Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity

Background: Adenosine triphosphate (ATP) is secreted from hepatocytes under physiological conditions and plays an important role in liver biology through the activation of P2 receptors. Conversely, higher extracellular ATP concentrations, as observed during necrosis, trigger inflammatory responses that contribute to the progression of liver injury. Impaired calcium (Ca2+) homeostasis is a hallmark of acetaminophen (APAP)-induced hepatotoxicity, and since ATP induces mobilization of the intracellular Ca2+ stocks, we evaluated if the release of ATP during APAP-induced necrosis could directly contribute to hepatocyte death. Results: APAP overdose resulted in liver necrosis, massive neutrophil infiltration and large non-perfused areas, as well as remote lung inflammation. In the liver, these effects were significantly abrogated after ATP metabolism by apyrase or P2X receptors blockage, but none of the treatments prevented remote lung inflammation, suggesting a confined local contribution of purinergic signaling into liver environment. In vitro, APAP administration to primary mouse hepatocytes and also HepG2 cells caused cell death in a dose-dependent manner. Interestingly, exposure of HepG2 cells to APAP elicited significant release of ATP to the supernatant in levels that were high enough to promote direct cytotoxicity to healthy primary hepatocytes or HepG2 cells. In agreement to our in vivo results, apyrase treatment or blockage of P2 receptors reduced APAP cytotoxicity. Likewise, ATP exposure caused significant higher intracellular Ca2+ signal in APAP-treated primary hepatocytes, which was reproduced in HepG2 cells. Quantitative real time PCR showed that APAP-challenged HepG2 cells expressed higher levels of several purinergic receptors, which may explain the hypersensitivity to extracellular ATP. This phenotype was confirmed in humans analyzing liver biopsies from patients diagnosed with acute hepatic failure. Conclusion: We suggest that under pathological conditions, ATP may act not only an immune system activator, but also as a paracrine direct cytotoxic DAMP through the dysregulation of Ca2+ homeostasis.