Increase, Preneoplastic foci (hepatocytes) leads to Increase, hepatocellular adenomas and carcinomas

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.

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.

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.