The Pennsylvania State University

The Graduate School

Department of Veterinary and Biomedical Science

IDENTIFICATION OF ENDOGENOUS MODULATORS FOR THE

ARYL HYDROCARBON RECEPTOR

A Thesis in

Genetics

by

Christopher R. Chiaro

© 2007 Christopher R. Chiaro

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

December, 2007

The thesis of Christopher R. Chiaro was reviewed and approved* by the following:

Gary H. Perdew John T. and Paige S. Smith Professor in Agricultural Sciences Thesis Advisor Chair of Committee

C. Channa Reddy Distinguished Professor of Veterinary Science

A. Daniel Jones Senior Scientist Department of Chemistry

John P. Vanden Heuvel Professor of Veterinary Science

Richard Ordway Associate Professor of Biology Chair of Genetics Graduate Program

*Signatures are on file in the Graduate School

iii ABSTRACT

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor capable of being regulated by a structurally diverse array of chemicals ranging from environmental carcinogens to dietary metabolites. A member of the basic helix-loop- helix/ Per-Arnt-Sim (bHLH-PAS) super-family of DNA binding regulatory proteins, the

AhR is an important developmental regulator that can be detected in nearly all mammalian tissues. Prior to ligand activation, the AhR resides in the cytosol as part of an inactive oligomeric protein complex comprised of the AhR ligand-binding subunit, a dimer of the 90 kDa heat shock protein, and a single molecule each of the immunophilin like X-associated protein 2 (XAP2) and p23 proteins. Functioning as chemosensor, the

AhR responds to both endobiotic and xenobiotic derived chemical ligands by ultimately directing the expression of metabolically important target genes. Primarily responsible for mediating the toxicological and biological effects of dioxin and other environmentally persistent carcinogens, the AhR was originally characterized for its role in orchestrating the adaptive metabolic response to xenobiotic compounds. Recently, however, the AhR has been identified as performing a critical role in a number of physiologically important life functions, including proper embryonic and liver development, immune system homeostasis, resolution of fetal vasculature, and maintenance of normal cardiac physiology. Currently, the most potent AhR agonists to be identified are of synthetic origin, yet an increasing number of natural compounds have been shown to activate the receptor. Although essential roles for the AhR in normal cellular biology have already been established and continue to evolve, no high iv affinity physiologically relevant endogenous ligand has been identified. Therefore, the

ultimate goal of this research project was to identify such ligands. The initial data

presented in this thesis confirms the presence of a putative endogenous ligand(s) for the

AhR in the CV-1 cell line, while demonstrating the existence of an AhR regulated

feedback mechanism functioning to control putative endogenous ligand levels. Derived

from the kidney epithelium of the African green monkey, the CV-1 cell line is an

immortalized cell culture line exhibiting minimal AhR expression. Consequentially, the

level of AhR-regulated cytochrome P450 metabolism is also compromised allowing for

subsequent accumulation of cellular metabolites, including potential intracellular

endogenous ligands for the AhR. However, the ectopic expression of AhR-regulated

cytochrome P450s from the 1A or 1B families effectively reduced the high level of

constitutive AhR activity observed in CV-1 cells. Meanwhile cytochrome P450 2E1, an

isoform not regulated by AhR, exhibited no significant effect. Furthermore, extracts of lung tissue prepared from Ahr-null mice clearly revealed, by the increased AhR activation potential compared to “wild-type” mice, the accumulation of an endogenous ligand for the AhR. Coupled with the high level of AhR-dependent CYP1A1 constitutive activity normally seen in lung, these observations support the existence, at least in mouse lung tissue, of an auto-regulatory loop between the AhR and CYP1A1

functioning to modulate endogenous ligand levels. Presented in subsequent chapters are

results identifying and characterizing the ability of several eicosanoid molecules to

activate AhR signaling. For instance, 12(R)-hydroxy- 5(Z),8(Z),10(E),14(Z)-

eicosatetraenoic acid (12(R)-HETE) and 5(S), 12(R)-dihydroxy-6(E),8(E),10(E),14(Z)-

eicosatetraenoic acid (6-trans-LTB4), two potent pro-inflammatory metabolites of v arachidonic acid, were discovered as indirect activators of the AhR, capable of activating AhR signaling, but failing to bind of the receptor. Surprisingly, other structurally similar isomers, such as 12(S)-HETE and LTB4 respectively, did not activate

AhR signaling. In addition, the 5,6- and 14,15- positional isomers of leukotriene A4

(LTA4) along with various dihydroxyeicosatetraenoic acid (DiHETE) metabolites were

also identified as potent activators of the AhR. Furthermore, conclusive evidence is

presented demonstrating that 5,6-DiHETE isomers, cellular metabolites of 5,6-LTA4, can serve as endogenous ligands for the AhR, capable of directly binding and transforming the receptor to its DNA binding form. Although several of these eicosanoid molecules function as direct ligands for the receptor, others appear to activate the AhR through an indirect mechanism. Nevertheless, lipoxygenase metabolites are physiologically important bioactive lipid mediators that now comprise an exciting new class of endogenous modulators for the AhR.

vi TABLE OF CONTENTS

LIST OF FIGURES ...... ix

LIST OF TABLES...... xii

ABBREVIATIONS ...... xiii

ACKNOWLEDGEMENTS...... xv

CHAPTER 1 ...... 1

INTRODUCTION ...... 2

1.1 THE ARYL HYDROCARBON (AH) RECEPTOR...... 2 1.1.1 Structural features of the AhR...... 3 1.1.1.1 The basic and helix-loop-helix domains ...... 3 1.1.1.2 The Per-Arnt-Sim (PAS) domain...... 5 1.1.1.3 The hsp90 and ligand binding domain ...... 6 1.1.1.4 The transactivation domain ...... 6

1.2 THE AH RECEPTOR SIGNAL TRANSDUCTION PATHWAY...... 7 1.2.1 The un-liganded AhR core complex...... 10 1.2.2 Transformation of the Ah receptor...... 10 1.2.2.1 Ligand binding ...... 11 1.2.2.2 Translocation of the AhR core complex ...... 11 1.2.2.3 AhR and ARNT Heterodimerization and DNA Binding ...... 12

1.3 LIGANDS FOR THE AH RECEPTOR...... 13 1.3.1 Halogenated aromatic hydrocarbons ...... 13 1.3.2 Polycyclic aromatic hydrocarbons ...... 17 1.3.3 Dietary ligands...... 21 1.3.4 Endogenous ligands...... 25

1.4 AHR MEDIATED BIOLOGICAL RESPONSES ...... 30 1.4.1 The AhR regulates xenobiotic metabolism ...... 30 1.4.1.1 AhR regulation of phase I enzymes ...... 31 1.4.1.2 AhR regulation of phase II enzymes ...... 37 1.4.2 The AhR mediates the carcinogenic and toxicological response to environmental pollutants...... 42

vii 1.5 PHYSIOLOGICAL ROLES FOR THE AHR...... 45 1.5.1 AhR influences normal vascular development...... 45 1.5.2 AhR influences cardiac development...... 48

1.6 EICOSANOIDS...... 49 1.6.1 Overview ...... 49 1.6.2 Arachidonic acid cascade ...... 51 1.6.3 Cyclooxygenase Metabolites...... 54 1.6.4 Lipoxygenase Metabolites...... 55 1.6.4.1 Arachidonate 5-LOX metabolites ...... 57 1.6.4.2 Arachidonate 8-LOX metabolites ...... 66 1.6.4.3 Arachidonate 12-LOX metabolites ...... 67 1.6.4.4 Arachidonate 15-LOX metabolites ...... 76 1.6.5 Cytochrome P450 monooxygenase metabolites...... 83

1.7 OBJECTIVES AND SIGNIFICANCE OF RESEARCH ...... 84 1.7.1 Identification of an endogenous ligand(s) for the Ah receptor...... 85 1.7.2 Characterization of the endogenous modulator responsible for the elevated AhR activity in CV-1 cells...... 85 1.7.3 Investigate the potential auto-regulatory pathway controlling AhR activity in CV-1 cells through modulation of endogenous ligand levels...... 86

CHAPTER 2 ...... 88

CHARACTERIZATION OF A PUTATIVE ENDOGENOUS LIGAND FOR THE AH RECEPTOR IN CV-1 CELLS...... 88

2.1 ABSTRACT ...... 89 2.2 INTRODUCTION ...... 90 2.3 EXPERIMENTAL PROCEDURES...... 94 2.4 RESULTS...... 104 2.5 DISCUSSION...... 128

CHAPTER 3 ...... 134

THE EICOSANOID METABOLITE, 12(R)-HETE, IS AN ACTIVATOR OF THE AH RECEPTOR ...... 134

3.1 ABSTRACT ...... 135 3.2 INTRODUCTION ...... 137 3.3 EXPERIMENTAL PROCEDURES...... 140 3.4 RESULTS...... 146 3.5 DISCUSSION...... 163 viii CHAPTER 4 ...... 167

5,6-DIHETE ISOMERS ARE ENDOGENOUS LIGANDS FOR THE AH RECEPTOR...... 167

4.1 ABSTRACT ...... 168 4.2 INTRODUCTION ...... 170 4.3 EXPERIMENTAL PROCEDURES...... 173 4.4 RESULTS...... 178 4.5 DISCUSSION...... 208

Summary and Conclusions ...... 213

References...... 222

ix LIST OF FIGURES

Figure 1.1: The aryl hydrocarbon receptor ...... 4

Figure 1.2: The aryl hydrocarbon receptor signal transduction pathway ...... 9

Figure 1.3: Halogenated aromatic hydrocarbon (HAH) ligands of the AhR...... 15

Figure 1.4: Polycyclic Aromatic Hydrocarbon (PAH) Ligands of the AhR...... 20

Figure 1.5: Dietary ligands of the AhR...... 22

Figure 1.6: Endogenous ligands for the AhR...... 28

Figure 1.7: The archidonic acid cascade...... 53

Figure 1.8: The 5-Lipoxygenase Pathway ...... 59

Figure 1.9: The Structures of Some Common Cysteinyl Leukotrienes...... 62

Figure 1.10: Hydrolysis products of leukotriene A4 (LTA4) ...... 64

Figure 1.11: Hydroxyeicosatetraenoic acids (HETEs) ...... 75

Figure 1.12: The Structures of Some Common Lipoxin Molecules...... 80

Figure 1.13: Summary of CV-1 Cell Line Characteristics and Initial Project Hypothesis ...... 87

Figure 2.1: Decreased AhR Activity Results from Expression of AhR-Regulated Cytochrome P450 Isoforms in CV-1 Cells...... 106

Figure 2.2: No Reduction in AhR Activity Results from the Expression of a Control P450 Isoform in CV-1 Cells...... 108

Figure 2.3: Tetramethoxystilbene (TMS) Inhibition of CYP1B1 Rescues AhR- Mediated Transcriptional Activity...... 110

Figure 2.4: The Effect of 2,4,3’5’-tetramethoxystilbene(TMS), a CYP1B1 Specific Inhibitor, on AhR Activity...... 112

Figure 2.5: Organic Extractions of CV-1 Cytosol Activate the AhR...... 116

Figure 2.6: Organic Extracts of CV-1 Cytosol Transform the AhR into its DNA- Binding Form...... 118

Figure 2.7: Digestion of CV-1 Cytosolic Protein using Proteinase K Treatment...... 120 x Figure 2.8: Protease Treatment of CV-1 Cytosol Does Not Alter DRE-Driven Luciferase Activity ...... 121

Figure 2.9: HPLC Fractionation of Putative Endogenous AhR Ligands from CV-1 Cells ...... 123

Figure 2.10: Examining the Level of Constitutive Ah Receptor Activity in Various Tissues from AhR+/+ and AhR-/- Mice...... 125

Figure 2.11: Comparing the Levels of CYP1A1 and CYP1B1 mRNA in Various Tissues from AhR+/+ and AhR-/- Mice ...... 127

Figure 2.12: Diagrammatic Representation of the Proposed AhR Auto-regulatory Mechanism...... 129

Figure 3.1: 12(R)-HETE Activates the AhR in Reporter Based Biological Activity Assays...... 147

Figure 3.2: Assessing the Biochemical Purity and Integrity of 12(R)-HETE ...... 149

Figure 3.3: Mass Spectrometric Analysis of 12(R)-HETE...... 151

Figure 3.4: 12(R)-HETE Fails to Transform the AhR into its DNA Binding Form....154

Figure 3.5: 12(R)-HETE Does Not Compete for AhR Binidng ...... 155

Figure 3.6: Metabolites of 12(R)-HETE Fail to Demonstrate AhR Activity ...... 157

Figure 3.7: 12(R)-HETE Can Activate AhR Target Genes in Multiple Human Cell Lines...... 159

Figure 3.8: RNA Interference (RNAi) Technology Demonstrates Effective Suppression of AhR Expression ...... 161

Figure 3.9: 12(R)-HETE Modulates AhR Signaling in a Receptor Dependent Manner...... 162

Figure 4.1: Leukotriene A4 (LTA4) Isomers Activate the AhR in Reporter Based Biological Activity Assays ...... 179

Figure 4.2: Enzymatically Derived Metabolites of Leukotriene A4 Fail to Activate the AhR in Biological Activity Assays...... 182

Figure 4.3: All trans Isomers of LTB4 Can Activate the AhR in Biological Activity Assays...... 185 xi

Figure 4.4: All trans Isomers of LTB4 Are Incapable of Directly Binding the AhR...... 187

Figure 4.5: Epimers of 5,6-DiHETE Can Activate AhR Signaling in Biological Activity Assays...... 190

Figure 4.6: 5,6-DiHETE Epimers Can Induce AhR Transformation and Heterodimerization ...... 192

Figure 4.7: Epimers of 5,6-DiHETE Are Capable of Directly Binding the AhR...... 194

Figure 4.8: Aged Preparations of 5(S),6(S)-DiHETE Demonstrate an Increased Ability to Activate the AhR in Biological Activity Assays...... 196

Figure 4.9: Aged Preparations of 5,6-DiHETE Exhibit an Enhanced Binding Affinity for the AhR ...... 198

Figure 4.10: Inactive Preparations of 5,6-LTA4 Methyl Ester Acquire AhR Activity Following Short-Term Storage at Elevated Temperature...... 201

Figure 4.11: The 11-trans 5(S),6(R)-DiHETE Isomer Produces a Comparatively Stronger Induction of Reporter Gene Activity...... 204

Figure 4.12: All trans 5(S),6(R)-DiHETE Exhibits an Increased Binding Affinity for the AhR ...... 206

xii LIST OF TABLES

Table 1-1: Biochemical Properties of 12-lipoxygenase Enzymes ...... 68

Table 4-1: Summary of Bioactive Lipid Modulators for the AhR...... 221

xiii ABBREVIATIONS

AA, arachidonic acid AhR, Aryl hydrocarbon receptor α-MEM, α-minimal essential medium ARNT, aryl hydrocarbon receptor nuclear translocator B[a]P, benzo[a]pyrene bHLH, basic helix-loop-helix CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid COPD, chronic obstructive pulmonary disorder COX, cyclooxygenase CYP, cytochrome P450 Cys-LT, cysteinyl leukotriene DiHETE, dihydroxyeicosatetraenoic acid DiHETrE, dihydroxyeicosatrienoic acid DMSO, dimethyl sulfoxide DRE, dioxin responsive element DTT, dithiothreitol EDTA, ethylene diaminetetraacetic acid EMSA, electrophoretic mobility shift assay EPA, eicosapentaenoic acid FBS, fetal bovine serum GR, glucocorticoid receptor GST-Ya, glutathione-S-transferase Ya subunit HAH, halogenated aromatic hydrocarbon HEPES, 4-(2-hydroxyethyl)-1-piperazineeethanesulfonic acid HETE, hydroxyeicosatetraenoic acid HETrE, hydroxyeicosatrienoic acid HpETE, hydroperoxyeicosatetraenoic acid hsp90, heat shock protein 90 I3C, indole-3-carbinol IBD, inflammatory bowel disease LBD, ligand binding domain LOX, lipoxygenase enzyme LT, leukotriene

LTA4, 5(S),6(S)-oxido-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid

LTB4, 5(S),12(R)-dihydroxy-6(Z),8(E),10(E),14(Z)-eicosatetraenoic acid

LTC4, 5(S)-hydroxy-6(R)-(S-glutathionyl)-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid

LTD4, 5(S)-hydroxy-6(R)-(S-cysteinylglycinyl)-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid xiv

LTE4, 5(S)-hydroxy-6(R)-(S-cysteinyl)-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid

MENG, 25mM MOPS, 2mM EDTA, 0.02% NaN3 and 10% glycerol (pH=7.5) NQO1, NAD(P)H:Quinone Oxidoreductase 1 PAH, polycyclic aromatic hydrocarbon PAS, Per-ARNT-Sim PBS, phosphate buffered saline PGD2, prostaglandin D2 PGE2, prostaglandin E2 PGF2, prostaglandin F2 PGG2, prostaglandin G2 PGH2, prostaglandin H2 PGHS, prostaglandin endoperoxide synthase PMNL, polymorphonuclear leukocytes SDS, sodium dodecyl sulfate SRS-A, slow reacting substances of anaphylaxis TAD, transactivation domain TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin TMS, 2, 4, 3′, 5′-tetramethoxystilbene Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine TSDS-PAGE, tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis TXA, thromboxane A2 UGT1A1, uridine diphosphate glucuronosyltransferase XAP2, hepatitis B virus X-associated protein 2 xv ACKNOWLEDGEMENTS

I would first like to thank my advisor and mentor, Dr. Gary Perdew, for the research opportunity I was granted and for his enthusiasm, expertise, and many recommendations during the duration of this thesis project. I also wish to express my deepest gratitude to Dr. A. Daniel Jones for educating me on the fundamentals of mass spectrometry in addition to providing me with valuable guidance and knowledge. Furthermore, I sincerely thank Dr. C. Channa Reddy and K. Sandeep Prahbu for their valuable advice, helpful suggestions, and technical expertise. I thank all current and former members of the Perdew laboratory at Penn State University, especially Rushang D. Patel for contributing RT-PCR analysis and J. Luis Morales for providing critical computer support and for prompting many intriguing discussions. Additionally, I am grateful to all committee members for the many thought provoking and insightful discussions contributing toward my development as a scientist. Perhaps, most importantly, I am thankful to my parents for their valuable advice, guidance, encouragement, patience, and support during this educational process. Finally, I wish to thank all of my friends and family for their continued support, especially Melissa for contributing valuable graphic design skills during the completion of this thesis.

CHAPTER 1

INTRODUCTION

2 1.1 THE ARYL HYDROCARBON (AH) RECEPTOR

The aryl hydrocarbon receptor (AhR) is a ligand activated, basic helix-loop-helix

(bHLH) transcription factor and member of the bHLH /PAS (Per-Arnt-Sim) protein superfamily of DNA binding regulatory proteins [1]. The transcriptional activity of this soluble intracellular receptor is regulated through high affinity interaction with numerous xenobiotic ligands, including many known toxins and carcinogens [2]. As a result, it is responsible for mediating most, if not all, of the diverse toxicological and carcinogenic effects associated with hydrophobic environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) [3]. The vast range of pleiotrophic effects mediated by the AhR include, but are not limited to, dermal toxicity, hepatotoxicity, immunotoxicity, teratogenesis, tumor promotion, and endocrine disruption [4]. The AhR is also believed to play a role in the pathogenesis of cardiovascular disease [5]. All of these seemingly unrelated biological effects appear to be mediated through sustained and/or inappropriate activation of AhR signaling, ultimately resulting in the perturbation of cellular homeostasis.

Albeit structurally distinct, the AhR remains functionally similar in many ways to members of the nuclear hormone receptor superfamily. Mechanistically, in terms of target gene regulation, the AhR closely resembles that of the glucocorticoid receptor

(GR). In addition many of the biochemical and physiochemical properties of the AhR, including molecular mass, sedimentation coefficient, and Stokes radius, are comparable with members of the steroid hormone receptor family [6-8]. 3 1.1.1 Structural features of the AhR

1.1.1.1 The basic and helix-loop-helix domains

Found in a relatively large class of sequence-specific transcriptional regulator proteins is the basic-helix-loop-helix (bHLH) domain. Proteins containing this domain are capable of binding DNA as either homo- or hetero-dimers, with the basic amino acid

(AA) region and HLH domains occupying critical roles in DNA binding and protein dimerization respectively [9]. Both the AhR and its dimerization partner ARNT (aryl hydrocarbon receptor nuclear translocator) contain a bHLH motif located near their

amino terminus (Figure 1.1). The basic region mediates the ability of the AhR to interact

with DNA while the HLH motif allows the AhR to dimerize with ARNT [10]. The basic

region of the AhR: ARNT heterodimer recognizes a specific DNA target sequence known

as the dioxin responsive element (DRE) or xenobiotic responsive element (XRE).

Composed of the nucleotide sequence 5’-T(C/T)GCGTG-3’, the DRE is typically located

upstream of the promoter region in AhR regulated genes [11]. The sequence is highly

conserved in mammals and mutational analysis of the DRE has revealed the 5’ half site

sequence T(C/T)GC is required for AhR interaction while ARNT recognizes the 3’ half

site GTG [12]. Interaction by both AhR and ARNT at their respective half site sequences

is required to maintain enhancer function. 4

Figure 1.1: The aryl hydrocarbon receptor. A diagrammatic representation of the known functional domains and their relative positions in the receptor. The domains and/or regions responsible for such critical functions as dimerization, DNA binding, ligand binding, and transactivation are indicated.

5 1.1.1.2 The Per-Arnt-Sim (PAS) domain

Originally discovered in eukaryotes, the PAS domain is named for its homology to the Drosophila regulatory protein Period (Per), the mammalian AHR and ARNT proteins, and the Drosophila regulatory protein Single-minded [13]. PAS domains are

found in a wide variety of proteins from bacteria to higher animals and plants where they

function as sensory domains monitoring changes in light, redox potential, oxygen levels,

overall cellular energy levels, and to signals generated during embryogenesis [14].

Furthermore, even though the AhR is the only characterized member of the bHLH/PAS

superfamily of proteins known to be ligand-activated [15], it is the PAS domain

contained in the AhR which is involved in ligand binding and thus responsible for

monitoring changes in cellular ligand concentration. The PAS region of homology is

located toward the carboxy terminal side of the bHLH domain in both the AhR and

ARNT proteins. It is comprised of two hydrophobic stretches of approximately 50 amino

acid residues, termed PAS A and PAS B, separated by a region of approximately 150

residues (Figure 1.1). The PAS domain of the receptor is responsible for mediating the

association of AhR with Hsp90 in the unliganded core complex. It is hypothesized that

Hsp90 interaction functions to keep the receptor in an appropriate conformation for high

affinity ligand binding while repressing any intrinsic DNA-binding ability [9, 16-18]. In

the ligand activated form of the AhR, the PAS domain participates in heterodimerization

with ARNT [16, 17, 19]. 6 1.1.1.3 The hsp90 and ligand binding domain

Partially overlapping with the PAS domain and extending toward the C-terminal end of the receptor is the AhR ligand binding and Hsp90 interaction domain (Fig 1.1).

Characterized through the use of deletion mapping studies, this region was demonstrated to be essential for AhR interaction with Hsp90 [18, 20]. In addition, radiolabeled ligand studies also established this region as comprising part of the ligand binding pocket.

Although this region extends outside of the PAS domain toward the C-terminus, only deletions within the PAS domain appear to affect ligand binding. Deletion of the PAS A repeat reduced ligand binding to approximately 30% while deletion of PAS B completely abolished ligand binding [21]. Recently, a three-dimensional model for the murine AhR ligand binding domain (LBD) has been predicted using known PAS domain folding patterns from other crystallized PAS proteins in conjugation with computer based modeling predictions [22]. These studies have identified critical amino acid residues important in determining receptor affinity for ligand.

1.1.1.4 The transactivation domain

Located at the carboxy terminus, the AhR contains a modular transactivation domain (TAD) (see Figure 1.1) responsible for communicating the induction signal from the enhancer to the promoter of AhR responsive genes. In humans, the TAD can be divided into three distinct subdomains classified according to the amino acid composition. The acidic subdomain extends between residues 500-600 while the glutamine (Q) rich subdomain, critical in transcriptional activation of dioxin responsive 7 genes, is located between amino acids 600-713. The proline/serine/threonine (P/S/T) rich subdomain, a potential inhibitor of transactivation potential, is contained in the final 135

C-terminal residues of the TAD (aa 713-848) [23]. Studies performed in cultured cells using yeast Gal4 fusion proteins demonstrated the C-terminal region as being critical to the transactivation potential of the AhR [24]. Although relatively low levels of activity have been reported for each of the individual TAD regions, combinations of domains lead to significantly higher levels of transcriptional activation, an observation consistent with synergistic activity between TAD subdomains [25]. Furthermore, it has been determined that the AhR TAD functions directly at the promoter region of target genes as opposed to the disruption of nucleosome structure at enhancer sequences [26]. At the promoter, the

TAD may function to activate transcription through direct interaction with basal transcription factors or through the recruitment of co-activators to the promoter complex.

1.2 THE AH RECEPTOR SIGNAL TRANSDUCTION PATHWAY

In the absence of ligand, the AhR is localized in the cytoplasm as heterotetrameric

9S protein complex [27, 28]. Occurring shortly after or concomitantly with ligand binding, the AhR is transformed into an active state allowing for translocation of the 9S core complex into the nucleus. Inside the nucleus, liganded AhR dissociates from the docking complex and heterodimerizes with ARNT, its dimerization partner, to form a high affinity DNA binding complex capable of recognizing specific DNA target sequences, known as the DRE [29-31]. AhR:ARNT heterodimer interaction with the

DRE sequence, commonly located in the upstream enhancer region of AhR regulated 8 genes, results in the alteration of target gene expression. Genes transcriptionally regulated by the AhR are primarily involved in foreign chemical metabolism, including various phase I and II xenobiotic metabolizing enzymes, in addition to genes encoding proteins involved in cell growth and differentiation [8]. Displayed in (Figure 1.2) is a pictorial representation of the AhR signal transduction pathway. 9

Figure 1.2: The aryl hydrocarbon receptor signal transduction pathway. Localized in the cytosol, the un-liganded AhR exists as part of a heterotetrameric protein complex. Contained in the stable untransformed 9S core complex are the AhR ligand binding subunit, a dimer of hsp90, and a single molecule of the immunophillin-like XAP2 protein. Following ligand binding the AhR undergoes a transformation process whereby the 9S core complex translocates to the nucleus, detaches from hsp90 and XAP2, and heterodimerizes with ARNT forming a transcriptionally active 6S complex. The active heterodimer can then bind to specific DNA sequences known as dioxin response elements or DREs in the upstream regulatory region of target genes, such as Cyp1A1, recruit co-activators to the transcription complex and thereby activate transcription.

10 1.2.1 The un-liganded AhR core complex

The results from several early studies indicated that prior to ligand binding the

AhR remained localized in the cytoplasm as part of a large, inactive, variably-sized protein complex [32, 33]. Chemical cross-linking analysis later confirmed the un- liganded AhR existed in the cytosol docked in an ~300 kDa protein complex with three other protein molecules [27, 28]. Contained in this core complex are the AhR ligand- binding subunit, a dimer of 90 kDa heat shock protein (hsp90) and a single molecule of the immunophilin-like XAP2 protein [28, 34].

1.2.2 Transformation of the Ah receptor

AhR transformation after ligand binding is a poorly defined process involving a series of steps that concludes upon heterodimerization with ARNT in the nucleus.

Transformation of the AhR is accompanied by a decrease in the apparent molecular weight of the core complex, which is reflected by a reduction in sedimentation values, from 9 S to 6 S, respectively [35]. Transformation results in the nuclear accumulation of receptor which is a prerequisite for heterodimerization and the subsequent formation of an active transcription complex. Several studies have suggested the temperature dependent transformation process centers on a ligand-induced conformational change that confers upon the receptor an increased affinity for ligand, the ability to translocate into the nucleus, dissociate from the core complex and form a heterodimer with ARNT [36]. 11 1.2.2.1 Ligand binding

The binding of ligand by the AhR initiates receptor transformation and ultimately results in the modulation of AhR target genes. The ability to activate the AhR has been demonstrated to correlate well with ligand affinity for the receptor [37]. Most high affinity AhR ligands are hydrophobic by nature and can easily traverse a cellular membrane, enter the cell, and bind to the unliganded AhR core complex. Alternatively, there exists a body of accumulating evidence suggesting that endogenous ligands for the

AhR may exist [38]. Such molecules could be generated inside the cell or by neighboring cell types and proceed to modulate receptor activity. Since the identification of an endogenous AhR ligand was the focus of this doctoral thesis, known AhR ligands of both natural and synthetic origin will be covered in substantial detail in a later section devoted to the topic.

1.2.2.2 Translocation of the AhR core complex

Using murine cell lines deficient in AhR activity, ARNT (aryl hydrocarbon

receptor nuclear translocator) was subsequently identified as the dimerization partner for

AhR and named for its hypothesized role in mediating nuclear translocation of the receptor [29, 30]. It was later revealed that ARNT is restricted to the nucleus of the cell, serving no role in nuclear translocation, and functioning only as a dimerization partner for the AhR [29]. Although the exact mechanism of AhR nuclear translocation still remains unclear, bipartite nuclear localization signals (NLS), identified in the N-terminal region of the receptor, have been shown to mediate the ability of the AhR to translocate 12 to the nucleus [39]. It is hypothesized that following ligand binding, as an essential part of the AhR transformation process, the NLS becomes exposed allowing importin recognition and thus initiating the translocation of the receptor core complex to the nucleus [40].

1.2.2.3 AhR and ARNT Heterodimerization and DNA Binding

The observed increase in Cyp1A1 mRNA levels following TCDD exposure suggested that ligand activated AhR could induce transcriptional activation of target genes through direct DNA binding [41]. UV-crosslinking experiments using a synthetic

DRE oligonucleotide demonstrated that TCDD induced AhR from rat liver bound to

DNA as a heteromeric complex. Further analysis of the crosslinked protein-DNA complex by SDS-PAGE indicated the presence of two proteins with approximate molecular weights of 100 and 110 kDa [11, 42]. Transcriptionally active AhR was eventually isolated from the nuclei of ligand treated cells existing in a heterodimeric protein complex with ARNT. As the functional dimerization partner for the AhR, ARNT serves as a structural component of the high affinity DNA-binding form of the receptor and is required for AhR function [30]. The ligand activated AhR/ARNT complex can proceed to activate target gene transcription through specific interaction with DNA sequences known as dioxin responsive elements (DREs) or xenobiotic responsive elements (XRE) [43].

13 The AhR:ARNT heterodimer recognizes the consensus nucleotide sequence 5’-

TNGCGTG-3’ [43-45] with the AhR recognizing the 5’-TNGC half-site of the DRE sequence while ARNT recognizes the GTG-3’ half-site [12, 46]. Methylation protection and interference assays have indicated that ligand activated AhR binds within the major groove of the DNA double helix, contacting both strands of DNA, and interacting with all four guanine residues present in the core DRE recognition motif [47].

1.3 LIGANDS FOR THE AH RECEPTOR

The AhR is a promiscuous receptor capable of being activated by a vast range of structurally diverse organic compounds of both natural and synthetic origin. To date, the highest affinity ligands characterized for this receptor belong to the organohalogen class of compounds. Most of these ligands () However, various other types of xenobiotic compounds including many of dietary origin are able to bind and activate this ligand dependent receptor. Several low affinity endogenous ligands have also been identified for the AhR. Known AhR ligands will be subdivided into four major groups and discussed accordingly.

1.3.1 Halogenated aromatic hydrocarbons

The halogenated aromatic hydrocarbons (HAHs) are a group of structurally related and highly toxic halogen-containing organic molecules comprised of the polychlorinated dibenzodioxins (PCDD), polychlorinated dibenzofurans (PCDF), and 14 polychlorinated biphenyls (PCB) (Figure 1.3). Ubiquitous in nature, these planar

lipophilic compounds, as a result of their chemical stability and increased biological half-

life, tend to persist in the environment for long periods of time resulting in their

bioaccumulation. HAHs can be rapidly absorbed through all major exposure routes

including inhalation, ingestion, and dermal absorption leading to their accumulation in

adipose tissue. Typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent

congener, these environmental toxicants can induce a broad spectrum of biochemical and

toxicological effects. Exposure may result in dermal toxicity, gastrointestinal toxicity,

hepatic toxicity, developmental and reproductive toxicity, central and peripheral neurotoxicity, immunosuppression and tumor promotion in humans [48-51]. Long term

exposure in rodents leads to the development of liver, lung, skin, and thyroid tumors [52].

The AhR-mediated toxic response, however, appears to be species-, tissue- and ligand-

specific. 15

Figure 1.3: Halogenated aromatic hydrocarbon (HAH) ligands of the AhR. The chemical structures of several common HAH compounds including the prototypical dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

16 Although never purposefully synthesized, PCDDs and PCDFs are planar lipophilic compounds produced as byproducts during the synthesis of commercially relevant organochlorine products like herbicides, pesticides and plastics. In addition, they are generated during iron ore sintering and steel production, through the chlorine bleaching of wood pulp, and during thermal reaction processes, such as municipal and industrial waste incineration, accidental fires, and the burning of fossil fuels [53] [4]. In general, the generation of PCDDs and PCDFs is likely whenever organic materials are combusted under oxygen deficient conditions while in the presence of a suitable chlorine donor [51].

Polychlorinated biphenyls (PCBs), on the other hand, were commercially produced in vast quantities for many years until their large scale synthesis was banned by the EPA in 1977 [54]. These toxic, man-made, organic soluble compounds were manufactured for their superior electrical and temperature insulating abilities. Industrial applications included use as components of electrical transformer and capacitor fluid, heat transfer and hydraulic fluids, plasticizers, paints and lubricating oils. [4] [55]. As a result of their widespread industrial use, PCB residue can now be identified in almost every component of the global ecosystem including the atmosphere, soil and waters of oceans, lakes and streams. These stable and persistent contaminants have been identified in plants, fish, birds, livestock, and wildlife in addition to being detected in samples of human blood, adipose tissue and breast milk [56-59].

17 Exposure to dioxin and other related chemicals can impart detrimental consequences on human health mediated primarily by the AhR. The toxicity of these compounds correlates well with their ability to activate the AhR [10, 15]. As high affinity ligands, these compounds can directly bind and activate the AhR effectively disrupting normal AhR signaling. Tissue-specific alterations in AhR dependent gene expression appears to be responsible for eliciting the adverse health effects. The AhR mediated toxicological response will be discussed in more detail in the following section.

1.3.2 Polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs), known also as polyarenes, are a group of planar, hydrophobic molecules built from a collection of fused aromatic rings. They may be comprised of four, five, six or seven member carbon rings, although those formed from five or six member carbon rings are most common. Rich in carbon these products of incomplete combustion are produced whenever organic material is burned [60]. As such, they are commonly found in diesel exhaust and cigarette smoke, generated during the processing of coal and crude oil, produced by industrial power generating plants, formed during the incineration of waste, the char-grilling of meats and the combustion of coal, oil, natural gas and wood products [61]. They can also be formed during the slow geological combustion process that produce the oil and coal deposits found in the earth

[62] as well during other natural events like forest fires and volcanic eruptions [61]. As a result of their ubiquitous nature, PAH compounds can be found in the air, food, and 18 drinking water making human exposure to these widespread environmental contaminants essentially unavoidable.

Many PAH compounds possess carcinogenic, mutagenic, teratogenic and atherogenic properties [5, 63]. The toxicity of PAH compounds is very dependent on structure. In general, the degree of nonplanarity correlates well with the carcinogenic and mutagenic potential of these molecules. Planar PAHs, because they are more stable and less reactive, exhibit a decreased potency and less overt toxicity when compared with their non-planar counterparts [61]. Conversely, the elevated level of toxicity observed with non-planar PAHs is believed to be a consequence of increased reactivity, resulting from the enhanced olefinic character found in some of their double bonds [64] For example, methyl group substitution of the ring system in 7,12-dimethylbenz[a]anthracene

(DMBA) results in distortion that decreases planarity and increases reactivity. This intermediate affinity AhR ligand [65] has a much higher carcinogenic potential than the more planar parent compound benz[a]anthracene [61]. The chemical structures of several common PAH compounds, including benzo[a]pyrene (B[a]P), 7,12- dimethylbenz[a]anthracene (DMBA) and 3-methylcholanthrene (3-MC) are depicted

(Figure 1.4). These molecules are well-defined AhR ligands capable of activating

receptor mediated gene expression through direct ligand binding [65-68]. Ultimately, they initiate their own metabolism and clearance through the induction of AhR regulated

xenobiotic metabolizing enzymes such as CYP1A1, CYP1A2 and CYP1B1 [69, 70].

CYP450 mediated metabolism of PAH compounds, although intended to facilitate their

clearance, may also result in the production of carcinogenic intermediates capable of 19 covalently binding to DNA. This will be discussed further in the following section dedicated to the AhR mediated toxicological response. 20

Figure 1.4: Polycyclic Aromatic Hydrocarbon (PAH) Ligands of the AhR. The chemical structures of several classical PAH compounds including B[a]P, DMBA, and 3- MC are shown. These compounds are carcinogenic ligands for the AhR.

21 1.3.3 Dietary ligands

Although dioxin and other structurally-similar environmental contaminants are the most frequently studied AhR ligands, many naturally occuring dietary compounds have been identified that can function as AhR ligands (Figure 1.5). For example, indole-

3-carbinol (I3C), a glucobrassicin metabolite found in cruciferous vegetables such as cabbage and broccoli, has been identified as a low affinity ligand for the AhR in vitro

[71]. Under acidic conditions, like that found in the stomach, I3C molecules can undergo acid-catalyzed condensation to form indolo[3,2b]carbazole (ICZ), a potent AhR ligand with an in vitro binding affinity similar to that of TCDD [71, 72]. The presence of ICZ in the murine intestinal tract and the production of high affinity AhR ligands in rat fecal suspensions of I3C indicate the conversion of dietary indoles, including I3C and tryptophan, into significantly more potent AhR ligands in vivo [71, 73] Both ICZ and its methylated derivative 5,11-dimethylindolo[3,2b]carbazole (MICZ) are potent activators of AhR-dependent gene expression capable of inducing transcription of a DRE-driven luciferase reporter construct in both human and murine hepatoma cell lines [74]. The production of these high affinity AhR ligands, from the much weaker I3C, may explain the strong induction in CYP1A1 catalytic activity observed with I3C.

Displaying close structural similarity with ICZ are 6-diformylindolo-[3,2b]- carbazole (FICZ) and 6,12-formylindolo[3,2-b]carbazole (dFICZ) (Figure 1.5). These

UV-light induced tryptophan derived photoproducts are high affinity AhR ligands [75].

Of particular interest is FICZ, a potent AhR ligand capable of 22

Figure 1.5: Dietary ligands of the AhR. Depicted are the chemical structures of several dietary compounds able to function as AhR ligands.

23 binding and activating the AhR in vitro with a binding affinity substantially greater than that observed for TCDD. Although FICZ is known to form in TRP solutions exposed to light, it has yet to be identified in tissues or plasma. In addition, FICZ is capable of regulating induction of CYP1A1 in human keratinocytes and murine dervied Hepa-1 cells and is itself rapidly metabolized by this P450 enzyme [76] Another group of tryptophan derived metabolites, the tryptanthrins, are AhR ligands biosynthesized from tryptophan and anthranilic acid derivatives by Candida lipolytica, a yeast commonly found in human food [77]. Derived from ubiquitous dietary precursor molecules, the unsubstituted tryptanthrin molecule is the first and only identified AhR ligand of microbial origin.

Produced during the cooking and broiling of protein rich foods, heterocyclic aromatic amines (HCA) are an another group of dietary molecules that can serve as ligands for the AhR. However, in comparison to high affinity ligands like B[a]P and

TCDD, these compounds have a weak potential for AhR activation [78]. This may be explained by the relatively small size of these molecules when compared with most other high affinity AhR ligands.

Curcumin, a naturally-occuring chemopreventive agent, is the prinicpal phenolic antioxidant and anti-inflammatory compound found in tumeric, a commonly used bright yellow spice from the ginger family. The anticarcinogenic effects of this compound have been demonstrated in numerous animal models for skin and gastrointestinal carcinoma

[79, 80]. Furthermore, curcumin is an AhR ligand possessing very unique properties in regards to AhR biology. Like all prototypical AhR ligands, it is capable of directly 24 binding the receptor initiating transformation and nuclear accumulation which ultimately results in increased CYP1A1 gene expression. However, curcumin is truly unique among

AhR ligands because it possess a distincitve ability to induce phase I xenobiotic metabolism while concurrently inhibiting the commonly observed CYP-mediated carcinogen bioactivation [81]. The ability to function as a potent cellular antioxidant, coupled with its unique ability to positively modulate effective carcinogen metabolism while inhibiting carcinogen bioactivation, make curcumin a powerful chemopreventive

AhR ligand.

Brevetoxins (PbTX) are a family of nonaromatic, lipophilic, polyether, marine neurotoxins produced by the red tide dinoflagellate Ptychodiscus brevis, formerly known as Gymnodinium breve [82]. Nine different brevetoxins (PbTx 1-9) are known to be produced, but only brevetoxin-6 (PbTx-6) is a ligand for the AhR. This epoxide containing molecule is able to directly bind and activate the AhR. It induces a concentration dependent gel shift and is capable of competing with radiolabeled TCDD for AhR binding [83].

Interestingly, there exist several other dietary compounds, unable to compete with radiolabeled TCDD for AhR binding, but nonetheless able to regulate CYP1A gene expression through an AhR-dependent mechanism. For example, the oxidized carotenoids canthaxanthin and β-apo-8′-carotenal, despite a lack of binding affinity for the AhR, have been shown to induce CYP1A gene expression through an AhR dependent mechanism [84]. Furthermore, omeprazole, a pharmacologically relevant benzimidazole 25 compound found in certain acid reflux medications, is capable of activating the AhR- signaling despite its apparent inability to function as a AhR ligand [85, 86].

1.3.4 Endogenous ligands

Accumulating evidence has demonstrated activation of the AhR in the absence of an exogenous ligand [38, 87, 88] Assuming ligand is required for receptor activation, these results support the existence of an endogenous ligand for the AhR. Perhaps the most convincing evidence for an endogenous ligand is derived from studies with AhR knockout mice. AhR null animals exhibit a broad spectrum of defects including lesions in the heart, liver, spleen, glandular stomach, large intestine, skin and uterus indicating a critical role for the AhR in normal developmental processes [89]. Over the last decade, there have been several endogenous ligands or modulators for the AhR identified. These structurally dissimiliar compounds are discussed below.

The degradation of cellular hemoproteins leads to the release of heme, an iron containing porphyrin molecule. Endogenous heme is metabolized enzymatically to biliverdin (BV) and subsequently to bilirubin (BR) [90]. Although BR demonstrates an increased affinity for the receptor, both molecules have been characterized as ligands for the AhR capable of binding the receptor and inducing expression of DRE-responsive genes. Nevertheless, these compounds are considered to be low affinity ligands capable of competing only weakly with [3H]TCDD for Ah receptor binding in vitro [91].

26 Indigo, a dark blue pigment commonly used to dye fabric, and indirubin, a red colored structural isomer of indigo, have been isolated from concentrated human urine and identified as AhR ligands. In a yeast based reporter assay, these indole-derived metabolites were able to activate the human AhR (hAhR) with potency equal to or exceeding that of TCDD [92]. However, it is uncertain if these molecules are true physiological ligands for the AhR generated from endogenous precursors, or whether they represent the urinary excretion products of an exogenous compound.

2-(1’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is a compound that has been isolated from acidified high temperature porcine lung extracts, and characterized as a high affinity AhR ligand capable of binding the human, murine and fish Ah receptors [93]. Although the endogenous production of this indole based molecule is still questionable, its formation is nevertheless conceivable through a condensation reaction involving tryptophan and cysteine. In addition, recent studies have established this molecule as a high affinity AhR ligand capable of inducing heterodimerization and DRE binding with a magnitude equal to TCDD, yet failing to elicit any the hallmark toxicity commonly associated with the more stable TCDD molecule [94].

Over the past several years various eicosanoid molecules have been indicated as modulators of AhR activity (Figure 1.6). For example, certain prostaglandins can serve

as AhR agonists stimulating receptor transformation and DNA binding in vitro while

inducing AhR-dependent gene expression in cultured cells. In a reporter-gene based 27 assay system, relatively high concentrations of PGH1, PGH2, PGG2, PGB3, PGD3, and

PGF3α were capable of inducing DRE-driven reporter gene expression. Furthermore, it

was reported that PGG2, a highly unstable cyclic endoperoxide intermediate, can induce

expression of an AhR-dependent reporter gene to a level exceeding that observed with a

maximal inducing dose of TCDD [95]. These results, if accurate, are quite logical given the ability of the AhR to regulate expression of prostaglandin endoperoxide H2- synthase-

2 gene (PGHS-2) coupled with the observation that TCDD is capable of altering

arachidonic acid metabolism. Another potential eicosanoid modulator of AhR activity is

lipoxin A4. This anti-inflammatory arachidonic acid metabolite has previously been

reported as a agonist for the AhR [96]. However, using a purified preparation of this

compound I have been unable to reproduce these same results, contradicting the ability of this molecule to serve as an Ah receptor ligand. 28

Figure 1.6: Endogenous ligands for the AhR. Depicted are the chemical structures of several endogenously formed compounds known to regulate AhR function.

29 Recently, it has been demonstrated that modified low density lipoprotein (LDL) activates the AhR using in vitro models of endogenous vascular physiology. Both sheared and oxidized LDL particles activated the AhR, although the exact mechanism of this activation still remains unclear. In addition, when compared to their heterozygous littermates, the sera from AhR null animals contain an elevated level of AhR-activating

LDL particles suggesting an endogenous role for the AhR in the physiological response to modified LDL [97].

Furthermore, 7-ketocholesterol (7-KC), an oxysterol present in blood and a major component of oxidized LDL [98], has previously been identified as an endogenous, physiological modulator of the AhR capable of antagonistic action against xenobiotic ligands. This oxidized cholesterol derivative is produced enzymatically from either 7α- or 7β-hydroxycholesterol through the action of 7-hydroxycholesterol dehydrogenase (7-

HCD). It can bind to the AhR and compete with high affinity ligands for receptor occupancy, resulting in the effective inhibition of TCDD-mediated transactivation by preventing receptor binding to DNA [99]. Highly expressed in the tissues of hamsters while essentially absent in rabbits, rats and mice, 7-HCD expression levels correlate closely with the observed degree of TCDD resistance in these organisms [100]. 30 1.4 AHR MEDIATED BIOLOGICAL RESPONSES

1.4.1 The AhR regulates xenobiotic metabolism

In order to effectively respond to chemical insults, most organisms posses a biological detoxification and defense mechanism. Operating through the induction of xenobiotic metabolizing enzymes, these detoxification systems generate an adaptive metabolic response to protect against foreign chemicals. Xenobiotics are compounds of natural or synthetic origin recognized as foreign by the human body and include compounds such as plant-derived or fungal derived secondary metabolites, a vast number of environmental pollutants, food additives and drugs such as antibiotics [101]. Serving as a potential xenobiotic chemosensor, the AhR is a ligand-activated transcription factor that can mediate the adaptive metabolism of xenobiotics through activation of specific drug metabolizing cytochrome P450 monooxygenases (P450’s) [102].

Enzymes responsible for the biotransformation of xenobiotics can be formally classified as either phase I or phase II metabolizing enzymes based on the type of catalyzed reaction chemistry. Phase I biotransformation, also known as functional group modification, effectively increases the hydrophilicity of a substrate molecule through incorporation or exposure of a polar moiety. Phase I modifications are most commonly achieved through oxidation, reduction, hydrolysis, dealkylation, deamination, dehalogenation, ring formation and ring opening reactions such as aromatic and aliphatic hydroxylation or epoxidation [103]. Phase II biotransformation is commonly refered to as conjugation because the previously oxygenated molecules are conjugated to small 31 hydrophillic endogenous molecules like glutathione, glucuronic acid or amino acids producing highly polar easily excretable metabolic products [103]. Collectively, phase I and phase II metabolism perform an important role in protecting the human body from toxins.

1.4.1.1 AhR regulation of phase I enzymes

Cytochrome P450’s are heme-containing monooxygenase enzymes comprising a superfamily of more than 3000 members. They found in organisms ranging from bacteria to vertebrates that are capable of catalyzing the monooxygenation of various endogenous and exogenous substrates [104]. Currently, there are 57 known cytochrome P450’s in humans, 15 of these being primarily involved in xenobiotic metabolism of which 4 are known to be directly regulated by the AhR, CYP1A1, -1A2, -1B1 and -2S1 [105].

Cytochrome P4501A1 (CYP1A1) is a well characterized, substrate inducible, microsomal enzyme contributing to the phase I metabolism of numerous xenobiotic compounds. In humans, CYP1A1 expression is primarily extra-hepatic being expressed mainly in the lung, placenta and peripheral blood cells [106]. CYP1A1 exhibits low basal mRNA expression, but transcriptional regulation of gene expression is highly inducible in response to ligand activated AhR. In fact, it appears that CYP1A1 is the most highly induced AhR regulated gene [107].

32 CYP1A1 occupies an essential role in initiating the detoxification of various hydrophobic pollutants and environmental carcinogens. CYP1A1 metabolism can lead to a decrease in the toxicity of these foreign chemicals provided they can be successfully processed into water-soluble derivatives. However, some pollutants and carcinogenic compounds are not effectively detoxified through P450 metabolism. Paradoxically, many of these environmental procarcinogens become activated by CYP1A1 monooxygenation and transformed into highly reactive genotoxic intermediates, capable of reacting irreversibly with cellular macromolecules [108]. These carcinogenic electrophiles, primarily phenolic products and epoxides, can form DNA adducts leading to mutations and the subsequent initiation of cancer [109]. Of particular importance in the activation of chemical carcinogens is CYP1A1. This AhR regulated enzyme is responsible for catalyzing the bioactivation of polycyclic aromatic hydrocarbons (PAHs) commonly found in cigarette smoke and exhaust emissions [110] Historically, it was this AhR mediated CYP1A1 metabolism that was primarily responsible for the enzymatic activity originally classified as aryl hydrocarbon hydroxylase (AHH) activity [111].

In addition to xenobiotic metabolism, CYP1A1 can also participate in the metabolism of numerous endogenous lipophilic compounds including eicosapentanoic acid (EPA), arachidonic acid (AA) and various eicosanoid compounds [107]. CYP1A1 is also involved in estrogen metabolism and can preferentially catalyze the hydroxylation of

17β-estradiol at the C-2 position to generate 2-hydroxy-estradiol [112]. Additional

CYP1A1 substrates include melatonin, a hormone and potent antioxidant, along with theophylline, a methylxanthine drug used to treat respiratory diseases [113]. 33 Classically, CYP1A1 induction was viewed as a marker of AhR activation and associated with potentially adverse effects. However, the detoxification and potential bioactivation of chemical carcinogens can not be visualized as the only role for CYP1A1 metabolism. Numerous endogenous substrates for CYP1A1 have been identified with the list expected to continue growing. For example, (). Therefore, it can be hypothesized that CYP1A1 induction, mediated by an endogenous AhR ligand, could result in the degradative and/or biosynthetic metabolism of important biologically active compounds, serving as a means to regulate cellular signaling pathways. Futhermore, experimental results obtained by myself and others indicate a potential role for AhR regulated P450’s, especially CYP1A family members, in modulating the level of an endogenous AhR ligand(s) and thus serving to regulate AhR activity through completion of an autoregulatory loop (see chapter 2).

Cytochrome P4501A2 (CYP1A2) is the second in a pair of enzymes comprising the human CYP1A subfamily of cytochrome P450 mixed function oxidases. This enzyme, also under AhR regulation, shares an approximate 70% sequence homology with

CYP1A1 [114]. In contrast to CYP1A1, however, this isoform is predominately expressed only in human liver accounting for approximately 15% of the total P450 content of this organ [115]. As a result, it is the only AhR-regulated P450 to play an important role in human drug metabolism catalyzing the complete or partial metabolism of more than twenty clinically used drugs [116]. CYP1A2 is also involved in the metabolism of food-derived heterocyclic amines, including caffeine, and responsible for the metabolism and elimination of many toxicologically significant foreign chemicals 34 such as aflatoxin B1 and acetaminophen [117]. In general, CYP1A2 catalyzes the metabolism of xenobiotics with aryl and heterocyclic amine structures [116]. As with

other xenobiotic metabolizing enzymes, however, CYP1A2 metabolism can often result

in the bioactivation of many environmental procarcinogens especially tobacco-specific nitrosamines, certain mycotoxins and aryl amines [118].

It is important to note that a significant variability in CYP1A2 mRNA levels can exist among individuals. Variations in mRNA levels, as large as 40-fold have been observed [119], leading to corresponding changes in enzyme activity and substrate metabolism [116]. However, the interindividual variability in CYP1A2 enzymatic activity appears to be influenced more by the intake of drugs and dietary compounds than by genetic factors [116]. Cigarette smoke, for example, through activation of the AhR can induce CYP1A2 resulting in increased catalytic activity that can subsequently effect the metabolism of numerous pharmacologically-relevant compounds. This is of clinical significance given the number of smokers and the importance of CYP1A2 in the metabolism of various drugs.

Cytochrome P4501B1 (CYP1B1) is a xenobiotic-metabolizing enzyme constitutively expressed in nearly all extra-hepatic tissues with the exception of lung

[120]. It is active in many ocular structures and appears to play a critical, but not yet well understood, endogenous role in the normal development and function of the eye [121]. It shares an approximate 40% amino acid sequence homology with CYP1A1 and overlaps in catalytic activity with both CYP1A1 and CYP1A2 [122]. Induction of CYP1B1 35 expression is dependent on AhR-activation with dioxin-mediated induction of CYP1B1 having been demonstrated for both mice and the MCF-7 breast cancer cell line [123,

124].

CYP1B1-mediated metabolism has been shown to be important for various xenobiotics such as ethoxyresorufin, theophylline, and caffeine, a commonly consumed

CNS stimulant [122]. In humans CYP1B1 plays a major role as a E2 hydroxylase

participating in the hydroxylation of 17β-estradiol at the C4 position to generate the

carcinogenic metabolite, 4-hydroxy-E2 [125]. Additional members of the CYP1 family

can also hydroxylate 17β-estradiol albeit with different regiospecificities. CYP1A1, for

example, will preferentially hydroxylate 17β-estradiol at the C2 as opposed to the C4

position [126]. Consequentially, because 4-hydroxy E2 is a carcinogenic molecule,

CYP1B1 metabolism of 17β-estradiol has been suggested to play a major role in

mammary carcinogenesis [125].

CYP1B1 is also an efficient metabolizer of polycyclic and nitro aromatic

hydrocarbons as well as aryl amines and thus is capable of bioactivating these

environmental procarcinogens. Because CYP1B1 is expressed extrahepatically in

steroidogenic tissues such as ovary, testis and adrenal gland along with steroid-responsive

tissues like breast, uterus, and prostate it may be occupy an important role in various

hormone responsive cancers. 36 Cytochrome P450 2S1 (CYP2S1) is a relatively recently identified member of the cytochrome P450 superfamily of heme containing monooxygenases. This novel AhR regulated P450, identified in both human and mouse, has been shown to possess a relatively high level of basal expression, but is still dioxin inducible in the classic AhR regulated manner [127]. It is expressed extrahepatically in the epithelial cells of tissues frequently exposed to xenobiotics. The highest expression levels are commonly observed in tissues serving as primary environmental defense barriors such as the skin along with the gastrointestinal, respiratory and urinary tracts [128]. Similar to other dioxin-inducible cytochrome P450’s, it is hypothesized that CYP2S1 may play a role in the metabolism of xenobiotic compounds and subsequent bioactivation of chemical carcinogens.

Surprisingly, however, it does not bioactivate benzo[a]pyrene (B[a]P), 4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), or 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP), three major cigarette smoke carcinogens [129], but does metabolize the toxic and potentially carcinogenic aromatic compound naphthalene

[130].

CYP2S1 is expressed throughout embryogenesis and is involved in the metabolism of important endogenous compounds, but aside from all-trans retinoic acid

(ATRA), the carboxylic acid form of vitamin A (retinol), the endogenous substrates for

CYP2S1 remain unknown. Although ATRA is a retinoid metabolite critical to embryonic development, it becomes teratogenic at high levels [131]. However, because

CYP2S1 enzymatic activity is inducible by retinoic acid, it prevents the accumulation of the fat soluble ATRA metabolite through increased oxidative metabolism of the 37 compound to the inactive metabolites, 4-hydroxy retinoic acid and 5,6-epoxy retinoic acid [128].

CYP2S1 expression is largely elevated in tumors of epithelial origin and its expression has been demonstrated to correlate with poor prognosis in colorectal cancer

[132]. Thus, upon further investigation, it is conceivable that this novel P450 enzyme, like other AhR regulated P450s, could become clinically relevant and consequential in maintaining human health.

1.4.1.2 AhR regulation of phase II enzymes

The AhR also regulates the expression of several Phase II xenobiotic metabolizing enzymes including UGTA1A1, GST-Ya subunit and NADPH Quinone

Reductase. These enzymes are responsible for increasing the hydrophilicity of phase I intermediates through conjugation with small water soluble endogenous molecules thus facilitating their excretion via bile or urine. This process is crucial to the detoxification of foreign chemicals and toxins.

UGT1A1: (Uridine diphosphate glucoronosyltransferase) Regulated by the AhR, this enzyme performs an essential physiological role in the glucuronidation of a large variety of molecules ranging from cellular waste products to environmental pollutants.

The transfer of glucuronic acid from uridine 5’-diphosphoglucuronic acid to various cellular and dietary metabolites functions to increase water solubility and enhance 38 elimination through excretion [133]. UGT1A1 contributes to phase II xenobiotic metabolism through conjugation of glucuronic acid with phase I metabolites.

Consequently, a reduction in functional UGT1A1 often results in a decreased ability of the liver to detoxify xenobiotics [134]. In addition, because only UGT1A1 can catalyze the glucuronidation of bilirubin, decreased enzymatic activity can result in the accumulation of unconjugated bilirubin in the blood stream resulting in hyperbilirubinemia disorders such as Crigler-Najar or Gilbert’s syndrome [135] [136].

The discovery of an XRE in the upstream regulatory region of the UGT1A1 structural gene suggests that this enzyme may be regulated by the AhR upon exposure to environmental pollutants in a manner similar to CYP1A1 [137]. Futhermore, results obtained in the human hepatoma HepG2 cell line indicate exposure to AhR ligands including TCDD, β-naphthoflavone (βNF) and B[a]P increases both UGT1A1 mRNA and protein levels. This Ah receptor ligand inducibility of UGT1A1 is lost if the XRE core region (CACGCA) is mutated or deleted confirming the role of the AhR in regulating human UGT1A1 induction [138].

GST-Ya: (Glutathione-S-transferase Ya subunit) Glutathione S-transferases

(GSTs) are a family of enzymes that can catalyze the nucleophilic attack of reduced glutathione on a variety of electrophilic substrate molecules. GST induction is an evolutionarily conserved biochemical response to oxidative stress with prooxidant exposure capable of inducing upregulation of this cellular detoxification system. Arising 39 primarily through oxidative phosphorylation, the endogenous production of reactive

- oxygen species (superoxide anion O2 , hydrogen peroxide H2O2, and the hydroxyl radical

HO) from partially reduced O2 is an unavoidable consequence of aerobic respiration.

Free radicals may also arise through lipoxygenase, cyclooxygenase, or cytochrome P450

catalyzed reactions. Reactive oxygen species can directly damage membrane lipids,

DNA, and protein, resulting in macromolecule breakdown and the generation of

cytotoxic degradation products capable of further indirect damage to cellular

components. GSTs are one of the many cellular enzyme systems that can protect against

the by-products of oxidative stress through formation of less reactive glutathione

conjugates. In addition, xenobiotic compounds such as drugs, pesticides, herbicides,

environmental pollutants and carcinogens can also serve as substrates for soluble GST

and be detoxified through conjugation and excretion (as reviewed in [139]).

Although regulated by the AhR, the upstream enhancer region of the GST-Ya

gene is considerably more complex than the UGT1A1 gene, containing several regulatory

sequences in its 5’ flanking region. The promoter region of this gene contains a distinct

CYP1A1-like DRE, a hepatocyte nuclear factor 1 (HNF1) recognition motif and a unique

β-NF responsive element unlike the CYP1A1 DRE [140]. This latter element is partially

responsible for controling basal gene expression in addition to regulating gene induction

in response to planar aromatic compounds such as 3-MC and βNF [140]. The results of

electrophoretic mobility shift assays (EMSA) have demonstrated that this element is

bound by a nuclear factor upon β-NF treatment. In the rat, for example, C/EBP-α binds 40 constitutively to this XRE element with coordination of AhR binding occuring upon induction with an AhR agonist thus suggesting that both AhR and C/EBP function cooperatively to induce transcription of this gene [141]. However, in another study performed in mice GST-Ya mRNA induction by both planar hydrocarbons and electrophilic intermediates was dependent on a single upstream regulatory element while also requiring functional AhR and CYP1A1 [142]. Taken together this seems to suggest that induction of the GST-Ya subunit gene in mice occurs through the formation of an electrophilic intermediate and that this upstream regulatory region between base pairs -

754 to -713 should more appropriately be considered an electrophile response element.

Nonetheless, despite some mechanistic differences, the GST-Ya subunit gene in both the mouse and rat appears to involve regulation by the AhR.

NAD(P)H:Quinone Oxidoreductase 1 (NQO1): In the cell the carbonyl group is a prevalent organic moiety occuring in many endogenous compounds such as steroid hormones and lipid mediators and frequently found in xenobiotics like food additives, drugs or environmental pollutants. Those of significant toxicological importance, due primarily to the intrinsic chemical reactivity of this functional group toward cellular macromolecules, are conjugated ketones, such as quinones, α/β unsaturated aldehydes or

α/β dicarbonyls [143]. NQO1 is a homodimeric flavoprotein essential in the cellular defense against reactive forms of oxygen and in the inhibition of neoplasia. This enzyme, induced under conditions of oxidative stress or in response to planar hydrocarbons, can provide the cell with several levels of protection against chemical insult. Primarily,

NQO1 functions in the reduction and detoxification of highly reactive quinones by 41 catalyzing a single stage two electron reduction. The product of this reaction is a hydroquinone that can be easily conjugated and excreted. NQO1 also acts to maintain endogenous lipid-soluble antioxidants, such as ubiquinone (co-enzyme Q) and α- tocopherol-quinone, in the their active reduced form. In addition, NQO1 has been implicated in influencing cell fate decisions through enhancing the stability of p53. This tumor suppressor is capable of being post-translationally modified by NQO1 resulting in the inhibition of proteosomal degradation, leading to greater p53 protein stability and an increased cellular half-life [144]. Ultimately, the accumulation of p53 can result in growth arrest or apoptosis in response to cellular stressors such as oxidant stress or DNA damage. [145, 146].

Two xenobiotic response elements (XRE) located in the 5’upstream regulatory region of this gene have been characterized. The first region between nucleotides (-392 to -352) contains an XRE with sequence identity similar to the classic XREs that mediate

CYP1A1 induction. Likewise this element also demonstrates inducibility by TCDD and

β-Naphthaflavone (βNF). A second region between nucleotides (-434 to -404) contains an XRE that more closely resembles the recently identified antioxidant response element

(ARE) of the GST-Ya subunit gene and displays inducibility by βNF and the phenolic antioxidant, t-butylhydroquinone [147]. It is important to note that CYP1A1 activity may be required for quinone reductase induction based on the decrease in NQO1 expression in response to CYP1A1 silencing by siRNA [148, 149]. In summary, regulation of quinone reductase can be mediated by three distinct mechanisms, AhR dependent induction, antioxidant mediated upregulation and CYP1A1 mediated activation. 42 Aldehyde dehydrogenase: Aldehyde dehydrogenases are NAD(P)+-dependent

enzymes involved in the oxidation of aldehydes to carboxylic acids. Aldehydes are

highly reactive molecules formed during many biological processes including retinoic

acid biosynthesis, metabolism of amino acids, lipids, carbohydrates and drugs. The

aldehyde dehydrogenase reaction is considered to be a cellular detoxification reaction and is also responsible for removing the electrophilic products of alcohol oxidation (as reviewed [150]).

ALDH3A1 is an AhR-regulated gene containing at least 7 DREs in its upstream regulatory region. Four of these appear to function co-operatively to induce transcription upon exposure to TCDD. 3-MC and βNF have also been shown to induce expression of

ALDH3A1 but antioxidants and electrophiles fail to elicit the same response [151].

1.4.2 The AhR mediates the carcinogenic and toxicological response to environmental pollutants

For nearly twenty-five years, the central role of the AhR in the adaptive metabolic response to PAH’s has been extensively studied, with many of the mechansitic details of

PAH toxicity having been elucidated. Formed during the incomplete burning of fossil fuels, combustion byproducts such as B[a]P, are wide-spread environmental carcinogens commonly found in cigarette smoke, chargrilled meats, and diesel exhaust. In addition, creosote, a wood preservative used on railroad ties and telephone poles, along with roofing tar are also known to contain PAH’s. The AhR-mediated toxicity of PAH 43 compounds results directly from their bioactivation by metabolic pathways designed to detoxify and eliminate foreign chemicals. For example, B[a]P is metabolized and bioactivated to the active diol expoxide form, benzo[a]pyrene-7,8-diol-9,10-epoxide

(BPDE), by the AhR-regulated cytochrome P450 monooxygenase, CYP1A1, an enzyme designed to prepare foreign chemicals for subsequent conjugation and excretion [152,

153]. This reactive metabolite is a mutagen known to exert its carcinogenic effect through the formation of covalent adducts with DNA [152]. As a result, the carcinogenicity of B[a]P and other related PAH compounds is directly connected with their ability to bind and activate the AhR, inducing target gene expression [66]. AhR regulated cytochrome P450 monooxygenase enzymes of the 1A and 1B families are primarily responsible for bioactivating the inducing PAH compounds into reactive electrophilic and subsequently carcinogenic molecules capable of initiating cell transformation and cancer [153, 154]. The carcinogenicity of PAH compounds could be greatly alleviated if their cellular bioactivation could be minimized. The lack of benzo[a]pyrene mediated carcinogenicity in AhR null mice confirms this theory [66].

In addition to mediating the carcinogenic potential of PAH’s, the AhR is considered to play a pivotal role in the biological pathways responsible for potentiating the toxic, teratogenic and carinogenic effects of such notorious environmental chemicals as TCDD, polychlorinated coplanar biphenyls, and dibenzofurans [155]. TCDD was first correlated with human illness in the mid-1950’s and although the molecular mechanism responsible for dioxin-mediated toxicity still remains poorly understood, it has been well documented that exposure to PAHs results in a myriad of diverse systemic and tissue- 44 specific pathological changes. These effects, all of which appear to be mediated through ligand binding and subsequent activation of the AhR, include tumor promotion, immune dysfunction, thymic atrophy, loss of body weight, embryonic teratogenesis, reproductive toxicity and several types of cancer [156-158]. Histopathological changes in such organs as the liver, lung, thymus, pancreas, adrenal glands, and central nervous system have also been observed [158] with the thymus and liver being the organs most consistently affected by dioxin exposure [159]. In addition, activation of the AhR by dioxins and other related HAH compounds is known to cause a disruption in almost every hormonal system ever examined [157].

Unlike PAH-induced toxicity where the genotoxic mechanism of carcinogenesis is well defined, dioxin-induced toxicity is not completely understood although alterations in AhR mediated gene expression are still believed to play a central role. In vivo dioxin, like B[a]P, is a carcinogenic compound but in stark contrast with the prototypical PAH compounds, dioxin is not directly genotoxic. Thus, even though dioxin exposure can cause mutations, it is not capable of directly binding to macromolecules like DNA.

Instead, because it is known to be both a promoter and initiator of cancer in humans, it is believed that dioxin-induced toxicity may result from disruption of signaling pathways.

Alternatively, dioxin toxicity may result through the production of a secondary toxicant capable of binding DNA [156]. TCDD, the prototypical dioxin, along with other HAHs are ubiquitous environmental contaminants which can bioaccumulate as a result of their biological stability. Consequently, human exposure to these compounds may be 45 percieved as chronic and wide-spread making these pollutants an important health concern.

1.5 PHYSIOLOGICAL ROLES FOR THE AHR

The most convincing evidence in support of a normal physiological role for the

AhR is derived from studies using Ahr null mice. These animals are completely devoid of cyp1a1 gene expression and lack cyp1a2 inducibility, are impervious to B[a]P induced carcinogenesis, and resistant to TCDD-induced toxicity [66, 160]. Interestingly, Ahr null mice display multiple abnormalities of the heart, liver and immune system not observed in their wild-type counterparts. These results confirm an important developmental and physiological role(s) for the AhR in normal cellular biology. A role(s) which, in theory, could be regulated in terms of timing, duration and magnitude by a high affinity endogenous ligand.

1.5.1 AhR influences normal vascular development

In an attempt to elucidate the physiological role of the AhR in normal vertebrate biology, gene targeting has been used by several different research groups to construct

Ahr knockout mice. However, the use of unique targeting strategies and differences in genetic background has resulted in the generation of Ahr null animals that share some common characteristics, yet possess distinctive differences in phenotype [161]. Three distinct Ahr null animals have been independently generated and are currently being 46 studied. The original Ahr null animals, generated in the laboratory of Dr. Frank

Gonzalez, harbor an Ahr null allele inactivated by targeted replacement of exon 1 with a neomycin resistance gene. In addition to disruption of the first exon, the translational start site for AhR expression has also been deleted along with a stretch of basic amino acids potentially important in DNA binding [162]. In an independent attempt, the

Bradfield laboratory produced a second Ahr null animal through targeted disruption of exon 2 of the AhR locus. Unlike the aforementioned approach, this strategy fails to remove the translational start site for the AhR, but does effectively disrupt the bHLH domain of the receptor which is essential in dimerization and DNA-binding [163].

Recently, a third Ahr null animal, also generated through targeted replacement of exon 2, was produced in the lab of Dr. Fujii-Kuriyama [66]. For simplicity in the following discussion, the Gonzalez knockouts will be referred to as Δ1/Δ1 animals while Δ2/Δ2 will be used to represent the Bradfield knockout animals.

Initial studies using the Δ1/Δ1 AhR null animals reported a 50% mortality rate among new born pups. However, even though half of the mice died shortly after birth, the remaining littermates survived to maturity and were fertile. This mortality was not seen with other AhR null animals. Studies performed with the Δ1/Δ1 animals suggested a biological role for the Ah receptor in the normal development of the liver and immune system. In general, the Δ1/Δ1 knockout animals demonstrated a decreased accumulation of peripheral lymphocytes, most notably in the spleen and lymph nodes, along with several hepatic defects, including an approximate 50% reduction in liver size and the 47 presence of bile duct fibrosis. The level of fibrosis increased with time and by one year of age adenomas and carcinomas could sometimes be found in the livers of Ahr null mice

[162]. These animals also exhibited several additional liver pathologies not seen in other

AhR -/- animals including glycogen depletion, eosinophilia of periportal hepatocytes and inflammation of bile ducts [161].

Despite differences, a consistent observation among Ahr null mice is the importance of the AhR to liver development, appearing to affect both function and size throughout the life of the organism. In agreement with initial results obtained using the

Δ1/Δ1 null animals, a reduction in liver size was also seen in the Δ2/Δ2 null mice.

Further analysis revealed the observed reduction in liver weight is directly related to a reduction in hepatocyte size caused by a decrease in cytoplasmic volume. This decrease in liver size was eventually determined to result from a diminished portal blood supply, a consequence of the massive portosystemic shunting present in these animals [164]. Also known as a portacaval shunt or portosystemic vascular shunt, this condition is the most common of congenital liver anomalies. Most often the condition is the result of blood flowing through the portal vein directly into the systemic vascular system and hence bypassing the liver. This bypass of the liver is a normal condition during fetal development that should resolve itself shortly after birth. If it remains through adulthood it is known as a patent ductus venosus [165]. Remnants of neonatal vascular structure were also found in the eye and kidney of AhR null animals in the form of a persistent and extensive hyaloid artery and an exaggerated limbal vessel expansion, respectively [164].

Thus, in mice, the AhR is required for closure of the Ductus Venosus and resolution of 48 other neonatal vasculature structures into the more mature vascularity normally seen in adult organisms [166]. In general all Ahr null mice exhibit a decrease in liver size, mild hepatic fibrosis, a decreased growth rate, reduced fertility, loss of both CYP1A1 and

CYP1A2 induction, and a decrease in CYP1A2 constitutive expression [161]. In addition

Ahr -/- mice were deficient in their ability to catabolize retinoic acid with the livers of Ahr null mice containing elevated levels of retinoids [167].

1.5.2 AhR influences cardiac development

The generation of Ahr null mice has also revealed a potential role for the receptor in cardiovascular homeostasis [89]. Inactivation of the AhR has a profound impact on the cardiovascular system resulting in hypertension, progressive cardiac hypertrophy, fibrosis of the myocardium and enlargement of the arteries and arterioles, suggesting of a role for the AhR in cardiovascular physiology and disease [168]. The development of cardiac hypertrophy in Ahr null mice, evident by 5 months of age, is accompanied by a significant increase in left ventricular (LV) mass and expression of cardiac hypertrophy marker genes, such as β-myosin heavy chain and β-myosin light chain 2V [168, 169].

As a compensatory response of the heart to hemodynamic overload, increased blood pressure is believed to be the major determinate responsible for an increase in LV mass [170], although other factors may be associated with the clinical onset of LV growth. Consistent with this theory, cardiac hypertrophy in Ahr -/- mice also correlates

with an elevated mean arterial blood pressure along with an increase in the levels of 49 endothelin-1 (ET-1) and angiotensin II (Ang II) [168], potent vasoconstricting peptides and direct acting mitogens previously found to be associated with cardiomyocyte hypertrophy [171, 172]. Both ET-1 and Ang II are cardiac mitogens believed to induce their growth-promoting effects on the heart through an increase in the production of reactive oxygen species (ROS) [173]. Recently, it was demonstrated that the observed cardiac hypertrophy in AhR null mice is indeed associated with an increase in the production of ROS in heart tissue. Mechanistically, it appears as though ET-1 mediated activation of the ETA receptor is responsible for the increase in oxidative stress through

induction of NAD(P)H oxidase activity, resulting in elevated levels of cardiac ROS [174,

175]. Although the exact mechanism by which ROS functions to induce cardiac

hypertrophy remains unclear, it is possible that these molecules mediate a hypertrophic

response in cardiac tissue by acting as regulators of gene expression functioning either

through direct activation of G-proteins [176] or by altering the activity of other growth-

promoting signaling pathways, such as MAPKs [177].

1.6 EICOSANOIDS

1.6.1 Overview

Warranted by the discovery that certain eicosanoids, in particular lipoxygenase

metabolites, can activate the AhR, it is essential that an overview of eicosanoid

biochemistry is presented. Although all branches of the arachidonic acid (AA) cascade

will be covered, the focus will be centered on the various bioactive lipid mediators 50 generated through lipoxygenase metabolism. Eicosanoids are a family of naturally occurring, biologically active, low molecular weight, oxidized lipid molecules frequently derived from 5,8,11,14-eicosatetraenoic acid, a 20-carbon ω-6 essential fatty acid commonly known as AA [178]. Although it may be the most abundant precursor to eicosanoid biosynthesis in humans, AA remains only one of several suitable fatty acid substrates. In theory, any 20 carbon polyunsaturated fatty acid containing the required cic,cis- 1,4-pentadienyl functionality, such as 5,8,11-eicosatrienoic acid (mead acid),

8,11,14-eicosatrienoic acid (DGLA) or 5,8,11,14,17-eicosapentaenoic acid (EPA), could serve as a precursor to eicosanoid biosynthesis [179]. In addition to utilizing multiple essential fatty acid precursors, mammalian cells also contain a variety of oxygenating enzymes capable of generating specific eicosanoid metabolites through the precise incorporation of detailed biochemical information. Therefore, a diverse array of eicosanoid molecules can, in theory, be synthesized as a direct result of the many cell types and enzymatic pathways through which these essential fatty acid substrates may be metabolized. Formed by most cells in the human body, these lipid signaling molecules are not stored but rather synthesized de novo in response to cellular stimuli including, but not limited to, mechanical trauma, cytokines or growth factors [180]. Eicosanoids, unlike endocrine-type hormones, are not transported in the blood and therefore function as potent local hormones signaling at (autocrine) or immediately adjacent to (paracrine) their site of synthesis [180, 181]. Critical to overall human health, the eicosanoids play a central role in inflammation as well as the homeostatic control of numerous biological functions. Essential to cellular and organ physiology, they are capable of exerting 51 powerful physiological and pathological effects on the cardiovascular, pulmonary, reproductive, and digestive systems.

1.6.2 Arachidonic acid cascade

Arachidonic acid (AA) is a 20-carbon polyunsaturated fatty acid containing four double bonds with the last double bond occurring 6 carbons from the ω-terminus (20:4,

ω-6). These four double bonds provide critical reaction sites for a number of enzymatic transformations that culminate in the production of various eicosanoids molecules of significant biological and medical importance [182]. In resting cells, the majority of arachidonic acid is stored within the cell membrane esterified at the sn-2 position in the glycerol backbone of hormonally-sensitive phospholipid pools. Fatty acids esterified at this position are commonly released through the action of phospholipase A2 (PLA2)

[183]. This 85 kDa enzyme can become activated by calcium, translocate to cellular membranes, catalyzing the hydrolysis and subsequent release AA [184]. Once released, multiple fates await the mobilized arachidonate molecule. For example, it may become reincorporated back into membrane phospholipids, diffuse to the outside of the cell facilitating cell-cell communication or undergo enzyme catalyzed metabolism [185].

There are three distinct enzymatic pathways of AA metabolism, the cyclooxygenase, lipoxygenase and cytochrome P450 monooxygenase, all resulting in the oxidative metabolism and subsequent chemical transformation of AA into various eicosanoid molecules such as prostanoids, leukotrienes, lipoxins, hydroxyeicosatetraenoic acids 52 (HETEs) and epoxyeicosatrienoic acids (EETs) [186]. These pathways which form the backbone of the arachidonic acid cascade are outlined in Figure 1.7. 53

Figure 1.7: The arachidonic acid cascade. Illustrated are the cyclooxygenase, lipoxygenase and cytochrome P450 monooxygenase pathways of arachidonic acid metabolism. Although depicted for arachidonic acid, any polyunsaturated fatty acid with the required 1,4-pentadienyl structural element may serve as a substrate in these enzyme catalyzed reaction pathways. The oxidative metabolism of polyunsaturated fatty acids results in the biosynthesis of potent lipid mediators with many diverse and widespread effects.

54 1.6.3 Cyclooxygenase Metabolites

Insertion of molecular oxygen into the arachidonic acid backbone catalyzed by the prostaglandin endoperoxide synthases (PGHS), also known as the cyclooxygenases

(COXs), results in the first committed step of prostanoid biosynthesis [187]. Collectively known as prostanoids, the prostaglandins, thromboxanes and prostacyclins are three classes of eicosanoid molecules produced by the cyclooxygenase pathway. First identified from semen in the 1930’s, the prostaglandins are named for the prostate gland thought to be their source of origin. However, their relatively low abundance and instability prevented identification of their true EFA origin for nearly thirty years [188].

Two COX isoforms, a constitutively expressed COX-1 and an inflammation inducible COX-2 isoform, have been identified. Prostaglandin production by COX-1 occurs in most tissues and appears to be involved in physiological regulation of tissue homeostasis, such as in the gastric mucosa and cardiovascular system. Alternatively,

COX-2 is selectively induced at a limited number of tissue sites by the action of inflammatory mediators such as IL-1 and LPS [189]. Both COX enzymes contain endoperoxide synthase and peroxide reductase activity allowing these isoforms to catalyze the same two metabolic transformations. Through the insertion of two molecules of oxygen, the endoperoxide synthase activity of COX catalyzes the oxidation and subsequent transformation of arachidonic acid into prostaglandin G2 (PGG2), an unstable cyclic endoperoxide intermediate. The peroxidase activity of the enzyme then catalyzes the enzymatic reduction of PGG2 to prostaglandin H2 (PGH2) [190]. PGH2 is a 55 reactive lipophilic intermediate with a reasonably long half-life (90-100 sec) that can serve as the substrate for cell-specific prostaglandin synthases. Several of these enzymes have been identified capable of isomerizing PGH2 into biologically active prostaglandin

molecules including PGE2, PGD2 and PGF2. Alternatively PGH2 may be metabolized by

prostacyclin synthase or thromboxane synthase resulting in formation of PGI2 and TXA2

respectively [191]. Both PGI2 and TXA2 are chemically unstable molecules that will

rapidly degrade to the biologically inactive compounds TXB2 and PGF1α, respectively.

1.6.4 Lipoxygenase Metabolites

Lipoxygenases (LOX) are a heterogeneous family of lipid-peroxidizing enzymes

capable of catalyzing the stereospecific addition of molecular oxygen into the carbon

backbone of polyunsaturated fatty acids containing a cis,cis-1,4-pentadiene system. This

oxidation reaction results in the formation of the corresponding fatty acid hydroperoxide

[192]. LOX enzymes are widely distributed among animals, plants and fungi and can

even be found in certain species of cyanobacteria, but have never been identified in

typical prokaryotic bacteria or yeast, organisms which also lack suitable polyunsaturated

fatty acid substrates [193, 194] LOX enzymes are classified with respect to their

positional specificity for molecular dioxygen insertion and in theory six LOX families

may be distinguished including 5-LOX, 8-LOX, 9-LOX, 11-LOX, 12-LOX and 15-LOX.

However, in mammals, only four of these isoforms, 5-LOX, 8-LOX, 12-LOX and 15-

LOX, have been discovered to date [194]. The eicosanoid portion of this literature

review will focus primarily on metabolites produced by the 5-LOX, 12-LOX and 15- 56 LOX pathways, as it was these metabolites that were carefully scrutinized for Ah receptor activity.

The LOX catalyzed insertion of molecular oxygen occurs in three consecutive steps. The initial rate-limiting step is the stereoselective abstraction of hydrogen from a bisallylic methylene group generating a carbon centered fatty acid radical. Because most natural occurring polyenoic fatty acids contain multiple methylene interrupted double bonds, the selectivity of hydrogen abstraction occurs on a compounded level. The regioselectivity of the LOX isoform determines the particular bis-allylic methylene group targeted for hydrogen abstraction while the enantioselectivity of the enzyme controls whether the pro-S or pro-R hydrogen is removed during radical formation. Subsequent rearrangement of the pentadienyl radical, via electron redistribution, can occur toward either the methyl terminus (+2 rearrangement) or carboxy terminus (-2 rearrangement) ultimately resulting in formation of a conjugated cis, trans diene hydroperoxide. Finally, stereospecific insertion of molecular oxygen into the rearranged pentadienyl radical can occur at either the C-1 or C-4 positions, depending upon radical rearrangement, generating a fatty acid hydroperoxy radical. This radical is then reduced to the corresponding anion as the enzyme bound non-heme iron cofactor becomes oxidized back to the ferric state (as reviewed in [192, 194] ). Generation of this hydroperoxyl intermediate is the first step in the biosynthesis of more complex eicosanoid molecules such leukotrienes, lipoxins, hepoxilins, HETE’s and DiHETE’s. 57 1.6.4.1 Arachidonate 5-LOX metabolites

The 5-lipoxygenase (5-LOX) pathway was discovered by Borgeat and

Samuelsson in the late 1970’s. It is the source of powerful pro-inflammatory lipid mediators originating primarily from various leukocyte cell types and possessing cellular actions related to immediate hypersensitivity and inflammation [195]. As would be expected of a system that generates potent inflammatory molecules, the 5-LOX pathway is highly expressed in cells of myeloid origin including neutrophils, esinophils, basophils, monocytes, mast cells and macrophages [196] in response to a variety of stimuli such as antigens, cytokines, oxidants, microbes and toxins [197]. Cell stimulation resulting in the elevation of intracellular cytoplasmic calcium can activate the 5-LOX pathway by inducing translocation of both cPLA2 and 5-LOX enzymes toward intracellular

membranes. [198] [199]. In addition to promoting translocation, calcium is also required for maximal 5-LOX enzymatic activity. Once associated with the membrane, cPLA2 can catalyze the release of AA allowing the liberated molecule to be delivered to 5-LOX through the action of 5-lipoxygenase activating protein (FLAP). This small membrane bound protein is critical to 5-LOX activity and appears to function as an arachidonic acid

binding and transfer protein [199].

Activated 5-LOX preferentially catalyzes the oxygenation of unesterified

arachidonic acid through insertion of molecular oxygen at C-5 to generate 5(S)- hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5(S)-HpETE) [200].

Occupying a bifurcated position in the 5-LOX pathway, this pivotal hydroperoxide 58 intermediate, partitions between reduction to the corresponding monohydroxy fatty acid,

5(S)-hydroxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5(S)-HETE) and further enzymatic transformation by 5-LOX into the allylic epoxide leukotriene A4 [201].

Possessing dual catalytic activity, 5-LOX is an enzyme with both oxygenase and LTA4 synthase activity, and thus, is capable of catalyzing the two-step conversion of arachidonic acid to LTA4 (Figure 1.8) [202]. 59

Figure 1.8: The 5-Lipoxygenase Pathway. Outlined above is the 5-lipoxygenase pathway of arachidonic acid metabolism. Depicted are the structures of key intermediates such as 5(S)-HpETE and 5,6-LTA4, a transient molecule that can be subsequently metabolized through both enzymatic and non-enzymatic pathways. The result is the production of various DiHETE and cysteinyl-conjugated leukotriene metabolites

60 Reduction of 5(S)-HpETE to 5(S)-HETE may occur enzymatically through the action of glutathione peroxidases or non-enzymatically through hydroperoxide decomposition. 5(S)-HETE, in turn, may be further metabolized into the closely related

5-oxo-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-oxo-EET) through the action of 5- hydroxyeicosanoid dehydrogenase (5-HEDH) (Figure 1.8). This microsomal enzyme is highly selective for eicosanoids containing a 5(S)-hydroxyl group followed by a 6-trans double bond and thus displays little activity for other monohydroxy fatty acids. 5-oxo-

EET may also be formed through the direct dehydration of 5-HpETE, a reaction that is promoted by heme containing compounds like methemoglobin [203]. The oxidation of

5(S)-HETE to 5-oxo-EET is reversible, as microsomes from human polymorphonuclear leukocytes (PMNL), in the presence of NADPH, can reduce the ketone functionality, stereospecifically regenerating the 5(S)- hydroxyl [204]. Both compounds, however, are potent pro-inflammatory molecules that possess chemotactic and proliferative effects.

5(S)-HETE has been shown to be important in various diseases of the lung where it can induce airway constriction and stimulate mucus secretion [205]. 5-oxo-EET is chemotactic for both esinophils and neutrophils, although its primary target appears to be the esinophil as it is the strongest lipid chemoattractant for this cell type [203]. It elicits a multitude of cellular responses in these cells including calcium mobilization, actin polymerization, integrin expression and degranulation. Both of these compounds can also stimulate proliferation of prostate tumor cells [203, 206].

Alternatively, 5-HpETE can undergo 5-LOX catalyzed dehydration and rearrangement to generate the unstable allylic epoxide 5(S)-trans-5,6-oxido- 61 7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (LTA4) (Figure 1.8). This labile epoxide is a pivotal intermediate possessing several metabolic fates. It can react enzymatically with glutathione via glutathione-S-transferase (LTC4 synthase) to generate leukotriene C4

(LTC4) [207]. Collectively termed the cysteinyl leukotrienes (cysteinyl-LTs), LTC4 along with its metabolites LTD4 and LTE4 (Figure 1.9) are peptide conjugated

leukotrienes (LTs) and potent inflammatory mediators that comprise the slow reacting

substances of anaphylaxis (SRS-A) [207, 208]. The cysteinyl-LTs possess a myriad of

diverse biological functions including contraction of smooth muscle, secretion of mucus,

modulation of cytokine production and recruitment of allergic inflammatory cells. These compounds are important in the pathogenesis of human bronchial asthma responsible for mediating airway inflammation and bronchoconstriction resulting in a reduction of airflow to the alveoli [209]. 62

Figure 1.9: The Structures of Some Common Cysteinyl Leukotrienes. Shown above are the structures of several cysteinyl conjugated leukotrienes metabolites.

63

Alternatively, hydrolysis of LTA4, mediated either enzymatically by LTA4 hydrolase or cytosolic epoxide hydrolase, or non-enzymatically through the net addition of water, results in the formation of multiple dihydroxy metabolites (Figure 1.10). LTA4 hydrolase is a cytosolic bifunctional zinc-containing metalloenzyme capable of hydrolyzing the epoxide functionality contained in LTA4, thus generating the potent

chemotactic and aggregatory molecule, 5(S),12(R)-dihydroxy-6(Z),8(E),10(E),14(Z)-

eicosatetraenoic acid (LTB4) [210, 211]. LTB4 is a potent pro-inflammatory molecule

that has been proposed to play a critical role in a number of acute and chronic inflammatory disease states such as chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD) [211], cystic fibrosis [212] rheumatoid arthritis and

psoriasis [213]. Recent research also indicates a potential role for LTB4 pro- inflammatory activity in the pathogenesis of myocardial infarction [214]. 64

Figure 1.10: Hydrolysis products of leukotriene A4 (LTA4). Shown are the various enzymatic and non-enzymatically derived hydrolysis products of LTA4.

65

An alternative route of LTA4 enzymatic metabolism is via cytosolic epoxide hydrolase which catalyzes the opening of the LTA4 epoxide ring to generate erythro-

5(S),6(R)-dihydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (5(S),6(R)-DiHETE)

[215]. This molecule, which was found to be the most abundant hydrolysis product of

LTA4 in rat kidney homogenates [216], has weak LTD4 receptor agonist activity in

guinea pig lung membranes and is a modest stimulus for protein kinase C [217] [218].

Furthermore, a 5,6-DiHETE molecule with an all trans conjugated triene chromophore,

namely 5(S),6(R)-dihydroxy-7(E),9(E),11(E),14(Z)-eicosatetraenoic acid, has been

identified in rat kidney [216]. Formation of this molecule from 5(S),6(R)-DiHETE

appears to be catalyzed by a membrane bound factor through direct cis-trans

isomerization of the C-11 double bond.

LTA4 is a highly unstable chemically reactive epoxide molecule possessing an

extremely short half-life (< 3 sec) in aqueous buffer at physiological pH (7.4) and

temperature (37oC) [219]. Because of its aqueous instability, non-enzymatic hydrolysis

of the LTA4 epoxide ring can result in the formation of multiple dihydroxy metabolites.

Shown in figure 1.10, the major non-enzymatic hydrolysis products of LTA4 are

5(S),12(R)-dihydroxy-6(E),8(E),10(E),14(Z)-eicosatetraenoic acid (6-trans-LTB4) and its

C-12 epimer 5(S),12(S)-dihydroxy-6(E),8(E),10(E),14(Z)-eicosatetraenoic acid (6-trans-

12-epi-LTB4) [220]. These 5,12-DiHETE metabolites, epimeric at C-12, are the all trans

triene isomers of LTB4. Non-enzymatic hydrolysis of LTA4 can result in the formation

of 5(S),6(R)-dihydroxy-7(E),9(E),11(Z),14(Z)-eicasotetraenoic acid (5(S),6(R)-DiHETE) and its C-6 epimer 5(S),6(S)-dihydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid 66 (5(S),6(S)-DiHETE) [217]. Thus, 5(S),6(R)-DiHETE is a dihydroxy metabolite that can be formed through both enzymatic and non-enzymatic transformation of LTA4.

However, the amount of non-enzymatic hydrolysis occurring in the cell, may in fact, be minimal. Enzymatically formed products such as LTB4 and cysteinyl-LTs appear to

predominate after the addition of exogenously added LTA4 with only minimal amounts of

non-enzymatic products such as 5(S),6(S)-DiHETE, 6-trans-LTB4 and 6-trans-12-epi-

LTB4 being observed [221].

1.6.4.2 Arachidonate 8-LOX metabolites

8(S)-lipoxygenase (8(S)-LOX) is a recently characterized mammalian

lipoxygenase shown to be expressed in mouse skin after irritation or treatment with tumor

promoters [222]. This enzyme catalyzes the stereospecific oxygenation of AA at position

C-8 forming an 8(S)-hydroperoxy intermediate that rapidly decomposes to generate 8(S)-

hydroxy-5(Z),9(E),11(Z),14(Z)-eicosatetraenoic acid (8(S)-HETE). 8(S)-HETE may also

be generated by non-enzymatic lipid peroxidation or through a P450 monooxygenase

catalyzed reaction [223, 224]. These sources are believed to be responsible for the 8(S)-

HETE detected in human neutrophils, psoriatic skin, human tracheal epithelial cells and

squamous head and neck carcinomas. 8(S)-LOX can also catalyze, with lower efficiency,

the insertion of oxygen into the 9(S) position of linoleic acid [225].

Although 8-HETE can be detected, the 8(S)-LOX enzyme, however, has never

been identified in human tissues. Nevertheless, based on approximate 78% sequence 67 identity along with the structure and chromosomal localization of the corresponding gene,

15(S)-LOX-2 is regarded as the human orthologue of murine 8(S)-LOX [226]. The obvious difference in positional specificity is believed to result from a change of only two amino acid residues responsible for determining the orientation of AA binding. If AA can penetrate head first into the binding pocket oxygenation will occur on C-8, while tail first binding places C-15 in position to be oxidized. So, if tyrosine 603 and histidine 604 of 8(S)-LOX are changed to asparagine and valine respectively, the corresponding residues in 15-LOX-2, the positional specificity of the enzyme is converted from 8(S) to

15(S) [225].

1.6.4.3 Arachidonate 12-LOX metabolites

Originally identified in human and bovine platelets, arachidonate 12- lipoxygenase (12-LOX) was the first documented lipoxygenase in the animal kingdom, although several different isoforms from various sources have since been discovered and characterized. Despite displaying heterogeneity with regard to factors such as substrate specificity and product profiles, all 12-LOX enzymes can catalyze the regiospecific insertion of molecular oxygen at carbon 12 of arachidonic acid to generate a 12- hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HpETE) intermediate [227]. However, because both 12(S)-and 12(R)-lox isoforms have been identified, the stereochemical orientation of the 12-HpETE intermediate depends upon the specificity of the lipoxgenase enzyme. A comparison of some of the biochemical properties of the different 12-lipoxygenase enzymes are shown (Table 1-1). 68

Table 1-1: Biochemical Properties of 12-lipoxygenase Enzymes

Table 1-2. A summary of the common biochemical properties attributed to the different 12-lipoxygenase enzymes including stereospecificity of oxygenation, tissue distribution in humans and mice, and preferred fatty acid substrates [227-229]. 69 Three 12(S)-lipoxygenase isoforms and a single 12(R)-lipoxygenase enzyme have thus far been identified. Named for the cells in which they were originally discovered, the 12(S)-lipoxygenase isoforms are classified as platelet, leukocyte, and epidermis . The murine leukocyte-type 12(S)-LOX is an enzyme widely distibuted among cells. It is capable of oxygenating a broad variety of substrates ranging from free polyenoic fatty acids of 18-20 carbon atoms, such as linoleic, linolenic and arachidonic acids, to more complex substrates like phopsholipids and cholesterol esters commonly found in cellular membranes and lipoprotein particles [227]. As a result of its broad substrate specificity, the product profile for this lipoxygenase is also quite complex.

Furthermore, in addition to producing the characteristic 12(S)-hydroperoxy intermediate from arachidonic acid like other 12-LOX enzymes, it can also generate a substantial amount of 15(S)-HpETE product. The ratio of these products, however, is enzyme dependent and varies considerably among species. Ratios of approximately 3:1 for murine leukocyte-type 12-LOX to 11:1 for bovine tracheal 12-lipoxygenase have been observed [230]. Based on similiarities in enzymatic structure and function, human 15-

LOX-1 also expressed in multiple tissues, is the human homologue of this murine lipoxygenase [231, 232].

In contrast, the platelet-type 12(S)-LOX enzyme of both human and mouse origin has a much more limited tissue distribution having only been identified in platelets and skin. It possesses a higher substrate specificity being essentially inactive towards complex esterified subtrates and 18 carbon fatty acids [233]. The platelet-type enzyme preferentially utilizes arachidonic acid as substrate catalyzing the formation of 12-HpETE 70 in a reaction that proceeds almost linearly for over 30 minutes. This is also different from the leukocyte-type 12(S)-LOX catalyzed reaction which is terminated after only a few minutes as the enzyme loses catalytic effiency due to suicide inactivation [228].

Remarkably, the murine epidermal 12(S)-LOX isoform prefers to metabolize arachidonic and linoleic fatty acid methyl esters (FAME), compounds unable to serve as high affinity substrates for other LOX isozymes, while demonstrating little if any activity toward the corresponding free fatty acids. Although this epidermal 12(S)-LOX isoform appears restricted in its tissue distribution compared with other murine LOX isoforms, its expression has been demonstrated in differentiated keratinocytes of mouse epidermis, the root sheath and bulb of hair follicles and the conjunctiva of the eyelid and sebaceous gland [234] However, due to the lack of a functional epidermal 12(S)-LOX gene, no expression of this isoform has been detected in humans with the exception of a psuedogene expressed in the skin and hair follicles [234, 235].

Recently a human 12(R)-lipoxygenase has been cloned and expressed [229].

This novel human enzyme is the first described lipoxygenase enzyme capable of generating hydroperoxy and subsequent hydroxy molecules of the (R) stereochemical configuration. 12(R)-HETE is an eicosanoid previously known to be synthesized via the

CYP450 pathway of arachidonic acid metabolism. It has been detected in human psoriatic skin lesions and it now appears as though its formation can be the result of either a lipoxygenase or CYP450 biosynthetic route.

71 With several different 12-LOX isoforms, each possessing its own unique substrate specificity, there are numerous eicosanoid molecules that can be generated by this pathway. Tissue specificity of the various isoforms also determines which 12-LOX metabolites a cell can produce. In this review I will focus only on the common 12-LOX metabolites.

Initiated upon production of a 12-HpETE molecule, the 12-LOX pathway immediately becomes bifurcate at this point. The primary hydroperoxy intermediate can easily decompose via any of several different routes, including both selenium dependent and independent glutathione peroxidase systems, to generate the corresponding hydroxy acid, 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE) [236]. Alternatively, enzymatic isomerization of 12-HpETE catalyzed by hepoxilin synthase can generate a pair of epoxy-hydroxy containing fatty acid molecules known as hepoxilin A3 (HxA3) and hepoxilin B3 (HxB3). These molecules have been detected in neutrophils, platelets

and pancreatic islet cells as well as brain, lung and aortic tissue [237]. Although, both

hepoxilin isomers contain a trans-epoxide functionality, only hepoxilin A3, 8 (S/R)-

hydroxy 11(S),12(S)-epoxy-5(Z), 9(E), 14(Z) eicosatrienoic acid, is biologically active

with no cellular activity having been demonstrated for hepoxilin B3, 10(S/R)-hydroxy-

11(S),12(S)-epoxyeicosa-5Z,8Z,14Z-trienoic acid [238, 239]. As a result of the oxido

ring, hepoxilin A3 is an intrinsically unstable molecule capable of being further

metabolized. Opening of the epoxide ring mediated by epoxide hydrolase or through acid

catalyzed hydrolysis will generate a trihydroxyeicosatrienoic acid (TriHETrE) molecule,

trivially termed trioxilin A3 [237, 238]. Although hepoxilin B3 is thought to be more 72 resistant to hydrolysis [238], its corresponding trihydroxy derivative, trioxilin B3, has been detected in normal human epidermis [240] with elevated levels having been observed in psoriatic lessions [241]. In addition to hydrolysis, the epoxide functionality of hepoxilin A3 can also undergo GST catalyzed conjugation with glutathione to generate

11-glutathionyl HxA3 (HxA3-C), a bioactive peptido-hepoxilin metabolite [242].

Recently the R-isomer of HxA3, 8(R)-hydroxy-11(R),12(R)-epoxy-5(Z),9(E),14(Z)-

eicosatrienoic acid, generated from 12(R)-HpETE via the isomerase action of mammalian epidermal LOX-type 3 (eLOX3) has been identified [243]. This enzyme, coexpressed along with 12(R)-LOX mainly in the skin, lacks the typical lipoxygenase catalytic

activity, but appears to still possess the epoxyalcohol (hepoxilin) synthase activity.

12(S)-HETE, such as that generated by platelets and leukocytes, can be further metabolized in several different ways. In human polymorphonuclear leukocytes

(PMNL’s), for example, ω-oxidation of 12(S)-HETE generates 12(S),20-DiHETE while a second oxidation event catalyzed by 5-LOX results in the formation of the dual lipoxygenase product 5(S),12(S)-dihydroxy-6(E),8(Z),10(E),14(Z)-eicosatetraenoic acid

(5(S),12(S)-DiHETE), a stereoisomer of LTB4 [244, 245]. A similar 5(S),12(S)-

DiHETE molecule containing an all trans conjugated triene chromophore (6-trans-12- epi-LTB4) can be generated by the non-enzymatic hydrolysis of LTA4 as discussed earlier. Additional 12(S)-HETE metabolites can be formed through enzyme catalyzed reduction of the 10,11 double bond resulting in the formation of 12-hydroxy-

5(Z),8(Z),14(Z)-eicosatrienoic acid (12(S)-HETrE), also known as 10,11-dihydro-12(S)-

HETE. This compound, along with its oxidized derivative 12-oxo-5(Z),8(Z),14(Z)- 73 eicosatrienoic acid (10,11-dihydro-12-oxo-ETE) have been identified in porcine PMNLs.

[246]. Finally, chain shortened metabolites such as 8(S)-hydroxy-4(Z),6(E),10(Z)- hexadecatrienoic acid (8(S)-HHxTrE), commonly known as tetranor 12(S)-HETE, can be formed through peroxisomal mediated β-oxidation of 12(S)-HETE [247].

Even though it may be generated from AA through 12(R)-LOX metabolism or as the product of an allylic oxidation catalyzed by a CYP450, the subsequent metabolism of

12(R)-HETE has been demonstrated to be similar to that of its 12-LOX produced (S) enantiomer. Many of the same metabolites generated from 12(S)-HETE can also be formed in the opposite stereochemical orientation from 12(R)-HETE. For example tetranor 12(R)-HETE and 12(R)-HETrE have been identified in corneal tissue of the eye

[248]. 12(R)-HETE may also undergo ω-terminal oxidation generating 12(R),20-

DiHETE or serve as a substrate for human 5-LOX, with the resulting product being

5(S),12(R)-dihydroxy-6(E),8(E),10(E),14(Z)-eicosatetraenoic acid (6-trans-LTB4) [236].

However, despite similiarities in the metabolism of both 12-HETE isomers, the biological activity of the two enantiomers can differ significantly. For example, although both molecules possess chemotactic and chemokinetic properties, 12(R)-HETE is much more potent, demonstrating approximately 20x more activity than 12(S)-HETE in vitro

[249]. Furthermore, unlike its 12(S)- enantiomer, only 12(R)-HETE can inhibit Na+-K+-

ATPase activity in the cornea, kidney, heart and blood vessels [250, 251] and modulate

vascular tone in rabbit aortic ring preparations [236]. Conversely, 12(S)-HETE has been

shown to be vital to the survival and metastatic process of certain cancer cells [252] with 74 the invasiveness of tumor cells being positively correlated with their ability to produce

12(S)-HETE [253]. Experimental evidence suggests that 12(S)-HETE functions to augment tumor cell metastatic potential through modulation of protein kinase C (PKC) signaling [254]. The structures of both 12(S)- and 12(R)-HETE are shown in

Figure 1.11 along with the structures of some other common HETE isomers for

comparison. 75

Figure 1.11: Hydroxyeicosatetraenoic acids (HETEs). Depicted are the chemical structures of several common mono-HETEs. Each of these molecules was investigated for their potential to activate the Ah receptor signaling.

76 1.6.4.4 Arachidonate 15-LOX metabolites

It was once believed that the arachidonate 15-lipoxygenase isoform did not exist in animal tissues. However, in 1975 the discovery of a 15-LOX enzyme from rabbit reticulocytes reversed this outstanding view [255]. This was followed over a decade later by the characterization of a human 15-LOX ortholog from eosinophils [256]. Because both human and rabbit reticulocyte-type 15-LOX (15-LOX-1) share a high level of similarity with leukocyte-type 12-LOX, they have been classified as a 12/15- lipoxygenase (12/15-LOX). In addition, although 15-LOX-1 oxygenates AA primarily at position 15, it will, to a lesser extent, also oxygenate the fatty acid at position 12.

Recently a second 15-LOX isoform, termed epidermis-type 15-LOX or 15-LOX-

2, has been identified from human root hairs. Expression of this enzyme has been demonstrated in tissues such as skin, prostate, lung and cornea [226]. It shares little sequence homology with reticulocyte-type 15-LOX and subsequently its enzymatic properties also strongly differ as it exclusively generates only the 15(S)-hydroperoxy intermediate from AA [257]. Unlike with 15-LOX-1, LA is a poor substrate for this enzyme and is subsequently less well metabolized [226].

Expressed in numerous animal tissues, 15-lipoxygenases (15-LOXs) are lipid peroxidizing enzymes capable of catalyzing with positional specificity the stereospecific introduction of molecular dioxygen into both free and esterified polyenoic fatty acids substrates to generate an ω-6 hydroperoxy fatty acid derivative [257, 258]. As with other 77 LOX isoforms, introduction of oxygen induces double bond migration and the subsequent formation a conjugated diene in the resulting hydroperoxy intermediate. Using arachidonic acid as a substrate, 15-LOX catalyzes oxygen insertion at position C-15 generating 15(S)-hydroperoxy-5(Z),8(Z),11(Z),13E-eicosatetraenoic acid (15-HpETE).

This unstable hydroperoxy intermediate is rapidly reduced to the corresponding hydroxy metabolite, 15(S)-hydroxy-5(Z),8(Z),11(Z),13E-eicosatetraenoic acid, more commonly referred to as 15(S)-HETE (Fig 1.11).

As a result of the intrinsic leukotriene synthase activity of reticulocyte 15-LOX,

15(S)-HpETE can be dehydrated and alternatively metabolized to 14(S),15(S)-trans- oxido-5(Z),8(Z),10(E),12(E)-eicosatetraenoic acid (14,15-LTA4) [259]. Enzymatic

hydrolysis of the leukotriene epoxide intermediate produces a single major product with

both hydroxyl groups originating from molecular oxygen. Formation of this dihydroxy

metabolite is catalyzed by cytosolic epoxide hydrolase cleavage of the trans epoxide ring

of 14,15-LTA4 in a trans manner to form, erythro-14(R),15(S)-dihydroxy-

5(Z),8(Z),10(E),12(E)-eicosatetraenoic acid (14(R),15(S)-DiHETE) [260]. In addition,

both this compound and its C-14 epimer threo-14(S),15(S)-dihydroxy-

5(Z),8(Z),10(E),12(E)-eicosatetraenoic acid (14(S),15(S)-DiHETE) can also be formed in

minor amounts through the non-enzymatic hydrolysis of 14,15-LTA4 [261]. These

products, however, contain hydroxyl groups at position C-14 derived from water rather

than molecular oxygen. Additional dihydroxy metabolites produced by non-enzymatic hydrolysis of the unstable allylic epoxide intermediate are 8(R),15(S)-dihydroxy-

5(Z),9(E),11(E),13(E)-eicosatetraenoic acid (8(R),15(S)-DiHETE) and 8(S),15(S)- 78 dihydroxy-5(Z),9(E),11(E),13(E)-eicosatetraenoic acid (8(S),15(S)-DiHETE). Both of these 8,15-DiHETE epimers contain an all trans conjugated triene chromophore with the

C-8 hydroxyl groups derived from water [262].

Because of the two doubly allylic methylene groups, 15(S)-HETE can serve as a lipoxygenase substrate and undergo additional oxygenation generating secondary metabolites such as DiHETEs and lipoxins respectively. Dihydroxy metabolites produced by dual lipoxygenase mechanism include 8(S),15(S)-dihydroxy-

5(Z),9(E),11(Z),13(E)-eicosatetraenoic acid (8(S),15(S)-DiHETE) produced through

8(S)-LOX oxygenation of 15(S)-HETE. It is important to note, the trans, cis, trans

(E,Z,E) conjugated triene chromophore contained in this compound differs from the all trans triene contained in the non-enzymatically produced 8,15-DiHETEs. Also produced through a dual lipoxygenase mechanism is 5(S),15(S)-dihydroxy-6(E),8(Z),11(Z),13(E)- eicosatetraenoic acid (5(S),15(S)-DiHETE) produced either by 5(S)-LOX oxygenation of

15(S)-HETE or 15(S)-LOX oxygenation of 5(S)-HETE [263].

First described in human leukocytes, the lipoxins are a group of inflammation resolving bioactive lipid mediators containing three hydroxyl groups and a conjugated tetraene chromophore, produced as a result of lipoxygenase interaction [264]. Their synthesis has since been demonstrated in cells from bovine, porcine and rat including basophils and macrophages [265]. Formation of these unique anti-inflammatory molecules can be accomplished through multiple biosynthetic pathways. Both the basic 79 chemical structure of the lipoxins shown in Figure 1.12 and the means of generating these

compounds appear to have been conserved throughout the course of evolution [264]. 80

Figure 1.12: The Structures of Some Common Lipoxin Molecules. Depicted are the structures of several common lipoxin metabolites. These anti-inflammatory molecules are produced through the interaction of multiple lipoxygenase enzymes.

81 Transcellular biosynthesis is an important cellular method of generating lipid- derived mediators. Oxygenated products of arachidonic acid metabolism can be transferred among cells during cell-cell interactions resulting in the formation of additional biologically active compounds. Initial studies performed with airway epithelial cells and monocytes indicated lipoxin biosynthesis as occurring through a 15-

LOX pathway with 15(S)-HpETE or 15(S)-HETE serving as a substrate for subsequent neutrophil 5-LOX oxygenation. The result of this dual lipoxygenase oxygenation is the formation of a 5(S),6(S)-epoxy-15(S)-hydroxy-7(E),9(E),11(Z),13(E)-eicosatetraenoic acid intermediate, which is ultimately resolved to produce 5(S),6(R),15(S)-trihydroxy-

7(E),9(E),11(Z),13(E)-eicosatetraenoic acid (LXA4) and its positional isomer

5(S),14(R),15(S)-trihydroxy-6(E),8(Z),10(E),12(E)-eicosatetraenoic acid (LXB4) [266].

Although the all trans isomers of these compounds have also been detected, LXA4 and

LXB4 remain the major lipoxin products formed in-vivo [267].

Occurring primarily in the vasculature, where platelets are accepted as adhering to

neutrophils, is a second pathway of lipoxin biosynthesis. Here 5-LOX produced LTA4 released from leukocytes can be rapidly absorbed by platelets and converted through 12-

LOX metabolism to the aforementioned 5(S),6(S)-epoxytetraene intermediate and subsequently into the lipoxins [264]. This pathway becomes increasingly more relevant when platelets are under hypoxic conditions or contain diminished levels of glutathione

[268].

82 In addition, an aspirin-triggered 15-epi-lipoxin pathway has recently been identified as a third major route to lipoxin biosynthesis. In this scenario, acetylation of cyclooxygenase-2 (COX-2) blocks prostaglandin production and induces a switch in enzymatic activity favoring the production of 15(R)-HETE. After its release from endothelial or epithelial cells, 15(R)-HETE can be further metabolized in a transcellular manner by leukocyte 5-LOX into the aspirin-triggered lipoxins (ATL). These compounds are the 15(R) epimers of LXA4 and LXB4 and share the same potent

inflammation resolving properties. However, the 15(R) lipoxins, as a result of their C-15

chirality, are enzymatically inactivated to the keto form at a much slower rate allowing

the molecule to remain active over a longer period of time. Thus, it appears as though

aspirin, in addition to its well characterized mechanism of inhibiting prostaglandin

synthesis, can also trigger the formation of novel biologically active lipid molecules (as

reviewed [268]). Contrasting in biological activity compared with most other bioactive lipids, the lipoxins, especially LXA4 and 15(R)-epi-LXA4 and are potent inflammation

resolving molecules providing counter-regulatory signals against the many endogenous

pro-inflammatory mediators.

Also generated by aspirin acetylated COX-2 are several of the resolvin molecules,

a new family of potent anti-inflammatory lipid mediator derived from long chain ω-3

PUFAs. Together with a related group of metabolites termed protectins, these pro-

resolving molecules are responsible, at least in part, for programming the termination of

an acute inflammatory response [268]. Distinctive and highly stereospecific, these

bioactive lipid mediators are characterized according to their chemical structure with 83 resolvin molecules of the 18(R) E series (RvE) being generated from eicosapentaenoic acid (EPA) by aspirin acetylated COX-2 oxygenation, while resolvins of the 17(R) D- series (AT-RvD) are made in a similar manner using docosahexaenoic acid (DHA) as the substrate molecule. Conversely, resolvin molecules of the 17(S) D-series are produced through lipoxygenase metabolism of DHA. Additionally, dihydroxy metabolites of DHA containing a conjugated triene structure, a docosatriene (DT), are termed protectins or neuroprotectins when generated in neural tissues [269]. Because of their unique ability to terminate inflammation while actively directing the molecular events involved in resolution, these recently discovered bioactive lipids provide exciting new therapeutic opportunities in the treatment and prevention of inflammatory diseases.

1.6.5 Cytochrome P450 monooxygenase metabolites

A third pathway of arachidonic acid metabolism, the cytochrome P450 monooxygenase pathway, is a family of greater than 100 isozymes that can catalyze the in vivo metabolism of arachidonic acid using NADPH and molecular oxygen in a 1:1 stoichiometry. Three types of cytochrome P450 catalyzed oxidative reactions are known to occur. Olefin epoxidation, also known as the epoxygenase reaction, generates 4 sets of regiospecific epoxyeicosatrienoic acids (EETs) that include 5,6-EET, 8,9-EET, 11,12-

EET and 14,15-EET. Each of these four EET regioisomers can be synthesized as either the R,S or S,R enantiomer [270]. These molecules can be subsequently hydrolyzed to their corresponding dihydroxy derivatives (DHETs) either specifically through the action of epoxide hydrolase or non-enzymatically [271]. Bis-allylic oxidation, a second type of 84 P450 catalyzed oxidative reaction, is a lipoxygenase-like reaction leading to the formation of midchain hydroxyeicosatetraenoic acids (HETEs) containing a cis,trans- conjugated dienol functionality [272]. Six regioisomeric HETEs with hydroxylations at positions 5, 8, 9, 11, 12, and 15 have all been identified. These metabolites have been demonstrated to form in liver microsomal fractions as near racemic mixtures with the clear exception of 12-HETE which was enantioselectively formed as (R) isomer [273].

The final type of cytochrome P450 catalyzed oxidation reaction are the omega (ω) and omega-1 (ω-1) terminal hydroxylations responsible for producing the C16-C20 alcohols of

arachidonic acid [270]. These omega terminal HETEs are produced by CYP1A and

CYP4A isozymes and as a result contain hydroxyl groups that are produced directly,

differing from lipoxygenase catalyzed hydroxylations which proceed through formation

of a hydroperoxy intermediate. In addition, the ω- and ω-1 hydroxylase enzymes lack the

requirement for the presence of cis double bonds.

1.7 OBJECTIVES AND SIGNIFICANCE OF RESEARCH

A role for the AhR in normal cellular biology has been previously postulated and

substantiating scientific evidence is constantly accumulating. It is nearly for certain that

the AhR plays a crucial biological role(s) in normal cellular metabolism and that one or

more high affinity endogenous ligands exist to modulate the timing, duration and

magnitude of its function in the cell. Although several endogenous compounds

possessing low affinity for the receptor have been identified, no high affinity endogenous 85 ligand has yet been discovered. Thus, a key question still remaining to be resolved is the identity of a true high affinity endogenous regulator for the AhR.

1.7.1 Identification of an endogenous ligand(s) for the Ah receptor

The identification of a true high affinity physiologically important endogenous ligand for the AhR will greatly improve our understanding of the biochemical role(s) for this orphan receptor in normal cellular metabolism. Studies conducted with AhR-/- mice support the hypothesis that the AhR performs critical functions in normal cellular growth and differentiation independent of its response to environmental pollutants. Identification of such endogenous compounds capable of regulating AhR activity will allow the physiological role(s) for the AhR to be more precisely elucidated. Furthermore, the identification of an endogenous AhR ligand will provide insight into the mechanism responsible for the deleterious effects seen with persistant AhR activation. Ultimately this information may lead to the development of new highly effective therapuetic agents.

1.7.2 Characterization of the endogenous modulator responsible for the elevated AhR activity in CV-1 cells

To thoroughly investigate the mechanism responsible for the high level of constituative AhR activity commonly observed in the CV-1 cell line. These cells derived from the kidney of the african green monkey display activated AhR in the absence of an obvious exogenous ligand. Theoretically, assuming that AhR activation requires ligand, these observations can be interpreted as indirect evidence for the existence of a potent 86 endogenous AhR ligand(s) in these cells. Purification and structural characterization of the endogenous ligand(s) or modulator(s) present in this cell line will be attempted.

1.7.3 Investigate the potential auto-regulatory pathway controlling AhR activity in CV-1 cells through modulation of endogenous ligand levels.

To investigate the effect of AhR regulated P450’s from the CYP1 family on the level of constitutive AhR activity observed in the CV-1 cell line. In theory, metabolic alteration of a putative endogenous ligand, would subsequently affect the level of constitutive AhR activity in these cells, thus supporting the existence of an AhR mediated cytochrome P450 auto-regulatory pathway capable of controlling Ah receptor activity through modulation of endogenous ligand(s) levels. It would also provide additional evidence supporting the existence of an endogenous ligand(s) for the AhR. In addition, the characterization of any identified feedback loops may provide insight into the types of molecules that maybe serving as endogenous ligands or modulators for the AhR. A summary of pertinent characteristics possessed by the CV-1 cell line and the subsequent hypothesi derived from this information that inspired the initial portion of this thesis research project are presented (Figure 1.13).

87

Figure 1.13: Summary of CV-1 Cell Line Characteristics and Initial Project Hypothesis. The CV-1 cell line exhibits a minimal level of AhR expression resulting in a decreased level of AhR-regulated CYP450 metabolic enzymes. Although these cells contain only a minimal level of receptor, they possess an AhR pool with high constitutive activity possibly due to an increased level of endogenous ligand(s) or modulator(s). Accumulation of metabolites could be expected under conditions of reduced CYP450 metabolic activity. Therefore, it was hypothesized that expression of AhR-regulated CYP450 enzymes would decrease the level of endogenous AhR activators resulting in decreased AhR activity.

88

CHAPTER 2

CHARACTERIZATION OF A PUTATIVE ENDOGENOUS LIGAND FOR THE AHR IN CV-1 CELLS

89 2.1 ABSTRACT

An essential role for the AhR in cellular biology has been previously established, but no high affinity, physiologically-relevant endogenous ligand has yet been identified.

We have confirmed the existence of a putative endogenous ligand(s) in CV-1 cells capable of being metabolized and inactivated by AhR-regulated cytochrome P450 metabolism. Expression of cytochrome P450s 1A1, 1A2 or 1B1 reduced AhR-mediated luciferase reporter activity, while cytochrome P450 2E1, an isoform independent of AhR regulation, exhibited no significant effect. Studies with 2,4,3’,5’-tetramethoxystilbene

(TMS), a potent and specific inhibitor of cytochrome P450 1B1, was partially blocked cytochrome P450 1B1-mediated reduction in reporter gene activity. These results provide evidence of the existence of a possible feedback mechanism in which AhR regulated cytochrome P450s from the CYP1A and CYP1B families are able to metabolically alter putative endogenous ligand(s). Several experiments were performed to provide initial characterization of these putative endogenous ligands including electrophoretic mobility shift assay (EMSA) analysis which demonstrated that these ligands can directly activate the AhR. Soluble extracts from various C57BL/6J and Ahr- null mouse tissues were also analyzed for the presence of AhR activators. Ahr-null mouse lung tissue demonstrated a four-fold increase in AhR-mediated reporter activity in cells. Quantitative PCR analysis revealed that lung tissue exhibits relatively high constitutive CYP1A1 mRNA levels. These results suggest that there is an autoregulatory feedback loop between the AhR and cytochrome P450 1A1 in mouse lung.

90 2.2 INTRODUCTION

The aryl hydrocarbon receptor (AhR) is a ligand-activated basic helix-loop-helix

(bHLH) transcription factor and member of the bHLH /PAS (basic helix loop helix Per-

Arnt-Sim) family of DNA binding regulatory proteins [9, 274, 275]. In the absence of ligand, the Ah receptor resides in the cytosol in an oligomeric 9S protein complex.

Contained in this core complex are the AhR ligand-binding subunit, a dimer of the 90 kDa heat shock protein and a single molecule of the immunophilin like X-associated protein 2 (XAP2) and p23 [28, 34, 276]. Ligand binding induces receptor translocation into the nucleus followed by dissociation of the 90 kDa heat shock protein. Once in the nucleus the ligand activated AhR dimerizes with ARNT (aryl hydrocarbon receptor

nuclear translocator) forming a high affinity DNA binding complex capable of binding

specific target DNA sequences known as dioxin responsive elements (DRE) in the

regulatory region of responsive genes [31]. Interaction with these response elements

results in the alteration of gene expression. Genes transcriptionally regulated by the AhR are primarily involved in foreign chemical metabolism and include the xenobiotic metabolizing cytochrome P450 enzymes from the 1A and 1B families [277, 278].

Additional AhR-regulated genes include glutathione S-transferase Ya subunit [279],

NAD(P)H-menadione oxidoreductase [147], UDP- glucuronosyltransferase [280],

aldehyde-3-dehydrogenase [151], and prostaglandin endoperoxide H synthase-2 gene

[281].

91 Induction of DRE-responsive genes results primarily from ligand-activation of the

AhR signal transduction cascade. There are many types of compounds, ranging from widespread and potentially toxic man-made environmental contaminants to dietary metabolites formed in the acidic environment of the stomach that can serve as regulators of the AhR. Halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons are two of the most potent classes of AhR ligands known and include such carcinogenic compounds as polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and benzo[a]pyrene. These compounds are produced through various industrial processes, including chlorine bleaching of wood pulp, waste incineration, incomplete combustion of fossil fuels, metal production and synthesis of organochlorine products [4]. In addition to synthetic exogenous ligands, it is well documented that dietary derived indole-containing compounds including indole-3-carbinol (I3C) [71], oxidized carotenoids such as canthaxanthin and astaxanthin [84], and heterocyclic amines produced during the cooking of meat [78] can all function as AhR ligands. Perhaps even more significant is the formation of indolo[3,2b]carbazole, a metabolite of I3C produced via acid condensation in the stomach, and capable of functioning as a potent AhR ligand possessing an affinity for the receptor only one order of magnitude less potent than the highly toxic carcinogen

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [71, 73]. Any toxicological or biological significance of long-term exposure to these rapidly metabolized dietary-derived ligands of the AhR remains to be determined.

An important question that remains unresolved is the identity of key endogenous regulators of the AhR. Several endogenous compounds possessing low 92 affinity for the receptor have been identified, including the heme breakdown products bilirubin and biliverdin [90, 91], 7-ketocholesterol, a cholesterol derivative [99], and several arachidonic acid metabolites, including lipoxin A4 [96], along with various prostaglandins [95]. However, no high affinity endogenous AhR ligand has yet been characterized that can activate the AhR at physiologically relevant conditions, despite the accumulation of evidence supporting their existence. For example, activation of AhR dependent processes in the absence of exogenously added ligand has been reported with animal cells in culture [38, 87, 88, 282]. Assuming that AhR activation requires ligand, these observations can be interpreted as indirect evidence for the existence of an endogenous ligand(s), present at sufficient concentrations to lead to significant levels of

AhR activation. Furthermore, studies utilizing Ahr-null mice have provided, quite possibly, the most conclusive evidence supporting an endogenous biological role for the receptor. Three independent laboratories have each generated Ahr-null mice and, although differences in phenotype have been reported, all of these animals share certain common defects. In general, these mice displayed multiple hepatic defects, including a decrease in liver size and weight due to a decrease in the cytoplasmic volume of hepatocytes along with mild bile duct fibrosis [162, 164]. Other aberrations included compromised immune system function due to a decrease in the number of splenocytes

[162], reproductive defects [157] and anomalies of the eye and kidney, including persistent neonatal vascular structures [164]. In addition to the aforementioned defects, mice harboring Ahr-null alleles exhibited decreased constitutive expression of the xenobiotic metabolizing enzyme cytochrome P4501A2, along with a complete loss of cytochrome P4501A1 induction normally seen in response to dioxin exposure [161]. 93 This specific phenotype has resulted in benzo[a]pyrene no longer being able to mediate skin carcinogenesis in these animals [66]. Taken together, these observations provide support for the existence of high affinity endogenous AhR ligands. However, it is also possible that the AhR is activated in a ligand-independent manner. Nevertheless, it is probable that the AhR plays a crucial biological role in normal cellular metabolism and that one or more high affinity endogenous ligand exist to modulate the timing, duration and magnitude of its function in the cell. Elucidating the structure of such a ligand(s) would enable a biological role for this enigmatic orphan receptor to be more precisely determined. Subsequently, the significance of excessive Ah receptor activation by environmental compounds could be more thoroughly evaluated and new molecular targets for therapeutic drugs might emerge. In this report we established the presence of putative high affinity endogenous Ah receptor ligand(s) isolated from CV-1 cells and also establish the fact that the compounds are also a substrate for AhR-regulated cytochrome

P450s. In addition, evidence is provided for an autoregulatory feedback loop between the

AhR and cytochrome P450 1A1, a mechanism which appears most active in murine lung tissue.

94 2.3 EXPERIMENTAL PROCEDURES

Chemicals and Reagents:

Glycerol, acrylamide, and bisacrylamide were purchased from Research Organics,

Inc. (Cleveland, OH). Optima grade high purity organic solvents meeting ACS standards were purchased from Fisher Scientific (Pittsburgh, PA) and used wherever applicable throughout the course of these studies. Glacial acetic acid and powdered anhydrous sodium sulfate, manufactured by EMD Chemical, were obtained from VWR (West

Chester, PA). Anhydrous dimethyl sulfoxide (DMSO) 99.9% purity was purchased from

Sigma-Aldrich (Milwaukee, WI). TCDD was a generous gift from Steve Safe (Texas

A&M University).

Plasmids and Bacterial Strains:

The construct pcDNA3-βmAhR was used to express the murine AhR while pGudLuc 6.1 was used for DRE-driven luciferase reporter gene expression in cells as previously described [283]. β-galactosidase was expressed utilizing the pDJM2-β-gal construct and served as a control for transfection efficiency [284]. Ectopic expression of cytochrome P450 isoforms 1A1, 1A2, 1B1, and 2E1 in CV-1 cells was achieved with the expression vectors pCMV4-hCYP1A1 and pCMV4-hCYP1A2 was obtained from Dr.

Robert Tukey (Univ. CA, San Diego, CA), pRcCMV-hCYP1B1 was obtained from Dr.

William F. Greenlee (CIIT, North Carolina), and pCI-hCYP2E1 was supplied by Judy

Raucy (Puracyp Inc., Carlsbad, CA). The hCYP2E1 cDNA was subcloned from the 95 EcoR1 site of pCR2.1 to pCI. Plasmids were transformed into E. coli DH5α competent bacterial cells (PGC, Frederick, MD). Transformed bacteria were grown overnight in

Luria broth supplemented with the appropriate antibiotic for selection.

Cell Lines and Cell Culture:

The CV-1 cell line was obtained from the American Type Culture Collection

(Rockville, MD). Trypsin-EDTA, PBS, α-MEM, penicillin, and streptomycin were all obtained from Sigma (St. Louis, MO). FBS was purchased from HyClone Laboratories

(Logan, UT). Opti-MEM was purchased from Life Technologies/Gibco BRL (Baltimore,

MD). CV-1 cells were grown in α-modified minimal essential media (α-MEM) supplemented with 10% fetal bovine serum (v/v), 100 IU/ml penicillin, and 0.1 mg/ml

o streptomycin at 37 C in a humidified atmosphere containing 6% CO2 / 94% room air.

Transient Transfection, Luciferase and β-Galactosidase Assays:

Transfection of CV-1 cells was performed in 6-well tissue culture plates with a total of 2.5 μg of plasmid DNA per well and delivered using LipofectAMINE reagent

(Life Technologies/Gibco BRL, Baltimore, MD) according to the method described by the manufacturer. Transient transfections utilized 500 ng pcDNA3/βmAhR, 200 ng of

pGudLuc 6.1, a DRE-driven reporter construct along with 200 ng of pDJM-βgal, the β-

galactosidase control plasmid. In addition to the aforementioned vectors, transfections

also included varying amounts of a cytochrome P450 expression vector or control 96 plasmid. Upon completion of the transfection process, lipofectAMINE complexes were removed and replaced with α-MEM containing 10% fetal bovine serum. 24 h post transfection, cells were thoroughly rinsed with PBS and lysed in 1X cell culture lysis buffer (2 mM CDTA, 2 mM DTT, 10% Glycerol, 1% Triton X-100). Lysates were centrifuged at 18,000 x g for 15 min. Cytosol was assayed for luciferase activity using the Promega luciferase assay system (Promega Corp., Madison, WI) as specified by the manufacturer. Light production was measured for 15 s at room temperature using a TD-

20e Luminometer (Turner Designs, Inc., Sunnyvale, CA). Cytosolic protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce,

Rockford, IL). β-Galactosidase assays were performed and spectrophotometrically monitored at 420 nm using the Promega β-Galactosidase assay system (Promega Corp.,

Madison, WI). Luciferase activity was expressed relative to β-galactosidase activity or protein concentration. Each bar represents the mean (+/-) the standard deviation of four separate determinations. Statistical analysis of treatments was performed on GraphPad

Prism 4 software (San Diego, CA) using one-way ANOVA and Tukey’s multiple comparison test (α=0.05). Values determined as being statistically significant from each other are indicated with different letters.

Inhibition of CYP1B1 by 2, 4, 3′, 5′-tetramethoxystilbene (TMS):

CV-1 cells were grown to ~80% confluency in 6-well tissue culture plates and then transfected with plasmid DNAs using Lipofectamine (Life Technologies/Gibco

BRL, Baltimore, MD), as described by the manufacturer. Control cells were transfected 97 with 500 ng pcDNA3-βmAhR, 250 ng pDJM2-β-gal, 200 ng pGudLuc 6.1 and pCI vector, for a total of 1.5 μg of DNA per well. Experimental cells received the same transfected DNA profile except pCI vector was replaced with 400 ng pRcCMV-CYP1B1.

Transfected cells were allowed 16 h of recovery in complete medium before being dosed with concentrations of TMS, ranging from 50 nM to 10 μM, for an additional 8 h prior to harvest. Cells were thoroughly rinsed with PBS prior to lysis in 1X cell culture lysis buffer. Cell lysates were centrifuged at 18,000 x g for 15 min. Cytosol was assayed for luciferase activity and was expressed as RLU per units of β-galactosidase activity. Each bar represents the mean (+/-) the standard deviation of three separate determinations.

Statistical analysis of treatments was performed on GraphPad Prism 4 software (San

Diego, CA) using one-way ANOVA with Dunnett’s multiple comparison test (α=0.05).

Values determined as being statistically significant from untreated control (no TMS) are indicated by the presence of an asterisk (*).

TMS Biological Activity Assay:

HepG2 40/6 reporter cells were plated into 24-well tissue culture plates (Falcon,) at a density of 5.0 x 105 cells/ well, and allowed 18 h of recovery before being treated for

6 h with increasing amounts of TMS, DMSO or TCDD. Upon completion of the

bioassay, cells were thoroughly rinsed with PBS followed by the addition of 1X cell

culture lysis buffer (2 mM CDTA, 2 mM DTT, 10% Glycerol, 1% Triton X-100). After

freezing overnight at -80oC, the lysates were thawed and centrifuged at 18,000g for 15

min. The resulting cytosol was assayed for luciferase activity using the Promega 98 luciferase assay system (Promega Corp., Madison, WI) as specified by the manufacturer.

Light production was measured using a TD-20e Luminometer (Turner Designs, Inc.,

Sunnyvale, CA). Cytosolic protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Luciferase activity was expressed relative to protein concentration. Statistical analysis of treatments was performed on GraphPad

Prism 4 software (San Diego, CA) using one-way ANOVA with Dunnett’s multiple comparison test (α=0.05). Values determined as being statistically significant from the solvent (S) control are indicated by the presence of an asterisk (*).

Initial Ligand Characterization:

C-18 Solid Phase Extraction columns (Phenomenex, Torrance, CA) were initially used in attempts to concentrate the putative AhR ligand from CV-1 cells. Eventually solid phase extraction methods were abandoned in favor of liquid extraction methods using high purity Optima grade organic solvents (Fisher, Pittsburgh, PA). Performing multiple (2-3) extractions of CV-1 cytosol in series with an excess (2.0-2.5 volumes) of organic solvent proved to be the most effective method for extracting the AhR modulating compounds. Cytosol was mixed together with organic solvent (hexane:ethyl acetate 1:1 v/v) in a 15 x 130 mm round bottom glass centrifuge tube. This solvent was used for all extractions. The tubes were capped with a solvent washed silicon stopper and the mixtures were vortexed vigorously for several min. To facilitate separation of the aqueous and organic phases, the samples were centrifuged at 1600 x g for 10 min using a

Beckman S4180 rotor in a Beckman Allegra 21 tabletop centrifuge at 15oC. Following 99 extraction, the organic phase was removed and dried under reduced pressure in a centrivap under argon gas sparge or by hand under a continuous gentle stream of filtered argon gas. Immediately upon reaching dryness, the samples were carefully resolubilized in DMSO and assayed using the HepG2 40/6 reporter cell line, which contains a stably integrated pGudLuc 6.1 vector [285]. Each bar represents the mean of three separate determinations (+/-) the standard deviation. Statistical analysis of treatments was performed on GraphPad Prism 4 software (San Diego, CA) using one-way ANOVA with

Tukey’s multiple comparison test (α=0.05). Values determined as being statistically significant from each other are indicated with different letters.

Electrophoretic Mobility Shift Assays:

DRE-specific EMSAs were performed using in-vitro translated AhR and ARNT proteins. Expression vectors for these proteins were translated using a TNT coupled transcription and translation rabbit reticulocyte lysate kit (Promega Corp., Madison, WI).

Varying amounts of CV-1 cytosol were extracted with hexane: ethyl acetate (1:1v/v), stripped of residual water through incubation with anhydrous sodium sulfate, evaporated to dryness under reduced atmospheric pressure using a vacuum centrifuge (centrivap), sparged with argon gas upon completion and resuspended in 1.5 μl DMSO. Proteins for the transformation reactions were mixed together at a 2:1 molar ratio in HEDGE buffer

(25 mM HEPES, 1mM EDTA, 10 mM sodium molybdate, 10% glycerol, pH 7.5), followed by the addition of either DMSO solubilized organic extract of CV-1 cytosol or

20 nM TCDD, which served as a positive control. All transformation assays were 100 incubated for 90 min at room temperature, followed by the addition of oligonucleotide buffer (42 mM Hepes, 0.33 M KCL, 50% glycerol, 16.7 mM DTT, 8.3 mM EDTA, 0.125 mg/ml CHAPS, 42 ng/μl poly dI:dC). After 15 min of incubation, ~200,000 cpm of 32P- labeled wild type DRE oligonucleotide was added to each reaction and incubated for an additional 15 min. A portion of each sample was then removed and electrophoretically separated on a 6% non-denaturing polyacrylamide gel. Wild type DRE oligonucleotides comprised of the nucleotide sequence 5’-GATCTGGCTCTTCTCACGCAACTCCG-3’ and 3’-ACCGAGAAGAGTGCGTTGAGGCCTAG-5’ were gifts from Dr. M.S. Denison

(Univ. CA, Davis).

Proteinase K Treatment:

Approximately 2.6 mg of CV-1 cytosol was incubated in the presence of proteinase K (1:1/w:w) for 90 min at room temperature. An equal amount of CV-1 cytosol, serving as a control, was left untreated and incubated under the same conditions.

Upon completion of proteinase K digestion, 20, 30 and 40 μg aliquots of cytosol were removed from both reactions. These samples were mixed with an equal volume of 2X tricine sample buffer, heated at 95oC for 5 min and resolved by Tricine sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (TSDS-PAGE) (3.96% stacking gel / 7.92%

running gel) to confirm complete digestion of cytosolic protein. The remaining cytosol

from both the treated and untreated reactions was divided into 125, 250 μg, and 500 μg

aliquots. Samples were then extracted with 3 volumes of hexane:ethyl acetate (1:1 v/v) .

The organic phase was removed and the extract dried over anhydrous Na2SO4, followed 101 by evaporation to completion under argon gas. The extracts were then dissolved in a minimal volume of DMSO and applied to HepG2 40/6 cells for bioassay analysis. After a 6 h incubation cells were harvested and assayed for luciferase reporter gene activity.

Results are displayed in bar graph format with each bar representing the mean of three separate determinations (+/-) the standard deviation. GraphPad Prism 4 software (San

Diego, CA) was used to compare protease treated and untreated samples for statistical significance using the Student’s t-test (α=0.05). Pairs determined to be statistically significant are indicated by the presence of asterisk (*) while pairs lacking statistical significance are indicated with a double asterisk (**).

Mice:

Adult wild-type C57BL/6J mice (~2 month) were purchased from the Jackson laboratory (Bar Harbor, ME). Ahr-null mice in a C7BL/6J background were obtained from Dr. Bradfield (McArdle Laboratory for Cancer Research, University of Wisconsin-

Madison Medical School). The mice had free access to water and diet and were maintained in a temperature and light-controlled facility. The standards for animal use and care set by the Pennsylvania State University’s animal research program were followed for all experiments. Carbon-dioxide inhalation method was used to sacrifice the mice. Tissue samples were collected, frozen in liquid nitrogen and stored at -80°C until processed further.

102 Murine Tissue Extraction:

Mouse tissues were mechanically homogenized in buffer and cytosolic extraction isolated as previously described [286]. The protein concentration of cytosolic preparations was determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford,

IL). Based on calculated protein values for each tissue, 1.85 mg of heart, liver, lung and kidney cytosolic preparation along with 1.23 mg of spleen were used in the organic extraction process. Tissue derived cytosolic samples were extracted twice using 3.0 volumes of hexane:ethyl acetate (1:1 v/v). To facilitate separation of the aqueous and organic phases, sample emulsions were centrifuged at 1,600 x g for 10 min at 15oC.

Following phase separation the organic component was removed and stripped of residual

water through incubation with anhydrous sodium sulfate and evaporated to completion

under a continuous gentle stream of filtered argon gas. Immediately upon reaching

dryness, the samples were resolubilized in DMSO and assayed using the HepG2 40/6

reporter cell line as described earlier. GraphPad Prism 4 software (San Diego, CA) was

used to plot and analyze data generated from tissue samples for statistical significance

using the Student’s t-test (α=0.05). Statistical significance between selected samples is

indicated by the presence of asterisk (*) while samples lacking statistical significance are

indicated with a double asterisk (**).

103 Real-time quantitative PCR (qPCR):

Total RNA was isolated from mice tissues using TRI Reagent® (Sigma-Aldrich) and was reverse transcribed using the High Capacity cDNA Archive® kit (Applied

Biosystems) according to the respective manufacturer’s protocols. The cDNA made from

25 ng of RNA was used for each qPCR reaction. qPCR was performed on DNA Engine

Opticon® system using the IQ™ SYBR® Green qPCR Kit purchased from Bio-Rad

Laboratories, Inc. GraphPad Prism 4 software (San Diego, CA) was used to plot and

analyze data generated from tissue samples for statistical significance using the Student’s

t-test (α=0.05). Statistical significance between selected samples is indicated by the

presence of asterisk (*) while samples lacking statistical significance are indicated with a

double asterisk (**).

104 2.4 RESULTS

CV-1 Cells Contain a Putative Endogenous Ligand that Appears to be Metabolized by AhR- Regulated Cytochrome P450 Monooxygenase Enzymes.

Previous studies examining AhR nuclear translocation and transcriptional activity

revealed that potentially high concentrations of a putative AhR endogenous regulator

existed in the CV-1 cell line (Chang and Puga, 1998). The CV-1 cell line is an

immortalized animal cell line derived from the kidney epithelium of the African green

monkey, a tissue type which expresses significant levels of AhR. Following transient co-

transfection of these cells with an AhR expression vector and a DRE-driven luciferase reporter gene construct, high levels of luciferase activity can be observed despite in the absence of any exogenous ligand. However, based on the fact that AhR activation may not solely be dependent upon ligand, this data does not thoroughly substantiate the existence of an endogenous AhR ligand. Further experimentation was needed to confirm the existence of a high affinity endogenous AhR ligand in the CV-1 cell line. After

confirming the observations of Chang and Puga in our own laboratory using the CV-1

cell line, we expanded upon this initial discovery through a series of transient transfections to determine the effect of various ectopically expressed cytochrome P450

isoforms on DRE-driven reporter gene activity. The goal was to confirm the presence of a putative endogenous Ah receptor ligand and test our hypothesis of the existence of a potential AhR mediated cytochrome P450 auto-regulatory pathway.

105 Expression vectors for cytochrome P450 isoforms CYP1A1, CYP1A2, CYP1B1, and CYP2E1 were individually co-transfected into CV-1 cells, along with an AhR expression vector and a DRE-driven luciferase reporter gene construct. The potential of the CYP1 family members to metabolically alter putative ligand activity was the primary focus of this experiment. CYP2E1 served as a control since it is capable of metabolizing substrates such as ethanol, which are smaller than known AhR ligands (i.e. M.W. > 220).

Transfection of all cytochrome P450 isoforms was performed in quadruplicate at concentrations between 50-400 ng of the appropriate expression vector. Changes in putative endogenous ligand concentration were determined through changes in DRE- driven luciferase reporter activity (Figure 2.1). Luciferase values were normalized to

protein concentration. Based on the observed induction in AhR-mediated luciferase

reporter activity, the results of this experiment suggest the possible existence of a putative

endogenous AhR ligand in CV-1 cells capable of undergoing CYP1 metabolism.

106

Figure 2.1: Decreased AhR Activity Results from Expression of AhR-Regulated Cytochrome P450 Isoforms in CV-1 Cells. Expression vectors for CYP1A1, CYP1A2 and CYP1B1 were transiently co-transfected into cultured CV-1 cells, along with an AhR expression vector (pcDNA3/βmAhR) and the DRE-driven luciferase reporter (pGudLuc 6.1). The subsequent effect of P450 expression on DRE-driven luciferase reporter activity was assayed. Values are presented as the percent change from the empty vector control. Each data point represents the mean (+/-) S.D. of four separate determinations. Statistical analysis was performed using Tukey’s multiple comparison test (α=0.05). Values determined as being statistically significant from each other are indicated with different letters.

107 To control for the proper functioning of the AhR signal transduction pathway, cells were transfected with plasmids expressing AhR, CYP1B1 and DRE-driven reporter followed by treatment with TCDD to demonstrate transient expression of CYP1B1 did not lead to inactivation of the AhR pathway (Figure 2.2). This experiment clearly indicates that TCDD induction still produces a dramatic increase in AhR transcriptional activity, even in the presence of CYP1B1 expression (panel A). Expression of CYP served as negative control for P450 expression in CV-1 cells and resulted in the lack of specific metabolic activity toward the putative endogenous AhR modulator (panel B).

Interestingly, expression of CYP2E1 resulted in a significant increase in AhR activity with the lower levels of transiently transfected expression vector. The reason for this is not known. Thus, it appears as though a level of specificity exists between this putative

AhR ligand and the P450s which are capable of metabolically altering its activity. These results provide evidence suggesting the existence of a possible feedback mechanism in which AhR regulated cytochrome P450 isoforms from the CYP1A and CYP1B families are able to metabolically alter putative endogenous ligand structure.

108

Figure 2.2: No Reduction in AhR Activity Results from the Expression of a Control P450 Isoform in CV-1 Cells. Serving as a transfection control, CYP2E1, an isoform independent of AhR regulation failed to produce a reduction in AhR mediated reporter gene activity (panel A). Values are presented as the percent change from an empty vector control. Each data point represents the mean (+/-) S.D. of three separate determinations. To validate the proper functioning of the AhR signaling pathway, CV-1 cells were transfected with 800 ng of CYP1B1 followed by a 12 h induction with 100 nM TCDD (panel B). Statistical analysis was performed using Tukey’s multiple comparison test (α=0.05). Values determined as being statistically significant from each other are indicated with different letters.

109 Inhibition of CYP1B1 by 2,4,3’5’-tetramethoxystilbene(TMS) Rescues AhR- Mediated Transcriptional Activity.

In order to confirm that a direct metabolic alteration of the putative endogenous ligand by CYP1B1 was responsible for the reduction in DRE-driven luciferase reporter gene activity, studies were performed with a cytochrome P450 1B1 specific inhibitor.

TMS, a methoxy derivative of 2,4,3’5’-tetrahydroxystilbene has been shown to be a potent and specific inhibitor of cytochrome P450 1B1 [125]. CV-1 cells were co- transfected with an AhR expression vector, a DRE-driven luciferase reporter construct and a β-galactosidase expressing control vector. The transfection procedure was terminated after 12 h through removal of lipofectamine complexes and the cells given several hours of recovery time in complete culture media. Eight hours prior to harvest, cells were treated with TMS in concentrations varying from 50 nM to 10 μM. Compared with cells not expressing pRc/CMV/CYP1B1, treatment with 50 μM TMS induced an approximate 70% recovery in DRE-driven luciferase reporter gene activity. Increasing the TMS concentration increased the reporter gene response in a dose-dependent manner up to 750 nM, suggesting that inhibition of CYP1B1 activity leads to an accumulation of an AhR ligand (Figure 2.3). However, even in the absence of ectopic CYP1B1

expression, TMS treatment of CV-1 cells still resulted in a dose dependent induction of

DRE-driven luciferase gene activity. This additional increase in reporter gene activity

could be mediated either through the ability of TMS to serve as an AhR agonist or the

possibility that TMS blocks endogenous metabolism of putative endogenous AhR

ligand(s) or modulator(s). 110

Figure 2.3: Tetramethoxystilbene (TMS) Inhibition of CYP1B1 Rescues AhR-Mediated Transcriptional Activity. CV-1 cells were co-transfected with an AhR expression vector, a DRE-driven luciferase reporter construct and a β-galactosidase expressing control vector both in the presence or absence of CMV/hCYP1B1 vector for 12 h. Following transfection and recovery, the cells were treated with the CYP1B1 specific inhibitor, 2,4,3’5’- tetramethoxystilbene (TMS). The observed increase in AhR activity following inhibition of CYP1B1 metabolism may result from accumulation of an endogenous AhR ligand or modulator. Each data point represents the mean (+/-) S.D. of four separate determinations. Values are presented as relative luciferase units and have been normalized to protein concentration. Statistical analysis of reporter values generated with TMS treatment in both CV-1 and CV-1 + CYP1B1 expressing cells was performed using Dunnett’s multiple comparison test (α=0.05). Values determined as being statistically significant from the respective untreated control (no TMS) are indicated by the presence of an asterisk (*).

111

In order to differentiate between these two possibilities, we tested the ability of TMS to act as an AhR agonist. Biological activity assays performed using the HepG2 40/6 reporter cell line indicate that TMS possess weak AhR agonist activity only at elevated concentrations (Figure 2.4.). 112

Figure 2.4: The Effect of 2,4,3’5’-tetramethoxystilbene(TMS), a CYP1B1 Specific Inhibitor, on AhR Activity. Biological activity assays performed in the human derived HepG2 40/6 reporter cell line indicate that 2,4,3’5’-tetramethoxystilbene (TMS) possess weak AhR agonist activity. In response to treatment with various concentrations of TMS ranging from 50nM to 1.0μM, reporter cells displayed weak dose-dependent activation of an AhR-activated luciferase reporter gene. Control (C) designates untreated cells while solvent (S) indicates vehicle (DMSO) treatment only. Values are presented as relative luciferase units and have been normalized to protein concentration. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical analysis of TMS treatments was performed using Dunnett’s multiple comparison test (α=0.05). TCDD controls were compared independently from TMS treatments. Values determined as being statistically significant from the solvent (S) control are indicated by the presence of an asterisk (*).

113 However, an elevation in reporter activity was observed only for TMS concentrations of

750 nM or greater. This result may indicate that in CV-1 cells at concentrations of TMS below 750 nM, the observed increase in reporter gene activity could result from an inhibition of putative AhR ligand metabolism, as opposed to the weak agonist properties of TMS. Furthermore, the ability of TMS to reverse CYP1B1-mediated inhibition of

DRE-driven reporter activity occurs at 50 nM TMS, well below the concentration needed to induce AhR-mediated reporter activity in the HepG2 40/6 cell line. These results taken together indicate that, at low concentrations, TMS is capable of inhibiting CYP1B1 metabolism, thus allowing for a recovery in DRE-driven reporter gene activity. This provides strong evidence that CYP1B1 plays a major metabolic role in modulating the activity of the AhR possibly through alterations in the structure, and hence activity, of a putative endogenous AhR ligand(s) in CV-1 cells.

Ligand Characterization.

In order to identify the putative endogenous AhR ligand(s) from CV-1 cells, a purification strategy needed to be developed, in the absence of any information on the chemical characteristics of the molecule. This necessitated making several initial assumptions, many of which were ultimately verified through experimental testing. We rationalized that an endogenous ligand for the AhR, like most xenobiotic ligands for this receptor, would be relatively hydrophobic in nature and thus able to partition into hydrophobic stationary phase or organic solvent system. It was further hypothesized that in order to activate the AhR in vivo, the endogenous ligand must be capable of 114 partitioning into the cytoplasm. Thus, the cytosolic fraction appeared to be the optimal source of starting material in the search for endogenous AhR ligands. Furthermore, to choose a fraction that contained membranes would lead to organic extracts laden with high concentrations of neutral lipids, known to interfere with our assay system.

Initially, solid phase extraction (SPE) methods were employed to concentrate and purify the active metabolites. After testing several hydrophobic sorbent materials, it was determined that a C-18 reversed phase resin was the most efficient, thus confirming our assumption that the active molecule was relatively hydrophobic. Preliminary purification attempts focused on using C-18 solid phase extraction columns to purify and concentrate the active compounds prior to HPLC analysis. However, this method was later replaced by liquid extraction using high purity organic solvents after discovering the C-18 sorbent material itself possessed significant AhR activity (data not shown). Various solvents and solvent combinations were tested in order to determine an efficient solvent system for the liquid-liquid extraction method. Preliminary data demonstrated that several solvents including hexane, ethyl acetate, dichloromethane and a mixture of hexane and diethyl ether (1:1 v/v) or hexane and ethyl acetate (1:1 v/v) were very effective in extracting the endogenous AhR activators from CV-1 cytosol (data not shown). It was also discovered that using excess anhydrous sodium sulfate to remove any residual trace of water from the organic extract greatly aids in preserving the integrity of the active compound. This was especially important for organic extracts made with protic solvents such as ethyl acetate which have higher water solubility and therefore generate extracts containing increased amounts of residual aqueous phase (Figure 2.5). Furthermore, it was 115 determined that solvent extraction efficiency was enhanced through acidification of cytosolic samples prior to organic extraction (data not shown). This result would appear to indicate the presence of one or more ionizable acidic functional groups in the structure of the putative endogenous ligand or modulator.

Based on this preliminary information a hexane-ethyl acetate (1:1 v/v) solvent system was chosen to extract acidified cytosolic fractions derived from either cultured cells or tissue samples. Although the initial information on the chemical properties of the putative, endogenous AhR ligand contained in CV-1 cells is strictly qualitative, it has proven extremely valuable, especially in performing large scale, high yield, organic extractions of cytosolic fractions with reproducible results.

116

Figure 2.5: Organic Extractions of CV-1 Cytosol Activate the AhR. Both hexane and ethyl acetate extracts of CV-1 cytosol were tested for DRE-driven luciferase activity in the HepG2 40/6 reporter cell line. AhR modulators from CV-1 cytosol were capable of partitioning into either solvent system. However, the AhR activity contained in organic extracts of CV-1 cytosol was further enhanced when all trace amounts of aqueous residue was removed using anhydrous sodium sulfate (Na2SO4). Values are presented as relative luciferase units and have been normalized to protein concentration. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical comparison of treatments was performed using Tukey’s multiple comparison test (α=0.05). Values determined as being statistically significant from each other are indicated with different letters.

117 Electrophoretic Mobility Shift Assays Confirm the Presence of an Endogenous AhR Ligand in CV-1 Cell Extracts.

DNA mobility shift assays were performed to demonstrate that the ability of these

CV-1 extracts to induce reporter activity in HepG2 40/6 cells was indeed the direct result of AhR activation by a true high affinity endogenous ligand. Liquid phase organic extracts of CV-1 cytosol were prepared by extracting the required volume of cytosol with three volumes of hexane:ethyl acetate (1:1 v/v). The organic phase was removed to a clean vessel and stripped of any residual water through a brief incubation with anhydrous sodium sulfate. The extract was then aliquoted, dried under reduced atmospheric pressure and solubilized in DMSO. Vectors for the AhR and ARNT proteins were in vitro translated in rabbit reticulocyte lysate. Translated AhR and ARNT proteins were mixed together in a 2:1 molar ratio. A solvent blank comprised of evaporated hexane:ethyl acetate served as a background control in addition to the standard negative and positive controls. The mobility of the 32P-DRE-AhR:ARNT complex from cytosol- transformed AhR was compared to that of TCDD-induced AhR. The results of

Figure 2.6 demonstrate that an organic extract of CV-1 cytosol contains an endogenous

AhR ligand capable of activating AhR signaling apparently through direct interaction

with the receptor.

118

Figure 2.6: Organic Extracts of CV-1 Cytosol Transform the AhR into its DNA- Binding Form. DNA mobility shift assays reveal the observed induction in reporter gene activity observed with organic extracts of CV-1 cytosol is the result of direct AhR activation by an endogenous ligand. The mobility of the 32P-DRE-AhR:ARNT complex from cytosolic extract transformed AhR was compared to that of TCDD induced AhR after electrophoresis on a non-denaturing polyacrylamide gel.

119 Proteinase K Treatment Does Not Alter DRE-Driven Luciferase Reporter Activity.

In order to differentiate between a low molecular weight endogenous ligand and the possibility of a bioactive peptide modulator, a protease treatment experiment was performed. CV-1 cytosol was treated with 1.0 μg of proteinase K per mg of cytosolic protein or left untreated for 90 min at room temperature. Aliquots from both protease- treated and untreated samples were resolved by TSDS-PAGE and visualized through coomassie blue staining. The protease treatment resulted in complete digestion of protein into amino acids and small peptides (Figure 2.7). However, protease treatment failed to

abolish or significantly alter DRE-driven luciferase reporter activity in organic solvent

extracts from protease digested cytosolic samples (Figure 2.8). This result suggests that

the induction in reporter gene activity observed with solvent-extracted CV-1 cytosol is

presumably not due to a peptide ligand or a modulatory protein molecule.

120

Figure 2.7: Digestion of CV-1 Cytosolic Protein using Proteinase K Treatment. Cytosol samples generated from CV-1 cells were either treated with 1.0 μg of proteinase K per mg of cytosolic protein or left untreated for 90 min at room temperature. Aliquots from both protease-treated and untreated samples were resolved by TSDS-PAGE and visualized through coomassie blue staining.

121

Figure 2.8: Protease Treatment of CV-1 Cytosol Does Not Alter DRE-Driven Luciferase Activity. Following protease treatment, samples of CV-1 cytosol were acidified to pH = 4.0 and extracted using hexane:ethyl actetate. The biological activity contained in each pair of samples was examined using the HepG2 40/6 reporter cell line. Values are presented as relative luciferase units and have been normalized to protein concentration. Each data point represents the mean (+/-) S.D. of three separate determinations. Shaded bars indicated untreated samples while open bars represent proteinase K treated cytosol. Statistical comparison for each pair of protease treatments was performed using the Student’s t-test (α=0.05). Pairs determined to be statistically significant are indicated by the presence of asterisk (*) while pairs lacking statistical significance are indicated with a double asterisk (**).

122 HPLC Fractionation of CV-1 Cytosol Indicates a Complex Mixture of AhR Modulating Compounds.

Initially the fractionation of cytosolic samples was performed using reverse-phase high performance liquid chromatography (HPLC). However, this approach was eventually replaced by a classic normal phase HPLC methodology in order to alleviate stability problems associated with exposing the unstable active samples to solvent systems containing water. Normal phase HPLC separation, using isocratic elution together with a solvent system comprised of hexane:isopropanol, revealed the presence of multiple peaks possessing AhR activation potential distributed over a wide range of retention times (Figure 2.9). These results confirm the complexity of AhR activity in the

organic extract, presumably due to the presence of multiple AhR ligands or modulators.

Alternatively, this activity profile may also be indicative of unresolved stability issues

involving a single ligand that is degrading into several active products. Nevertheless, the

activity is capable of being separated into distinct peaks, allowing a high level of purity to potentially be obtained, which is a necessity if subsequent identification of the fractions through the use analytical techniques like LC-MS and tandem MS-MS is to be attempted. 123

Figure 2.9: HPLC Fractionation of Putative Endogenous AhR Ligands from CV-1 Cells. Organic solvent extracts of CV-1 cell cytosol were prepared and fractionated using normal phase HPLC. Multiple fractions were collected and analyzed for biological activity in the HepG2 40/6 reporter cell line. DRE-driven luciferase reporter activity is plotted as a function of time.

124 Endogenous Modulators for the AhR Accumulate in the Lung of Ahr-Null Mice.

Based on the results obtained in CV-1 cells, we hypothesized that in the absence of AhR expression potential AhR ligands would accumulate in some tissues because they would fail to induce their own cytochrome P450 mediated metabolism. To test this hypothesis cytosolic extracts were generated from various C57BL6/J and Ahr-null mouse tissues and extracted using a hexane-ethyl acetate solvent system. The level of AhR transcriptional activity induced by the various cytosolic extracts was assessed using the

HepG2 40/6 reporter cell line. Although the majority of AhR-null tissues examined displayed minimal differences in activity when compared with their respective wild-type counterparts, a striking increase in AhR activity was observed with lung tissue extracts generated from AhR-null mice. The lungs of AhR-null mice appear to accumulate significantly elevated levels of endogenous AhR ligands and/or modulators based on the observed four-fold increase in AhR transcriptional activity induced by cytosolic extracts of this tissue (Figure 2.10). Unexpectedly, a decrease in AhR activity was seen with

extracts of heart tissue from Ahr-null mice, a result unique in its own right and worthy of

more thorough investigation in the future. Most important, however, are the results

obtained with murine lung tissue extracts. These results support our hypothesis that in

the absence of AhR expression, tissues from Ahr-null mice would accumulate elevated

levels of endogenous AhR ligands and/or modulators that are normally metabolized by

AhR-regulated CYP450 enzymes. 125

Figure 2.10: Examining the Level of Constitutive Ah Receptor Activity in Various Tissues from AhR+/+ and AhR-/- Mice. Hexane:ethyl acetate (50:50 v/v) extraction of cytosol prepared from various mouse tissues produces organic extracts capable of driving the expression of a DRE-controlled luciferase reporter gene. Extracts derived from the tissues of both “wild-type” and null mouse strains are examined for differences in their ability to modulate AhR activity. The experiment was repeated twice, independently, and revealed the same results. Values are presented as relative luciferase units and have been normalized to protein concentration. Each data point represents the mean (+/-) S.D. of three separate determinations. Closed bars represent wild-type mice, while open bars represent AhR null mice. Statistical comparison for each pair of tissues was performed using the Student’s t-test (α=0.05). Pairs determined to be statistically significant are indicated by the presence of asterisk (*) while pairs lacking statistical significance are indicated with a double asterisk (**).

126 Mouse Lung Exhibits Relatively High Constitutive CYP1A1 Expression.

The constitutive level of CYP1A1 and CYP1B1 mRNA was determined in various mouse tissues isolated from C57BL6/J and Ahr-null mice. Interestingly, murine lung exhibited a four-fold increase in CYP1A1 mRNA levels over that observed for heart, the tissue with the second highest level of CYP1A1 mRNA expression in

C57BL6/J mice (Figure 2.11). In contrast, CYP1B1 levels were relatively low in lung

tissue. The level of constitutive CYP1A1 mRNA expression was dependent on AhR

expression. Surprisingly, CYP1B1 mRNA expression was essentially the same in both

C57BL6/J and Ahr-null mice indicating that constitutive expression of CYP1B1 is not dependent on the AhR. The results obtained with CYP1A1 suggest that in mouse lung the

AhR is constitutively active.

127

Figure 2.11: Comparing the Levels of CYP1A1 and CYP1B1 mRNA in Various Tissues from AhR+/+ and AhR-/- Mice. The constitutive expression of CYP1A1 and CYP1B1 mRNA in AhR-KO and “wild-type” mouse tissues is examined. The RNA obtained from different mouse tissues was reverse-transcribed and analyzed for Cyp1a1 (panel A) or Cyp1b1 (panel B) mRNA abundance using real-time RT-PCR. The graphs represent data obtained from 2 animals of each genotype (wild-type and AhR-null). The experiment was repeated twice, independently, and revealed the same results. Error bars represent standard deviation. Closed bars represent wild-type mice, while open bars represent AhR-null mice. Statistical comparison for each pair of tissues was performed using the Student’s t-test (α=0.05). Pairs determined to be statistically significant are indicated by the presence of asterisk (*) while pairs lacking statistical significance are indicated with a double asterisk (**).

128 2.5 DISCUSSION

This study is one of the first attempts to isolate putative endogenous ligands from biological extracts. The results provide strong evidence supporting the existence of a putative high affinity endogenous ligand(s) for the AhR using CV-1 cells as a model and is consistent with previously published observations [38]. These ligands appears to be capable of modulating receptor activation through direct binding and are able to induce

DRE-driven luciferase reporter gene expression to a level greater than or equal to a saturating dose of TCDD. Furthermore, it appears as though the putative ligand(s) is capable of relatively high affinity interaction with the AhR, thus allowing the receptor to mediate the metabolism of its own putative endogenous ligand(s). This would suggest the existence of a negative feedback auto-regulatory pathway in which ligand activation of the AhR would lead to activation of genes in the Ah battery, including cytochrome

P450 1A1, 1A2 and 1B1 (Figure 2.12). Expression of these P450 enzymes could then

mediate metabolism of the putative endogenous ligands, resulting in the attenuation of

the AhR pathway. In addition, considering the modest amount of transfected plasmid

needed in Fig. 1 to almost completely suppress Ah receptor constitutive activity, it would

appear that even a modest increase in P450 expression may be capable of completing this

feedback loop, especially CYP1A1, which appears to exhibit the highest efficiency of

ligand metabolism. The ability of CYP1A1 to autoregulate its own expression in response

to exogenous AhR ligands, such as polycyclic aromatic hydrocarbons, has been well

documented [96, 287] so it is reasonable to expect that this type of regulation could occur

in cellular homeostasis. 129

Figure 2.12: Diagrammatic Representation of the Proposed AhR Auto-regulatory Mechanism. Activated by an endogenous ligand or modulator, the AhR induces expression of various target genes including several metabolic enzymes such as CYP1A1, CYP1A2 or CYP1B1. Expression of AhR-regulated CYP450 enzymes appears to result in the metabolism and subsequent inactivation of the putative endogenous ligand(s) or modulator(s). In this proposed feedback mechanism, the receptor regulates metabolism of its own putative endogenous ligand(s) or modulator(s) resulting in attenuation of the AhR signaling pathway.

130 Derived from the kidney epithelium of the African green monkey, the CV-1 cell line is an immortalized cell line. With minimal AhR expression present in these cells, expression levels of AhR-mediated cytochrome P450s would be extremely low and subsequent accumulation of cellular metabolites, including potential intracellular endogenous AhR ligands. Thus, an increased level of a relatively high affinity endogenous ligand may explain the elevated levels of DRE-driven luciferase reporter gene activity observed in these cells when transfected with an AhR expression vector and a DRE-driven luciferase reporter gene construct. This may also explain the ability of purified cytosolic extracts to induce luciferase reporter gene expression to a level equal or greater than that observed for a saturating dose of TCDD (data not shown). Similar observations have also been reported in bioassay experiments using purified prostaglandins, especially PGG2 [95]. It is also possible that, in addition to being a potent

high affinity AhR agonist, the putative endogenous ligand in CV-1 cells may also be

capable of stimulating the AhR pathway by an indirect mechanism.

The possibility of a cell culture media contaminant producing the observed

induction in DRE-driven luciferase reporter activity upon treatment with organic solvent

extract of CV-1 cell cytosol was addressed in several ways. All bioassay experiments

performed have included as one of several controls, a media blank in which HepG2 40/6

cells receive no treatment other than the cell culture media in which they are routinely

grown. No induction in reporter gene activity has ever been observed with media

incubation alone (data not shown). Second, a time course assay was performed using the

HepG2 40/6 reporter cell line. Media was incubated in a cell culture incubator for 48 h 131 prior to the start of the experiment. Every 6 h for an 18 h period media was removed and added to HepG2 40/6 cells. An induction in reporter gene activity was never observed with conditioned media. In a similar experiment to test the possibility of a secreted metabolite, media conditioned for 48 h by actively growing CV-1 cells was added to the

HepG2 40/6 reporter cell line every 6 h for an 18 h period. Once again, no induction was observed in DRE-driven luciferase reporter gene activity.

Our results have revealed the presence of a relatively potent, putative endogenous ligand in CV-1 cells capable of activating the AhR pathway by directly binding and transforming the receptor to its DNA-binding form. In addition, this putative endogenous ligand can be metabolized by AhR regulated cytochrome P450s. This non-peptide ligand is easily extracted into aprotic solvents after acidification, suggesting that the compound is hydrophobic in nature with one or more ionizable acidic groups. In addition, the presence of multiple peaks of activity upon HPLC fractionation of extracts from CV-1 cell cytosol would suggest that more than one AhR ligand exists in CV-1 cells. Attempts to further purify these individual peaks have been complicated by their apparent instability (data not shown).

To further explore the significance of the data obtained in CV-1 cells we wanted to test whether an AhR-cytochrome P450 auto-regulatory loop exists in vivo. To accomplish this, cytosolic samples of five tissues from “wild-type” and Ahr-null mice were extracted with organic solvent and tested for their ability to activate the AhR in a reporter cell line. Interestingly, lung extracts from Ahr-null mice revealed a four-fold 132 increase in AhR activation potential compared to “wild-type” mice. This observation, coupled with the relatively high AhR-dependent CYP1A1 constitutive activity seen in lung, 50-fold higher than any other tissue tested, would support the assertion that an auto- regulatory loop between the AhR and CYP1A1 exists. It is interesting to note that the constitutive level of CYP1B1 in lung appears to be quite low. This data would indicate that lung from Ahr-null mice would be a rich source of putative endogenous AhR ligands.

Whether this activity is due to the previously identified compound, 2-(1′H-indole-3′- carbonyl)-thiazole-4-carboxylic acid methyl ester from porcine lung will require further investigation [93]. Considering that the lung is a complex tissue made up of a number of cell types, future studies will focus on the specific cell type(s) in Ahr-null mice that accumulate AhR ligands. While no quantitative differences in the presence of AhR ligands was seen in other tissues, it is possible that the method chosen to assess the presence of ligands may not be examining all AhR ligands, such as more polar compounds. The ability of AhR ligands to accumulate in tissues of Ahr-null mice may be attenuated by the presence of constitutively expressed CYP1B1, CYP1A2, or other P450s that are not totally dependent of AhR to mediate expression. These observations also suggest that exogenous AhR ligands in the diet are not accumulating in Ahr-null mice. If this were the case we would expect to detect increases in AhR ligands in Ahr-null mouse tissues such as liver. In summary, the in vivo data clearly supports the presence of an

AhR-mediated cytochrome P450 auto-regulatory loop in mouse lung.

Efforts to identify the compound using various complementary methods of mass spectrometry (MS) and tandem mass spectrometry (MS-MS) are underway in both CV-1 133 cells and in Ahr-null mouse lung. Results of these experiments should reveal the identity of this putative AhR ligand(s) and provide insight into the types of compounds that can serve as high affinity endogenous ligands for the AhR. This information will greatly improve our understanding of the physiological role of the AhR and the subsequent biochemical mechanism of action of this receptor.

CHAPTER 3

THE EICOSANOID METABOLITE, 12(R)-HETE, IS AN ACTIVATOR OF THE AH RECEPTOR

3.1 ABSTRACT

The aryl hydrocarbon receptor (AhR) is a ligand-regulated transcription factor that can be activated by structurally diverse chemicals, ranging from environmental carcinogens to dietary metabolites. Evidence supporting a necessary role for the AhR in normal biology has been established; however, no high affinity endogenous ligand/activator has been identified. Here we report the ability of 12(R)-hydroxy-

5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12(R)-HETE), an arachidonic acid metabolite produced by either a lipoxygenase or cytochrome P-450 pathway, to act as a potent indirect modulator of the AhR signaling pathway. In striking contrast, neither

12(S)-HETE, an enantiomer of 12(R)-HETE, nor the structurally similar regioisomers such as 5-HETE or 15-HETE demonstrated any significant activation of the AhR.

Electrophoretic mobility shift assays, together with ligand competition binding experiments, have demonstrated that 12(R)-HETE does not bind or directly activate the

AhR to a DNA binding species in vitro. However, cell-based DRE-driven luciferase reporter assays indicate the ability of 12(R)-HETE to modulate AhR activity, while induction of an endogenous AhR target gene confirmed the ability of 12(R)-HETE to activate AhR-mediated transcription in human hepatoma (HepG2) and keratinocyte

(HaCaT) derived cell-lines. One possible explanation for these results is that a metabolite of 12(R)-HETE is acting as a direct ligand for the AhR. However, several known cellular metabolites of 12(R)-HETE such as 12-oxo-ETE, tetranor-12(R)-HETE, and 10,11- dihydro-12(R)-HETE (12(R)-HETrE) failed to exhibit AhR activity. Nevertheless, because siRNA- mediated suppression of AhR expression also decreased the ability of 136 12(R)-HETE to mediate induction of AhR target genes, it was concluded receptor expression is required for 12(R)-HETE to activate AhR signaling. Conclusively, these results indicate that 12(R)-HETE, a potent pro-inflammatory molecule, is a potent activator of AhR signaling despite its inability to directly bind the receptor. Furthermore, these results suggest that during certain inflammatory disease states such as psoriasis, in which 12(R)-HETE is produced, the AhR would likely be activated.

137 3.2 INTRODUCTION

The aryl hydrocarbon receptor (AhR) is a ligand-activated basic helix-loop- helix/Per-ARNT-Sim transcription factor expressed in most of the cell and tissue types found in vertebrates [158]. In the absence of ligand, the AhR resides in the cytosol in a heterotetrameric protein complex. Contained in this core complex are the AhR ligand- binding subunit, a dimer of the 90 kDa heat shock protein and a single molecule of the immunophilin-like X-associated protein 2 (XAP2, also referred to as AIP or ARA9) [28,

34]. After ligand binding, the receptor translocates into the nucleus and forms a high affinity DNA binding complex upon heterodimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT). The AhR/ARNT complex can interact with specific DNA target sequences known as dioxin-responsive elements (DRE) in the regulatory region of AhR responsive genes, resulting in altered gene expression [31].

Most AhR target genes are primarily involved in foreign chemical metabolism and include the xenobiotic metabolizing cytochrome P450 enzymes from the CYP1A and

CYP1B families [41], along with NAD(P)H-quinone oxido-reductase 1, an aldehyde dehydrogenase, and several phase II conjugating enzymes, including glutathione-S- transferase Ya and UDP-glucuronosyltransferase 1A1 [288, 289] In addition, the AhR regulates genes involved in growth and cellular homeostasis, such as epiregulin and

Hairy and Enhancer Split homolog 1 (HES-1) [290, 291].

The AhR plays an important role in the adaptive metabolic response to xenobiotic exposure; this response can be modulated by a diverse range of compounds, including 138 many potentially toxic man-made environmental contaminants, such as halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons [69, 292]. In addition to xenobiotic metabolism, the AhR has emerged as playing an important physiological role in vascular development in the liver [164, 293]. However, a key question still remaining unresolved is the identity of endogenous modulators of AhR activity. Evidence supporting the existence of an endogenous physiological role for AhR has been steadily accumulating, with the strongest support obtained from studies with AhR null mice.

These animals displayed multiple hepatic defects, including a decrease in liver size and weight [162], resulting presumably from the presence of a portosystemic shunt [164], a persistent unresolved remnant of fetal vasculature [294]. Additional aberrations included compromised immune system function [162], reproductive defects [157], altered kidney vasculature, and vascular anomalies in the eye, including the presence of a persistent hyaloid artery [161]. AhR null mice also exhibited a decrease in constitutive expression of cytochrome P4501A2, along with a complete loss of cytochrome P4501A1, induction normally seen only in response to AhR activation [161]. Collectively, these observations indicate a crucial biological role(s) for the AhR in normal physiology. Furthermore, assuming AhR activation requires ligand, these observations provide indirect evidence supporting the existence of one or more high affinity endogenous AhR ligands, existing to modulate the timing, duration and magnitude of AhR function in the cell. Identifying such a ligand(s) would enable the biological role(s) for this enigmatic orphan receptor to be more precisely determined, thus allowing the significance of excessive Ah receptor activation by environmental compounds to be more thoroughly evaluated. Previously, several eicosanoid molecules, including lipoxin A4 [96], along with various 139 prostaglandins [95] have been reported as AhR ligands. During a screening of eicosanoids, in particular those produced by lipoxygenase or cytochrome P450 metabolic pathways, several molecules possessing AhR activity were identified. In this report we characterize 12(R)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12(R)-HETE), an arachidonic acid metabolite of either lipoxygenase or cytochrome P-450 origin, as an activator of the AhR signal transduction pathway.

140 3.3 EXPERIMENTAL PROCEDURES

Chemicals and Enzymes.

12(S)-Hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12(S)-HETE) and

12(R)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12(R)-HETE) were purchased from BIOMOL (Plymouth Meeting, PA). Additional quantities of both 12-

HETE isomers along with 12-oxo-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12-

KETE) were purchased from Cayman Chemical Company (Ann Arbor, MI). 12(R)-

hydroxyeicosatrienoic acid (12(R)-HETrE), also known as 10,11-dihydro-12(R)-HETE,

was a gift from Dr. John R. Falck (University of Texas, Southwest Medical Center,

Dallas,TX). Optima grade high purity organic solvents were purchased from Fisher

Scientific (Pittsburgh, PA) and used in all chromatographic separations. Anhydrous

dimethyl sulfoxide (DMSO) (99.9% purity) was purchased from Sigma-Aldrich

(Milwaukee, WI). TCDD was a generous gift from Dr. Steven Safe (Texas A&M

University). 32P-γATP was purchased from PerkinElmer (Boston, MA) while T4

polynucleotide kinase was from Promega (Madison, WI). PolydI:dC was purchased from

Amersham Biosciences (Piscataway, NJ) and pre-cast 6% non-denaturing polyacrylamide

gels were from Invitrogen (Carlsbad, CA).

Cell Lines and Cell Culture.

The HepG2 40/6 reporter cell line [285] was generated as described previously,

while the Hepa 1.1 reporter cell line [295] was a kind gift from Dr. Michael S. Denison 141 (University of California, Davis). Trypsin-EDTA, PBS, α-MEM, penicillin, and streptomycin were all obtained from Sigma (St. Louis, MO). FBS was purchased from

HyClone Laboratories (Logan, UT). Reporter cell lines were grown in α-MEM supplemented with 10% fetal bovine serum (v/v), 100 IU/ml penicillin, and 0.1 mg/ml

o streptomycin at 37 C in a humidified atmosphere containing 5% CO2/95% room air.

Clonal selection of reporter cell lines was maintained through the use of 300 μg/ml of

G418 (GibcoBRL, Carlsbad, CA).

Cell-based Reporter Assay.

Reporter cell lines were plated into 24-well tissue culture plates (Falcon,) at a

density of 5.0 x 105 cells/ well, and allowed 18 h of recovery before beginning a 6 h

treatment with increasing amounts of 12-HETE or a 12-HETE metabolite. Following

completion of the dosing regiment, cells were thoroughly rinsed with PBS prior to

addition of 1X cell culture lysis buffer (2 mM CDTA, 2 mM DTT, 10% Glycerol, 1%

Triton X-100). After being frozen overnight at -80oC, the lysates were then thawed and

centrifuged at 18,000g for 15 min. The resulting cytosol was assayed for luciferase

activity using the Promega luciferase assay system (Promega Corp., Madison, WI) as

specified by the manufacturer. Light production was measured using a TD-20e

Luminometer (Turner Designs, Inc., Sunnyvale, CA). Cytosolic protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL).

Luciferase activity was expressed relative to protein concentration. Statistical analysis of

treatments was performed on GraphPad Prism 4 software (San Diego, CA) using one- 142 way ANOVA with Dunnett’s multiple comparison test (α=0.05). Values determined as being statistically significant from control are indicated by the presence of an asterisk (*).

Electrophoretic Mobility Shift Assay.

DRE-specific EMSAs were performed using in vitro translated AhR and ARNT proteins. Expression vectors for these proteins were translated using a TNT coupled transcription and translation rabbit reticulocyte lysate kit (Promega Corp., Madison, WI).

A solution of 12-hydroxyhexadecatetraenoic acid in ethanol was evaporated under argon gas and resolubilized in DMSO to achieve a stock solution of appropriate concentration.

Serial dilutions of this stock were made in DMSO to achieve the various working stocks needed to perform a dose-response curve. Proteins for the transformation reactions were mixed together at a 1:1 molar ratio in HEDG buffer, followed by addition of either 0.5 μl

DMSO solubilized 12-HETE isomer, 12-HETE metabolite or TCDD. All transformation assays were incubated for 90 min at room temperature, followed by the addition of oligonucleotide buffer (42 mM Hepes, 0.33 M KCl, 50% glycerol, 16.7 mM DTT, 8.3 mM EDTA, 0.125 mg/ml CHAPS, 42 ng/μl poly dI:dC). Following 15 mins of incubation time in oligonucleotide buffer, ~200,000 cpm of 32P-labeled wild type DRE

was added to each reaction. Samples were mixed with an appropriate amount of 5X loading dye and separated on a 6% non-denaturing polyacrylamide gel. Wild type DRE

oligonucleotides of the sequence 5’-GATCTGGCTCTTCTCACGCAACTCCG-3’ and

3’-ACCGAGAAGAGTGCGTTGAGGCCTAG-5’ were a gift from Dr. M.S. Denison.

143 AhR Ligand Competition Binding Assay.

2-Azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin was synthesized in our laboratory according to the procedure described previously, and was stored in methanol

protected from light [296]. Hepa-1 cell cytosol, a source of mouse AhR, was prepared in

MENG buffer (25 mM MOPS, 2 mM EDTA, 0.02% sodium azide, 10% glycerol, pH 7.4)

and diluted to a final protein concentration of 1.0 mg/ml. All binding experiments were

carried out in the dark with 150 μg of soluble Hepa-1 cytosolic protein incubated with

increasing concentrations of 12-HETE for 30 min. Next a saturating concentration of the

AhR photoaffinity ligand, 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin (0.10 pmol;

i.e. 4 x 105 cpm) was added and incubated for an additional 30 min at 23oC to achieve

equilibrium binding. The samples were photolysed at >302 nm for 4 min at a distance of

8 cm using two 15-Watt UV lamps (Dazor Mfg. Corp. St. Louis, MO). After irradiation,

each sample was mixed with an equal volume of 2X tricine sample buffer (TSB) (0.9 M

Tris, pH 8.45, 24% glycerol, 12% w/v SDS, 0.015% w/v Coomassie Blue G, 0.005% w/v

phenol red) and heated to 95oC for 5 min. Equal amounts of each sample were loaded onto a 9% Tricine-SDS-PAGE gel and subjected to denaturing electrophoresis overnight

at 15mA/gel. The gels were fixed in destain (25% isopropyl alcohol, 10% acetic acid,

10% glycerol), dried under vacuum and exposed at -80oC to a sheet of X-OMAT-Blue

film (Eastman Kodak Co.). Following autoradiogram generation, the radioactive AhR

bands were excised from the acrylamide gel and quantified through scintillation counting

using a LKB-Wallac RiaGamma (model 1274) automatic gamma counter (Waltham,

MA). Statistical analysis of binding data was performed with GraphPad Prism 4 software 144 (San Diego, CA) using one-way ANOVA and Tukey’s multiple comparison testing

(α=0.05). Values determined as being statistically significant from empty vector control are indicated by the presence of different letters.

HPLC Purification and Spectral Analysis.

HPLC purifications were performed using a Waters 600E multi-solvent delivery unit and controller coupled with a Waters 996 photodiode array detector. The system was integrated and operated with the use of Waters Millenium32 software. Normal phase

HPLC purification of 12-HETE was performed on a Supelco LiChrosphere 5 micron

Silica-60 (4.6 mm x 250 mm) column using a hexane: isopropanol: acetic acid solvent

system (989:10:1 v/v/v) applied at 1ml/min with a linear gradient increase in isopropanol

concentration of 0.8% per min over 30 min. High resolution mass determination for

12(R)-HETE and the MS/MS product ion spectrum for the molecular ion (m/z 319) were

both acquired on a Waters QT of Ultima mass spectrometer using an electrospray

ionization source operated in negative ion mode. A Waters 2795 HPLC pump and autosampler were used for sample introduction. Instrument parameters were as follows: capillary voltage = 3.0 KV, source temperature = 90 C; desolvation temperature = 200 C;

cone voltage = 35 V; The product ion spectrum was generated using argon as the

collision gas at a pressure of 3 x 10-3 mbar as measured at the collision cell manifold;

collision energy was 30 eV. Flow injection analyses were performed for both MS and

MS/MS modes, with 0.25 mL/min of 75% acetonitrile/25% water transporting the

injected material to the mass spectrometer ion source. For both MS and MS/MS modes, 145 spectra were acquired by accumulating transients for 0.5 seconds with a 0.1 second interscan delay. All mass spectrometry analysis was performed by Dr. A. Daniel Jones formerly of the Pennsylvania State University now serving as the director of the RTSF mass spectrometry facility at Michigan State University.

Quantitative RT-PCR analysis.

Total RNA was isolated from cells using TRI Reagent® (Sigma-Aldrich) and was reverse transcribed using the High Capacity cDNA Archive® kit (Applied Biosystems) according to the respective manufacturer’s protocols. The cDNA made from 25 ng of

RNA was used for each qPCR reaction. qPCR was performed on a DNA Engine

Opticon® system using the IQ™ SYBR® Green qPCR Kit purchased from Bio-Rad

Laboratories, Inc. Data was analyzed and plotted using GraphPad Prism 4 software (San

Diego, CA). Each bar represents the mean (+/-) S.D. of three separate determinations.

Statistical analysis of 12(R)-HETE dose-response data was made using one-way ANOVA

with Dunnett’s multiple comparison test (α=0.05). Values determined as being

statistically significant from solvent (S) control are indicated by the presence of an

asterisk (*). Comparison of 12(R)-HETE treatments from siRNA experiments was

performed using the Student’s t-test (α=0.05). Values determined as being statistically

significant are indicated by the presence of an asterisk (*).

146 3.4 RESULTS

12(R)-HETE Can Activate the AhR In Cell-based Assays.

The CV-1 cell line upon ectoptic expression of AhR exhibits high levels of constitutive receptor activity [38]. Preliminary studies with extracts generated from these cells indicated the possibility of endogenous bioactive lipids as mediators of AhR activity

(unpublished data). The molecular weight of many known potent AhR ligands is between

250-360 Da, as are the majority of eicosanoids, bioactive lipids formed from arachidonic acid. In addition, these molecules are capable of adopting a structural conformation that could be accommodated by the AhR ligand binding pocket. Therefore, key metabolites of the 5, 12, and 15-lipoxygenase pathways were screened for AhR activity using cell culture based biological activity assays. Bioassays performed using the HepG2 40/6 cell line, a human liver derived reporter cell line containing a stably integrated copy of a

DRE-driven luciferase reporter construct, were used to screen various hydroxyeicosatetraenoic acid (HETE) metabolites for potential AhR transcriptional activity. This approach led to the discovery of 12(R)-hydroxy-5(Z),8(Z),10(E),14(Z)- eicosatetraenoic acid (12(R)-HETE) as a modulator of Ah receptor activity in cells, capable of stimulating the AhR signaling pathway in a dose-dependent manner

(Figure 3.1). Activation of the AhR by 12(R)-HETE, although only moderate in

magnitude, appears to be highly specific, when compared with the lack of activity seen

among all other HETE isomers examined. Interestingly, analysis of the isomer activity

profile indicates that both position and stereochemical orientation of the hydroxyl group

plays an important role in the ability of these molecules to modulate AhR activity. 147

Figure 3.1: 12(R)-HETE Activates the AhR in Reporter Based Biological Activity Assays. Various HETE isomers were screened via bioassay in the human derived HepG2 40/6 reporter cell line for their ability to activate the AhR. Analyzing both the (S) and (R) enantiomers of each positional HETE isomer reveals 12(R)-HETE is capable of inducing a potent AhR activation. Control (C) represents untreated reporter cells while solvent control (S) indicates a negative control comprised of vehicle (DMSO) treatment only. Values are presented as relative luciferase units and have been normalized to protein concentration. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical analysis of treatments was performed using Dunnett’s multiple comparison test (α=0.05). TCDD controls were compared independently of HETE samples. Values determined as being statistically significant from the solvent (S) control are indicated by the presence of an asterisk (*).

148 Analysis of the Biochemical Purity of 12(R)-HETE Preparations.

Several commercially-available preparations of specific eicosanoid molecules produced false positive results in initial assays, probably due to trace levels of hydrophobic contamination. Therefore, the purity and structural integrity of 12(R)-HETE preparations needed to be analyzed prior to further experimental use. To remove any possible contaminating impurities, especially the early eluting hydrophobic material identified as possessing significant AhR activity in some commercially prepared HETE preparations (data not shown), 12(R)-HETE preparations were subjected to normal phase

HPLC purification (Figure 3.2). Eluting with a retention time (TR) of 12.25 min (panel

A), the peak representing the purified 12(R)-HETE fraction was collected and further

analyzed before being used in any experimental applications. Evaporation of the HPLC

mobile phase followed by immediate solublization in degassed absolute methanol

rendered the sample preparation available for subsequent analytical techniques. Spectral

analysis of purified 12(R)-HETE preparations generated spectra displaying a

characteristic absorbance maximum at 235 nM (panel B), indicating the presence of a

conjugated diene functionality.

149

Figure 3.2: Assessing the Biochemical Purity and Integrity of 12(R)-HETE. To remove contaminating impurities, 12(R)-HETE preparations were subjected to straight phase HPLC purification (panel A). UV spectral analysis of purified 12(R)-HETE preparations generates spectra displaying a characteristic absorbance maximum at 235nm, indicating the presence of a conjugated diene functionality (panel B).

150 Additional confirmation regarding the purity and integrity of the 12(R)-HETE sample was achieved via mass spectrometry analysis performed on a Waters QTof Ultima mass spectrometer using electrospray ionization operated in negative ion mode (ESI-)

(Figure 3.3). After loss of a proton, sample molecules exhibit the characteristic

molecular ion for 12(R)-HETE at mass-to-charge ratio (M/Z) of 319. Furthermore,

accurate mass determination of the molecular ion resulted in a measured mass of

319.2272, a value that correlates well with the theoretical calculated mass for the

compound of 319.2279. Differing by only 2.19 ppm the accurate mass value is in

agreement for a compound comprised of the elemental composition C20 H31 O3 (panel A).

Furthermore, MS-MS analysis of the molecular ion produced daughter fragments of

M/Z= 301 and 257 consistent with HETE decomposition via loss of H2O and the loss of

both CO2 and H2O, respectively. The presence of the peak at M/Z= 179, characteristic

for 12-HETE, is produced through cleavage of the carbon backbone between C-11 and C-

12 and supports the position of a hydroxyl group on carbon 12 (panel B). With the purity

of 12(R)-HETE preparations being firmly established only after re-purification, it became

necessary to use freshly re-purified preparations in all subsequent analyses.

151

Figure 3.3: Mass Spectrometric Analysis of 12(R)-HETE. Negative mode electrospray ionization (ESI-) spectra of 12(R)-HETE displaying the 12-HETE molecular ion at M/Z=319. Exact mass determination of the molecular ion confirms an elemental composition of C20 H31 O3, indicative of an ionized mono-HETE molecule (panel A). MS/MS analysis of the molecular ion generates the characteristic 12-HETE daughter fragment at M/Z= 179. Specific to 12-HETE, this fragment is produced through carbon backbone cleavage via breakage of the C-11 to C-12 bond. In addition, fragments at M/Z= 301 and 257 can also be seen consistent with HETE decomposition via loss of H2O and the loss of both CO2 and H2O, respectively. (panel B).

152 12(R)-HETE Does Not Directly Bind to the Ah Receptor.

Despite its ability to activate the AhR pathway in cell culture, 12(R)-HETE failed to induce AhR heterodimerization or DRE-binding as determined by electrophoretic mobility shift assay. Using 12(S)-HETE as a negative control, both 12-HETE enantiomers were tested for AhR activity and compared against a TCDD-induced AhR positive control. Neither of the 12-HETE isomers induced transformation of the AhR to its DNA binding form, as evident by the complete lack of shifted radiolabeled complex

(32P-DRE-AhR:ARNT) after electrophoresis on a non-denaturing polyacrylamide gel.

The lack of heterodimer complex formation, even at 10 μM and 25 μM where a

substantial induction in reporter gene activity was observed, clearly indicates the inability of 12(R)-HETE to serve as a direct AhR ligand (Figure 3.4). Further evidence for lack of

12(R)-HETE binding to the AhR was obtained through the use of in vitro ligand

competition binding assays. Using cytosolic preparations of AhR generated from the

Hepa-1.1 cell line, increasing concentrations of 12(R)-HETE failed to demonstrate any

significant ability to compete with radiolabeled photoaffinity ligand for AhR binding

when compared with 15(R)-HETE, a negative control (Figure 3.5). Although weak non-

specific displacement of radioligand arises from lipid treatment in general, no significant

dose-dependent displacement was observed with 12(R)-HETE, confirming the inability

of this compound to serve as an AhR ligand. In contrast, a positive control comprised of

300 nM benzo[a]pyrene treatment exhibited potent displacement of the photoaffinity

ligand (panel A). The quantified level of radioactivity in each receptor gel band after

displacement with 12(R)-HETE or controls is presented (panel B). Collectively, the 153 results of these binding experiments reveal the inability of 12(R)-HETE to activate the

AhR to its DNA binding form or compete with a radiolabeled dioxin-like photoaffinity ligand for AhR binding. Although this clearly demonstrates the lack of competitive AhR binding by 12(R)-HETE, it does not demonstrate a complete lack of binding, as the possibility for low affinity interaction of 12(R)-HETE with the receptor still remains.

However, the total absence of heterodimer formation in the gel-shift assay does support the complete lack of 12(R)-HETE binding affinity for the AhR. 154

Figure 3.4: 12(R)-HETE Fails to Transform the AhR into its DNA Binding Form. Using 12(S)-HETE as a lipid control (panel B), both 12-HETE enantiomers were tested for AhR activity and compared against a positive control of 20 nM TCDD induced AhR. 12(R)-HETE failed to activate the AhR to its DNA binding form (panel A). Gel shift controls included a negative (-) control containing no ARNT, a background (B) control comprised of only AhR and ARNT to control for background heterodimerization, and a solvent (S) control to access the effects of vehicle on heterodimer formation.

155

Figure 3.5: 12(R)-HETE Does Not Compete for AhR Binding. Ligand competition binding experiments reveal the inability of 12(R)-HETE to specifically compete with a radiolabeled photoaffinity ligand for AhR binding. Compared with 15(R)-HETE, serving as a negative control (NC), no significant displacement of the radiolabled dioxin molecule could be seen in response to increasing concentrations of 12(R)-HETE. Conversely, potent displacement of radioligand is observed with a positive control comprised of 300nM benzo[a]pyrene (B[a]P). The solvent control (C) represents vehicle (DMSO) treatment of Ah receptor (panel A). A graphical representation of the ligand competition binding data is presented (panel B). Statistical analysis of data was performed using Tukey’s multiple comparison test (α=0.05). Values determined as being statistically significant from each other are indicated by different letters.

156 Metabolites of 12(R)-HETE Fail to Activate AhR.

Because purified 12(R)-HETE was unable to compete with ligand or activate the

AhR in vitro, yet displayed AhR activity in cell based assay systems, it was hypothesized that a downstream metabolite of 12(R)-HETE may be responsible for the observed activity of this compound. Therefore, in an attempt to identify the active metabolite, known 12(R)-HETE metabolic pathways were screened for compounds capable of activating AhR transcription (Figure 3.6). In most cells, metabolism of 12(R)-HETE is

limited to peroxisomal-mediated β-oxidation or cytochrome P450 catalyzed ω-oxidation

(panel A). These reactions will generate chain shortened metabolites, such as tetranor

12(R)-HETE (8(R)-HHxTrE), or ω−hydroxylated metabolites such as 12(R), 20-DiHETE

respectively [297, 298]. Oxidation of 12-HETE to a keto intermediate (12-oxo-EET)

followed by keto-reduction to the 10,11- dihydro metabolite, 12(R)-hydroxy-5,8,14-

eicosatrienoic acid (12(R)-HETrE) has been described in porcine neutrophil and bovine

corneal epithelial microsomes [299]. Using the HepG2 40/6 reporter cell line, several

12(R)-HETE metabolites were analyzed for their ability to modulate AhR transcriptional

activity. Neither tetranor-12(R)-HETE (8(R)-HHxTrE) nor 12-oxo-ETE (12-KETE), two

common metabolites of 12(R)-HETE, could activate AhR signaling (panel B). In

addition, the dihydro metabolite 12(R)-HETrE also failed to activate AhR-mediated

transcription (data not shown). Unfortunately, we were unable to obtain 12(R), 20-

DiHETE for testing. Nevertheless, the 12(R)-HETE metabolites tested failed to

significantly activate the AhR. These results further underscore the high level of

specificity in 12(R)-HETE mediated receptor activation. 157

Figure 3.6: Metabolites of 12(R)-HETE Fail to Demonstrate Significant AhR Biological Activity. Common biochemical pathways of 12(R)-HETE metabolism are displayed (panel A). Using the HepG2 40/6 reporter cell line, several 12(R)-HETE metabolites were screened for AhR activity. The results obtained with tetranor-12(R)- HETE (8(R)-HHxTrE) and 12-oxo-ETE (12-KETE), two common metabolites of 12(R)- HETE, are depicted (panel B). Values are presented as relative luciferase units and have been normalized to protein concentration. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical analysis of treatments was performed using Dunnett’s multiple comparison test (α=0.05). TCDD treatments were compared independently from 12(R)-HETE metabolite treatments. Values determined as being statistically significant from solvent (S) control are indicated by the presence of an asterisk (*).

158 12(R)-HETE Activates Transcription of an Endogenous AhR Target Gene in Multiple Human Cell Lines.

The ability of 12(R)-HETE to induce expression of CYP1A1, an endogenous AhR target gene almost totally dependent on activated AhR for expression, was analyzed using a pair of distinct human cell lines originating from different tissue types. Using this system, 12(R)-HETE mediated transcription of CYP1A1 was confirmed in both HepG2 and HaCaT cells, lineages derived from liver and epidermal origin respectively

(Figure 3.7). 12(R)-HETE treatment of Hep G2 cells resulted in a dose-dependent

induction of CYP1A1 mRNA levels, with a maximal-observed induction of approximately 20-fold occurring in response to 4.0 μM 12(R)-HETE treatment (panel A).

Similar results were obtained with HaCaT cells, which demonstrated an approximate 16- fold induction in response to the 4.0 μM 12(R)-HETE (panel B). However, statistically significant induction was observed at a much lower concentration for the HaCaT cell line, occurring in the nanomolar range, with 250 nM 12(R)-HETE treatment displaying an approximate 2-fold induction and 500 nM 12(R)-HETE treatment generating an approximate 3-fold induction in CYP1A1 mRNA levels. Thus, in the human-derived

HaCaT cell line, nanomolar concentrations of 12(R)-HETE can produce a statistically significant induction of AhR target gene mRNA levels.

159

Figure 3.7: 12(R)-HETE Can Activate AhR Target Genes in Multiple Human Cell Lines. The ability of 12(R)-HETE to activate AhR driven transcription of cyp1A1 was confirmed in two different human cell lines. 12(R)-HETE treatment of HepG2 cells resulted in a dose-dependent induction of cyp1A1 message levels (panel A), while a similar result was also obtained with HaCaT cells (panel B). Solvent (S) indicates the negative control comprised of vehicle (DMSO) treatment only. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical comparison of 12(R)-HETE treatments with solvent (S) control was performed using Dunnett’s multiple comparison test (α=0.05). Comparison of TCDD treatments with control was performed independently using the Student’s t-test (α=0.05). Values determined as being statistically significant from the solvent (S) control are indicated by the presence of an asterisk (*).

160 12(R)-HETE Modulates AhR-Mediated Transcription in a Receptor Dependent Manner.

Considering that 12(R)-HETE failed to demonstrate direct binding to the AhR, yet retains the ability to regulate AhR target genes in cells, it was necessary to determine whether the observed activation was occurring through an AhR dependent mechanism.

The level of AhR protein expression in HaCaT cells was significantly reduced in response to siRNA targeted against the AhR (Figure 3.8). AhR protein levels were

examined by western blot (panel A). Quantification of the blot through phosphor image

analysis revealed the levels of AhR protein expression were reduced by ~75% (panel B).

Under suppressed AhR protein levels, we can evaluate the effect of 12(R)-HETE on

AhR-mediated gene expression. Quantitative RT-PCR was used to analyze the

expression of three AhR target genes, CYP1A1, CYP1B1 and Ah receptor repressor, in

response to 12(R)-HETE in both AhR siRNA treated and control siRNA treated HaCaT

cells (Figure 3.9). 12(R)-HETE mediated activation of CYP1A1 is significantly

decreased in the AhR siRNA treated cells, indicating a required role for the receptor in

mediating the observed induction in CYP1A1 message levels (panel A). Similar results

were obtained for CYP1B1 (panel B) and Ah receptor repressor (panel C). These results

demonstrate that 12(R)-HETE-mediated activation of endogenous AhR target genes

functions through a receptor dependent mechanism. 161

Figure 3.8: RNA Interference (RNAi) Technology Demonstrates Effective Suppression of AhR Expression. In order to examine the role of the AhR in 12(R)- HETE mediated activation of AhR target genes, the effectiveness of RNA interference (RNAi) technology to suppress AhR gene expression in HaCaT cells first needed to be confirmed. Western blot analysis reveals a significant reduction in AhR expression levels in response to siRNA treatment (panel A). Phosphorimager analysis of the radioactive bands indicates a 75% reduction in AhR protein levels (panel B).

162

Figure 3.9: 12(R)-HETE Modulates AhR Signaling in a Receptor Dependent Manner. 12(R)-HETE mediated activation of cyp1A1, a classic AhR target gene is significantly squelched in the siRNA treated cells (panel A). Similar results were also obtained for cyp1B1 (panel B) and AhRR (panel C) thus confirming a role for the receptor in the activation of AhR target genes by 12(R)-HETE. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical analysis of the indicated 12(R)-HETE treatments was performed using the Student’s t-test (α=0.05). Values determined as being statistically significant are depicted by the presence of an asterisk (*).

163 3.5 DISCUSSION

To appreciate the significance of 12(R)-HETE mediated activation of the AhR requires an understanding of 12(R)-HETE production and the subsequent potential for this molecule and its metabolites to elicit an inflammatory response. 12(R)-HETE is generated by enzymatic oxidation of arachidonic acid via a 12(R)-lipoxygenase or cytochrome P450 mediated pathway, and serves as a proinflammatory lipid mediator

[236]. In addition, this compound can also be formed through enzymatic reduction of a

12-oxo-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12-oxo-ETE) molecule [300].

12(R)-HETE is a major eicosanoid metabolite identified in bovine, rabbit and human ocular tissue, where corneal epithelial microsomes can convert arachidonic acid to 12(R)-

HETE in the presence of NADPH [301, 302]. As the predominant lipoxygenase product in ocular tissue during inflammation, 12(R)-HETE is produced in the cornea after injury most likely by CYP4B1 [303]. This HETE is also produced upon treatment with vasopressin or after short-term contact lens wear in ocular tissue [304, 305]. In the cornea 12(R)-HETE is a potent chemotactic and angiogenic factor that may contribute to the growth of new blood vessels during chronic inflammation [306]. Exposure to 12(R)-

HETE also leads to decreased intraocular pressure and this physiologic endpoint is related to inhibition of Na,K-ATPase activity [307]. Indeed, this enzymatic activity can be efficiently inhibited by 12(R)-HETE, while 12(S)-HETE largely fails to inhibit this enzyme [250, 301]. However, further investigation will be required to determine if this ability of 12(R)-HETE to inhibit the Na, K-ATPase plays a role in AhR activation. The presence of the AhR in ocular tissue has not been determined; but CYP1A1 expression, 164 which is dependent on AhR activity for expression, has been detected in murine, rat and porcine tissues [308-310]. Interestingly, a significant constitutive level of CYP1A1 mRNA was detected in 3 to 5-week old rat extra-lenticular tissue [309]. Thus, it is likely that ocular inflammation would lead to AhR activation.

A second site of significant 12(R)-HETE production is in human skin during psoriasis and other inflammatory dermatoses [311, 312]. Unlike in the cornea, the enzyme responsible for 12(R)-HETE production is the skin-specific 12(R)-lipoxygenase

[229, 313]. The 12(R)-lipoxygenase is expressed in neonatal skin and during skin development [229]. In addition, it has been recently recognized that 12(R)-lipoxygenase deficiency in mice disrupts epidermal barrier function and thus establishes the importance of 12(R)-HETE production to normal skin development [314]. In humans, a mutation in the 12(R)-lipoxygenase gene has been associated with the genetic disorder non-bullous congenital ichthyosiform erythroderma, further supporting the significance of this lipoxygenase [315]. During psoriasis 12-HETE formation is elevated with 12(R)-HETE being the predominant form detected [316, 317]. In contrast, normal skin predominantly synthesizes the S-enantiomer of 12-HETE [318]. Neutrophil infiltration is a common characteristic of psoriasis and topical application of 12(R)-HETE produces erythema and neutrophil accumulation [249, 319]. These results coupled together support the conclusion that 12(R)-HETE is a major mediator of skin inflammation. Interestingly, a transgenic mouse expressing a constitutively active form of the AhR in keratinocytes exhibited postnatal inflammatory skin lesions [320]. This suggests that the AhR may play a role in the inflammatory responses observed in human skin diseases. 165 The actual mechanism of 12(R)-HETE-mediated activation of the AhR remains to be determined, but there are several possibilities. First, 12(R)-HETE might be metabolized into an AhR ligand. However, the primary characterized metabolites of

12(R)-HETE could not activate the AhR. A second possible mechanism is that 12(R)-

HETE activates a pathway leading to the production or stimulating the release of an endogenous ligand found in the cell. A third plausible mechanism involves 12(R)-HETE- mediated activation of a cellular signaling pathway resulting in ligand-independent activation of the AhR. Irregardless of the actual mechanism of AhR activation by 12(R)-

HETE, the level of specificity is clearly remarkable, considering other monohydroxylated

HETEs and 12(R)-HETE metabolites demonstrate essentially no ability to activate AhR signaling. Further investigations are warranted to explore the unique properties of 12(R)-

HETE. Currently, inhibition of Na,K-ATPase activity is the only specific biochemical mechanism of action observed for 12(R)-HETE. Future studies will focus on determining the mechanism of AhR activation.

The AhR is still considered an orphan receptor despite identification of several endogenous compounds that can serve as direct AhR ligands. The orphan receptor status of the AhR persists primarily because the potential endogenous ligands which have been identified are either relatively weak or fail to demonstrate a physiological function.

Recently, a relatively high affinity compound, 2-(1′H-indole-3′-carbonyl)-thiazole-4- carboxylic acid methyl ester, was isolated from acid treated cytosolic lung extracts [93].

However, it was never demonstrated if this apparent tryptophan and cysteine product actually exists in tissue extracts under physiological conditions. A tryptophan 166 photoproduct, 6-formylindolo[3,2b]carbazole, can form within cultured cells, but has not been shown to form in vivo [321]. Indirubin isolated from human urine has been found to be a relatively potent AhR ligand [92]. However, while indirubin can be formed from indole through cytochrome P450 metabolic activity and subsequent dimerization, it remains unknown if this compound plays a role in normal physiological processes [322].

Both bilirubin and 7-ketocholesterol, two physiologically relevant compounds, have been demonstrated to be ligands for the AhR [91, 99]. However, because these compounds are relatively weak receptor ligands and occur only under very restrictive in vivo conditions, they are unlikely important AhR ligands. Hydrodynamic shear stress has also been shown to activate CYP1A1 activity in Hep G2 cells, possibly resulting from the release of an AhR ligand within the cell [323, 324]. Interestingly, treatment with arachidonic acid in combination with hydrodynamic shear stress resulted in an approximate 3-fold increase in CYP1A1 activation compared to stress alone. This would suggest that a possible arachidonic acid metabolite might be involved in activating AhR signaling which would ultimately result in the activation of CYP1A1. Overall, the aforementioned results described in this chapter indicate for the first time the ability of a lipoxygenase product, commonly found in various tissues under inflammatory conditions, to activate the AhR. Furthermore, these results provide a firm link between inflammation, 12(R)-

HETE production and AhR activation within a variety of tissues. This work also provides important clues to a possible physiological function for the AhR in inflammatory signaling.

CHAPTER 4

5,6-DIHETE ISOMERS ARE ENDOGENOUS LIGANDS FOR THE AHR

168 4.1 ABSTRACT

The aryl hydrocarbon receptor (AhR), in addition to orchestrating an adaptive metabolic response to xenobiotic compounds, is critical in proper liver development, the resolution of fetal vasculature, and the maintenance of normal cardiac physiology. A member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) superfamily of developmental regulatory proteins, the AhR is a ligand-activated transcription factor capable of being regulated by a structurally diverse group of chemicals ranging from carcinogenic environmental pollutants, to dietary metabolites, to endogenously formed bioactive lipid molecules. Expanding the list of lipid mediators for the AhR, we report the identity of several eicosanoid molecules, including the common positional isomers of

LTA4 and various DiHETE metabolites, as capable of activating AhR signaling.

Furthermore, conclusive evidence is presented demonstrating that 5,6-

dihydroxyeicosatetraenoic acid isomers (5,6-DiHETEs), cellular metabolites of 5,6-

LTA4, can serve as endogenous ligands for the AhR. Produced during lipoxygenase

metabolism of arachidonic acid, 5,6-DiHETE isomers are formed upon hydrolysis of

leukotriene A4 (LTA4), an unstable allylic epoxide intermediate. LTA4 hydrolysis, which

can proceed through both enzyme catalyzed as well as non-enzymatic reaction

mechanisms, results in the formation of multiple dihydroxy isomers. Using a cell-based

DRE-driven luciferase reporter assay, various LTA4 metabolites, including several 5,6- and 5,12-DiHETE products, were analyzed for AhR activity with only the 5,6-DiHETE isomers demonstrating the ability to directly bind and activate AhR signaling.

Electrophoretic mobility shift assays (EMSA) demonstrated the ability of these molecules 169 to directly bind the AhR activating it to a DNA-binding species in vitro. Furthermore, ligand competition binding experiments confirm, via positive displacement of a radiolabeled photoaffinity ligand for the receptor, the ability of these compounds to directly bind in the ligand binding domain (LBD) of the AhR. Interestingly, aged preparations of 5,6-DiHETE isomers produced an enhanced level of AhR activation while demonstrating an increase in binding affinity for the receptor. Although the reason for this has not been fully determined, evidence supporting the formation of geometric isomers in the conjugated triene region of these molecules is believed to be responsible for the observed increase in activity.

170 4.2 INTRODUCTION

The aryl hydrocarbon receptor (AhR) is a ligand activated environmental sensory protein belonging to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) superfamily of DNA-binding transcriptional regulatory proteins [10, 325]. Initiated by ligand binding, the activated AhR undergoes a transformation process involving translocation to the nucleus and subsequent heterodimerization with ARNT (aryl hydrocarbon receptor nuclear translocator) to form a functional DNA-binding regulatory complex [8, 9, 326,

327]. This active heterodimeric AhR/ARNT complex regulates target gene expression through selective interaction with specific xenobiotic regulatory sequences, known as dioxin response elements or (DREs), typically contained in the upstream enhancer region of AhR target genes [45, 328-330]. Functioning like a chemosensor, the AhR is a cellular regulatory protein that can be activated by a structurally diverse group of chemicals ranging from carcinogenic environmental pollutants to dietary metabolites, to endogenously formed bioactive lipid molecules [331]. Historically, the study of AhR biology has centered on the ability of this soluble receptor to mediate the adaptive metabolic response to xenobiotic compounds. By modulating the induction of cytochrome P450s from the 1A and 1B families, the AhR is able to effectively induce the metabolism of many foreign chemical insults, decreasing their biological half-life by facilitating their excretion [105, 288, 332]. Recently, through the study of AhR null mice, the receptor has been identified as critical in the proper development of fetal vasculature [166, 333] in addition to occupying an important, yet still undefined, role in cardiac physiology [168, 169, 334]. Thus, as the AhR emerges as a critical regulator of 171 normal physiologic and developmental processes, the identification of key endogenous modulators of receptor activity becomes essential.

Previously, we demonstrated that Ah receptor activity can be activated by 12(R)-

HETE, an inflammatory lipid produced by the arachidonic acid cascade. Here we report the ability of several additional eicosanoid molecules, various 5,6- dihydroxyeicosatetraenoic acid isomers (5,6-DiHETEs), to serve as true endogenous ligands for the Ah receptor. Produced during lipoxygenase metabolism of arachidonic acid, 5,6-DiHETE isomers are formed upon hydrolysis of leukotriene A4 (LTA4), an unstable reactive allylic epoxide intermediate [335, 336]. With an extremely short half- life of approximately 3 sec under physiological conditions [219], LTA4 is a transient

molecule that, if not further metabolized will rapidly decompose via non-enzymatic pathways. Enzymatic metabolism of LTA4 includes the GST-transferase catalyzed

conjugation of reduced glutathione (GSH), via a sulfoether linkage, resulting in the

opening of the epoxide ring and the subsequent formation of the cysteinyl leukotrienes,

LTC4, LTD4 and LTE4. These peptide conjugated bioactive lipids are the potent

inflammatory mediators comprising the slow reacting substance of anaphylaxis (SRS-A)

[337-339]. Additional LTA4 metabolites are produced through hydrolysis of the strained

oxirane ring, a reaction capable of proceeding through both enzyme-catalyzed as well as non-enzymatic reaction mechanisms, ultimately resulting in the formation of various dihydroxy products. For example, LTA4 hydrolase catalyzes the formation of leukotriene

B4 (LTB4) [340, 341], while the generation of 5(S),6(R)-DiHETE is mediated by a

soluble epoxide hydrolase [215, 342]. Although the enzyme catalyzed reactions are 172 stereospecific, producing only a single reaction product, the non-enzymatic addition of water to LTA4 results in the formation of multiple dihydroxy positional and stereoisomers. Produced are 5(S),6(R)-dihydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (5(S),6(R)-DiHETE) and 5(S),6(S)-dihydroxy-7(E),9(E),11(Z),14(Z)- eicosatetraenoic acid (5(S),6(S)-DiHETE), a pair of dihydroxy fatty acid enantiomers epimeric at position C-6. In addition, non-enzymatic hydrolysis of LTA4 also generates the more prevalent 5(S),12(R)-dihydroxy-6(E),8(E),10(E),14(Z)-eicosatetraenoic acid (6- trans LTB4) and 5(S),12(S)-dihydroxy-6(E),8(E),10(E),14(Z)-eicosatetraenoic acid (6- trans-12-epi-LTB4), isomers of enzymatically produced LTB4 [335, 336, 343].

The studies performed in this section were driven by our initial findings that

suggested LTA4 possessed potent AhR activity, but was theoretically too unstable to

serve as an AhR ligand. Therefore, we performed an in-depth examination of LTA4 metabolites including the commonly formed 5,6- and 5,12-DiHETE isomers along with several cysteinyl-LT’s for their ability to bind and activate the AhR. Although several active metabolites were identified only the 5,6-DiHETEs demonstrated the ability to serve as ligands the for AhR.

173 4.3 EXPERIMENTAL PROCEDURES

Chemicals and Enzymes.

Preparations of methyl-5(S),6(S)-oxido-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid

(LTA4 methyl ester) were obtained through the generous contributions of Dr. C. Channa

Reddy (Dept. of Veterinary and Biomedical Sciences, The Pennsylvania State University,

University Park, PA). Additional amounts of LTA4 methyl ester along with racemic 5-HETE

were purchased from BIOMOL (Plymouth Meeting, PA). 5(S),6(R)-dihydroxy-

7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (5(S),6(R)-DiHETE) and 5(S),6(S)-

dihydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (5(S),6(S)-DiHETE) in addition

to 5-oxo-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-KETE) and still further

quantities of LTA4 methyl ester were obtained from Cayman Chemical Company (Ann

Arbor, MI). Optima grade high purity organic solvents were purchased from Fisher

Scientific (Pittsburgh, PA) and used in all chromatographic separations. Anhydrous

dimethyl sulfoxide (DMSO) (99.9% purity) was purchased from Sigma-Aldrich

(Milwaukee, WI). TCDD was a generous gift from Dr. Steven Safe (Texas A&M

University). 32P-γATP was purchased from PerkinElmer (Boston, MA) while T4

polynucleotide kinase was from Promega (Madison, WI). PolydI:dC was purchased from

Amersham Biosciences (Piscataway, NJ) and pre-cast 6% non-denaturing polyacrylamide

gels were from Invitrogen (Carlsbad, CA).

174 Cell Lines and Cell Culture.

The HepG2 40/6 reporter cell line [285] was generated as described previously, while the Hepa 1.1 reporter cell line [295] was a kind gift from Dr. Michael S. Denison

(University of California, Davis). Trypsin-EDTA, PBS, α-MEM, penicillin, and streptomycin were all obtained from Sigma (St. Louis, MO). FBS was purchased from

HyClone Laboratories (Logan, UT). Reporter cell lines were grown in α-MEM supplemented with 10% fetal bovine serum (v/v), 100 IU/ml penicillin, and 0.1 mg/ml

o streptomycin at 37 C in a humidified atmosphere containing 5% CO2/95% room air.

Clonal selection of reporter cell lines was maintained through the use of 300 μg/ml of

G418 (GibcoBRL, Carlsbad, CA).

Cell-based Reporter Assay.

Reporter cell lines were plated into 24-well tissue culture plates (Falcon,) at a

density of 5.0 x 105 cells/ well, and allowed 18 h of recovery before beginning a 6 h

treatment with increasing amounts of LTA4 methyl ester, LTA4 metabolite (5,6- or 5,12-

DiHETE), control HETE, DMSO or TCDD. Upon completion of the dosing regiment,

cells were thoroughly rinsed with PBS prior to addition of 1X cell culture lysis buffer (2

mM CDTA, 2 mM DTT, 10% Glycerol, 1% Triton X-100). After being frozen

overnight at -80oC, the lysates were then thawed and centrifuged at 18,000g for 15 min.

The resulting cytosol was assayed for luciferase activity using the Promega luciferase

assay system (Promega Corp., Madison, WI) as specified by the manufacturer. Light 175 production was measured using a TD-20e Luminometer (Turner Designs, Inc.,

Sunnyvale, CA). Cytosolic protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Luciferase activity was expressed relative to protein concentration. Statistical analysis of treatments was performed with GraphPad

Prism 4 software (San Diego, CA) using Dunnett’s multiple comparison test (α=0.05).

Values determined as being statistically significant from the solvent (S) control are indicated by the presence of an asterisk.

Electrophoretic Mobility Shift Assay.

DRE-specific EMSAs were performed using in vitro translated AhR and ARNT proteins. Expression vectors for these proteins were translated using a TNT coupled transcription and translation rabbit reticulocyte lysate kit (Promega Corp., Madison, WI).

All eicosanoid preparations were evaporated under argon gas and resolubilized in DMSO to achieve a stock solution of appropriate concentration. Serial dilutions of this stock were made in DMSO to achieve the various working stocks needed to perform a dose- response curve. Proteins for the transformation reactions were mixed together at a 1:1 molar ratio in HEDG buffer, followed by addition of either 0.5 μl DMSO solubilized

LTA4 methyl ester, LTA4 metabolite (5,6- or 5,12-DiHETE isomer), control HETE or

TCDD. All transformation assays were incubated for 90 min at room temperature,

followed by the addition of oligonucleotide buffer (42 mM Hepes, 0.33 M KCL, 50%

glycerol, 16.7 mM DTT, 8.3 mM EDTA, 0.125 mg/ml CHAPS, 42 ng/μl poly dI:dC).

Following 15 min of incubation time in oligonucleotide buffer, ~200,000 cpm of 32P- 176 labeled wild type DRE oligonucleotide was added to each reaction. Samples were then mixed with an appropriate amount of 5X loading dye and separated via electrophoresis separated on a 6% non-denaturing polyacrylamide gel. Wild-type DRE oligos comprised of the nucleotide sequence 5’-GATCTGGCTCTTCTCACGCAACTCCG-3’ and 3’-

ACCGAGAAGAGTGCGTTGAGGCCTAG-5’ were a gift from Dr. M.S. Denison.

AhR Ligand Competition Binding Assay.

2-Azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin was synthesized in our laboratory according to the procedure described previously, and was stored in methanol protected from light [296]. Hepa-1 cell cytosol, a source of mouse AhR, was prepared in

MENG buffer (25 mM MOPS, 2 mM EDTA, 0.02% sodium azide, 10% glycerol, pH 7.4) and diluted to a final protein concentration of 1.0 mg/ml. All binding experiments were carried out in the dark with 150 μg of soluble Hepa-1 cytosolic protein incubated with increasing concentrations of 12-HETE for 30 min. A saturating concentration of the AhR photoaffinity ligand, 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin (0.10 pmol; i.e.

4 x 105 cpm) was added and incubated for an additional 30 min at 23oC to achieve

equilibrium binding. The samples were photolysed at >302 nm for 4 min at a distance of

8 cm using two 15-Watt UV lamps (Dazor Mfg. Corp. St. Louis, MO). After irradiation, each sample was mixed with an equal volume of 2X tricine sample buffer (TSB) (0.9 M

Tris, pH 8.45, 24% glycerol, 12% w/v SDS, 0.015% w/v Coomassie Blue G, 0.005% w/v phenol red) and heated to 95oC for 5 min. Equal amounts of each sample were loaded onto a 9% Tricine-SDS-PAGE gel and subjected to denaturing electrophoresis overnight 177 at 15mA/gel. Proteins were transferred to p (PDVF) membrane using constant amperage

(500mA) for 4 hr in a Genie electro blot apparatus. The membrane was then exposed overnight at -80oC to X-OMAT-Blue film (Eastman Kodak Co.). Following autoradiogram generation, the radioactive AhR bands were excised from the acrylamide gel and quantified through scintillation counting using a LKB-Wallac RiaGamma (model

1274) automatic gamma counter (Waltham, MA). Statistical analysis of binding data was performed with GraphPad Prism 4 software (San Diego, CA) using one-way ANOVA with Tukey’s multiple comparison test (α=0.05). Values determined as being statistically significant from empty vector control are indicated with different letters.

178 4.4 RESULTS

Leukotriene A4 (LTA4) Isomers Activate AhR Signaling in Reporter Based Biological Activity Assays.

Driven by the initial observation that several non-selective lipoxygenase

inhibitors could squelch constitutive AhR activity in the CV-1 cell line, a methodical

screening of lipoxygenase products for potential AhR ligands was initiated. Screening

for biological activity in the human derived HepG2 40/6 reporter cell line, positional

isomers of LTA4 were the first LOX products identified to possess AhR activity.

Preparations of both 5,6-LTA4 and 14,15-LTA4, obtained from the laboratory of Dr. C.

Channa Reddy were capable of activating AhR signaling in a dose-dependent manner

with the maximum observed induction for each isomer equal to or greater than that

observed with 1nM TCDD (Figure 4.1). However, due to the extreme instability and

short half-life of these reactive allylic epoxide intermediates, downstream metabolites

were immediately suspected as being responsible for the observed induction in biological

activity following treatment with these compounds. Because 5,6-LTA4 is the more

common and better characterized of the leukotriene A4 isomers, our efforts and attention

became focused on screening the 5-LOX pathway for potential AhR endogenous ligands. 179

Figure 4.1: Leukotriene A4 (LTA4) Isomers Activate the AhR in Reporter Based Biological Activity Assays. Positional isomers of LTA4 were screened for ability to activate the Ah receptor. Both 5,6-LTA4 and 14,15-LTA4 are capable of activating AhR signaling in a dose-dependent manner (panel A). The solvent (S) control is a negative control comprised of vehicle (DMSO) treatment only. Values are presented as relative luciferase units and have been normalized to protein concentration. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical analysis of treatments was performed using Dunnett’s multiple comparison test (α=0.05). TCDD treatments were compared independently from LTA4 treatments. Values determined as being statistically significant from the solvent (S) control are indicated by the presence of an asterisk. The 5-LOX metabolic pathway is outlined in detail below (panel B).

180 Derived through 5-LOX catalyzed insertion of molecular oxygen into arachidonic acid followed by subsequent 5-LOX catalyzed dehydration of the hydroperoxy intermediate, formation of 5,6-LTA4 is at the center of the 5-LOX pathway. Capable of being further metabolized via both enzymatic and non-enzymatic pathways, metabolism of 5,6-LTA4 can result in the production of various DiHETE positional and stereoisomers

as well as in the formation of leukotriene B4 (LTB4) and cysteinyl leukotrienes (LTC4,

LTD4 and LTE4).

Enzymatically Derived Metabolites of Leukotriene A4 Fail to Activate the AhR in Biological Activity Assays.

5(S),12(R)-dihydroxy-6(Z),8(E),10(E),14(Z)-eicosatetraenoic acid, more

commonly known as LTB4, is a dihydroxy metabolite of LTA4, formed enzymatically by

the action of LTA4 hydrolase. Also formed through enzymatic metabolism of LTA4 is

the cysteinyl leukotriene (cysLT) LTE4 or 5(S)-hydroxy-6(R)-(S-cysteinyl)-

7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid. Formed from LTD4 through the action of

dipeptidase [344], LTE4 was chosen for testing because of the relatively small size of its

conjugated peptide moiety. However, neither of these enzymatically formed metabolites

was capable of activating AhR signaling when screened via bioassay in the human

derived HepG2 40/6 reporter cell line (Figure 4.2). Nonetheless, in order to be thorough,

all available cysLT’s including LTC4, LTD4, LTE4 and 11-trans LTE4 were examined for

their ability to bind and activate the AhR via DNA electrophoretic mobility shift assay, 181 but as expected, none of these peptide conjugated leukotriene metabolites demonstrated any ability to activate the receptor (data not shown).

182

Figure 4.2: AhR Activity Possessed by Common Enzymatically Derived Metabolites of Leukotriene A4. Leukotriene B4 (LTB4), a dihydroxy metabolite of LTA4, and Leukotriene E4 (LTE4), a sulfidopeptide leukotriene, were screened via bioassay in the human derived HepG2 40/6 reporter cell line for their ability to activate the Ah receptor. LTE4 was unable to activate AhR signaling while LTB4 produced a weak induction in AhR activity. The solvent (S) control is a negative control comprised of vehicle (DMSO) treatment only. Luciferase values have been normalized to protein concentration and are presented as fold induction compared to solvent (S) control. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical analysis of treatments was performed using Dunnett’s multiple comparison test (α=0.05). Comparison of TCDD treatments with control were made independently from other treatments. Values determined as being statistically significant from solvent (S) control are indicated by the presence of an asterisk (*).

183

All trans Isomers of LTB4 Activate the AhR in Biological Activity Assays.

Having identified essentially no AhR activity among the common enzymatic

metabolites of LTA4, various non-enzymatically formed DiHETEs were analyzed for the

potential to activate AhR signaling. 6-trans LTB4 and 6-trans-12-epi LTB4, a pair of C-

12 stereoisomers produced through non-enzymatic hydrolysis of LTA4, demonstrated the ability to induce DRE-driven luciferase reporter gene activity in a dose-dependent manner when administered to the human derived HepG2 40/6 reporter cell line.

Although both of these non-enzymatically formed isomers of LTB4 produced a

significant induction in AhR activity, the effect observed with 6-trans LTB4, known more

specifically as 5(S),12(R)-dihydroxy-6(E),8(E),10(E),14(E)-eicosatetraenoic acid, was

more potent resulting in a 3-fold induction of activity at 1.0 μM and a maximum

induction of 27-fold, equal to approximately 75% of that observed with 1nM TCDD,

occurring with 25.0 μM treatment. Treatment with 6-trans-12-epi LTB4, an identical

molecule except for the opposite stereochemical orientation of the C-12 hydroxyl group,

produced a less potent response (Figure 4.3). Although these molecules are considered to

form primarily as the result of non-enzymatic metabolism of LTA4, it is important to

mention that 6-trans LTB4 can also be produced through at least two additional

biochemical pathways. Although the physiological relevance is not clear, oxidative

decomposition of cysteinyl leukotrienes in the presence of myeloperoxidase and

hypochlorous acid can also result in the formation of 6-trans LTB4 [345]. Furthermore, a

cellular factor with double bond isomerase activity has been identified in rat kidney 184 homogenates and shown to be capable of enzymatically converting LTB4 into 6-trans

LTB4 [346]. 185

Figure 4.3: All trans Isomers of LTB4 Activate the AhR in Reporter Based Biological Activity Assays. 6-trans LTB4 and 6-trans-12-epi LTB4, a pair of stereoisomers produced through non-enzymatic hydrolysis of LTA4, stimulate a dose-dependent induction of DRE- driven luciferase gene in the human derived HepG2 40/6 reporter cell line. The solvent (S) control is a negative control comprised of vehicle (DMSO) treatment only. Luciferase values have been normalized to protein concentration and are presented as fold induction compared to background. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical analysis of treatments was performed using Dunnett’s multiple comparison test (α=0.05). Comparison of TCDD treatments with control were made independently from LTB4 treatments. Values determined as being statistically significant from solvent (S) control are indicated by the presence of an asterisk (*).

186

All trans Isomers of LTB4 Fail to Induce AhR Transformation.

Despite their ability to activate the AhR pathway in cell culture, neither of the 6-

trans LTB4 isomers was able to induce considerable DRE-binding when analyzed by

electrophoretic mobility shift assay and compared against a positive control comprised of

20 nM TCDD-induced AhR. After electrophoresis of the samples on a non-denaturing

polyacrylamide gel and overexposure of the X-ray film, a miniscule amount of

heterodimer complex formation could be seen in response to 6-trans LTB4 treatment at

both 10.0 and 25.0 μM (Figure 4.4). The observed band, if genuine, appears to be far less

than what would be expected given the bioassay results for this compound. Furthermore,

6-trans-12-epi LTB4 treatment produced even less heterodimer formation than 6-trans

LTB4, despite the ability of this compound to demonstrate a substantial induction in

reporter gene activity at 25.0 μM. Because neither of these isomers were capable of

inducing significant AhR transformation, evident by an essential lack in shifted

radiolabeled complex (32P-DRE-AhR:ARNT) formation, it was concluded that neither of the compounds could directly transform the receptor to its DNA-binding form. However,

additional studies would be required to determine if these compounds are capable of

competing for AhR binding.

187

Figure 4.4: All trans Isomers of LTB4 Fail to Induce Transformation of the AhR. Both 6-trans LTB4 (panel A) and 6-trans-12-epi LTB4 (panel B) isomers were tested for their ability to bind and transform the AhR and compared to a positive control comprised of 20 nM TCDD induced receptor. When analyzed at increasing concentrations ranging from 1.0 to 25.0 μM, neither isomer could induce significant heterodimerization, indicating a relative inability of these compounds to bind and transform the AhR into its DNA binding form. Gel shift controls included a negative (-) control containing no ARNT, a background (B) control comprised of only AhR and ARNT to control for background heterodimerization, and a solvent (S) control to access the effects of vehicle on heterodimer formation.

188 Persisting in our effort to identify a true endogenous ligand for the AhR, one capable of activating the receptor through direct ligand binding, additional screening of

LTA4 metabolites was performed. Still holding steadfast to the assumption that LTA4 was too unstable to be directly responsible for the observed induction in reporter gene activity, the only remaining untested metabolites of LTA4, the 5,6-DiHETE isomers,

were subsequently screened for AhR activity. However, the neither significance nor the

biological role of these molecules has been fully elucidated and the extent of their

formation in the cell remains ambiguous.

5,6-DiHETE Epimers Activate AhR Signaling in Biological Activity Assays.

The 5,6-DiHETEs are pair of dihydroxy metabolites, epimeric at C-6, produced

through the metabolism of LTA4 [335]. 5(S),6(R)-dihydroxy-7(E),9(E),11(Z),14(Z)-

eicosatetraenoic acid (5(S),6(R)-DiHETE) is a molecule whose formation is feasible

through multiple pathways including the enzymatic transformation of LTA4 by liver cytosolic epoxide hydrolase [347]. Alternatively, non-enzymatically hydrolysis of the unstable epoxide ring contained in LTA4 can also give rise to formation of 5(S),6(R)-

DiHETE. Furthermore, 5(S),6(R)-DiHETE may also be formed from 5(S)-HETE through the 6(R)-oxygenase activity contained in enzymes such as porcine leukocyte 5-

LOX [348]. Alternatively, its C-6 epimer, 5(S),6(S)-dihydroxy-7(E),9(E),11(Z),14(Z)- eicosatetraenoic acid (5(S),6(S)-DiHETE), is a molecule known only to be formed through non-enzymatic hydrolysis of LTA4 [336]. When tested in-vivo using the human

derived HepG2 40/6 reporter cell line, both 5(S),6(R)-DiHETE and 5(S),6(S)-DiHETE 189 demonstrated the ability to activate the Ah receptor (Figure 4.5). Although 5(S),6(S)-

DiHETE produced a more potent response, treatment with increasing concentrations of either stereoisomer resulted in the dose-dependent activation of AhR signaling as analyzed through induction of a DRE-driven luciferase reporter gene. For comparison purposes, the molecular structure of both 5,6-DiHETE isomers are depicted (panel B). 190

Figure 4.5: Epimers of 5,6-DiHETE Activate AhR Signaling in Reporter Based Biological Activity Assays. Treatment of the human derived HepG2 40/6 reporter cell line with 5(S),6(R)-DiHETE and 5(S),6(S)-DiHETE produced a dose-dependent activation of AhR signaling (panel A). The solvent (S) control is a negative control comprised of vehicle (DMSO) treatment only. Values are presented as relative luciferase units and have been normalized to protein concentration. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical analysis of treatments was performed using Dunnett’s multiple comparison test (α=0.05). Comparison of TCDD treatments with control were made independently from DiHETE treatments. Values determined as being statistically significant from solvent (S) control are indicated by the presence of an asterisk (*). The structures of both 5,6-DiHETE isomers are depicted (panel B).

191 5,6-DiHETE Epimers Induce AhR Transformation and Heterodimerization.

Using electrophoretic mobility shift assays (EMSA), it was confirmed that the observed induction in reporter gene activity observed with the 5,6-DiHETE isomers is the result of direct AhR binding and activation. Using 20 nM TCDD treated receptor as a positive control, the formation of 32P-DRE-AhR:ARNT complexes in response to 5,6-

DiHETE treatment was examined after electrophoresis on a non-denaturing

polyacrylamide gel (Figure 4.6). Both 5,6-DiHETE isomers induced AhR transformation and DRE binding in a dose-dependent manner with the 5(S),6(S)-DiHETE epimer displaying a significantly increased potency, consistent with the results obtained in biological activity assays. 192

Figure 4.6: 5,6-DiHETEs Induce AhR Transformation and Heterodimerization. DNA electrophoretic mobility shift assays (EMSA) reveal the ability of 5,6-DiHETE isomers to directly bind and activate the AhR to its DNA-binding form in a dose- dependent manner at concentrations ranging from 1.0 to 25.0μM. The signal observed with 20nM TCDD treated receptor served as a positive control for the formation of 32P- DRE-AhR:ARNT complexes. Additional gel shift controls included a negative (-) control containing no ARNT, a background (B) control comprised of only AhR and ARNT to control for background heterodimerization, and a solvent (S) control to access the effects of vehicle on heterodimer formation.

193 5,6-DiHETE Epimers Compete for AhR Binding.

In order to further analyze the ability of the 5,6-DiHETE isomers to bind the

AhR, ligand competition binding experiments were performed. Only a slight displacement of the radiolabeled dioxin molecule could be seen in response to increasing concentrations of 5(S),6(R)-DiHETE, while the 5(S),6(S) epimer demonstrated a much more potent ability to displace the photoaffinity ligand. Racemic 5-HETE, serving as a negative control (NC), demonstrated no significant ability to displace radioligand, while a positive control comprised of 300 nM benzo[a]pyrene (B[a]P) exhibited strong photoaffinity ligand displacement (Figure 4.7). The radiolabeled AhR bands were

quantified through phosphoimager analysis. A graphical representation of the ligand

competition binding data indicates an approximate 50% displacement of radiolabeled

photoaffinity ligand by 5(S),6(S)-DiHETE at the highest assay concentration. 194

Figure 4.7: Epimers of 5,6-DiHETE Are Capable of Directly Binding the AhR. Ligand competition binding experiments reveal the ability of 5,6-DiHETE isomers to directly compete with a radiolabeled photo-affinity ligand for AhR binding (panel A&B). DiHETE binding affinity was analyzed through photo-affinity ligand displacement with the 5(S),6(S)-DiHETE epimer demonstrating the greatest affinity for the AhR. Both DiHETE isomers were examined at increasing concentrations ranging from 1.0 to 25.0μM. Racemic 5-HETE, a negative control (NC), demonstrated no significant ability to displace radioligand at a concentration of 25.0μM, while 300 nM benzo[a]pyrene (B[a]P), serving as the positive control, exhibited strong photoaffinity ligand displacement. The solvent (S) control represents vehicle (DMSO) treatment of Ah receptor. A graphical representation of the binding data is presented (panel C&D). Statistical analysis of data was performed using Tukey’s multiple comparison test (α=0.05). Values determined as being statistically significant from each other are indicated by different letters.

195 Aged Preparations of 5(S),6(S)-DiHETE Demonstrate an Increased Ability to Activate the AhR in Biological Activity Assays.

5,6-DiHETE preparations, especially the 5(S),6(S) epimer, experience a time- dependent gain in biological activity when stored for prolonged periods of time under argon sparge at -80oC. Treatment of the human derived HepG2 40/6 reporter cell line with increasing concentrations of aged1 preparations of 5(S),6(S)-DiHETE resulted in a significantly enhanced dose-dependent activation of AhR signaling (Figure 4.8).

Specifically, treatment with 10.0uM aged 5(S),6(S)-DiHETE produced an induction in

reporter gene activity that exceeded that observed with 1nM TCDD, while that recorded

in response to 25.0uM treatment rivaled that observed with a saturating dose of TCDD.

Compared with results obtained in a similar experiment using a freshly purchased preparation of 5(S),6(S)-DiHETE (Figure 4.5), where a 10.0uM treatment produced an

induction only 40% of that seen with 1nm TCDD, the results obtained here demonstrate

the significant increase in biological activity that is gained among aged preparations of

5(S),6(S)-DiHETE.

1 An aged DiHETE preparation is defined as having been used and stored under argon gas sparge at -80oC for approximately six months or more before being reused, as opposed to a fresh sample which was used immediately or within several days of arrival in the laboratory. 196

Figure 4.8: Aged Preparations of 5(S),6(S)-DiHETE Demonstrate an Increased Ability to Activate the AhR in Biological Activity Assays. 5,6-DiHETE preparations experience a time-dependent gain in activity when stored for prolonged periods of time under argon sparge at -80oC. Treatment of the human derived HepG2 40/6 reporter cell line with increasing concentrations of aged preparations of 5(S),6(S)-DiHETE resulted in a significantly enhanced dose-dependent activation of AhR signaling. The solvent (S) control is a negative control comprised of vehicle (DMSO) treatment only. Values are presented as relative luciferase units and have been normalized to protein concentration. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical analysis of treatments was performed using Dunnett’s multiple comparison test (α=0.05). Comparison of TCDD treatments with control were made independently from DiHETE treatments. Values determined as being statistically significant from solvent (S) control are indicated by the presence of an asterisk (*).

197 Aged Preparations of 5,6-DiHETE Exhibit an Enhanced Binding Affinity for the AhR.

Likewise, preparations of the 5,6-DiHETE stereoisomers acquire an improved binding affinity for the AhR over time as determined through DNA mobility shift assays.

Using 20nM TCDD treated receptor as a positive control, the formation of 32P-DRE-

AhR:ARNT complexes formed in response to treatment with aged preparations of 5,6-

DiHETEs was examined after electrophoresis on a non-denaturing polyacrylamide gel.

An enhancement in the AhR transformation and DRE binding capability of 5,6-DiHETE

is observed among samples which have undergone prolonged periods of storage under

argon sparge at -80oC. This effect is most striking with the 5(S),6(S)-DiHETE epimer

(Figure 4.9). Gel shift controls included a negative (-) control containing no ARNT, a

background (B) control comprised of only AhR and ARNT to control for background

heterodimerization, and a solvent (S) control to access the effects of vehicle on

heterodimer formation. 198

Figure 4.9: Aged Preparations of 5,6-DiHETE Exhibit an Enhanced Binding Affinity for the AhR. Over time preparations of the 5,6-DiHETE stereoisomers acquire an improved binding affinity for the AhR as determined through DNA mobility shift assays. 20 nM TCDD treated receptor served as a positive control for 32P-DRE- AhR:ARNT complex formation. Samples were examined after electrophoresis on a non- denaturing polyacrylamide gel. Gel shift controls included a negative (-) control containing no ARNT, a background (B) control comprised of only AhR and ARNT to control for background heterodimerization, and a solvent (S) control to access the effects of vehicle on heterodimer formation.

199 Although the exact mechanism responsible for the increase in activity observed among aged preparations of 5,6-DiHETE is still not fully understood, double bond isomerization and the formation of geometric isomers appears to be involved. However, this explanation was solely derived based on the interpretation of results obtained through both HPLC and UV-spectral analysis of DiHETE samples. Comparison of HPLC chromatograms generated during the re-purification of aged 5(S),6(S)-DiHETE revealed the formation of two miniscule peaks with negligible optical absorbance at 272nM. These peaks eluted ahead of the parent DiHETE compound and were not present in fresh preparations, at least not at detectable levels. Careful spectral analysis of these two new peaks revealed the presence of a UV-spectrum characteristic in shape for a DiHETE molecule, but containing a slightly shifted λmax for the conjugated triene. Compared with

a λmax value of 270nm for 5,6-DiHETE in hexane based HPLC mobile phase, both of the new peaks displayed hypsochromically (blue) shifted λmax values of 268.8nm and

264.1nm when recorded in the same manner. The observed shift in λmax values could

potentially indicate isomerization of the conjugated triene to the all trans orientation or

formation of a conjugated tetraene, respectively. Therefore, because these two additional

peaks were the only observable difference between aged and fresh preparations of 5,6-

DiHETE, it was hypothesized that formation of a geometric isomer was responsible for

the gain in activity among the aged preparations.

Interestingly, a similar phenomenon has been observed with positional isomers of

LTA4. Depending on the source, sample preparations exhibited a great deal of variation 200 in the amount of biological activity displayed. For example, commercially available preparations of 5,6-LTA4 obtained from BIOMOL (Plymouth Meeting, PA), even after

further purification in our laboratory, consistently demonstrated significant AhR activity,

while Cayman Chemical Company (Ann Arbor, MI) preparations exhibited little or no

activity. However, samples of Cayman 5,6-LTA4 ME stored for extended periods of time at -80oC under argon sparge demonstrated significant AhR activity upon reuse in gel shift

assays (Figure 4.9 panel C). In addition, if the inactive Cayman 5,6-LTA4 sample preparations were stored at room temperature under argon gas sparge for several days, they acquired the ability to bind and activate the AhR (Figure 4.10), while still retaining the characteristic LTA4 spectra. Furthermore, comparison of UV-spectra generated from the active Cayman LTA4 sample with that produced from an inactive preparation proved

to be identical, as both samples displayed spectra with peaks at 271 nm, 280 nm, 292 nm.

(Figure10B). 201

Figure 4.10: Inactive Preparations of 5,6-LTA4 Methyl Ester Acquire AhR Activity Following Short-Term Storage at Elevated Temperature. Inactive preparations of 5,6-LTA4 ME suddenly acquire the ability to bind and activate the AhR following a brief storage period (5-7 days) at room temperature (25oC) under argon gas sparge. Following elevated temperature storage, the 5,6-LTA4 methyl ester preparation was tested at increasing concentrations ranging from 0.5 to 25.0μM for AhR activity. 20nM TCDD transformed AhR served as a positive control (panel A). Interestingly, LTA4 Me preparations stored at -80oC remained inactive, yet UV-spectra generated from both sets of samples proved to be identical displaying the characteristic LTA4 spectra with peaks at 271 nm, 280 nm, 292 nm and no observable differences (panel B).

202

Similarly, BIOMOL preparations of 14,15-LTA4 demonstrated strong AhR

activity, while samples prepared by Larodan Fine Chemicals (Malmo, Sweden), the only

other commercially available source of the compound, were inactive. Furthermore, although the original preparations of both 5,6- and 14,15-LTA4 synthesized several years

earlier in the laboratory of Dr. C. Channa Reddy produced a strong induction in AhR

activity, all subsequent synthesis attempts by myself and others resulted in the

preparation of inactive compounds (data not shown).

Collectively, the observations made with 5,6-LTA4 together with those made with

aged preparations of 5,6-DiHETE suggested the possible formation of a geometric isomer

in these preparations as potentially being responsible for the observed increase activity.

In support of this rationale is the fact that BIOMOL preparations of LTA4 are listed as containing 3-5% of the 11-trans 5,6-LTA4 isomer. Furthermore, a slow temperature-

dependent isomerization of the C-11 double bond has been observed with such

compounds as LTC4, LTD4, LTE4 and 5,6-DiHETEs during low temperature storage. In

addition, the C-11 trans isomer of several common leukotrienes and DiHETEs, including

11-trans LTC4, 11-trans LTD4, 11-trans LTE4, and 11-trans 5(S),6(R)-DiHETE, have

been shown to form in-vivo [216, 349, 350]. Thus, isomerization of the C-11 double

bond in the stereoisomers of 5,6-DiHETE maybe responsible for the increase in activity

associated with the storage of these molecules. However, with neither of the 11-trans

5,6-DiHETE epimers readily available and time of the essence, custom synthesis became the only viable option and because it was financially feasible to only synthesize one 203 compound, the 11-trans 5(S),6(R)-DiHETE epimer was chosen because its formation in- vivo had already been demonstrated [216].

All trans 5(S),6(R)-DiHETE Demonstrates a Comparatively Stronger Induction of Reporter Gene Activity in Biological Activity Assays.

Using the murine derived Hepa-1.1 reporter cell line, a direct comparison of the biological activity contained in each of C-11 geometric isomers of 5(S),6(R)-DiHETE was performed. Under identical experimental conditions, treatment with an increasing concentration of pure 11-trans 5(S),6(R)-DiHETE resulted in an enhanced dose- dependent activation of AhR signaling when compared with its C-11 cis double bond isomer. While neither isomer produced a strong induction in DRE-driven luciferase reporter gene activity, a substantial improvement was achieved solely through isomerization of the C-11 double bond (Figure 4.11). This alone provides an important

piece of structure activity relationship (SAR) information about the compound and

potential eicosanoid ligands for the AhR in general and although the change may appear

subtle, it can be visualized as having a pronounced effect on the conformation of the

molecule (panel B) Double bond isomerization at C-11 should, in theory, allow the

olefinic region of the molecule to adopt an altered structural conformation, one more

rectangular shape, which could potentially allow for an increase in AhR binding. 204

Figure 4.11: The 11-trans 5(S),6(R)-DiHETE Isomer Produces a Comparatively Stronger Induction of Reporter Gene Activity. Treatment of the murine derived Hepa- 1.1 reporter cell line with increasing concentrations of pure 11-trans 5(S),6(R)-DiHETE resulted in an enhanced dose-dependent activation of AhR signaling when compared with its C-11 geometric isomer. The solvent control (S) is a negative control comprised of vehicle (DMSO) treatment only. Values are presented as relative luciferase units and have been normalized to protein concentration. Each data point represents the mean (+/-) S.D. of three separate determinations. Statistical analysis of treatments was performed using Dunnett’s multiple comparison test (α=0.05). Comparison of TCDD treatments with control were made independently from DiHETE treatments. Values determined as being statistically significant from solvent (S) control are indicated by the presence of an asterisk (*).

205 All trans 5(S),6(R)-DiHETE Displays an Increased Binding Affinity for the AhR.

Ligand competition binding experiments confirm the all trans geometric isomer of 5(S),6(R)-DiHETE exhibits a much stronger binding affinity for the AhR than its 11- cis isomer. Compared with 5(S),6(R)-DiHETE which resulted in minimal radioligand displacement, increasing concentrations of 11-trans 5(S),6(R)-DiHETE produced a much more potent effect, demonstrating the ability to displace approximately 70% of photoaffinity ligand binding in response to 25.0 μM treatment (Figure 4.12). Meanwhile,

5-oxo-ETE, serving as a negative control (NC), demonstrated no significant ability to

displace radioligand, while a positive control comprised of 300 nM benzo[a]pyrene

(B[a]P) exhibited strong photoaffinity ligand displacement. The solvent control (C)

represents vehicle (DMSO) only treatment of Ah receptor. A complete graphical

representation of the ligand competition binding data is presented (panel C&D). 206

Figure 4.12: All trans 5(S),6(R)-DiHETE Exhibits an Increased Binding Affinity for the AhR. Ligand competition binding experiments reveal the 11-trans geometric isomer of 5(S),6(R)-DiHETE possesses an increased binding affinity for the AhR. Compared with its 11-cis isomer (panel A), increasing concentrations of 11-trans 5(S),6(R)-DiHETE exhibit a significantly more potent displacement of radiolabeled photoaffinity ligand (panel B). 5-oxo- ETE served as a negative control (NC) and demonstrated no significant ability to displace radioligand while a positive control comprised of 300 nM benzo[a]pyrene (B[a]P) exhibited strong photoaffinity ligand displacement. The solvent control (C) represents vehicle (DMSO) treatment of Ah receptor. A graphical representation of the ligand competition binding data is presented (panel C&D). Statistical analysis of data was performed using Tukey’s multiple comparison test (α=0.05). Values determined as being statistically significant from each other are indicated by different letters.

207 With double bond isomerization at C-11 as the only difference between epimers, the increase in binding affinity observed with the 11-trans isomer can most logically be perceived as resulting from a change in conformation that allows the molecule to bind the

AhR more effectively. However, even with a substantial increase in binding affinity for the AhR, 11-trans 5(S),6(R)-DiHETE only demonstrated a marginal increase in the ability to activate transcription of a DRE-driven luciferase reporter gene (Figure 11A).

Furthermore, when CYP1A1 mRNA levels were analyzed in HaCaT cells using R.T.-

PCR, treatment with all trans 5(S),6(R)-DiHETE resulted in the same level of target gene induction as its 11-cis counterpart (data not shown). Thus, despite its enhanced AhR binding affinity, 11-trans 5(S),6(R)-DiHETE still appears as a weak AhR ligand, at least in terms of regulating induction of CYP1A1, a classic AhR target gene.

208 4.5 DISCUSSION

Taken collectively, the results presented in this chapter demonstrate that 5- lipoxygenase products, in particular 5,6-leukotriene A4 and its subsequent 5,6-

dihydroxyeicosatetraenoic acid metabolites, are capable of regulating AhR signaling.

This demonstrates, for the first time, a connection between pro-inflammatory lipid

mediators produced through the 5-LOX pathway and AhR activation. This relationship,

however, appears to be quite complicated, still convoluted by many unresolved details

regarding the exact double bond geometry of the most active metabolites. Nevertheless,

it indicates a potentially important role for the AhR during the inflammatory process.

However, unlike other studies which have previously drawn a correlation between AhR

activation and inflammation [320], our results provide definitive evidence linking

specific endogenously formed lipoxygenase metabolites with AhR activation and cellular

inflammation by confirming the ability of these pro-inflammatory eicosanoid molecules

to also serve as direct AhR ligands. The identification of bioactive lipid mediators as

endogenous ligands for the AhR not only provides added insight into the potential physiological role(s) for the receptor, but may also allow for a better understanding of the toxicity associated with chronic AhR activation. Furthermore, activation of AhR signaling by pro-inflammatory lipids could develop into an issue of paramount medical importance, especially considering the number of disease conditions that result from, or are worsened by, a state of chronic inflammation, such as atherosclerosis [192, 351], pulmonary fibrosis [352], Alzheimer's disease [353], the many different autoimmune disorders [354, 355], and countless forms of cancer [356-359]. 209 Having firmly demonstrated the ability to directly bind and activate the AhR, it is clearly apparent that 5,6-DiHETEs are indeed endogenous ligands for this orphan cellular receptor. Conversely, upon initial observation, it may be argued that the overall concentrations needed to produce a substantial level of luciferase reporter gene activation seem quite elevated. However, one must consider the very polar nature of these dihydroxy organic acids and their inefficient cellular uptake compared with much more hydrophobic molecules like TCDD, which can efficiently translocate across cellular membranes. Therefore, in actuality, the concentration of DiHETE molecules reaching the inside of the cell is expected to be far lower than the concentration established in the culture media [360]. In fact, comparing the activity of an endogenous lipid mediator for the AhR against TCDD mediated gene induction results in a biased comparison for several reasons. In addition to the aforementioned uptake issues, it is also highly unlikely that an endogenous activator would need to produce gene induction of a magnitude equivalent to that seen with TCDD, the most potent of the HAH congers. Furthermore, it is likely that the extremely high levels of gene induction seen in response to TCDD exposure, contribute to the toxicity and carcinogenicity associated with this compound.

More realistic is to view a biologically relevant induction of target gene expression as occurring in response to much smaller increases in gene expression levels. The selective induction of a target gene(s) in a highly coordinated manner, in which the timing, duration and magnitude of expression were precisely controlled, by an endogenous regulator for instance, could result in a biologically significant response with only several fold induction in gene expression levels. Also, unlike TCDD which is highly resistant to metabolic degradation and thus continues to drive gene expression, metabolism of 5,6- 210 DiHETE compounds would be expected to begin immediately after uptake, thus effectively reducing their cellular concentration. Although the metabolic events responsible for the degradation of many hydroxylated polyunsaturated fatty acids is still largely unknown, termination of 5,6-DiHETE biological activity could be expected to occur via metabolic inactivation in a manner similar to the 6-trans epimers of LTB4

[361].

Perhaps the biggest issue surrounding the magnitude of the biological response observed with any these molecules is the presence of geometric isomers. It appears highly unlikely that the time dependent increase in activity, consistently observed among aged preparations of LTA4 and 5,6-DiHETE, is the result of anything other than the

formation of a highly active geometric isomer. This assumption, however, is solely based

on the lack of significant biochemical differences observed between fresh and aged

preparations of compound. With no visible changes in the UV-spectra for the highly active aged preparations, it is highly unlikely that any significant amount of the

molecules have undergone change. Yet, the biological activity of these samples is greatly

enhanced. Only after careful fractionation were minor subtleties in the peak profiles of

the aged preparations detected in the form of two minor peaks eluting ahead of the parent

compound. If either of these newly formed peaks is responsible for the increased activity of the aged preparations, it would suggest the formation of an extremely high affinity

AhR ligand based on the large gain in biological activity despite the trace amount of new material. Therefore, it seems quite possible that much lower concentrations of 5,6-LTA4 or 5,6-DiHETE would result in AhR activation once the most active isomer is identified. 211 As discussed earlier, because the novel peaks identified in the aged DiHETE preparations were still characteristic in shape for a DiHETE molecule, but displayed hypsochromically shifted λmax values, geometric isomers in the conjugated triene

functionality were hypothesized. But, in order to avoid potential issues regarding the biological relevance of the non-enzymatically formed 5(S),6(S)-DiHETE epimer, only geometric isomers of the epoxide hydrolase formed 5(S),6(R)-DiHETE were

investigated. Specifically, because its formation in-vivo had been previously

demonstrated, the 11-trans isomer of 5(S),6(R)-DiHETE was analyzed, and although it

displayed improved biological activity, it still produced only a modest activation of AhR

signaling. However, if indeed, the increase in AhR activity of aged DiHETE preparations

is resulting from cis - trans double bond isomerization at C-11, then it stands to reason

that the pure 11-trans isomer of 5(S),6(S)-DiHETE should produce the greatest level of

AhR activation, considering that aged 5(S),6(S)-DiHETE preparations demonstrated the

greatest gain in biological activity during storage. Similarly, the presence of a highly

active geometric isomer may also explain the differences in the activity observed among

different sources of 5,6-LTA4, while the formation of such an isomer could also explain

the relatively rapid gain in activity when inactive LTA4 preparations are allowed to stand

at room temperature under an inert atmosphere.

Conversely, there is still the possibility that a small amount of oxidized, degraded

or rearranged product is responsible for producing the elevation in activity levels

observed with aged DiHETE samples, but the likelihood of that would seem quite

improbable given the storage conditions. Oxidation and oxidative degradation should be 212 minimized in samples stored under an inert atmosphere at -80oC. Furthermore, because

the formation of the 11-trans geometric isomer of 5,6-DiHETE has been observed during

low temperature storage [216], this appears to be the most logical explanation for the

enhanced activity observed with the aged DiHETE preparations. Nevertheless, the

results presented here indicate the ability of bioactive lipid metabolites produced through

lipoxygenase metabolism of AA, to serve as activators of the AhR indicating a

biochemical role for the receptor as a selective lipid sensor. In the future, through

continued identification of lipophilic endogenous ligands and activators for the AhR, the

biological rational underlying this lipid discerning capability of the AhR should be

revealed.

213 4.6 Summary and Conclusions

Accumulating scientific evidence indicates an important cellular role for the AhR in normal vertebrate physiology. Driven by these observations, the primary objective of this doctoral thesis was to identify and characterize a physiologically relevant high affinity endogenous ligand(s) for the AhR. Through the identification of such molecules, it was anticipated that valuable information about the biological role for the AhR and the subsequent consequences of inappropriate and/or excessive activation would be revealed.

Initially, our search for an endogenous AhR ligand began with the CV-1 cell line, an immortalized animal cell culture line derived from the kidney epithelium of the African green monkey. In particular, these cells were chosen because they naturally express only a minimal amount of AhR, resulting in the reduction of AhR-regulated cytochrome P450 metabolism. Thus, under these conditions, it was hypothesized that cellular metabolites, including potential intracellular endogenous ligands for the AhR, would be expected to accumulate. Furthermore, this cell line had been previously demonstrated by another laboratory, while studying receptor nuclear localization, to contain elevated levels of constitutively active AhR. Taken collectively, these observations indicated that CV-1 cells potentially contained elevated levels of putative endogenous ligand(s) for the AhR, thus providing an enriched source of starting material from which to identify putative endogenous AhR ligands and P4501A substrates.

The second chapter of this thesis presents data confirming the existence of a putative endogenous ligand(s) for the AhR in CV-1 cells, as organic extracts prepared 214 from CV-1 cytosol demonstrated the ability to directly bind and activate the AhR in gel shift assays. Although it was already established that CV-1 cells contained an elevated level of constitutively active AhR, this initial result provided strong evidence supporting the presence of endogenous ligand(s) for the AhR as being responsible for the observed activity. Subsequent HPLC fractionation of cytosol later revealed the presence of multiple activity peaks indicating a complex mixture of putative AhR ligands and/or indirect activators. It was during HPLC method development, while trying to determine optimal extraction and purification conditions, that several pieces of crucial qualitative information about the putative endogenous ligand(s) were obtained. For example, because acidification of CV-1 cytosol was required for optimal extraction of biological activity into organic solvent, it appeared to indicate the presence of an ionizable functional group(s) in the putative endogenous ligand(s). Additionally, switching from reverse phase to straight phase HPLC produced drastic improvements in chromatographic reproducibility and consistency of biological activity profiles. Still even greater enhancements in reproducibility and biological activity were achieved after implementing the use of anhydrous sodium sulfate to remove trace amounts of water from all organic solvents. Collectively, these results suggest the presence of one or more potentially labile functional groups in the putative endogenous ligand molecule(s). Furthermore, it is likely the molecule(s) is quite hydrophobic based on its ability to partition into organic solvents like hexane, ethyl acetate and dichloromethane. Finally, one last piece of speculative information about the identity of the putative endogenous ligand(s) can be obtained from the initial mass spectrometry results. Even though numerous attempts to characterize active fractions by LC-MS proved to be arduous and largely unsuccessful, sample 215 analysis performed independently by three different laboratories consistently demonstrated that the vast majority of biological active fractions contained an abundance of fragments with M/Z ratios between 300 and 400 in. This size range, albeit common to many small molecules, is also consistent with most eicosanoid molecules, and studies performed using inhibitors of eicosanoid biosynthesis, such as NDGA or ketoconazole, produced dramatic reductions in the level of constitutive AhR activity in CV-1 cells.

Therefore, taken together, I believe the aforementioned information indicates the presence of a bioactive lipid mediator functioning as an endogenous ligand for the AhR in CV-1 cells. In the future, attempts to identify this molecule(s) should be met with greater success due to powerful new improvements in separation science, namely the advent of ultra- performance liquid chromatography (UPLC), together with technological advancements in mass spectroscopy instrumentation.

The results presented in chapter 2 also provide strong evidence supporting the existence of a possible ligand activated feedback mechanism, where AhR regulated cytochrome P450s from the CYP1A and CYP1B families are able to metabolically alter the biological activity of the putative endogenous ligand(s), thus squelching AhR signaling. This model is supported by studies that demonstrated the expression of cytochrome P450s 1A1, 1A2 or 1B1 reduced AhR-mediated luciferase reporter activity; while the non-AhR regulated cytochrome P450 2E1 exhibited no significant effect. In addition, an increase in AhR activity is observed among soluble extracts generated from the lung tissue of Ahr-null mice, a result consistent with perturbation of an autoregulatory feedback loop between the AhR and cytochrome P450 1A1 that allows for the subsequent 216 accumulation of AhR ligands. This discovery provides a clear understanding that certain tissues from Ahr null animals, especially lung tissue, accumulate elevated levels of putative endogenous ligand(s) will provide future researchers with a new platform in the search for endogenous AhR ligands.

Chapter three identifies 12(R)-HETE, an arachidonic acid metabolite capable of being produced through either lipoxygenase or cytochrome P-450 metabolism, as a potent indirect activator of the AhR pathway. While gel shift assays together with ligand competition binding experiments demonstrated the inability of 12(R)-HETE to directly bind or activate the AhR to a DNA binding species, DRE-driven luciferase reporter assays indicated the ability of 12(R)-HETE to induce AhR activity. Likewise, quantization of CYP1A1, CYP1B1, and AhRR mRNA levels by RT-PCR confirmed the ability of 12(R)-HETE to activate AhR transcription in the human derived HepG2 and

HaCaT cell line, while siRNA knock-down of AhR levels confirmed that expression of receptor is indeed required for 12(R)-HETE mediated induction of AhR target genes.

Perhaps the most striking result, however, was the lack of AhR activity observed with structurally similar mono-HETEs such as 5-HETE, 12(S)-HETE and 15-HETE which failed to exhibit any significant activation of the AhR in cell culture. Although cellular metabolism of 12(R)-HETE into a direct AhR ligand is the most likely explanation for these results, none of the available metabolites of 12(R)-HETE demonstrated any capability to activate AhR signaling, and while continued examination of 12(R)-HETE metabolites could provide valuable information, it would require an extensive amount of organic synthesis. Perhaps a more relevant approach to understanding the underlying 217 mechanism of 12(R)-HETE mediated AhR activation is through examination of the known physiological effects of 12(R)-HETE formation. Previously demonstrated in ocular tissue to be a potent inhibitor of the Na-K-ATPase, the ability of 12(R)-HETE to modulate this biochemical equivalent of the Na:K pump could provide a potential mechanistic explanation for 12(R)-HETE mediated activation of the AhR. In the future it will be essential to design experiments addressing the effects of various chemical inhibitors of the Na-K-ATPase on AhR activity. Furthermore, it will be important to examine the effects of eicosatrienoic acid metabolites such as 5,6-EpETrE, 11,12-

EpETrE and 11,12-DiHETrE on AhR activity as these compounds have also been demonstrated to inhibit Na-K-ATPase activity [362]. Finally, it is important to mention that cAMP, a secondary cellular messenger serving as a mediator for hormone, neurotransmitter, and prostaglandin signaling has been shown to activate the AhR [363].

Ironically, elevated cellular concentrations of cAMP also inhibit the Na-K-ATPase.

Thus, taken together this information suggests that regulation of the Na-K-ATPase is in some way linked with AhR activation and through subsequent investigation of this link a potential mechanistic explanation for 12(R)-HETE activation of the AhR may be revealed. However, despite the currently ill-defined mechanism, these results nevertheless demonstrate an important ability of 12(R)-HETE to serve as a potent ligand- independent activator of the AhR, and also suggest the likelihood of AhR activation during cellular inflammation in tissues where 12(R)-HETE is produced.

Although ligand-independent activation of the AhR may be a novel concept, this type of regulatory mechanism has been previously demonstrated with the estrogen 218 receptor (ER), a member of the nuclear hormone receptor superfamily. It is well established that ER-mediated transcription can be activated in ligand-independent manner through a mechanism involving second-messenger signaling pathways. For example, in response to activation of tyrosine kinase receptors by extracellular stimuli, a signal transduction cascade is initiated culminating in activation of the mitogen-activated protein kinase (MAPK) pathway. Activated MAPK directly regulates ER phosphorylation on Ser-118 and indirectly controls phosphorylation of the ER on Ser-167 through regulation of a downstream kinase, p90 ribosomal S6 kinase (Rsk) [364].

Phosphorylation of these serine residues in the AF-1 domain of the ER appears to influence ER-mediated transcription by enhancing the recruitment of transcriptional coactivators [365, 366].

Collectively, the results presented in chapter 3 not only indicate the ability of

12(R)-HETE to serve as a potent indirect activator of the AhR, but also suggest the likelihood of AhR activation during cellular inflammation in tissues where 12(R)-HETE is produced. Elucidating the mechanism of 12(R)-HETE mediated activation of the AhR may ultimately reveal a novel mechanism of AhR regulation with widespread biological implications. However, it is also quite possible that 12(R)-HETE mediated activation of

AhR signaling functions through a mechanism involving receptor phosphorylation in a manner similar to that characterized for ligand-independent activation of the ER.

The final chapter of this thesis identifies several additional lipoxygenase derived metabolites, including the common positional isomers of LTA4 along with various 219 DiHETE molecules, as modulators of AhR signaling. Furthermore, conclusive evidence is presented demonstrating that 5,6-DiHETEs, cellular metabolites of 5,6-LTA4, can serve as true endogenous ligands for the AhR, capable of directly binding and activating the receptor. Specifically, the ability these metabolites to drive transformation of the receptor into a DNA-binding species is authenticated through EMSA analysis, while ligand competition binding experiments confirmed, via displacement of a radiolabeled photoaffinity ligand for the receptor, the ability of these DiHETEs to directly bind the

LBD of the AhR. Interestingly, aged preparations of 5,6-DiHETE isomers produced an enhanced level of AhR activation while demonstrating an increased binding affinity for the receptor. Likewise, inactive preparations of LTA4 exhibited a time dependent gain in

AhR activity during storage under an inert atmosphere. Although the reason for these anomalies has not been fully determined, evidence supporting the formation of geometric isomers in the conjugated triene region of these molecules is believed to be responsible.

Clearly, additional studies will be needed to determine the identity of the highly active

“aged” metabolite. However, building upon valuable preliminary information set forth in this thesis should allow for the successful identification of this molecule.

Conclusively, this research has confirmed the existence of a putative endogenous ligand(s) for the AhR in the CV-1 cell line, while also establishing the presence of an auto-regulatory mechanism capable of modulating AhR activity in these cells through the control of ligand metabolism. Perhaps even more importantly, we have demonstrated the ability of various pro-inflammatory eicosanoid metabolites, in particular those generated through lipoxygenase metabolism, to regulate AhR signaling. Summarized in Table 4-1 220 are the biologically active lipid molecules identified during the course of this research project to function as ligands or activators for the AhR. Also presented is the lowest statistically relevant dose needed for either biological activity or receptor occupancy, as determined through luciferase reporter assay or competition binding assay, respectively

(Table 4-1). Although several of these bioactive lipids appear to mediate AhR activity

through an indirect mechanism, others demonstrated, through their ability to directly bind and activate the receptor, the capability to serve as endogenous ligands for the AhR.

Perhaps most importantly, this thesis establishes the AhR as an intracellular receptor capable of functioning as a bioactive lipid sensor, at least with regard to select lipid metabolites. Finally, in the future, by deciphering the global effect of these bioactive lipids on AhR mediated gene expression, it can be determined whether the AhR is functioning to mediate pro-inflammatory signaling or conversely participating in an inflammation resolving role. 221

Table 4-1: Summary of Bioactive Lipid Modulators for the AhR

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Vita

Christopher R. Chiaro

Education December, 2007: Ph.D. Genetics The Pennsylvania State University, University Park, PA

May, 1997: Bachelor of Science in Biology (genetics option) Molecular and cellular biology (minor) The Pennsylvania State University, University Park, PA Publications Chiaro CR, and Perdew GH. (2007) 5,6-DiHETE Isomers are Endogenous Ligands for the Ah Receptor. (Manuscript in preparation)

Chiaro CR, Patel RD, Perdew GH. (2007) 12(R)-HETE, an Arachidonic Acid Derivative, is an Activator of the Aryl Hydrocarbon (Ah) Receptor. (Manuscript submitted PNAS)

Chiaro CR, Patel RD, Marcus CB, Perdew GH. (2007) Evidence for an aryl hydrocarbon receptor-mediated cytochrome P450 autoregulatory pathway. Mol. Pharmacol. 72(5):1369-79.

Yakhnin AV, Trimble JJ, Chiaro CR, Babitzke P. (2000) Effects of mutations in the L- tryptophan binding pocket of the Trp RNA-binding attenuation protein of Bacillus subtilis. J Biol Chem. 275(6):4519-24.

Selected Abstracts CR Chiaro, RD Patel, and GH Perdew. 12(R)-HETE is a Potent Activator of the Aryl hydrocarbon Receptor. Eicosanoids and other bioactive lipids in cancer, inflammation and related diseases 10th International conference, Montreal, Canada (2007).

CR Chiaro, AD Jones, Y Cao, J Born, C Marcus and GH Perdew. 5,6-Leukotriene A4 as a Potent Activator of the Aryl hydrocarbon Receptor. Society of Toxicology 44th Annual Meeting, New Orleans, LA (2005).

CR Chiaro, C Marcus, and GH Perdew. Characterization of a High Affinity Putative Ah Receptor Endogenous Ligand from CV-1 Cells. 21st Summer Symposium in Molecular Biology, The Pennsylvania State University, University Park, PA (2002).

CR Chiaro, C Marcus, and GH Perdew. Evidence for the Presence of a Putative Ah Receptor Ligand in CV-1 Cells. Society of Toxicology 39th Annual Meeting, Philadelphia, PA (2000).

Awards 2003 COAS graduate student competitive grant writing program winner