Current issues with acetaminophen hepatotoxicity--a clinically relevant model to test the efficacy of natural products

Abstract

There is a significant need to evaluate the therapeutic potential of natural products and other compounds purported to be hepatoprotective. Acetaminophen-induced liver injury, especially in mice, is an attractive and widely used model for this purpose because it is both clinically relevant and experimentally convenient. However, the pathophysiology of liver injury after acetaminophen overdose is complex. This review describes the multiple steps and signaling pathways involved in acetaminophen-mediated cell death. The toxicity is initiated by the formation of a reactive metabolite, which depletes glutathione and binds to cellular proteins, especially in mitochondria. The resulting mitochondrial oxidant stress and peroxynitrite formation, in part through amplification by c-jun-N-terminal kinase activation, leads to mitochondrial DNA damage and opening of the mitochondrial permeability transition pore. Endonucleases from the mitochondrial intermembrane space and lysosomes are responsible for nuclear DNA fragmentation. Despite the oxidant stress, lipid peroxidation is not a relevant mechanism of injury. The mitochondrial dysfunction and nuclear DNA damage ultimately cause oncotic necrotic cell death with release of damage-associated molecular patterns that trigger a sterile inflammatory response. Current evidence supports the hypothesis that innate immune cells do not contribute to injury but are involved in cell debris removal and regeneration. This review discusses the latest mechanistic aspects of acetaminophen hepatotoxicity and demonstrates ways to assess the mechanisms of drug action and design experiments needed to avoid pitfalls and incorrect conclusions. This review should assist investigators in the optimal use of this model to test the efficacy of natural compounds and obtain reliable mechanistic information.

Figure 1. Metabolic activation of acetaminophen (APAP)

More than 80-90% of an administered dose of APAP is conjugated with glucuronic acid or sulfate and excreted. A small fraction is metabolized by cytochrome P450 enzymes to form the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which can be conjugated and detoxified by glutathione (GSH). After an overdose of APAP, excess NAPQI is formed leading to cellular GSH depletion and consequently covalent binding of NAPQI to cellular proteins, which is the initiating step of toxicity. Any therapeutic intervention (natural products, solvents) administered before APAP has the potential to inhibit cytochrome P450 enzymes and effectively protect against APAP toxicity.

Figure 2. Glutathione depletion kinetics in rodent liver after a high dose of APAP

A hypothetical graph based on Knight et al. 2001 and Saito et al. 2010b,. Glutathione (GSH) levels fall quickly after 200–600 mg/kg APAP in the absence of a metabolism inhibitor during the first 20–30 min leading to hepatic GSH depletion of about 90% of baseline values (solid line). In the presence of dimethyl sulfoxide (DMSO) or another inhibitor of APAP metabolism, NAPQI formation and consequently the kinetics of GSH depletion are delayed (dashed line). The length of the delay and the extent of inhibition will depend upon the clearance rate of the inhibitor and the dose of APAP (among other considerations). At doses of 200–300 mg/kg APAP, a small amount of DMSO may sufficiently inhibit metabolism and cause only moderate GSH depletion, which is insufficient to initiate toxicity. In contrast, a high overdose (500–600 mg/kg APAP) may lead to delayed depletion (dashed line) and consequently less toxicity compared to a saline-treated mouse receiving APAP. The critical point is that measuring GSH levels at two hours would not detect this metabolic inhibition.

Figure 3. Mitochondrial oxidant stress and peroxynitrite formation during APAP hepatotoxicity

Metabolism of APAP causes formation of the reactive metabolite NAPQI, which binds to mitochondrial proteins and initiates mitochondrial oxidative stress. This results in formation of peroxynitrite within mitochondria as well as activation and translocation of JNK to the mitochondria from the cytosol. The activation of JNK triggers an amplification of mitochondrial oxidative stress and peroxynitrite formation, ultimately resulting in induction of the mitochondrial permeability transition. As a consequence, mitochondrial factors such as cytochrome c, endonuclease G and the apoptosis inducing factor (AIF) are released from the mitochondrial intermembrane space. Endonuclease G and AIF then translocate to the nucleus to initiate nuclear DNA fragmentation.

Figure 4. Mode of cell death

Fundamental differences in cell morphology between apoptotic cell death and oncotic necrosis. Hematoxylin & eosin-stained sections of livers obtained from animals treated with either 700 mg/kg galactosamine/100 μg/kg endotoxin (left panel) or 300 mg/kg acetaminophen (right panel) for 6 hours. Hepatocellular apoptosis is characterized by apoptotic body formation, chromatin condensation and cell shrinkage (arrows in left panel). Oncotic necrosis is characterized by karyorrhexis, karyolysis, cell swelling and loss of membrane integrity (arrows on right panel). X400 (all panels)

Figure 5. Innate immune response during acetaminophen hepatotoxicity

During APAP-induced hepatotoxicity damaged hepatocytes release cellular contents including DAMPs. DAMPs activate innate immune cells resulting in an inflammatory response including cytokine/chemokine production and immune cell recruitment. These innate immune cells are present to maintain host defense against invading pathogens and to remove cellular debris thereby promoting liver regeneration but in this model they are not actively participating in the injury process. Abbreviations: DAMPs, Damage-Associated Molecular Patterns; TLRs, Toll-like Receptors; HMGB1, High mobility group box-1 protein; HSPs: Heat shock proteins; MIP-2, Macrophage inflammatory protein-2 (CXCL2); MCP-1, Monocyte chemoattractant protein-1 (CCL2); IL-6, Interleukin-6; TNF-α, Tumor necrosis factor-α