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DOI: 10.1080/01926230701311351
Genomic Profiling in Nuclear Receptor-Mediated Toxicity
1 Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599, USA Correspondence: Address correspondence to: Dr. Ivan Rusyn, 0031 Michael Hooker Research Center, CB #7431, Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7431, USA; e-mail:iir{at}unc.edu
Nuclear receptors (NRs) are attractive drug targets due to their role in regulation of a wide range of physiologic responses. In addition to providing therapeutic value, many pharmaceutical agents along with environmental chemicals are ligands for NRs and can cause adverse health effects that are directly related to activation of NRs. Identifying the molecular events that produce a toxic response may be confounded by the fact that there is a significant overlap in the biological processes that NRs regulate. Microarrays and other methods for gene expression profiling have served as useful, sensitive tools for discerning the mechanisms by which therapeutics and environmental chemicals invoke toxic effects. The capability to probe thousands of genes simultaneously has made genomics a prime technology for identifying drug targets, biomarkers of exposure/toxicity and key players in the mechanisms of disease. The complex intertwining networks regulated by NRs are hard to probe comprehensively without global approaches and genomics has become a key technology that facilitates our understanding of NR-dependent and -independent events. The future of drug discovery, design and optimization, and risk assessment of chemical toxicants that activate NRs will inevitably involve genomic profiling. This review will focus on genomics studies related to PPAR, CAR, PXR, RXR, LXR, FXR, and AHR.
Key Words: Toxicogenomics • nuclear receptor • transcription profiling • PPAR • CAR • PXR • AHR Abbreviations: AHR, aryl hydrocarbon receptor • CAR, constitutive androstane receptor • CYP450s, cytochrome P450s • FXR, farnesoid x receptor • LXR, liver x receptor • PPs, peroxisome proliferators • PPARs, peroxisome proliferator activated receptors • PB, phenobarbital • PXR, pregnane x receptor • RXR, retinoid x receptor • TCDD, 2', 3', 7, 8'-tetrachlordibenzo-p-dioxin
Nuclear receptors are a group of ligand-activated transcription factors, which are responsible for regulating expression of genes associated largely with metabolism, developmental function, and cell differentiation. Nuclear receptor-targeted pharmaceuticals are estimated to be 10–15% of the $400 billion global pharmaceutical market. A number of therapeutic compounds including antibiotics, anticonvulsants, hypolipidemics, and cancer therapies target nuclear receptors. They are prime candidates as drug targets for several reasons. Because the ligands are small and lipophilic, they resemble endogenous inducer compounds. Many nuclear receptors have tissue-specific expression that is crucial for the specificity of drug action. Also, nuclear receptors play key roles in many physiological processes where phase I and II metabolism genes are involved. These include synthesis and metabolism of steroids, vitamin D, cholesterol, lipids, and bile acids (Waxman and Azaroff, 1992; Mangelsdorf and Evans, 1995; Honkakoski and Negishi, 2000). Nuclear receptors are also inadvertent targets of numerous anthropogenic environmental toxicants (e.g., phthalates, dioxins). While the mechanisms by which these chemical agents mediate toxicity are still largely unknown, nuclear receptors have been shown to play key roles in their pathophysiological effects. Thus, determining the human health risk, particularly of low-dose chronic exposures to nuclear receptor ligands is of increasing priority to environmental regulatory agencies. The nuclear receptor superfamily of proteins is subdivided into 6 subfamilies, based on their amino acid sequence similarities (Mangelsdorf et al., 1995; Aranda and Pascual, 2001). The general structure of all nuclear receptors is very similar, and encapsulates three functional domains: (1) a ligand-binding/dimerization domain, (2) a DNA-binding/weak dimerization domain, and (3) transactivation domains. The ligand-binding and dimerization domain at the carboxy terminus of the receptor is where endogenous or xenobiotic ligands bind resulting in activation of the receptor. Many receptors require heterodimerzation with retinoid x receptor (RXR) (Mangelsdorf and Evans, 1995). The DNA-binding domain of the receptor is responsible for recognition of a receptor-specific response element consisting of a specific sequence of nucleotides in the promoter region of the target gene. The transactivation domains consist of a ligand-independent transcription activation function (AF)-1 and a ligand–dependent transcription AF-2. AF-1 is located at the amino acid terminus of the receptor and is a target for kinase mediated phosphorylation cascades, which can affect transcriptional activity in the absence of ligand-binding. AF-2 is located at the carboxy terminus that also harbors the ligand-binding site. AF-2 can interact with other transcription factors to form a complex of co-activators, which regulate histone acetyltransferase activity, or co-repressors, which regulate histone deacetylase activity (Glass and Rosenfeld, 2000; Steinmetz et al., 2001). In common among most nuclear receptors is that they transcriptionally regulate a number of proteins involved in xenobiotic metabolism, particularly phase I cytochrome P450 (CYP450) enzymes (Figure 1) (Waxman, 1999; Honkakoski and Negishi, 2000; Johnson et al., 2002). Because of the redundancy in regulation of CYP450s and many other genes, identifying individual nuclear receptors that are responsible for a specific therapeutic or toxic effect can be challenging. Not only do nuclear receptors share transcriptional targets, but many serve as transcriptional inducers of one another. Also, ligands are often not selective for one particular nuclear receptor target, but rather are partial or full agonist for a number of receptors.
In light of these complexities, new methods have been developed for studying global chemical-induced changes in macromolecules (genes, proteins, metabolites) that collectively define an organ’s response. Genomic profiling involves use of microarray technology (but has recently been broadened to include multiplex RT-PCR assays) to measure transcript levels in tissue or cell culture. Because of the high-throughput capabilities and data-rich output, gene expression profiling is an efficient and useful alternative to traditional methods in toxicology.
Microarrays as a Tool for Mechanistic and Predictive Toxicology For chemical compounds in which the mechanism of toxicity is well-characterized and key players are known, the more focused view that traditional assays offer is suitable; however, for most chemical toxicants, the molecular events leading to toxicity or involved in disease progression are not well-defined. In these cases, the genome-wide view of gene expression that microarrays offer is more ideal. Because toxicogenomics is a relatively novel technology, there are a number of limitations that must be resolved before array data is widely accepted. Microarray studies have been touted as being highly sensitive for detecting toxic responses at much earlier time points and/or at lower doses than histopathology, clinical chemistry or other traditional toxicological assays can detect (Heinloth et al., 2004; Morgan et al., 2005). However, based on the nature of the assay, measurements of extreme levels of gene expression—low or high—are thought to be unreliable. Also, the reproducibility of microarray experiments has raised concerns. "Batch effects" based on the day, user, and laboratory environment have been observed in array datasets (Baker et al., 2004; Bammler et al., 2005). To address these concerns, confirmation of microarray-derived gene expression profiles is typically performed using quantitative real-time polymerase chain reaction (RT-PCR) or Northern blot analysis. Another limitation to widespread use of genomics technology is the high cost. The price and availability of microarrays in early years of the technology were prohibitive. With the establishment of genomic core facilities at universities and research institutes and growing competition in the private sector, the cost of array experiments is now more reasonable. Given the high return of data on investment, it is likely that this technology will continue to be used widely. Finally, improvements should continue to be made on statistical analysis and presentation of microarray data such that it is easy to interpret. Prior to the current advances in bioinformatics, the most common way of reporting results of microarray studies involved listing differentially expressed genes, with little information about the statistical significance or biological pathways with which the genes are associated. New mathematical and graphical approaches have been developed to improve data presentation and interpretation. Also, curated web-based tools and software applications have been developed to provide information on cellular location, physiological function, or disease association of a given gene. These approaches to analyzing array data, coined "pathway mapping" provide more biological relevance to the analyses. Toxicogenomics studies thus far have largely supported what is already known for many chemical compounds, though opposing and inconclusive results have been presented. Until many of the above-mentioned shortcomings (i.e., cost, reproducibility, and data presentation/interpretation) and others are addressed, the great potential for toxicogenomics as a predictive and mechanistic tool in risk assessment may not be fully realized. This review considers the current body of knowledge involving use of genomic profiling to understand the role of nuclear receptors, primarily as they relate to chemical-induced liver toxicity. Many of these studies have furthered our understanding of molecular mechanisms underlying xenobiotic-induced liver injury and ability to predict toxicity. Several nuclear receptors that are currently of great pharmacological or toxicological relevance, which include peroxisome proliferator activated receptors (PPARs), constitutive androstane receptor (CAR), pregnane X receptor (PXR), retinoid X receptor (RXR), liver X receptor (LXR), and farnesoid X receptor (FXR) are detailed in this review. The aryl hydrocarbon receptor (AHR), though not a nuclear receptor, will also be taken into consideration here. The estrogen receptor will not be discussed, since a comprehensive review on use of toxicogenomics to understand molecular mechanisms of toxicity by xeno-estrogens was recently published (Moggs, 2005).
In general, PPARs are known for their role in fatty acid metabolism and glucose homeostasis (Dreyer et al., 1992; Schmidt et al., 1992). For this reason, they have been identified as useful drug targets for hyperlipidemia, diabetes and obesity. Of the 3 known isoforms of PPARs—  , β/ and  —PPAR and PPAR have been studied most widely.
Pharmaceutical compounds that target PPAR
PPAR plays a major role in adipocyte differentiation and glucose and insulin homeostasis. It is mainly found in adipose tissue and cells of the immune system, although it is expressed to some extent in the intestines and liver (Kliewer et al., 1994; Braissant et al., 1996). Compounds that activate PPAR, such as thiazoladinediones have proven to be a good treatment for conferring insulin-sensitivity to insulin-resistant diabetics (Alarcon et al., 2004; Vasudevan and Balasubramanyam, 2004). Endogenous ligands of PPAR include fatty acid derivatives such as 15-deoxy-delta12,14-prostaglandin J2 (15d-PJ2) and eicosapentaenoic acid (Forman et al., 1995; Yu et al., 1995).
PPARβ/
Gene expression profiling to investigate molecular mechanisms associated with toxic effects resulting from PPAR activation has been carried out to a large extent for PPAR
Gene Expression Profiling Supports a Nongenotoxic Mechanism of Action of PPAR An induction of cell proliferation, oxidative stress, and suppression of apoptosis are generally accepted as key steps in the mode of action of non-genotoxic carcinogens (Butterworth, 1990). Indeed, these responses were demonstrated in the acute, sub-acute and sub-chronic gene expression studies that were conducted to identify gene signatures associated with peroxisome proliferator-induced effects in liver.
Gene expression profiling along with pathway mapping as an unbiased way to identify gene signatures and temporal associations was used by several research groups to study acute and sub-acute effects of DEHP on mouse liver. Currie et al. (2005) reported that acute exposure to DEHP induces a 2-stage transcriptional response, one occurring at early time points (2 and 8 hours), and another occurring later (24 and 72 hours). This study suggests that many PPAR
Gene expression data from subchronic studies feeding clofibrate and ciprofibrate also confirmed well-known effects of PPs. Kramer et al. evaluated dose-dependent changes in rat liver gene expression that resulted from 5 days of clofibrate (up to 80 mg/kg) treatment (Kramer et al., 2003). Analysis of transcriptional profiles obtained from cDNA arrays identified 163 genes whose expression was altered by clofibrate treatment. A majority of the genes were associated with metabolism. PPAR
Surprisingly, a strong induction in genes associated with cell proliferation was not observed by Kramer et al. (2003), even though immunohistochemical detection of proliferating cell nuclear antigen (PCNA) showed a dose-dependent induction of cell replication. The lack of a transcriptional signature for cell cycle regulation could be explained by the fact that peroxisome proliferator-induced cell proliferation is thought to rapidly increase, then decline after 4–7 days of treatment (Marsman et al., 1988). In addition, genes involved in apoptosis were found to be both up- and down-regulated and immunohistochemical analysis of caspase 3 (Casp3) revealed no changes in apoptosis. While this study reported a dose response in the transcriptional changes, cell proliferation and clinical chemistry, it is not possible to conclude whether PPAR
Microarray studies in mouse liver following 2 weeks of dietary treatment of potent peroxisome proliferator, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY-14,643) revealed similar results with altered expression of genes involved in lipid and glucose metabolism, transcription, apoptosis and cell cycle (Cherkaoui-Malki et al., 2001). A novel set of 27 peroxisome proliferator-regulated genes were identified and included cell death inducing DNA fragmentation factor Transcriptional changes in genes involved in lipid and carbohydrate metabolism, cell proliferation, stress response, immune and inflammatory responses and transcription were identified in rat liver following subchronic (60 days) ciprofibrate treatment (Yadetie et al., 2003). Though only 8% of the 5,000 genes that were probed on the cDNA arrays demonstrated a significant change in expression, the pathways that were altered corroborate previously observed phenotypic changes associated with long-term peroxisome proliferators treatment (Lalwani et al., 1981; Marsman et al., 1988).
Genomic Analysis of PPAR Genomic studies have further demonstrated that the mechanism of carcinogenesis by peroxisome proliferators in rodents may not be relevant to humans who appear to be insensitive to peroxisome proliferator-mediated liver effects (Bentley et al., 1993; Gonzalez et al., 1998). Gene expression changes in clofibrate-induced hepatocellular carcinomas (HCC) were compared to 6 other mouse models of HCC and human HCCs in an effort to determine what molecular events, if any, are common among human and mouse cancers (Lee et al., 2004). Interestingly, gene expression profiles revealed that clofibrate induced HCCs and those spontaneously formed in Aox–null mice were the least similar to human HCC. Identifying acute markers of hepatocarcinogenesis is important for improving mechanistic understanding and diagnostic capabilities. In a recent genomics study, early gene markers were identified in clofibrate-induced liver cancer. Diethylnitrosamine (DEN) was administered in a single dose as an initiating agent, followed by dietary feeding of clofibrate for up to 20 months (Michel et al., 2005). Microarray and RT-PCR analysis of liver tissue (including tumors) at early and later time points for up to 20 months found that expression of transferring growth factor beta-induced transcript, Tsc22, a previously identified potential marker of hepatocarcinogenesis, was consistently down-regulated over the 20 months. This effect was reversed when clofibrate diet was removed (Kramer et al., 2004). While the function of Tsc22 is not completely known, previous studies with anti-cancer agents have shown an induction of Tsc22 (Uchida et al., 2000; Hino et al., 2002).
Three other genes with similar expression patterns were identified as possible acute markers of hepatocarcinogenesis. These include fibroblast growth factor receptor subtype 4 (Fgfr4), dual specificity protein phosphatase 1 (Dusp1, also called Mkp1), and small conductance calcium activated potassium channel (Kcnn2), all of which have demonstrated modulation by glucocorticoids (Riva et al., 1998; Brem et al., 1999; Kotev-Emeth et al., 2002; Engelbrecht et al., 2003). These findings suggest that clofibrate not only directly regulates genes through PPAR
Gene Expression Profiling Reveals PPAR Principle component analysis (PCA) along with a pairwise correlation analysis of the treatment groups confirmed the greater similarity in gene expression profiles among compounds within the same class than between the two classes. Additionally, temporal changes in expression were observed as a result of peroxisome proliferators or phenobarbital treatment. Genes that demonstrate a delayed response to treatment (higher expression at 2 weeks) were identified as good candidate markers of toxicity and adaptation to exposure, while transiently altered genes (induced at 2 hours) likely constitute the initial response in liver following the first insult. Different mammalian species have demonstrated varying degrees of sensitivity to PPs (Dirven et al., 1993; Graham et al., 1994; Mukherjee et al., 1994; Roberts et al., 2000). In a recent genomics study, cynomolgus monkeys were exposed to ciprofibrate for 4 or 15 days (Cariello et al., 2005). Similar to rodents, a dose-dependent increase in liver weight and number of peroxisomes was observed. Though rhesus monkey arrays are available and would have been the more appropriate choice, the authors chose to use human arrays assuming that a large number of transcripts would be still detected. Pathway mapping of differentially expressed genes found reported in the 15-day, high-dose group revealed that processes involving ribosomes, proteasomes, fatty acid metabolism, tryptophan metabolism and oxidative phosphorylation were significantly up-regulated.
Down-regulation in coagulation cascades was also observed which is consistent with the therapeutic effects of fibrates and confirms previous reports of reduced inflammatory response caused by peroxisome proliferator treatment (Yadetie et al., 2003). In contrast to rodents, regulatory genes such as Nf Comparisons of gene expression of β-oxidation between species demonstrated a greater induction in rodents than in primates. Also, decreased mRNA for growth response genes and induction of those involved in apoptosis suggests an anti-proliferative, pro-apoptotic response in primates. These mRNA expression data correlate well with the phenotypic response, as no proliferation was observed in primate livers measured by immunohistochemical detection of Ki-67 and by mitotic activity (Hoivik et al., 2004). This genomic study confirms the disparate response in rodents and primates to PPs, and further demonstrates the idea that primates are a less sensitive species.
In vitro genomic profiling has also confirmed many of the responses observed in vivo. Studies performed in rat (FaO) and human HepG2 liver-derived cell lines following 6 hours of treatment with WY-14,643 (50 µM) revealed few similarities in gene expression (Vanden Heuvel et al., 2003). Induction of lipid metabolism and suppression of signaling and growth factor response was observed in rat cell lines. A number of novel genes that have not previously been identified as being PP-responsive, including kinases and phosphotases were also regulated by WY-14,643 treatment in FaO cells. Mkp1 was identified as a target of PPAR
A genomics study in immortalized hepatocytes from Ppar
Previous studies conducted in vitro using primary cells have demonstrated that hepatocytes, in absence of Kupffer cells or TNF
Overall, gene expression profiles for PPAR
CAR and PXR are expressed in liver, intestine, lung, and other tissues where they play an important role in xenobiotic sensing and act as master regulators of detoxifying phase I and II enzymes (Waxman, 1999). Because of these two traits, CAR and PXR have been characterized as "xeno-sensors" that protect the liver and other organs from potentially harmful compounds (Huang et al., 2003; Kretschmer and Baldwin, 2005). Physiological ligands of PXR (also known as steroid and xenobiotic receptor, or SXR) include corticosterone, progesterone, and precursors to pregnenolone (Kliewer et al., 1998; Lehmann et al., 1998; Jones et al., 2000). PXR also has a large number of exogenous ligands, many of which have been identified as endocrine-disrupting chemicals. The ligand-binding pocket of PXR is also larger than most other nuclear receptors, which may help explain its promiscuity (Watkins et al., 2001, 2003). PXR is located in the nucleus and has low basal activity (Moore et al., 2003). In the nucleus, it dimerizes with RXR and binds to the a xenobiotic response element of PXR target genes (Blumberg and Evans, 1998). The primary role of PXR as an activator of xenobiotic metabolism became evident from PXR-mediated induction of Cyp3a4 (Moore and Kliewer, 2000). CYP3A4 is the most abundant CYP450 in human liver (~30%) and is responsible for metabolism of about 50% of pharmaceuticals (Rendic and Di Carlo, 1997; Kliewer et al., 1999). CAR is a less promiscuous receptor than PXR, with fewer known ligands (Moore and Kliewer, 2000; Moore et al., 2003). Phenobarbital (PB) is the prototypic CAR-activator, though it does not directly bind the receptor (Zelko et al., 2001). Unlike PXR, CAR is located in the cytoplasm and is constitutively expressed in absence of endogenous ligand. A number of other PB-like inducers of CAR activate a signal transduction pathway that causes CAR to translocate from the cytoplasm to the nucleus. There it heterodimerizes with RXR to effect transcription of target genes. In general the chemicals that activate CAR and PXR receptors vary widely in structure, although many share common features, such as their hydrophobic nature, low molecular weight and presence of either a ketone or hydroxyl group (Waxman and Azaroff, 1992; Schuster and Langer, 2005;). It is well established that PXR and CAR regulate metabolism and elimination of many xenobiotics and endogenous compounds by inducing CYP450s, primarily CYP3A4 and CYP2B10, respectively (Waxman, 1999; Honkakoski and Negishi, 2000). As shown in Figure 3, genes encoding other CYP450s along with phase II enzymes, glutathione–S-transferases (GSTs), UDP-glucuronosyltransferases (UGTs) and sulfotransferase (SULTs) are regulated by CAR and PXR. Transporters such as multi-drug resistance protein 1 (Mdr1) and organic anion transporter polypeptide (Oatp2) are induced by PXR-agonists, while multi-drug resistance-associated protein (Mrp2) gene expression increases as a result of CAR-activation (Geick et al., 2001; Hagenbuch et al., 2001; Synold et al., 2001; Kast et al., 2002). CAR- and PXR-mediated induction of these transporters, often collectively referred to as ATP-binding cassette (ABC) proteins, results in efflux of xenobiotics to the gut lumen or uptake into bile for ultimate elimination from the liver.
Regulation/Autoregulation of Nuclear Receptors by CAR and PXR A number of genomics studies have been conducted to assess the role of CAR and PXR in normal liver physiology and in chemical-induced liver toxicity. Most have confirmed that CAR and PXR mediate induction of phase I and II enzymes and transporters (Xie et al., 2000; Maglich et al., 2002; Wei et al., 2002). In a study using RT-PCR, expression of about 40 genes involved in xenobiotic metabolism was measured to identify receptor-specific and tissue-specific gene signatures in mouse liver and intestines (Maglich et al., 2002). Expression profiles from Pxr-null and Car-null mice that were treated for 28 hours with either a PXR-agonist, pregnenolone-16a-carbonitrile (PCN, 100 mg/kg), or a CAR-agonist, 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP, 0.3 mg/kg), revealed that a number of enzymes and transporters induced in the small intestines were regulated by PXR, not CAR. Similarly, several genes induced in liver were regulated by CAR, but not PXR. Autoregulation of PXR and cross-talk with CAR in mouse liver was demonstrated by PCN-induced expression of PXR and CAR mRNA in wild-type, but not PXR-null mice. Additional gene expression profiles were obtained from human hepatocytes of 2 different donors. Interestingly, 48 hours of hPXR-specific activator, rifampicin (10 µM) was able to increase AHR-regulated Cyp1a1 and Cyp1a2 mRNA levels by as much as 24-fold over control, while phenobarbital induced both genes, but only 3-fold over control. PXR and CAR activators were also able to induce expression of AHR. The fact that PXR-activation by PCN did not induce Ahr or CYP1a1 in mouse liver confirms that there are substantial species differences between human and mouse PXR receptor. PXR’s autoregulatory response that was observed in mouse liver was much weaker in human hepatocytes and regulation of CAR by PXR was not observed.
Recent gene expression studies using CAR-activators further demonstrate the diverse role of CAR in normal liver function. Microrarray analysis of liver tissue from wild-type and Car-null mice treated with PB for 12 hours identified 144 significant genes, which grouped into distinct categories based on their dependence on CAR for altered expression (Ueda et al., 2002). Genes that were CAR-dependent for induction by PB were those associated with xenobiotic metabolism, including Cyp2b10, aldhehyde dehydrogenase (Ald1) and flavin containing monooxygenase 5 (Fmo5). Another group of genes, which required CAR for suppression, spanned a wide range of functions including signal transduction, fatty acid oxidation, energy metabolism, and cell surface communication. A third group of genes which were induced by PB in Car-null mice included Cyp4a10 and Cyp4a14, which are PPAR The enzymes encoded by these 2 genes are major microsomal peroxidases and may contribute to oxidative stress in the liver (Leclercq et al., 2000). Since Cyp4a10 and Cyp4a14 were suppressed in wild-type mice, CAR may act as a suppressor for oxidative stress, an idea consistent with previous findings (Sugatani et al., 2001). Finally, another set of genes were induced or repressed by PB, in a CAR-independent manner. Within this group of genes was aminolevulinate synthase 1 (Alas-1), a key gene in heme biosynthesis. Heme supply is an essential factor of CYP450 activity, and is thought to be coordinated with CYP450 induction (Iba et al., 1999). CAR-independent induction of Alas-1 has been confirmed in other studies (Yamamoto et al., 2004). These results suggest that CAR-agonists elicit other pathways that regulate hepatic expression in addition to those mediated by CAR.
A network of genes regulated by PXR was identified in a study that used a humanized mouse model that expressed either a full-length or transcriptionally active variant of hPXR (Rosenfeld et al., 2003). These mice, termed VP-hPXR constitutively regulate PXR target genes, which eliminates the added variables that using ligand activators can create. From the 8700 sequence cDNA microarrays, 150 unique transcripts that were differentially expressed were identified. A number of distinct CYP450s, particularly those associated with
In an attempt to find overlapping genes, these expression data were compared to results from the abovementioned studies by Ueda et al. (2002) and Maglich et al. (2002). Only half of the chosen transcripts had synonymous expression among the 2 datasets, which could be due to the varying dosing regimens and different platforms used in the 3 studies. When these three data sets are considered collectively, PXR and CAR appeared to induce many common genes, including Cyp2a4, Cyp2b10, and Cyp3a11, Aldh1a1 and Ald1a7, all of which are involved in Phase I metabolism, Gsta1, Gstm1 and 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (Papss2), which are involved in phase II conjugation, and Mrp3 of phase III elimination. Suppression of PPAR
CAR and PXR in Drug Induced Liver Injury This mechanism of toxicity explains why pretreatment with CYP450 inducers such as alcohol or phenobarbital exacerbates APAP-induced liver injury in both humans and rodents (Burk et al., 1990; Kostrubsky et al., 1997; Pirotte, 1984; Sinclair et al., 1998). Similar studies using other hepatotoxicants such as carbon tetrachloride (CCl4) have also demonstrated a role for CAR in mediating toxicity (Yamazaki et al., 2005). To date, there is no published report of a genomic study that would specifically address the role of CAR in APAP- or CCl4-induced liver injury. A group of environmental chemicals whose toxicity is thought to be a result of PXR and CAR-activation includes azole fungicides, also known as conazoles. These compounds were developed as pharmaceuticals for treating fungal infections and are currently used as pesticides (Sheehan et al., 1999). Previous reproductive toxicity tests in rats using a number of conazoles reported testicular atrophy, prostate atrophy, reproductive failure and cancers of the thyroid and pituitary gland and liver. Conazoles have demonstrated CYP450 modulation, which led to the idea that their toxicity may be nuclear receptor-mediated (Hurley, 1998; Nelson et al., 2004). Two recent toxicogenomics studies were conducted to assess the effects of 4 triazoles (a subset of conazoles) on expression of CYP450s and other genes in rat liver and testis (Tully et al., 2006), and in mouse liver (Goetz et al., 2006). Rats treated with fluconazole (up to 50 mg/kg), propiconazole (up to 150 mg/kg), myclobutanil (up to 150 mg/kg) or triadimefon (up to 115 mg/kg) for 14 days had significantly enlarged livers, a response which was not observed at the lowest of the 3 doses. No changes in testis weight were observed. Global gene profiles showed that expression of 376 of 1137 genes was altered in liver and 357 of 2249 in testis by at least one treatment group. In both liver and testis, the majority of differentially expressed genes were selectively induced by only 1 triazole. Strong induction of only 2 genes, Ces2 and UDP-glucuronyltransferase (Udpgtr) was observed across all treatments. Induction by multiple triazoles of 4 of 6 genes encoding CYP3A enzymes was detected in liver using microarrays and RT-PCR. High concordance in liver expression of genes encoding CYP450 enzymes was observed across all triazoles. Also, clustering of 26 differentially expressed genes in liver that are regulated by CAR or PXR revealed a very homogenous profile across all triazoles, suggesting a common mechanism of action among the fungicides that may involve CAR or PXR activation. Neither CYP450s nor other CAR/PXR-regulated genes were uniformly expressed across all treatments in testis, providing some evidence that the mechanism of toxicity in this organ may not be nuclear receptor-mediated, at least directly. In mouse liver, following the same dosing regimen as used in rats, expression of genes encoding CYP450s and other CAR or PXR-regulated genes varied by triazole treatment (Goetz et al., 2006). Comparison of mouse and rat data from these 2 studies revealed a common induction of Ces2, solute carrier organic anion transporter 1a4 (Slco1a4), and genes encoding CYP3A family enzymes by at least 3 of the 4 triazoles, but few other similarities were observed. From these genomics studies it may be concluded that triazoles induce compound specific pathways in rat liver, but also uniformly induce pathways involving CYP450s and CAR/PXR-associated enzymes and transporters that are likely important to the mechanism of action of triazoles. The role of CAR and PXR in mouse liver injury by these agents is less conclusive, given the variable induction of genes related to xenobiotic metabolism and requires further studies.
Elucidating the Mechanism of CAR-Mediated Carcinogenesis and Identifying Markers for Predictive Toxicology Studies of CAR-mediated hepatocarcinogenesis, using TCPOBOP or PB in rats and mice demonstrated a strong correlation between Cyp2b1 induction and tumor promotion (Diwan et al., 1992). Additionally, PB was an effective inducer and tumor promoter in mice and rats, while TCPOBOP was effective only in mice. Car-null mice have unequivocally shown that hypertrophic, hyperplastic, and carcinogenic effects of these compounds are CAR-mediated (Yamamoto et al., 2004). Because of the species difference in receptor ligands and target genes, humanized CAR mice have recently been developed to better understand the human relevance of hepatic effects of PB-like compounds that are observed in mice. The hCAR mice, when treated with PB (0.05%) for 1 week exhibit increased proliferation, liver enlargement and induction of Cyp2b10 and mouse double minute 2 (Mdm2), a gene that is involved in inhibiting p53-mediated cell growth arrest (Huang et al., 2005). These new studies suggest that humans may be a sensitive and susceptible species to hepatocarcinogenesis by PB and PB-like compounds. Although there are reports which support these animal studies and link long-term PB treatment with liver cancer (Vazquez and Marigil, 1989), PB and PB-like compounds are not typically associated with increased cancer incidence in humans.
While it is clear that CAR is involved in the mechanism of action of PB-induced rodent carcinogenesis, the molecular events leading to cancer are still not known. Cell proliferation is an important event in carcinogenesis because it is a critical mechanism for driving clonal expansion of mutated or differentiated cells (Columbano et al., 1981; Butterworth, 1990). Microarray studies have helped to identify genes that are involved in the proliferative response associated with CAR-activation. Genes with a strong immediate-early induction in response to proliferation-inducing treatments, partial hepatectomy or TCPOBOP have been identified using cDNA arrays. An induction in Gadd45β occurred only 3 hours after treatment. Gadd45 is a growth arrest and DNA-inducible gene with anti-apoptotic activity that is often mediated by NF- Oxidative stress and induction of metabolizing enzymes are also thought to play a key role in the mode of action of nongenotoxic carcinogens (Klaunig and Kamendulis, 2004). Genomic profiling using amplified fragment length polymorphism (ALFP) in rat liver following sub-chronic (13 weeks) revealed 168 sequence "contigs," overlapping clones that represent a continuous region of DNA, with altered expression of 2-fold or greater (Elrick et al., 2005). Pathway mapping confirmed an induction of genes encoding for CYP2B family enzymes, UGTs and a number of other xenobiotic metabolizing enzymes. Expression of a select group of these genes was confirmed by RT-PCR. Transcription profiles from ALFP, when compared with 5 day PB-treated rat liver expression profiling by a standard microarray procedure (Kramer et al., 2004), revealed concordant expression of genes involved in xenobiotic metabolism and stress response, strengthening the argument that oxidative stress as a result of enzyme induction plays a major role in hepatocarcinogenesis by nongenotoxic compounds. Gene expression patterns associated with cell cycle, apoptosis and cellular metabolism genes, contrasted between the 2 studies. These differences may be explained by the shift in cell turnover rate that occurs between subacute and chronic treatment with nongenotoxic carcinogens (Marsman et al., 1988). These genomics studies have significantly added to the body of knowledge on CAR and PXR by confirming the role of PXR and CAR in xenobiotic metabolism, identifying other nuclear receptor co-regulators, providing important insight into the mechanism of action of CAR- or PXR-activators and identifying novel and early-responding genes that may be involved in CAR-mediated liver toxicity or carcinogenesis.
RXR is a common binding partner for a number of receptors, including PPAR, CAR, PXR, LXR, and FXR and is also capable of forming homodimers (Mangelsdorf and Evans, 1995). For these receptors, transcription of target genes requires the formation of an RXR-NR complex, which binds to the response element in the promoter region of the target gene. PPAR and FXR, among others have been identified as permissive binding partners with RXR. In this case, a heterodimer can be activated independently by an agonist for the primary receptor (i.e., PPAR, CAR, FXR, etc), by an RXR-agonist or by both to cause synergistic effects. RXR heterodimers that contain nonpermissive partners can only be activated by the partner receptor’s agonist but not by RXR agonists.
Three distinct RXR isoforms (-
RXR as Mediator of Glutathione Homeostasis Glutathione (GSH) is an important endogenous antioxidant that is responsible for scavenging electrophiles produced as a result of phase I metabolism. A role for RXR in regulating GSH homeostasis was demonstrated using a mouse model for Rxr deficiency in hepatocytes (Wu et al., 2004). This hepatocyte-specific knockout whole animal model was developed as a result of embryonic lethality caused by total gene knockout (Kastner et al., 1994; Sucov et al., 1994; Kastner et al., 1996). To further assess Rxr–regulated pathways, cDNA microarray analysis was performed. Over 280 of 15,000 expressed sequence tags had significantly altered expression in Rxr-deficient hepatocytes as compared to wild-type cells (Wan et al., 2000a). A small subset of genes associated with GSH synthesis was significantly down-regulated in Rxr-deficient mice and hepatic GSH was greatly reduced. Cyp1a2 and Cyp3a11 mRNA was also significantly lower compared to wild-types. Rxr-deficient hepatocytes were more sensitive to oxidative stress by t-butylhydroperoxide compared to wild-types.
Interestingly, the Rxr
In a similar study confirming the role of RXR
Role of RXR
From studies that measured expression of PPAR
LXR and FXR serve as master regulators of cholesterol and bile acid homeostasis, respectively. Endogenous ligands for LXR primarily include oxysterols (e.g., 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol) (Janowski et al., 1996; Lehmann et al., 1997). This receptor is widely expressed in the liver and intestines (Lu et al., 2001). LXR forms a heterodimer with RXR, which can be activated by both LXR- and RXR-specific ligands. It is thought that the heterodimer is prebound to DNA but is complexed with co-repressors. Binding of RXR or LXR agonists releases co-repressors and recruits co-activators for initiation of transcription (Chen and Evans, 1995; Glass and Rosenfeld, 2000). FXR is activated by primary and secondary bile acids such as cholic acid (CA) and chenodeoxycholic acid (CDCA) (Makishima et al., 1999; Parks et al., 1999). It, too, is widely expressed in the liver and intestines, which is consistent with its physiological function and activates transcription of its target genes in a fashion much like LXR. LXR and FXR are rather connected in their functions. As a modulator of oxysterol levels, LXR is responsible for regulating cholesterol synthesis and metabolism. Accumulation of cholesterol can lead to a number of deleterious hepatic, cardiovascular, and neurological effects (Carleton et al., 1991; Dietschy and Turley, 2004; Miller and Chacko, 2004; Pauli-Magnus et al., 2005). The endogenous mechanism for eliminating potentially toxic sterols in the liver is by converting them to bile acids. Amphiphilic bile acids solubilize cholesterol and eliminate it in bile (Chiang, 2003). Bile acids are also important for absorption of fat-soluble vitamins, A, D, E, and K. Because accumulation of bile salts in liver is also toxic and can lead to interhepatic cholestasis, tight regulation of their synthesis and circulation ensures that a nontoxic intracellular level is maintained (Pauli-Magnus et al., 2005). FXR accomplishes this by inducing feedback repression and feed-forward regulatory loops to suppress bile acid synthesis (Eloranta and Kullak-Ublick, 2005). It is not then surprising that targets of LXR and FXR, shown in Figure 5 are largely those associated with bile acid synthesis, efflux transport, lipoprotein metabolism, and fatty acid metabolism. A role for LXR in immune response has also been reported (Castrillo et al., 2003; Joseph et al., 2004).
Diverse Roles of LXR and FXR in Lipid, Bile Acid and Glucose Metabolism One of the most important gene targets of both LXR and FXR is Cyp7a1, which catalyzes the rate-limiting step in the pathway of cholesterol conversion into bile acids. LXR has an inductive effect on the enzyme, while FXR activation is suppressive. Many other transcription factors, mostly nuclear receptors, transcriptionally regulate Cyp7a1 (Lehmann et al., 1997; Parks et al., 1999; Marrapodi and Chiang, 2000; Chen et al., 2001) FXR and LXR also regulate expression of transporters but for differing purposes. LXR modulates reverse transport of cholesterol by up-regulation of ABC transporters. ABCA1 induces apolipoprotein-mediated efflux of cholesterol and lipid-loaded macrophages back to the liver to prevent accumulation on arterial walls as foam cells. Oxysterols induce ABCA1 to a greater extent in the intestines than in the liver (Repa et al., 2000; Singaraja et al., 2001). FXR regulates transport of bile salts between the liver and intestines during enterohepatic circulation. Hepatic efflux involves the bile salt export pump (BSEP, also known as ABCB11). In the intestinal lumen, activation of FXR suppresses sodium-dependent bile salt transporters, effectively decreasing bile acid absorption. Finally, bile salts are reabsorbed from portal circulation into the hepatocyte. Sodium-dependent taurocholate co-transporting peptides (NTCO) and sodium-independent organic anion transporting peptides (OATPs) are responsible for facilitating hepatic uptake. Through tight regulation of these transporters, FXR-activation can prevent bile-acid induced liver toxicity (Kalaany and Mangelsdorf, 2006).
There are few gene expression profiling studies in liver to date that involve LXR or FXR. One study, by Anderson et al. (2004), used oligonucleotide arrays to identify a gene network regulated by RXR, LXR and PPAR
Both LXR and FXR have also been implicated in glucose metabolism. Previous studies show that LXR or FXR activation in liver results in suppression of gluconeogenesis. In fact, one study demonstrated that activation of LXR improves insulin sensitivity of diabetic insulin-resistant rats (Cao et al., 2003). Genomics studies were conducted to further investigate this response and to identify novel LXR-regulated genes in liver, white adipose or brown adipose (Stulnig et al., 2002) that may be involved in glucose synthesis. Mice treated with T0901317 for 7 days exhibited a significant increase in genes associated with sterol biosynthesis/metabolism, lipid metabolism, heme synthesis, and detoxification. An induction, as high as 8-fold, in genes associated with peroxisomes support previous findings that LXR and PPAR |
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Abstract
Nuclear Receptors and Their...
-oxidation of fatty acids (
" and highlighted blue) or oxidative stress (denoted by "
" and highlighted red) were identified by searching for these terms in each proteins’ GO Biological Processes using PathStudio software.
b, Jun and Cmyc, which are thought to be involved in peroxisome proliferator-induced growth and anti-apoptotic effects, were down-regulated in cynomolygus monkey liver (
