Acetaminophen Hepatotoxicity: Paradigm for Understanding Mechanisms of Drug-Induced Liver Injury

Abstract

Acetaminophen (APAP) overdose is the clinically most relevant drug hepatotoxicity in western countries, and, because of translational relevance of animal models, APAP is mechanistically the most studied drug. This review covers intracellular signaling events starting with drug metabolism and the central role of mitochondrial dysfunction involving oxidant stress and peroxynitrite. Mitochondria-derived endonucleases trigger nuclear DNA fragmentation, the point of no return for cell death. In addition, adaptive mechanisms that limit cell death are discussed including autophagy, mitochondrial morphology changes, and biogenesis. Extensive evidence supports oncotic necrosis as the mode of cell death; however, a partial overlap with signaling events of apoptosis, ferroptosis, and pyroptosis is the basis for controversial discussions. Furthermore, an update on sterile inflammation in injury and repair with activation of Kupffer cells, monocyte-derived macrophages, and neutrophils is provided. Understanding these mechanisms of cell death led to discovery of N-acetylcysteine and recently fomepizole as effective antidotes against APAP toxicity.

Keywords: autophagy; drug discovery; drug-induced liver injury; modes of cell death; oxidant stress; sterile inflammation.

Figure 1.. Adaptive Responses after APAP overdose.

Acetaminophen hepatotoxicity after an overdose mainly affects centrilobular hepatocytes where cytochrome P450 2E1 (Cyp2E1) generates the reactive metabolite NAPQI, which is scavenged by hepatic glutathione stores. This results in a depletion of glutathione, which allows formation of NAPQI-protein adducts. This molecular modification activates autophagic pathways which remove adducted proteins and attempt to limit cellular damage. When this adaptive pathway is overwhelmed, NAPQI-adducts on mitochondrial proteins induce directional release of superoxide into the cytosol from respiratory complex III which activates a MAP kinase cascade culminating the activation of JNK and its mitochondrial translocation. This is accompanied by a decrease in mitochondrial membrane potential, which induces a reversible change in mitochondrial morphology. The mild cytosolic oxidant stress also likely activates antioxidant responses mediated by Nrf2 which detaches from its binding partner Keap1 to translocate to the nucleus and induce transcription of genes involved in glutathione resynthesis after binding to the antioxidant response element (ARE). Mitochondrial JNK translocation also induces mitochondrial membrane depolarization and induction of mitophagy which attempts to remove damaged organelles and maintain cellular function (Figure created with Biorender.com).

Figure 2.. Mitochondrial JNK translocation amplifies mitochondrial dysfunction.

Persistent JNK activation and translocation to the mitochondria inhibits mitochondrial electron transport and respiration, causing release of superoxide from respiratory complex I, which affects the mitochondrial matrix proteins unlike the earlier superoxide generation from complex III. Reaction of superoxide with nitric oxide generates highly reactive peroxynitrite which modifies mitochondrial proteins by tyrosine nitration, a process requiring lysosomal iron taken up into the mitochondria. The formation of nitrotyrosine on mitochondrial proteins ultimately causes complete dissipation of mitochondrial membrane potential and induction of Drp1-mediated mitochondrial fission. Ultimately, activation of the mitochondrial permeability transition results in release of endonuclease G (EndoG) and apoptosis inducing factor (AIF) which translocate to the nucleus, causing DNA fragmentation and ultimately hepatocyte necrosis (Figure created with Biorender.com).

Figure 3.. Mechanistic timeline for therapeutic options after an APAP overdose.

Since oral overdose of APAP is most common in humans, the earliest intervention typically administered in the ER on presentation is typically activated charcoal to prevent absorption of the drug. However, this would only be relevant if the patient presents immediately after the overdose before the drug has been completely absorbed. For patients presenting up to 10 h after an overdose, N-acetylcysteine (NAC) would be effective since it provides cysteine for glutathione resynthesis enabling scavenging of the reactive metabolite NAPQI generated from APAP. For severe overdoses, 4-methyl pyrazole (4MP) provides additional benefit since it is mechanistically distinct from NAC, inhibiting NAPQI formation by inhibition of Cyp2E1. 4MP would likely be beneficial for patients presenting beyond the 10 h window, since it protects through additional mechanisms such as inhibition of JNK activation which would prevent amplification of mitochondrial oxidant stress. Recent data suggests that HGF/EGF delivery and Wnt agonists can also provide benefit in patients with delayed presentation, where necrosis has destroyed hepatocytes surrounding the central vein. These interventions could enhance the regenerative response to facilitate repopulation of areas of necrosis and enhance recovery (Figure created with Biorender.com).