Delayed Treatment With 4-Methylpyrazole Protects Against Acetaminophen Hepatotoxicity in Mice by Inhibition of c-Jun n-Terminal Kinase

Abstract

Acetaminophen (APAP) overdose is the most common cause of hepatotoxicity and acute liver failure in the United States and many western countries. However, the only clinically approved antidote, N-acetylcysteine, has a limited therapeutic window. 4-Methylpyrazole (4MP) is an antidote for methanol and ethylene glycol poisoning, and we have recently shown that cotreatment of 4MP with APAP effectively prevents toxicity by inhibiting Cyp2E1. To evaluate if 4MP can be used therapeutically, C57BL/6J mice were treated with 300 mg/kg APAP followed by 50 mg/kg 4MP 90 min later (after the metabolism phase). In these experiments, 4MP significantly attenuated liver injury at 3, 6, and 24 h after APAP as shown by 80%-90% reduction in plasma alanine aminotransferase activities and reduced areas of necrosis. 4MP prevented c-Jun c-Jun N-terminal kinase (JNK) activation and its mitochondrial translocation, and reduced mitochondrial oxidant stress and nuclear DNA fragmentation. 4MP also prevented JNK activation in other liver injury models. Molecular docking experiments showed that 4MP can bind to the ATP binding site of JNK. These data suggest that treatment with 4MP after the metabolism phase effectively prevents APAP-induced liver injury in the clinically relevant mouse model in vivo mainly through the inhibition of JNK activation. 4MP, a drug approved for human use, is as effective as N-acetylcysteine or can be even more effective in cases of severe overdoses with prolonged metabolism (600 mg/kg). 4MP acts on alternative therapeutic targets and thus may be a novel approach to treatment of APAP overdose in patients that complements N-acetylcysteine.

Keywords: N-acetylcysteine; acetaminophen hepatotoxicity; autophagy; c-Jun N-terminal kinase; galactosamine-endotoxin; mitochondria.

Figure 1.

Delayed 4-methylpyrazole (4MP) treatment protects against acetaminophen (APAP)-induced liver injury. Animals were treated with 300 mg/kg APAP followed by 50 mg/kg 4MP 90 min after APAP. A, Plasma alanine aminotransferase (ALT) at 3 and 6 h after APAP. B, Representative hematoxylin and eosin (H&E)-stained liver section 3 h post-APAP. C, Three hours post-APAP and 4MP. D, Six hours post-APAP. E, Six hours post-APAP and 4MP (×50 magnification). F, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (×50 magnification) shown for animals treated with APAP for 6 h and (G) for animals treated with APAP and 4MP for 6 h. Data represent means ± SEM of n = 11 animals per group. *p < .05 (compared with animals treated with APAP alone).

Figure 2.

Delayed 4-methylpyrazole (4MP) treatment prevented acetaminophen (APAP)-induced sustained c-Jun n-terminal kinase (JNK) activation. Animals were treated with 300 mg/kg APAP followed by 50 mg/kg 4MP 90 min after APAP. Cytosolic fraction and isolated mitochondrial fraction were subjected to western blotting for P-JNK and JNK after 3 (A) and 6 h (B) after APAP. Three animals were analyzed per group.

Figure 3.

Delayed 4-methylpyrazole (4MP) treatment prevented acetaminophen (APAP)-induced mitochondrial dysfunction independent of reactive metabolite formation. Animals were treated with 300 mg/kg APAP followed by 50 mg/kg 4MP 90 min after APAP. A, APAP-cysteine adducts were quantified by HPLC-ECD in liver homogenate 3 and 6 h post-APAP treatment. B, Cytosolic fractions were subjected to western blotting for apoptosis-inducing factor (AIF) and β-actin. C, Total glutathione (GSH) was measured in liver tissue homogenate at 6 h post-APAP. D, The ratio of glutathione disulfide (GSSG) to total GSH was calculated. Data represent means ± SEM of n = 6 animals per group. *p < .05 (compared with animals treated with APAP alone).

Figure 4.

4-Methylpyrazole (4MP) inhibits c-Jun n-terminal kinase (JNK) activation induced by glutathione (GSH) depletion and oxidant stress and injury and JNK activation in the D-galactosamine/endotoxin (Gal/ET) model. A, C57BL/6J mice were treated with 100 mg/kg phorone followed by 1 mmol/kg tBHP 1 h after phorone and 50 mg/kg 4MP 1 h after tBHP. At 6 h after tBHP cytosolic fractions were subjected to western blotting for P-JNK and JNK. For the Gal/ET model, C57BL/6J mice were cotreated with 700 mg/kg galactosamine, 100 µg/kg endotoxin, and 50 mg/kg 4MP for 1 and 6 h. B, Plasma alanine aminotransferase (ALT) at 6 h after Gal/ET. C, Cytosolic fractions were subjected to western blotting for P-JNK and JNK at 1 and 6 h after Gal/ET. D, TNF-α mRNA expression in liver samples at 1 h after Gal/ET. Data represent means ± SEM of n = 6 animals per group. *p < .05 (compared with control). #p < .05 (compared with Gal/ET).

Figure 5.

4-Methylpyrazole (4MP) docks into the active site and destabilize both c-Jun n-terminal kinase (JNK)1 and JNK2. Snapshots of 4MP molecular docking results. A, Binding site and pose of docked 4MP (yellow sticks) compared with previously published structure of JNK1 (gray surface) bound to an ATP analog (green/dark gray sticks). B, 4MP (yellow/light gray sticks) docked into the ATP binding site of JNK2 (gray surface), overlaid with a structure of the inhibitor BIRB796 (green/dark gray sticks), a compound demonstrated to prevent ATP binding. C, Binding site and orientation of docked 4MP (yellow/light gray sticks) compared to the published structure of Cyp2e1 (gray surface) with 4MP and the heme moiety (green/dark gray sticks). Each structure has portions of the enzyme removed for clarity, and PDB codes 3E4E, 3V3V, 3NPC were used. Control docking experiments. D and E, Overlays from 2 angles of a published structure of quercetagetin (green/dark gray sticks) bound to JNK1 (gray surface) with the docked binding mode (yellow/light gray sticks). F and G, Two views of a structure of JNK2 (gray surface) bound to BIRB796 (green sticks) superimposed with the docked binding mode of BIRB796 (yellow/light gray sticks). H, Detection of soluble protein in the supernatant fraction by Western blot and aggregation temperature curve from cellular thermal shift assay (CETSA) assay to evaluate 4MP-JNK binding in vitro. I, Hepatic JNK enzyme activity was assayed in vitro in the presence or absence of 2 mM 4MP using a c-Jun recombinant protein as the substrate.

Figure 6.

Efficacy of 4-methylpyrazole (4MP) versus N-acetylcysteine (NAC). C57BL/6J mice were treated with 300 mg/kg acetaminophen (APAP) for 6 and 24 h followed by 50 mg/kg 4MP, 200 mg/kg 4MP, or 500 mg/kg NAC alone or in combination 90 min after APAP. A, Plasma alanine aminotransferase (ALT) of 6 and 24 h post-APAP. B, Animals were treated with 600 mg/kg APAP followed by 50 mg/kg 4MP 90 min later. Plasma ALT 24 h post-APAP. Data represent means ± SEM of n = 9 animals per group. *p < .05 (compared with APAP alone). #p < .05 (compared with APAP  + NAC).