2003 Progress Report: Species-Specific Xenobiotic Metabolism Mediated by the Steroid and Xenobiotic Receptor SXREPA 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, 2003 through December 31, 2004
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 of 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.
During Year 1 of the project, we made progress on Objectives 1 and 4. The mammalian xenobiotic response is mediated primarily by two broad-specificity sensors: the orphan nuclear receptors SXR/PXR and constitutive androstane receptor. SXR/PXR plays a critical role in the regulation of phase I (CYP), phase II (conjugating), and phase III (ABC family transporters) detoxifying enzymes, coordinately regulating steroid, drug, and xenobiotic clearance in the liver and intestine. SXR/PXR is activated by a diverse group of steroid hormones, dietary compounds (e.g., phytoestrogens), prescription drugs (e.g., taxol, rifampicin), medicinal herbs (e.g., St. John’s Wort), and xenobiotics (e.g., organochlorine pesticides) that are all substrates for the SXR-induced enzymes. Because SXR/PXR exhibits species-specific differences in its response to phytoestrogens, clinically important drugs, and xenobiotics, we infer that the metabolism of some compounds will be correspondingly different between humans and model organisms.
Species-Specific Antagonism of SXR by PCBs
We extended our preliminary results regarding the differential activation of SXR and PXR by polychlorinated biphenyls (PCBs) and xenobiotic compounds. We tested a variety of PCBs for their ability to activate human, rabbit, rat, and mouse receptors and noted a distinct species preference. We found that more highly chlorinated PCB congeners were able to activate rodent PXR but not human SXR. These same PCBs were able to bind directly to human SXR and antagonize its activation and target gene induction in primary human hepatocytes and LS180 human colon carcinoma cells. Using SXR transcriptional activation and antagonism data, a predictive molecular model for PCB binding to SXR was formulated, tested, and used to identify additional antagonistic PCBs. A total of 15 PCBs were identified that activate rodent PXR but antagonize human SXR. These PCBs include some of the most abundant congeners identified in human tissues (PCB 138, 153) and are among the most persistent and abundant congeners in human tissues and show striking differences in their potential to be metabolized in rodents and humans. PCBs strongly induce their own metabolism in rodents, whereas our findings suggest that they antagonize their own metabolism, and that of other xenobiotics, dietary compounds, and endogenous steroids, in humans. Thus, the use of rats to predict the risk of human exposure to these PCBs or mixtures that contain them likely will lead to erroneous conclusions. These findings suggest more broadly that the literature concerning the effects of xenobiotic chemicals and the attendant risks for human and wildlife populations will need to be reevaluated where the behavior of xenobiotic sensors differs across species. The differential metabolism of drugs, xenobiotics, and dietary compounds mediated by SXR/PXR provides both an explanation and a molecular tool with which to address the often contradictory and controversial literature on the effects of dietary and environmental chemicals on human health.
We have provided an extensive set of activation data to our collaborator Dr. William Welsh of the University of Medicine and Dentistry of New Jersey for quantitative structure-activity relationships (QSAR) model building. The training set included compounds that activated the receptors across species as well as species-specific agonists and antagonists. Dr. Welsh and his colleagues will use this dataset to develop QSAR models of how SXR and PXR are activated and antagonized by xenobiotic compounds. The first fruit of these studies is included in the PCB paper published in Environmental Health Perspectives.
Identification of SXR Target Genes
We have begun treating primary human hepatocytes with SXR activators and collecting RNA for microarray analysis of SXR target genes. These studies are expected to continue in Year 2 of the project.
Identification of SXR Homologs From Other Species
We developed PCR primers that can detect SXR homologs from all known species and used these to identify SXR from several species not previously published. In collaboration with Dr. Taisen Iguchi at the National Institute of Basic Biology, Okazaki, Japan, we obtained cDNA libraries prepared from several species of monkeys (Japanese monkey, Crab eating monkey, marmoset), dog (Beagle), Japanese quail, and medaka. We cloned SXR orthologs from these animals and used 3' rapid amplification of cDNA ends to obtain the entire ligand binding domain sequences. These will be cloned into pCMX-GAL4 for activation analysis in transfected cells.
Tissue Specific Activation of SXR
During our testing of xenobiotic compounds described above, we noted that many compounds did not equally induce SXR target genes in different tissues. For example, bisphenol A could induce the expression of the SXR target gene CYP3A4 in liver cells but not in intestinal LS-180 cells where it induced the expression of the phase II gene UGT1A1 and the phase III gene MDR1. We tested this further and noted a general pattern in which strong SXR activators could induce genes of all three phases of xenobiotic metabolism in intestine, whereas only phase I genes such as CYP3A genes were induced in liver. Weaker SXR activators could only induce phase II and phase III genes in intestine. We currently are investigating the mechanism underlying this tissue-specific regulation of target genes and hope to submit a manuscript in Year 2 describing our findings.
During Year 2 of the project, we aim to accomplish the following:
- Publish a manuscript describing the tissue-specific regulation of SXR target genes.
- Design PCR and sequencing primers and develop conditions for genomic sequencing of SXR from human blood samples and PXR from mouse liver samples.
- Begin sequencing to detect single nucleotide polymorphisms (SNPs) and recreate any SNPs detected in the human and mouse receptors.
- Continue identifying SXR homologs from model organisms of interest and begin testing these for transcriptional activation by known and suspected SXR agonists.
- Continue analysis of SXR target genes by microarray analysis.
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|
||Tabb MM, Kholodovych V, Grün F, Zhou C, Welsh WJ, Blumberg B. Highly chlorinated PCBs inhibit the human xenobiotic response mediated by the steroid and xenobiotic receptor (SXR). Environmental Health Perspectives 2004;112(2):163-169.||