To the extent possible under law, AOP-Wiki has waived all copyright and related or neighboring rights to KE:1393

Event: 1393

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Hepatocytotoxicity

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Hepatocytotoxicity

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Cellular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
hepatocyte

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Organ term
liver

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Cyp2E1 Activation Leading to Liver Cancer KeyEvent Francina Webster (send email) Open for citation & comment EAGMST Under Review

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
rodents rodents High NCBI
Homo sapiens Homo sapiens High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
All life stages High

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Term Evidence
Mixed High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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).

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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).

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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

Evidence for Perturbation by Stressor

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

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.