Key Event Title
|Level of Biological Organization|
Key Event Components
|signaling||nuclear receptor subfamily 1 group I member 3||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|CAR activation- Hepatocellular tumors||MolecularInitiatingEvent|
|dog||Canis lupus familiaris||High||NCBI|
|All life stages||High|
Key Event Description
The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor that is expressed primarily in the liver (and to a lesser extent in kidneys, intestines and stomach), which can be activated by xenobiotics or by certain endogenous cellular metabolites. CAR normally is tethered in the cytoplasm via a set of specific proteins including heat shock protein 90 (HSP90) and other chaperones. Chemical ligands bind to the ligand binding site of CAR, and a conformational change frees CAR from the tethering proteins and facilitates its transport into the nucleus. In addition, indirect CAR activators (e.g. phenobarbital) can bind to the EGF receptor to initiate a series of steps that eventually dephosphorylate a critical Threonine-38 residue in CAR, allowing it to migrate into the nucleus. Inside the nucleus, CAR dimerizes with RXRα and this CAR-RXR complex binds to specific response elements on the DNA to activate transcription of specific CAR-responsive genes. CAR is unique among nuclear receptors, in that it is constitutively active when in the nucleus, i.e. it will spontaneously dimerize with RXR and alter gene expression, even without an activator bound to its ligand binding domain. When activated and translocated to the nucleus, CAR alters the transcription of multiple genes, and it is the altered levels of these gene transcripts (i.e. mRNA levels) that produce the downstream biological effects following activation of CAR (Omiecinski et al., 2011b; Omiecinski et al., 2011a; Reschly and Krasowski, 2006; Swales and Negishi, 2004).
How It Is Measured or Detected
Activation of CAR by a chemical substance is often detected in an in vitro system, using a reporter construct that is transiently transfected into a model cell line. The reporter readouts are typically luminescent (e.g. luciferase-based) (Omiecinski et al., 2011b; Stanley et al., 2006). Because CAR is constitutively active, many traditional reporter assay approaches can be confounded due to high background activity when the cytoplasmic tethering complex for CAR is inadequate in the cell line being used. Omiecinski et al. (2011b) were able to develop a successful reporter assay for CAR from mouse, rat, human and dogs by inserting a 5 amino acid modification into the different species' CAR, in conjunction with a luciferase reporter construct driven by a human CYP2B6 response element. This system showed strong responses to model CAR activators that were selective for each species' CAR, which is an important consideration since the ability of a particular chemical to activate the CAR receptor is very species-specific (Omiecinski et al., 2011b). Other groups have used a similar strategy to develop sensitive reporter assays by inserting a single amino acid residue into human CAR (Chen et al., 2010).
With in vivo testing, activation of CAR by a chemical substance is most readily detected by indirect methods, considering the complex set of processes that are involved. Typically, expression of a small subset of genes in a tissue of interest (e.g. liver) that are known to be regulated by CAR can be measured via RT-PCR methods (reverse transcripase - polymerase chain reaction), or for the whole animal transcriptome by microarrays or RNAseq methodologies (Currie et al., 2014; Peffer et al., 2018a; Peffer et al., 2018b). In these experiments, treatment of animals for 7 or 14 days and comparison of the response in control vs. treated tissue is assessed; CAR-responsive genes in mice might include Cyp2b10, Gadd45b, Ki67, Cyp2c55 and Gstm3 (Oshida et al., 2015a; Peffer et al., 2018a; Tojima et al., 2012), but an appropriate set of genes for the species and strain being tested would need to be devised based on the literature. Oshida et al. (2015a) have developed a CAR signature in mice that represents the combined change in an 83-gene signature derived from multiple CAR activating compounds given to groups of mice for 30 days. A compound's response compared to the CAR signature can be compared for both the direction and magnitude of all 83 genes, and a statistically significant match evaluated via Correlation Engine (Illumina). When a known CAR activator (Peffer et al.; Tamura et al., 2013) that was not part of the training set was tested and evaluated, it also gave a clear statistically significant confirmation as a CAR activator (Peffer et al., 2018b).
A more generic in vivo measurement approach that may be applicable in a wider array of species, is to look for increases in enzyme activity or protein levels for CAR-responsive enzymes, such as CYP2B or CYP3A induction (Burke et al., 1985; Burke et al., 1994; Sun et al., 2006). While this approach gives some evidence that the chemical tested is a CAR activator, it must be recognized that other nuclear receptors can also induce the same enzymes to varying extents, so evidence by these methods is suggestive of CAR activation but not definitive. More definitive evidence that a substance is a CAR activator, can be attained in vivo by experiments in CAR null mice or rats, which lack the gene for the CAR molecule. Absence of responses in CAR null mice or rats for the gene expression, CYP2B enzyme induction, liver hepatocellular hypertrophy and increases in liver weight, and presence of these responses in treated wild-type animals, is a convincing proof that these effects were mediated by activation of CAR in the wild-type animals.
Domain of Applicability
CAR (NR1I3) is evolutionarily conserved across mammalian species, but is not present in other vertebrate species (Moore et al., 2006; Omiecinski et al., 2011b; Reschly and Krasowski, 2006). The related NR1I nuclear receptors PXR (NR1I2) and VDR (NR1I1) are found in diverse vertebrate species from fish to mammals, and evidence suggests that CAR arose from a duplication of an ancestral PXR gene (Reschly and Krasowski, 2006). CAR exhibits a low sequence conservation of amino acids between species, including the residues of amino acids within the ligand-binding pocket. As a result, different species' CAR receptors have very different abilities to bind and become activated by CAR-activating chemicals (Omiecinski et al., 2011b). In different mammalian species, the role of CAR has been most actively studied in the liver, where it plays a central role in activation of CYP enzymes, Phase II conjugation enzymes, lipid and glucose metabolism and detoxification of bile acids. CAR is also found at lower levels in the intestine, stomach and kidneys (Moore et al., 2006). In rats and mice, CAR has been shown to also stimulate genes responsible for hepatocellular proliferation, and as a result, these species can eventually develop hepatocellular adenomas and carcinomas that do not develop in other mammalian species such as hamsters and humans (Elcombe et al., 2014; Lake, 2018).
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Stressors of CAR (i.e. chemicals that activate CAR) can be quite species-specific, due to the heterogeneity of the amino acid sequence of CAR, particularly in the ligand binding domain. TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene, 3,3′,5,5′-tetrachloro-1,4-bis(pyridyloxy)benzene) is a very potent direct CAR activator in the mouse (Huang et al., 2005; Omiecinski et al., 2011b). In reporter assays for CAR from mouse, rat, human and dog, TCPOBOP was shown to be a strong activator of mouse CAR (28-fold), but it showed no activation of rat and dog CAR, and only a minimal activation of human CAR (3-fold) (Omiecinski et al., 2011b). Consistent with this potent activation in mice, studies in wild-type and CAR-null mice showed that absence of the CAR receptor effectively blocked CAR-mediated downstream effects including gene activation (Tojima et al., 2012), cell proliferation (Huang et al., 2005; Wei et al., 2000) and formation of liver tumors (Diwan et al., 1992; Huang et al., 2005).
Metofluthrin is a potent activator of CAR in rats, and produces liver tumors in chronic toxicity studies in male and female Wistar rats, but no liver tumors in male and female CD-1 mice (Yamada et al., 2009). In rats, metofluthrin produced significant increases in gene expression of CAR-responsive genes Cyp2b1/2 and Cyp3a1, increased cell proliferation and increased indirect markers of CAR activation such as CYP2B protein levels and hepatocellular hypertrophy. While metofluthrin was not tested in CAR-null rats, isolated rat hepatocyte cultures treated with metofluthrin and siRNA for CAR (a gene silencing technique) caused a knockdown of Car mRNA expression and resulting suppression of the response to metofluthrin in terms of Cyp2b1 mRNA and Car mRNA levels (Deguchi et al., 2009). This suppression indicates that metofluthrin produces its in vivo effects in rats via activation of CAR.
Phenobarbital is an indirect activator of CAR, in that it does not bind directly to the ligand binding domain, but instead binds to an EGF receptor to initiate a series of steps that eventually dephosphorylate a critical Threonine-38 residue in CAR, allowing it to migrate into the nucleus and alter gene expression (Mutoh et al., 2009; Mutoh et al., 2013; Swales and Negishi, 2004). Downstream events following CAR activation by phenobarbital have been shown to be essentially the same as with direct CAR activators, and include Cyp2b10 gene expression, cell proliferation and increases in mouse liver tumors (Huang et al., 2005), which are prevented by treatment of CAR-null mice lacking the CAR receptor (Huang et al., 2005; Wei et al., 2000; Yamamoto et al., 2004). Thus, lack of these downstream key events in CAR-null mice, including an initiation-promotion study, has been used to demonstrate that phenobarbital activates the CAR receptor in mice.
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