Upstream eventIncrease, Mitogenic cell proliferation (hepatocytes)
Increase, Preneoplastic foci (hepatocytes)
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|
Life Stage Applicability
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.
Uncertainties and Inconsistencies
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.
Quantitative Understanding of the Linkage
Increases in altered foci (primarily eosinophilic, or mixed) have typically occurred at the same dose levels where the preceding key event was observed (increased cell proliferation), and where the subsequent adverse outcome occurred (increased hepatocellular adenomas and carcinomas). With phenobarbital in C57BL/10J mice, 1000 ppm (113 mg/kg/day) produced increases in BrdU labelling index, eosinophilic foci and liver tumors, whereas 200 ppm (22 mg/kg/day) had no effects on any of these findings (Table 3) (Jones et al., 2009). With metofluthrin in male Wistar rats, 900 ppm and 1800 ppm produced increases in BrdU labelling index, altered foci (mixed or eosinophilic) and liver tumors, and these dose levels also produced the earlier key events in the proposed AOP with metofluthrin (Table 5) (Deguchi et al., 2009; Yamada et al., 2009). In these rat studies, 200 ppm metofluthrin 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 MOA for metofluthrin including cell proliferation. 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
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.
[see reference list at end of this AOP; it includes all cited references]