Fasting of mice inhibits apoptosis during paracetamol-induced hepatotoxicity - critical role of hepatic ATP

Daniel Antoine, Dominic Williams, Anja Kipar, B.Kevin Park

University of Liverpool, Liverpool, UK

Drug-induced liver injury represents a major impediment to drug development and is one of the most prevalent hepatic adverse effects seen in the clinic. Paracetamol (APAP) hepatotoxicity accounts for a significant proportion of cases of acute liver failure in the USA and UK and is reproducible in animal models (Ostapowicz et al., 2002). It is regarded that necrosis is the major form of cell death but despite intense research, inconsistencies remain concerning the existence or role played by apoptosis during APAP hepatotoxicty. Literature analysis revealed that when animals had been fasted prior to experimentation; no evidence for APAP-induced apoptosis could be found. In contrast, studies that provide experimental evidence for APAP-induced apoptosis, animals had free access to food prior to investigation (Ray et al., 1996, Cover et al., 2005). The aim of this study was to assess the impact of fasting on the toxicological response associated with APAP, in particular the role of apoptosis, which could help explain the inconsistencies observed in the literature. Male CD-1 (20-30g) mice were either fasted 24hr prior to experimentation or had free access to food. Mice were dosed APAP (530mg kg-1 + 5μCi [14C]-APAP; 5hr; i.p) Some mice received diethyl maleate (DEM; 4.7mmol kg-1; i.p) or glucose / glycine (1000 / 250 mg kg-1; i.p) 1hr prior to APAP administration. Hepatotoxicity was determined through histological examination and serum alanine aminotransferase (ALT) measurements. Hepatic GSH, ATP and APAP covalent binding was also measured. Serum High Mobility Group Box protein 1 (HMGB1) was determined by LC-MS and ELISA as a marker of necrosis. Determination of cytokeratin-18 (CK18) caspase dependent cleavage by LC-MS and ELISA, pro-caspase processing and DNA laddering were utilised as markers of hepatic apoptosis. Necrosis was the major form of cell death induced by APAP (ALT; 2646.4±1012.3U l-1**. HMGB1; 210.2±55.1ng ml-1**). However, hepatocyte apoptosis was also observed by assessing pro-caspase 3 processing, DNA laddering a cleavage of CK18 (801.4±198.1pmol ml-1*). Overnight fasting blocked CK18 cleavage (232.4±201.6pmol ml-1†), pro-caspase 3 processing and DNA laddering. Hepatic necrosis was higher in fasted compared to fed mice (HMGB1; 295.8±20.3ng ml-1†). Metabolic activation of APAP, determined by covalent binding, was unaffected by overnight fasting (fed; 1.87±0.45nmol mg-1: fast; 1.95±0.44nmol mg-1). Overnight fasting, however, resulted in a depletion of both basal levels of hepatic GSH and ATP content. Pre-depletion of GSH with DEM did not abolish the apoptotic response induced by APAP in fasted mice. However, in fasted mice where hepatic ATP content was restored by glucose / glycine administration prior to APAP, apoptosis was observed when determined by CK18 cleavage (498.6±80.5pmol ml-1), DNA laddering and pro-caspase 3 processing. Depletion of hepatic ATP content by fasting animals is critical in determining the balance between APAP hepatotoxicity being a mixed apoptotic / necrotic event or just necrosis. Fasting of animals prior to experimentation has important implications for the cellular outcome of mechanistic toxicological investigations and requires careful consideration. †p<0.05 compared to fed mice. *p<0.05, **p<0.01 compared to control vehicle dosed mice.

Ostapowicz et al., 2002 Ann Intern Med 137:947-54. Ray et al., 1996 JPET 279: 1470-83. Cover et al., 2005 JPET 315: 879-87. Strubelt et al., 1981 Tox App Pharm 60:66-77.