Upstream eventAltered expression of hepatic CAR-dependent genes
Increase, Mitogenic cell proliferation (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||Moderate|
Life Stage Applicability
|All life stages||Moderate|
Key Event Relationship Description
The altered expression of mouse and rat genes that are related to increased cell proliferation will, by definition, produce measurable changes in cell proliferation in hepatocytes (Elcombe et al., 2014; Yang and Wang, 2014). Hepatocytes have the ability to regenerate when properly stimulated, such as following partial hepatectomy. The CAR-responsive genes such as Gadd45b, Ki67 and the Cdc20 are necessary gene targets that are part of this synchronized response that results in progress out of the quiescent cell cycle stage (G0), resulting in DNA replication during S-phase (a measurable marker of cell proliferation), and eventual cell division.
Evidence Supporting this KER
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).
Uncertainties and Inconsistencies
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).
Quantitative Understanding of the Linkage
The Moderate quantitative understanding supporting the linkage between CAR-responsive gene expression changes and measurable changes in cell proliferation (increased BrdU labeling or Ki67 labelling index) has been established in a limited number of studies with ample dose-response data (Deguchi et al., 2009; Geter et al., 2014; Jones et al., 2009). Typically, these dose-response studies show that effects on gene expression (e.g. increased Cyp2b10 mRNA) and associated events related to liver metabolism (e.g. increased PROD activity) can occur at lower, non-tumorigenic dose levels, and changes in markers of cell proliferation including cell cycle pathway changes and BrdU or Ki67 labelling index are seen at somewhat higher dose levels. For example the study of Geter et al. (2014) with phenobarbital showed that in male CD-1 mice, the most sensitive parameters measured was PROD activity (BMD = 0.2 – 2.4 mg/kg/day), cell proliferation by BrdU labeling index occurred at higher dose levels (BMD = 13 – 14 mg/kg/day) and in general for this particular study, significant changes in gene expression pathways were only seen at dose levels slightly higher than these apical markers.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
The pathway leading from CAR-mediated gene expression changes to an increase in cell proliferation (BrdU, PCNA or Ki67 labeling index) has strong evidence indicating it is selectively observed in mice and rats, but not in other mammalian species (Deguchi et al., 2009; Foster, 2000; LeBaron et al., 2013; Parzefall et al., 1991; Plant et al., 1998). Recently a chimeric mouse model that had >90% replacement of mouse hepatocytes with human hepatocytes was used to examine the comparative in vivo effects of 3-5 dietary concentrations of phenobarbital in CD-1 mouse hepatocytes vs. the chimeric human hepatocytes (Yamada et al., 2014). In the liver cells of CD-1 mice after 1 week of treatment, increased mRNA levels of Cyp2b10, Cyp3a11, Ki67 and Gadd45b were observed, along with increased BrdU labeling index. In the human liver cells of chimeric mice, increased mRNA levels of CYP2B and CYP3A were observed, but there were no differences from control for Ki67 and GADD45B mRNA levels nor any increases in BrdU labeling index (Yamada et al., 2014). In summary, a large amount of experimental data including both in vitro and in vivo studies has demonstrated that the downstream key event of increased cell proliferation following treatment with CAR activators occurs selectively in mice and rats, and is correlated with CAR-mediated gene expression changes that reflect pathways of cell cycle control and cell proliferation.
Chen, T., Tompkins, L. M., Li, L., Li, H., Kim, G., Zheng, Y. and Wang, H. (2010), A single amino acid controls the functional switch of human constitutive androstane receptor (CAR) 1 to the xenobiotic-sensitive splicing variant CAR3. J Pharmacol Exp Ther 332, 106-15, 10.1124/jpet.109.159210.
Currie, R. A., Peffer, R. C., Goetz, A. K., Omiecinski, C. J. and Goodman, J. I. (2014), Phenobarbital and propiconazole toxicogenomic profiles in mice show major similarities consistent with the key role that constitutive androstane receptor (CAR) activation plays in their mode of action. Toxicology 321, 80-8, 10.1016/j.tox.2014.03.003.
Deguchi, Y., Yamada, T., Hirose, Y., Nagahori, H., Kushida, M., Sumida, K., Sukata, T., Tomigahara, Y., Nishioka, K., Uwagawa, S., Kawamura, S. and Okuno, Y. (2009), Mode of action analysis for the synthetic pyrethroid metofluthrin-induced rat liver tumors: evidence for hepatic CYP2B induction and hepatocyte proliferation. Toxicol Sci 108, 69-80, 10.1093/toxsci/kfp006.