Potential Cross-Talk between the Aryl Hydrocarbon Receptor (AHR) and the Constitutive Androstane Receptor (CAR) Public

Abstract

Potential Cross-Talk between Aryl Hydrocarbon Receptor (AHR) and the Constitutive Androstane Receptor (CAR)

Cytochrome P450s (CYPs) are a major class of hemoproteins responsible for the oxidation of endogenous and exogenous compounds as well as the metabolism of over 70% of commercially available drugs. The induction of different CYPs is regulated by several transcription factors. The aryl hydrocarbon receptor (AHR), the constitutive androstane receptor (CAR), and the pregnane X receptor (PXR) are three ligand-activated transcription factors that regulate the transcription of cytochrome P450s-CYP1A, CYP2B, and CYP3A respectively. While there has been extensive evidence of the cross-talk between CAR and PXR there has been very limited proof of cross-talk between AHR and CAR. Using combinations of AHR, CAR, and PXR agonists in primary rat hepatocytes we studied gene expression at the RNA and protein level for CYP1A, CYP2B, and CYP3A. This work provides evidence of an AHR and CAR cross-talk phenomenon. Specifically, AHR activation suppresses phenobarbital-mediated induction of CYP2B at the RNA and protein level with a significant attenuation at 48 hours. This down-regulation of CY2B occurred with two structurally different AHR agonists, beta-napthoflavone (β-NF) and 3-methylcholanthrene (3MC). Additionally, the attenuation of CYP2B expression is via an AHR: CAR interaction and not an AHR: PXR interaction. Attempts to chemically inhibit AHR activation with the AHR antagonist 6, 2, 4-trimethoxyflavone (TMF) were futile, suggesting ligand specific antagonism. When investigating CAR translocation during exposure to AHR agonists and PB, initial experiments showed no change in the amount of nuclear CAR. However, future studies are needed to further elucidate CAR translocation during co-treatment. Evidence gained from this study as well as other studies looking at cross-talk and/or repression of nuclear factors can be used to speculate how AHR inhibits CAR's regulation of CYP2B. Finally, understanding any potential cross-talk between AHR and CAR could reveal any potential for drug-drug interactions as well as further our understanding of the regulation of cytochrome P450s via nuclear factors.

Table of Contents

Table of Contents
ABSTRACT...iv
ACKNOWLEDGEMENTS...vi
1. INTRODUCTION...1

1.1 Cytochrome P450 and Xenobiotic Clearance...1

1.2 Aryl Hydrocarbon Receptor (AHR)...2

1.3 Constitutive Androstane Receptor (CAR)...4

1.4 Pregnane X Receptor (PXR)...7

1.5 Drug-Drug Interactions...8

1.6 Proposal...9

2. MATERIALS AND METHODS

2.1 Materials and Reagents...11
2.2 Preparation of Rat Hepatocytes and Cell Culture...11
2.3 RNA Extraction and Reverse Transcriptase Real-time qPCR...12
2.4 Protein Extraction and Immunoblotting...13
2.5 Preparation of Nuclear and Cytosolic Fractions for CAR blotting...13
2.6 Statistical Analysis...13

3. RESULTS

3.1 Effect of β-NF on CYP2B1 RNA levels after pretreatment with PB...15
3.2 Effect of CAR agonists with or without β-NF on CYP2B and CYP1A mRNA...15

3.3.1 Concentration dependence of Exogenous AHR agonist β-NF's Effects on CYP2B1 and CYP1A2 Gene Expression...17
3.3.2 Effect of the Exogenous AHR ligand 3MC on CYP2B and CYP1A Gene Expression...23
3.3.3 Effect of Endogenous AHR ligand KA on CY2B and CYP1A Gene Expression...26
3.4.1 CYP3A2 mRNA Expression...29
3.5 PXR and AHR: Effect of AHR agonist on CYP3A2 expression induced by PXR ligands...31

3.5.1 Dexamethasone...31
3.5.2 Pregnenolone-16α-carbonitrile (PCN)...31

3.6 CAR Translocation...34
3.7 Chemical Inhibition of AHR with TMF...36

4. Discussion and Conclusions...38
5. References...45-52

Index of Figures
Figure 1.1: Nomenclature for Cytochrome P450s...2
Figure 1.2a: Mechanism of aryl hydrocarbon receptor activation and subsequent transcription of target genes...3
Figure 1.2b: Structures of AHR agonist beta-napthoflavone (β-NF) and 3-methylcholanthrene (3MC)...4
Figure 1.3a: Mechanism of constitutive androstane receptor (CAR) activation directly by TCPOBOP and indirectly by PB...6
Figure 1.3b: Proposed mechanisms of indirect activation of CAR by phenobarbital (PB)...6
Figure 1.4a: Structure of PXR agonists' dexamethasone (DEX) and pregnenolone 16-alpha-carbonitrile (PCN)...7
Figure 1.4b: Overlap between CAR and PXR...8
Figure 3.1: of PB and β-NF co-treatment after 48 hr pretreatment with PB on CYP2B mRNA levels...16
Figure 3.2a: Effect of simultaneous treatment with β-NF on CYP2B1 induction by CAR activators...18
Figure 3.2b: Effect of simultaneous treatment with β -NF on CYP2B2 induction by CAR activators...19
Figure 3.2c: Effect of simultaneous treatment with β-NF on CYP1A2 induction by CAR activators...19
Figure 3.3a: Dose-dependent Response of β-NF on CYP1A2 mRNA expression...21
Figure 3.3b: Effect of concomitant treatment with 1 mm PB and increasing concentrations of β-NF on CYP2B1 mRNA expression...21
Figure 3.3c: Effect of PB and β-NF co-treatment on CYP2B protein expression...22
Figure 3.3d: Dose-dependent response of 3MC on CYP1A2 mRNA expression...24
Figure 3.3e: Effect of concomitant treatment with 1 mm PB and increasing concentrations of 3MC on CYP2B1 mRNA expression...24
Figure 3.3f: Effect of PB and 3MC co-treatment on CYP2B protein expression...25
Figure 3.3g: Dose-dependent response of KA on CYP1A2 mRNA expression...27
Figure 3.3h: Effect of concomitant treatment with 1 mm PB and increasing concentrations of KA on CYP2B1 mRNA expression...27
Figure 3.3i: Effect of PB and KA co-treatment on CYP2B protein expression...28
Figure 3.4a: Effect of simultaneous treatment with β-NF on CYP3A2 induction by CAR activators...30
Figure 3.4b: Effect of treatment with AHR agonist and PB on CYP3A2 mRNA Expression...30
Figure 3.5a: Effect of 20 µM dexamethasone (PXR ligand) with or without 10 µM β-NF (AHR ligand) on CYP3A2...32
Figure 3.5b: Effect of 10 µM PCN (PXR ligand) with or without 10 µM β-NF (AHR ligand) on CYP3A2...33
Figure 3.6: PB-dependent CAR translocation with β-NF co-treatment...35
Figure 3.7a: 6,2,4-trimethoxyflavone (TMF) an antagonist of AHR...36
Figure 3.7b: Effect of TMF and β-NF co-treatment on CYP1A2 mRNA expression...37
Figure 3.7c: Effect of TMF and 3MC co-treatment on CYP1A2 mRNA expression...37
Figure 4: Plausible Mechanism for attenuation of CYP2B...44
Scheme 3.1: Experimental design to observe the effect of β-NF on CYP2B1 mRNA levels after pretreatment with PB...16
Scheme 3.2: Experimental Design for simultaneous co-treatment with CAR activators with or without β-NF...18
Scheme 3.3: AHR agonists were treated with or without 1 mM PB to see their relative potencies...20
Scheme 3.7: Experimental Design for AHR antagonist (TMF) coupled with AHR agonist (3MC or β-NF)...36
Table 1: Primer sequences used...12

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	BA
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Health Sciences, Pharmacology
Mot-clé	drug metabolism Nuclear Receptors Cytochrome P450s
Committee Chair / Thesis Advisor	Morgan, Edward T, Emory University
Committee Members	Liotta, Dennis C, Emory University Ortlund, Eric, Emory University

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