Purinergic receptor antagonist A438079 protects against acetaminophen-induced liver injury by inhibiting p450 isoenzymes, not by inflammasome activation

Abstract

Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in the western world. Controversy exists regarding the hypothesis that the hepatocyte injury is amplified by a sterile inflammatory response, rather than being the result of intracellular mechanisms alone. A recent study suggested that the purinergic receptor antagonist A438079 protects against APAP-induced liver injury by preventing the activation of the Nalp3 inflammasome in Kupffer cells and thereby preventing inflammatory injury. To test the hypothesis that A438079 actually affects the intracellular signaling events in hepatocytes, C57Bl/6 mice were treated with APAP (300 mg/kg) and A438079 (80 mg/kg) or saline and GSH depletion, protein adduct formation, c-jun-N-terminal kinase (JNK) activation, oxidant stress, and liver cell necrosis were determined between 0 and 6 h after APAP administration. APAP caused rapid GSH depletion, extensive protein adduct formation in liver homogenates and in mitochondria, JNK phosphorylation and mitochondrial translocation of phospho-JNK within 2 h, oxidant stress, and extensive centrilobular necrosis at 6 h. A438079 significantly attenuated GSH depletion, which resulted in a 50% reduction of total liver and mitochondrial protein adducts and substantial reduction of JNK activation, mitochondrial P-JNK translocation, oxidant stress, and liver injury. The same results were obtained using primary mouse hepatocytes. A438079 did not directly affect JNK activation induced by tert-butyl hydroperoxide and GSH depletion. However, A438079 dose-dependently inhibited hepatic P450 enzyme activity. Thus, the protective effect of A438079 against APAP hepatotoxicity in vivo can be explained by its effect on metabolic activation and cell death pathways in hepatocytes without involvement of the Nalp3 inflammasome.

FIG. 1.

Acetaminophen-induced liver injury in C57Bl/6 mice with or without A438079. Animals were pretreated with 2mg/mouse A438079 or saline and then 1h later with 300mg/kg APAP or vehicle control. (A) Plasma ALT at 0, 2, and 6h. (B) Representative H&E-stained liver sections (×50 magnification) and TUNEL staining (×50 magnification) are shown for controls and animals treated with APAP for 6h. Data represent means ± SE of n = 6 animals per group. *p < 0.05 (compared with controls, t = 0). #p < 0.05 (compared with APAP/saline).

FIG. 2.

Liver GSH and GSSG. Animals were pretreated with 2mg/mouse A438079 or saline and then 1h later with 300mg/kg APAP or vehicle control. Total GSH was measured in liver tissue homogenate at 0, 2, and 6h post-APAP (A). GSSG was measured at the same times (B) and the ratio of GSSG to total GSH is shown (C). Data represent means ± SE of n = 6 animals per group. *p < 0.05 (compared with control, t = 0). #p < 0.05 (compared with APAP/saline).

FIG. 3.

JNK phosphorylation after APAP with and without A438079 pretreatment. Animals were pretreated with 2 mg/mouse A438079 or saline and then 1 h later with 300 mg/kg APAP or vehicle control. At 0, 2, and 6 h after APAP, cytosolic factions were subjected to Western blotting for phosphorylated JNK, total JNK, and beta-actin (A). Western blotting was also performed on the isolated mitochondrial fraction for phosphorylated JNK, total JNK, and porin (B). Densitometry was performed on these blots and the P-JNK-to-JNK ratio was calculated for the different time points in the cytosol (C) and in the mitochondria (D). *p < 0.05 (compared with control, t = 0). #p < 0.05 (compared with APAP/saline)

FIG. 4.

Effects of A438079 on APAP-protein adduct formation. Animals were pretreated with 2mg/mouse A438079 or saline and then 1h later with 300mg/kg APAP or vehicle control. APAP-cysteine adducts were quantified by HPLC-ECD in liver homogenate (A) and in the mitochondrial fraction (B) at 0, 0.5, 2, and 6h post-APAP. Data represent means ± SE of n = 3–6 animals per group. *p < 0.05 (compared with control, t = 0). #p < 0.05 (compared with APAP/saline).

FIG. 5.

Effect of A438079 on GSH depletion kinetics in vivo and on P450 activities. Animals were pretreated with 2mg/mouse A438079 or saline and then 1h later with 300mg/kg APAP or vehicle control. (A) Total liver GSH was quantified at very early times (0, 10, 20, and 30min post-APAP). Data represent means ± SE of n = 3–5 animals per group. *p < 0.05 (compared with APAP/saline). (B) Cytochrome P450 activities were measured in the 14,000 × g supernatant of mouse liver homogenate using the 7EFC deethylase assay. Various concentrations of A438079 or the classical P450 inhibitor piperonyl butoxide were added. Data are expressed in percent compared with untreated control samples (100%); data represent means ± SE of n = 3 samples per concentration. *p < 0.05 (compared with control). #p < 0.05 (compared with piperonyl butoxide).

FIG. 6.

Effects of A438079 on APAP toxicity in mouse hepatocytes. Primary mouse hepatocytes were pretreated for 1h with A438079 (100µM) and then APAP was added (5mM). (A) Cell death was measured by LDH release at 9h. (B) APAP protein adducts were determined at 1 and 3h after APAP. (C) P-JNK and JNK protein expression was evaluated by Western blotting in untreated cells and at 9h. (D) Densitometric analysis of the Western blots and calculation of the P-JNK-to-JNK ratio. Data represent means ± SE of n = 3–5 separate experiments. *p < 0.05 (compared with control). #p < 0.05 (compared with APAP/saline).

FIG. 7.

Effect of A438079 on JNK phosphorylation. Mice were treated with phorone (PH) and tBHP to cause GSH depletion and oxidant stress, respectively. Animals were either pretreated with 2mg/mouse of A438079 or saline. One hour after PH/tBHP, JNK phosphorylation was assessed by Western blotting (A) and the P-JNK-to-JNK was ratio was calculated (B). Data represent means ± SE of n = 3 animals per group. *p < 0.05 (compared with vehicle control).