ACETAMINOPHEN HEPATOTOXICITY IN PRIMARY HUMAN HEPATOCYTES AND MICE

Abstract

Acetaminophen is the most prevalent cause of acute liver failure (ALF) and drug-induced liver injury (DILI) in western countries. Extensive studies have revealed important intracellular events during the pathogenesis after APAP in vivo and in vitro. However, no detailed mechanistic research has been conducted in freshly isolated primary human hepatocytes (PHH), which are the gold standard to test drug-induced toxicity. To that end, the detailed injury time course, dose-response curves and signaling events were characterized. The overall time course and sequence of events mirror the clinical situation in APAP overdose patients, but occur significantly more slowly than in APAP-treated rodents, emphasizing cautious data extrapolation across experimental models. In addition, c-Jun N-terminal kinase (JNK) inhibitor moderately attenuated cell death after APAP, suggesting a detrimental role of JNK in injury progression. Although APAP-induced hepatotoxicity is reproducible in the murine model, it is not the case for AMAP, a regioisomer of APAP. AMAP was considered for long to be non-hepatotoxic in mice, primary mouse hepatocytes (PMH), hamsters and hepatoma cell lines. The lack of toxicity was largely due to the significantly less mitochondrial protein adduct formation after AMAP compared with APAP. In PHH, significant cell death was observed after AMAP, accompanied by a loss of mitochondrial membrane potential and the absence of JNK activation or P-JNK translocation to mitochondria. Further investigation indicated that AMAP toxicity was readily explained by mitochondria protein adducts formation in primary human but not mouse hepatocytes, highlighting the critical role of mitochondrial protein arylation in determining APAP or AMAP hepatotoxicity. Additional studies were performed to investigate the toxicity of ATP in vitro. ATP released from necrotic hepatocytes is considered a damage-associated molecular pattern (DAMP) molecule which could elicit innate immune responses, and therefore contributes to cell death. A recently published paper also suggested a direct toxicity of ATP. However, experiments in four different hepatocyte types including PHHs demonstrated an absence of toxicity directly by ATP. The fourth study focuses on characterization of APAP metabolites and adducts formation in APAP overdose patients, suggesting the importance of profiling both metabolites and protein adduct formation in the clinical diagnosis of APAP overdose. Given the importance of c-Jun N-terminal kinase (JNK) in APAP-induced liver injury, two pharmacological inhibitors of apoptosis signal-regulating kinase 1 (ASK1), an upstream kinase of JNK, were tested in mice. The ASK1 inhibitor attenuated liver injury both as a pre-treatment and as a 1.5h post-treatment by blocking JNK activation and P-JNK translocation to mitochondria. Importantly, inhibiting ASK1 activity did not affect liver regeneration.