Key Event Title
|Level of Biological Organization|
Key Event Components
|regulation of gene expression||abnormal|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|CAR activation- Hepatocellular tumors||KeyEvent|
|dog||Canis lupus familiaris||NCBI|
|All life stages|
Key Event Description
This key event describes the gene expression changes specific to activation of the constitutive androstane receptor transcription factor (CAR; NR1I3) in the hepatocytes of mammalian species that have been have been exposed to xenobiotics or endogenous activators. Changes in mRNA concentrations, protein concentrations, or protein activity may be used to measure CAR-dependent gene regulation.
CAR is maintained in a multiprotein complex in the cytoplasm in its inactive state. CAR may be activated by direct binding of a ligand (e.g. TCPOBOP) or by indirect mechanisms that do not involve ligand-CAR interactions (e.g., activation by phenobarbital). Activated CAR is released from the protein complex in the cytoplasm and is translocated to the nucleus, where it binds as a dimer with the retinoid X receptor alpha (RXRα) to regulatory DNA elements of target genes. CAR-RXR binding to specific target genes is achieved via a highly conserved DNA-binding domain on the CAR molecule, and results in the stimulation or suppression of target gene transcription (Omiecinski et al., 2011b; Tolson and Wang, 2010).
CAR is primarily expressed in the liver and small intestine and is activated by a variety of xenobiotics and by endobiotics such as bilirubin. CAR-dependent expression profiles identified in the livers of all mammalian species include genes involved in phase I and phase II xenobiotic metabolism and transport, glucose metabolism, and lipid metabolism (Omiecinski et al., 2011a; Tolson and Wang, 2010). In rats and mice, but not other mammalian species, additional CAR-responsive genes that regulate cell proliferation and apoptosis have been identified (Peffer et al., 2018b). Some xenobiotics will induce several transcription factors simultaneously, and these transcription factors may regulate the expression of some of the same genes. For instance, there is some overlap between the gene expression profiles of CAR-responsive genes and pregnane X receptor (PXR)-responsive genes. Both PXR and CAR can dimerize with RXRα and have been shown to influence the expression of common genes involved in functions such as xenobiotic metabolism, apoptosis, and cell signaling, although each transcription factor also independently controls the expression of genes specific to that nuclear receptor as well (Cui and Klaassen, 2016; Tojima et al., 2012; Tolson and Wang, 2010).
Hepatic CAR-dependent genes have been identified by comparing the transcriptional profiles of wild-type mice to those of CAR-null mice after xenobiotic stimulation. These include genes involved in functions such as apoptosis, lipid metabolism, xenobiotic metabolism and transport, cholesterol biosynthesis, and cell cycle regulation (Aleksunes and Klaassen, 2012; Tojima et al., 2012).
The induced expression of several genes have been used as indicators of hepatic CAR activation in mice and rats, but not all of these genes are specific to CAR activation. In particular, CAR activation leads to the induction of xenobiotic-metabolizing enzymes belonging to the cytochrome P450 CYP2B and CYP3A subfamilies, namely the increased expression of Cyp2b10 and Cyp3a11 in mice, Cyp2b1/2 and Cyp3a1 in rats, and CYP2B6 and CYP3A4 in humans (Cui and Klaassen, 2016; Deguchi et al., 2009; Geter et al., 2014; Oshida et al., 2015a; Peffer et al., 2018b). Cross-talk can occur between xenobiotics, and PXR activation can also induce CYP2B and CYP3A isoforms; however, unlike prototypical CAR activators, PXR activators will generally induce expression of CYP3A isoforms to a greater extent than CYP2B isoforms. Other metabolizing and transporter genes that are upregulated by CAR activation include UGT1A1, MRP2, SLC1A6, GSTA1 and GSTA2, and sulfotransferase enzymes have been found to be activated or suppressed by CAR activation with extensive variations based upon the species and sex of the animals (Tolson and Wang, 2010). This induction may be assayed at the mRNA level or the enzyme activity level (Elcombe et al., 2014; Felter et al., 2018; Peffer et al., 2018b). The induction of Ki67 and Gadd45b, genes that are implicated in cell proliferation, have also been used as indicators of CAR activation in mice and rats, although the expression of these genes is not strictly CAR-dependent (Columbano et al., 2005; Peffer et al., 2018a; Peffer et al., 2018b). Gadd45b can play an anti-apoptotic role, and its induction coincides with entry into an active cell cycle. It has been classified as a marker of immediate early phase of hepatocyte cell proliferation (Columbano et al., 2005). Ki67 is one of several cell cycle control genes that are transcriptionally altered in response to CAR activation to ultimately result in a pro-proliferative, anti-apoptotic environment (Peffer et al., 2018b).
A gene expression biomarker signature of CAR activation has been developed based on the genomic responses of livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators, namely phenobarbital, TCPOBOP, or CITCO, for three days. This resulted in a gene expression signature containing 83 genes (76 upregulated, 7 downregulated) that can be used to reliably predict CAR activation in the mouse liver using a Running Fisher’s algorithm (Oshida et al., 2015a). This signature was shown to exhibit a prediction accuracy of 97% when tested against chemicals that are known to be positive and negative for CAR activation in the mouse.
How It Is Measured or Detected
Changes in CAR-dependent gene expression is most directly determined by quantitating mRNA levels of the genes of interest isolated from chemical-exposed cultured hepatocytes or the livers of chemically-treated animals. Changes in mRNA levels are then statistically compared to levels detected in the appropriate controls, such as the livers of vehicle-treated animals, chemical-treated CAR-null animals, or vehicle-treated hepatocytes, to determine if there is a statistically significant change in CAR-dependent gene expression.
Recognizing that not all mRNAs are translated into protein, CAR-dependent gene expression changes may also be determined at the protein level or by quantitating changes in protein (e.g., enzyme) activity. The appropriateness of the method used to query for CAR-dependent gene expression is dependent on the design and goals of a particular experiment. For instance, high-throughput chemical screening for CAR activation may be best accomplished by evaluating the transcriptional response of query chemicals against a CAR mRNA expression signature (Oshida et al., 2015a), whereas protein level/activity determinations may be more appropriate in experiments in which a small number of proteins are of particular interest, such as experiments where the mode of action for a specific adverse effect in the liver are being investigated.
The measurement of mRNA levels of genes of interest can be accomplished using one of several well-established methods that are widely accepted by the scientific community. Methods such as quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), microarray expression profiling, and next generation sequencing methods (whole transcriptome RNA sequencing, e.g., RNA-seq) are highly specific, sensitive, and generate reproducible results. Microarrays and RNA-seq technologies in particular allow for the rapid and simultaneous quantitation of many transcripts. Advances in several of these techniques have enabled the reliable analysis of differential gene expression in a variety of sample types including cell lysates, frozen tissues, and formalin-fixed, paraffin-embedded (FFPE) tissues.
The quantitation of proteins of interest from cell lysates can be performed using immunostaining techniques such as enzyme-linked immunosorbent assays (ELISAs) or denaturing gel electrophoresis followed by Western blot analysis. These techniques are well-established and well-accepted in the scientific community. Changes in specific protein activity may also be conducted if suitable assays are available. For instance, changes in activity level of enzymes can be determined using biochemical fluorometric assays. For CYP2B isoforms, the formation of resorufin from pentoxyresorufin (pentoxyresorufin-O-depentylation; PROD) or from benzyloxyresorufin (benzyloxyresorufin-O-debenzylation; BROD) can be assayed, although there is some contribution from other Cyps, including those in the CYP3A family (Burke et al., 1985; Burke et al., 1994; Gervot et al., 1999; Lake, 2009; reviewed by Peffer et al., 2018b; Sun et al., 2006). Benzyloxyquinolone metabolism (BQ = benzyloxyquinoline-O-debenzylation) and testosterone 6-β-hydroxylase activity can also be used to measure CYP3A activity (Chovan et al., 2007; Renwick et al., 2001; Stresser et al., 2002).
Domain of Applicability
Gene expression can be modulated by nuclear receptor activation in all living systems, but the CAR nuclear receptor is only found in mammalian cells (Moore et al., 2006; Omiecinski et al., 2011b), so therefore, the altered expression of CAR-responsive genes is specific to mammals. Evidence of CAR activation and altered gene expression has been studies in humans, as well as in rats, mice, hamsters, dogs and monkeys (Diwan et al., 1986; Hasmall and Roberts, 1999; Lake, 2018; Lake, 2009; Omiecinski et al., 2011b; Peffer et al., 2018b; Reschly and Krasowski, 2006). As discussed in prior sections, the specific genes and the processes controlled by them are somewhat common following CAR activation across the mammalian species, but individual species differences are clearly observed. For example, cell proliferation and apoptosis control genes have been shown to be altered by CAR activation in mice and rats, but not in humans or hamsters (Peffer et al., 2018b).
Evidence for Perturbation by Stressor
1. The ability of PB to induce members of the CYP2B and CYP3A subfamilies of cytochrome enzymes in the livers of rats and mice as well as a number of other genes involved in xenobiotic metabolism, cell proliferation, energy metabolism and lipid metabolism is well-established and has been demonstrated in multiple studies (Elcombe et al., 2014; Peffer et al., 2018b; Whysner et al., 1996; Yamada et al., 2014). Examination of gene expression profiles of in CAR null mice revealed that not all PB-induced genes are CAR-dependent (Ueda et al., 2002).
2. PB, TCPOBOP, CITCO were used to develop a CAR-dependent gene expression signature (83 genes) in the mouse liver that can be used to predict CAR activation. TCPOBOP (3 mg/kg body weight) was administered (i.p.) to wild-type and CAR-null mice once, followed by injection with vehicle for 2 consecutive days; CITCO (30 mg/kg body weight) and PB (100 mg/kg body weight) were administered once daily for three consecutive days; livers were isolated from mice 6 h after the last injection (Chua and Moore, 2012; Oshida et al., 2015a). Genes that are upregulated in the CAR signature include Gadd45b and many associated with xenobiotic metabolism, including Cyp2b10 (Oshida et al., 2015a).
1. The hepatic gene expression profiles of male rats administered 3600 ppm metofluthrin in the diet for one week had similar gene expression profiles to those of male rats administered 1000 ppm NaPB (a known CAR activator) for one week, including the modulation of xenobiotic metabolism genes. For example, a statistically-significant, dose-dependent induction of the mRNA levels of Cyp2b1/2 and Cyp3a1 was observed in the livers of male and female rats administered metofluthrin in the diet for one week (Deguchi et al., 2009; Yamada et al., 2009). Additional experiments used RNA interference (RNAi) where CAR-specific small interfering RNA (siRNA) was co-incubated along with metofluthrin. Exposure of rat hepatocytes to metofluthrin plus the CAR siRNA in vitro resulted in a statistically significant reduction in the Cyp2b1 mRNA levels compared to hepatocytes treated with metofluthrin without CAR knockdown (Deguchi et al., 2009).
1. Hepatic CAR-dependent genes were identified by comparing the gene expression profiles of wild-type and CAR/PXR single and double-knockout mice after intraperitoneal (i.p.) injection with TCPOBOP and/or PCN (pregnenolone 16alpha-carbonitrile; PXR activator) for 12 h prior to sacrifice (3 mg/kg body weight) (Tojima et al., 2012). Both common and unique gene target genes for CAR and PXR were identified. CAR-dependent gene targets included those involved in xenobiotic metabolism, apoptosis, cholesterol metabolism and lipid metabolism. A similar experiment in wild-type C57BL/6N mice administered TCPOBOP (i.p., 3 mg/kg) or PCN once daily for four days identified common and unique CAR- and PXR-target genes (Cui and Klaassen, 2016).
2. The hepatic gene expression profiles of CAR-null, PXR-null, and PXR/CAR-null and WT rats after single treatment (i.p.; 16 h after injection) with vehicle (corn oil), PXR agonist PCN (100 mg/kg) and/or TCPOBOP (12.5 mg/kg) were compared. The results identified that PXR and CAR are involved in the regulation and expression of both common and different drug metabolizing genes in the rat liver (Forbes et al., 2017). As an example, induction of rat Cyp2b2 expression was avidly expressed in wild-type and PXR-null rats, but was blocked in the CAR-null and PXR/CAR-null rats.
3. Comparison of gene expression profiles of wild-type and CAR null mice exposed to TCPOBOP (i.p.; 3 mg/kg bw/day) for 3 consecutive days and seven weeks revealed that TCPOBOP induced the expression of prototypical CAR-responsive genes (e.g. Cyp2b10) and lipogenic genes such as Fasn, Elovl6, Gpat and Pnpla3 (Marmugi et al., 2016).
1. CITCO was identified as a CAR activator using an in vitro fluorescence-based CAR activation assay, and incubation of primary human hepatocytes isolated from two donors with 1 µM CITCO for 48 h resulted in the transcriptional upregulation of selected genes involved in xenobiotic metabolism, including the prototypical CAR target gene CYP2B6 (Maglich et al., 2003).
2. Genome-wide transcriptional profiling performed on primary human hepatocytes isolated from six individual donors exposed to 1 µM CITCO for 24 h resulted in the modulation of genes primarily involved in xenobiotic metabolism including CYP2B6, CYP2B7P1 and CYP2A7 (Kandel et al., 2016).
3. WT and hCAR-KO HepaRG cells (human hepatic cell line) were exposed to vehicle or 1 µM CITCO for 24 h, and mRNA was subjected to global gene expression analysis. Comparative analyses identified both previously identified CAR-responsive genes and novel genes that were associated with hCAR activation (Li et al., 2015). These experiments also demonstrated that CITCO achieved CYP2B6 induction predominantly via hCAR activation, whereas phenobarbital induced CYP2B6 by both hCAR and hPXR activation. Many of the genes that were differentially expressed via hCAR activation reflected an inhibition of cell cycle progression (i.e. cell proliferation) in these human HepaRG cultures. These cell cycle/proliferation related pathways repressed by hCAR activation included the TGF-β, p53 and Jak-STAT pathways. In addition, the known role of activators of mCAR in mouse liver for controlling lipogenesis via gene expression changes was not apparent in human HepRG cells after treatments with known hCAR activators.
4. In contrast to the results in HepaRG cells (above), immortalized human hepatocytes (IHH) incubated with 1 µM CITCO for 48 h resulted in the induction of prototypical CAR-responsive gene CYP2B6, and genes/transcription factors involved in lipid metabolism that could suggest a lipogenic response in human liver (Marmugi et al., 2016).
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