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.