Investigation of the role of inflammation in drug-induced hepatotoxicity

• Main Author: Benson, Craig Andrew
• Published: University of Liverpool 2011
• Subjects: 616.36207
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.569176

Drug-induced liver injury (DILI) is the most frequent cause for withdrawal of approved drugs and attrition of drug development. Although the cellular mechanisms of DILI leading to overt tissue and organ damage are still not fully defined, model hepatotoxins such as acetaminophen (paracetamol; AP AP) have been used in animal models to provide important insight into the relationship between chemical structure, drug metabolism and cellular response. The overall extent of APAP injury is thought to be dependent on the activation of cell-cell signalling pathways, in particular between hepatocytes and cells of the innate immune system. However, the extent of the role of the innate immune and inflammatory response in the modulation of liver injury remains poorly defined, with conflicting evidence in the literature. The work presented in this thesis has used model hepatotoxins AP AP and galactosamine (GaIN) and the innate immune stimulator lipopolysaccharide (LPS) as tools to explore the mechanism of inflammation associated with DILl with the aim to further understand the various inflammatory pathways during DILl and their contribution to the progression ofliver injury. Male C57BLl6 mice were used to investigate the inflammatory response associated with AP AP, GaIN and LPS exposure. Inflammation was measured as serum levels of TNF -u, IL-6 and IL- 1β by ELlSA and neutrophil infiltration as assessed by histological analysis. Hepatotoxicity in the AP AP model was characterised by a dose and time-dependent increase in serum ALT and was confirmed by histological analysis (O-lOOOmg/kg; 0-24hr). Significant elevations in levels of TNF -α, IL-6 and IL-l β were observed in the serum of mice following APAP overdose (530mg/kg; 0-24hr). Overnight fasting of mice led to a greater hepatotoxic and inflammatory response compared to non-fasted mice post AP AP treatment. The LPS model was characterised by having no hepatotoxicity, as serum ALT activity remained at control levels (control - 41.37±8.45U/l; LPS - 50.57±5.56U/I; 5mg/kg; 24hr) and having a potent inflammatory response as determined by cytokine release (TNF-α 513.13±50.15pg/ml; IL-6 - 1201.12±157.20pg/ml; IL-lβ - 227.08±39.93pg/ml; 5mg/kg; 24hr) and neutrophil infiltration. The GaIN model was characterised by mild hepatotoxicity (ALT activity 875.30±144.81U/l; 800mg/kg; 24hr) and a mild inflammatory response, with increase in cytokines (TNF-α - 56.44±5.68pg/ml; IL-6 - 63.22±8.41pg/ml; IL-lβ- 40.72±3.64pg/ml; 800mg/kg; 24hr) and neutrophil infiltration. Concurrent administration of suppressors of either metabolism (DMSO), or the innate immune response (aspirin, ethyl pyruvate and anti-HMGBl antibody) was also investigated. DMSO inhibited APAP metabolism, whereas aspirin had no effect on AP AP toxicity or the inflammatory response. Delayed administration of ethyl pyruvate reduced toxicity and serum HMGB 1 content post APAP. Expression of RAGE mRNA over a 24hr time course was measured in whole livers of fasted and fed mice treated with APAP by RT-PCR. Full-length (flRAGE), endogenous secretory (esRAGE) and total (tRAGE) isoforms of RAGE increased in fed mice between l hr and 3hr. Fasted mice showed a more prolonged increase in expression of all 3 isoforms of RAGE, culminating in a peak increase at 5hr. Western blot analysis determined that there were no changes in RAGE protein expression in whole liver over a 24hr period of AP AP iv exposure. In situ hybridisation revealed a nuclear accumulation of RAGE mRNA III hepatocytes following AP AP exposure. In summary, the studies described in this thesis have provided further evidence for the role of inflammation during DILl by using and developing a variety of in vivo models. These models assessed both basic and novel mechanisms of inflammation, such as cytokine release, hepatic neutrophil infiltration and RAGE expression, and tested potential therapeutic inhibitors such as aspirin and EP.