SNAPSHOT

Event: 785: Activation, Androgen receptor

Short Name: Activation, Androgen receptor

AOPs Including This Key Event

Stressors

Biological Context

Event: 716: Increase, Mitogenic cell proliferation (hepatocytes)

Short Name: Increase, Mitogenic cell proliferation (hepatocytes)

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

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).

Event: 774: Increase, Preneoplastic foci (hepatocytes)

Short Name: Increase, Preneoplastic foci (hepatocytes)

AOPs Including This Key Event

Biological Context

Cell term

Event: 719: Increase, Adenomas/carcinomas (hepatocellular)

Short Name: Increase, Adenomas/carcinomas (hepatocellular)

AOPs Including This Key Event

Biological Context

Organ term

Relationship: 787: Activation, Androgen receptor leads to Increase, Mitogenic cell proliferation (hepatocytes)

AOPs Referencing Relationship

Relationship: 773: Increase, Mitogenic cell proliferation (hepatocytes) leads to Increase, Preneoplastic foci (hepatocytes)

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Studies in various species, or in isolated hepatocytes from various mammalian species including humans, have demonstrated that CAR activators such as phenobarbital or metofluthrin produce a cell proliferation response that is seen in mice or rats, but not in hamsters, guinea pigs or humans (Hasmall and Roberts, 1999; Hirose et al., 2009; James and Roberts, 1996; Yamada et al., 2014; Yamada et al., 2009).   Accordingly, phenobarbital and other CAR activators do not produce liver tumors in long term studies in hamsters (Diwan et al., 1986; Elcombe et al., 2014). Consistent with the lack of effects on proliferation, Diwan et al. (1986) also reported that in Syrian hamsters, phenobarbital treatment at 500 ppm in the drinking water did not produce any increases in preneoplastic foci of cellular alteration compared to groups that received an initiator alone. Therefore, this key event of increased foci in the liver has strong data indicating it is specific to mice and rats, the species which also develop hepatocellular tumors in response to known CAR activators.

Key Event Relationship Description

Based on altered gene expression under the influence of CAR activation, an increase in cell proliferation of hepatocytes leads to a greater chance of normal, spontaneous errors in DNA replication and thus a higher proportion of altered hepatocytes. The hepatocytes with abnormal DNA can exhibit cell-cell communication differences from normal hepatocytes, and experience greater cell division even in the presence of contact inhibition with other hepatocytes. The islands of more actively dividing hepatocytes can be detected via histology based both on the larger numbers of cells (hyperplasia) and possibly a characteristic staining property of the clonally expanded cells (foci of cellular alteration – either eosinophilic, basophilic or clear cell). Thus, a higher rate of proliferation in the rodent liver leads to greater prevalence of altered hepatocytes, which clonally expand to generate an increase in preneoplastic foci.

Evidence Supporting this KER

The increased cell replication rate in the liver due to CAR activation (i.e. via a mitogenic signaling) is similar to other well-understood modes of action where an increase in cell proliferation leads to an eventual increase in preneoplastic foci, such as PPARα activating ligands and AhR activating ligands, which also lead to an increase in preneoplastic foci via clonal expansion of transformed hepatocytes.  In mice lacking the CAR receptor, including initiation-promotion assays, the upstream events (e.g. CAR activation, altered gene expression, and increased cell proliferation) and the downstream events (e.g. preneoplastic foci) are all blocked, providing strong support for the biological plausibility of this Key Event Relationship (Huang et al., 2005; Tamura et al., 2015; Tamura et al., 2013; Yamamoto et al., 2004).

The observed increase in numbers of preneoplastic foci, usually with eosinophilic staining properties, is observed with great regularity in mode of action work of CAR activating xenobiotics where histopathology at later times has been examined. This increase in foci (mixed or eosinophilic) after 2 years was observed at tumorigenic dose levels with metofluthrin in male rats (Deguchi et al., 2009), and at tumorigenic dose levels in mice treated with phenobarbital (Jones et al., 2009). With TCPOBOP in mice, multiple eosinophilic foci were reported to co-occur along with an increased incidence of eosinophilic adenomas and carcinomas after 60 weeks of treatment (Diwan et al., 1992). With well-studied CAR activators such as phenobarbital and TCPOBOP, increased cell proliferation has been detected at similar dose levels where increased altered foci are seen (Geter et al., 2014; Huang et al., 2005; Kolaja et al., 1996a; Kolaja et al., 1996b) (Tables 2 and 3); therefore, there is strong support for the linkage of these earlier key events with CAR activators leading to an increase in pre-neoplastic foci.

The incidence of altered foci, and their histological staining properties (e.g. eosinophilic, basophilic, clear cell, mixed) are not always reported in published studies of carcinogenicity with CAR activating compounds. In addition, the timing of interim or final sacrifices and histopathology data may possibly miss a window of time (for certain molecules) where the increase in preneoplastic foci can be quantified. However, the consistent findings with well-known CAR activating compounds and their absence in CAR knockout mouse studies provide a strong basis for their existence in the CAR AOP.

References

[see reference list at end of this AOP; it includes all cited references]

Relationship: 774: Increase, Preneoplastic foci (hepatocytes) leads to Increase, Adenomas/carcinomas (hepatocellular)

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Phenobarbital and other CAR activators do not produce liver tumors in long term studies in hamsters (Diwan et al., 1986; Elcombe et al., 2014). Consistent with the lack of effects on proliferation and on tumor development, Diwan et al. (1986) also reported that phenobarbital treatment at 500 ppm in the drinking water did not produce any increases in preneoplastic foci of cellular alteration compared to groups that received an initiator alone. Further, treatment of CAR knockout mice lacking the CAR nuclear receptor with phenobarbital or TCPOBOP produced none of the early key events (e.g. altered expression of CAR-responsive cell cycle genes, increased cell proliferation) and no increases in altered foci or tumors (Huang et al., 2005; Yamamoto et al., 2004). Therefore, the development of increased foci in the liver in response to treatment with CAR activators has strong data indicating it is specific to mice and rats, the species which also develop hepatocellular tumors in response to known CAR activators.

Key Event Relationship Description

Clonally expanded cells (foci of cellular alteration – either eosinophilic, basophilic or clear cell) have been shown to be increased at tumorigenic dose levels of CAR activators such as phenobarbital, TCPOBOP and metofluthrin. As discussed for earlier key events, the CAR-mediated events that lead to an increase in altered foci lead to a greater abundance of cells with mutations in their DNA that are less responsive to normal cell-cell signaling and control mechanisms. As a result, these foci are considered preneoplastic lesions, and can progress with time into adenomas and carcinomas. The continued CAR-mediated stimulus for increased cell proliferation within these foci (e.g. as demonstrated in studies by Kolaja et al., 1996b) will also provide an environment where the mutant cells can survive and develop into tumors.

Evidence Supporting this KER

The development of liver tumors in rodents, whether spontaneously or induced by a non-genotoxic carcinogen, has consistently included the development of altered foci as a precursor step to hepatocellular adenomas and carcinomas (Goldsworthy and Fransson-Steen, 2002; Tamura et al., 2015). These foci are considered preneoplastic lesions, and their ability to progress to form adenomas and/or carcinomas in rodents has been previously recognized. In the case of CAR activators, an increased incidence of preneoplastic foci has been consistently shown to precede tumor development, and there is a high biological plausibility for this Key Event Relationship (Elcombe et al., 2014; Goldsworthy and Fransson-Steen, 2002; Jones et al., 2009; Lake, 2009).

The observed increase in numbers of preneoplastic foci, usually with eosinophilic staining properties, is observed with great regularity in mode of action work of CAR activating xenobiotics where histopathology at later times has been examined. This increase in foci (mixed or eosinophilic) after 2 years was observed at tumorigenic dose levels with metofluthrin in male rats (Deguchi et al., 2009), and at tumorigenic dose levels in mice treated with phenobarbital (Jones et al., 2009). With TCPOBOP in mice, multiple eosinophilic foci were reported to co-occur along with an increased incidence of eosinophilic adenomas and carcinomas after 60 weeks of treatment (Diwan et al., 1992).

In addition, experiments where the MIE (CAR activation) is blocked have been performed with these model CAR activators. For phenobarbital and TCPOBOP in mice, the early key events and the progression to increased altered foci and hepatocellular tumors were all blocked in CAR knockout mice (Huang et al., 2005; Yamamoto et al., 2004). Foci of cellular alteration in CAR knockout mice were also prevented in an initiation-promotion model using the CAR activators cyproconazole and fluconazole (Tamura et al., 2015), and the incidence of adenomas and carcinomas was similarly decreased (Tamura et al., 2015). Thus, there is strong support for the involvement of CAR activation in these mechanisms, and that the stated sequence of key events following CAR activation leads to an increase in pre-neoplastic foci and then liver tumors in mice and rats.

The incidence of altered foci, and their staining properties (e.g. eosinophilic, basophilic, clear cell, mixed) are not always reported in published studies of carcinogenicity with CAR activation compounds. However, the consistent findings with well-known CAR activating compounds and their absence in CAR knockout mouse studies provide a strong basis for their existence in the CAR AOP.

References

[see reference list at end of this AOP; it includes all cited references]