAP]) hepatotoxicity is a proc ess characterized by Ca2+ deregulation. Cellular functions utilizing C a2+ as a second messenger molecule affect both cytosolic and nuclear s ignal transduction. Many studies have independently shown Ca2+-related effects on target molecules in response to toxic doses of APAP; howev er the primary Ca2+ target resulting in liver necrosis has not been de termined. We hypothesize that Ca2+-dependent DNA damage is a critical event in liver necrosis caused by alkylating hepatotoxins, In this stu dy, Ca2+-dependent endonuclease activity was determined hom DNA single -strand lesions measured by fluorometric analysis of DNA unwinding. Th e status of cytosolic Ca2+ was determined by measuring Ca2+-dependent activation of glycogen phosphorylase a. Primary cultures of mouse hepa tocytes exposed to a toxic concentration of APAP showed twofold and gr eater increases in glycogen phosphorylase a stimulation at 6 hours, wh ich was reversible with Ca2+-chelating agents. Cell death was preceded by a large decline in intact, double-stranded DNA. Following toxic ad ministration 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 hepatoblast oma HepG2 cell line by lactate dehydrogenase (LDH) release and tetrazo lium reduction using the 3-4,5-dimethylthiazole-2-yl-2,5-diphenyl zoli um bromide thiazol blue(MTT) assay. The Ca2+ chelator, ethylene glycol -bis (beta-aminoethyl ether) N',N',N', N'-tetraacetic acid (EGTA), blo cked 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 lit tle or no contribution of the cAMP pathway to phosphorylase a stimulat ion during APAP-induced necrotic death. Results with these in vitro mo dels 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 t arget for APAP hepatotoxicity.