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Event: 716

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Increase, cell proliferation (hepatocytes)

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. More help
Increase, cell proliferation (hepatocytes)
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Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help
Level of Biological Organization

Cell term

The location/biological environment in which the event takes place.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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Cell term

Organ term

The location/biological environment in which the event takes place.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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  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 signaling 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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Process Object Action
cell proliferation mitogenic signaling cell increased
hepatocyte proliferation hepatocyte increased

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
CAR activation- Hepatocellular tumors KeyEvent Kristin Lichti-Kaiser (send email) Open for citation & comment Under Review
AR- HCC KeyEvent Charles Wood (send email) Open for adoption Under Development
PPARalpha-dependent liver tumors in rodents KeyEvent Chris Corton (send email) Under development: Not open for comment. Do not cite Under Development

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.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
rat Rattus norvegicus NCBI
mouse Mus musculus NCBI
human Homo sapiens NCBI
Hamster Hamster NCBI
dog Canis lupus familiaris NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help
Life stage Evidence
All life stages High

Sex Applicability

An indication of the the relevant sex for this KE. More help
Term Evidence
Unspecific 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. More help

Key Event Description:

Cell proliferation in the livers of rats and mice occurs through exposure to a mitogen and is characterized by liver enlargement without evidence of necrosis. In contrast, regenerative/compensatory proliferation occurs following loss of liver parenchymal cells from necrosis or hepatectomy.

In mammals, the nature of the hepatocyte proliferative response is shaped by the identity of the mitogen, the time course and dose of administration, and the species and strain of the test animal (Columbano and Shinozuka, 1996).

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.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). Do not provide detailed protocols. More help

Mitogenic proliferation in vitro and in vivo is measured by the incorporation of labeled nucleosides or nucleoside analogs into newly synthesized DNA (Peffer et al., 2018b), the detection of endogenous markers of proliferation such as antigen Ki-67 or proliferating cell nuclear antigen (PCNA) (Kee et al., 2002;  Muskhelishvili et al., 2003;  Wood et al., 2015), and other immunohistochemical techniques to detect proliferating cells. For each of these methods, a labeling index (fraction of labeled cell population/total number of cells in population) is calculated, and this index can be statistically compared between different groups (Wood et al., 2015).

Nucleoside and nucleoside analog labeling. Actively proliferating cells undergo DNA synthesis in a highly regulated process during the S (synthesis) phase of the cell cycle. Once the DNA of a cell is replicated during S phase, the cell undergoes mitosis. This results in two cells, each of which has a complete copy of the genome. The DNA replication that occurs in S phase may be detected by the incorporation radiolabeled (e.g., 3H-thymidine) into the newly synthesized DNA, which can be detected from isolated livers using standard autoradiographic techniques. Nucleoside analogs may also be incorporated into the newly-synthesized DNA, including 5-bromo-2-deoxyuridine (BrdU) or 5-ethyl-2’-deoxy uridine (EdU), which may be detected using standard immunohistochemical and biolabeling techniques, respectively (Cavanagh et al., 2011). Drawbacks of the use of nucleoside analogs include concerns regarding the proper administration (dose, route of administration and length of exposure) to animals that allow for adequate labeling without inducing considerable toxicity (Cavanagh et al., 2011;  Cohen, 2010). In addition, nucleoside/nucleoside analog incorporation techniques are not specific for the detection of proliferation but may also identify cells that are undergoing DNA synthesis during apoptosis or DNA repair.

Endogenous markers of proliferation. Ki-67 and PCNA are endogenous proteins expressed by mammalian cells that are in active phases of the cell cycle (G1, S, G2, M) and are not expressed in quiescent (G0) cells (Dietrich, 1993;  Eldrige et al., 1993;  Scholzen and Gerdes, 2000). They are detected in hepatocytes using standard immunohistochemical techniques. The advantage of using endogenous markers is that they do not require administration of exogenous markers for labeling, and they can be used for both prospective and retrospective cell proliferation analysis. A direct comparison of BrdU, Ki67 and PCNA labeling in various proliferating tissues of male Sprague-Dawley rats (Muskhelishvili et al., 2003) has indicated that Ki67 and BrdU immunohistochemistry methods gave similar labelling index results, whereas PCNA immunohistochemistry was not concordant with these methods and gave highly variable results. These authors suggested that PCNA is less accurate as a measure of cell proliferation because it has a long half-life and can be retained in cells that are not dividing, and is more involved in DNA repair mechanisms than Ki67. As a result, Ki67 has emerged as a more preferred endogenous marker for assessing cell proliferation in hepatocytes in recent years compared to PCNA.

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help

Epidermal growth factor (EGF) is one of several extracellular ligands of the epidermal growth factor receptor (EGFR). The EGFR signaling pathway is conserved in most animals, in which it controls processes such as cell proliferation, differentiation, adhesion, and migration (Barberan and Cebria, 2018).

EGFR is a transmembrane protein that is classified as a tyrosine kinase receptor. EGFR has several structural domains: 1) an N-terminal extracellular domain that binds ligands such as EGF, 2) a transmembrane domain, 3) an intracellular domain containing tyrosine kinase activity, and 4) a C-terminal region that contains tyrosine residues that are the sites of autophosphorylation. Ligand binding results in a cascade of events that include EGFR homo-or heterodimerization, activation of the tyrosine kinase domain, tyrosine autophosphorylation, and ultimately the activation of downstream signaling cascades that control various processes in the liver such as proliferation, survival, differentiation, response to injury, and repair (Berasain and Avila, 2014;  Komposch and Sibilia, 2015).

EGF has been used as an agent to stimulate proliferation of rat, mouse, and human hepatic cells in culture (Bowen et al., 2014;  Haines et al., 2018c;  Hodges et al., 2000;  Parzefall et al., 1991).

Other mitogenic agents produce a cell proliferation response in rats and mice, but not other mammalian species such as humans, hamsters or dogs.  These agents include phenobarbital (a model CAR activator) (Haines et al., 2018c;  Hirose et al., 2009;  Parzefall et al., 1991), WY-14,643 (pirinixic acid) (a model PPARalpha activator) (Corton et al., 2018) and TCDD (a model AhR activator) (Becker et al., 2015;  Budinsky et al., 2014).


List of the literature that was cited for this KE description. More help

Barberan, S. and Cebria, F. (2018), The role of the EGFR signaling pathway in stem cell differentiation during planarian regeneration and homeostasis. Semin Cell Dev Biol, 10.1016/j.semcdb.2018.05.011.

Becker, R. A., Patlewicz, G., Simon, T. W., Rowlands, J. C. and Budinsky, R. A. (2015), The adverse outcome pathway for rodent liver tumor promotion by sustained activation of the aryl hydrocarbon receptor. Regul Toxicol Pharmacol 73, 172-90, 10.1016/j.yrtph.2015.06.015.

Berasain, C. and Avila, M. A. (2014), The EGFR signalling system in the liver: from hepatoprotection to hepatocarcinogenesis. J Gastroenterol 49, 9-23, 10.1007/s00535-013-0907-x.

Bowen, W. C., Michalopoulos, A. W., Orr, A., Ding, M. Q., Stolz, D. B. and Michalopoulos, G. K. (2014), Development of a chemically defined medium and discovery of new mitogenic growth factors for mouse hepatocytes: mitogenic effects of FGF1/2 and PDGF. PLoS One 9, e95487, 10.1371/journal.pone.0095487.

Budinsky, R. A., Schrenk, D., Simon, T., Van den Berg, M., Reichard, J. F., Silkworth, J. B., Aylward, L. L., Brix, A., Gasiewicz, T., Kaminski, N., Perdew, G., Starr, T. B., Walker, N. J. and Rowlands, J. C. (2014), Mode of action and dose-response framework analysis for receptor-mediated toxicity: The aryl hydrocarbon receptor as a case study. Crit Rev Toxicol 44, 83-119, 10.3109/10408444.2013.835787.

Cavanagh, B. L., Walker, T., Norazit, A. and Meedeniya, A. C. (2011), Thymidine analogues for tracking DNA synthesis. Molecules 16, 7980-93, 10.3390/molecules16097980.

Cohen, S. M. (2010), Evaluation of possible carcinogenic risk to humans based on liver tumors in rodent assays: the two-year bioassay is no longer necessary. Toxicol Pathol 38, 487-501, 10.1177/0192623310363813.

Columbano, A. and Shinozuka, H. (1996), Liver regeneration versus direct hyperplasia. FASEB J 10, 1118-28.

Corton, J. C., Peters, J. M. and Klaunig, J. E. (2018), The PPARalpha-dependent rodent liver tumor response is not relevant to humans: addressing misconceptions. Arch Toxicol 92, 83-119, 10.1007/s00204-017-2094-7.

Dietrich, D. R. (1993), Toxicological and pathological applications of proliferating cell nuclear antigen (PCNA), a novel endogenous marker for cell proliferation. Crit Rev Toxicol 23, 77-109, 10.3109/10408449309104075.

Eldrige, S. R., Butterworth, B. E. and Goldsworthy, T. L. (1993), Proliferating cell nuclear antigen: a marker for hepatocellular proliferation in rodents. Environ Health Perspect 101 Suppl 5, 211-8, 10.1289/ehp.93101s5211.

Haines, C., Elcombe, B. M., Chatham, L. R., Vardy, A., Higgins, L. G., Elcombe, C. R. and Lake, B. G. (2018c), Comparison of the effects of sodium phenobarbital in wild type and humanized constitutive androstane receptor (CAR)/pregnane X receptor (PXR) mice and in cultured mouse, rat and human hepatocytes. Toxicology 396-397, 23-32, 10.1016/j.tox.2018.02.001.

Hirose, Y., Nagahori, H., Yamada, T., Deguchi, Y., Tomigahara, Y., Nishioka, K., Uwagawa, S., Kawamura, S., Isobe, N., Lake, B. G. and Okuno, Y. (2009), Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes. Toxicology 258, 64-9.

Hodges, N. J., Orton, T. C., Strain, A. J. and Chipman, J. K. (2000), Potentiation of epidermal growth factor-induced DNA synthesis in rat hepatocytes by phenobarbitone: possible involvement of oxidative stress and kinase activation. Carcinogenesis 21, 2041-7.

Jones, H. B., Orton, T. C. and Lake, B. G. (2009), Effect of chronic phenobarbitone administration on liver tumour formation in the C57BL/10J mouse. Food Chem Toxicol 47, 1333-40, 10.1016/j.fct.2009.03.014.

Kee, N., Sivalingam, S., Boonstra, R. and Wojtowicz, J. M. (2002), The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. J Neurosci Methods 115, 97-105.

Kolaja, K. L., Stevenson, D. E., Johnson, J. T., Walborg, E. F., Jr. and Klaunig, J. E. (1996a), Subchronic effects of dieldrin and phenobarbital on hepatic DNA synthesis in mice and rats. Fundam Appl Toxicol 29, 219-28.

Komposch, K. and Sibilia, M. (2015), EGFR Signaling in Liver Diseases. Int J Mol Sci 17, 10.3390/ijms17010030.

Muskhelishvili, L., Latendresse, J. R., Kodell, R. L. and Henderson, E. B. (2003), Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA. J Histochem Cytochem 51, 1681-8.

Parzefall, W., Erber, E., Sedivy, R. and Schulte-Hermann, R. (1991), Testing for induction of DNA synthesis in human hepatocyte primary cultures by rat liver tumor promoters. Cancer Res 51, 1143-7.

Peffer, R. C., LeBaron, M. J., Battalora, M., Bomann, W. H., Werner, C., Aggarwal, M., Rowe, R. R. and Tinwell, H. (2018b), Minimum datasets to establish a CAR-mediated mode of action for rodent liver tumors. Regul Toxicol Pharmacol 96, 106-120, 10.1016/j.yrtph.2018.04.001.

Scholzen, T. and Gerdes, J. (2000), The Ki-67 protein: from the known and the unknown. J Cell Physiol 182, 311-22, 10.1002/(sici)1097-4652(200003)182:3<311::aid-jcp1>;2-9.

Wood, C. E., Hukkanen, R. R., Sura, R., Jacobson-Kram, D., Nolte, T., Odin, M. and Cohen, S. M. (2015), Scientific and Regulatory Policy Committee (SRPC) Review: Interpretation and Use of Cell Proliferation Data in Cancer Risk Assessment. Toxicol Pathol 43, 760-75, 10.1177/0192623315576005.

Yamada, T., Okuda, Y., Kushida, M., Sumida, K., Takeuchi, H., Nagahori, H., Fukuda, T., Lake, B. G., Cohen, S. M. and Kawamura, S. (2014), Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver. Toxicol Sci 142, 137-57, 10.1093/toxsci/kfu173.