Upstream eventIncrease, Preneoplastic foci (hepatocytes)
Increase, Adenomas/carcinomas (hepatocellular)
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat||adjacent||High||High|
|Androgen receptor activation leading to hepatocellular adenomas and carcinomas (in mouse and rat)||adjacent|
|Chronic cytotoxicity leading to hepatocellular adenomas and carcinomas (in mouse and rat)||adjacent|
Life Stage Applicability
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.
Uncertainties and Inconsistencies
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.
Quantitative Understanding of the Linkage
In studies where their incidences are reported, increases in altered foci (primarily eosinophilic, or mixed) have typically occurred at the same dose levels where increases in hepatocellular adenomas and carcinomas occurred. With phenobarbital in male C57BL/10J mice, 1000 ppm (113 mg/kg/day) produced an increase in eosinophilic and clear cell foci and an increase in liver tumors, whereas 200 ppm (22 mg/kg/day) had no effects on either finding (Table 3) (Jones et al., 2009). With metofluthrin in male Wistar rats, 900 ppm and 1800 ppm produced increases in mixed foci and eosinophilic foci, respectively, and an increased incidence of liver tumors was observed at 900 ppm and above (Table 5) (Deguchi et al., 2009; Yamada et al., 2014). For metofluthrin in rats, 200 ppm represented a No Effect Level for both altered foci and hepatocellular tumors, and it also failed to produce any of the earlier key events in the proposed AOP for metofluthrin. Thus, for these well-studied CAR activators that have ample dose-response data, a strong quantitative understanding of this linkage is available in mice and in rats.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
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.
[see reference list at end of this AOP; it includes all cited references]