Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity

Abstract

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.

Figure 1

Extracellular ATP signaling enhances APAP-induced hepatotoxicity. (A) Liver intravital microscopy showing sinusoids (in red) and GFP-expressing neutrophils (in green). Mice were treated with acetaminophen (500 mg/Kg; 24 h) and a group received apyrase (25 U/mice) 2 hours after APAP treatment. Control mice were wild type C57 or Lysm-eGFP (in intravital microscopy studies). No significant differences regarding liver injury were observed between C57 and C57-Lysm-eGFP expressing mice. Note the reduced liver injury and neutrophil recruitment in apyrase-treated mice. (B) H&E stained liver sections confirmed liver injury and partial protection promoted by extracellular ATP metabolism by apyrase. (C) Circulating levels of liver transaminase (ALT) and (D-E) pro-inflammatory cytokines (TNF-α and IL-1β). (F) Serum ALT levels of APAP-challenged mice treated with different purinergic receptors antagonists TNP-ATP (selective P2X, 1 mg/Kg) and oxi-ATP (selective P2X7, 9 mg/Kg). * P < 0.05 in comparison to control group and ** in comparison to vehicle-treated group. N = 5/group. Data are mean ± SEM. Scale: 100 μm.

Figure 2

APAP-induced remote lung injury was not reduced following extracellular ATP cleavage or P2 receptors blockage. (A) Lung histology from control mice showing normal morphological findings, while APAP-treated mice presented marked lung inflammation, similarly to apyrase treated group (25 U/mice). Representative images from five different mice/group. (B) Leukocyte numbers in bronchial-alveolar lavage (BAL) from APAP treat mice showing that liver injury triggered remote lung inflammation, which was not prevented by dampening ATP sensing. (C) Macrophages were the most frequent cell type found in BAL. * - P < 0.05 in comparison to control group, ANOVA followed by Bonferroni post-test. Data are mean ± s.e.m. N = 5/group. Data are mean ± SEM. Scale: 100 μm.

Figure 3

ATP is released and accounts to additional cell death during APAP incubation. (A-B) Dose and time-response curves of APAP challenged HepG2 cells (24 h). (C-E) Acridine orange/ethidium bromide viability test confirmed APAP cytotoxicity. (F) ATP and ADP quantification in HepG2 supernatant (HPLC) in different timepoints after APAP incubation (20 mM). (G-H) Effects of apyrase (10 U/ml) or different P2, P2X and P2Y blockers (0.1 mM, 100 μM and 30 μM, respectively) on APAP-mediated cell death. (I-J) Direct cytotoxic effect of ATP and ADP on “naïve” HepG2 cells, confirmed by acridine orange/ethidium bromide viability test. Controls (medium alone) were not different throughout the experiments. * P < 0.05 in comparison to control group and ** in comparison to vehicle or medium treated group.

Figure 4

Hyper-responsiveness to ATP increased intracellular Ca^{2 +}signal during APAP challenge. (A) Ca²⁺ signal induced by ATP and ADP (10 μM) in HepG2 cells. (B-C) Culture medium from necrotic/suffering cells triggered intracellular Ca²⁺ signal in “naïve” HepG2 cells, which was abolished by apyrase (10 U/ml) or P2 blocker suramin (0.1 mM). (D-E) Snapshots from confocal microscopy showing Ca²⁺ signal in HepG2 cells treated or not with APAP at 20 mM. (F-G) ATP-triggered Ca²⁺ signals from cells incubated or not with APAP for 24 hours. (H) Ca²⁺ sequestration by BAPTA-AM (1 nM) partially reverted APAP-mediated cell death. * P < 0.05 in comparison to control group and ** in comparison to vehicle or medium treated group.

Figure 5

APAP-challenged primary hepatocytes presented sustained and repeated intracellular calcium signal due to exogenous ATP administration. (A) Primary mouse hepatocytes (PMH) were isolated and loaded with a fluorescent calcium probe (Fluo4-AM). (B) Following APAP incubation (18 h), PMH viability decreased in a dose-dependent manner, reaching 50% of survival when 20 mM of APAP was used. Therefore, this dose was used to subsequent experiments. (C) ATP administration (10 μM) caused calcium signal in naïve hepatocytes, which returned to baseline values after 60 seconds. Six replicates are represented in the graph. (D) However, APAP-treated cells (20 mM; 6 h) developed a hyper-responsive behavior to the same ATP dose, displaying higher and sustained calcium signal, which was also prolonged for longer periods (200 seconds). (E) Treatment with an unspecific P2 antagonist (suramin, 0.1 mM) completely abrogated APAP effects over ATP stimulation. (F) Representative cells of each group were displayed together. (G) Snapshots from live calcium signal recording using confocal microscopy. Videos were rendered in “rainbow pallet” to facilitate fluorescence observation (generated by Fluo4-AM). Following 110 seconds of ATP stimulation, APAP treated cells remaining responsive with increased intracellular calcium signal, while control cells returned to baseline values after 55–70 seconds. Scale = 20 μm. (H) Incubation of primary hepatocytes with exogenous ATP in the dose range found during necrosis (10-100 μm, 18 h) significantly reduced cell viability. * P < 0.05 in comparison to control group. Data are mean ± SEM.

Figure 6

Up-regulation of several purinergic receptors may explain hyper-responsiveness to ATP during hepatotoxicity. (A) Quantitative PCR for purinergic receptors from cells cultivated in the presence or absence of APAP (20 nM; 24 h). Untreated HepG2 cells (C) determined baseline expression. (B-C) HepG2 incubation with adenosine in the presence or absence of APAP. (D) Serum ALT levels of APAP-challenged mice treated with theophylline (P1 receptor blocker, 20 mg/Kg). (E) Quantitative PCR analysis of liver samples from acute hepatitis patients in comparison to healthy volunteers. Baseline expression was determined by choosing a healthy volunteer as control. (F) Changes in expression of different P2 receptors were compared to serum ALT levels from healthy and acute hepatitis patients. Note that increased P2R expression was correlated with higher serum ALT levels in several patients. Pearson’s correlation was calculated comparing fold increase of P2R expression to serum ALT levels (r). * P < 0.05 in comparison to control group and ** in comparison to vehicle treated group.

Figure 7

Proposed mechanism: 1: Under physiological conditions, extracellular ATP regulates several intracellular signaling pathways, which involves also calcium compartmentalization. 2–3: Acetaminophen incubation directly causes hepatocyte necrosis, calcium imbalance and further ATP release. 4: In parallel, challenged viable hepatocytes up-regulate several purinergic receptors, probably as a regulatory homeostatic strategy, causing ATP hyper-responsiveness. Binding of extracellular ATP to purinergic receptors increases intracellular Ca²⁺ and pulses, which accounted to additional cell necrosis, reverberating APAP-induced death. Dampening of extracellular ATP signalling or reducing intracellular Ca²⁺ availability significantly reduced hepatocyte necrosis. Data from FHF patients suggest that a similar necrosis-amplification pathway may be involved in organ injury progression.