Hepatocytotoxicity

Taxonomic Applicability: Hepatotoxicity can occur in any species that has a liver.

Sex applicability: It can occur in both sexes

How it works: Hepatotoxicity occurs through three main mechanisms: apoptosis, necrosis, and necroptosis. (1) During programmed cell death, apoptotic cells are ‘disassembled’ and cellular components ‘bleb’ off as large vacuoles, which can be eliminated by phagocytosis. Apoptosis is activated via the extrinsic pathway (mediated through a death receptor, Tumor Necrosis Factor Receptor; TNFR) or intrinsic pathway (mediated through the mitochondria), each of which activate the caspase cascade (Riedl and Shi 2004). (2) Necrosis is an unregulated, accidental form of cell death that occurs when severe damage to cellular components causes the cell to die abruptly and spill its contents into the extracellular space.  Released cellular components include damage-associated molecular patterns (DAMPs) that trigger an inflammatory response. (3) The third type of cell death is necroptosis, or programmed necrosis, which uses the same death receptor that is upstream to the extrinsic pathway of apoptosis, but signaling results in a necrotic outcome. The decision for TNFR to signal for apoptosis or necroptosis is thought to depend on the receptor protein kinases 1 and 3 (RIP1, RIP3), which are part of the protein complex that forms on the intra-cellular portion of the TNFR. Activation of caspase-8 cleaves the RIP1-RIP3 complex and favours apoptosis, whereas inhibition of caspase-8 favours the RIP1-RIP3 complex (called the ‘necrosome’). As per standard necrosis, necroptosis results in DAMP release, which triggers inflammation. Necroptosis has been reviewed (Vandenabeele, et al. 2010). Cell death mechanisms in the liver and in liver disease have also been reviewed (Eguchi, et al. 2014, Luedde, et al. 2014).

The mitochondrial permeability transition (MPT) is an important process that leads to necrosis or apoptosis. When the mitogen activated protein kinase (MAPK) cascade is triggered (ASK1MKK4JNK), Bcl-2-associated X protein (Bax)  is recruited to the outer mitochondrial membrane (Youle and Strasser 2008). Bax triggers the opening of the mitochondrial permeability transition pore (MTP), through which cytochrome c is released, which triggers the caspase cascade and apoptosis. Alternatively, when the MTP opens across the inner and outer mitochondrial membranes, mitochondrial swelling and decoupling of oxidative phosphorylation (i.e., loss of ATP generation) leads to cell death by necrosis (Pessayre, et al. 2010, Rasola and Bernardi 2007).

In vivo (liver):

• Hematoxylin and eosin (H&E)-stained liver sections can be examined by a pathologist for the presence of cytotoxicity;
• Serum levels of alanine aminotransferase (ALT) can be used as an indicator of hepatotoxicity. Serum levels of aspartate aminotransferase (AST) can also be used; however, AST is considered to be less ‘liver specific’ than ALT. Therefore, an AST/ALT ratio is often used. ALT and AST are typically measured using a commercial kit (e.g., from Sigma Aldrich or Roche); protocol: www.bio-protocol.org/e931;
• Additional serum biomarkers of liver cell death have been reviewed in: (Eguchi, et al. 2014), and include: miRNAs (including mir-122), soluble death receptors (sTNFR, sTRAIL, sFas), microparticles (small vesicles released from dying cells), and other soluble proteins (including High mobility group box 1, HMGB1, and cleaved keratin 18, K18);

In vivo or in vitro:

• Lactate dehydrogenase (LDH) leakage. LDH leakage is a measure of necrotic cell death, which can be detected using a colorimetric absorbance assay based on MTT reduction (Chan, et al. 2013).
• Trypan Blue Exclusion. Trypan blue is a commercially available dye that only stains dead cells;
• Apoptosis can be assayed by measuring caspase activation. There are a number of commercially available caspase assay kits:
  • The TUNEL assay is commonly used to measure DNA fragmentation that results from apoptotic signaling cascades (Lozano, et al. 2009)
    • This assay measures the presence of nicks in the DNA that are identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that catalyzes the addition of dUTPs that are secondarily labeled with a marker;
• In the MTT assay in which viable cells (with active metabolism) convert MTT into a purple compound (formazan), whereas dead cells remain colourless (Riss, et al. 2004);
• Trypan blue assay: non –viable cells take-up trypan blue, whereas viable cells remain colourless (Strober 2015).

Cytotoxicity can occur in any species from bacteria through to humans. Hepatocytotoxicity can occur in any species with a liver.

Chan, F.K., Moriwaki, K., De Rosa, M.J., 2013. Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol. Biol. 979, 65-70.

Eguchi, A., Wree, A., Feldstein, A.E., 2014. Biomarkers of liver cell death. J. Hepatol. 60, 1063-1074.

Lozano, G.M., Bejarano, I., Espino, J., Gonzalez, D., Ortiz, A., Garcia, J.F., Rodriguez, A.B., Pariente, J.A., 2009. Relationship between caspase activity and apoptotic markers in human sperm in response to hydrogen peroxide and progesterone. J. Reprod. Dev. 55, 615-621.

Luedde, T., Kaplowitz, N., Schwabe, R.F., 2014. Cell death and cell death responses in liver disease: mechanisms and clinical relevance. Gastroenterology 147, 765-783.e4.

Pessayre, D., Mansouri, A., Berson, A., Fromenty, B., 2010. Mitochondrial involvement in drug-induced liver injury. Handb. Exp. Pharmacol. (196):311-65. doi, 311-365.

Rasola, A., Bernardi, P., 2007. The mitochondrial permeability transition pore and its involvement in cell death and in disease pathogenesis. Apoptosis 12, 815-833.

Riedl, S.J., Shi, Y., 2004. Molecular mechanisms of caspase regulation during apoptosis. Nat. Rev. Mol. Cell Biol. 5, 897-907.

Riss, T.L., Moravec, R.A., Niles, A.L., Duellman, S., Benink, H.A., Worzella, T.J., Minor, L., 2004. Cell Viability Assays, in: Sittampalam, G.S., Coussens, N.P., Nelson, H., Arkin, M., Auld, D., Austin, C., Bejcek, B., Glicksman, M., Inglese, J., Iversen, P.W., Li, Z., McGee, J., McManus, O., Minor, L., Napper, A., Peltier, J.M., Riss, T., Trask OJ, J., Weidner, J. (Eds.), Assay Guidance Manual, Bethesda (MD).

Strober, W., 2015. Trypan Blue Exclusion Test of Cell Viability. Curr. Protoc. Immunol. 111, A3.B.1-3.

Vandenabeele, P., Galluzzi, L., Vanden Berghe, T., Kroemer, G., 2010. Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat. Rev. Mol. Cell Biol. 11, 700-714.

Youle, R.J., Strasser, A., 2008. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47-59.