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 (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 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.