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Activation, Constitutive androstane receptor leads to Altered expression of hepatic CAR-dependent genes
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat||adjacent||High||Moderate||Brendan Ferreri-Hanberry (send email)||Open for citation & comment||EAGMST Under Review|
Life Stage Applicability
|All life stages||High|
Key Event Relationship Description
Activation of CAR by an endogenous or foreign substance leads to translocation of the CAR-RXR heterodimer into the nucleus, and this dimer binds to DNA recognition elements in the regulatory region of CAR-responsive genes. CAR activation thus alters gene expression and upregulates xenobiotic-metabolizing enzymes such as CYP2B, CYP2C, CYP3A, sulfotransferases, UDP-glucuronyltransferases and glutathione transferases, as well as xenobiotic transporters such as Mrp2 and Mrp4 (Omiecinski et al., 2011a). In addition, CAR alters genes involved in lipid homeostasis, glucose utilization and energy metabolism. In rats and mice, the expression of additional genes involved in cell proliferation and apoptosis control are altered; Gadd45beta and Cdc20 are examples of genes that function in this way and are upregulated in mice within hours of treatment with a CAR activator (Peffer et al., 2018a; Peffer et al., 2018b; Tojima et al., 2012).
Evidence Supporting this KER
For the MIE of CAR activation, direct measurement of this step after exposure to a stressor in vivo is difficult to attain, because of the complex sequence of intermediate steps that follow. Nuclear translocation of a fluorescently tagged CAR molecule following activation by a stressor such as phenobarbital has been observed in vitro in hepatocytes, and occurs fairly rapidly (i.e. within a few hours) (Chen et al., 2010). More commonly, indirect measurements of downstream effects, and absence of those effects in CAR-null mice or rats, are employed to demonstrate that a particular molecule or other stressor can activate CAR (Huang et al., 2005; Peffer et al., 2018b; Peffer et al., 2007; Yamamoto et al., 2004).
Following the activation of CAR by stressors such as phenobarbital, TCPOBOP or metofluthrin in rats or mice, the in vivo and in vitro data are strong that demonstrate a characteristic pattern of changes in CAR-responsive genes. The data for these example molecules are described further within this KER.
It is highly plausible that activation of CAR would produce changes in expression of specific genes, since transcription of genes specifically associated with CAR response elements is how this nuclear receptor achieves its biological effects (Tien and Negishi, 2006). In mice, rat and human hepatocytes, CAR activation produces changes in genes related to CYP2B and CYP3A enzymes, certain sulfotransferase and glucuronyltransferase enzymes, hepatic transport proteins and genes related to lipid and glucose metabolism (Maglich et al., 2003; Omiecinski et al., 2011b; Omiecinski et al., 2011a; Tien and Negishi, 2006). In mice and rats, CAR activation has been shown to also achieve alterations in genes that give an overall pathway change associated with increased progression through the cell cycle such as Gadd45b and Cdc20 (Deguchi et al., 2009; Geter et al., 2014; Ross et al., 2010; Tojima et al., 2012).
Uncertainties and Inconsistencies
In general, CAR activators show very consistent, large fold-increases for the characteristic expression of Cyp2b isoforms across in vivo studies in multiple species and with many different molecules. While certain genes related to a pro-proliferative effect appear to be CAR-mediated and reproducibly impacted in multiple studies (Currie et al., 2014; Deguchi et al., 2009; Geter et al., 2014; Peffer et al., 2007; Tojima et al., 2012), there are examples where changes in a specific gene was not observed. For example, Ross et al. (2010) tested 80 mg/kg/day (ip dosing) phenobarbital for 4 days in WT C57BL/6J mice, and they observed a 15.8-fold increase in Cdc20, but did not see an increased expression of Gadd45b. Mapping of a specific genes’ changes following activation of CAR by a particular CAR activator may be affected by the species, strain, dose level and time point examined, as well as the other non-CAR effects of that molecule. Examining for a significant pathway change is likely to be a more reliable measure of this Key Event Relationship (Oshida et al., 2015a), but this is also somewhat dependent on the experimental design, the species and duration of treatment, and the pathway analysis tools.
Because the extent or degree of activation of CAR is not readily measurable with in vivo studies, the nature of the relationship between CAR activation and altered mRNA expression (e.g. linear, exponential, other) cannot be stated. In addition, the direction of change in certain CAR-responsive genes is variable, in that CAR activation causes an increase in expression of some genes and a decrease in expression of other genes. For example, in mice treated with TCPOBOP by ip injection, Tojima et al. (2012) showed large increases in expression of Cyp2b10 (151x), Cdc20 (37x) and Gadd45b (14x), plus decreases in expression of lipid-related genes Acsl5 (0.5x), Slc21a1 (0.2x) and Hmgcs2 (0.3x). The different fold-change values for specific genes also indicates that the magnitude of the gene expression differences can be very dependent on the properties of the individual gene, and whether it is normally active or quiescent prior to treatment with a CAR activator.
The onset of CAR activation (upstream KE) and the time scale of measurable differences in gene expression (downstream KE) are influenced by variables such as pharmacokinetics and route of administration of an administered stressor. However, it is established that exposure of the livers of mice to a CAR activator (TCPOBOP) by intraperitoneal injection, thus bypassing the intestinal transit and absorption processes, was able to achieve large changes in gene expression in the liver after 12 hours (Tojima et al., 2012). The cascade of normal biological events following CAR activation by a ligand (i.e. binding, nuclear translocation, altered gene expression, and post-transcriptional modifications of mRNA) likely takes several hours to be completed, after which a measurable difference vs. control in mRNA levels is detectable by RT-PCR or microarrays. After the initial perturbation by a stressor of CAR, the time course of changes in gene expression is indefinite. That is, some gene expression changes can continue to be observed for as long as the CAR activator is present. For example, in mice treated with phenobarbital for up to 32 weeks following initiation with a single dose of diethylnitrosamine, differences in gene expression of CAR-responsive genes such as Gadd45b and Cdc2 continued to be observed throughout the entire treatment period in both non-neoplastic liver tissue and in liver tumors of treated vs. untreated animals (Phillips et al., 2009a).
Known modulating factors
Activation of PXR (NR1I2), a related nuclear receptor to CAR (NR1I3), is a possible confounding factor that may be operative for certain substances. There is much cross-talk between CAR and PXR, and similar responsive genes, and a particular agent could produce a mixed set of gene expression response by activating both PXR and CAR (Tojima et al., 2012; Stanley et al., 2006).
Known Feedforward/Feedback loops influencing this KER
In terms of this specific KER1 of activation of CAR (MIE) leading directly to altered expression of CAR-responsive genes (KE1), there are no known feedback loops that would affect this overall process. As an example of this lack of feedback alteration, when mice were treated long-term with phenobarbital, they continued to show significant changes in expression of CAR-responsive genes up through 32 weeks of treatment (Phillips et al., 2009).
Domain of Applicability
CAR receptors are present in the livers of virtually all mammalian species; however, there are important differences in protein sequence and thus ligand binding properties (Omiecinski et al., 2011b; Reschly and Krasowski, 2006). In reporter assays for mouse, rat, dog and human CAR, clear qualitative as well as quantitative differences in the ability of suspected CAR activators to activate CAR from the different species were demonstrated (Omiecinski et al., 2011b). In terms of the specific KER of CAR activation directly leading to altered gene expression specific to CAR activation, in vitro hepatocyte experiments indicate that human hepatocytes have only partial overlap with mice and rats in terms of the genes that are affected. In particular, genes that are related to CYP induction (e.g. Cyp2b isoforms) show increases in expression across mouse, rat and human if the CAR molecule for that species is activated, but the pro-proliferative gene pathways have been shown to be activated only in mice and rats (Elcombe et al., 2014; Hasmall and Roberts, 1999; Hirose et al., 2009; Lake, 2009). For example, the total number of altered genes in livers of chimeric mice that reflected human hepatocytes (293) compared to livers of similarly treated CD-1 mice (846) was much lower, and only 10 differentially expressed genes (primarily CYP genes) were common to both species’ liver samples following treatment with phenobarbital at dose levels of 1000 – 2500 ppm in the diet (Yamada et al., 2014).
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