Emerging and established modes of cell death during acetaminophen-induced liver injury

Abstract

Acetaminophen (APAP)-induced liver injury is an important clinical and toxicological problem. Understanding the mechanisms and modes of cell death are vital for the development of therapeutic interventions. The histological and clinical features of APAP hepatotoxicity including cell and organelle swelling, karyolysis, and extensive cell contents release lead to the characterization of the cell death as oncotic necrosis. However, the more recent identification of detailed signaling mechanisms of mitochondrial dysfunction, the amplification mechanisms of mitochondrial oxidant stress and peroxynitrite formation by a mitogen-activated protein kinase cascade, mechanisms of the mitochondrial permeability transition pore opening and nuclear DNA fragmentation as well as the characterization of the sterile inflammatory response suggested that the mode of cell death is better termed programmed necrosis. Additional features like mitochondrial Bax translocation and cytochrome c release, mobilization of lysosomal iron and the activation of receptor-interacting protein kinases and the inflammasome raised the question whether other emerging modes of cell death such as apoptosis, necroptosis, ferroptosis and pyroptosis could also play a role. The current review summarizes the key mechanisms of APAP-induced liver injury and compares these with key features of the newly described modes of cell death. Based on the preponderance of experimental and clinical evidence, the mode of APAP-induced cell death should be termed programmed necrosis; despite some overlap with other modes of cell death, APAP hepatotoxicity does not fulfill the characteristics of either apoptosis, necroptosis, ferroptosis, pyroptosis or autophagic cell death.

Keywords: Acetaminophen; Apoptosis; Cell death; Drug hepatotoxicity; Ferroptosis; Necrosis.

Figure 1:. Forms of cell death discussed in the context of acetaminophen hepatotoxicity.

The classical pathways for apoptosis are mediated by the Fas or TNF receptor, whose activation results in their trimerization and activation of caspase 8. While caspase 8 directly activates effector caspases such as caspase 3 in type I cells such as thymocytes, it cleaves the protein Bid in the cytosol in type II cells such as hepatocytes. Truncated Bid (tBid) then translocates to the mitochondria, where along with other Bcl2 family member proteins such as Bax, Bad and Bak it induces mitochondrial permeability transition and release of cytochrome c into the cytosol. This results in assembly of the apoptosome consisting of APAF1, cytochrome c, ATP and procaspase 9 and subsequent activation of caspase 9, which then activates caspase 3. Activation of caspase 3 then results in the characteristic caspase activated DNAse (CAD) mediated nuclear DNA fragmentation and nuclear condensation, ultimately resulting in apoptotic cell death. In a scenario where TNF activation occurs when caspase 8 is inhibited, however, the signaling shifts to formation of a necrosome complex consisting of the receptor interacting kinases 1 & 3 and the mixed lineage kinase domain like pseudokinase (MLKL). The phosphorylation and activation of MLKL on this complex results in its translocation to the plasma membrane, and disruption of the membrane resulting in cell death by necroptosis. Acetaminophen (APAP) mediated hepatocyte cell death has some features of both these modes of cell death, but lacks certain defining features of each, which puts it in a class apart. Cytochrome P450 mediated metabolism of APAP results in formation of the reactive metabolite NAPQI, which induces mitochondrial oxidant stress and activation of the MAP kinase JNK. Translocation of activated JNK as well as Bax to the mitochondria induces an oxidant stress, which amplifies mitochondrial dysfunction and also upregulates RIP3 kinase levels. The subsequent induction of the mitochondrial permeability transition results in release of cytochrome c, endonuclease G and apoptosis inducing factor into the cytosol. However, cytochrome c release does not result in assembly of the apoptosome and endonuclease G and AIF translocation to the nucleus causes nuclear DNA fragmentation and programmed necrosis.

Figure 2:. Pyroptosis.

Pyroptosis is cell death induced by inflammatory mediators such as bacteria, viruses or toxic microbial material mediated by caspase 1, 4, 5 and 11 in humans. Identification of these inflammatory material by cells results in activation of inflammation sensing complexes such as the NALP3 inflammasome, which results in activation of caspase 1. This is complemented by direct activation of caspases 4, 5 and 11 by bacterial lipopolysaccharides (LPS), where caspase 11 can then activate the NALP3 inflammasome. The activated caspases then cleave gasdermin D, and the insertion of the resulting protein fragments into the plasma membrane form channels which cause cell swelling and necrosis.

Figure 3:. Ferroptosis.

Ferroptosis is a mode of cell death mediated by cellular free iron and lipid peroxidation in the context of decreased glutathione peroxidase activity. Intracellular reactive oxygen species such as hydrogen peroxide react with cellular free iron through the Fenton reaction to generate the reactive hydroxyl radical, which attacks lipid membranes to induce lipid peroxidation and membrane instability, which can ultimately result in leakage of cellular material and cell death. This process can be inhibited by compounds such as ferrostatin or chelation of free cellular iron to prevent lipid peroxidation