Lack of Direct Cytotoxicity of Extracellular ATP against Hepatocytes: Role in the Mechanism of Acetaminophen Hepatotoxicity

Abstract

Background Acetaminophen (APAP) hepatotoxicity is a major cause of acute liver failure in many countries. Mechanistic studies in mice and humans have implicated formation of a reactive metabolite, mitochondrial dysfunction and oxidant stress as critical events in the pathophysiology of APAP-induced liver cell death. It was recently suggested that ATP released from necrotic cells can directly cause cell death in mouse hepatocytes and in a hepatoma cell line (HepG2). Aim To assess if ATP can directly cause cell toxicity in hepatocytes and evaluate their relevance in the human system. Methods Primary mouse hepatocytes, human HepG2 cells, the metabolically competent human HepaRG cell line and freshly isolated primary human hepatocytes were exposed to 10-100 μM ATP or ATγP in the presence or absence of 5-10 mM APAP for 9-24 h. Results ATP or ATγP was unable to directly cause cell toxicity in all 4 types of hepatocytes. In addition, ATP did not enhance APAP-induced cell death observed in primary mouse or human hepatocytes, or in HepaRG cells as measured by LDH release and by propidium iodide staining in primary mouse hepatocytes. Furthermore, addition of ATP did not cause mitochondrial dysfunction or enhance APAP-induced mitochondrial dysfunction in primary murine hepatocytes, although ATP did cause cell death in murine RAW macrophages. Conclusions It is unlikely that ATP released from necrotic cells can significantly affect cell death in human or mouse liver during APAP hepatotoxicity. Relevance for Patients Understanding the mechanisms of APAP-induced cell injury is critical for identifying novel therapeutic targets to prevent liver injury and acute liver failure in APAP overdose patients.

The impact of sterile inflammation in acute liver injury

“…Treatment with P2XR7 pharmacological antagonist yielded more protection than the corresponding gene knockout of P2XR7 [21], and the inhibitor was subsequently shown to inhibit APAP metabolism, and thus its protective effects were independent of its alleged pharmacological effects on inflammasome activation [208]. Furthermore, evidence that extracellular ATP directly exacerbates APAP- induced cell death [205] has also been un-repeatable in both rodent and human hepatocytes [209]. Most importantly, human plasma samples from APAP overdose patients did not have any evidence of enhanced IL-1ß formation at any point over the first seven days of injury [5].…”