Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity

Abstract

BackgroundAdenosine 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.ResultsAPAP 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.ConclusionWe 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.

Hepatic Overexpression of ATP Synthase β Subunit Activates PI3K/Akt Pathway to Ameliorate Hyperglycemia of Diabetic Mice

“…P2 receptors are also constitutively expressed in liver cells and HepG2 cells (24,34). Exposure to exogenous ATP has been reported to elevate the cytosolic free calcium level in HepG2 cells and primary cultured mouse hepatocytes (35). PPADS is a specific inhibitor for the P2X subtype, whereas suramin is considered to be a broad-spectrum P2 receptor inhibitor (36).…”

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

“…Although we could not confirm a role of caspase-1, the Nalp3 inflammasome and IL-1β (Williams et al, 2010b, 2011), or the purinergic receptor P2X7 (Xie et al, 2013) in the mechanism of APAP hepatotoxicity, a recent paper introduced a novel hypothesis for the role of ATP released by necrotic cells. Amaral et al (2013) provided evidence that human hepatoma cells (HepG2) exposed to APAP release ATP into the culture supernatant. In addition, the authors further demonstrated that primary mouse hepatocytes and HepG2 cells can be directly killed by exposure to ATP at concentrations between 10 and 100 μM (Amaral et al, 2013).…”

“…Amaral et al (2013) provided evidence that human hepatoma cells (HepG2) exposed to APAP release ATP into the culture supernatant. In addition, the authors further demonstrated that primary mouse hepatocytes and HepG2 cells can be directly killed by exposure to ATP at concentrations between 10 and 100 μM (Amaral et al, 2013). Thus, in addition to activating the inflammasome, ATP may directly contribute to aggravation of APAP-induced liver injury as a cytotoxic agent (Amaral et al, 2013).…”

Purinergic Signaling in Early Inflammatory Events of the Foreign Body Response: Modulating Extracellular ATP as an Enabling Technology for Engineered Implants and Tissues

“…Similarly, in a mouse model of acetaminopheninduced hepatotoxicity, overdose resulted in liver necrosis and massive neutrophil infiltration; again, apyrase administration significantly attenuated this response. 116 Apyrase treatment has also shown improvement of outcome and reduction of inflammation in a number of other models including lipopolysaccharide-induced lung inflammation, 117 laryngeal airway hyperreactivity, 118 asthma, 119 mechanical ventilation-induced acute lung injury, 120 and cardiac xenograft. 121 In the case of implants, we have observed reductions in inflammatory cell infiltrate and coagulative necrosis of muscle tissue when apyrase was applied in a rat submuscular implant model (Fig.…”