2004 Progress Report: Species-Specific Xenobiotic Metabolism Mediated by the Steroid and Xenobiotic Receptor SXR

EPA Grant Number: CR830686
Title: Species-Specific Xenobiotic Metabolism Mediated by the Steroid and Xenobiotic Receptor SXR
Investigators: Blumberg, Bruce
Institution: University of California - Irvine
EPA Project Officer: Louie, Nica
Project Period: January 1, 2003 through December 31, 2005 (Extended to December 31, 2007)
Project Period Covered by this Report: January 1, 2004 through December 31, 2005
Project Amount: $949,986
RFA: Issues in Human Health Risk Assessment (2001) RFA Text |  Recipients Lists
Research Category: Health Effects , Human Health , Human Health Risk Assessment , Health


The overall aim of this research project is to aid in providing a molecular basis for understanding the commonalities and differences in how humans and model animals respond to chemical exposure. We hypothesize that activation of the nuclear receptor steroid and xenobiotic receptor/pregnane X receptor (SXR/PXR) and consequent effects on metabolism is the mechanism underlying the differential susceptibility of humans and laboratory animals to environmental chemicals. The specific objectives of this research project are to: (1) characterize the commonalities and differences in the response of human and rodent SXR/PXR; (2) identify functional differences in the activation and/or regulation SXR/PXR among humans and between commonly used strains of laboratory mice; (3) determine whether the compounds are metabolized in vivo; and (4) identify target genes regulated by SXR/PXR as a response to environmental chemical exposure.

Progress Summary:

During Year 2 of the project, we made progress on Objectives 1, 2, and 4 and identified a new and important research direction, the mutually inhibitory cross-regulation of SXR and nuclear factor-kappa B (NF-κB) signaling pathways. In addition, we have undertaken development of a mouse that is “fully humanized” with respect to SXR. Two peer-reviewed manuscripts were published, and two others are in preparation.

Tissue-Specific Regulation of SXR Target Genes

SXR is expressed at high levels in the liver and intestine where it acts as a xenobiotic sensor that regulates the expression of cytochrome P450 enzymes such as CYP3A4 and CYP2C8; conjugation enzymes such as UGT1A1; and ABC family transporters such as MDR1 and MRP2. SXR is thus a master regulator of xenobiotic clearance, coordinately controlling steroid and xenobiotic metabolism.

As mentioned in the previous annual report, we noted that several SXR activators showed tissue-specificity in their ability to induce SXR target genes. In our studies of SXR activation by natural and xenobiotic compounds, we discovered that certain forms of vitamin E were good SXR activators. The induction of drug and xenobiotic metabolizing enzymes by dietary compounds may play an important role in the differential susceptibility of people and laboratory animals to chemical insult and suggested to us that this topic warranted further investigation. We tested all eight forms of vitamin E and found that tocotrienols could bind to and activate human SXR, whereas none of the tocopherols could activate SXR or induce expression of SXR target genes. Although tocotrienols were weaker activators of SXR than rifampicin (RIF), they act as selective modulators of the CYP3A4 gene in that they induce expression of CYP3A4 in hepatocytes but not in intestinal cells. Therefore, tocotrienols are a new class of SXR ligands that are able to selectively regulate expression of its target gene-CYP3A4 in different tissues. Tocotrienols induce expression of UGT1A1 and MDR1 in intestinal cells but not in hepatocytes. Therefore we next studied the mechanism through which this tissue-specific activation occurred.

We investigated the expression of known SXR coregulators in two different cell lines, human primary hepatocytes and LS180 intestinal cells. The nuclear receptor corepressor (NCoR) was expressed at similarly high levels in both cell types but LS180 cells have a much lower coactivator/corepressor ratio compared with primary hepatocytes. This may explain the stronger activation we observed in hepatic cells in our transfection assays. However, altering the coactivator/corepressor ratio by steroid receptor coactivator-1, overexpression failed to enhance the ability of tocotrienols to induce CYP3A4. Therefore, the different coactivator/corepressor ratios in hepatic versus intestinal cell lines cannot solely account for the tissue-selective activation of CYP3A4 by SXR that we observed. We next tested the effects of modulating corepressor function.

Because NCoR is expressed at relatively high levels in LS180 cells, we inhibited the endogenous NCoR by transfection of a dominant negative NCoR (DN-NCoR) into this cell line. Transfection of DN-NCoR resulted in enhanced induction of CYP3A4 expression by tocotrienols. However, transfection of DN-NCoR did not further induce UGT1A1 and MDR1 gene expression in response to tocotrienols. This may be caused by the different promoter contexts of UGT1A1 and MDR1 compared to CYP3A4. These results suggest that SXR coregulators, especially NCoR, play important roles in determining tissue-specific effects of selective modulators of SXR, such as tocotrienols. The ability of tocotrienols to regulate SXR target genes in a tissue-specific manner suggests that future development of compounds that selectively activate SXR target genes in certain tissues will be possible.

Drug-drug interactions are a common problem in medical practice, but drug-nutrient interactions are less widely considered when prescribing medications. Although drug-nutrient interactions are not as common as drug-drug interactions, there is evidence to suggest that vitamin supplements can affect the absorption and effectiveness of drugs. Vitamin E is taken daily by more than 35 million people in the United States. Our data suggest that tocotrienols can bind to and activate SXR and induce SXR target gene expression at low micromolar concentrations. Pharmacokinetic studies show that the plasma concentration of tocotrienols can exceed 2000 ng/ml (> 5 mM) following oral administration of a single dose of 300 mg mixed tocotrienols. This concentration may be even higher in the liver. This would be expected to lead to SXR activation in vivo, and therefore to increase CYP3A4 expression in the liver and UGT1A1 and MDR1 expression in the intestine. Therefore, the increased expression of drug metabolizing enzymes by vitamin E would be expected to cause a drug-nutrient interaction.

Identification of SXR Homologs From Other Species

We continued to identify SXR orthologs from other species. In addition to the monkey, dog, quail, and medaka sequences previously identified, we have cloned SXR homologs from zebrafish, fathead minnow, carp, fugu, axolotl, and American alligator. Notably, we identified homologs of both SXR and constitutive androstane receptor (CAR) from carp, which clearly indicates that these receptors diverged during the evolution of bony fishes. Previously it had been thought that SXR and CAR diverged after birds because the chicken xenobiotic receptor, CXR, has characteristics of both SXR and CAR. We also noted that the fish receptors are nearly as divergent from each other as they are from the mammalian receptors. All of our SXR “zoo” have been cloned into pCMX-GAL4. We are undertaking receptor activation assays to determine the similarities and differences among these receptors in how they respond to ligands. This aspect of the project will be completed in Year 3.

Identification of SXR Target Genes

We have continued to treat primary human hepatocytes with SXR activators and perform microarray analysis of the genes induced. To date, we have identified a variety of xenobiotic metabolizing enzymes and are in the process of validating other candidate target genes to determine which are bona fide SXR targets. Other studies in our laboratory have identified target genes for the retinoic acid receptor, so we are confident that this task can be accomplished in Year 3.

Identification of SNPs in Human and Mouse SXR

We designed and constructed a set of primers that can amplify the human SXR gene (in two fragments) and primers that selectively amplify each exon of the gene. It has taken some time to work out the details of genomic sequencing, but this technology is now established in the laboratory and sequencing is proceeding. In parallel with our single nucleotide polymorphism (SNP) sequencing effort, we are constructing plasmids that express all of the known SXR SNPs for testing purposes. The response of these constructs, as well as any SNPs we identify, will be tested against a panel of natural and xenobiotic SXR activators to test our hypothesis that polymorphisms in the receptor can account for some of the individual differences in response to chemicals.

Construction of a Mouse With a “Fully Humanized” SXR Response

An important tool for the understanding of xenobiotic metabolism was the development of the so-called humanized mouse. This animal is deficient in the endogenous PXR and transgenic for the human SXR gene expressed in the liver. This model demonstrates convincingly that SXR is the key regulator of CYP3A expression and that it is the ligand binding domain of this receptor, rather than the DNA-binding domain or target DNA-binding elements that controls the selective activation of target genes in response to species-specific activators. This animal was shown to be sensitive to human-specific activators such as RIF and proposed to be a suitable model for human responses to chemicals. Although this mouse is a valuable tool for some studies, it also has shortcomings that make it unsuitable as a general model for the human xenobiotic response. First, the SXR cDNA is expressed only in the livers of these mice. It is well known that first pass metabolism in the gut is very important in drug metabolism, particularly for orally administered drugs. These first generation humanized mice do not express the transgene or the endogenous gene in the gut. Second, the cDNA is highly expressed as a transgene, obviating any endogenous mechanisms for the feedback regulation of the normal gene. Third, because the cDNA is not expressed from the promoter of the orthologous mouse gene, there will be no alternative promoter usage or splicing as is the case for the endogenous gene. Fourth, we have shown that both the human and mouse genes may play a role in bone development, and it is known that SXR is expressed in kidney and breast tissue, among others. This suggests that the first generation humanized mice will lack appropriate responses in tissues not generally thought of as sites of xenobiotic metabolism, which could lead to erroneous or incomplete results.

To overcome these difficulties, we have collaborated with colleagues in Japan to construct a mouse that is predicted to show a fully humanized response to human SXR activators by knocking in the ligand-binding domain of the human receptor into the mouse gene. The 5' flanking region, promoter and the 3' flanking regions of the mouse gene are all preserved in this construct. The targeted allele includes the first three exons and two introns of the mouse gene. Therefore, we expect that the expression of this gene will be regulated identically to the mouse gene, including any alternative promoter usage or alternative splicing at the 5' end, thereby creating an animal that more faithfully reflects the function of the endogenous gene. Although this receptor is only humanized in the ligand-binding domain, it has been demonstrated that the response of endogenous CYP promoters normally regulated by the SXR pathway faithfully follows the pharmacology of the receptor. It also has been demonstrated that changing four residues in the ligand binding domain of the mouse receptor is sufficient to humanize its response to ligands. We expect that our knock-in animal (hSXRki) will show a response profile to ligands that mirrors human SXR, thus making it a good model for predicting the effects of human exposure to xenobiotic chemicals. It also has the advantage that any SNPs we identify that substantially alter the ligand response can be readily transferred to the mouse model for extensive testing.

Mutually Inhibitory Interactions Between SXR and NF-κB Signaling Pathways

RIF is a macrocyclic antibiotic first used as an antituberculosis agent and now used as a component in the multidrug treatment of a variety of diseases caused by Mycobacterium tuberculosis and its relatives. RIF also is used to treat a wide array of infections caused by Gram-positive and Gram-negative bacteria, Chlamydia, Legionella, and some fungi. RIF therapy is complicated by its propensity to cause drug interactions by inducing hepatic drug metabolizing enzymes such as CYP3A4 through transcriptional activation of SXR. RIF also acts as an immunosuppressant in liver cells and its immunosuppressive role has been well described in humans. We were intrigued by the immunosuppressant effects of RIF, particularly because one of the most widespread phenotypes observed in wildlife exposed to xenobiotic chemicals is an apparently nonspecific inhibition of the immune system that increases susceptibility to parasites and cancers.

Figure 1. Strategy for Generating a Mouse With a Fully-Humanized SXR Response. Top, structure of the wild type mouse gene. The targeting vector fuses the ligand binding domain of the human cDNA to the corresponding place in the mouse gene, in exon 3, just after the DNA-binding domain. The Cre-recombined allele is the final product.

Interestingly, several other pharmaceutical agents such as phenytoin and RU486 also activate SXR and exert immunosuppressive side effects, and it has also long been known that inflammation and infection reduce hepatic CYP expression. We found that activation of SXR by RIF and other agonists antagonizes the activity of NF-κB, a key regulator of inflammation and the innate and adaptive immune responses. The NF-κB family consists of five members, namely p65 or Rel A, Rel B, c-Rel, p50, and p52. NF-κB normally remains in the cytoplasm bound to the inhibitory protein IκB. Activating signals, such as proinflammatory cytokines, reactive oxygen species, and viral products lead to phosphorylation and degradation of IκB, allowing NF-κB to translocate to the nucleus and directly regulate the expression of its target genes. Mice deficient in the SXR ortholog PXR show increased expression of NF-κB target genes in multiple tissues as well as increased inflammation in the small intestine. Not only does SXR inhibit NF-κB activity, but activation of NF-κB inhibits SXR activity and the expression of SXR target genes. Inhibition of NF-κB also enhances the activity of SXR and the expression of its target genes. Thus, the negative cross-talk between SXR and NF-κB not only reveals the possible mechanism underlying the immunosuppressive effects of RIF but also explains the decreased expression of hepatic CYP genes during inflammation or infection, which has been known for decades. These observations reveal SXR’s novel function as a negative mediator of inflammation and immunity and suggest an important relationship between xenobiotic metabolism and the inflammatory and immune responses. We propose that inhibition of NF-κB activity by xenobiotic SXR activators in wildlife plays an important role in the apparent immunosuppression observed.

Future Activities:

In Year 3 of the project, we aim to accomplish the following:

  • Publish a manuscript detailing the cross-talk between SXR and NF-κB.
  • Finish SNP sequencing in human and mouse SXR and test the individual receptors for their ability to respond to a panel of SXR activators.
  • Conduct receptor activation assays to determine the commonalities and differences in the response of the “SXR zoo” to ligands.
  • Complete the identification and validation of SXR target genes.
  • Validate the fully humanized SXR mouse as related to how it responds to test ligands and how primary hepatocytes metabolize known human and rodent-selective activators.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 39 publications 8 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article Zhou C, Tabb MM, Sadatrafiei A, Grün F, Blumberg B. Tocotrienols activate the steroid and xenobiotic receptor, SXR, and selectively regulate expression of its target genes. Drug Metabolism and Disposition 2004;32(10):1075-1082. CR830686 (2004)
CR830686 (2006)
not available

Supplemental Keywords:

xenobiotic, species-specific response, pollution, pesticide,, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Biochemistry, Risk Assessment, chemical exposure, xenobiotics, steroids, animal model, human exposure, exposure assessment, human health risk

Progress and Final Reports:

Original Abstract
  • 2003 Progress Report
  • 2005
  • 2006 Progress Report
  • Final