Investigation Of Apoptotic & Necrotic Dynamics Through HMGB 1 & Cytokeratin-18 in Drug Induced Liver Injury

Daniel Antoine1, Dominic Williams1, Sophie Regan1, Jean Sathish1, John Foster2, B.Kevin Park1
1University of Liverpool, Liverpool, United Kingdom, 2AstraZeneca, Alderley Park, Cheshire, United Kingdom

Liver failure represents one of the most prevalent drug induced adverse effects seen in the clinic and is a major cause of attrition of new chemical entities in development. Activation of cell signalling pathways between hepatocytes and innate immune cells are important in the modulation of paracetamol (APAP) hepatotoxicity (Lui et al., 2004). HMGB1 (high mobility group box protein 1) is a potent activator of innate immune cells. It is specifically released from necrotic but not apoptotic hepatocytes and activated immune cells. It has also been shown to be important in linking necrotic cell death to the inflammatory response during APAP hepatotoxicity modulation (Scaffidi et al., 2002). The role of apoptosis in APAP hepatotoxicity remains controversial. Cytokeratin-18 (CK-18) is an early target of caspase cleavage during apoptosis. CK-18 fragments accumulate in the blood and provide a non-invasive marker of drug induced apoptosis (Cummings et al., 2005).The aim of this study was to utilise the model hepatotoxin, APAP to further investigate the role of apoptosis and inflammatory signalling in drug induced hepatotoxicity through CK-18 and HMGB1 and to validate and quantify HMGB1 and caspase cleaved CK-18 as non-invasive, sensitive, mechanism based serum indicators of liver injury development in animal models of APAP hepatotoxicity. Male CD-1 mice (20-30g) were dosed APAP (0-1000 mgkg-1; 0-24hr; i.p). Some mice were pre-dosed the P450 inhibitor 1-aminobenzotriazole (ABT; 100 mgkg-1; 1hr pre-dose; i.p) or the caspase inhibitor Z-VAD-fmk (10 mgkg-1; 15min pre-dose; i.v). Hepatotoxicity was determined through histological examination and serum alanine aminotransferase (ALT) measurments. Hepatic glutathione (GSH) was also measured and serum HMGB1 and CK-18 fragments determined by ELISA and validated by MALDI-TOF and LC-MS. APAP (530 mgkg-1; 5hr) induced a dose dependent increase in serum ALT and HMGB1 content and a decrease in hepatic glutathione. APAP induced CK-18 cleavage (702±105 U/l) was also dependent upon dose (300-530 mgkg-1). APAP (530 mgkg-1) showed a time dependent increase in serum ALTs (2215±516 U/l; 5hr) and an accumulation in CK-18 fragment levels upto 24hr. Within this time course hepatic GSH decreased until 3hr and returned to control levels at 24hr. Serum HMGB1 levels peaked significantly at 3-5hrs (198±18 ng/ml) and returned to control levels at 24hr (7.2±2.9 ng/ml). This was reflected by histopathological examination showing a peak in hepatotoxicity at 5hr and a hepatic physiological adaptation/repair at 24hr. The P450 inhibitor, ABT prevented the release of ALTs, HMGB1, CK-18 fragments and GSH depletion showing that these are dependent upon APAP reactive metabolite formation. Modulation of APAP hepatotoxicity with Z-VAD-fmk prevented CK-18 fragmentation but increased serum HMGB1 (305±10 ng/ml), ALTs (3420±326 U/l) and apparent worsening of liver injury compared to APAP alone. Dosing mice with the APAP clinical antidote NAC (N-acetyl-cysteine) showed that by looking at serum HMGB1 and CK-18 levels NAC was effective 2hr post dose but ALT determination revealed NAC was effective 3hr post APAP dose. This data demonstrates that both serum HMGB1 and CK-18 fragment determination are sensitive and informative mechanism based markers for drug induced liver injury. Apoptosis also appears to occur in parallel with necrosis at early time points during APAP hepatotoxicity, perhaps as a protective mechanism to prevent the released of harmful inflammatory mediators.



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