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Event: 715

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Activation, Constitutive androstane receptor

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. More help
Activation, Constitutive androstane receptor
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Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help
Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Organ term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor).  Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description.  To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons.  If a desired term does not exist, a new term request may be made via Term Requests.  Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Process Object Action
signaling nuclear receptor subfamily 1 group I member 3 increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
CAR activation- Hepatocellular tumors MolecularInitiatingEvent Brendan Ferreri-Hanberry (send email) Open for citation & comment EAGMST Under Review

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
Hamster Hamster High NCBI
human Homo sapiens High NCBI
dog Canis lupus familiaris High NCBI
Monkey Monkey High NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help
Life stage Evidence
All life stages High

Sex Applicability

An indication of the the relevant sex for this KE. More help
Term Evidence
Unspecific High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

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., 2011bOmiecinski et al., 2011aReschly and Krasowski, 2006Swales and Negishi, 2004).

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

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., 2011bStanley 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., 2014Peffer et al., 2018aPeffer 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., 2015aPeffer et al., 2018aTojima 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., 1985Burke et al., 1994Sun 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

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help

CAR (NR1I3) is evolutionarily conserved across mammalian species, but is not present in other vertebrate species (Moore et al., 2006Omiecinski et al., 2011bReschly 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., 2014Lake, 2018).

References

List of the literature that was cited for this KE description. More help

Burke, M. D., Thompson, S., Elcombe, C. R., Halpert, J., Haaparanta, T. and Mayer, R. T. (1985), Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem Pharmacol 34, 3337-45.

Burke, M. D., Thompson, S., Weaver, R. J., Wolf, C. R. and Mayer, R. T. (1994), Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem Pharmacol 48, 923-36.

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.

Diwan, B. A., Lubet, R. A., Ward, J. M., Hrabie, J. A. and Rice, J. M. (1992), Tumor-promoting and hepatocarcinogenic effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in DBA/2NCr and C57BL/6NCr mice and an apparent promoting effect on nasal cavity tumors but not on hepatocellular tumors in F344/NCr rats initiated with N-nitrosodiethylamine. Carcinogenesis 13, 1893-901.

Elcombe, C. R., Peffer, R. C., Wolf, D. C., Bailey, J., Bars, R., Bell, D., Cattley, R. C., Ferguson, S. S., Geter, D., Goetz, A., Goodman, J. I., Hester, S., Jacobs, A., Omiecinski, C. J., Schoeny, R., Xie, W. and Lake, B. G. (2014), Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator. Crit Rev Toxicol 44, 64-82, 10.3109/10408444.2013.835786.

Huang, W., Zhang, J., Washington, M., Liu, J., Parant, J. M., Lozano, G. and Moore, D. D. (2005), Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor. Mol Endocrinol 19, 1646-53, 10.1210/me.2004-0520.

Lake, B. G. (2018), Human relevance of rodent liver tumour formation by constitutive androstane receptor (CAR) activators. Toxicology Research, 10.1039/c8tx00008e. http://dx.doi.org/10.1039/C8TX00008E

Moore, D. D., Kato, S., Xie, W., Mangelsdorf, D. J., Schmidt, D. R., Xiao, R. and Kliewer, S. A. (2006), International Union of Pharmacology. LXII. The NR1H and NR1I receptors: constitutive androstane receptor, pregnene X receptor, farnesoid X receptor alpha, farnesoid X receptor beta, liver X receptor alpha, liver X receptor beta, and vitamin D receptor. Pharmacol Rev 58, 742-59, 10.1124/pr.58.4.6.

Mutoh, S., Osabe, M., Inoue, K., Moore, R., Pedersen, L., Perera, L., Rebolloso, Y., Sueyoshi, T. and Negishi, M. (2009), Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3). J Biol Chem 284, 34785-92, 10.1074/jbc.M109.048108.

Mutoh, S., Sobhany, M., Moore, R., Perera, L., Pedersen, L., Sueyoshi, T. and Negishi, M. (2013), Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling. Sci Signal 6, ra31, 10.1126/scisignal.2003705.

Omiecinski, C. J., Coslo, D. M., Chen, T., Laurenzana, E. M. and Peffer, R. C. (2011b), Multi-species analyses of direct activators of the constitutive androstane receptor. Toxicol Sci 123, 550-62, 10.1093/toxsci/kfr191.

Omiecinski, C. J., Vanden Heuvel, J. P., Perdew, G. H. and Peters, J. M. (2011a), Xenobiotic metabolism, disposition, and regulation by receptors: from biochemical phenomenon to predictors of major toxicities. Toxicol Sci 120 Suppl 1, S49-75, 10.1093/toxsci/kfq338.

Oshida, K., Vasani, N., Jones, C., Moore, T., Hester, S., Nesnow, S., Auerbach, S., Geter, D. R., Aleksunes, L. M., Thomas, R. S., Applegate, D., Klaassen, C. D. and Corton, J. C. (2015a), Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium. Nucl Recept Signal 13, e002, 10.1621/nrs.13002.

Peffer, R. C., Cowie, D. E., Currie, R. A. and Minnema, D. J. (2018a), Sedaxane-Use of Nuclear Receptor Transactivation Assays, Toxicogenomics, and Toxicokinetics as Part of a Mode of Action Framework for Rodent Liver Tumors. Toxicol Sci 162, 582-598, 10.1093/toxsci/kfx281.

Peffer, R. C., LeBaron, M. J., Battalora, M., Bomann, W. H., Werner, C., Aggarwal, M., Rowe, R. R. and Tinwell, H. (2018b), Minimum datasets to establish a CAR-mediated mode of action for rodent liver tumors. Regul Toxicol Pharmacol 96, 106-120, 10.1016/j.yrtph.2018.04.001.

Peffer, R. C., Moggs, J. G., Pastoor, T., Currie, R. A., Wright, J., Milburn, G., Waechter, F. and Rusyn, I. (2007), Mouse liver effects of cyproconazole, a triazole fungicide: role of the constitutive androstane receptor. Toxicol Sci 99, 315-25, 10.1093/toxsci/kfm154.

Reschly, E. J. and Krasowski, M. D. (2006), Evolution and function of the NR1I nuclear hormone receptor subfamily (VDR, PXR, and CAR) with respect to metabolism of xenobiotics and endogenous compounds. Curr Drug Metab 7, 349-65.

Stanley, L. A., Horsburgh, B. C., Ross, J., Scheer, N. and Wolf, C. R. (2006), PXR and CAR: nuclear receptors which play a pivotal role in drug disposition and chemical toxicity. Drug Metab Rev 38, 515-97, 10.1080/03602530600786232.

Sun, G., Thai, S. F., Lambert, G. R., Wolf, D. C., Tully, D. B., Goetz, A. K., George, M. H., Grindstaff, R. D., Dix, D. J. and Nesnow, S. (2006), Fluconazole-induced hepatic cytochrome P450 gene expression and enzymatic activities in rats and mice. Toxicol Lett 164, 44-53, 10.1016/j.toxlet.2005.11.015.

Swales, K. and Negishi, M. (2004), CAR, driving into the future. Mol Endocrinol 18, 1589-98, 10.1210/me.2003-0397.

Tamura, K., Inoue, K., Takahashi, M., Matsuo, S., Irie, K., Kodama, Y., Ozawa, S., Nishikawa, A. and Yoshida, M. (2013), Dose-response involvement of constitutive androstane receptor in mouse liver hypertrophy induced by triazole fungicides. Toxicol Lett 221, 47-56, 10.1016/j.toxlet.2013.05.011.

Tojima, H., Kakizaki, S., Yamazaki, Y., Takizawa, D., Horiguchi, N., Sato, K. and Mori, M. (2012), Ligand dependent hepatic gene expression profiles of nuclear receptors CAR and PXR. Toxicol Lett 212, 288-97, 10.1016/j.toxlet.2012.06.001.

Wei, P., Zhang, J., Egan-Hafley, M., Liang, S. and Moore, D. D. (2000), The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature 407, 920-3, 10.1038/35038112.

Yamada, T., Uwagawa, S., Okuno, Y., Cohen, S. M. and Kaneko, H. (2009), Case study: an evaluation of the human relevance of the synthetic pyrethroid metofluthrin-induced liver tumors in rats based on mode of action. Toxicol Sci 108, 59-68, 10.1093/toxsci/kfp007.

Yamamoto, Y., Moore, R., Goldsworthy, T. L., Negishi, M. and Maronpot, R. R. (2004), The orphan nuclear receptor constitutive active/androstane receptor is essential for liver tumor promotion by phenobarbital in mice. Cancer Res 64, 7197-200, 10.1158/0008-5472.can-04-1459.