Progress 01/01/03 to 09/30/07
Outputs
OUTPUTS: Our studies of the mechanisms regulating pesticide and environmental contaminant metabolism and detoxification have continued in the past year. Models systems have been developed to test if environmental contaminants are ligand activators of the pregnane X receptor (PXR), also known as the steroid and xenobiotic receptor (SXR). PXR is a transcription factor that regulates many key metabolizing enzymes in the liver and small intestine including cytochrome P4503A4 (CYP3A4). A human PXR.1 expression plasmid was transfected into the COS 1 or HepG2 cell lines with a CYP luciferase reporter plasmids. We continued to test if environmental contaminants including phthalates, pyrethroids, endosulfan, polybrominated diphenyl ethers (PBDEs) and the chemotherapeutic drugs paclitaxel and docetaxel induce CYP luciferase reporter activity in a PXR-dependent manner. This model system will allow identification of environmental contaminants that are PXR activators and will provide information
suggesting the mechanisms of compound toxicity. The coding region for PXR.2 isoform has been cloned and we have initiated studies to determine its transcriptional activity compared to PXR.1. We are also investigating the levels and regulation of human PXR variants in freshly isolated human liver cells (hepatocytes), and discovered that PXR levels are induced by glucocorticoids. We are also investigating glucocorticoid induction of PXR protein leads to synergistic induction of CYP3A4 by environmental contaminants in human hepatocytes and a H4IIE rat hepatoma model system. In order to understand the regulation of PXR expression at the molecular level, we are studying the human PXR gene. We continued to investigate the human PXR gene by initiating a number of studies to determine if PXR 1 and 2 are the products of separate mRNAs or if their expression is driven by separate promoters. Studies are continuing using primer extension analysis. Cloning of PXR promoter elements into the pGL4
(Promega) luciferase reporter vector were also started. We are continuing to investigate the PXR.1 and PXR.2 promoter regions and will be investigating the regulation of these promoters. Deletion analysis of the PXR promoters will localize those regulatory elements critical for regulation of PXR expression. These studies are significant because changes in PXR expression can lead to changes in the expression of many key-metabolizing enzymes. Lastly, to serve as an in vivo model, we have recently obtained wild type and PXR knockout or gene ablated mice. Our future studies in these mice will investigate the toxic and metabolic effects of phthalates such as di(2-ethylhexyl) phthalate (DEHP), taxanes, and other environmental contaminants to determine the role of PXR. These studies were done while mentoring Beth Cooper (Masters student), Leslie Tompkins (Ph.D. student), and the undergraduate students Andrew Sarine, Ashley Smith, Evan Brewer, and Peter Thompson.
PARTICIPANTS: Andrew D. Wallace served as PI for this project. Dr. Ernest Hodgson and the late Dr. Randy L. Rose served as collaborators from NCSU. Dr. Gregory DeKrey was a collaborator from University of Northern Colorado. Students included Leslie M. Tompkins (Ph.D), Beth W. Cooper (MS) and the undergraduates Andrew Sarine, Ashley Smith, Evan Brewer, and Peter Thompson all from NCSU.
Impacts
Our studies of the mechanisms regulating pesticide and environmental contaminant metabolism and detoxification have determined that a number of compounds bind PXR and regulate cytochrome P4503A4 (CYP3A4). Using our HepG2 human hepatoma model system we have determined that DEHP, its metabolite MEHP, pyrethroids, endosulfan, polybrominated diphenyl ethers (PBDEs) and the chemotherapeutic drugs paclitaxel and docetaxel induce CYP3A4 luciferase reporter activity in a PXR-dependent manner. PXR.1 and PXR.2 are both activated by these compounds. Further studies have determined that a number of these compounds induce CYP3A4 protein levels and activity in human hepatocytes. Also, PXR protein levels were found to be induced by the glucocorticoid dexamethasone (Dex) in both human hepatocytes and H4IIE cells. We have also determined that synergistic induction of CYP3A4 promoter activity was induced by Dex and DEHP, which was abrogated by GR siRNA. Our studies investigating the
human PXR gene have determined that PXR 1 and 2 are the products of separate mRNAs and their expression are driven by different promoters. These studies utilized primer extension analysis which was a superior method than the previous attempted RACE experiments. Lastly, in collaboration with Dr. Greg DeKrey (University of Northern Colorado) we have determined in vivo that endosulfan significantly reduced sleep times of wild type and human PXR mice treated with a sedative that is a CYP3A substrate. In PXR knockout or gene ablated mice no change in sleep times were observed. This suggests that PXR is critical in the endosulfan induction of metabolizing enzymes.
Publications
- Cooper, B. W., Cho, T. M. and A. D. Wallace (2007). Phthalate Induction of CYP3A4 is Dependent on Glucocorticoid Regulation of PXR Expression. Toxicological Sciences Submitted.
- Tompkins, L. M., Sit, T. L., and Wallace, A. D. (2007). Unique Transcription Start Sites and Distinct Promoter Regions differentiate the Pregnane X Receptor (PXR) isoforms PXR 1 and PXR 2. Drug Metab Dispos Submitted.
- Tompkins, L. M., and Wallace, A. D. (2007). Mechanisms of cytochrome P450 induction. J Biochem Mol Toxicol 21, 176-81.
- Wallace, A.D. and S. A. Meyer. (2007) Hepatotoxicity. In Introduction to Biochemical Toxicology, Fourth Edition. Eds. Robert Smart and Ernest Hodgson. John Wiley and Sons, Inc. New York. Submitted.
- Wallace, A.D. and J. B. Tarnoff. (2007).Nephrotoxicity. In Introduction to Biochemical Toxicology, Fourth Edition. Eds. Robert Smart and Ernest Hodgson. John Wiley and Sons, Inc. New York. Submitted.
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Progress 10/01/05 to 09/30/06
Outputs
Our studies of the mechanisms regulating pesticide and environmental contaminant metabolism and detoxification have continued in the past year. Models systems have been developed to test if environmental contaminants are ligand activators of the steroid and xenobiotic receptor (PXR). PXR is a transcription factor that regulates many key metabolizing enzymes in the liver and small intestine including cytochrome P4503A4 (CYP3A4). In these model systems, a human PXR.1 expression plasmid is transfected into the COS 1 or HepG2 cell lines with a CYP luciferase reporter plasmids. We have established that environmental contaminants including phthalates, chlordane, endosulfan, polybrominated diphenyl ethers (PBDEs) and the chemotherapeutic drugs paclitaxel and docetaxel induce CYP luciferase reporter activity in a PXR-dependent manner. This model system will allow identification of environmental contaminants that are PXR activators and will provide information suggesting the
mechanisms of compound toxicity. We have also determined that a second message is produced from the PXR gene, which we will refer to as PXR.2. The coding region for PXR.2 isoform has been cloned and we have initiated studies to determine its transcriptional activity. We have also initiated to investigate the mRNA and protein levels of the different human PXR isoforms. We have demonstrated that glucocorticoid induction of PXR protein leads to synergistic induction of CYP3A4 by environmental contaminants. We are also investigating the levels and regulation of human PXR variants in freshly isolated human liver cells (hepatocytes), and discovered that PXR levels are induced by glucocorticoids. Additionally, human, rat, and mouse isoforms have been detected with our PXR antibodies in isolated liver samples. Studies are continuing to determine the levels of PXR protein expression after various hormone treatments. In order to understand the regulation of PXR expression at the molecular
level, we are studying the human PXR gene. We have determined that the PXR 1 and 2 are the products of separate mRNAs and their expression is driven by different promoters. We have done 5' rapid amplification of 5' complementary DNA ends (5' RACE), RNase protection assays, and primer extension assays to determine the transcriptional start sites for PXR.1 and PXR.2. Promoter elements for both PXR.1 and human PXR.2 promoter were cloned into the pGL3-basic (Promega) luciferase vector. We are continuing to investigate the PXR.1 and PXR.2 promoter regions and will be investigating the regulation of these promoters by glucocorticoids. Deletion analysis of the PXR promoters will localize those regulatory elements critical for regulation of PXR expression. These studies are significant because changes in PXR expression can lead to changes in the expression of many key-metabolizing enzymes. To serve as an in vivo model, we have recently obtained wild type and PXR knockout or genes ablated
mice. Our future studies in these mice will investigate the toxic and metabolic effects of phthalates such as Di(2-ethylhexyl) phthalate (DEHP), taxanes, and other environmental contaminants to determine the role of PXR.
Impacts
Information from these studies of the Pregnane X receptor (PXR) transcription factor and various environmental contaminants will advance our understanding of the mechanisms involved in protecting organisms from these harmful compounds. By studying the PXR signal transduction pathway findings will further define the mechanisms by which toxicants from environmental and occupational exposures impact human health.
Publications
- Casabar, R. C., Wallace, A. D., Hodgson, E., and Rose, R. L. 2006. Metabolism of endosulfan alpha by human liver microsomes and its utility as a simultaneous in vitro probe for CYP2B6 and CYP3A4. Drug Metab Dispos 34, 1779-85.
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Progress 10/01/04 to 09/30/05
Outputs
Our studies of the mechanisms regulating pesticide and environmental contaminant metabolism and detoxification have continued in the past year. Models systems have been developed to test if environmental contaminants are ligand activators of the steroid and xenobiotic receptor (SXR). SXR is a transcription factor that regulates many key metabolizing enzymes in the liver and small intestine including cytochrome P4503A4 (CYP3A4). In these model systems, a human SXR.1 variant expression plasmid is transfected into the COS 1 or HepG2 cell lines with a CYP 3A4 luciferase reporter plasmid. We have established that environmental contaminants including phthalates, chlordane, endosulfan, polybrominated diphenyl ethers (PBDEs) and the chemotherapeutic drugs paclitaxel and docetaxel induce CYP3A4 luciferase reporter activity in an SXR-dependent manner. This model system will allow identification of environmental contaminants that are SXR activators and will provide information
suggesting the mechanisms of compound toxicity. Studies have been initiated to investigate the mRNA and protein levels of the different human SXR isoforms. Using the SXR antibodies we have produced, rat SXR protein expression was detected in the H4IIE-C3 rat hepatoma cell line, and SXR levels were elevated by glucocorticoid treatment. We have demonstrated that glucocorticoid induction of SXR protein leads to synergistic induction of CYP3A4 by environmental contaminants. We are also investigating the levels and regulation of human SXR variants in freshly isolated human liver cells (hepatocytes), and discovered that SXR levels are induced by glucocorticoids. Additionally, human, rat, and mouse isoforms have been detected with our SXR antibodies in isolated liver samples. Studies are continuing to determine the levels of SXR protein expression after various hormone treatments. In order to understand the regulation of SXR expression at the molecular level, we are studying the human SXR
gene. We have determined that the SXR variants 1 and 2 are the products of separate mRNAs and their expression is driven by two different promoters. A 2 kilobase portion of the human SXR.2 promoter was cloned into the pGL3-basic (Promega) luciferase vector (hSXR2Kb-Luc). Co-transfection of the glucocorticoid receptor (GR) and hSXR2Kb-Luc reporter was determined to repress SXR promoter activity. We are continuing to investigate the SXR.1 and SXR.2 promoter regions and will be investigating the regulation of these promoters by glucocorticoids. Deletion analysis of the SXR promoters will localize those regulatory elements critical for regulation of SXR expression. These studies are significant because changes in SXR expression can lead to significant changes in the expression of many key-metabolizing enzymes including CYP3A4. To serve as an in vivo model, we have recently obtained wild type and SXR knockout or genes ablated mice, and are working to establish breeding colonies. Our future
studies will investigate the toxic and metabolic effects of phthalates such as Di(2-ethylhexyl) phthalate (DEHP), taxanes, and other environmental contaminants in these mice to determine the role of SXR.
Impacts
Information from these studies of the steroid and xenobiotic receptor (SXR) transcription factor and various environmental contaminants will advance our understanding of the mechanisms involved in protecting organisms from these harmful compounds. By studying the SXR signal transduction pathway findings will further define the mechanisms by which toxicants from environmental and occupational exposures impact human health.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
Studies of the mechanisms regulating pesticide and environmental contaminant metabolism and detoxification have been initiated. A model system has been developed to test if environmental contaminants are ligand activators of the steroid and xenobiotic receptor (SXR). SXR is a transcription factor that regulates many key metabolizing enzymes in the liver and small intestine including cytochrome P4503A4 (CYP3A4). In this model system, a human SXR variant 1 expression plasmid is transfected into the COS 1 or HepG2 cell lines with a CYP 3A4 luciferase reporter plasmid. As a positive control, we have established that the antibiotic rifampicin, a known SXR ligand, induces CYP3A4 luciferase reporter activity in an SXR-dependent manner. We have tested numerous environmental contaminants including Di(2-ethylhexyl) phthalate (DEHP), chlordane, endosulfan, and deltrametrin along with the chemotherapeutic drugs taxol and docetaxel. Ongoing studies have determined these compounds
activate human SXR and induce CYP3A4 promoter activity. This model system will allow identification of environmental contaminants that are SXR activators and will provide information suggesting the mechanisms of compound toxicity. Studies have been initiated to investigate the mRNA and protein levels of the three different human SXR isoforms. To add in the testing of the SXR antibody that we developed we are cloning the hSXR variant 2 and variant 3 isoforms. Using our SXR antibody, SXR protein expression has been detected in the H4IIE-C3 rat hepatoma cell line. We are also investigating the levels and regulation of human SXR variants in freshly isolated human liver cells (hepatocytes), and how SXR levels correlate with CYP3A4 protein and activity levels. Additionally, human, rat, and mouse SXR isoforms have been detected with our SXR antibody. We have also undertaken cloning of the mouse and rat SXR isoforms to do comparative studies of species differences in the ability to activate
SXR and induced CYP3A. Studies are ongoing to determine the levels of SXR protein expression after various hormone treatments, and the dynamics of SXR protein turnover. In order to further study the regulation of SXR expression at the molecular level, a 2 kilobase portion of the human SXR promoter was cloned into the pGL3-basic (Promega) luciferase vector (hSXR2Kb-Luc). Within the 2Kb SXR promoter a number of candidate hepatic nuclear factor 4 alpha (HNF4a) regulatory elements were identified. Along with this hSXR2Kb-Luc reporter, an HNF4a expression plasmid was co-transfected into COS1 cells, which resulted in elevated SXR promoter activity. Co-transfection of the glucocorticoid receptor (GR) and hSXR2Kb-Luc reporter was determined to repress SXR promoter activity. Deletion analysis of the SXR promoter will localize those regulatory elements critical for regulation of SXR expression. These studies are significant because changes in SXR expression can lead to significant changes in
the expression of many key-metabolizing enzymes including CYP3A4.
Impacts
Information from these studies of the steroid and xenobiotic receptor (SXR) transcription factor and various environmental contaminants will advance our understanding of theInformation from these studies of the steroid and xenobiotic receptor (SXR) transcription factor and various environmental contaminants will advance our understanding of the mechanisms involved in protecting organisms from these harmful compounds. By studying the SXR signal transduction pathway findings will further define the mechanisms by which toxicants from environmental and occupational exposures impact human health. mechanisms involved in protecting organisms from these harmful compounds. By studying the SXR signal transduction pathway findings will further define the mechanisms by which toxicants from environmental and occupational exposures impact human health.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
Studies of the mechanisms regulating pesticide and environmental contaminant metabolism and detoxification have been initiated. A model system has been developed to test if environmental contaminants are ligand activators of the steroid and xenobiotic receptor (SXR). SXR is a transcription factor that regulates many key metabolizing enzymes in the liver and small intestine. In this model system, a human SXR expression plasmid is transfected into the COS 1 cell line with a cytochrome P450 3A4 luciferase reporter plasmid. As a positive control we have established that the antibiotic rifampicin, a known SXR ligand, induces CYP3A4 luciferase reporter activity in an SXR-dependent manner. We have initiated testing of the pesticides N, N-diethyl-m-toluamide (DEET) and fipronil along with the chemotherapeutic drug taxol. Ongoing studies will determine if these compounds, and other environmental contaminants, activate SXR and induce CYP3A4 expression. This model system will
allow identification of compounds that are SXR activators and, will provide information suggesting the mechanisms of compound toxicity. In our studies of SXR protein and mRNA expression, testing continues of the SXR antibody that we developed. Using this newly developed antibody, SXR protein expression has been detected in H4IIE rat hepatoma cells. Additionally human, rat, and mouse SXR isoforms have been detected with this antibody. Studies are ongoing to determine the levels of SXR protein expression after various hormone treatments, and the dynamics of SXR protein turnover. Data suggests that SXR protein degradation occurs by a protease known as the proteasome. These studies will provide valuable data concerning how the activated SXR receptor induces transcriptional activity and how SXR activation is terminated. In order to further study the regulation of SXR expression at the molecular level, the human SXR promoter will be cloned from human genomic DNA into the pGL3-basic
(Promega) luciferase vector. Using this luciferase reporter plasmid, studies will identify the regulatory elements leading to induction and repression of SXR expression. Deletion analysis of the SXR promoter will localize those regulatory elements critical for regulation of SXR expression. These studies are significant because changes in SXR expression can lead to significant changes in the expression of many key-metabolizing enzymes including CYP3A4.
Impacts
Information from these studies of the steroid and xenobiotic receptor (SXR) transcription factor and various environmental contaminants will advance our understanding of theInformation from these studies of the steroid and xenobiotic receptor (SXR) transcription factor and various environmental contaminants will advance our understanding of the mechanisms involved in protecting organisms from these harmful compounds. By studying the SXR signal transduction pathway findings will further define the mechanisms by which toxicants from environmental and occupational exposures impact human health. mechanisms involved in protecting organisms from these harmful compounds. By studying the SXR signal transduction pathway findings will further define the mechanisms by which toxicants from environmental and occupational exposures impact human health.
Publications
- No publications reported this period
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