Regulation of PXR Expression and Activation by Xenobiotics Induces Human Metabolizing Enzymes

REGULATION OF PXR EXPRESSION AND ACTIVATION BY XENOBIOTICS INDUCES HUMAN METABOLIZING ENZYMES

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

HATCH

Reporting Frequency

Annual

Accession No.

0211853

Grant No.

(N/A)

Cumulative Award Amt.

(N/A)

Proposal No.

(N/A)

Multistate No.

(N/A)

Project Start Date

Oct 1, 2007

Project End Date

Oct 1, 2013

Grant Year

(N/A)

Program Code

[(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Toxicology

Non Technical Summary
The human body is exposed to a wide variety of toxic substances via food intake, inhalation, or absorption by the skin. In response to exposure to xenobiotics from the environment, such as insecticides or herbicides, the body activates defense mechanisms to metabolize and eliminate these potentially harmful compounds. These studies will determine if the biological response is appropriate to toxic chemical expsosure, leading to elimination, or if activation is occurring which could lead to unwanted toxic side effects.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3999	1030	10%
305	5220	1030	10%
305	5220	1150	10%
305	7010	1150	10%
306	3999	1150	10%
314	7010	1150	10%
314	7010	1180	10%
723	3999	1030	10%
723	3999	1150	10%
314	5220	1150	10%

Knowledge Area
314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals; 305 - Animal Physiological Processes; 723 - Hazards to Human Health and Safety; 306 - Environmental Stress in Animals;

Subject Of Investigation
3999 - Animal research, general; 7010 - Biological Cell Systems; 5220 - Pesticides;

Field Of Science
1180 - Pharmacology; 1030 - Cellular biology; 1150 - Toxicology;

Keywords

insecticides

herbicides

pregnane x receptor (pxr)

glucocorticoids

cytochrome p450

xenobiotic metabolism

cyp3a4

phthalates

endosulfan

Goals / Objectives
The objectives of this research project are to identify pharmaceuticals and environmental contaminants such as insecticides, herbicides, and phthalates that are pregnane X receptor (PXR) activators, characterize the dynamics of PXR promoter activity and protein expression, and determine the magnitude of the changes in metabolizing enzymes and their activity. While a number of xenobiotics and environmental contaminants that activate PXR have been identified, the mechanisms controlling PXR expression are largely undetermined. Our findings will further define the mechanisms by which toxicants from environmental and occupational exposures impact human health.

Project Methods
The ability of xenobiotics to activate the PXR signaling pathway and the induction of metabolizing enzymes will be initially studied using appropriate model cell lines. To identify potential PXR activators or ligands, we are constructing a GAL4-human PXR expression plasmid that will be co-transfected with a GAL4-Luciferase reporter into African green monkey kidney cells (COS1). Identified compounds that are shown to activate PXR will be tested using freshly isolated human liver hepatocytes and in vivo using rodent models including wild type mice, PXR knockout mice, and human PXR transgenic mice. To examine if hormones regulate PXR mRNA and protein levels, studies will be done in cell lines, hepatocytes, and rodent models. In order to understand the regulation of human PXR expression at the molecular level, we are studying the PXR gene by identification and characterizing of the PXR promoter region. Also a number of key metabolizing enzymes will be investigated for the magnitude of induction by environmental contaminants that activate PXR in the presence of co-administered hormones. To identify new genes regulated by PXR activation by pesticides and environmental contaminants microarray analysis will be done.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Activities. The ability of pesticides, phthalates, and pharmaceuticals to alter promoter activity of the CYP2B6 and CYP3A4 xenobiotic-metabolizing enzymes was assessed using human and mammalian liver and kidney cell lines. CYP2B6 and CYP3A4 changes in protein levels and enzyme activity by these compounds were determined using freshly isolated human hepatocytes (liver cells). In vivo studies were conducted using wild type and transgenic mice expressing human pregnane X receptor (PXR) (hPXR-mice) and to determine if induction CYP3A enzymatic activity was dependent on PXR expression and if it was species specific. The promoter of the human PXR gene was further analyzed to identify PXR transcriptional start sites and a number of human PXR reporter luciferase plasmids were constructed based on these results. Human PXR-luciferase promoter activity was assessed after co-transfection with liver transcription factor expression plasmids. The pesticide endosulfan, phthalates, and their metabolites were assessed for their ability to alter PXR, CAR, and other receptor activities. Collaborative studies with Ernest Hodgson (North Carolina State) were also undertaken to determine the human variation in metabolism of the pesticides in regards to the ability to make toxic or non-toxic metabolites. Also collaborative studies were undertaken with Dr. Lifang Hou (Northwestern University) to investigate pesticides linked to leukemia and lymphoma development by the NIH Agricultural Health Study (AHS), and we are assessing if these pesticides act via epigenetic mechanisms. Results have been presented at the 2010 Society of Toxicology annual meeting, 2010 International Society for the Study of Xenobiotics annual meeting, and at North Carolina State Undergraduate Research Symposiums. PARTICIPANTS: Dr. Andrew D. Wallace, Dr. Ernest Hodgson, Dr. Edward Croom, Dr. Parikshit Das, Mr. Yan Cao, Ms. Nneze Eluka, Mr. Morgan Miller, Ms. Joonhee Park, Mr. Chad Hunter, Ms. Ivey Rice, Ms. Sindhu Chandramouleeeswaran, Adam Ward, and Ben Lyles. Department of Environmental and Molecular Toxicology, North Carolina State University. Collaborators: Drs. Gregory K DeKrey and Catherine S. Gardiner, School of Biological Sciences, College of Natural and Health Sciences, University of Northern Colorado, Greenly, Colorado. Dr. Lifang Hou Northwestern, Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL. Training and professional development: Graduate student training was provided for Dr. Edward Croom. Undergraduate research training was provided for Ms. Nneze Eluka, Mr. Morgan Miller, Ms. Joonhee Park, Mr. Chad Hunter, Ms. Ivey Rice, Adam Ward, and Ben Lyles. TARGET AUDIENCES: Target audiences that were specifically served by this project's goals include farmers and pesticide applicators. Efforts to deliver knowledge gained by this project include peer-viewed publication of the findings, presentation of findings at local and national scientific meetings, and collaboration with the NC Agromedicine Institute to disseminate findings. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
New findings concerning the ability of phthalates to induce PSA promoter, CYP3A4 promoter, CYP3A4 protein levels and enzymatic activity have extended our earlier results. The mechanisms of induction were investigated using antagonists and siRNAs to block this response by the phthalates and pharmaceuticals determined that the glucocorticoid receptor and PXR expression were critical. The PXR 1 and PXR 2 promoter reporter luciferase plasmids were further studied and PXR 1 promoter activity was found to be highly induced by hepatic nuclear factor 4 alpha (HNF4α) and peroxisome proliferator-activated receptor (PPAR). The ability of the pesticide endosulfan was found to significantly induce CYP3a11 in studies using wild type and transgenic mice. Induction of CYP3A by endosulfan was found to be dependent on PXR based on these studies. Human liver metabolism of the pesticide chlorpyrifos was found to be highly variable with some individuals forming more of a toxic metabolite while other individuals formed more of a non-toxic metabolite.

Publications

Casabar RT, Das PC, DeKrey GK, Gardiner CS, Cao Y, and Wallace AD. 2010. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor. Toxicol Appl Pharmacol. 245(3): 335-43
Croom EL, Wallace AD, and Hodgson E. 2010. Human variation in CYP-specific chlorpyrifos metabolism. Toxicology. 276(3): 184-91.
Tarloff JB and Wallace AD. 2010. Nephrotoxicity. In: A Textbook of Modern Toxicology, Fourth Edition. Ed Hodgson E. John Wiley and Sons, Inc, New York, NY. pp 291-302.
Hannas BR, Wang YH, Baldwin, WS, Li Y, Wallace AD, and LeBlanc, GA. 2010. Interactions of the crustacean nuclear receptors HR3 and E75 in the regulation of gene transcription. Gen Comp Endocrinol. 167(2): 268-7
Wallace AD, Cao Y, Chandramouleeswaran S, and Cidlowski JA. 2010. Lysine 419 targets human glucocorticoid receptor for proteasomal degradation. Steroids. 5(12): 1016-1023.
Wallace AD and Hodgson E. 2010. Chemical and Physiological Effects on Xenobiotic Metabolism. In: A Textbook of Modern Toxicology, Fourth Edition. Ed Hodgson E. John Wiley and Sons, Inc, New York, NY. pp173-211.
Wallace AD and Meyer SA. 2010. Hepatotoxicity. In: A Textbook of Modern Toxicology, Fourth Edition. Ed Hodgson E. John Wiley and Sons, Inc, New York, NY. pp 277-289.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The ability of pesticides, phthalates, and pharmaceuticals to alter promoter activity of the CYP2B6 and CYP3A4 xenobiotic-metabolizing enzymes was assessed using human and mammalian liver and kidney cell lines. CYP2B6 and CYP3A4 changes in protein levels and enzyme activity by these compounds were determined using freshly isolated human hepatocytes (liver cells). In vivo studies were conducted using wild type, transgenic mice expressing human pregnane X receptor (PXR) (hPXR-mice), and PXR knockout (PXR-KO) mice to determine if induction CYP3A enzymatic activity was dependent on PXR expression and if it was species specific. The promoter of the human PXR gene was analyzed to identify PXR transcriptional start sites and a number of human PXR reporter luciferase plasmids were constructed based on these results. Human PXR-luciferase promoter activity was assessed after co-transfection with liver transcription factor expression plasmids. The ability of the pesticide endosulfan, phthalates, and their metabolites were assessed for their ability to alter PXR, CAR, and other receptor activities. Results have been presented at the 2009 Society of Toxicology annual meeting, 2009 International Society for the Study of Xenobiotics annual meeting, and at North Carolina State Undergraduate Research Symposiums PARTICIPANTS: Individuals: Dr. Andrew D. Wallace, Dr. Ernest Hodgson, Dr. Edward Croom, Dr. Parikshit Das, Mr. Yan Cao, Ms. Nneze Eluka, Mr. Morgan Miller, Ms. Joonhee Park, Mr. Chad Hunter, Ms. Ivey Rice, Ms. Jennifer Wong, and Ms. Sindhu Chandramouleeeswaran. Department of Environmental and Molecular Toxicology, North Carolina State University. Collaborators: Drs. Gregory K DeKrey and Catherine S. Gardiner, School of Biological Sciences, College of Natural and Health Sciences, University of Northern Colorado, Greenly, Colorado. Training and professional development: Graduate student training was provided for Dr. Edward Croom. Undergraduate research training was provided for Ms. Nneze Eluka, Mr. Morgan Miller, Ms. Joonhee Park, Mr. Chad Hunter, Ms. Ivey Rice, and Ms. Jennifer Wong. TARGET AUDIENCES: Target audiences that were specifically served by this project's goals include farmers and pesticide applicators. Efforts to deliver knowledge gained by this project include peer-viewed publication of the findings, presentation of findings at local and national scientific meetings, and collaboration with the NC Agromedicine Institute to disseminate findings. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
New findings concerning the ability of phthalates to induce PSA promoter, CYP3A4 promoter, CYP3A4 protein levels and enzymatic activity have extended our earlier results. The mechanisms of induction were investigated using antagonists and siRNAs to block this response by the phthalates and pharmaceuticals determined that the glucocorticoid receptor and PXR expression were critical. The PXR 1 and PXR 2 promoter reporter luciferase plasmids were further studied and PXR 1 promoter activity was found to be highly induced by hepatic nuclear factor 4 alpha (HNF4α) and peroxisome proliferator-activated receptor (PPAR). The ability of the pesticide endosulfan was found to significantly induce CYP3a11 in studies using wild type and transgenic mice. Induction of CYP3A by endosulfan was found to be dependent on PXR based on these studies. CYP2B6 levels were found to be highly variable, but were expressed in children throughout development.

Publications

Wallace A.D. and Hodgson E. 2009. Chemical and Physiological Effects on Xenobiotic Metabolism. A Textbook of Modern Toxicology, Fourth Edition. Ed. Hodgson E. John Wiley and Sons, Inc, New York, NY. Submitted
Wallace A.D. and Meyer S.A.. 2009. Hepatotoxicity. A Textbook of Modern Toxicology, Fourth Edition. Ed. Hodgson E. John Wiley and Sons, Inc, New York, NY. Submitted
Wallace A.D. and Tarloff J.B., Nephrotoxicity. 2009. A Textbook of Modern Toxicology, Fourth Edition. Ed. Hodgson E. John Wiley and Sons, Inc, New York, NY. Submitted.
Croom, E. L., J.C. Stevens, R.N. Hines, A.D. Wallace and E. Hodgson. 2009. Human Hepatic CYP2B6 Developmental Expression: The Impact of Age and Genotype. Biochem Pharmacol. 78: 184-90. PMID: 19464434

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Activities. The ability of pesticides, phthalates, and pharmaceuticals to alter promoter activity of the CYP2B6 and CYP3A4 xenobiotic-metabolizing enzymes was assessed using human and mammalian liver and kidney cell lines. CYP2B6 and CYP3A4 changes in protein levels and enzyme activity by these compounds were determined using freshly isolated human hepatocytes (liver cells). In vivo studies were conducted using wild type, transgenic mice expressing human pregnane X receptor (PXR) (hPXR-mice), and PXR knockout (PXR-KO) mice to determine if induction CYP3A enzymatic activity was dependent on PXR expression and if it was species specific. The promoter of the human PXR gene was analyzed to identify PXR transcriptional start sites and a number of human PXR reporter luciferase plasmids were constructed based on these results. Human PXR-luciferase promoter activity was assessed after co-transfection with liver transcription factor expression plasmids. The ability of the pesticide endosulfan and phthalates were assessed for their ability to alter PXR, CAR, and other receptor activities. Results have been presented at the Society of Toxicology Annual meeting (Seattle, WA) and at North Carolina State Undergraduate Research Symposiums PARTICIPANTS: Individuals: Dr. Andrew D. Wallace, Dr. Ernest Hodgson, Dr. Leslie M. Tompkins, Beth W. Cooper, Mr. Yan Cao, Mr. Peter M. Thompson, Mr. Morgan Miller, Mr. Chad Hunter, Ms. Ashley Smith, Ms. Ivey Rice, Ms. Jennifer Wong, Mr. Evan Brewer, and Ms.Sindu Chandramouleeswaran, Department of Environmental and Molecular Toxicology, North Carolina State University. Collaborators: Drs. Gregory K. DeKrey and Catherine S. Gardiner,School of Biological Sciences, College of Natural and Health Sciences, University of Northern Colorado, Greeley, Colorado. Training or professional development: Graduate student training was provided for Dr. Leslie M. Tompkins and Beth W. Cooper. Undergraduate research training was provided for Mr. Peter M. Thompson, Mr. Morgan Miller, Mr. Chad Hunter, Ms. Ashley Smith, Ms. Ivey Rice, Ms. Jennifer Wong, Mr. Evan Brewer, and Ms. Sindu Chandramouleeswaran. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
New findings concerning the ability of phthalates to induce CYP3A4 promoter, protein levels, and enzymatic activity have extended our earlier results. The mechanisms of induction were investigated using antagonists and siRNAs to block this response by the phthalates DEHP and MEHP,and determined that the glucocorticoid receptor and PXR expression were critical. The transcriptional start sites of the human PXR gene were investigated and it was determined that the PXR isoforms PXR 1 and PXR 2 are unique mRNA transcripts generated from alternative promoters. PXR 1 and PXR 2 promoter reporter luciferase plasmids were generated and PXR 1 promoter activity was found to be highly induced by hepatic nuclear factor 4 alpha (HNF4a). PXR 1 and PXR 2 expression vectors were created and the ability of each isoform to active CYP3A4 promoter activity was assessed in luciferase assays. PXR 1 and PXR 2 both demonstrated similar abilities to induce CYP3A4 promoter activity by a number of different ligands. The ability of the pesticide endosulfan was found to significantly induce CYP3A4 and CYP2B6 in human hepatocytes. Induction of CYP3A by endosulfan was found to be dependent on PXR based on in vitro and in vivo studies.

Publications

Tompkins LM, Sit TL, Wallace AD 2008 Unique transcription start sites and distinct promoter regions differentiate the pregnane X receptor (PXR) isoforms PXR 1 and PXR 2. Drug Metab Dispos 36:923-9.
Cooper BW, Cho TM, Thompson PM, Wallace AD 2008 Phthalate induction of CYP3A4 is dependent on glucocorticoid regulation of PXR expression. Toxicol Sci 103:268-77.
Wallace AD, Meyer SA 2008 Hepatotoxicity. In: Molecular and Biochemical Toxicology, Fourth Edition. Eds Smart RC, Hodgson E. John Wiley and Sons, Inc, New York, NY, pp 671-692.
Tarloff JB, Wallace AD 2008 Biochemical Mechanisms of Renal Toxicity. In: Molecular and Biochemical Toxicology, Fourth Edition. Eds Smart RC, Hodgson E. John Wiley and Sons, Inc, New York, NY, pp 693-724.