Altered expression of hepatic CAR-dependent genes leads to Increase, cell proliferation (hepatocytes)

The CAR-mediated changes in expression of pro-proliferative genes such as Gadd45b, Ki67 and Cdc20 and the genomic pathways such as cell proliferation or cell cycle progression have been demonstrated to occur with multiple CAR activators (Currie et al., 2014; Deguchi et al., 2009; Geter et al., 2014; Oshida et al., 2015a; Ross et al., 2010; Tojima et al., 2012), and markers of cell proliferation such as BrdU labeling index or Ki67 labeling index have also been demonstrated to occur in mice and rats exposed to these same CAR activators. It is highly plausible that gene expression changes lead to increased cell proliferation signals in hepatocytes, as genes in the Gadd45 family are known to interact with cyclins, cyclin-dependent kinase inhibitors and p53 to alter progression through the cell cycle (Liebermann and Hoffman, 2008). Other CAR-mediated changes in gene expression such as those related to metabolism enzymes (e.g. CYP2B isoforms) lead to associative events in the liver such as increased CYP2B activity and/or protein, hepatocellular hypertrophy and increase liver weight.

Strong support for this linkage comes from in vivo and in vitro studies with model CAR activators, and via the absence of the same effects including specific gene expression changes and cell proliferation changes (by BrdU, PCNA or Ki67 labeling index) in CAR knockout mice lacking the CAR receptor. In a study with 850 ppm dietary phenobarbital in C3H wild-type and CAR knockout mice, increases in Cyp2b10 and Gadd45b gene expression were associated with increases in cell proliferation by Ki67 labeling index after 3 days or 7 days of treatment in wild-type mice; the increases in gene expression were abolished or substantially reduced in CAR knockout mice, and no effects on Ki67 labeling index were observed (Peffer et al., 2007). In Fischer 344 rats, Ozawa (Ozawa et al., 2011) showed that phenobarbital at 500 ppm in the diet produced a sustained increase in Gadd45b expression and a decrease in Gadd45g expression; these directions of change have been previously associated with enhanced cell proliferation. Also, doses of 500 – 1000 ppm phenobarbital have previously been shown to produce a cell proliferation response in rat liver (Deguchi et al., 2009; Kolaja et al., 1996a).

One minor uncertainty in this relationship is related to the different time courses of the two linked key events. With most CAR activators, the increase in cell proliferation (e.g. by BrdU, Ki67 or PCNA labeling index) is an early event (days) that is transient when one measures the response across the whole liver. For example, phenobarbital produced an increased BrdU labeling index in B6C3F1 mice that was maximal at 7 days and gradually dissipated at days 14, 21 and 28; no difference was observed vs. control at day 90 (Kolaja et al., 1996a) Phenobarbital in F344 rats produced an increase in labelling index at day 7 that was only marginally affected at day 14 and returned to control levels from day 21 onward (Kolaja et al., 1996a). Currie (Currie et al., 2014) compared cell proliferation by BrdU and cell proliferation gene pathways by IPA. Phenobarbital and the direct CAR activator propiconazole given to male CD-1 mice produced significant changes in the cell cycle/cell proliferation IPA pathways at both 4 and 30 days of treatment, whereas BrdU labeling index was maximal at 1 or 2 days of treatment, with no difference from controls at 14 days and beyond. The work of Kolaja (1996a; 1996b) has demonstrated that the cell proliferation in the liver does persist for longer time intervals in mice, if one examines the response within altered foci in an initiation-promotion model, with 500 ppm dietary phenobarbital as the promoter. Therefore, a genomic response to CAR activators in rodent liver (as a whole) can be measured and shown to produce a sustained response of enhanced signaling for increased cell proliferation, but the downstream key event of increased cell proliferation (as measured via BrdU or Ki67 labeling) is often insufficiently sensitive to detect a sustained difference vs. controls in the whole liver. The time course of these related key events can be very dependent on the zones of the liver examined (periportal, midzonal, centrilobular; Kolaja et al., 1996a), and on the specific CAR activator and the species/strain of rodent that is tested (Elcombe et al., 2014; Huang et al., 2005; Kolaja et al., 1996a).