Event: 716

Key Event Title


Increase, Mitogenic cell proliferation (hepatocytes)

Short name


Increase, Mitogenic cell proliferation (hepatocytes)

Biological Context


Level of Biological Organization

Cell term


Cell term

Organ term


Organ term

Key Event Components


Process Object Action
cell proliferation mitogenic signaling cell increased
hepatocyte proliferation hepatocyte increased

Key Event Overview

AOPs Including This Key Event


AOP Name Role of event in AOP
CAR activation- Hepatocellular tumors KeyEvent
AR- HCC KeyEvent
PPARalpha-dependent liver cancer KeyEvent



Taxonomic Applicability


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


Life stage Evidence
All life stages High

Sex Applicability


Term Evidence
Unspecific High

Key Event Description


Key Event Description:

One of the mechanisms known to induce cell proliferation in the livers of rats and mice occurs through exposure to a mitogen. Mitogenic cell proliferation is characterized by liver enlargement without evidence of necrosis, as opposed to regenerative/compensatory proliferation, in which the liver parenchyma is restored after loss due to necrosis or hepatectomy.

In mammals that have been administered a mitogenic xenobiotic, several factors impact the nature of the hepatocyte proliferative response. These include the identity of the mitogen, the time course and dose of administration, and the species and strain of the test system. The effects on the liver may be confined to certain lobes, or may be observed throughout the organ (Columbano and Shinozuka, 1996).

How It Is Measured or Detected


There are several well-characterized and well-accepted techniques that have been used to detect mitogenic proliferation in vitro and in vivo (Peffer et al., 2018b). These include the detection of labeled nucleosides or nucleoside analogs that have been incorporated into newly synthesized DNA, or 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). Several of these techniques may involve immunohistochemical techniques to detect proliferating cells, thus allowing for the detection of proliferation within specific tissue sections. 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


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).

Evidence for Perturbation by Stressor



1.         NaPB treatment has been shown to increase replicative DNA synthesis in cultured mouse (Haines et al., 2018c) and rat hepatocytes (Haines et al., 2018c;  Hirose et al., 2009).

2.         NaPB treatment (1 week 500-2500 ppm in the diet) was shown to significantly increase the BrdU labeling index in the livers of male CD-1 mice and male Wistar rats compared to their respective vehicle-treated controls (Yamada et al., 2014).

3.         An increase in replicative DNA synthesis was observed in male and female mice administered 1000 ppm NaPB in the diet for 1 month (Jones et al., 2009).

4.         PB at 0, 10, 50, 100 and 500 mg/kg (ppm) in the diet was administered to 8 week old male rats and male mice for 90 days. A significant induction of hepatic replicative DNA synthesis (as determined by [3H]-thymidine incorporation) was observed in the rat liver at 7 days, but had returned to control levels by 14 days. In mice, there was a significant increase in hepatic replicative DNA synthesis throughout treatment (Kolaja et al., 1996a). In both species, the most pronounced effect was observed in the centrilobular region.

Epidermal growth factor

Epidermal growth factor

1.         Human epidermal growth factor (hEGF) treatment was shown to significantly increase replicative DNA synthesis, and Ki-67 mRNA levels in human hepatocytes of chimeric mice with humanized livers (human hepatocyte chimeric livers) (Yamada et al., 2014).

2.         EGF has been shown to increase the proliferation of mouse (Bowen et al., 2014;  Haines et al., 2018c), rat (Bowen et al., 2014;  Haines et al., 2018c;  Hodges et al., 2000), and human (Haines et al., 2018c;  Parzefall et al., 1991) hepatocyte cultures as determined by increase in replicative DNA synthesis compared to appropriate controls.

pirinixic acid

WY-14,643 (pirinixic acid)

  1. WY-14,643 (pirinixic acid) is a potent PPARα activator, and its ability to stimulate cell proliferation has been reviewed in Corton et al. (2018).

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)

  1. TCDD is a potent AhR activator, and its ability to stimulate cell proliferation has been reviewed in Becker et al. (2015).  



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>3.0.co;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.