Increase, cell proliferation (hepatocytes) leads to Increase, Preneoplastic foci (hepatocytes)

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.

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.

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.