API

Event: 715

Key Event Title

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Activation, Constitutive androstane receptor

Short name

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Activation, Constitutive androstane receptor

Biological Context

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Level of Biological Organization
Molecular

Cell term

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Organ term

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Key Event Components

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Process Object Action
signaling nuclear receptor subfamily 1 group I member 3 increased

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
CAR activation- Hepatocellular tumors MolecularInitiatingEvent

Stressors

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Taxonomic Applicability

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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

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Life stage Evidence
All life stages High

Sex Applicability

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Term Evidence
Unspecific High

Key Event Description

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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

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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

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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).


Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

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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., 2005Omiecinski 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., 2005Wei et al., 2000) and  formation of liver tumors (Diwan et al., 1992Huang 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., 2009Mutoh et al., 2013Swales 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., 2005Wei et al., 2000Yamamoto 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.



References

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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.

 

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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.

 

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