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, [need to mention other tissues and organs here] 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 alterated levels of these gene transcripts (i.e. mRNA levels) that produce the downstream biological effects following activation of CAR (Omiecinski et al., 2011a).
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) (Stanley et al., 2006; Omiecinski et al., 2011b). 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, likely due to differences in the amino acid residues of each species' CAR protein (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., 2013; Peffer et al., 2017; Peffer et al., 2018). 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 (Tojima et al., 2011; Oshida et al., 2015a; Peffer et al., 2017), but an appropriate set of genes for the species and strain being tested will 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 threshold derived via Correlation Engine (Illumina). When a known CAR activator (cyproconazole; Peffer et al., 2007; 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., 2018).
A more generic in vivo approach that may be applicable in a wider array of species, is to look for increases in enzyme activity or protein levels for CAR-responsIive 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 induced the same enzymes to varying extents, so evidence by these methods is suggestive but not definitive by itself. 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
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event