Novel Mechanisms of Hepatic CYP2C11 Regulation by Aromatic Hydrocarbons

NOVEL MECHANISMS OF HEPATIC CYP2C11 REGULATION BY

AROMATIC HYDROCARBONS

RANA SAWAYA

A thesis submitted in conformity with the requirements for the degree of

Doctor of Philosophy

Graduate Department of Pharmacology and Toxicology

University of Toronto

395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada

Your file Votre reference ISBN: 978-0-494-44801-4 Our file Notre reference ISBN: 978-0-494-44801-4

NOTICE: AVIS: The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats.

The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission.

In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these.

While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada ABSTRACT

Novel mechanisms of hepatic CYP2C11 regulation by aromatic hydrocarbons

Rana Sawaya, Ph.D. 2008

Department of Pharmacology and Toxicology, University of Toronto.

The aryl hydrocarbon receptor (AHR) mediates most of the toxic and adaptive responses elicited by aromatic hydrocarbons. These chemicals suppress the transcription of the growth hormone-regulated, male-specific rat hepatic cytochrome P450 2C11 gene

(CYP2C11) via an unknown mechanism. I hypothesize that the promoter and 5'-flanking region of this gene mediate suppression following aromatic hydrocarbon treatment.

Extended lengths of the CYP2C11 5'-flank were cloned into a promoterless luciferase plasmid. Suppression of CYP2C11 constructs was not observed upon treatment of transfected rat 5L, BP8, or mouse Hepa-1 cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD) or 3-methylcholanthrene (MC). In human HepG2 cells, a construct containing

10.1-kb of the 5'-flank displayed a pronounced 6-fold induction by TCDD, which was blocked by a-naphthoflavone, an AHR antagonist/partial agonist. Deletion analysis identified a region between -1.8 and -1.3-kb, which contains a dioxin-responsive element

(DRE) previously demonstrated to bind the AHR, that contributes to the paradoxical induction. A critical role for the DRE was confirmed by site-directed mutagenesis. The

CYP2C11 constructs were studied in living rats, where an intact endocrine system and the full complement of transcription factors needed for suppression of the endogenous gene are present. Using hydrodynamic-based injections to deliver plasmid DNA to the liver of live rats, we studied the MC-responsiveness of luciferase constructs containing 10.1-kb,

5.6-kb and 2.4-kb of the CYP2C11 5'-flank. MC suppressed CFP2C7i-luciferase

ii activity of the 10.1-kb and 5.6-kb constructs to below 50% of vehicle levels by 24 h and

72 h. Luciferase activity of the 2.4-kb construct was decreased to 63% of vehicle levels

24 h after MC treatment, but no suppression was detected by 72 h. This suggests that

negative regulatory element(s) responsible for CYP2C11 reporter suppression by MC

exist in the first 2.4-kb of this gene; however, additional cis-acting elements located

between -5.6 and -2.4-kb mediate persistent reporter suppression. Cy7>2Ci7-luciferase

constructs display a potentially misleading cell-specific paradoxical induction in vitro

that is AHR-dependent and DRE-mediated. This is the first demonstration of aromatic

hydrocarbon-mediated suppression of a CTP2C7i-luciferase construct, suggesting that

the 5'-flanking region and promoter mediate down-regulation of this gene in the intact rat.

in ACKNOWLEDGEMENTS

There are many people to whom I owe much gratitude for their contributions in making my thesis work possible.

• I would like to thank my supervisor Dr. David Riddick for his scientific direction,

support, encouragement and enduring patience at all times over the years. Dr.

Riddick is a remarkable mentor and I am truly grateful for the opportunity of

having been his graduate student.

• Many thanks to the members of my advisory committee: Dr. Patricia Harper, Dr.

Rachel Tyndale and Dr. Peter Wells; for all their useful suggestions, valuable

feedback, and interest in my work.

• Special thanks to the other members of Dr. Riddick's laboratory who I have spent

so much time with these past years. I want to thank Ms. Chunja Lee and Ms.

Anne Mullen Grey for their friendship, support, and scientific input. They made

each day in the laboratory enjoyable.

I also want to acknowledge the people in our department who contributed to this research in many different capacities.

• Thanks to Dr. Philip Seeman, Dr. Denis Grant, Dr. Rachel Tyndale, Dr. Allan

Okey and Dr. Peter McPherson for their generosity in sharing laboratory

equipment.

• I would like to thank Ms. Diana Clark, Ms. Phyllis Thawe, Ms. Sylvia Kawar and

Ms. Fiona Smillie for all their organization and help.

IV • I want to acknowledge Dr. Mary Erclik, Dr. Kim Sugamori, Dr. Ivy Moffat and

Ms. Ewa Hoffmann for their assistance with protocols and valuable scientific

discussions.

I am very grateful to the following individuals for providing useful resources and

materials needed for me to complete this research:

• Dr. Anahita Bhathena for use of plasmids: (-2.4-2C11), (-1.3-2C11) and

(-0.2-2C11).

• Mr. Paul Boutros for bioinformatics support.

• Dr. Michael Denison (University of California, Davis, CA) for providing the

pGudlucl.l plasmid.

• Dr. Martin Gottlicher (Institute of Toxicology and Genetics, Karlsruhe,

Germany) for providing the 5L and BP8 cells.

• Dr. Edward Morgan (Emory University, Atlanta, GA) and Dr. Harry Gelboin

(National Cancer Institute, Bethesda, MD) for the generous gift of antibodies.

• The staff at the Division of Comparative Medicine within the University of

Toronto. In particular, I want to acknowledge Mr. Frank Giuliano and Ms.

Dorothy Donn for their incredible skill and assistance with the hydrodynamics-

based injections.

• Funding from the Canadian Institutes of Health Research (Grant MOP-42399 to

Dr. Riddick) and the University of Toronto made this research possible.

I also want to thank those people outside the world of research who are the centre of my world, and whose love keeps me going. I would like to thank my first science teacher: a very knowledgeable man who taught me the value of education and whom I admire

v immensely, my father. Many thanks to my mother, who gives me constant love and endless comfort. My parents went to great lengths so that I may have every opportunity that I worked hard enough to achieve, and for this I am eternally grateful. Thanks to my siblings: my incredible brother Samer, for always knowing the right thing to say at the right time, and for his technical assistance in dealing with my continual computer mishaps; and my beautiful sister Sara, who is so full of life and always makes me laugh.

Many thanks to my loving grandmother Claire, who kept my stomach full so that I might have the energy to write this thesis. Above all, I thank Jesus; through Him all things are possible.

VI TABLE OF CONTENTS

ABSTRACT ii ACKNOWLEDGEMENTS iv TABLE OF CONTENTS vii LISTOFTABLES xi LISTOFFIGURES xii LIST OF ABBREVIATIONS xiv

1.0 INTRODUCTION 1

1.1 Statement of Research Problem 1

1.2 Aromatic hydrocarbons 2 1.2.1 Source of exposure and environmental contamination 2 1.2.1.1 Polycyclic aromatic hydrocarbons 2 1.2.1.2 Halogenated aromatic hydrocarbons 4 1.2.2 Toxicity of aromatic hydrocarbons 5

1.3 Aryl hydrocarbon receptor 9 1.3.1 Discovery and structure-function 9 1.3.2 ARNT and AHRR: other members of the bHLH/PAS superfamily 14 1.3.3 Function of the AHR 18 1.3.3.1 Role of the AHR in aromatic hydrocarbon-mediated toxicity 18 1.3.3.2 Developmental and endogenous roles of the AHR 19 1.3.4 Dioxin-responsive elements 21 1.3.5 Regulation of gene expression 24 1.3.5.1 Adaptive role of the AHR in the Induction of P450 enzymes 24 1.3.5.2 Adaptive role of the AHR in the Suppression of P450 enzymes.... 28 1.3.5.3 Genes involved in cell growth and differentiation 29 1.3.6 Natural and endogenous AHR ligands 30 1.3.7 AHR antagonists 32

1.4 Cytochrome P450 35 1.4.1 Role of CYP1 family members in aromatic hydrocarbon-mediated toxicity 39 1.4.2 Hormonal regulation of sexually dimorphic rat P450s 40

1.5 In vivo reporter gene studies 43

1.6 CYP2C11 56 1.6.1 CYP2C11 5'-flanking region and promoter 56 1.6.2 Physiological CYP2C11 regulation 60

vn 1.6.2.1 Onset of CYP2C11 expression at puberty 61 1.6.2.2 Mechanisms involved in GH transcriptional regulation of CYP2C11 62 1.6.2.3 CYP2C11 expression and pathophysiological states 72 1.6.2.4. CYP2C11 regulation by inflammatory cytokines 74 1.6.3 CYP2C11 levels following xenobiotic treatment 76 1.6.4 Down-regulation of CYP2C11 by aromatic hydrocarbons 79 1.6.4.1 Evidence for a transcriptional mechanism of action 82 1.6.4.2 Role of the AHR in CYP2C11 suppression 87 1.6.5 Possible mechanisms involved in aromatic hydrocarbon-mediated CYP2C11 suppression 89

1.7 Hypothesis and objectives 91 1.7.1 Research Rationale and Experimental Approaches 91 1.7.2 Hypothesis 92 1.7.3 Objectives 92

2.0 MATERIALS AND METHODS 94

2.1 Luciferase reporter constructs 94 2.1.1 Construction of reporter plasmids 94 2.1.2 Restriction enzyme digestion and agarose gel electrophoresis 99 2.1.3 Purification of DNA from agarose gel 100 2.1.4 DNA ligation 101 2.1.5 Plasmid preparation 101 2.1.6 Bioinformatic analysis of the CYP2C11 5'-flanking region 105 2.1.7 Sequencing of PCR-amplified CYP2C11 5'-flank inserts 106

2.2 Site-Directed Mutagenesis 107

2.3 Culture of continuous cell lines 108

2.4 Detection of CYP1A1, CYP2C11, AHR, a-tubulin and p-actin mRNA 108 2.4.1 RNA isolation 108 2.4.2 Reverse-transcription reactions Ill 2.4.3 Detection of mRNA by RT-PCR 111 2.5 Transient transfection in continuous cell lines 113 2.5.1 Optimization of transfection efficiency 113 2.5.2 Transient transfection 114 2.5.3 Chemical treatment of continuous cell lines 115 2.5.4 Isolation of cell extracts 115 2.5.5 Bradford protein assay 115

2.6 Animals and treatment 116

viii 2.7 In vivo AHR antagonist studies 118

2.8 Analysis of CYP2C11 and p-actin mRNA by real time RT-PCR 119

2.9 Immunoblot anlaysis 126

2.10 Reporter gene assays 128 2.10.1 Dual luciferase reporter assays for in vitro transfections 128 2.10.2 Dual luciferase reporter assays for in vivo transfections 129 2.11 Statistical analysis 129 2.11.1 Statistical analysis for in vitro studies 129 2.11.1 Statistical analysis for in vivo studies 130

3.0 RESULTS 131

3.1 Cloning, sequencing and bioinformatics 131 3.1.1 Bioinformatic analysis of putative TF binding sites located in the CYP2C11 5'-flanking region 131 3.1.2 Luciferase reporter constructs: confirmation by diagnostic restriction digestion 133 3.1.3 Luciferase reporter constructs: confirmation by DNA sequencing 133

3.2 In vitro studies 139 3.2.1 Optimization of transfection efficiency 139 3.2.2 CYP2C11 mRNA levels in rat hepatoma 5L and BP8 cells 139 3.2.3 AHR mRNA levels in rat hepatoma 5L and BP8 cells 139 3.2.4 Transient transfections in 5L, BP8 and Hepa-1 cells 141 3.2.5 Transient transfections and analyses of "transcriptionally active" region within the CYP2C11 5'-flank in HepG2 cells 144 3.2.6 Effect of site-directed mutagenesis of CYP2C11-DRE3 on TCDD- induced luciferase activity in HepG2 cells 149 3.2.7 Effect of the AHR antagonist a-NF on TCDD-induced luciferase activity in HepG2 cells 150

3.3 In vivo studies 154 3.3.1 Hepatic luciferase activity in rats receiving hydrodynamics-based injections 154 3.3.2 Endogenous CYP1A1 levels in rats receiving hydrodynamics-based injections 157 3.3.3 Endogenous CYP2C11 levels in rats receiving hydrodynamics-based injections 157 3.3.4 Correlation studies of endogenous CYP2C11 protein and mRNA levels in rats receiving hydrodynamics-based injections 161 3.3.5 Correlation studies of endogenous CYP2C11 mRNA levels and luciferase activity in rats receiving hydrodynamics-based injections 164

IX 3.3.6 Effect, of oc-NF on MC-induced endogenous CYP1A1 levels in vivo 168

4.0 DISCUSSION 170

4.1 Luciferase reporter assays in continuous cell lines 170 4.1.1 Lack of suppression of CYP2C11 reporter constructs in continuous cell lines 170 4.1.2 Role of the AHR in paradoxical induction of CYP2C11 -luciferase constructs in HepG2 cells 170

4.2 Luciferase reporter assays in vivo 175 4.2.1 Considerations when using the hydrodynamics-based approach 175 4.2.2 Transgene expression 177 4.2.3 Effects of MC on endogenous CYP2C11 expression 180 4.2.4 Time-course of CYP2C11 reporter suppression 185 4.2.5 Possible mechanisms mediating CYP2C11 suppression by MC in vivo.... 187 4.2.5.1 Role of the AHR 187 4.2.5.2 Interference with GH signaling pathways by MC 188 4.2.5.3 Involvement of inflammatory cytokines 190

4.3 Overall summary of findings 192

4.4 Future studies 194 4.4.1 Additional deletion and mutant constructs introduced to rats by hydrodynamics-based injections 194 4.4.2 Studying CFP2C7i-luciferase constructs in hypophysectomized rats 194 4.4.3 Additional in vivo AHR antagonists 195 4.4.4 Chromatin immunoprecipitation assays to study the binding of TFs of interest to specific regions of the CYP2C11 5'-flank in a chromosomal context 196 4.4.5 Related studies in a mouse model 197 4.4.5.1 Mouse P450s down-regulated by aromatic hydrocarbons 197 4.4.5.2 Regulation of CyP2Cii-luciferase constructs by aromatic hydrocarbons in wild-type andAhr-mill mice 197

4.5 Physiological and global relevance of studying CYP2C11 down-regulation 198

5.0 REFERENCES 202

6.0 LIST OF PUBLICATIONS AND ABSTRACTS 238

x LIST OF TABLES

Table 1.1 Summary of selected aromatic hydrocarbon-responsive P450s outside the CYP1 family 26

Table 1.2 Sexually dimorphic P450s in rat liver 42

Table 1.3 Summary of selected in vivo studies of P450 regulation using in situ or hydrodynamics-based transfection of luciferase reporter plasmids 52

Table 2.1 Oligonucleotides and thermal cycling conditions used for PCR-based cloning of fragments of the CYP2C11 5'-flanking region 98

Table 2.2 Primer sequences and thermal cycling conditions used for analysis of

steady-state mRNA levels by RT-PCR 112

Table 2.3 Study design 1: In vivo AHR antagonist time-line 120

Table 2.4 Study design 2: In vivo AHR antagonist time-line 120 Table 2.5 Primer and probe sequences used for measurements of steady-state mRNA levels in rat liver by real-time quantitative RT-PCR 121

Table 3.1 Effect of MC on hepatic reporter gene activity in rats receiving hydrodynamics-based injections 158

Table 3.2 Variation in endogenous CYP2C11 protein and mRNA levels for vehicle- treated rats receiving hydrodynamics-based injections 163

XI LIST OF FIGURES

Figure 1.1 Chemical structure of AHR agonists from representative classes of aromatic hydrocarbons. 3

Figure 1.2 Schematic diagram of the AHR's mechanism of action as a ligand- activated transcription factor 11

Figure 1.3 Schematic structural diagram of bHLH superfamily members 16

Figure 1.4 Structurally diverse AHR antagonists 33

Figure 1.5 Summary of putatively functional human and rat CYP1, CYP2 and CYP3 family members 38

Figure 1.6 Developmental regulation of rat P450s by GH 44

Figure 1.7 Nucleotide sequence of the CYP2C11 5'-flanking region 58

Figure 1.8 Schematic representation of the endocrine regulation of hepatic CYP2C11 expression 64

Figure 1.9 Schematic diagram of GH-activated signaling leading to CYP2C11

transcriptional regulation 67

Figure 1.10 Schematic illustration of potential levels of P450 suppression 83

Figure 2.1 Schematic representations of luciferase reporter constructs used in this thesis 95 Figure 2.2 Visualization of 28S and 18S rRNA in total RNA samples prepared by the acid guanidinium thiocyanate-phenol-chloroform extraction method 110

Figure 2.3 Co-administration of firefly and Renilla luciferase plasmid DNA for hydrodynamics-based hepatocyte transfection 117

Figure 2.4 CYP2C11 and P-actin amplicons from RT-PCR amplification using real-time RT-PCR primers specific for each gene 123

Figure 2.5 Representative CYP2C11 and (3-actin standard curves generated by real-time RT-PCR 124

Figure 3.1 Location of putative transcription factor binding sites in the proximal 10.1-kbofthe CYP2C11 5'-flanking region 132

Xll Figure 3.2 Diagnostic restriction digestion analysis of CYP2C1 i-luciferase

reporter constructs 134

Figure 3.3 Endogenous CYP2C11 mRNA is undetectable in BP8 and 5L cells 140

Figure 3.4 Effects of TCDD and MC on CYP2C11 reporter gene activity in transiently transfected cells 142 Figure 3.5 Effects of TCDD and MC on CYP2C11 deletion constructs in transiently transfected HepG2 cells 146

Figure 3.6 Site-directed mutagenesis reveals that induction of CYP2CZi-lucfierase activity by TCDD in HepG2 cells is at least partially mediated by the CyP2Cii-DRE3 element 151

Figure 3.7 Effects of a-NF on TCDD-induced luciferase activity in HepG2 cells.. 152

Figure 3.8 Effect of MC on hepatic reporter gene activity in rats receiving hydrodynamics-based injections 155

Figure 3.9 Immunoblot analysis of endogenous hepatic CYP1 Al and CYP2C11 protein levels following vehicle or MC treatment in rats receiving hydrodynamics-based injections 159

Figure 3.10 Effect of MC administration on endogenous hepatic CYP1A1 and CYP2C11 mRNA levels in rats receiving hydrodynamics-based Injections 160

Figure 3.11 Optimization of CYP2C11 immunoblot assay 162

Figure 3.12 Correlation between endogenous hepatic CYP2C11 protein and mRNA levels in rats following hydrodynamics-based injections 165

Figure 3.13 Correlation between hepatic luciferase activity and endogenous CYP2C11 mRNA levels in rats receiving hydrodynamics based injections 166

Figure 3.14 CYP1 Al induction by MC is not affected by a-NF in vivo 169

Figure 4.1 Possible transcription factor binding sites mediating in vivo CYP2C11 reporter suppression by MC 189

xill LIST OF ABBREVIATIONS

A adenine AHR aryl hydrocarbon receptor AHH aryl hydrocarbon hydroxylase AIP aryl hydrocarbon receptor-interacting protein ARNT aryl hydrocarbon receptor nuclear translocator AHRR aryl hydrocarbon receptor repressor AHRE aryl hydrocarbon response element ALT alanine transaminase ANOVA analysis of variance AST aspartate aminotransferase ATP adenosine triphosphate B[a]P benzo[a]pyrene bHLH basic helix-loop-helix BIS N,N'-methylenebisacrylamide BLAST Basic local alignment search tool BLAT Blast-like alignment tool bp base pair BSA bovine serum albumin C cytosine °C degrees Celsius CAR constitutive androstane receptor CBP/p300 cAMP-responsive element-binding protein (CREB)-binding protein/p300 CDK8 cyclin-dependent kinase 8 cDNA complementary deoxyribonucleic acid CH-223191 2-methyl-2#-pyrazole-3-carboxylic acid ChIP chromatin immunoprecipitation CIS cytokine inducible SH2 protein CO corn oil

Ct threshold cycle CYP cytochrome P450

xiv DEPC diethylpyrocarbonate DEX dexamethasone DHEA dehydroepiandrosterone 3',4'-DMF 3',4'-dimethoxyflavone DMSO dimethylsulfoxide DNA deoxyribonucleic acid DNase deoxyribonuclease dNTP deoxynucleoside triphosphate DRB 5,6-dichlorobenzimidazole riboside DRE dioxin-responsive element DTT dithiothreitol ECL enhanced chemiluminescence EDTA ethylenediaminetetraacetic acid EMSA electrophoretic mobility shift assay ER estrogen receptor EROD ethoxyresorufin-O-deethylase FBS fetal bovine serum G guanine ug microgram GAS interferon-y-activated site GH growth hormone GHR growth hormone receptor GHRH growth hormone releasing hormone GRE glucocorticoid response element h hour HAH halogenated aromatic hydrocarbon HAT histone acetyltransferase HCB 3,4,5,3',4\5'-hexachlorobiphenyl HEP hypoxia-inducible factor HNF hepatocyte nuclear factor HRE hypoxia response element

xv Hsp90 90 kDa heat shock protein H7W Han/Wistar HVTV high-volume tail vein HYPX hypophysectomized IARC International Agency for Research on Cancer

IKB nuclear factor-KJB inhibitory factor ICZ indolo- [3,2-Z?]-carbazole iDRE inhibitory dioxin-responsive element IL interleukin i.p. intraperitoneal In vivo in the living animal In vitro in controlled environment outside the living organism JAK2 Janus kinase 2 kb kilobase kDa kilodalton kg kilogram uL microliter LARII luciferase assay reagent II L-E Long-Evans LPS lipopolysaccharide luc luciferase M molar uM micromolar MAPK mitogen-activated protein kinase MC 3-methylcholanthrene MCDF 6-methyl-1,3,8-trichlorodibenzofuran Med220 mediator subunit of 220 kDa MEM a-minimum essential medium mg milligram min minute mL milliliter

XVI MMLV Moloney murine leukemia virus MMTV mouse mammary tumor virus 3'M4'NF 3' -methoxy-4' -nitroflavone mRNA messenger ribonucleic acid MSG monosodium glutamate NADPH nicotinamide adenine dinucleotide phosphate (reduced) NCoA-2 nuclear coactivator 2 a-NF a-naphthoflavone P-NF P-naphthoflavone NF-KB nuclear factor-KB nmol nanomole NE Normalized expression Nrf2 nuclear factor E2-related factor 2 P450 cytochrome P450 PAH polycyclic aromatic hydrocarbon PAS Period-ARNT-Single Minded PB phenobarbital PBS phosphate-buffered saline PCB polychlorinated biphenyl p/CIP p300/CBP-interacting protein PCR polymerase chain reaction PKC protein kinase C PPAR peroxisome proliferator activated receptor PPRE peroxisome proliferator response element PREX positive regulatory element for XRE-mediated gene expression PXR pregnane X receptor RNA ribonucleic acid RNase ribonuclease rpm revolutions per minute rRNA ribosomal ribonucleic acid RT reverse transcription

XVll RT-PCR reverse transcription coupled polymerase chain reaction RXR retinoid X receptor s second SAR structure-activity relationship SD standard deviation SDS sodium dodecyl sulfate SH2 Src homology 2 siRNA small interfering ribonucleic acid SOCS suppressor of cytokine signaling SRC steroid receptor coactivator STAT5 signal transducer and activator of transcription 5 SV40 simian virus 40 T thymine T3 triiodothyronine T4 thyroxine 2,4,5-T 2,4,5-trichlorophenoxyacetic acid TAE 40 mM Tris-acetate/ 1 mM EDTA, pH 8.0 TBE 0.09 M Tris-borate/ 2 mM EDTA, pH 8.3 TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin TE8 10 mM Tris-HCl, ImM EDTA, pH 8.0 TEMED N,N,N'N',-tetramethylethylenediamine TGF-p transforming growth factor-P TF transcription factor TNF tumor necrosis factor Tris Tris(hydroxymethyl)aminomefhane UV ultraviolet V vehicle V volt w/v weight per volume XAP2 hepatitis B virus X-associated protein 2 x g times gravitational force

xviii X-gal 5-bromo-4-chloro-3-indolyl-P-galactopyranoside XRE xenobiotic response element

xix 1.0 INTRODUCTION

1.1 Statement of Research Problem

Aromatic hydrocarbons are environmental contaminants that pose a threat to the health of human beings and wildlife. Growing scientific interest in aromatic hydrocarbons stems from the toxic and endocrine-disrupting potential of these chemicals.

It is therefore important to gain insights into the mechanisms by which aromatic hydrocarbons elicit toxicity. Of particular interest are mechanisms implicated in the modulation of the expression of genes involved in the metabolism of xenobiotics and endogenous substrates: cytochromes P450 (CYPs or P450s). Aromatic hydrocarbons elicit most of their biological effects by binding to the aryl hydrocarbon receptor (AHR).

In turn, the AHR mediates toxic and adaptive effects by altering the expression of genes such as P450s. P450s constitute a superfamily of enzymes that play a major role in the hepatic metabolism of foreign and endogenous chemicals. Changes in the expression of genes encoding P450s leads to altered enzyme levels, which may change the metabolic capability of an organism leading to toxic outcomes. Aromatic hydrocarbons cause both the up- and down-regulation of P450 gene expression through the AHR. Mechanisms of

AHR-mediated induction of P450s are at least partially understood as they have been studied extensively; however, the mechanisms behind P450 suppression are far less characterized.

Previous investigations indicate that aromatic hydrocarbons suppress the expression of CYP2C11 in the liver of male rats via an unknown transcriptional mechanism that has been a central focus of our laboratory (Jones and Riddick, 1996; Lee and Riddick, 2000). This gene encodes the major P450 found in the male rat liver, and

1 the main physiological regulator of CYP2C11 expression is the male pulsatile pattern of

growth hormone (GH) release. The studies presented in this thesis aim to clarify

mechanisms involved in the down-regulation of CYP2C11 in response to aromatic

hydrocarbons by studying luciferase reporter constructs that contain varying lengths of

the CYP2C11 5'-flanking region and promoter in both continuous cell lines and living

rats.

1.2 Aromatic hydrocarbons

1.2.1 Source of exposure and environmental contamination

Aromatic hydrocarbons are environmental contaminants that are present in almost

every aspect of the global ecosystem (Franzen et al., 1988; Green et al., 2004; Tanabe et

al., 2004). There are various chemical classes of aromatic hydrocarbons including

polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons

(HAHs). Chemical structures of representative compounds from these two classes of

aromatic hydrocarbons are shown in Fig. 1.1.

1.2.1.1 Polycyclic aromatic hydrocarbons

Non-halogenated PAHs, represented by the laboratory chemical 3-

methylcholanthrene (MC), are formed during the incomplete combustion of organic

materials. PAHs are found in automobile exhaust (Mahadevan et al., 2004), furnace gas,

cigarette smoke (Saeki et al., 2003), and grilled meat (Kazerouni et al., 2001), with diet being the primary source of human exposure (Reinik et al., 2007). Benzo[a]pyrene

(B[a]P) is a widely studied PAH and is a potent animal carcinogen [reviewed in:

(Kazerouni et al., 2001)]. B[a]P has been detected in a wide spectrum of dietary products including grilled meat, grains, fruits, vegetables, seafood, oils and drinking water

2 CI (X CI

CI O CI 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD)

3-MethylchoIanthrene (MC)

D X X X X

X X X X

Benzo[a]pyrene (B[a]P) Polychlorinated biphenyls (PCBs) X=ClorH

Figure 1.1. Chemical structure of AHR agonists from representative classes of aromatic hydrocarbons. (A) TCDD is an example of a halogenated aromatic hydrocarbon; (B) MC and (C) B[a]P are examples of polycyclic aromatic hydrocarbons; (D) PCBs are halogenated aromatic hydrocarbons with the chemical identity of specific congeners determined by the positions of chlorine or hydrogen (X) substitution.

3 [reviewed in: (Kazerouni et al., 2001; Reinik et al., 2007)]. The presence of this chemical in seafood is likely due to aquatic pollution, whereas B[a]P in meat products is formed during pyrolysis in the grilling process (Kazerouni et al., 2001; Reinik et al., 2007). The total daily mean dietary intake of B[a]P is estimated to be 124.55 ng per human being

(Reinik et al., 2007).

In addition to dietary intake, occupational exposure of humans to PAHs is well- documented (Boffetta et al., 1997; Lemm et al., 2004). Examples include aluminum production, carbon electrode production, chimney sweeping, road paving, roof installation and graphite electrode production.

Unlike HAHs, PAHs are readily metabolized in the cell and products of their metabolism may exert toxic outcomes. For example, B[a]P is metabolized to four carcinogenic diol epoxides that covalently bind deoxyribonucleic acid (DNA), proteins or lipids [reviewed in: (Lee and Shim, 2007)]. The major metabolites of MC are 1-hydroxy-

3-MC, l-keto-3-MC, cholanthrene (Myers et al., 1989; Myers and Flesher, 1990), 2- hydroxy-3-MC and 3-hydroxy-MC (Shou and Yang, 1996). 3-Hydroxy-MC has been implicated as an important contributor to mutagenesis, although both toxic and non-toxic metabolites are formed during the metabolism of MC (Shou and Yang, 1996).

1.2.1.2 Halogenated aromatic hydrocarbons

Chlorinated dibenzo-/?-dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD or "dioxin"), are formed as by-products during the combustion of chlorine- containing materials and other processes including the manufacturing of paper and pulp, wood combustion, metal processing, pesticide manufacturing and waste incineration

[reviewed in: (Grassman et al., 1998; Baccarelli et al., 2004; Hu et al., 2005; Schecter et

4 al., 2006)]. Since dioxins are highly lipophilic, they tend to bioaccumulate in the food

chain. The daily consumption of contaminated foods (Natsume et al., 2004) such as beef

(Anyanwu et al., 2003) and fish (Schmidt, 2004), leads to the accumulation of dioxin in

the human body (Yoshida and Nakanishi, 2003; Lai et al., 2004). TCDD was first

brought to public attention in the 1970s. It was formed as a by-product of the

commercial synthesis of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), which is a chemical

found in Agent Orange, a herbicide and defoliant used in the Vietnam War [reviewed in:

(Landers and Bunce, 1991; Frumkin, 2003)].

Polychlorinated biphenyls (PCBs) are a group of structurally related chemicals

that were widely used in numerous industrial manufacturing processes due to their flame

resistance and electrical insulating properties. Health concerns arising from accidental

exposures and environmental contamination of these chemicals led to their banning in the

United States in 1979 [reviewed in: (Ross, 2004)].

1.2.2 Toxicity of aromatic hydrocarbons

PAH exposure can cause deleterious effects to the health of humans. Studies

reveal that populations exposed to PAHs have an increased risk of developing cancers

(Pavanello et al., 1999), particularly cancers of the lung, stomach (Lee and Shim, 2007;

Wang et al., 2007), skin and bladder (Boffetta et al., 1997). The International Agency for

Research on Cancer (IARC) classifies B[a]P as a Group 1 chemical indicating it is a known human carcinogen.

PAHs consist of 2 or more fused aromatic rings (Fig. 1.1) and their bioactivation by the CYP1A subfamily and other enzymes results in the production of reactive metabolites that covalently bind cellular macromolecules (Shimada and Fujii-Kuriyama,

5 2004; Lee and Shim, 2007) to initiate carcinogenesis (Xu et al., 2005b). B[a]P is found in cigarette smoke and undergoes metabolism by CYP1A1, CYP2A6 and CYP2E1

(Oyama et al., 2007). Oyama et al. (2007) suggest a relationship between the expression of these P450 enzymes and the development of lung cancer in the bronchial epithelium of heavy smokers, a finding which supports the carcinogenic potential of B[a]P in human cancers. High levels of PAH metabolites covalently bound to DNA are present in coke oven workers (Wang et al., 2007), populations living in polluted urban regions, fire fighters (Kriek et al., 1998), and psoriatic patients who receive dermal coal tar treatment

(Paleologo et al., 1992). The formation of such adducts leads to DNA damage, which may result in gene mutations and carcinogenesis.

Unlike PAHs, HAHs do not form reactive intermediates that interact with DNA and, therefore, are non-genotoxic. Instead, they act primarily by modulating gene expression, which typically translates to altered protein levels (Pastorelli et al., 2006).

TCDD controls genes at both transcriptional and post-transcriptional levels, including epigenetic modifications (Ray and Swanson, 2004; Wu et al., 2004) and alterations in messenger ribonucleic acid (mRNA) stability (Shimba et al., 2000; Henley et al., 2004).

TCDD has less of an effect on microRNA levels (Moffat et al., 2007).

TCDD and its congeners produce a wide spectrum of adverse effects that are species- and sex-dependent (Enan and Matsumura, 1995). Toxic effects [reviewed in:

(Devito and Birnbaum, 1994)] include a wasting syndrome (Fetissov et al., 2004), neurotoxicity (Williamson et al., 2005), chloracne (Baccarelli et al., 2005), hepatotoxicity and immunotoxicity (Tomita et al., 2003; Nagai et al., 2005). Dioxins possess anti estrogenic activity, which accounts for their reputation as endocrine-disruptors

6 (Landrigan et al., 2003). Numerous estrogen-responsive genes are modulated by TCDD

(Watanabe et al., 2004; Tanaka et al., 2007; Boverhof et al., 2008), and the reproductive

systems of both genders are profoundly affected by this chemical (Aitken et al., 2004;

Miyamoto, 2004; Warner et al., 2004; Wolff et al., 2005). TCDD can increase the

incidence and severity of endometriosis (Birnbaum and Cummings, 2002; Rier and

Foster, 2002), and is implicated in causing embryotoxicity and teratogenicity (Thomae et

al, 2004; Thomae et al., 2006; Kransler et al., 2007).

The IARC classifies TCDD as a known human carcinogen based on mechanistic

information about the AHR and data from animal studies. This classification has led to

much controversy in the field of dioxin research. Although TCDD is a recognized

carcinogen in rats, mice and hamsters [reviewed in: (Birnbaum, 1994)], its role in human

cancers remains controversial (Bertazzi et al., 1997; Bodner et al., 2003; Cole et al.,

2003; Frumkin, 2003; Starr, 2003; Baccarelli et al., 2004; Steenland et al., 2004).

Similarly, PCBs are known animal carcinogens, but their ability to initiate and/or promote human cancers is not as clear. PCBs are endocrine disruptors in humans and exposure may lead to a variety of hormonal disorders [reviewed in: (Ross, 2004)].

Earlier studies suggest some coplanar PCBs can exert toxicities similar to those produced by TCDD (Safe et al., 1985).

Knowledge of human health outcomes in response to dioxins depends largely on cases of occupational, accidental and intentional exposures. The hallmark of dioxin toxicity in humans is chloracne, a severe skin condition mainly confined to the face. The highest recorded level of dioxins in a human is 144,000 pg/g blood lipids, which was mainly associated with symptoms of fatigue and chloracne (Schecter et al., 2006).

7 Olestra, a fat substitute used to facilitate the removal of dioxins from humans exposed to this toxic chemical (Geusau et al., 1999), was used in this particular case. An industrial accident in Seveso, Italy in 1976 left residents exposed to TCDD, many of which developed chloracne (Baccarelli et al., 2005). The biological half-life of dioxin in humans is 5-10 years (Poiger and Schlatter, 1979; Pirkle et al., 1989; Starr, 2003;

Baccarelli et al., 2004), and plasma TCDD levels remain elevated in Seveso residents more than 20 years following the accident (Baccarelli et al, 2004). The deliberate poisoning of the Ukranian president Viktor Yushchenko was widely publicized in

September 2004. Yushchenko suffered from severe chloracne that continues to improve with time (Schecter et al., 2006). Overall, dioxin toxicity in humans appears largely confined to the dermatological effects of chloracne (Baccarelli et al., 2005).

Consequently, some researchers believe the labeling of TCDD as a Group 1 human carcinogen by the IARC should be reconsidered (Cole et al., 2003). The effects of low- dose, chronic human exposure to dioxins remains unknown (Schecter et al., 2006).

Humans appear to be less susceptible to TCDD toxicity compared to most laboratory animals. When examining CYP1A mRNA levels and ethoxyresorufin-O- deethylase (EROD) activity, human hepatocytes and human HepG2 cells were 10- to

1,000-fold less sensitive to TCDD compared to rat or monkey hepatocytes (Silkworth et al., 2005). Human AHR and the AHR nuclear translocator (ARNT) mRNA expression in embryonic palates is 346- and 135-fold lower, respectively, than levels found in mice

(Abbott et al., 1999a). The human AHR has a similar TCDD binding affinity as the

DBA/2 "non-responsive" mouse strain (Ema et al., 1994). Section 1.3.1 contains additional mechanistic insight into the reduced sensitivity of humans to TCDD's effects.

8 Aside from the essential role of TCDD binding to the AHR, we still do not have a

clear picture of how the toxicity of this chemical is manifested (Walker, 2007). Judging

by the wide range of adverse outcomes resulting from exposure to aromatic

hydrocarbons, it is important that researchers continue to investigate mechanisms that

convey this toxicity.

1.3 The Aryl hydrocarbon receptor

1.3.1 Discovery and structure-function

Early studies revealed that PAH administration to the C57BL/6 mouse strain

resulted in the induction of hepatic aryl hydrocarbon hydroxylase (AHH), a microsomal

catalytic activity largely attributed to CYP1A enzymes, and these mice were susceptible to PAH toxicity; conversely, the "non-responsive" DBA/2 strain was more tolerant

(Benedict et al., 1973; Kouri, 1976). The "responsive" C57BL/6 strain differed from the

DBA/2 strain at the Ah locus, now known to encode the cytosolic AHR protein (Nebert et al., 1972; Thomas et al., 1972). C57BL/6 mice carry the Ahb allele, while DBA/2 mice carry the Ahd allele [reviewed in: (Poland and Knutson, 1982)]. Interestingly, treatment of the "non-responsive" strain with a 10-fold higher concentration of TCDD results in similar AHH enzymatic induction as the "responsive" strain, although "non-responsive" mice fail to respond to higher concentrations of MC (Poland and Glover, 1975; Okey et al., 1989). This suggested that differences between these two mouse strains in their responses to TCDD and MC was likely a result of an induction receptor site with reduced binding affinity in the DBA/2 strain (Poland et al., 1974). Since TCDD is 30,000-times more potent than MC at inducing AHH activity in rodent liver (Poland and Glover,

1974), the maximal induction response can be achieved in "non-responsive" mice by

9 increasing the TCDD dose, whereas effective inducing concentrations of MC, a less

potent and more rapidly metabolized compound, cannot be achieved in "non-responsive" mice. The AHR was discovered in 1976 by demonstrating saturable, high affinity binding sites for [3H]TCDD in hepatic extracts of C57BL/6 mice (Poland et al., 1976).

Competitive binding studies using radiolabeled TCDD and MC support the existence of a common receptor for these two compounds (Okey and Vella, 1982). The molecular basis for the differences in responsiveness between C57BL/6 and DBA/2 mice was clarified by the finding that the AHR encoded by the Ahb allele has approximately 10-fold higher affinity for TCDD than the AHR encoded by the Ahd allele (Okey et al., 1989). The molecular properties of the AHR have been characterized and a clear role for the AHR in mediating TCDD- and MC-induced toxicities has been established [reviewed in: (Okey et al., 1994)].

Fig. 1.2 shows the AHR's mechanism of action following ligand binding. The unbound AHR resides in the cytoplasm in a complex with two molecules of the 90 kDa heat shock protein (hsp90) (Denis et al., 1988; Perdew, 1988; Wilhelmsson et al., 1990),

AHR-interacting protein (AIP) or hepatitis B virus X-associated protein 2 (XAP2)

(Carver and Bradfield, 1997; Ma and Whitlock, 1997; Meyer et al., 1998), and p23 (Nair et al., 1996; Kazlauskas et al., 1999). These chaperone proteins function to localize and stabilize the unliganded AHR in the cytoplasm [reviewed in: (Cox and Miller, 2004)].

Aromatic hydrocarbons enter the cell by passive diffusion and bind to the AHR (Landers and Bunce, 1991). The complex migrates to the nucleus where the AHR dissociates from hsp90 molecules to interact with its dimerization partner, ARNT (McGuire et al., 1994;

Eguchi et al., 1997; Heid et al, 2000). The AHR/ARNT heterodimer recognizes and

10 Cytoplasm

Ligand

Developmental Adaptive Altered expression of Toxic endpoints arising Altered P450 levels genes involved in the from alterations in P450- caused by xenobiotics. maintenance of normal mediated metabolism and Altered metabolism of growth, differentiation altered expression of xenobiotics and and development genes involved in cellular endogenous substances homeostasis

Figure 1.2. Schematic diagram of the AHR's mechanism of action as a ligand-activated transcription factor.

11 binds to dioxin-responsive elements (DREs) located in the 5'-flanking region of various target genes (Lai et al., 1996), including CYP1A1 (Fujisawa-Sehara et al., 1987;

Matsushita et al., 1993), CYP1A2 (Black and Quattrochi, 2004), CYP1B1 (Zhang et al.,

1998), UGT1A1 and ALDH3A1 (Nebert and Gonzalez, 1987; Nebert et al., 2000).

The AHR is a ligand-activated transcription factor (TF) belonging to the basic helix-loop-helix (bHLH)/Period-ARNT-Single Minded (PAS) superfamily [reviewed in:

(Denison et al., 2002; Kewley et al., 2004)]. Members of this superfamily, such as the

AHR and hypoxia inducible factor (HIF), respond to environmental stimuli including xenobiotics and hypoxia by initiating signaling pathways. BHLH/PAS proteins are critical sensors that respond to stimulating cues by increasing or decreasing the expression of their target genes. Prior to functional DNA binding, bHLH/PAS proteins must first dimerize. The AHR is the only ligand-activated member of this superfamily and is transformed into its DNA-binding conformation only after ligand binding.

Dimerization occurs through the PAS homology domain (consisting of PAS A and PAS

B repeats), and the bHLH region. The PAS domain of the AHR facilitates ligand binding and interactions with hsp90, while the bHLH region mediates DNA binding, nuclear localization and nuclear export [Fig. 1.3; reviewed in: (Denison et al., 2002; Kewley et al., 2004)]. The C-terminus transactivation domain contains a Q-rich region which facilitates the assembly of co-activators and co-repressors, ultimately influencing the activity of the basal transcriptional machinery at the promoter region of target genes. The

AHR is widely expressed in tissues of vertebrates and homologues lacking ligand- binding activity have been identified in invertebrate species, which suggests evolutionary conservation of the receptor (Hahn et al., 1994; Hahn and Karchner, 1995; Qin and

12 Powell-Coffman, 2004). Generally, the AHR's N-terminus shares high sequence

homology between species, while the C-terminus is less conserved (Ramadoss and

Perdew, 2005).

Polymorphisms of the human AHR gene have been identified (Racky et al., 2004),

[reviewed in: (Harper et al., 2002; Okey et al., 2005)]. These polymorphisms may

modulate CYP1A1 inducibility as shown by the assessment of EROD activity in human

lymphocytes (Smart and Daly, 2000), and CYP1A1 mRNA levels following the

introduction of expression vectors encoding AHR variants into AHR-deficient Hepa-1

cells (Wong et al., 2001). A correlation between AHR polymorphisms and cancer

susceptibility has yet to be determined (Kawajiri et al., 1995; Cauchi et al., 2001). The

rat AHR is also polymorphic as shown by comparisons of the "TCDD-sensitive" Long-

Evans (L-E) and "TCDD-resistant" Han/Wistar Kuopio (H/W) strains, which display

more than a 1,000-fold difference in susceptibility to lethality following dioxin exposure

(Pohjanvirta et al, 1998). An intronic point mutation within the AHR of H/W rats results in altered RNA splicing such that 38 or 43 amino acids are deleted from the C-terminus

(Pohjanvirta et al., 1998). The H/W AHR is -98 kDa in size compared to -106 kDa in other rat strains.

Numerous factors regulate the expression and function of the AHR including xenobiotics, hormones, inflammatory cytokines, physiological state, cell cycle state and growth factors [reviewed in: (Harper et al., 2006)]. In turn, the AHR regulates the transcription of numerous genes by interacting with a series of co-activators. Using chromatin immunoprecipitation (ChIP) assays, the following co-activators were shown to assemble on the mouse Cyplal enhancer region following TCDD treatment: steroid

13 receptor co-activator-1 (SRC-1), nuclear co-activator-2 (NCoA-2) and p300/cAMP- responsive element-binding protein (CREB)-binding protein (CBP) co-integrator protein

(p/CIP) [reviewed in: (Hankinson, 2005)]. These proteins are pi60 histone acetyltransferase (HAT) co-activators. Another study found that the TCDD-dependent recruitment of mediator subunit of 220 kDa (Med220), cyclin-dependent kinase 8

(CDK8) and p300/CBP to the Cyplal enhancer leads to the assembly of RNA polymerase II at the Cyplal promoter (Wang et al., 2004). An interaction between the

AHR and histone H4, a component of the nucleosomal unit, has been demonstrated

(Dunnetal., 1993).

1.3.2 ARNT and AHRR: other members of the bHLH/PAS superfamily

The domain organization of the AHR's dimerization partner, ARNT (also known as HBF-ip), is shown in Fig. 1.3. ARNT is ubiquitously expressed and its deletion in mice results in embryonic lethality (Kozak et al., 1997). This protein can form heterodimers with several members of the bHLH superfamily including HIF-lcc. Under hypoxic conditions, HIF-la/ARNT can bind to hypoxia response elements (HREs) found within gene regulatory sequences (Semenza, 1998). The AHR and HIF-la can compete for ARNT heterodimerization (Gradin et al., 1996), with physiological consequences that remain unclear.

Cyp2sl is inducible by both dioxins and hypoxia (Rivera et al., 2007). This is at least partly mediated by a region of the gene that contains overlapping HREs and DREs, which recruit HIF-la/ARNT and AHR/ARNT heterodimers following hypoxic conditions and dioxin treatment, respectively. ARNT can also form homodimers that were recently shown to drive Cyp2a5 transcription in primary mouse hepatocytes and

14 Hepa-1 cells by binding to an E-box element located in the 5'-flanking region of this

gene (Arpiainen et al., 2007).

Although ARNT regulates the transcription of numerous genes, expression of

ARNT itself seems to be relatively refractory to modulation. Recent studies show that

ARNT levels can be increased by xenobiotic exposure. For instance, dexamethasone

treatment can increase hepatic ARNT mRNA levels 6- to 9-fold in rats as early as 3 h

post-treatment (Mullen Grey and Riddick, manuscript in preparation). Microarray

analysis shows ARNT mRNA levels are elevated 5-fold in liver tissue isolated from

pyrazole-treated mice (Nichols and Kirby, 2008).

Furthermore, ARNT acts as a co-activator for estrogen receptor (ER)-dependent

gene transcription, which may result in competition between the AHR and ER for ARNT

as a cofactor (Ruegg et al., 2008). This study shows that transfection of HeLa cells with

small interfering (si) RNA targeting ARNT decreases the activity of an estrogen- responsive reporter gene. TCDD treatment of HC11 or HeLa cells also down-regulates estrogen-responsive reporter gene activity. ChIP analyses in MCF-7 cells show reduced

ARNT occupancy at the promoter of an estrogen-regulated gene following TCDD treatment. Since ARNT is recruited for heterodimerization with the AHR following

TCDD treatment, this mechanism may contribute to the anti-estrogenic properties of

TCDD since less ARNT is available for ER co-activation.

The aryl hydrocarbon receptor repressor (AHRR) is another member of the bHLH/PAS superfamily (Fig. 1.3). This protein lacks PAS B and Q-rich domains, which prevents it from binding ligands and activating transcription. The AHRR was first discovered in mice (Mimura et al., 1999), but has homologues in humans (Tsuchiya et

15 AHR Highly conserved between species Variable between species

N-terminus C-terminus

-ARNT dimerization -ARNT dimerization (PAS A) -Facilitates gene transcription -Nuclear localization -AHR ligand binding (PAS B) -Assembles co-activators and co- and export signals -Hsp90 binding (PAS B) repressors/ promoter -DNA binding communication -Hsp90 interaction

AHRR

N-terminus PAS A C-terminus

-ARNT dimerization -Lacks PAS B domain -Lacks transactivation domain -DNA binding -No ligand binding domain -No Hsp90 domain

ARNT

N-terminus C-terminus in

-DNA binding -Heterodimerization -Facilitates gene transcription -Nuclear localization -Assembles co-activators and co- signal repressors/ promoter communication

Figure 1.3. Schematic structural diagrams of bHLH superfamily members. [Adapted from Kewley et al. 2004; Denison et al. 2002].

16 al., 2003), rats (Korkalainen et al., 2004), and fish (Evans et al, 2005). The AHRR

contains the following TF consensus binding sites: nuclear factor-KB (NF-KB), DRE

(both type I and II as defined in Section 1.3.4), and glucocorticoid response element

(GRE) (Nishihashi et al., 2006). Even in the absence of a ligand, the AHRR is located in

the nucleus where it competes with the AHR for their common binding partner ARNT,

thus negatively regulating AHR function. The AHRR/ARNT complex binds DREs

without activating transcription. AHRR expression is induced by AHR/ARNT binding to

DREs located in this gene's 5'-flanking region (Mimura et al., 1999). Protein

interactions appear to be a major mechanism by which the AHRR controls gene

transcription. A recent report argues that the ability of the AHRR to act as a repressor is

not solely due to competition with the AHR for ARNT or DRE binding, but also from

protein-protein interactions between the AHRR's N-terminus and the AHR (Evans et al.,

2008). Furthermore, a recent study shows that CYP1A1 is kept in an inactive state

through the recruitment of co-repressors by the AHRR/ARNT heterodimer (Oshima et

al., 2007). A/irr-knockout mice have been generated by targeted disruptions of exon 2

and intron 2 (Hosoya et al., 2008). This will enable researchers to examine the endogenous role of the AHRR in vivo.

Recent evidence suggests a role for ARNT and AHRR in the pathology of

selected diseases. Decreased ARNT levels are linked to the occurrence of human type 2 diabetes (Gunton et al., 2005). A study reports down-regulation of AHRR in numerous human tumors (Zudaire et al., 2008), implicating a possible role for this protein as a tumor suppressor.

17 1.3.3 Function of the AHR

Biological responses to AHR activation have been categorized into three pathways: toxic, developmental and adaptive (Walisser et al., 2004a; Walisser et al.,

2004b). The toxic and adaptive pathways are initiated following binding of exogenous ligands, nuclear translocation and ARNT heterodimerization. Toxic responses refer to the harmful consequences of exposure to these chemicals, while adaptive responses involve the metabolism of substrates after aromatic hydrocarbon exposure.

Developmental responses involve the AHR's endogenous role in the growth and differentiation of various organs. Section 1.3.3.1 will address the AHR's participation in toxic pathological endpoints; Section 1.3.3.2 will examine developmental pathways; and

Section 1.3.5 will discuss adaptive pathways of the AHR.

1.3.3.1 Role of the AHR in aromatic hydrocarbon-mediated toxicity

The AHR is implicated in mediating almost every aspect of toxicity that is dioxin- related [reviewed in: (Bock and Kohle, 2006; Okey, 2007)]. Early structure-activity relationship (SAR) studies first showed that aromatic hydrocarbons with the highest AHR binding affinities also display the highest toxic potencies (Safe et al., 1985; Safe, 1990).

Studies of AAr-knockout mice reaffirm the role of the AHR in dioxin toxicity. Three independent groups have generated Ahr-mxW. mice (Fernandez-Salguero et al., 1995;

Schmidt et al., 1996; Mimura et al., 1997), which have served as useful models to study the function of the AHR in the three pathways mentioned previously. These knockout mice are no longer susceptible to TCDD-induced teratogenesis (Thomae et al., 2004) or

B[a]P-initiated carcinogenesis (Shimizu et al., 2000), suggesting that these toxic outcomes require the presence of the AHR. Transgenic mice harboring a constitutively

18 active AHR display a high incidence of hepatocarcinogenesis, which suggests die AHR

may promote tumors in specific tissues (Moennikes et al., 2004). Mice with a mutation

in the nuclear localization sequence of the AHR are resistant to TCDD-induced

hepatotoxicity, cleft palate and thymic atrophy (Bunger et al., 2003), indicating these

toxic endpoints depend on the AHR/ARNT pathway functioning as a ligand-activated TF.

TCDD-mediated toxicity is not observed when ARNT is expressed at low levels in mice

(Walisser et al., 2004a), further supporting the involvement of the AHR/ARNT

heterodimer in the toxic pathway.

Despite decades of study, mechanisms involved in AHR-mediated TCDD toxicity

are far from clear. TCDD is known to disrupt the expression of AHR-regulated genes. In hopes of identifying genes that confer toxicity, numerous laboratories have used microarrays to search for genes whose levels are altered by dioxin exposure [reviewed in:

(Okey, 2007)].

1.3.3.2 Developmental and endogenous roles of the AHR

The involvement of the AHR in developmental pathways can be examined using the A/zr-null mouse model. These mice exhibit abnormal physiological and developmental characteristics including a small liver due to the failure of the ductus venosus to close (Lahvis et al., 2005), impairment in immune responses (Rodriguez-Sosa et al., 2005), and liver fibrosis characterized by high retinoid levels (Andreola et al.,

2004; Corchero et al., 2004). These findings indicate that intact immune responses, normal liver development and the maintenance of retinoid homeostasis require the AHR.

Cardiac hypertrophy is also evident in A/zr-null mice, suggesting a role for the AHR in cardiovascular development and physiology (Lund et al., 2003). Female Ahr-rmll mice

19 display a series of reproductive deficiencies including small litter size, poor pup survival

and difficulties in pregnancy and lactation, which link the AHR to normal reproductive

physiology [reviewed in: (Abbott et al., 1999b; Pocar et al., 2005)]. The AHR appears to

contribute to normal renal development and nephrogenesis by regulating the expression

of Wtl, an important gene involved in kidney development. Studies comparing renal

cultures isolated from A/zr-null and wild-type mice reveal key histological differences

which may be attributed to the absence of the AHR in tissues isolated from knockout mice (Ramos et al., 2007). The numerous mechanisms by which the AHR participates in developmental pathways are only partially understood. Mice carrying a mutation in the nuclear localization sequence of the AHR exhibit the same developmental defects as Ahr- null mice, indicating that AHR-dependent nuclear events are required for normal development (Bunger et al., 2003).

The ability of the AHR to participate in developmental pathways may also occur through biological cross-talk with nuclear receptors. Nuclear receptors function to regulate biological processes and maintain homeostasis by modulating transcription of numerous genes including CYP2, CYP3 and CYP4 family members (Gronemeyer et al.,

2004; Fujii-Kuriyama and Mimura, 2005; Perissi and Rosenfeld, 2005). Nuclear receptors may share ligands, transcriptional co-activators, transcriptional co-repressors, and even cis-acting elements (Pascussi et al., 2004). Functional cross-talk between the

ER and the AHR has been the subject of much recent interest. ERcc is recruited to the

CYP1A1 promoter following treatment with AHR ligands, thus functioning as a positive

(Kato et al., 2005; Matthews et al., 2005; Matthews et al., 2007) or negative (Beischlag

20 and Perdew, 2005) co-regulator of AHR-mediated transcriptional activation and/or transcriptional repression.

Functional interactions between the AHR and nuclear receptors such as the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), are critical in controlling xenobiotic and endogenous chemical metabolism (Pascussi et al, 2004;

Petrick and Klaassen, 2007). Recent studies show that the AHR contributes to the regulation of CAR expression. TCDD induces CAR mRNA levels in mice (Petrick and

Klaassen, 2007), and another AHR agonist P-naphthoflavone (0-NF) up-regulates CAR mRNA levels in Ahr wild-type but not Ahr-null mice (Patel et al., 2007). The peroxisome proliferator-activated receptor a (PPARa) ligand WY-14643 increases AHR mRNA levels in Caco-2 cells and PPARa wild-type but not PPARa-null mice at least partly via a peroxisome proliferator response element (PPRE) located in the Ahr promoter

(Villard et al., 2007).

The AHR also plays an endogenous role in cell cycle progression. This will be discussed in Section 1.3.5.3.

1.3.4 Dioxin-responsive elements

The AHR/ARNT heterodimer recognizes and binds specific DNA sequences known as "dioxin-responsive elements" (DREs), "xenobiotic response elements" (XREs) or "aryl hydrocarbon response elements" (AHREs) (Denison et al., 1988). The DRE binding consensus is defined as follows: 5'-GCGTGNN(A/DNNN(C/G)-3', based on the binding of the activated AHR/ARNT complex to the Cyplal 5'-flanking region (Yao and Denison, 1992). This binding consensus contains the four invariant "core" nucleotides (CGTG) that are essential for receptor binding. ARNT is known to bind the

21 GTG nucleotides within the core sequence, while the AHR binds to nucleotides located

on the 5'-side of this site (Bacsi et al., 1995). Mutations outside the core sequence

diminish AHR/ARNT DNA binding affinity, while mutations within the CGTG core motif completely abolish AHR/ARNT binding to DNA (Shen and Whitlock, 1992). The

DRE enhancer functional consensus sequence [5'-(T/G)NGCGTG(A/C)(G/C)A-3'], is capable of not only binding the AHR/ARNT heterodimer, but can also initiate transcription following DNA binding (Lusska et al., 1993). Putative DREs occur frequently in human, rat and mouse genomic sequences (Sun et al., 2004). Although

DREs are typically located in the 5'-flanking regions of genes (Lai et al., 1996), recently this binding element was identified within the first intron of the human AHRR gene

(Haarmann-Stemmann et al., 2007).

ALDH3A1 encodes a class 3 aldehyde dehydrogenase enzyme whose expression is up-regulated by AHR ligands. Reporter gene studies in rat hepatoma cells revealed a novel role for DREs in maintaining constitutive ALDH3A1 levels (Reisdorph and

Lindahl, 2007). MC-inducible reporter gene expression is also mediated by the same

DREs. A DRE within the mouse Cyplbl 5'-flanking region can also support both basal and TCDD-induced expression of a reporter construct (Zhang et al., 2003a).

The human CYP1A1 and CYP1A2 genes are located in a head-to-head orientation separated by 23-kb of DNA; an arrangement also evident in the mouse orthologues.

CYP1A1 and CYP1A2 share a common bidirectional regulatory region which contains a cluster of DREs required to drive transcription of these genes following p-NF and MC treatment in HepG2 cells (Ueda et al., 2006). Despite sharing a common 5'-flanking region, expression of these two genes is regulated differently [reviewed in: (Fujii-

22 Kuriyama and Mimura, 2005)]. The liver expresses constitutive CYP1A2 levels which are up-regulated by aromatic hydrocarbon treatment, while CYP1A1 is not basally expressed in the liver but can be induced in numerous organs following exposure to AHR ligands. While the CYP1A1 5'-flank harbors multiple DREs which mediate up-regulation of this gene following aromatic hydrocarbon treatment, the role of DREs in the induction of CYP1A2 is not as clearly defined (Black and Quattrochi, 2004). A few years ago, a novel role for the AHR/ARNT complex in mediating CYP1A2 induction following aromatic hydrocarbon exposure was found. The heterodimer is recruited to a novel site bound by an unknown protein on the CYP1A2 5'-flanking region (Sogawa et al., 2004).

In this sense, the AHR/ARNT complex acts as a co-activator of rat CYP1A2 gene induction following MC treatment. The novel cis-acting sequence identified [5'-

CATGNNNNNNC(T/A)TG-3' (Boutros et al., 2004; Sogawa et al., 2004)] is distinct from the classical DRE sequence and was designated AHRE-II or DRE-II or XRE-II.

For clarity, the classical DRE sequence discussed above is now referred to as DRE-I or simply DRE.

DRE sequences may function to negatively regulate gene expression by behaving as "inhibitory DREs" (iDREs). Such elements do not necessarily differ in sequence from classical DREs. AHR/ARNT binding to iDREs interferes with the binding of positive trans-acting factors (required to maintain constitutive gene expression), to nearby enhancer elements, thus leading to decreased gene expression. IDREs are implicated in the down-regulation of numerous estrogen-responsive genes by TCDD including cathepsin D, c-fos and pS2. The AHR/ARNT complex directly binds to the core DRE sequence CGTG within these genes, interfering with the binding of Spl or AP-1 that are

23 required for basal gene transcription (Gillesby et al., 1995; Krishnan et al., 1995; Duan et

al., 1999; Wang et al., 2001). AHR binding to iDREs within these genes is attributed as

one mechanism by which TCDD elicits its antiestrogenic effects. Suppression of these

genes by TCDD is reversed upon treatment with the AHR partial agonist/antagonist a-

naphthoflavone (cc-NF), and transfection of antisense targeting AHR or ARNT

expression (Wang et al., 2001).

1.3.5 Regulation of gene expression

Typically aromatic hydrocarbon exposure is associated with the induction of

CYP1 family members. Although CYP1 isoforms show the greatest magnitude of

induction compared to other P450s following aromatic hydrocarbon treatment, the

literature shows that members of the CYP2 and CYP3 families may also respond to AHR

ligands. Table 1.1 summarizes selected P450s outside the CYP1 family that are

responsive to aromatic hydrocarbons.

1.3.5.1 Adaptive role of the AHR in the Induction of P450 enzymes

Functional DREs exist in the regulatory region of numerous P450 genes

[reviewed in: (Fujii-Kuriyama and Mimura, 2005)]. In an uninduced state, the mouse

Cyplal gene is assembled in a nucleosomal configuration, which limits the accessibility

of TFs, co-activators and co-repressors to gene regulatory regions. The ligand-activated

AHR/ARNT complex disrupts the nucleosome, thus altering chromatin structure and making DNA more accessible to trans-acting factors (Okino and Whitlock, 1995). The

AHR/ARNT heterodimer binds to several enhancer sequences in the Cyplal 5'-flanking region. Examination of protein-DNA interactions at the Cyplal enhancer and promoter following introduction of AHR wild-type or defective mutants, reveals that the AHR's

24 transactivation domain transmits a poorly understood "induction signal" from the enhancer to the promoter (Ko et al., 1997). This leads to subsequent promoter occupancy and gene transcription.

The AHR may play a role in the regulation of other P450s which are responsive to aromatic hydrocarbon treatment (summarized in Table 1.1). The hamster CYP2A8 is strongly induced following MC treatment (Sunouchi et al., 1988). Reporter gene studies identify a sequence designated PREX (positive regulatory element for XRE-mediated gene expression), which enhances DRE-mediated CYP2A8 inducibility (Kurose et al.,

1999). CYP2S1 is markedly up-regulated by TCDD via a mechanism that requires the presence of both the AHR and ARNT as shown by studies in mutant Hepa-1 cell lines

(Rivera et al., 2002). Three overlapping DREs located near the mouse Cyp2sl promoter recruit the AHR/ARNT heterodimer in a dose-dependent manner following TCDD treatment (Rivera et al., 2007). Another study shows that MC treatment slightly induces rat CYP2A1 protein levels in vivo (Thomas et al, 1981). In rat lungs, CYP2A3 mRNA expression increases 3-fold following MC administration (Kimura et al., 1989).

Examination of mouse liver slices following treatment with TCDD or P-NF shows 2- to

3-fold increases in CYP2A5 catalytic activity (Gokhale et al., 1997). The role of the

AHR in Cyp2a5 regulation was recently investigated in C57BL/6 and DBA/2 mouse strains (Arpiainen et al., 2005). The concentration of TCDD required for maximal

CYP2A5 mRNA induction is 10 uM in cultured hepatocytes derived from DBA/2 mice and only 10 nM in C57BL/6 cultured hepatocytes. Furthermore, CYP2A5 mRNA levels are markedly up-regulated in C57BL/6 mice treated with MC compared to the DBA/2 strain. Cyp2a5-luciferase reporter activity is increased by TCDD in wild-type Hepa-1

25 TABLE 1.1. Summary of selected aromatic hydrocarbon-responsive P450s outside the CYP1 family.

Gene Biological system Aromatic hydrocarbon Level of P450 expression change Direction and fold-change Reference administered of P450 expression

CYP2A1 rat liver MC (5 days) protein T~2 Thomas etal. 1981

CYP2A3 rat lung MC (1 day) mRNA T3 Kimura et al. 1989

Cyp2a5 mouse liver slices P-NF (3 days) coumarin-7-hydroxylase T2 Gokhale etal. 1997 TCDD (3 days) t3 1 ° mouse hepatocyte culture TCDD (1 day) protein T2.4 Arpianinen et al. 2005 TCDD (1 day) coumarin-7-hydroxylase T2.9 mouse liver MC (4 days) mRNA T strong

CYP2A8 hamster liver MC (4 days) protein T Sunouchi etal. 1988 1° hamster hepatocyte culture MC (1 day) reporter gene activity t10-20 Kurose et al. 1999

Cyp2sl mouse hepatoma cells TCDD (1 day) mRNA tio Rivera et al. 2002 mouse liver TCDD (1 day) mRNA t slight

CYP2A9 hamster liver MC (3 days) mRNA 4 Kurose et al. 1998 testosterone 7cc-hydroxylase 4 1.5

CYP2C8 1° human hepatocyte culture MC (1 day) mRNA 42-5 Ning et al. 2008

Cyp2d9 mouse liver MC (single injection) mRNA (day 7)* 4 1.4 Lee etal. 2006 protein (days 4,7) * 4 1.7 testosterone 16oc-hydroxylase (day 4) * 4 1.4

Cyp2d9/ll mouse liver MC (3 days) protein 4-1.1-1.3 Jenkins et al. 2006 Cyp2dl0

26 TABLE 1.1. cont.

Gene ^logical system Aromatic hydrocarbon Level of P450 expression change Direction and fold-change Reference administered of P450 expression

a hepatocyte culture MC (1 day) mRNA 42-5 Ning et al. 2008

Rat relative rat liver TCDD (13 weeks) mRNA 4 3.4 Ovando et al. 2006 of Cyp2j9 TCDD (1 day) mRNA 4 3.3

CYP3A rat liver MC (single injection) CYP3A2 mRNA (days 1-5)* 42 Jones et al. 1996 protein (day 1)* 43.6 testosterone 6p-hydroxylase (day 2)* 4 1.4

Cyp3a mouse liver MC (3 days) protein 4 slight Jenkins et al. 2006 mouse liver MC (single injection) CYP3A11 mRNA (days 1-2)* 4 1.5 Lee et al. 2006 protein (day 2)* 4 1.7-7f

CYP3A9 rat liver TCDD (13 weeks) mRNA (real-time RT-PCR) 41000 Vezinaetal. 20O4 mRNA (microarray analysis) 435

Rat relative rat liver TCDD (13 weeks) mRNA 4 86 Ovando et al. 2006 of Cyp3al3 TCDD (1 day) mRNA 46

If no value is given for the fold-change in P450 expression, the magnitude of change was not provided in the publication referenced. * Corresponds to the day maximal suppression of P450 levels was detected following a single i.p. MC injection, t Values include the range of suppression detected by four independent antibodies.

27 cells, but not COS-1 cells or Hepa-1 mutant lines lacking either the AHR or ARNT. Site-

directed mutagenesis of a DRE in the 5'-flanking region of Cyp2a5 abolishes dioxin-

inducibility of reporter gene activity. These studies illustrate the requirement of the

AHR/ARNT complex in Cyp2a5 induction by aromatic hydrocarbons.

1.3.5.2 Adaptive role of the AHR in the Suppression of P450 enzymes

P450 genes can also be down-regulated by aromatic hydrocarbon exposure

(summarized in Table 1.1). Unlike the partially understood mechanisms involved in

aromatic hydrocarbon-mediated P450 induction, there is much less known about

mechanisms underlying P450 suppression.

MC administration suppresses rat hepatic CYP3A protein expression and catalytic

activity, while trends for suppression are observed at the mRNA level for CYP3A2

(Jones and Riddick, 1996). Other P450s that are down-regulated following aromatic hydrocarbon treatment by unknown mechanisms include: rat CYP3A9 (Vezina et al.,

2004), hamster CYP2A9 (Kurose et al., 1998), rat P450s related to mouse CYP3A13,

CYP2J9 (Ovando et al., 2006), and mouse CYP2D9, CYP2D10, CYP2D11 and CYP3A

(Jenkins et al., 2006). A recent in vivo mouse study from our laboratory shows that MC decreases CYP2D9 protein, mRNA and catalytic activity, while triggering a marked loss of CYP3A apoprotein levels (Lee et al., 2006). A focus of our laboratory has been the down-regulation of rat CYP2C11 by aromatic hydrocarbons [reviewed in: (Riddick et al.,

2003; Riddick et al., 2004)]. This will be discussed in Section 1.6.4.

Although P450 down-regulation in response to aromatic hydrocarbon treatment occurs in several animal species, effects of these toxicants on the expression of the major human constitutive P450s remains largely unexplored. A recent study in human

28 hepatocytes showed that CYP2C8 and CYP2E1 mRNA levels are suppressed following

MC treatment (Ning et al., 2008).

1.3.5.3 Genes involved in cell growth and differentiation

There is growing evidence for a role of the AHR in the regulation of genes

involved in cell cycle control. Whether the receptor acts as an inhibitor or promoter of

cellular proliferation appears to be context-dependent [reviewed in: (Marlowe and Puga,

2005)]. Over-expression of the AHR suppresses cell growth in Leydig cells (Iseki et al.,

2005), while constitutively active AHR inhibits T cell growth by altering the expression

of genes involved in cell cycle arrest and apoptosis (Ito et al., 2004). The AHR may lead

to cell cycle inhibition by mediating the down-regulation of E2F-regulated genes known

to promote cell cycle progression (Marlowe et al., 2004).

DREs are present in the regulatory regions of several genes involved in cell

growth and differentiation [reviewed in: (Bock and Kohle, 2006)]. DREs located in the promoter of the oncogene c-myc likely mediate the repression of a reporter construct in human breast carcinoma cells that naturally express high levels of the AHR (Yang et al.,

2005). Treatment with AHR ligands leads to changes in the expression of genes involved in cell cycle control as observed for E2F (Marlowe et al., 2004), and the pro-apoptotic gene Box (Matikainen et al., 2001). TCDD treatment in Hepa-1 cells up-regulates the expression of NF-KB and AP-1 (TFs involved in the regulation of genes that participate in cell cycle control), through an AHR-dependent mechanism (Puga et al., 2000a). The

AHR can directly interact with NF-KB (Tian et al., 1999; Kim et al., 2000), and the retinoblastoma tumor suppressor protein (Marlowe et al., 2004). The AHR can function via mitogen-activated protein kinase (MAPK) signaling to increase levels of the proto-

29 oncogene c-jun (Weiss et al., 2005). Cross-talk between the AHR and Mdm2 or the

tumor suppressor p53 contributes to cell cycle control (Paajarvi et al., 2005).

In recent years, the potential application of AHR agonists in anticancer therapies

has been investigated (Westwell, 2004). AHR agonists can stimulate P450-dependent

generation of cytotoxic metabolites in cancer cells, resulting in cell death and regression

of tumors.

1.3.6 Natural and endogenous AHR Iigands

Although the majority of AHR Iigands are xenobiotics or synthetic chemicals,

there are also many endogenous and naturally-occurring Iigands [reviewed in: (Jeuken et

al., 2003; Nguyen and Bradfield, 2008)]. The majority of natural AHR Iigands are

derived from dietary sources. These include: flavonoids, carotenoids, phenolics and

indoles [reviewed in: (Denison et al., 2002; Jeuken et al., 2003; Fukuda et al., 2007)].

Numerous AHR agonists/antagonists are found in vegetables, teas, herbs and fruits

(Amakura et al., 2002; Fukuda et al., 2004). For example, indole-3-carbinol is found in

members of the Brassica family including Brussel sprouts, cauliflower and broccoli,

while the AHR antagonist resveratrol is a phenolic compound found in wines, nuts and berries (Casper et al., 1999). Flavonoids such as 3',4'-dimethoxyflavone (3',4'-DMF)

and 5,7-dimethoxyflavone are naturally-ocurring, plant-derived compounds that possess

AHR antagonist activity (Lee and Safe, 2000; Wen and Walle, 2007).

Generally, naturally-ocurring AHR Iigands have weak binding affinities compared to aromatic hydrocarbons such as TCDD; however, dietary indoles and tryptophan derivatives are quite potent. Treatment of HepG2 cells with TCDD or indirubin (10 nM) results in similar gene expression changes (Adachi et al., 2004).

30 Indirubin is synthesized as a by-product of indigo and is found in human urine and fetal

bovine serum (FBS). Both indirubin and indigo prove to be high-affinity AHR-binding

ligands (Guengerich et al., 2004; Sugihara et al., 2004). Indolo-[3,2-6]-carbazole (ICZ)

also possesses high AHR binding affinity (Chen et al., 1995c). Similarly, tryptophan and

tryptophan derivatives produced via ultraviolet (UV) light treatment display potent AHR

binding activity [reviewed in: (Nguyen and Bradfield, 2008)].

Evidence suggests the existence of endogenous, physiological AHR ligands. Cow

milk-based infant formula may contain a physiological AHR ligand since treatment of

HepG2 cells with this formula results in increased CYP1A protein and mRNA levels via the AHR (Xu et al., 2005a). Transfection of an AHR expression vector to AHR-devoid rat hepatoma BP8 cells leads to the up-regulation of CYP1A1 and CYP1B1 mRNA expression in the absence of an exogenous ligand (Roblin et al., 2004). This suggests the presence of an endogenous factor(s) in BP8 cells which is capable of driving AHR- dependent gene transcription. Furthermore, 7-ketocholesterol, which is moderately expressed in human tissue, can modulate AHR activity (Savouret et al., 2001). Lipoxin

A4 is another endogenous AHR ligand. This compound is a product of arachidonic acid metabolism and can drive AHR-dependent gene expression [reviewed in: (Denison et al.,

2002)]. Heme metabolites such as bilirubin may also function as endogenous AHR ligands since they can increase CYP1A1 activation in mouse hepatoma cells (Kapitulnik and Gonzalez, 1993; Sinai and Bend, 1997). McMillan and Bradfield (2007) recently reported that modified low-density lipoprotein can stimulate AHR activity. An endogenous AHR agonist known as 2-(rH-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester, has been identified from tissue isolated from porcine lung (Song et al.,

31 2002). Lastly, the physiological abnormalities apparent in Ahr-mx\\ mice further support

the existence of an endogenous AHR ligand(s) since the functional AHR is required for

normal physiological development (Denison et al., 2002).

1.3.7 AHR antagonists

Compounds that bind to the AHR are highly diverse in structure. A promiscuous

AHR may be functionally advantageous to an organism since it will likely detect a large

range of xenobiotics, perhaps providing some degree of protection from the deleterious

responses to more potent, toxic ligands such as TCDD (Jeuken et al., 2003). Upon the

examination of chemical structures of known AHR antagonists (Fig. 1.4), there are no

obvious common structural similarities. Despite the identification of numerous AHR

antagonists, it is difficult to find a compound that is a pure antagonist which displays no

agonist-like activity.

Functional in vivo AHR antagonists are of particular interest in this thesis. 3'-

Methoxy-4'-nitroflavone (3'M4'NF) is a synthetic flavone which has been used

successfully as an AHR antagonist in in vivo mouse studies (Dertinger et al., 2000;

Nazarenko et al, 2001). 3'M4'NF is able to compete with TCDD for AHR binding, but does not lead to nuclear translocation of the AHR complex (Dertinger et al., 2000).

Although it was thought that 3'M4'NF possessed very little or no agonist-like activity, other studies show that the delicate balance between this chemical's AHR agonist/antagonist abilities is concentration- and gene-dependent (Zhou and Gasiewicz,

2003; Zatloukalova et al., 2007). It is important to keep in mind that the effects of

3'M4'NF may not be solely AHR-related.

32 a-Naphthoflavone (oc-NF)

B CH.

H N—C N OH^O ii "V O CIL

2-MethyI-2#-pyrazole-3-carboxylic acid (CH-223191)

D ci NO, Cl o o OCH,

o Cl CH o'

6-Methyl-1,3,8-trichlorodibenzofuran 3'-Methoxy-4'-nitroflavone(3'M4'NF) (MCDF)

Figure 1.4. Structurally diverse AHR antagonists.

33 a-NF is a known in vitro AHR partial agonist/antagonist depending on its concentration

[reviewed in: (Santostefano and Safe, 1996)]. At concentrations of <1 nM, a-NF acts as

an AHR antagonist (Merchant et al., 1992), while at higher concentrations (10 uM), oc-

NF displays AHR partial agonist activity (Santostefano et al., 1993). a-NF binds to the

AHR, but the resulting complex does not enter the nucleus (Merchant et al., 1992), and is

inactive in DNA binding (Gasiewicz and Rucci, 1991). This compound has been used as

an in vivo AHR antagonist in mice and rats; however, endpoints measured in such studies

include ovarian follicle survival (Thompson et al., 2005), ovarian AHH activity and

primordial oocyte number (Mattison and Thorgeirsson, 1979). We were not able to find

any studies that assess the in vivo effects of a-NF on more direct AHR endpoints such as

hepatic CYP1A induction.

6-Methyl-l,3,8-trichlorodibenzofuran (MCDF) is another AHR antagonist that

has been studied in vivo. MCDF binds to the AHR and the complex translocates to the

nucleus where it competes with TCDD-activated AHR for DRE binding (Merchant et al.,

1993). In rats this compound partially antagonizes TCDD-induced hepatic AHH and

EROD activity (Astroff and Safe, 1988). Studies in mice show that MCDF can prevent

TCDD-induced immunotoxicity and causes a small reduction in elevated hepatic AHH

and EROD activity (Bannister et al., 1989). The use of MCDF is associated with anti

estrogenic activity which may hinder the interpretation of studies using this chemical as

an AHR antagonist (Astroff and Safe, 1988).

A recent study identified a novel compound, 2-methyl-2i/-pyrazole-3-carboxylic

acid (CH-223191), as a potent in vivo AHR antagonist in mice (Kim et al., 2006). CH-

223191 prevents TCDD binding to the AHR, nuclear translocation, DNA binding and

34 TCDD-induced AHR-dependent gene expression. This is a promising compound, as it

shows no detectable AHR agonist-like activity to date.

1.4 Cytochrome P450

There are two phases of drug biotransformation that generally function to convert

lipophilic drugs into polar metabolites that are readily excreted from the organism.

Phase I includes oxidation, reduction and hydrolysis reactions of exogenous compounds,

endogenous compounds or pharmacological agents, resulting in bioactivation, de

activation or no change in the activity of the parent compound (Wauthier et al, 2007).

Cytochrome P450s, which are found in all living organisms including bacteria, plants and

animals, play a major role in Phase I oxidation reactions.

P450s are named based on their spectral properties [reviewed in: (Estabrook,

2003)]. In the late 1950's, a pigment in mammalian liver microsomes was found to bind carbon monoxide (Garfinkel, 1958; Klingenberg, 1958). The pigment was identified as a hemoprotein and named cytochrome P450 since an absorbance spectrum peaking at 450 nm is formed when the reduced form of P450 binds carbon monoxide (Omura and Sato,

1964). The P450 catalytic cycle (Estabrook et al., 1971) is summarized below [reviewed in: (Guengerich, 1993)]. This reaction mediates drug hydroxylation by incorporating one atom of oxygen into the drug substrate, while the other oxygen atom forms water. The overall equation is as follows, with the drug represented as (RH):

+ + RH + 02 + NADPH + H -»• ROH + H20 + NADP

Briefly, the drug binds to the P450 enzyme, which has its heme prosthetic group in its oxidized ferric form (Fe3+). Within the membrane of the endoplasmic reticulum, an electron is donated to the RH/P450 complex from the reducing cofactor nicotinamide

35 adenine dinucleotide phosphate (NADPH) via the membrane-bound NADPH-cytochrome

P450 reductase. As a result, P450 is reduced to its ferrous state (Fe2+), which can bind

molecular oxygen (O2). A second electron is donated from NADPH or via cytochrome

bs, and this contributes to the activation of oxygen and splitting of the 0-0 bond. One

oxygen atom contributes to the formation of water, while the other is incorporated into

the drug substrate to form a hydroxylated product (ROH), which is then released from the

active site of the enzyme. The P450 is once again in its ferric form. Since one atom of

oxygen is incorporated into the drug substrate, the P450 enzymes are known as

monooxygenases.

All microsomal P450 enzymes receive electrons from NADPH-cytochrome P450

reductase. Deletion of this gene results in loss of microsomal P450 catalytic function as

well as embryonic lethality (Shen et al., 2002; Otto et al., 2003). This suggests an

essential role for microsomal P450s in normal physiological development. P450s have

several important endogenous substrates. For examples, P450s can oxidize physiological

steroid hormones in a regio- and stereo-selective manner (Waxman, 1988). This supports

a role for P450s in the metabolism of endogenous regulators of homeostasis (Dannan et

al., 1986) and provides a useful way to distinguish between various P450 isoforms.

Most P450s are found embedded in the endoplasmic reticulum membrane. Upon homogenization of tissues or cells, the smooth endoplasmic reticulum forms small circular vesicles known as microsomes [reviewed in: (Guengerich, 1993)]. P450s can also be found associated with the mitochondrial membrane [reviewed in: (Nebert and

Gonzalez, 1987)]. The first P450s to be crystallized were bacterial P450s; since they are not associated with organelle structures they are more readily soluble [reviewed in:

36 (Nebert and Gonzalez, 1987; Guengerich, 2004)].

Vertebrate P450 evolution has been occurring for -420 million years and Basic

Local Alignment Search Tool (BLAST) searches are continuously used to assist in the assembling of a P450 phylogenetic tree based on sequence similarity (Nelson, 2003).

P450 nomenclature is based on amino acid sequence homology (Nebert and Gonzalez,

1987; Nelson et al., 2004). P450s with >40% amino acid sequence homology are in the same gene family (ie. CYP1), while >55% sequence similarity results in P450s being placed within the same gene subfamily (ie. CYP1A). Members of the CYP1, CYP2 and

CYP3 families are major players in drug metabolism. Fig. 1.5 shows a summary of these family members found in humans and rats. According to David Nelson's Cytochrome

P450 homepage (http://drnelson.utmem.edu/CytochromeP450.html), the human genome currently contains 18 cytochrome P450 gene families and 44 subfamilies, totaling 57 known putatively functional P450 genes. The 2004 publication of the rat genome supported the existence of 84 rat cytochrome P450s (Gibbs et al., 2004). According to the Cytochrome P450 homepage, the current estimate is 88 putatively functional rat

P450s.

Phase II biotransformation involves conjugation reactions such as glucuronidation, glutathione conjugation and sulfation, which further aid in excretion by increasing the polarity of a compound or metabolite. These reactions are also known as detoxification reactions since the drug often loses its pharmacological activity. Drugs may also be excreted unchanged without the need for metabolism or may undergo phase

II conjugation without the need for phase I metabolism.

37 Human

CYPl CYP2 CYP3 CYP1A1, 1A2 CYP2A6,2A7,2A13 CYP3A4, 3A5, 3A7, 3A43 CYP1B1 CYP2B6 CYP2C8,2C9, 2C18, 2C19 CYP2D6 CYP2E1 CYP2F1 CYP2J2 CYP2R1 CYP2S1 CYP2U1 CYP2W1

Rat

CYPl CYP2 CYP3 CYP1A1,1A2 CYP2A1, 2A2, 2A3 CYP3A1> 3A2> 3A9> 3A18' 3A23 CYP1B1 CYP2B1, 2B2,2B3,2B12,2B21, 2B31 CYP2C6, 2C7, 2C11, 2C12, 2C13, 2C22, 2C23, 2C24, 2C79, 2C80, 2C81 CYP2D1, 2D2, 2D3, 2D4, 2D5 CYP2E1 CYP2F4 CYP2G1 CYP2J3,2J4,2J10,2J13,2J16 CYP2R1 CYP2S1 CYP2T1 CYP2U1 CYP2W1

Figure 1.5. Summary of putatively functional human and rat CYPl, CYP2 and CYP3 family members. [Adapted from Wauthier et al. 2007; http://dxnelson.utmem.edu/CytochromeP450.html]

38 1.4.1 Role of CYP1 family members in aromatic hydrocarbon-mediated toxicity

Metabolic activation of PAHs is a key step in the initation of mutagenesis.

Studies suggest that sensitivity to PAH-induced carcinogenicity may be attributed to

endogenous CYP1A1 and CYP1B1 expression. Cyplbl-mx\\ mice are unable to

metabolize 7,12-dimethylbenzo[a] anthracene and are resistant to the carcinogenicity of

this PAH (Buters et al., 1999). Furthermore, topical or subcutaneous B[a]P treatment

leads to the development of skin tumors in Ahr wild-type but not A/ir-null mice (Shimizu

et al., 2000). CYP1A1 expression is not found in the skin of A/zr-null mice,

demonstrating that this P450 contributes to tumor formation.

The presense of CYP1A1 may protect, or cause toxicity in mice following

aromatic hydrocarbon exposure. Cyplal -null mice are protected against TCDD-induced

lethality, wasting syndrome, as well as other toxic markers (Uno et al., 2004b), which

suggests CYP1A1 can participate under certain circumstances to toxicities produced by high doses of TCDD. On the other hand, CYP1 Al may play a protective role against

B[a]P-induced mortality. Cyplal wild-type mice can survive oral B[a]P administration, while Cyplal-mill mice display an accumulation of B[a]P DNA adducts and do not

survive (Uno et al., 2004a). By increasing the metabolism and subsequent de-activation

of B[a]P carcinogenic metabolites, CYP1A1 may protect mice by accelerating B[a]P detoxification.

Cypia2-null mice are more resistant to TCDD-induced hepatotoxicity and uroporphyria compared to wild-type mice (Smith et al., 2001), yet they are still susceptible to thymic atrophy. This suggests that only tissues that normally express

CYP1A2, such as the liver, are protected from toxicity in Cvjt?ia2-null mice (Uno et al.,

39 2004b). A recent study found that AHR expression is higher in gastric cancers compared to non-cancerous tissues (Ma et al., 2006). The authors speculate that increased CYP1A1 levels resulting from high AHR expression, may contribute to tumor formation by converting PAHs to reactive intermediates and/or metabolizing pro-carcinogens to their active forms. Other studies have also found an association between CYP1 inducibility and the occurrence of cancers of the lung, larynx, bronchi and oral cavity, while cancers of the ureter and bladder did not show this correlation [reviewed in: (Nebert et al., 2004;

Ma and Lu, 2007)]. The notion that CYP1A1 induction results in toxicological outcomes in humans is being challenged. Omeprazole is the first clinical drug known to induce human CYP1A levels. Since human CYP1A inducibility is highly variable, this raised concerns regarding the likelihood of unpredictable drug-drug interactions and increased bioactivation of toxic chemicals. In over 20 years of use, this drag has not been associated with cancer occurrence [reviewed in: (Ma and Lu, 2007)]. Whether increased

CYP1A levels are detrimental or beneficial on balance to human health remains debatable.

CYP1A1 can also be induced in the absence of an exogenous ligand. Retinoids act through retinoic acid responsive elements to induce CYP1A1 expression (Vecchini et al., 1994) and may be involved in cross-talk with the AHR. Studies also link protein kinase C (PKC) signaling to elevated CYP1A1 levels [reviewed in: (Delescluse et al.,

2000)].

1.4.2 Hormonal regulation of sexually dimorphic rat P450s

Many rat P450s are expressed in a sexually dimorphic pattern. Evidence of this dimorphism was first observed in rats treated with sodium amytal to induce the anesthetic

40 state. Nicholas and Barron (1932) found a major sex difference in the tolerability of this

drug in rats. Such early evidence of gender differences in drug metabolism is now

supported by mechanistic insights into the hormonal regulation of specific P450 forms.

Sex-specific P450s are evident in only one sex, while sex-predominant P450s are

found in both sexes but are expressed at greater levels in one sex. Microarray analysis

shows that GH is the major regulator of sexually dimorphic gene expression in the rat

liver (Ahluwalia et al., 2004). Sexual differences in the pattern of GH release controls

sex-dependent liver gene expression, including P450s. GH regulates such genes at the

transcriptional level (Sundseth et al., 1992), by various GH-initiated signaling pathways

including MAPK pathways and the Janus kinase (JAK)-signal transducer and activator of

transcription (STAT) pathway. The mechanisms by which GH regulates sex-dependent

hepatic P450 gene expression will be discussed in Section 1.6.2.2.

Agrawal and Shapiro (2001) have extensively studied sexually dimorphic P450

expression and the effect of hypophysectomy (hypx) on these rat isoforms. Hypx is the

removal of the pituitary gland, the master endocrine organ that secretes GH and several

other hormones. This procedure abolishes many sex differences in hepatic gene

expression. Table 1.2 summarizes sexually dimorphic rat P450s and the change in

expression levels following hypx in the male rat. The male pulsatile pattern of GH

release stimulates expression of the male-specific CYP2C11 gene, whose expression is

dramatically decreased following hypx. CYP2A2, CYP3A2 and CYP4A2 are also male-

specific genes whose expression is highest in the presence of the male pattern of GH

secretion but remains high in hypx rats. CYP2C13 is another male-specific P450 which is maximally expressed upon exposure to the male pattern of GH secretion or in the

41 absence of GH. The feminine pattern of continuous GH release suppresses CYP2C11,

CYP2C13, CYP2A2, CYP3A2 and CYP4A2 expression (Agrawal and Shapiro, 2001).

Female-specific CYP2C12 requires the female pattern of GH secretion for optimal expression. Hepatic CYP2C6 is a female-predominant gene, which is expressed in males

TABLE 1.2. Sexually dimorphic P450s in rat liver.

Gene Sex difference Change in expression following hypophysectomy in male rats

CYP1A2 F>M T2-5

CYP2A1 F>M 42

CYP2A2 M tl.25

CYP2C6 F>M t2

CYP2C7 F>M •I to almost undetectable levels

CYP2C11 M 44

CYP2C12 Remains undetectable

CYP2C13 M Maximally expressed

CYP2E1 F>M T<2

CYP3A2 M Tl.5

CYP4A2 M Maintains high expression

The estimated change in expression of each gene in the male rat liver following hypx is expressed as a fold-change in the level of mRNA and/or protein. [Adapted from Kato and Yamazoe, 1993; Agrawal and Shapiro, 2001].

42 at 60% of female levels. Hypx male rats show an induction in CYP2C6 hepatic

expression. CYP2A1 is also female-predominant. Levels of this gene are reduced in

hypx male rats. Another female-predominant isoform is CYP2C7, which is expressed at

levels 3- to 4-fold greater in females than males. CYP2C7 expression is barely detectable

in male hypx rats. Since not all sexually dimorphic P450s respond similarly to GH or

hypx, it is likely that each isoform is regulated by distinct mechanisms. "Neonatal

P450s" are expressed at high levels after birth when GH concentrations are low and the

sexually-differentiated pattern of GH secretion has not yet been established; their

expression declines during puberty. "Pubertal P450" levels increase during puberty when

GH secretion increases at -30 days of age in rats and the sexually-differentiated pattern

of GH secretion emerges [Fig. 1.6; (Kato and Yamazoe, 1993)].

1.5 In vivo reporter gene studies

Our CYP2C11 luciferase reporter constructs were hydrodynamically introduced

into rat hepatocytes in vivo. This technique is used to study gene regulation in the intact

organism rather than in an isolated cell culture system. It was first introduced in the

1990's when direct injection of nucleic acids into skeletal muscle, heart, liver or thyroid

resulted in gene expression at these sites [reviewed in: (Liu et al., 1999; Wolff and

Budker, 2005)]. Such procedures are invasive since they involve direct local

administration of the foreign DNA at the administration site. For many anatomical sites

(e.g. liver), this requires anesthesia, surgery, plasmid injection, and subsequent recovery

from the surgical procedure. To overcome such limitations, systemic administration techniques were sought to replace regional plasmid administration. The use of the tail

vein as the injection site for the rapid delivery of high volumes of DNA-containing

43 "Neonatal P450s" "Pubertal P450s"

c »»..•• """"w.".""" -0^ CYP3A2 © / CYP1A2 (male) / CYP2A1, CYP2B1 / CYP2C7, CYP2C11 CYP2B2 ' CYP2C12, CYP2C13 CYP2E1, CYP2C22 CYP3A2 V / GH not yet / GH is sexually / sexually dimorphic y dimorphic

0 15 30 45 Days after birth

Figure 1.6. Developmental regulation of rat P450s by GH. "Neonatal P450s" are expressed soon after birth and achieve maximal levels within 3 weeks. Many "neonatal P450s" are decreased in expression at puberty but some forms maintain their expression into adulthood (e.g. CYP3A2 in males). The sexually-differentiated pattern of GH secretion emerges during puberty. "Pubertal P450s" appear in the liver at approximately weeks and continue to increase until reaching maximal levels in adulthood. [Adapted from Kato and Yamazoe, 1993].

44 solutions results in high expression levels of DNA uptake and expression in hepatocytes

(Liu et al., 1999; Zhang et al., 1999). This technique is known as the hydrodynamics-

based or the high-volume tail vein (HVTV) injection method. Since the liver is an

important organ in drug metabolism, the hydrodynamics-based approach is an efficient

and convenient tool allowing researchers to study hepatic gene regulation.

When a large volume of solution is injected through the tail vein, it accumulates in the inferior vena cava. Since the tail vein injection rate exceeds cardiac output, the

DNA solution flows backwards from the inferior vena cava to the liver via the hepatic vein, resulting in high levels of plasmid DNA expression in hepatocytes [reviewed in:

(Liu et al., 1999; Budker et al., 2000; Maruyama et al, 2002; Hodges and Scheule,

2003)]. Foreign DNA is predominantly expressed in hepatocytes, but also in sinusoidal endothelial and Kupffer cells (Budker et al., 2006). Although the liver is the major site of transfection, other organs directly linked to the inferior vena cava such as kidneys, lungs and heart can also express reporter gene activity (Liu et al., 1999; Maruyama et al., 2002;

Kameda et al., 2003).

The exact mechanism of DNA uptake into hepatocytes is heavily debated

(Hodges and Scheule, 2003; Budker et al., 2006; Sebestyen et al., 2006). Briefly, the possible mechanisms under consideration are theories which suggest DNA enters via disruptions in the membrane versus entrance of DNA through receptor-mediated endocytosis [reviewed in: (Budker et al., 2000)]. The first hypothesis refers to the direct entry of plasmid DNA into the cytoplasm of hepatocytes by the formation of transient membrane pores or large-scale membrane disruptions. Some researchers suggest the pressure of the high-volume injection causes the fenestrae on the surface of liver

45 endothelial cells to enlarge. Ordinarily the fenestrae are too small to allow the passage of

large molecules such as DNA, but their enlargement facilitates the movement of DNA

through liver endothelial cells to hepatocytes (Zhang et al, 2004a). Zhang's studies

using scanning electron microscopy report the appearance of membrane pores in mouse

liver immediately following hydrodynamics-based injections. These pores reseal

approximately 10 min post-injection. The membrane pore theory explains why

hydrodynamics delivery to hepatoyctes is a non-specific process allowing for the entry of

DNA regardless of size, charge or shape (Sebestyen et al., 2006). Other studies using

light and electron microscopy report the appearance of large vesicles in hepatocytes

within 5 min following hydrodynamics-based injections (Budker et al., 2006). These

studies show the presence of plasmid DNA within the vesicles of swollen hepatocytes.

Since some vesicles appear linked to the extracellular space, it has been suggested that uptake of plasmid DNA may occur during the formation of these vesicles (Budker et al.,

2006). Such a mechanism is consistent with the known hepatic uptake of structurally diverse molecules. A study utilizing transmission electron microscopy demonstrates that hydrodynamics-based injections lead to high levels of fluid endocytosis into hepatocytes

(Crespo et al., 2005). The endocytotic vesicles possess large diameters and some researchers hypothesize that DNA penetrates into hepatocytes through permeable sites within these vesicles (Crespo et al., 2005). Transgene expression is reduced in fibrotic rats, which may be due to collagen formation in the extracellular matrix (Yeikilis et al.,

2006). This may limit fluid movement, minimizing the pressure created by the high- volume injection, which would decrease the occurrence of membrane disruptions and ultimately the entrance of DNA into the cell. A major weakness in theories that support

46 entry of foreign DNA through membrane disruptions is the failure to explain why the

presence of plasmid DNA is largely restricted to hepatocytes.

Other investigators speculate that DNA uptake is an active, receptor-mediated

process. A receptor-mediated pathway is supported by the observation that transgene

expression is a saturable process (Liu et al., 1999; Maruyama et al., 2002; Hodges and

Scheule, 2003; Sebestyen et al., 2006). Co-injection of an excess amount of polyanions

along with radiolabeled plasmid DNA decreases hepatic radioactivity, suggesting

receptors that recognize polyanions (such as DNA), mediate hepatic plasmid DNA uptake

(Kawabata et al., 1995). This theory is supported by the presence of an unidentified 75-

80 kDa oligonucleotide receptor on the surface of hepatocytes which may mediate

plasmid DNA uptake (Vlassov et al., 1994). Foreign DNA is commonly taken up and

expressed in only about half of hepatocytes (Liu et al., 1999), which suggests that uptake

of DNA is a selective process mediated by cell-specific receptors. Others argue this may be due to structural differences within hepatic sinusoids leading to transfection of hepatocytes at accessible locations within the perivenous sinusoids (Zhang et al., 2004a).

Many researchers argue against the receptor-mediated theory since DNA uptake seems to be a non-specific process. Plasmids of all types and sizes are uptaken by hepatocytes including various vectors carrying luciferase, P-galactosidase and green fluorescence protein reporter genes (Zhang et al., 2004a). Although most hydrodynamics-based studies involve the use of plasmid DNA, other smaller macromolecules that range in size and charge such as peptides, fragments from polymerase chain reactions (PCR) and RNA oligonucleotides, can also be uptaken by hepatocytes (Hodges and Scheule, 2003;

Kameda et al., 2003; Sebestyen et al., 2006).

47 Regardless of which mechanism is correct, plasmid DNA enters the cell before it

is substantially degraded by serum or cellular nucleases (Kawabata et al., 1995). The

mechanism by which DNA enters the nucleus remains unknown. The force used to drive

plasmid DNA into the cytoplasm may also drive the plasmid into the nucleus (Budker et

al., 2006). This theory is consistent with the presence of plasmid DNA in the nucleus of

some hepatocytes within 5 min (Budker et al., 2006), and transgene expression as early as

2 h post-injection (Liu et al., 1999).

Although the hydrodynamics-based approach is predominantly performed in

mice, it can also be achieved successfully in rats (Maruyama et al., 2002; Higuchi et al,

2003; Kameda et al, 2003; Maruyama et al., 2004; Gardmo and Mode, 2006; Yeikilis et

al., 2006). HVTV injections in rats or large mice (Budker et al., 2006) are facilitated by

the use of anesthetics. Overall, rats do not tolerate injections as well as mice (Hodges

and Scheule, 2003). Following high-volume injections, rats may have difficulty breathing due to complications associated with anesthesia or fluid entry into the lungs.

Three factors are critical in performing a successful HVTV injection: injection

speed, injection volume and quantity of plasmid DNA injected. An injection speed of

<5 s in mice and <15 s in rats yields highest transfection efficiencies, while injection times exceeding these durations result in markedly reduced foreign DNA expression (Liu et al., 1999; Maruyama et al., 2002; Feng et al., 2004). Without rapid tail vein injection, the DNA will enter the systemic circulation where it is vulnerable to degradation by blood nucleases (Liu et al., 1999). An injection volume equivalent to 8-12% of the animal's body weight is needed for successful transfections (Liu et al., 1999; Maruyama et al., 2002). In rats (body weight 250 g), DNA injected in a volume of 25 mL yields

48 maximal transgene expression, while transfection efficiency increases dose-dependently as the quantity of plasmid DNA injected is increased from 10-800 ug (Maruyama et al.,

2002). An optimal dose of 3 mg of DNA/ kg body weight is suggested for rats (Hodges and Scheule, 2003). Assessment of luciferase activity in mice 8 h following hydrodynamics-based injections, shows a dose-dependent increase in hepatic luciferase activity with increasing amounts of plasmid DNA that saturates at 5 fig (Liu et al., 1999).

Numerous conditions including consciousness, sex, injection volume, injection speed and DNA-carrier fluid can affect the HVTV transfection efficiency (Feng et al.,

2004). The use of Ringer's solution, rather than saline or phosphate-buffered saline

(PBS) as the DNA carrier fluid, increases the efficiency of the injection and results in less liver damage. Since the ionic chemical composition of Ringer's solution is comparable to that of blood, this is suggested to facilitate in vivo transfections. Ringer's solution also contains calcium chloride, which may precipitate foreign DNA allowing for more efficient cellular uptake as shown in vitro. Male mice show higher levels of foreign DNA expression compared to females. This coincides with lower plasma alanine transaminase

(ALT) levels in males, which suggests less hepatic damage results in higher transfection efficiency. The state of consciousness can also affect transfection efficiency.

Anesthetized mice express higher levels of foreign DNA compared to conscious mice.

Feng et al. (2004) speculate that reduced cardiac output resulting from anesthesia, may facilitate movement of the DNA solution from the inferior vena cava to the hepatic vein and ultimately the liver.

Transgene expression is promoter-dependent [reviewed in: (Maruyama et al.,

2002; Al-Dosari et al, 2006)]. A recent study examined the luciferase activity driven by

49 14 different promoters upon in vivo transfection of constructs into mouse hepatocytes.

By 8 h post-injection, it is obvious that luciferase activity varies with the promoter

strength of constructs (Al-Dosari et al., 2006). Mutations in the gene regulatory sequence

may diminish transgene expression (Kameda et al., 2003). Furthermore, the use of a

species-specific gene may prevent a host immune response (Kameda et al., 2003).

Foreign gene transcription can occur fairly soon following in vivo transfections.

Depending on the promoter, luciferase activity can be detected as early as 2 h following

injections and often peaks at 8 h (Liu et al., 1999). Promoter differences will affect the

duration of gene expression as shown by different time courses in a study that introduced

luciferase and rat erythropoietin plasmids to rats by hydrodynamics-based injections

(Kameda et al., 2003). Typically, the levels of proteins resulting from transgene expression peak in <24 h following in vivo transfection [reviewed in: (Rivera-Rivera et

al., 2003)]. A time course study of luciferase activity following hydrodynamics-based injections reveals a two-phase decline in transgene expression (Al-Dosari et al., 2006).

The first phase involves a rapid decrease in luciferase activity which occurs 5-7 days post-injection, while second phase decline is much slower. Southern and Northern blot analyses suggest that the rapid decline within the first phase is due to promoter silencing.

In vivo bioluminescence imaging of animals is an efficient and sensitive way to measure transgene expression at multiple time-points in the same organism. Rats or mice receive an intraperitoneal (i.p.) injection with a suitable firefly luciferase substrate such as luciferin. Animals are then anesthetized and placed on the imager (Zhang et al.,

2004b). Upon oxidation, the substrate emits photons that can penetrate the living tissue and are quantified by an in vivo imaging system. Recently, a study found that the firefly

50 luciferase substrate D-luciferin, may also be a substrate for membrane-bound pumps from the ATP-binding cassette family. The Renilla luciferase substrate coelenterazine is a substrate for the MDR1 P-glycoprotein pump. Administration of these substrates prior to in vivo imaging may result in photon emission that is influenced by the expression/ activity of drug transporters and unrelated to transgene expression (Zhang et al., 2007).

Previously, in situ transfections were used to study reporter genes in vivo

(summarized in Table 1.3). In situ transfection involves direct injection of the desired plasmid into the liver at multiple sites. This approach has been used to identify phenobarbital (PB)-responsive elements within the rat CYP2B1 and CYP2B2 genes (Park et al., 1996; Liu et al., 2001). The involvement of CAR in PB-mediated induction of rat

CYP2B1 has also been examined in situ (Yoshinari et al., 2001). In situ transfection was used to assess the in vivo transcriptional regulation of the mouse Cyp3al6 gene

(Nakayama et al., 2001). Mechanisms involving hormonal regulation of gene expression are best-studied in vivo since endocrine factors are at physiological levels and signaling pathways are intact. Kamataki's group directly injected CYP2C12 reporter constructs into rat liver to elucidate mechanisms of GH-mediated induction of this gene (Sasaki et al., 1999; Endo et al., 2005). Although in situ transfection allows for the study of reporter genes in the living animal, there are disadvantages associated with this technique. Direct injection of plasmid DNA into the liver involves abdominal incision and the placement of clamps around the liver to prevent outflow of the injected solution

[reviewed in: (Hodges and Scheule, 2003)]. Such a procedure is invasive and can lead to low transfection efficiency if the DNA-containing solution leaks out of the liver. These problems do not arise with the hydrodynamics-based method, which results in 40% of

51 TABLE 13. Summary of selected in vivo studies of P450 regulation using in situ or hydrodyanmics-based transfection of luciferase reporter plasmids.

Gene Method of in vivo transfection Xenobiotic treatment Time point(s) studied Conscious state of animal Reference post-injection during gene delivery

Mouse Cyp3al6 In situ transfection into mouse liver No treatment 24 h Anesthetized with ether Nakayama et al. 2001

Rat CYP2B1 In situ transfection into rat liver PB 24 h Anesthetized with ether Yoshinari et al. 2001

Rat CYP2B1 In situ transfection into rat liver PB 24 h Anesthetized with ether Park etal. 1996 Rabbit CYP2C1

Rat CYP2C12 In situ transfection into rat liver recombinant human GH 24h" Anesthetic not specified Endo et al. 2005

Rat CYP2C12 In situ transfection into rat liver recombinant human GH 7 days Anesthetic not specified Sasaki et al. 1999

Rat CYP2B2 In situ transfection into rat liver PB 24 h Anesthetized with ether Liu etal. 2001

Human CYP3A4 Hydrodynamics-based transfection into dexamethasone, MC variable Conscious Schuetz et al.2002 Rat CYP1A1, 2B1 mouse liver rifampin, and others,

Hydrodynamics-based transfection into PB -24 h Conscious Jackson et al. 2004 Mouse Cyp2c29 mouse liver phenytoin

Hydrodynamics-based transfection into garlic oil or 30-42 h Conscious Fisher etal. 2007 Human CYP2B6 mouse liver constituitents

Mouse Cyp2b9 Hydrodynamics-based transfection into PB 24h Conscious Rivera-Rivera et al. 2003 Mouse Cyp2bl0 mouse liver

52 Table 1.3. cont.

Gene Method of in vivo transfection Xenobiotic treatment Time point(s) studied Conscious state of animal Reference post-injection during gene delivery

Human CYP1A2,2C9, Hydrodynamics-based transfection into No treatment 8h-14days Conscious Al-Dosari et al. 2006 2C18, 2D6, 3A4 mouse liver Mouse Cyp2bl0

Q Human CYP3A4 Hydrodynamics-based transfection into dexamethasone 22-88 h Conscious Zhang et al. 2003b mouse liver rifampicin

All chemical treatments were administered at approximately the same time of in vivo transfections unless otherwise indicated. a Rats were administered PB at 4 h following surgery. b Rats were treated with recombinant human GH for 7 days prior to in vivo transfections. c Recombinant human GH was administered 24 h following direct DNA injection into the liver by a continuous infusion for 6 days. d Mice were treated with chemicals for 4 days and sacrificed 24 h later. Tail vein injections were performed on the 4th day of drug treatment. e Mice were treated with garlic oil or constituents 18 h following tail vein injections and imaged 12 h or 24 h later. f Chemical treatment was administered 6 h post- tail vein injection and rats sacrificed 18 h later. g Mice were treated with chemicals 16 h following hydrodynamics-based injections and imaged 6-72 h later.

53 hepatocytes expressing the foreign DNA (Liu et al., 1999; Zhang et al., 2004a), and the

quick recovery of animals following the procedure. Furthermore, expression of the

transgene is occurring at the same time the body is healing from the in situ procedure,

and this may affect the expression of the transgene. In some instances dexamethasone is

administered during in situ surgeries to prevent inflammatory responses at the site of

surgery (Park et al., 1996; Yoshinari et al., 2001; Rivera-Rivera et al., 2003).

Dexamethasone may affect the regulation of the transfected gene. Unfortunately both in

situ and hydrodynamics-based transfections require large quantities of plasmid DNA

(~300 {xg per injection), which can be challenging to generate when using a large number

of rats for in vivo studies.

The hydrodynamics-based injection technique is less invasive and has been used

in recent years to characterize gene regulatory sequences that mediate xenobiotic-

responsiveness. Numerous genes have been studied in living rodents using this

technique, including several P450 genes (summarized in Table 1.3). Plasmids containing

the human CYP3A4, rat CYP1A1 and CYP2B1 genes have been hydrodynamically

introduced to mice. Their responsiveness to xenobiotic treatment and the role of PXR in

CYP3A4 reporter induction were examined (Schuetz et al., 2002). Hydrodynamics-based

injections of Cyp2c29 reporter constructs into mouse hepatocytes was used to

characterize the role of CAR in the phenytoin-responsiveness of this gene (Jackson et al.,

2004). A recent study involving hydrodynamics-based injections in mice reports that induction of the human CYP2B6 promoter by garlic oil constituents is mediated by CAR

and nuclear factor E2-related factor 2 (Nrf2) acting on antioxidant response elements within this gene (Fisher et al., 2007). Transcriptional responses of mouse Cyp2b9 and

54 Cyp2bl0 genes to PB were examined in vivo upon hydrodynamics-based introduction of

reporter constructs to mice (Rivera-Rivera et al., 2003). The human CYP3A4 promoter

was hydrodynamically introduced into mouse hepatocytes, and its response to

dexamethasone or rifampicin measured (Zhang et al., 2003b). To date, all

hydrodynamics-based studies of P450 reporter constructs have been conducted in mice;

this thesis research is the first such study of P450 regulation in rats.

Plasmid DNA introduced by hydrodynamics-based injections is not integrated into the host genome (Kameda et al., 2003). However, transgenic Cypla2-luc and

CYP3A4-\\xc mice were created to study the transcriptional regulation of P450 reporter genes incorporated into the host genome (Zhang et al., 2003c; Zhang et al., 2004b).

Zhang's group challenged these transgenic mice with various xenobiotics to study the response of 13-kb of the human CYP3A4 promoter and 8.4-kb of the mouse Cypla2 promoter. Interestingly, the human CYP3A4 promoter is regulated differently in transgenic mice and rats harboring luciferase reporters (Zhang et al., 2004c). Transgenic mice harboring reporter genes (other than luciferase) driven by P450 genomic regulatory elements have also been studied. PB responsiveness of human GH reporters controlled by the rat CYP2B2 5'-flanking region was characterized in transgenic mice (Ramsden et al., 1999). The spatial and temporal activation of the AHR by TCDD was studied in transgenic lacZ reporter mice harboring a synthetic promoter containing two DREs

(Willey et al., 1998). Induction of human CYP3A4 has been studied extensively in transgenic lacZ reporter mice (Robertson et al., 2003).

Systemic administration of foreign DNA may have additional experimental and, ultimately, clinical therapeutic applications [reviewed in: (Hodges and Scheule, 2003)].

55 Hydrodynamics-based injections can be used as an efficient alternative to the creation of

knockout or transgenic mice since siRNA can be delivered by this technique to decrease

the expression of a desired gene. This method can be used to knock down tissue-specific

gene expression as opposed to knockout mouse models, which eliminate the expression

of a gene in all tissues (Hodges and Scheule, 2003). Furthermore, hydrodynamics-based injections of siRNA targeting genes whose deletion results in embryonic lethality is a useful substitute for the creation of conditional knockout mice.

1.6 CYP2C11

1.6.1 CYP2C11 5'-flanking region and promoter

The proximal 2.3-kb of the CYP2C11 5'-flanking region was the first part of this gene to be cloned and characterized (Morishima et al., 1987; Yoshioka et al., 1987; Strom et al., 1994). CYP2C11 has a single start site for transcription as shown by two independent laboratories (Morishima et al., 1987; Strom et al., 1994). CYP2C11 is located along a 35-kb stretch of genomic DNA on rat chromosome 1. Isolation of

CYP2C11 cDNA clones reveals a single open reading frame containing an ATG codon identifying the start of translation and a 3'-poly(A) tail (Yoshioka et al., 1987). Yoshioka et al. (1987) found that the transcribed message consists of 1,853 nucleotides in addition to a poly (A) tail. The migration of CYP2C11 mRNA on formaldehyde-agarose gels is consistent with a size of -2.1 kb. This gene contains 9 exons, which encode a polypeptide containing 500 amino acids (Morishima et al., 1987), with a predicted molecular weight of -57 kDa (Yoshioka et al., 1987). The migration of CYP2C11 protein on denaturing polyacrylamide gels is consistent with a molecular weight of -50 kDa. Gunn rats express a variant form of CYP2C11 cDNA, in which three of the total

56 500 amino acids are mutated. This results in 90% decreased CYP2C11 enzymatic

activity with no corresponding changes in apoprotein expression (Biagini and Celier,

1996). The half-life of CYP2C11 mRNA in primary rat hepatocytes is between 9.8 h

(Iber et al., 2001) and 16 h (Bhathena et al., 2002), while the in vivo protein half-life has

been estimated at 20 h (Shiraki and Guengerich, 1984).

A summary of selected putative TF binding sites present in the proximal 1.8-kb of

the CYP2C11 5'-flanking region is shown in Fig. 1.7. A TATA box sequence is located

29-bp upstream of the transcriptional start site and a GC- and purine-rich region is found

within the proximal 700-bp of the CYP2C11 5'-flank (Morishima et al, 1987). A putative GRE exists between -149 and -144-bp of the 5'-flanking region (Morishima et al., 1987). Two negative regulatory regions located at -1244 to -1202-bp (SIL1200) and

-395 to -354-bp (SIL400) on the CYP2C11 5'-flank can reduce the activity of a reporter construct under the control of a heterologous promoter (Strom et al., 1994). Strom's group speculated that de-repression of the silencer elements by GH may induce CYP2C11 transcription. Using electrophoretic mobility shift assays (EMSA), this group also identified a region between -103 and -91-bp, which shows reduced protein binding in nuclear extracts from male hypx rats treated with the male pattern of GH secretion compared to hypx rats administered continuous GH or no GH treatment (Strom et al.,

1994). This particular sequence is similar to the hepatocyte nuclear factor-1 (HNF-1) consensus binding element, although the protein bound to this region is not HNF-1 a.

EMSA shows that STAT5b binds to the CYP2C11 5'-flank at positions -1169 to

-1161-bp (Park and Waxman, 2001; Timsit and Riddick, 2002); a finding recently confirmed by ChIP analysis (Thangavel and Shapiro, 2007). The exact role of this

57 -1827 AGG AGGCACAGCC TTATTTGAAA GAAAAAGCAA CTGGCATAAA GTGGTGGATT

-1774 ATGTCACTTT GTGTGATGGT AGTAATCACT TAATTATGTA AACCAGAGTA TCCTATTGCA

-1714 CACCTTAAAT GTAGGCAATA AAAGTAAAAC TTTAAGCAGT AAAAAATACT GTAGAGGCCA NF-KB DRE DRE -1654 CCGCCATGCC ATCCAGACTG AGGAAGACCC GGAAAWfcG GGGCBBJTG ifcbCACAGCp I DRE

-1594 | ACGGTOpCAT TGGTAAGCAC CGCAAGCACC CAGGAGGCCG CGGGAATGJST GGAGGjjjgiC |

-1534 | AAJTTACCACA GGATCAACTT TGACAAATAT CATCCTGGTT ACTTTGGCAA AGTCGGCATG

-1474 AGTCATTACC ACTTGAAGAG GAGCCAGAGC TTCTGCCCAA CTGTCAACCT GGATAAAT§|g HNF-3 DRE DRE

-1414 TGGACGTTGG TCAGCpAtSfcA ^AiSA6atf<3Jp AAfrSqhGCAA AAAACAAGAG TGGAGCTGCT

-1354 CCCATCATTG ATGTTTTTCC AATCAGGCTA CTACAAAGTT CTGAGGAAGG GGAAGCTTCC

-1294 TAAGCAACCT GTCATCGTGA AGGCCAAATT CTTCAGCAGA AGAGCTGAAG SIL 1200 -1234 GGGTGTTGGA GGTGCCTGT-T'CTGGTGGCTT AAAGTCACTT CAGAGGTTAA TTAAATGCAA STAT5 -1174 ACATTTTCCA TGAAAAAAAA ATATTGTAGA AATGAATTAG CAGTTAAGAG CACTGGCTGC

-1114 TCTTCCAGAG AATCTCACCT GTTTGTAACT CTAGTTCCAC AGCTTCTGAG ACCCTCACAC

-1054 AGACATACAT GGAGGCAAAA CAAGGTTGAA CTATAAGATA AAAAACCAGT GTGGTGTATA

-994 TCCCAAGTGA TGTCTGTGAG TGGCATTATG ACCATTCTGT TATCCCGCAT TCTCTGCTAA

-934 AAGGGCTTTG TATGCAAAAG GTACCTTTAT TATATGAGCT AATTACATGA CTTAATTGGT

-874 GAGTAATCAT GGCTAATATT ACTATAGCAT GCTAAAGGAA GTATCCTAAC AACTGGGAAG

-814 ATTTACTGTG TAAACATTAC TTTATTTACA AGTATATGGT TTACCTTCAC TGCTAAAACT

-754 CAGTCTAAGA ATGCAATGAT CTTTGGATAA CATGATTTAC TAGATTAGCC CTGAGTTTAG

-694 ATTGTATGTT GAAACTCTCT GGGGTATTTG AAAAAAAGAA AAAAAAAGAC AGTAAATGAG

-634 TGATATGGGA GGGGGTGCCT TAGTTGGCTC ATGCTGAGGC ACACCTGCCC ACATCTCCCC

58 -574 CTTCTCCCCT CCCCCGAGGT ACCAGCCATA TATGGGTCTA GTATAAAAGA GAGCTTATTT

-514 GGAGGCATGG GAAAGGGGGT TGAGAAGGAA GTTGAGGCAG AGAAAGAGAA GTACAGAGAG

-454 AGAGAGAGGA AAGAAAGGAG AAGAGGCAGG CCAGGAATAT GTGGAGAGGA TGAGAGAGGJj SIL 400 -394 .."- ',"-.:>r\A v.-•: 3i J-IGAGGOA :\GGGAGAGA AGGAGAGAAG

-33 4 GAGAGAGGGA GAGAGAGAGA GGAAGGTTAG AGAGCAAAGA GAGGCTAGTC ATTTTTATGG

-274 CAAGCCAGGC TCTTGTCTGG ATGTTGCTAT GTAACAGTTG GGTAGAGCCA AGAAGGAATG

-214 CTAAGAAGCT AACTTTGGGT CCCACCCCTG GTTAGTTACA CAGATTTGTG GTAGTAGAAA

GRE HNF-1-Iike -154 GAGGAAGAAC'AGTTTTCACT TGTGTATTTT AACAGGTCAG GGTCCACAAA G|gjj|j|jj^ PP-responsive region HNF-3-like -94 TAAAGCATAT CTAQTigktf eGtCACXXUS-GTATCAGAAG CTCATjgJTJ*A%r#§3&%CT

TATA box NF-KB -34 AGCAT%A3S&-AAGTCCTGGA CAGCAAGCTC ACAGdSA^lW r-tGCrTGAGAAG GCTGCCiSP +1

Figure 1.7. Nucleotide sequence of the CYP2C11 5'-flanking region. Putative binding sites for the indicated transcription factors are depicted by grey highlighting. DREs are surrounded by a box and the core nucleotides required for AHR/ARNT binding are highlighted in grey. Nucleotides within the "transcriptionally active" region between -1827 and -1303-bp are bolded in black. Most transcription factor binding sites within this region are either putative sites as determined by bioinformatics analyses or confirmed binding sites of unknown function; an exception is the DRE3 located between -1546 to -1533-bp, which was shown to be functional by site-directed mutagenesis in the current thesis research. Numbering of nucleotide positions is according to version 3.4 of the November 2004 rat genomic assembly found on the UCSC Genome Browser website [http://genome.ucsc.edu/cgi-bin/hgGateway], relative to +1 denoting the G residue of the transcription initiation site. The ATG highlighted in grey (+23) indicates the translational start site.

59 STAT5 element in the GH control of CYP2C11 expression remains unclear, but it seems

that STAT5b may act as a component of a cascade involving other liver-enriched TFs

(e.g. HNFs). Co-transfection of CYP2C11 reporter constructs with STAT5 and HNF-3p

expression plasmids results in inhibitory cross talk between both TFs, a response that

may involve protein-DNA interactions at HNF binding sites located near the STAT5

element (Park and Waxman, 2001).

Our laboratory has previously identified several DRE-like sequences located in

the CYP2C11 5'-flanking region, one of which (CYP2C11-DRE3, -1546 to -1533) can

bind the activated AHR with relatively high affinity (Bhathena et al., 2002). Evidently,

the CYP2C11 5'-flank is enriched with TF binding sites that enable this gene to respond

to a wide range of xenobiotics, inflammatory mediators and endogenous hormones.

1.6.2 Physiological CYP2C11 regulation

CYP2C11 is the major male-specific constitutive P450 present in the rat liver. In past papers it has been referred to as: P-450 Ml, P-450 2c, P-450h, P-450 UT-A and

P-450-RLM5 [reviewed in: (Yeowell et al., 1987)]. It is essentially the only enzyme that can hydroxylate testosterone in the 2a position, and accounts for ~ 90% of testosterone

16a- hydroxylation in liver microsomes from untreated male rats (Waxman, 1988;

Biagini and Celier, 1996). Recombinant CYP2C11 in heterologous expression systems can hydroxylate vitamin D (Rahmaniyan et al, 2005) and convert arachidonic acid into epoxide metabolites that are important in vascular and renal regulation (Barbosa-Sicard et al., 2005; Yu et al., 2006).

CYP2C11 immunoreactive protein has been identified in microsomes isolated from extrahepatic tissues such as the lung, kidney and to a smaller degree the testes

60 (Ryan et al, 1993). The highest levels of CYP2C11 are found in liver tissue at ~2 nmol/mg microsomal protein (Biagini and Celier, 1996). Recently, CYP2C11 protein and mRNA expression have been identified in spleen, thymus and bone marrow tissue in male rats (Thangavel et al., 2007). The expression of this gene in these hematopoietic tissues is sexually dimorphic and GH-dependent. Transcript levels in the spleen and bone marrow are greater in males than the low levels in female rats. Hypx does not affect splenic CYP2C11, but increases CYP2C11 expression in bone marrow and induces a sexually dimorphic expression of this gene in the thymus that is not observed in intact rats. This suggests differences in the mechanism(s) of CYP2C11 regulation by GH in the liver and hematopoietic tissues.

1.6.2.1 Onset of CYP2C11 expression at puberty

Numerous studies in hypx and castrated rats have enhanced our understanding of the physiological regulation of CYP2C11. Expression of this enzyme is relatively low until puberty (Waxman et al., 1985). CYP2C11 is expressed in males at 30 days of age and dramatically increases until it reaches adult levels at -54 days (Gabriel et al., 1992).

Androgen production in neonatal male rats establishes the male pattern of GH secretion that is responsible for inducing CYP2C11 at puberty (Morgan et al., 1985a; Waxman et al., 1985; Dannan et al., 1986). Rats castrated at birth do not express CYP2C11 in later life; however, CYP2C11 expression is intact if castration occurs 10 weeks after birth

(Waxman et al., 1985), suggesting neonatal androgen imprinting is critical for normal expression of this P450 at puberty. Aromatic hydrocarbon administration during neonatal development results in P450 expression changes in the adult rat. TCDD exposure during the neonatal period in male rats alters testosterone metabolism, which modifies hormonal

61 imprinting and results in over-expression of CYP2C11 at puberty (Ishizuka et al., 2003).

Rats injected with a single dose of B[a]P as neonates display higher CYP2C11 catalytic activity and protein levels than control adult rats (Fujita et al., 1995).

Androgens also have effects beyond the neonatal period that alter CYP2C11 levels. Dannan et al. (1986) reported that testosterone injections given to post-pubertal male rats castrated at birth increase CYP2C11 expression regardless of neonatal exposure to testosterone. This indicates that neonatal androgen imprinting is not the only mechanism responsible for adult CYP2C11 expression. Exposure of female rats to testosterone during puberty increases CYP2C11 mRNA levels even after testosterone treatment is discontinued (Chang and Bellward, 1996). Treatment of ovariectomized female rats with a synthetic testosterone derivative during puberty leads to elevated levels of CYP2C11 protein and catalytic activity, which remain elevated 41 days after treatment is ceased (Anderson et al., 1998).

Circulating androgens maintain the expression profile of CYP2C11 in adult life

(Morgan et al., 1985a). Treatment of adult male rats with the anti-cancer drug cisplatin, which is known to decrease serum testosterone levels, down-regulates CYP2C11 protein expression and catalytic activity to 10-21% of intact levels (LeBlanc and Waxman,

1988). Overall, the effects of androgens on CYP2C11 expression appear mainly indirect, acting through hypothalamic-pituitary signaling pathways to modulate GH secretion

(Morgan et al., 1985a).

1.6.2.2 Mechanisms involved in GH transcriptional regulation of CYP2C11

GH is a polypeptide hormone produced in somatotrophs found in the anterior pituitary gland. The hypothalamus controls the GH pathway by secreting GH releasing

62 hormone (GHRH), which stimulates pituitary GH release, or somatostatin, which inhibits this process (summarized in Fig. 1.8). Androgens participate in GH regulation by acting on the hypothalamus [reviewed in: (Wiwi and Waxman, 2004)]. GH is secreted from the pituitary gland in a sexually dimorphic fashion. In adult male rats, it is secreted in pulses separated by 3.5-4 h time intervals, resulting in peak plasma GH levels 200-300 ng/mL and interpulse troughs where plasma GH concentrations are at or below limits of detection. In adult female rats, GH is secreted in a more continuous pattern, resulting in plasma concentrations of 15-40 ng/mL. Expression of the male-specific CYP2C11 gene is stimulated by the male pattern of GH release. To achieve maximal CYP2C11 expression, an intact pituitary is required since expression of this P450 is markedly reduced in hypx male rats (Yamazoe et al., 1986; Agrawal and Shapiro, 2001). Although all pituitary hormones are affected by hypx, studies in dwarf rats and monosodium glutamate (MSG)-treated rats provide evidence that the absence of GH alone is responsible for CYP2C11 suppression following hypx. Dwarf rats have no measurable

GH concentrations, but display intact levels of all other pituitary hormones. CYP2C11 expression in dwarf rats is only 30% of levels found in Sprague-Dawley rats (Shimada et al., 1997), which is consistent with residual CYP2C11 expression following hypx (Verma et al., 2005). Exposure of dwarf rats to the male pattern of intermittent GH secretion recovers CYP2C11 expression. Neonatal MSG administration selectively decreases GH levels in the adult rat without affecting other pituitary hormones. Neonatal MSG treatment decreases CYP2C11 expression in adult male rats (Waxman et al., 1990).

Administration of exogenous GH to hypx male rats can restore intact CYP2C11 expression. Restoration of 5% the normal amplitude of the male characteristic GH

63 Hypothalamus

+ GHRH + Somatostatin Androgens j i Pituitary Gland / X 3 Male rat $ Female rat

GH plasma levels

Time Time

\ /

CYP2C11

Liver

Figure 1.8. Schematic representation of the endocrine regulation of hepatic CYP2C11 expression. The hypothalamus secretes GH releasing hormone (GHRH), which stimulates pituitary GH secretion, and somatostatin, which inhibits GH release. Upon stimulation, the hypothalamus will secrete the sex-specific GH profile which induces hepatic CYP2C11 expression in males, and suppresses this gene in females. The testes secrete androgens required for neonatal imprinting of the GH profile which induces CYP2C11 in males at puberty. Androgens are also involved in the maintenance of CYP2C11 expression in the adult rat. [Adapted from Riddick et al., 2003].

64 profile to hypx rats can restore CYP2C11 to intact levels (Agrawal and Shapiro, 2000).

The same study showed that restoration of only 2.5% the normal GH amplitude to male hypx rats can elevate CYP2C11 expression to 50% of intact levels. This may be possible since GH at 5 ng/ml can saturate GH receptors (GHRs) in the liver (Leung et al., 1987).

Re-introducing exogenous GH to hypx male rats in a pattern that shortens the interpulse interval to 1.6 h is ineffective in restoring CYP2C11 levels. This reveals that it is the length of the interpulse period between GH pusles that is critical in effectively inducing normal CYP2C11 expression, even if the amplitude of the pulse is diminished (Agrawal and Shapiro, 2001). In senescent male rats, the length of the interpulse period is shortened, which leads to the down-regulation of male-specific P450s and induction of female P450s (Dhir and Shapiro, 2003).

Nuclear run-on assays using nuclei isolated from primary rat hepatocytes and rat liver tissue demonstrate that GH regulates CYP2C11 expression at the transcriptional level (Legraverend et al., 1992; Sundseth et al., 1992). In vitro footprinting analyses showed the presence of protein-DNA interactions on the CYP2C11 5'-flank that display sex- and GH-dependence (Sundseth et al., 1992); however, the precise role of specific

TFs in the physiological regulation of CYP2C11 is not established. The pulsatile pattern of GH secretion is the main physiological signal regulating CYP2C11 expression, and this regulation occurs at the transcriptional level at least in part by the JAK2/STAT5b pathway (Waxman and O'Connor, 2006).

The mammalian STAT family of TFs consists of seven members that respond to various stimuli including cytokines, interleukins (ILs) and epidermal growth factor

(Clodfelter et al., 2006). STAT1, STAT3 and STAT5 are all responsive to GH, but

65 STAT5 mediates the effects of GH on male-specific gene transcription through the

JAK2/STAT5b pathway. Both STAT5a and STAT5b are activated by the male GH profile (Choi and Waxman, 1999; Gebert et al., 1999), but knockout studies in mice show that STAT5b is responsible for maintaining sexually dimorphic hepatic gene expression in males (Udy et al., 1997). The JAK2/STAT5b pathway is summarized in Fig. 1.9

[reviewed in: (Waxman and O'Connor, 2006)]. A single molecule of GH binds to a GHR dimer located at the cell membrane. This leads to activation of the receptor-associated tyrosine kinase JAK2, which is phosphorylated at tyrosine sites. In turn, JAK2 phosphorylates the cytoplasmic domain of the GHR at tyrosine residues. STAT5b binds to these residues through its Src homology 2 (SH2) domain, and is then phosphorylated by JAK2. Tyrosine-phosphorylation enables the formation of STAT5b homodimers, which translocate to the nucleus and bind to interferon-y-activated site (GAS) consensus elements within gene regulatory sequences.

Mechanisms involved in the deactivation of this pathway following the GH pulse have been investigated (Gebert et al., 1999). Termination of STAT5b nuclear activity is achieved by dephosphorylation via phosphotyrosine phosphatases, which results in the relocation of STAT5b to the cytoplasm where it awaits the next GH pulse. Likewise, the

GHR/JAK2 complex is deactivated by dephosphorylation, but also through an additional pathway involving the inhibitory proteins: suppressors of cytokine signaling (SOCS) and cytokine inducible SH2 protein (CIS). SOCS and CIS proteins form a group of inhibitory molecules that are induced by cytokines and the GH pulse (Matsumoto et al., 1997;

Davey et al., 1999). STAT5 binding sites located in the 5'-flanking region of the CIS gene are thought to contribute to the induction of this protein by GH. In vivo studies

66 Male pulsatile GH secretion

Figure 1.9. Schematic diagram of GH-activated signaling leading to CYP2C11 transcriptional regulation. GH binds to GH receptor dimers. JAK kinases associated with the GH receptor are then activated and phosphorylate the receptor (phosphorylation is symbolized by a yellow circle). Phosphorylation recruits STAT5b to the receptor. STAT5b proteins are then phosphorylated by JAK, form dimers and translocate to the nucleus where they bind their consensus binding sequence in target genes including CYP2C11. The exact mechanism(s) of HNF activation by GH are unknown. GH can negatively regulate HNFs by C/EBPa (CCAAT/enhancer binding proteins). HNF-40C positively regulates C/EBPa and HNF-la, while negatively regulating HNF-3P (Wiwi and Waxman 2004). HNF-la and HNF-3P are known to bind their responsive elements located in the CYP2C11 5'-flanking region. [Adapted from Park and Waxman 2001 • Wiwi and Waxman 2004].

67 further support a role for STAT5 in the mRNA induction of selective SOCS family members (Davey et al., 1999). The SOCS/CIS proteins inhibit GH signaling by inactivating JAK2 and competing with STAT5b for GHR binding, which may signal proteasome-dependent degradation of the GH/GHR/JAK2/CIS complex [reviewed in:

(Thangavel and Shapiro, 2007)]. CIS over-expression decreases phosphorylation of

STAT5 (Matsumoto et al., 1997), which ultimately prevents STAT5 from entering the nucleus and modulating gene transcription. SOCS proteins can interact with JAK kinases to suppress their activity, thus preventing STAT5 phosphorylation (Naka et al., 1997). A recent study shows that only male hepatocytes display increased SOCS/CIS mRNA levels after the GH pulse, while females persistently express these proteins due to the constant presence of GH (Thangavel and Shapiro, 2007). This may contribute to the absence of CYP2C11 in females since JAK2/STAT5 signaling may be constantly muted, while in males this pathway cycles between activation and deactivation with each GH pulse.

STAT5b binding sites are found in the promoters of several rat male-specific genes including CYP2C11, CYP2A2 and CYP4A2 (Park and Waxman, 2001). StatSb-mOl mice lose sex-dependent liver P450 expression (Udy et al., 1997), which supports a critical role for STAT5b in the regulation of sexually dimorphic P450s in the male rat liver. Recently, the degree to which CYP2C11 hormonal regulation depends on the

JAK2/STAT5b pathway has been questioned (Murray et al., 2005; Verma et al, 2005;

Mode and Gustafsson, 2006; Wauthier et al., 2006a). STAT5 activity contributes to

CYP2C11 expression since peri-pubertal rats display increased STAT5 activity when the pulsatile pattern of GH begins and CYP2C11 is first expressed (Choi and Waxman,

68 2000). This indicates that STAT5 activity is linked to the onset of CYP2C11 expression

following the establishment of the male characteristic GH pulse. However, this study

shows that STAT5 activation is similar in pre- and post-pubertal hypx rats following exogenous male-characteristic GH administration, although CYP2C11 is only expressed in post-pubertal rats. This indicates that additional factors normally absent in pre pubertal rats contribute to the onset of CYP2C11 following the GH pusle. Verma et al.

(2005) report that hypx male rats express ~ 25-35% of normal CYP2C11 levels despite no detectable JAK2 or STAT5b activation. This group also showed that restoration of

GH at 10% of the normal amplitude to hypx male rats induces CYP2C11 expression without activating JAK2, although STAT5b is activated. This suggests that the

JAK2/STAT5b pathway alone is not sufficient for inducing GH-activated CYP2C11 transcription. Similarly, dwarf rats, which carry a point mutation in their GH gene, express 30% of intact CYP2C11 levels despite having no detectable GH in the pituitary gland (Shimada et al., 1997). Furthermore, decreased CYP2C11 levels are found in senescent rats compared to young rats, despite no detectable changes in phosphorylated

STAT5b protein or nuclear translocation (Wauthier et al., 2006b). However, the authors in this study did not assess STAT5-DNA binding activity, which may be altered.

On the other hand, numerous studies support a key role for the JAK2/STAT5b pathway in mediating GH-regulated CYP2C11 transcription. Treatment of hypx female rats with the male GH profile induces CYP2C11 expression to 60-65% of male levels

(Dhir et al., 2007). This coincides with lower levels of activated JAK2 and STAT5b, and

50% less STAT5b binding to the CYP2C11 5'-flank, suggesting that JAK2/STAT5b contributes to GH's transcriptional control of CYP2C11.

69 MAPK signaling may contribute to CYP2C11 regulation by GH since female rats display lower levels of activated MAPK (Dhir et al., 2007). Verma et al. (2005) report a

2- to 3-fold elevation of nuclear MAPK levels following restoration of 10% of the physiological GH pulse amplitude to hypx rats. This GH dose can induce CYP2C11 expression without activating JAK2.

It is not clear whether STAT5b regulates P450s by direct transcriptional effects or indirectly through cross-talk with transcriptional activators and repressors involved in

P450 regulation (Wauthier et al., 2006a). P450 promoters also contain consensus binding sequences for numerous HNFs including HNF-la, HNF-3, the orphan nuclear receptor

HNF-4a and HNF-6 [reviewed in: (Akiyama and Gonzalez, 2003; Wiwi and Waxman,

2004)]. HNFs, while not solely confined to the liver, are highly expressed in liver tissue and regulate gene expression during development by binding to their consensus sequences located within gene regulatory regions (Park and Waxman, 2001). HNFs can bind to P450 genes including CYP2C11, which is partly regulated by HNF-la and HNF-

3p (Park and Waxman, 2001), and CYP2C12, which is regulated by HNF-3P, HNF-4 and

HNF-6 (Sasaki et al., 1999; Delesque-Touchard et al., 2000; Endo et al., 2005). GH- activated STAT5b does not regulate CYP2A2 expression; however, multiple HNFs can act to either activate or repress transcription of this gene (Wiwi and Waxman, 2005).

Liver-specific deletion of HNF-4a in mice results in the down-regulation of sex- dependent male Cyp expression (Wiwi et al., 2004; Holloway et al., 2006). HNFs can also act alongside STAT5 to modulate GH-mediated transcription as observed for

CYP2C11 (Park and Waxman, 2001), and mouse Cyp2d9 (Wiwi and Waxman, 2005) regulation. Regulation of CYP2C11 by GH is likely mediated by a combination of direct

70 and indirect actions involving the JAK2/STAT5b pathway and HNFs (Holloway et al.,

2006; Wauthier et al., 2006a; Waxman and O'Connor, 2006).

The role of GH in CYP2C11 regulation is also evident when one examines the cultured rat hepatocyte model. Upon culturing hepatocytes on matrigel, endogenous

CYP2C11 mRNA levels drastically decline within the first day of culture. By 4-5 days in culture on matrigel, CYP2C11 expression is stabilized to <25% of intact levels

(Legraverend et al., 1992; Liddle et al., 1992). In the past, administration of GH pulses directly to hepatocytes has proved unsuccessful in restoring constitutive CYP2C11 expression (Liddle et al., 1992). It was not until recently that pulsatile administration of species-specific GH was shown for the first time to induce CYP2C11 to intact levels

(Thangavel et al., 2006).

Thyroid hormones may also contribute to CYP2C11 regulation. Hypothyroidism decreases CYP2C11 mRNA levels by up to 80%, although this suppression is not reversed by thyroxine (T4) replacement, indicating that suppression was not a direct result of thyroid hormones (Waxman et al., 1991). The authors suggest that CYP2C11 suppression may result from altered GH levels that occur secondary to this condition. In primary rat hepatocytes, triiodothyronine (T3) administration suppresses CYP2C11 mRNA levels by 50%, while concurrent treatment with GH results in additional

CYP2C11 suppression (Liddle et al., 1992).

In a wider context beyond CYP2C11, microarray studies in rats and mice have clearly established that sex-dependent liver gene expression is extensive, GH is the major determinant of sexually dimorphic gene expression, and GH's effects are primarily

71 mediated by STAT5b at the transcriptional level (Ahluwalia et al, 2004; Clodfelter et al.,

2006).

1.6.2.3 CYP2C11 expression and pathophysiological states

Diet, nutrition and disease state are known modulators of cytochrome P450s

(Yang et al., 1992). Ischemic preconditioning increases CYP2C11 protein and mRNA

levels in the brain (Liu and Alkayed, 2005). These authors show that hypoxia-activated

HIF-la contributes to the induction of CYP2C11 through binding to HREs located in the

CYP2C11 5'-flanking region as shown by EMSA. The epoxidation of arachidonic acid to

epoxyeicosatrienoic acids by CYP2C11 can protect against hypoxia (Yu et al., 2006).

Stress can modulate CYP2C11 expression as shown by altered levels of this P450

in response to glucocorticoids. Adrenalectomy suppresses hepatic CYP2C11 mRNA

levels, which suggests a positive role for glucocorticoids in the regulation of this gene

(Liddle et al., 1992). CYP2C11 mRNA levels in hepatocytes respond to glucocorticoid treatment in a biphasic manner. Low concentrations of dexamethasone induce

CYP2C11, while high concentrations (>10~7 M) suppress CYP2C11 expression (Iber et

al., 1997). This study shows a similar trend upon administration of the endogenous glucocorticoid corticosterone. The CYP2C11 response to glucocorticoids is reversed upon administration of the glucocorticoid receptor antagonist RU486. Since Liddle et al.

(1992) demonstrate that administration of dexamethasone (1 nM) increases CYP2C11 mRNA levels in primary rat hepatocytes, it is likely that glucocorticoids regulate

CYP2C11 by a direct transcriptional mechanism that can occur in an isolated culture system. A direct transcriptional mechanism is supported by the presence of a putative

72 GRE located in the CYP2C11 5'-flank (Morishima et al., 1987), although the functional

importance of this element remains unknown.

Diet also affects CYP2C11 levels. Under fasting conditions, CYP2C11 activity is

suppressed compared to fed rats as measured by hydroxylation of the non-steroidal anti

inflammatory flurbiprofen (Shimizu et al., 2003). CYP2C11 protein and mRNA levels

are down-regulated in streptozotocin-induced diabetic rats (Donahue et al., 1991). GH

and insulin can restore CYP2C11 expression to varying degrees, which suggests both

GH-dependent and -independent mechanisms are involved in the suppression of this gene during the diabetic state. In primary rat hepatocytes, glucagon, which is elevated in diabetic rats, can suppress CYP2C11 mRNA levels through the intracellular messenger cyclic AMP (Iber et al., 2001). Sodium intake can also modulate CYP2C11 catalytic activity and protein expression (Liu et al., 2003). Rats consuming a zinc-deficient diet during development have decreased levels of CYP2C11 protein, mRNA and catalytic activity post-puberty (Xu et al., 2001). Intake of a vitamin A-deficient diet in rats decreases testosterone 16a-hydroxylation and CYP2C11 protein levels by 40% and 20% from control levels, respectively (Martini and Murray, 1994). These authors found that the introduction of excess vitamin A into the diet did not reverse this effect.

Cholestasis is a condition associated with impaired bile secretion and the accumulation of bile acids in the liver. Li rats this condition can be modeled by bile duct ligation. Bile duct ligation dramatically suppresses CYP2C11 protein, mRNA and catalytic activity, perhaps as a result of increased estradiol serum levels in these rats

(Chen et al., 1995b). Microvesicular steatosis is the accumulation of lipid in hepatocytes.

73 Ingestion of orotic acid can mimic this condition in the rat, and results in suppression of

CYP2C11 mRNA at a pre-translational level (Su et al., 1999).

1.6.2.4. CYP2C11 regulation by inflammatory cytokines

Inflammation leads to the activation of macrophages which produce pro

inflammatory cytokines that function to regulate the inflammatory response

(Monshouwer and Hoebe, 2003). Cytokines interact with cell surface receptors to

mediate cellular and molecular inflammatory reactions. Numerous P450s including

CYP2C11, are down-regulated at the pre-translational level during inflammation, a

phenomenon mediated by both cytokines and endotoxins. Bacterial lipopolysaccharide

(LPS) is a well-characterized model of inflammation which stimulates the release of pro inflammatory cytokines by macrophages and monocytes. Injection of rats with LPS decreases CYP2C11 mRNA to 20% of control levels (Iber and Morgan, 1998), due to decreased CYP2C11 transcription as shown by nuclear run-on analysis (Cheng et al.,

2003).

Since the inflammatory response caused by activated cytokines may result in the release of reactive oxygen species and nitric oxide [reviewed in: (Morgan et al., 2008)], the role of nitric oxide in the suppression of CYP2C11 by inflammatory cytokines has been investigated. LPS and IL-1 treatment suppress CYP2C11 mRNA levels even in the presence of a nitric oxide synthase inhibitor, indicating suppression occurs by a nitric oxide-independent mechanism (Sewer and Morgan, 1998). A few years later, it was determined that a combination of several inflammatory cytokines mediate the down- regulation by stimulating NF-KB binding to a DNA element flanking the CYP2C11 transcriptional start site (Iber et al., 2000). This binding element also mediates

74 suppression of CYP2C11 reporter constructs in primary rat hepatocytes treated with IL-1.

EL-6 can suppress CYP2C11 reporter gene activity (Chen et al., 1995a; Iber et al, 2000), but unlike IL-1 or LPS, does not utilize the NF-KB responsive sequence (Iber et al.,

2000). Binding of NF-KB to its consensus sequence may interfere with the assembly of the transcriptional initiation complex or may recruit co-repressors which interfere with transcriptional activation (Iber et al., 2000).

NF-KB is a TF which is involved in the regulation of many genes, including several that regulate cell proliferation (Lu et al., 2004b). It is comprised of two subunits: p50 and p65, and is sequestered in the cytoplasm in complex with the NF-KB inhibitory factor (IKB) [reviewed in: (Iber et al., 2000)]. Upon activation by inflammatory cytokines, the inhibitory protein is degraded and NF-KB released. Upon release, this TF can enter the nucleus and bind to its consensus DNA sequence thus altering gene expression (Li and Stark, 2002). TCDD treatment can activate the AHR to form a complex with the p65 subunit of NF-KB, sequestering the complex in the cytoplasm (Tian et al., 1999). This allows p50 homodimers to enter the nucleus where they generally repress gene transcription, as observed in the down-regulation of CYP2C11 by NF-KB

(Iber et al., 2000).

IL-ip treatment leads to sphingomyelin hydrolysis which results in the production of ceramide and sphingosine in hepatocytes (Sewer and Morgan, 1997; Merrill et al,

1999). This pathway is implicated in CYP2C11 mRNA down-regulation following IL-ip treatment in primary rat hepatocytes, which may be attributed to alterations in AP-1 binding. Evidently, inflammatory cytokines suppress CYP2C11 expression by numerous mechanisms that are cytokine-specific.

75 A recent study suggests that the AHR may participate in the down-regulation of

CYP1A2 levels during sepsis in male rats (Zhou et al., 2008). Sepsis down-regulates

AHR expression within 6 h of onset and induces translocation of the AHR to the nucleus.

These findings suggest that CYP1A2 levels are decreased due to lowered AHR levels or

by AHR binding to iDREs in the CYP1A2 5'-flanking region.

The finding that infection suppresses the expression of numerous drug

metabolizing enzymes and drug transporters has serious clinical consequences since the

ability of a patient to respond to drug therapy is altered, resulting in an increased risk of

adverse drug reactions (Morgan et al., 2008).

1.6.3 CYP2C11 levels following xenobiotic treatment

An interesting phenomenon relating to CYP2C11 is that it is generally refractory to conventional P450 inducers. Expression of this gene is down-regulated by exposure to a diverse range of chemicals including: dexamethasone (Iber et al., 1997; Caron et al.,

2005), methoxychlor (Oropeza-Hernandez et al., 2003), tamoxifen (Kawai et al., 1999),

PB (Emi and Omura, 1988; Ryan et al., 1993; Kot and Daniel, 2007), cannabinoids

(Yamamoto et al., 1995), ethylbenzene (Bergeron et al., 1998), cyclosporine (Brunner et al., 1998), pyridine (Caron et al., 2005), and pregnenolone-16a-carbonitrile (Ryan et al.,

1993; Kot and Daniel, 2007).

Peroxisomes are organelles that participate in lipid homeostasis. They proliferate in response to chemicals known as "peroxisome proliferators", which act through the

PPAR [reviewed in: (Corton et al., 1998)]. PPARa is the predominant isoform expressed in the liver and binds to PPREs as a heterodimer with the retinoid X receptor (RXR).

PPARa ligands suppress CYP2C11 levels as observed by treatment of male rats with

76 clofibric acid (Su et al., 1999; Shaban et al., 2005), WY-14,643, di-n-butyl phthalate and

gemfibrozil (Corton et al., 1998). Since Corton's study shows that WY-14,643 also

down-regulates CYP2C11 in primary rat hepatocytes, a direct suppressive mechanism

may be occurring (Corton et al., 1998). Deletion analyses of CYP2C11 constructs in

HepG2 cells identified a region between -181 to -64-bp of the 5'-flanking region that

confers down-regulation of reporter gene activity by the peroxisome proliferators

nafenopin and dehydroepiandrosterone (DHEA; Ripp et al., 2003). Site-directed

mutagenesis within this region reverses this suppression, indicating that both nafenopin

and DHEA suppress CYP2C11 by acting through regulatory sequences within the 5'-

flanking region of this gene. Since gel shift studies do not identify PPARa/RXRa binding to this region, negative regulation may occur via PPARoc cross-talk with TFs that

act at this sequence rather than direct binding of PPARoc to the CYP2C11 5'-flank.

Studies support the existence of inhibitory cross-talk between the AHR and

PPARoc for their gene targets (Shaban et al., 2004). Co-treatment with clofibric acid and the AHR ligand Sudan III leads to an additive inhibitory effect on CYP2C11 protein, mRNA and catalytic activity that is more pronounced than the effect of each ligand alone

(Shaban et al., 2005). This may be a result of distinct mechanisms acting separately to suppress CYP2C11 levels, or through common mechanistic pathways shared by the AHR and PPARoc. Sudan III decreases PPARoc-target genes from the CYP4A subfamily in vivo and suppresses PPARoc and RXRoc protein levels in HepG2 cells (Shaban et al.,

2004), indicating negative cross-talk between the AHR and PPARoc. PPARoc activation can inhibit JAK2/STAT5b signaling, which may contribute to the suppression of

CYP2C11 expression by peroxisome proliferators (Shipley and Waxman, 2003).

77 Administration of the pesticide methoxychlor to male rats results in altered

CYP2C11 protein levels compared to vehicle treatment (Oropeza-Hernandez et al.,

2003). CYP2C11 mPvNA is decreased by 70% in rats treated for 24 h with styrene, a chemical used in the production of plastics and rubber, while CYP1A2, CYP2B, CYP2E1 and CYP3A2 levels are increased (Hirasawa et al., 2005).

Chronic ethanol exposure down-regulates hepatic and renal CYP2C11 protein and mRNA levels, which coincides with less activated STAT5b in these tissues (Badger et al.,

2003). Since ethanol can alter the male-specific intermittent GH profile, this may result in disruption of JAK2/STAT5 signaling and subsequent CYP2C11 transcriptional suppression.

Anti-cancer agents and other therapeutics can also suppress CYP2C11 expression.

Cyclosporine is an immunosuppressant drug that decreases CYP2C11 protein and catalytic activity, possibly as a result of altered GH or prolactin secretion (Brunner et al.,

1998; Lu et al., 2004a). Since co-treatment with bromocriptine, a known inhibitor of prolactin secretion, does not reverse CYP2C11 down-regulation (Lu et al., 2004a), and cyclosporine does not alter plasma GH levels (Lu et al., 2003), an alternative mechanism of suppression is likely responsible. Chronic low-dose exposure to the anti-cancer drug

5-fluorouracil in male rats results in suppression of CYP2C11 protein and catalytic activity, an effect that coincides with the depletion of plasma testosterone levels by this drug (Stupans et al., 1995). Tamoxifen, used in the treatment of breast cancer, suppresses

CYP2C11 protein and catalytic activity by 30-40%, possibly by decreasing GH secretion

(Ickenstein et al., 2004). The anti-cancer drug cisplatin lowers CYP2C11 protein and catalytic activity by 70-90% from control levels, an effect that is associated with

78 depletion of circulating androgens (LeBlanc and Waxman, 1988). A single i.p. injection

of cyclophosphamide also leads to decreased CYP2C11 protein and mRNA levels in rats

(LeBlanc and Waxman, 1990). In vivo treatment of rats with the antibiotic

chloramphenicol suppresses CYP2C11 protein, mRNA and catalytic activity by a pre-

translational mechanism in Sprague-Dawley rats but not in Fischer 344 rats (Kraner et al.,

1994), which suggests the existence of strain differences in the response of CYP2C11 to

certain chemicals. A pre-translational mechanism is also implicated in the down-

regulation of CYP2C11 protein, mRNA and catalytic activity following treatment of rats

with the anesthetic phencyclidine (Shelnutt et al., 1997). Treatment of rats with the

anesthetic propofol also decreases CYP2C11 protein and catalytic activity (Yamazaki et

al, 2006).

Evidently, numerous chemical and pathophysiological signals lead to the down-

regulation of CYP2C11 expression. Since very little is known about the mechanisms

mediating this suppression, it remains unclear whether distinct or common mechanistic

pathways are responsible for this response. It is difficult to speculate on the

physiological significance of CYP2C11 down-regulation; it has been proposed that this is

an adaptive homeostatic response that allows for the control of the production of reactive

oxygen species and nitric oxide release during stress (Riddick et al., 2004).

1.6.4 Down-regulation of CYP2C11 by aromatic hydrocarbons

Although numerous chemicals down-regulate CYP2C11 expression, Yeowell et al. (1987) noted that AHR agonists are particularly effective in suppressing this P450.

Early studies demonstrated that exposure of male rats to aromatic hydrocarbons results in hepatic CYP2C11 suppression by unknown mechanisms. The earliest of these studies

79 found that markers of CYP2C11 catalytic activity are decreased by TCDD (Hook et al.,

1975; Gustafsson and Ingelman-Sundberg, 1979). Following the development of antibodies specific for detecting this P450 isoform, these findings were followed by studies which detected CYP2C11 suppression by aromatic hydrocarbon treatment at the protein level (Guengerich et al., 1982; Dannan et al., 1983; Kamataki et al., 1986; Celier and Cresteil, 1989; Waxman et al., 1991; Ryan et al., 1993; Yuan et al., 1995).

Small monocyclic aromatic hydrocarbons, which do not activate the AHR, also suppress CYP2C11 expression. Treatment of rats with organic solvents such as toluene, benzene and trichloroethylene suppresses CYP2C11 catalytic activity when measured 6 h post-treatment (Wang et al., 1996). Another study reports that simple aromatics such as benzene, ethylbenzene and ra-propyl-benzene induce CYP1A1 and CYP2B protein levels, yet suppress CYP2C11 protein by 50% (Backes et al., 1993). Toluene, or methylbenzene, is a known inducer of CYP1A and CYP2B isoforms, but suppresses

CYP2C11 expression in vivo [reviewed in: (Nakajima and Wang, 1994)]. Bergeron et al.

(1988) report decreased CYP2C11 protein and catalytic activity by administration of ethylbenzene to rats.

Originally, CYP2C11 down-regulation was thought to result from competition between this enzyme and inducible P450s for a limited supply of heme, leading to degradation of CYP2C11 protein. The observation that CYP2C11 is also down-regulated at the mRNA level (Yeowell et al., 1987; Shimada et al., 1989; Yuan et al., 1995), dismissed this theory and pointed towards a pre-translational mechanism of suppression.

Yeowell et al. (1987) assessed CYP2C11 mRNA levels to gain insights into the mechanisms mediating protein down-regulation by MC and 3,4,5,3',4',5'-

80 hexachlorobiphenyl (HCB). A single i.p. injection of MC (50 mg/kg) suppressed

CYP2C11 mRNA levels by 65% when measured 17 h post-treatment. Overall, this group

found that suppression of CYP2C11 protein and catalytic activity was reflected at the

transcript level, supporting the involvement of a pre-translational mechanism. Emi and

Omura (1987) detected suppression of CYP2C11 mRNA levels as early as 3 h following

injection of rats with PB or MC, while maximal suppression occurs at 12 h. Another

study reported down-regulation of CYP2C11 mRNA levels to 25% of vehicle levels by

efhylbenzene, which suggests that decreased transcript levels at least partly account for

suppression of the protein (Yuan et al., 1995). This study did not report changes in

CYP2C11 mRNA levels by benzene, toluene, n-propyl-benzene, m-xylene and p-xylene;

although these chemicals down-regulate protein expression. Serron et al. (2001) also showed suppression of CYP2C11 protein, mRNA and catalytic activity following treatment of rats with ethylbenzene.

A previous study found that CYP2C11 protein expression is increased in the prostate and liver of rats treated with MC, although no response was observed following p-NF administration (Matsuda et al., 1995). To my knowledge, there have been no other reports of CYP2C11 induction by PAHs or HAHs.

Previous studies in our laboratory also report CYP2C11 suppression following treatment with both PAHs and HAHs. In cultured rat hepatocytes, TCDD decreases

CYP2C11 protein levels by 66% (Safa et al., 1997) and mRNA levels by 30% (Bhathena et al., 2002). Our laboratory has performed a time course study examining CYP2C11 expression over 14 days following a single i.p. injection of rats with MC (Jones and

Riddick, 1996). This study showed that the same dose of MC (50 mg/kg) that induces

81 CYP1A1 and EROD activity, concurrently suppresses hepatic CYP2C11 mRNA, protein and catalytic activity, further supporting a pre-translational mechanism for the suppression of this gene. Maximal suppression of CYP2C11 at all levels occurs 3-5 days following MC treatment, times that coincided with maximal CYP1A1 and EROD induction.

In more recent studies, Sudan III administration to rats decreased CYP2C11 expression at a pre-translational level via an unknown mechanism (Shaban et al., 2005).

Caron et al. (2005) confirm findings from our laboratory that MC suppresses CYP2C11 mRNA levels in vivo. CYP2C11 catalytic activity was also suppressed in rats treated with (3-NF (Kot and Daniel, 2007).

1.6.4.1 Evidence for a transcriptional mechanism of action

The regulation of gene product levels can occur at various stages ranging from gene transcription to post-translational protein modifications (summarized in Fig. 1.10).

Induction or suppression of a gene at the mRNA level does not necessarily imply transcriptional regulation. Changes in mRNA levels may be a result of altered mRNA stability or processing. A chemical may act to stabilize transcript levels, leading to increased mRNA and protein expression without altering the rate of gene transcription.

This is observed in the induction of CYP2E1 in response to fasting. Although CYP2E1 mRNA levels are elevated, nuclear run-on experiments, which measure the rate of gene transcription, do not indicate increased transcription of this gene (Hong et al., 1987). To determine whether mRNA stability is affected by a xenobiotic, one can quantify mRNA levels at several time points following addition of the transcriptional inhibitor 5,6- dichlorobenzimidazole riboside (DRB) to chemical- or vehicle- treated cells. If the

82 Transcription (a) Transcription

1. Pre-translational Pre-mRNA regulation

(b) RNA processing RNA-processing

\ (c) RNA stability

mRNA

2. Translational Translation regulation

(a) Post-translational protein Protein modifications

3. Post-translational regulation (b) Mechanism-based/ suicide inactivation

Figure 1.10. Schematic illustration of potential levels of P450 suppression. ••i represents the gene's promoter; • represents exons. 1. Pre-translational suppression may involve one or more of the following mechanisms: (a) decreased rate of gene transcription; (b) alterations in RNA processing; or (c) decreased mRNA stability. 2. Translational suppression involves decreased efficiency in the translation of mRNA into protein. 3. Post-translational suppression can occur by one or more of the following mechanisms: (a) post-translational protein modifications (e.g. phosphorylation, ubiquitination) that decrease protein stability or enzyme activity; (b) mechanism- based or suicide inactivation results in permanent loss of enzyme activity due to irreversible binding of a reactive metabolite. In this case one would observe decreased catalytic activity of the enzyme, but not necessarily suppressed protein levels. [Adapted from Yang et al. 1992].

83 mRNA half-life is similar in both treatment groups following DRB addition, this suggests

the gene is regulated by a transcriptional mechanism.

In situations where protein levels are altered without corresponding changes in the message, it is likely that a xenobiotic is affecting protein stability. Both alterations in protein degradation and translational efficiency can ultimately modulate protein levels.

Suppression of P450 protein levels and catalytic activity without an accompanying change at the mRNA level, may occur due to enzymatic inactivation. A xenobiotic may inactivate a P450 by irreversibly binding to the heme component or an amino acid residue

(Yang et al., 1992). This is referred to as "suicide" or "mechanism-based" inactivation and is always characterized by decreased enzymatic activity, but may or may not result in decreased protein levels. Alternatively, down-regulation of protein with no change in mRNA may result from decreased protein synthesis or protein de-stabilization. Post- translational covalent modifications added to a protein may affect its catalytic activity and/or stability and consequently its biological half-life.

Long-term or permanent silencing of a gene in a particular tissue or developmental state often involves epigenetic, covalent modifications (e.g. DNA methylation). On the other hand, short-term and reversible transcriptional changes that occur in response to environmental stimuli can involve several mechanisms described below [reviewed in: (Levine and Manley, 1989; Renkawitz, 1990; Jackson, 1991; Courey and Jia, 2001)]. The first mechanism of transcriptional suppression is known as competition or inhibition of DNA binding. This mechanism often involves steric hindrance resulting in the inability of a transcriptional activator to bind an enhancer sequence due to the presence of a negative factor(s). This negative factor may also

84 interfere with transcription by preventing the assembly of the transcription initiation complex at the gene's promoter. This appears to be the mechanism by which some estrogen-responsive genes are suppressed following TCDD-induced AHR binding to iDREs. In this example, the AHR acts as the negative factor, and binding to iDREs prevents activators from binding to nearby or adjacent sequences (Gillesby et al., 1995;

Krishnan et al., 1995; Wang et al., 2001). Competition for DNA binding may also occur when positive and negative TFs compete for the same cis-acting elements within the gene's regulatory region. The next transcriptional suppressive mechanism is sequestering, which involves the formation of an inactive complex upon the binding of a negative factor to an activator. This complex formed via protein-protein interactions prevents the positive factor from interacting with its target DNA sequence and driving transcription of the gene. An example of sequestering is observed when I-KB forms an inactive complex with NF-KB. Upon degradation of I-KB, NF-KB is free to enter the nucleus and modulate gene transcription. Sequestration of TFs or co-activators via protein-protein interactions is sometimes referred to as "squelching". Activation masking (or "quenching") is another form of transcriptional suppression. In this mechanism, the "positive-acting" TF is bound to its target DNA sequence, yet is unable to transmit the positive signal to the transcription initation complex. This interference can be a result of DNA-bound or free repressors that can interact with the transactivation domain of the positive-acting TF to prevent transcriptional activation. Silencing involves the repression of gene transcription due to direct binding of a repressor or negative-acting

TF to its consensus nucleotide sequence. By definition, a silencer consensus sequence confers gene suppression in a distance- and orientation-independent manner since it

85 shares many properties displayed by enhancer sequences. Some TFs can act to both positively and negatively regulate gene transcription in a context-dependent fashion. In this sense, it is plausible that the AHR mediates at least a component of CYP2C11

suppression by aromatic hydrocarbons, thus acting as a "dichotomous regulator" (Roberts and Green, 1995) to activate transcription in some genes while repressing transcription in others.

There is evidence that supports a transcriptional mechanism of CYP2C11 suppression by aromatic hydrocarbons. MC administration decreases CYP2C11 mRNA levels as early as 3 h post-treatment (Emi and Omura, 1988). Although this very early response has not been consistently observed by other investigators, suppression at such an early time-point is indicative of a direct transcriptional mechanism (Hanlon et al.,

2005). Furthermore, TCDD can decrease CYP2C11 mRNA levels in primary rat hepatocytes without altering the mRNA half-life (Bhathena et al., 2002). This finding has two major implications related to a transcriptional mechanism of suppression.

Firstly, TCDD does not decrease CYP2C11 mRNA stability; therefore, CYP2C11 mRNA down-regulation is likely due to decreased gene transcription. In addition, since this response was detected in cultured hepatocytes, at least a component of CYP2C11 suppression seems to be a direct effect of TCDD acting on the gene, rather than an indirect effect involving interference with hormonal signaling pathways. However, since primary rat hepatocytes only express 25% of intact CYP2C11 levels (Liddle et al., 1992), the response of this gene to TCDD treatment in an isolated culture system may not accurately represent CYP2C11 regulation in living rats where pituitary factors are present and stimulate full basal CYP2C11 expression. In primary rat hepatocytes, TCDD can

86 only decrease CYP2C11 levels that are expressed in the absence of endocrine control

(Safa et al., 1997). Nuclear run-on analyses demonstrate that the rate of CYP2C11 transcription is decreased by 51% within 6 h of MC administration to male rats, indicating that suppression of this gene at least partially occurs at the level of gene transcription (Lee and Riddick, 2000). Although these studies implicate a transcriptional mechanism of suppression, this may not be the sole mechanism involved in the down- regulation of CYP2C11 by aromatic hydrocarbons.

1.6.4.2 Role of the AHR in CYP2C11 suppression

The suppression of CYP2C11 by AHR ligands that are known CYP1A inducers may implicate a common pathway that mediates these opposite responses. The notion that the AHR may act as a "dichotomous" regulator (Roberts and Green, 1995), is a useful concept in considering how this TF can induce expression of CYP1 members while reducing CYP2C11 levels. The notion that the AHR is involved in CYP2C11 suppression was first introduced by SAR studies that correlated the suppression of this gene by PAHs to the extent of AHR-mediated responses (Yoshihara et al., 1982; Dannan et al., 1983). The suppression of CYP2C11 catalytic activity by PCBs and polychlorinated dibenzofurans coincides with AHR-mediated toxicities such as thymic atrophy (Yoshihara et al., 1982). Decreased CYP2C11 expression following in vivo treatment of rats with MC and polybrominated biphenyls correlates with induction of

CYP1A isoforms (Dannan et al., 1983). This study shows that chemicals most effective in inducing CYP1A members are also the most active down-regulators of CYP2C11 protein. SAR studies within our laboratory support this concept since the ability of several PAHs of the anthracene class to suppress CYP2C11 protein levels correlates with

87 both the AHR binding affinity and the AHR transformation potency of these compounds

(Safa et al., 1997). Our laboratory also reported that the transformed AHR can bind with high affinity to the CYP2C11-DRE3 element located at positions -1546 to -1533-bp in the

CYP2C11 5'-flank (Bhathena et al., 2002). Although no function has yet been linked to this sequence, this binding element may behave as an iDRE-like sequence in transcriptional suppression (Bhathena et al., 2002; Riddick et al., 2003).

Furthermore, oc-NF, an AHR antagonist/partial agonist, does not alter CYP2C11 mRNA levels (Emi and Omura, 1988), suggesting that AHR activation may be important in CYP2C11 suppression.

There is also contradictory evidence suggesting that CYP2C11 suppression and

CYP1A induction are regulated by different pathways following aromatic hydrocarbon treatment. Emi and Omura (1988) found that CYP2C11 mRNA levels are maximally down-regulated by MC at 12 h post-treatment while inducible P450s reach a maximum at

24 h, perhaps suggesting separate pathways of regulation based on different time courses of response to treatment. Yeowell et al. (1987) report that the time course of CYP2C11 suppression by HCB is different from that required for CYP1A1 induction, suggesting that HCB alters the expression of these two P450s by distinct mechanisms. Also, this study showed that the dose leading to maximum CYP2C11 mRNA suppression is higher than that required to induce CYP1A1 mRNA levels. Although differences in time-course and dose response may suggest different mechanisms for CYP1A induction versus

CYP2C11 suppression, these observations may also relate to differences in the sensitivities of the two responses and differences in the half-lives of the respective mRNAs or proteins.

88 1.6.5 Possible mechanisms involved in aromatic hydrocarbon-mediated CYP2C11 suppression

Our laboratory continues to be interested in both DIRECT and INDIRECT mechanisms involved in CYP2C11 down-regulation by aromatic hydrocarbons. Some possibilities of interest include: (i) direct involvement of AHR binding to a DRE-like sequence; (ii) disruption of normal endocrine pathways required for the maintenance of

CYP2C11 expression; (iii) modulation of cytokine signaling pathways. These potential mechanisms are not necessarily mutually exclusive and they do not rule out the involvement of other mechanisms.

The AHR may mediate at least a component of CYP2C11 suppression. This would explain the correlation between induction of CYP1A subfamily members and suppression of CYP2C11 levels by aromatic hydrocarbons as observed in several studies mentioned in Section 1.6.4.2. Since iDREs are involved in TCDD-mediated suppression of some estrogen-responsive genes, it is possible that the AHR may DIRECTLY suppress

CYP2C11 by interacting at similar sequences.

INDIRECT mechanisms, such as aromatic hydrocarbon-induced alterations in hormonal signaling, may also suppress this gene. An earlier theory suggested that

CYP2C11 suppression results as a consequence of decreased testosterone levels caused by some aromatic hydrocarbons. Yeowell et al. (1987) found that 5 days following administration of HCB (50 mg/kg) to rats, both CYP2C11 mRNA and serum testosterone levels are significantly reduced. However, this study also showed that low doses of HCB that do not alter testosterone plasma concentrations can still effectively suppress

CYP2C11 mRNA levels, thus refuting the androgen theory.

89 Alternatively, PAHs such as MC and simple aromatic hydrocarbons such as ethylbenzene may interfere with the ability of GH to maintain hepatic CYP2C11 transcription. Studies of exogenous GH administration to hypx rats support this mechanism (Timsit and Riddick, 2000; Serron et al., 2001). This theory is also supported by the demonstration that components of the GH signaling pathway (e.g. GHR) and other

GH target genes can be modulated by aromatic hydrocarbon treatment (Nukaya et al.,

2004; Lee et al., 2006). Interestingly, MC does not seem to disrupt JAK2-STAT5b signaling in rat liver or rat hepatoma cells (Timsit and Riddick, 2002).

Another INDIRECT mechanism that may lead to decreased CYP2C11 expression is the modulation of cytokine signaling pathways by aromatic hydrocarbons. Cytokine treatment can result in transcriptional suppression of CYP2C11 through activation of NF-

KB (Iber et al., 2000). Different scenarios can cause NF-KB activation. Aromatic hydrocarbon treatment may lead to the production of reactive oxygen species (Shertzer et al., 1998; Puga et al., 2000a; Hassoun et al., 2003; Matsumura, 2003; Li et al, 2004;

Shertzer et al., 2004), which are known to activate NF-KB (Puga et al., 2000a; Weng et al., 2004; Xu et al., 2004). Alternatively, aromatic hydrocarbons may induce production of cytokines (Li et al., 2002; Villard et al., 2007), which can also promote NF-KB activation. Following activation of the AHR by ligand binding and activation of NF-KB by cytokines, these two TFs can interact through the p65 subunit of NF-KB and remain in the cytoplasm (Tian et al., 1999). This interaction would allow p50 subunits to enter the nucleus, where they are typically associated with repressing transcription of target genes.

Activated p50 subunits can suppress transcription of CYP2C11 by binding to consensus sequences on the promoter of this gene as previously shown (Iber et al., 2000), or

90 potentially through AHR cross-talk with NF-KB (Zhao and Ramos, 1998). The AHR/NF-

KB complex can bind NF-KB-responsive elements in the regulatory region of the c-myc gene to activate transcription (Kim et al., 2000), so it is possible this complex could bind to the CYP2C11 5'-flank to suppress transcription under certain conditions. Cytokines may also INDIRECTLY suppress CYP2C11 transcription by altering GH signaling pathways (this will be discussed in Section 4.2.5.3).

1.7 Hypothesis and objectives

1.7.1 Research Rationale and Experimental Approaches

CYP2C11 expression is down-regulated in the liver of male rats by aromatic hydrocarbons via a transcriptional mechanism that is not yet understood (Jones and

Riddick, 1996; Lee and Riddick, 2000). To a limited extent, this can be reproduced in primary rat hepatocytes cultured on matrigel since TCDD decreases CYP2C11 protein and mRNA levels without altering the mRNA half-life (Safa et al., 1997; Bhathena et al.,

2002). However, studies in hepatocytes are limited in their ability to faithfully mimic the in vivo endocrine control of this gene. SAR investigations suggest that the AHR is involved in CYP2C11 suppression (Safa et al., 1997), a finding that is consistent with the ability of the transformed AHR to bind with high affinity to the CYP2CU-DRE3 element located at positions -1546 to -1533-bp in the CYP2C11 5'-flanking region (Bhathena et al., 2002).

Since both GH and aromatic hydrocarbons commonly alter gene expression by transcriptional mechanisms, our studies of CYP2C11 suppression are focused on this gene's 5'-flanking region and promoter. To date, luciferase reporter plasmids encompassing the proximal 2390-bp of the CYP2C11 5'-flanking region have not been

91 down-regulated by TCDD in liver-derived cell lines and primary rat hepatocytes

(Bhathena et al., 2002). Lack of suppression of CTP2Ci7-luciferase constructs by

TCDD in previous cell culture studies may be due to: (i) the need to study this gene in an

in vivo context and/or (ii) the absence of key upstream regulatory elements in the reporter

constructs employed to date. This thesis addresses both possibilities through

investigations executed in vitro and in vivo. My in vitro studies examine the second

possibility by examining regulation of luciferase reporter constructs under the control of

an extended CYP2C11 5'-flanking region in hepatoma cells of rat, mouse and human

origin. As a prelude to studying these novel reporter constructs in living rodents, the goal of this part of the thesis was to understand how extended regulatory regions of this gene influence its response to TCDD and MC treatment in isolated cellular systems. My in vivo work addresses both possibilities by studying the regulation of reporter constructs containing extended stretches of the CYP2C11 5'-flanking region in living rats using the hydrodynamics-based approach for plasmid delivery.

1.7.2 Hypothesis

In vivo suppression of CYP2C11 by aromatic hydrocarbons is mediated by the promoter and 5'-flanking region of the gene.

1.7.3 Objectives

My specific goals are classified into two sections: in vitro studies and in vivo studies.

In vitro studies:

[1] To clone and characterize extended stretches of the CYP2C11 5'-flanking region.

[2] To test the activity and response to aromatic hydrocarbons of CYP2C11 -luciferase

92 constructs in continuous hepatoma cell models.

In vivo studies:

[3] To confirm the in vivo suppression of endogenous hepatic CYP2C11 by MC.

[4] To study the response of CYP2C1 i-luciferase constructs to MC treatment in living rats using the hydrodynamics-based plasmid delivery approach.

93 2.0 MATERIALS AND METHODS

2.1 Luciferase reporter constructs

2.1.1 Construction of reporter plasmids

Schematic diagrams of all firefly luciferase constructs used in both in vitro and in vivo studies are shown in Fig. 2.1. All numbering of nucleotide positions is according to version 3.4 of the November 2004 rat genomic assembly found on the University of

California Santa Cruz (UCSC) Genome Browser website (Karolchik et al., 2003; http://genome.ucsc.edu/cgi-bin/hgGateway), relative to +1 denoting the G residue of the transcription initiation site (Morishima et al., 1987).

The following plasmids from Promega (Madison, WI) were used as controls: the promoterless pGL3-Basic, pGL3-Promoter under the control of the simian virus 40

(SV40) heterologous promoter, pSV-P-galactosidase and pRL-TK (Renilla luciferase) used to normalize for transfection efficiency. The positive control for AHR activation is pGudlucl.l (Dr. Michael Denison, University of California, Davis, CA). The luciferase gene in this plasmid is driven by the mouse mammary tumor virus (MMTV) promoter and regulated by a 480-bp fragment from the mouse Cyplal 5'-flanking region which contains four DREs (Garrison et al., 1996).

I used the UCSC database as the source of rat genomic sequence corresponding to extended regions of the CYP2C11 5'-flank. Bioinformatics analysis of this DNA region revealed new insights into potential TF binding sites that may contribute to the regulation of this gene. I proceeded to design three sets of primers to amplify extended stretches of the CYP2C11 5'-flanking region from rat genomic DNA, with the goal of generating novel luciferase constructs. Restriction enzyme sites were engineered into the 5'- end of

94 pGudlucl.l -| Cypiall-I MMTV

pGL3-Basic

pGL3-Promoter SV40

-10,061 ±20 (-10.1-2C11) # 11 t17 (-7.1-2C11) # t17 (-5.6-2C11) JL _+2/ (-2.4-2C11)

•1,301 -+21 (-1.3-2C11) t-Dsai -196 +21 (-0.2-2C11) TTTT

-2,344 -1,303 (-2.4 to -1.3-2Cll)-Promotei: EZJ SV40 -2,344 -1,303 -1M +21 (-2.4 to -1.3-2C1D-0.2 — EZJ -2,344 -1,813 -1M +21 (-2.4 to -1.8-2Cll)-0.2 —

-1,827 : 13Cl im, +21 (DRE-2C1D-0.2 — rm -10,061 •i I" i .+20 (-10.1-2C11)MUT -I #? JL +21 (-2.4-2Cll)MUT __ 7 \ -2,344 -7,303 -196, +21 (-2.4 to -1.3-2C11)-0.2MUT- I IX t -r,303 -

Figure 2.1. Schematic representation of luciferase reporter constructs used in this thesis. "D" represents CYP2C11-DRE3; i i represents the CYP2C11 5'-flanking region; represents the luciferase gene; MMTV and SV40 represent heterologous promoters.

95 each primer to facilitate cloning. The restriction digestion sites chosen were also located within the multiple cloning site of pGL3-Basic, but not within the targeted rat genomic sequence to be amplified. Forward primers were designed at 10.1-kb, 7.1-kb and 5.6-kb upstream of the CYP2C11 transcriptional start site on the rat genome, while reverse primers spanned the transcriptional start site and ended several nucleotides before the start codon (ATG) located at +23-bp. Primers were designed using the Primer 3 program

(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and had to fulfill criteria for proper length, GC content, and melting temperature. All PCR primers were obtained from Integrated DNA Technologies Inc. (Coralville, IA). Primer specificity and in silico

PCR analyses were assessed using the Primer UniGene Specificity (PUNS) database

(Boutros and Okey, 2004). The last step in primer design was to verify the likelihood of secondary structures forming between primers and primer pairs. This was tested online using the SciTools option provided by Integrated DNA Technologies

(http://www.idtdna.com/SciTools/SciTools.aspx). All three of our novel primer sets displayed satisfactory criteria for these parameters.

Extended regions of the CYP2C11 5'-flanking region were amplified by PCR using the Expand Long PCR system (Roche Diagnostics, Laval, Quebec, Canada) for plasmids:(-10.1-2Cll), (-7.1-2C11) and(-5.6-2Cll). Each PCR reaction contained

100 ng Sprague-Dawley rat genomic DNA (Clontech, Mountain View, CA) as a template, 0.4-0.5 uM primers, 0.4 mM of each 2'-deoxynucleoside 5'-triphosphate

(dNTP, Invitrogen Corporation, Carlsbad, CA), IX PCR buffer containing 27.5 mM

MgCt and 5 U of the Expand Long enzyme mixture containing Tag and Tgo DNA

96 polymerases. The PCR primer sequences, restriction endonucleases (New England

Biolabs, Ipswich, MA), and cycling parameters are described in Table 2.1.

Following amplification, PCR products were fractionated by agarose gel electrophoresis and purified using the QIAquick Gel Extraction Kit (Qiagen Inc.,

Valencia, CA). PCR products were then cloned into pCR-XL-TOPO or pCR-4-TOPO vectors using the TOPO XL PCR or TOPO TA Cloning Kits (Invitrogen Corporation).

These cloning vectors contain a single 3'-deoxythymidine (T) overhang and are covalently bound to the topoisomerase enzyme. Since PCR amplification with Taq polymerase adds a single deoxyadenosine (A) to the 3'-end of the amplified PCR product, this enables the insert to efficiently ligate with the linearized cloning vector.

The topoisomerase enzyme rejoins the two DNA strands. Following partial DNA sequencing of ~1000-bp on both ends of the PCR products to confirm their identity, PCR inserts were excised from the TOPO vectors by digestion with the restriction endonucleases indicated in Table 2.1. The digestion products were separated by agarose gel electrophoresis and purified using the QIAquick Gel Extraction Kit. Purified products were ligated into the promoterless pGL3-Basic luciferase expression vector as described in Section 2.1.4.

Plasmid: (-2.4-2C11), (-1.3-2C11) and (-0.2-2C11) were generated as described previously (Bhathena et al., 2002), and these plasmids were named (-2390-2C1 l)-pGL3,

(-1311-2Cll)-pGL3 and (-196-2Cll)-pGL3, respectively, in the original publication.

The plasmid (-2.4 to -1.3-2Cll)-Promoter contains a PCR-amplified region from

-2344 to -1303-bp digested with Mlul and Bglll, gel-purified and ligated into pGL3-

Promoter. The plasmid (-2.4 to -1.3-2C11)-0.2 contains the PCR-amplified region from

97 TABLE 2.1. Oligonucleotides and thermal cycling conditions used for PCR-based cloning of fragments of the CYP2C11 5'- flanking region.

Plasmid Forward and reverse primer sequences CYP2C11 genomic Restriction Thermal cycling parameters product length endonucleases (denature; anneal; extend) x cycles

(-10.1-2C11) 5'-TTGAATGCTGAGGTCACAGCTGAATGC (FP) 10,081-bp Sacl; Mlul (94730s; 657 30s; 68710min) x 34 5'-CAGCCTTCTCAGGGAGACTCCTGTGA (RP)

(-7.1-2C11) 5'-GCTTrGGTCTrCACGATTCCTGT (FP) 7,084-bp Mlul (94715s; 64730s; 6876min) x 34 5'-CCTTCTCAGGGAGACTCCTGTGA (RP)

(-5.6-2C11) 5'-CACACTTCCTCACTTCCTCCAAC (FP) 5,633-bp Mlul; Ncol (947 15s; 647 30s; 687 7min) x 34 5'-CCTTCTCAGGGAGACTCCTGTGA (RP)

(-2.4 to -1.3-2C11)-Promoter/ 5'-GATCTGGTGGGATGG(FP) 1,042-bp Mlul; BglU (947 30s; 577 30s; 727 60s) x 29 (-2.4 to -1.3-2Cll)-0.2 5'-CCCCTTCCTCAGAAC (RP) Sack, Mlul

(-2.4to-1.8-2Cll)-0.2 5'-GATCTGGTGGGATGG (FP) 532-bp Sad; Mlul (947 30s; 587 30s; 727 60s) x 29 5'-AAGGCTGTGCCTCCT (RP)

(DRE-2Cll)-0.2 5'-AGGAGGCACAGCCTT (FP) 525-bp Sacl; Mlul (947 30s; 587 30s; 727 60s) x 29 5'-CCCCTTCCTCAGAAC (RP)

FP, forward primer; RP, reverse primer Note: the above primer sequences show only the nucleotides matching CYP2C11 genomic sequences; docking nucleotides and restriction enzyme sites are not shown.

98 -2344 to -1303-bp digested with Sad and Mlul, gel-purified and ligated into (-0.2-2C11);

(-0.2-2C11) contains the region from -196 to +21-bp of the proximal CYP2C11 5'- flanking region and promoter cloned into pGL3-Basic.

Using the PCR primers listed in Table 2.1 followed by ligation into (-0.2-2C11), two new constructs were generated in order to examine the 5'- and 3'-halves of the region from -2344 to -1303-bp: (DRE-2C11)-0.2 and (-2.4 to -1.8-2C11)-0.2. The plasmid (DRE-2C11)-0.2 spans from -1827 to -1303-bp and encompasses the previously characterized CYP2C11-DKE3 (Bhathena et al., 2002) located at positions -1546 to

-1533-bp. The plasmid (-2.4 to -1.8-2Cll)-0.2 includes the region from -2344 to

-1813-bp.

PCR reactions for the generation of (-2.4 to -1.3-2Cll)-Promoter, (-2.4 to -1.3-

2C11)-0.2, (-2.4 to -1.8-2C11)-0.2 and (DRE-2C11)-0.2 contained 2.5 U Taq DNA polymerase (Invitrogen Corporation), IX PCR buffer [20 mM Tris; 50 mM KC1; 3 mM

MgCk], 0.4-0.5 uM primers and 0.4 mM of each dNTP. Each reaction used 1 ug of the plasmid (-5.6-2C11) as the template for amplification.

2.1.2 Restriction enzyme digestion and agarose gel electrophoresis

Restriction digestion of DNA was performed under conditions recommended by

New England Biolabs. Reactions contained 0.5-1 ug of plasmid DNA, 2 uL of 10X NE

Buffer 1-4 (as recommended for each restriction enzyme used), 1 uL restriction enzyme

(10-20 U), and water to make the reaction volume 20 uL. This reaction was incubated for 1-1.5 h at 37°C.

Following incubation, digestion products were separated on 0.8-1.5% (w/v) agarose gels containing 40 mM Tris-acetate; 1 mM efhylenediaminetetraacetic acid

99 (EDTA, pH 8.0); 0.5 ng/mL ethidium bromide. Running buffer was IX TAE (40 mM

Tris-acetate; 1 mM EDTA, pH 8.0) and gels were run at 80-100 V for 30-60 min

depending on the time needed for adequate separation of DNA bands. DNA fragments

were visualized on the BioDoc-It UV Transilluminator (Upland, CA) or an UV light box

(Vilber Lourmat, InterSciences Inc., Markham, ON). Gels imaged with the UV light box

were photographed with a Polaroid Direct Screen Instant camera DS34 (International

Biotechnologies Inc., New Haven, CT).

2.1.3 Purification of DNA from agarose gel

For subsequent ligation reactions, DNA digestion products were fractionated on agarose gels and the fragment of interest excised using a sharp, clean scalpel. Excised gel pieces were placed in a microfuge tube and the QIAquick Gel Extraction Kit was used to extract DNA from the gel. The excised gel was weighed, and 3 volumes of

Buffer QG added to 1 volume of the gel. The gel was dissolved by incubation at 50°C for 10 min. If the DNA segment of interest was between 500-bp and 4-kb in size, 1 gel volume of isopropanol was added to the sample and vortexed. Samples were then applied to a QIAquick spin column and centrifuged at 12,000 rpm for 1 min, allowing the

DNA to bind to the column. The column was washed with Buffer PE (containing ethanol), and centrifuged at 12,000 rpm for 1 min. DNA was eluted in pre-heated Buffer

EB (20-30 uL; 50°C; lOmM Tris-Cl, pH 8.5) applied to the center of the column, and allowed to stand for 1 min before centrifugation. Gel-purified DNA fragments were fractionated on 0.8-1.5% agarose gels to confirm the purification of the DNA fragments of predicted size.

100 2.1.4 DNA ligation

Ligations were performed in a volume of 20 uL. This volume contained 5 U of

T4 DNA Ligase (Invitrogen Corporation), 4 uL of 5X DNA Ligase Reaction Buffer [250

mM Tris-HCl (pH 7.6); 50 mM MgCl2; 5 mM ATP; 5 mM dithiothreitol (DTT); 25%

polyethylene glycol-8000], and water to make the reaction volume 20 uL. DNA

fragments (-30-75 ng) were added in a molar ratio of 3:1 (insert:vector backbone).

Ligation reactions were incubated overnight at room temperature.

2.1.5 Plasmid preparation

All bacterial work was performed using sterile technique. Agarose, bacto-yeast

extract, bacto-tryptone and bacto-agar were obtained from Difco (Detroit, MI). Plasmids:

(-10.1-2C11), (-7.1-2C11) and (-5.6-2C11) were transformed using XLIO-Gold

Ultracompetent bacterial cells (Stratagene, La Jolla, CA). Bacterial cells (100 uL) were

thawed on ice in pre-chilled 14-mL Falcon polypropylene round-bottom tubes. P-

Mercaptoethanol (4 uL) was added to bacterial cells in Falcon tubes incubated on ice for

10 min. The ligation mixture (1-2 uL) was added to the cells and incubated for 30 min on ice. Tubes were heat-pulsed for 30 s in a 42°C water bath, followed by a 2 min

+ incubation on ice. NZY broth (0.9 mL; LB broth containing 0.012 mM MgCl2; 0.012 mM MgS04; 0.01 M glucose, pH 7.5) was added to the tube and incubated at 37°C for

1 h with shaking at 225 rpm. The transformation mixture (50 u.L) was plated on LB agar plates [1% (w/v) bactotryptone; 0.5% (w/v) bacto-yeast extract; 1.5% (w/v) bacto-agar;

172 mM NaCl] containing 50 p,g ampicillin/mL and incubated inverted overnight at

37°C.

101 For all other plasmids, competent DH5a bacterial cells (Life Technologies,

Gaithersburg, MD) were used for transformation. Bacteria (50 JLLL) were chilled on ice in a pre-chilled microfuge tube. The ligation mixture (1-2 uL) was added to bacterial cells and incubated for 30 min on ice. Tubes were heat-shocked for 30 s in a 42°C water bath, followed by a 2 min incubation on ice. SOC medium was added to bring the volume of the reaction to 500 \iL, and tubes incubated at 37°C for 1 h with shaking at 225 rpm.

Transformed bacteria (50-100 |J.L) were plated on LB agar plates containing 50 |ig ampicillin/mL and incubated inverted overnight at 37°C.

Using an autoclaved toothpick, individual colonies from the LB-ampicillin plates were inoculated in 5 mL LB medium [1% (w/v) bactotryptone; 0.5 % (w/v) bacto-yeast extract; 172 mM NaCl], containing 50 ug ampicillin/mL. This culture was incubated in an orbital shaker at 37°C for 8-16 h, with shaking at 225 rpm. Approximately 1.5 mL of the 5 mL inoculation culture was centrifuged at 14,000 rpm for 2 min on a table top centrifuge. For small-scale plasmid DNA preparation, the QIAprep Spin Miniprep Kit

(Qiagen Inc.) was used as described in the manufacturer's protocol. This procedure uses a modified alkaline lysis method, followed by high-salt binding and subsequent elution of plasmid DNA from the QIAprep membrane. The bacterial pellet was resuspended in

Buffer PI (250 uL). Buffer P2 (250 uL; contains sodium hydroxide) was added to lyse bacterial cells and the reaction inverted 6 times. Buffer N3 (350 uL; contains guanidine hydrochloride and acetic acid) was added, and mixed immediately by inverting the tube

10 times. The reaction was centrifuged for 10 min at 12,000 rpm. The supernatant was applied to a QIAprep spin column and centrifuged for 1 min. The column was washed by adding Buffer PE (750 uL; containing ethanol) and centrifuged for 1 min. Buffer EB

102 (30-50 uL; 10 mM Tris-Cl, pH 8.5) was applied to elute the plasmid DNA from the

column.

The EndoFree plasmid purification Maxi kit (Qiagen Inc.) was used for large-

scale plasmid preparation for subsequent transfections. LB broth (100-150 mL)

containing ampicillin was inoculated with 1.5 mL of the original 5 mL culture described

previously. Bacteria were grown for -16 h and harvested by centrifugation at 2,500 rpm

for 30 min at 4°C. Plasmid DNA was then purified using a QIAFilter, which utilizes a

modified alkaline lysis procedure followed by plasmid DNA binding to an anion-

exchange resin and subsequent elution. Resuspension Buffer PI (10 mL) was added to

the bacterial pellet. Following complete resuspension of the pellet, Lysis Buffer P2

(10 mL) was added and the tube inverted 6 times. Following a 5 min incubation at room

temperature, pre-chilled Buffer P3 (10 mL) was mixed with the lysate by immediately

inverting the reaction tube 6 times. The lysate was then poured into the barrel of the

QIAFilter cartridge and incubated for 10 min at room temperature. A plunger was

inserted into the cartridge, and the lysate filtered into a 50-mL Falcon tube. Endotoxin- removal buffer (2.5 mL) was added and the tube incubated on ice for 30 min. The

supernatant was then applied to a pre-equilibrated QIAGEN-tip 500 and allowed to enter the column by gravity flow. The QIAGEN-tip was washed with Buffer QC, and the

DNA eluted with Buffer QF (12 mL). DNA was then precipitated by the addition of isopropanol (10.5 mL), and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was removed and the pellet was washed with 70% endotoxin-free ethanol

(5 mL), followed by centrifugation at 12,000 rpm for 10 min at 4 °C. The supernatant

103 was discarded and the pellet was air-dried for 10 min, followed by resuspension in TE

buffer (100-200 uL).

For in vivo transfections, the EndoFree plasmid purification Giga kit (Qiagen Inc.)

was used to generate very large quantities of plasmid DNA. The following plasmids

delivered by hydrodynamics-based injections were prepared using this kit: pGL3-Basic,

(-5.6-2C11), (-2.4-2C11) and pRL-TK. LB broth (-2.5 L) containing ampicillin was

inoculated with 2.5 mL of the 5 mL culture described previously. Bacteria were grown

for -16 h and harvested by centrifugation at 2,500 rpm for 30 min at 4°C. The method of purification is similar to the Maxi plasmid purification protocol described above, with a few modifications. The bacterial pellet was resuspended in Buffer PI (125 mL), lysed in

Buffer P2 (125 mL), and precipitated with pre-chilled Buffer P3 (125 mL). The lysate was then poured into the QIAFilter Giga cartridge and incubated at room temperature for

10 min. A vacuum was used to filter the lysate from the precipitate. Buffer FWB2

(50 mL) was added to the cartridge, and the precipitate gently stirred using a sterile spatula. The vacuum was used to pull the liquid through the filter. Endotoxin-removal

Buffer (30 mL) was added to the filtered lysate and the reaction incubated for 30 min on ice. The filtered lysate was then applied to a pre-equilibrated QIAGEN-tip 10,000 and allowed to enter the resin by gravity flow. Buffer QC was used to wash the QIAGEN-tip, and the DNA eluted with Buffer QN (100 mL). DNA was precipitated by the addition of isopropanol (70 mL). This solution was then centrifuged at 12,000 rpm for 30 min at

4°C. The supernatant was discarded and the pellet was washed with endotoxin-free 70% ethanol (10 mL), followed by centrifugation at 12,000 rpm for 10 min at 4°C. The

104 supernatant was decanted and the pellet was air-dried for 15 min. The pellet was then resuspended in endotoxin-free Buffer TE (0.3-1.5 mL).

The pGudlucl.l and (-10.1-2C11) endotoxin-free plasmids used in in vivo transfections were prepared and purified at large-scale by Aldevron (Fargo, ND), since I was not able to obtain sufficient yields of these two plasmids required for in vivo work using the previously described methods. Following purification, both plasmids were resuspended in buffer TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and stored at -20°C until use.

DNA yield and purity were assessed by measuring the absorbance at 260 and 280 nm on a Beckman DU-65 spectrophotometer (Beckman Instruments Inc., Fullerton, CA).

2.1.6 Bioinformatic analysis of the CYP2C11 5'-flanking region

Searches for putative TF binding sites in rat genomic sequence encompassing positions -22,344 to +56-bp of the CYP2C11 gene on chromosome 1 (UCSC Genome

Browser), were performed using MacMolly Tetra version 3.10 (Soft Gene GmbH).

Sequences were searched in both the forward and reverse complementary orientations.

Consensus sequences for the listed TF binding sites of interest were defined as follows: AHRE-I (DRE): must have the invariant core CGTG plus <2 mismatches from the AHR binding consensus [5'-GCGTGNN(A/T)NNN(C/G)-3'] defined by Yao and

Denison (1992); NF-KB: <1 mismatch from the consensus sequence

[5'-GGGANT(C/T)(CYr)CC-3']; HNF-3: <2 mismatches from the consensus sequence

[5'-TATTGA(C7T)TT(A/T)G-3']; AHRE-II: exact match to the consensus sequence defined by Sogawa et al. (2004) and Boutros et al. (2004)

[5'-CATGNNNNNNC(A/T)TG-3']; STAT-5: exact match to the consensus sequence

105 [5'-TTCNNNGAA-3']; AP-1: exact match to the consensus sequence

[5'-TGA(C/G)T(A/C)A-3']. The number of mismatches permitted for each consensus

binding sequence is an arbitrary number, and these sequences represent standard

"working definitions" for bioinformatic searches conducted in our laboratory.

2.1.7 Sequencing of PCR-amplified CYP2C11 5'-flank inserts

The CYP2C11 PCR-amplified products were sequenced to confirm their identity.

This was performed using a capillary-based fluorescent method by The Centre for

Applied Genomics at The Hospital for Sick Children (Toronto, Ontario).

Forplasmid: (-10.1-2C11), (-7.1-2C11), (-5.6-2C11), (-2.4-2C11), (-1.3-2C11),

(-2.4 to -1.3-2Cll)-Promoter, (-2.4 to -1.3-2Cll)-0.2, (-2.4 to -1.8-2Cll)-0.2 and

(DRE-2Cll)-0.2, the sequencing primer RVprimer3 (Promega; 5'-

CTAGCAAAATAGGCTGTCCC-3') was used to sequence PCR product inserts from the

5'- ends. This primer hybridizes upstream of the pGL3-Basic multiple cloning site and the sequencing reaction proceeds in the clockwise direction. The 3'-ends of the

CYP2C11 inserts were sequenced using GLprimer2 (Promega; 5'-

CTTTATGTTTTTGGCGTCTTCCA-3'), which hybridizes downstream of the multiple cloning site and sequences these inserts in the counterclockwise direction. Plasmid:

(-5.6-2C11), (-2.4-2C11) and (-1.3-2C11) were sequenced in the counterclockwise direction using GL-Primer2-RS (5' -CTTCATAGCCTTATGCAGTT-3'), which I designed specifically to hybridize further downstream of the multiple cloning site in pGL3-Basic in comparison to the GLprimer2 hybridization site. Since these latter three

CYP2C11 inserts are ligated into pGL3-Basic at the Ncol site (further downstream than the 3' restriction sites used for the other plasmids), GL-Primer2-RS is able to amplify this

106 ligation site, while GLprimer2 hybridizes too close to the Ncol site to allow for accurate

sequencing in this region.

The four site-directed mutant constructs, (-10.1-2C11)MUT, (-2.4-2C11)MUT,

(-2.4 to -1.3-2C11)-0.2MUT and (DRE-2C11)-0.2MUT, were sequenced in a uni

directional reaction spanning a 500-bp region which encompasses the desired DRE

mutation corresponding to positions -1539 to -1537-bp of the CYP2C11 5'-flanking

region. (DRE-2C11)-0.2MUT was sequenced using RVprimer3; (-2.4-2C11)MUT was

sequenced using GLprimer2; (-2.4 to -1.3-2C11)-0.2MUT and (-10.1-2C11)MUT were

sequenced using the following primer: 5'-AGGAGGCACAGCCTT-3', which

corresponds to positions -1827 to -1813-bp of the CYP2C11 5'-flank.

Each sequencing primer allows for the accurate sequencing of ~500-bp on each

end of the PCR product insert. However, the entire length of the longest construct,

(-10.1-2C11), was sequenced using a "walk on template" approach. This process begins

with the sequencing of ~500-bp on both the 5'- and 3'- ends of the 10.1-kb insert. Using the sequence information generated, new sequencing primers were designed to facilitate the sequencing of the next 500-bp on both ends of the insert. In this manner, the entire length of the insert was sequenced in multiple reactions performed on ~500-bp at a time on both ends.

2.2 Site-Directed Mutagenesis

Using the QuikChange site-directed mutagenesis kit (Stratagene), mutations were introduced into four CYP2C11 constructs: (-10.1-2C11), (-2.4-2C11), (-2.4 to -1.3-

2C11)-0.2 and (DRE-2C11)-0.2. The previously characterized CYP2CU-DKE3

(Bhathena et al., 2002) located at positions -1546 to -1533-bp [5'-

107 TTGCGTGCCTCCAG-3', invariant core nucleotides underlined] was targeted for mutagenesis by converting the AHR-binding core [5'-CGTG-3'] to a non-functional sequence [5'-CTAT-3'] (Yao and Denison, 1992). The forward primer was

5'-gccgcgggaatgctggaggATAGcaattaccacagg-3', and the reverse primer was

5'-cctgtggtaattgCTATcctccagcattcccgcggc-3\ in which the AHR-binding core nucleotides are capitalized and the mutated nucleotides are underlined. The mutations were confirmed by DNA sequencing and the newly generated plasmids were named:

(-10.1-2C11)MUT, (-2.4-2Cll)MUT, (-2.4 to -1.3-2C11)-0.2MUT and (DRE-2C11)-

0.2MUT.

2.3 Culture of continuous cell lines

The Hepa-lclc7 mouse hepatoma cell line and the HepG2 human hepatocellular carcinoma cell line were obtained from the American Type Culture Collection

(Manassas, VA). The 5L rat hepatoma cell line and the AHR-deficient BP8 derivative were obtained from Dr. Martin Gottlicher (Institute of Toxicology and Genetics,

Karlsruhe, Germany). oc-Minimum essential medium (MEM) and PBS were prepared by

Media Preparation Services at the University of Toronto. Cells were grown as monolayer cultures in oc-MEM supplemented with 10% FBS (Invitrogen Corporation) in an atmosphere of 5% C02 and 95% air at 37°C.

2.4 Detection of CYP1A1, CYP2C11, AHR, a-tubulin and p-actin mRNA

2.4.1 RNA isolation

Total RNA was isolated from rat liver tissue and cultured cells (5L and BP8) by the acid guanidinium thiocyanate-phenol-chloroform extraction method using TRI-

Reagent (Sigma Chemical Company, St. Louis, MO). All reagents and materials used in

108 RNA studies were ribonuclease (RNase)-free and only diethylpyrocarbonate (DEPC)-

txeated water was used. RNase-free water was purchased from Invitrogen Corporation.

For RNA isolation from cultured cells, 5L or BP8 cells (4 x 106 ) were plated per 60 mm

culture dish and cultured for 24 h. Cells were rinsed with PBS, and Tri-Reagent (1 inL)

was added. Cells were pipetted up and down and transferred to microfuge tubes. For

RNA isolation from liver tissue, Tri-Reagent (1 mL) was added to liver (-0.1 g), and

homogenized with 7 strokes using a Potter-Elvehjem apparatus.

Chlorophorm (200 uL) was added to homogenized liver tissue or pipetted

cultured cells, and tubes were mixed thoroughly and incubated at room temperature for

10 min. Samples were centrifuged at 14,000 rpm for 15 min at 4°C on a table top

centrifuge. The upper aqueous phase was carefully transferred to a new microfuge tube.

Isopropanol (500 uL) was added to precipitate the RNA. Samples were then centrifuged

at 14,000 rpm for 10 min at 4°C. The supernatant was decanted, and the RNA pellet

washed with 75% ethanol in DEPC-treated water (1 mL). Samples were centrifuged at

14,000 rpm for 5 min at 4°C. The supernatant was removed and the pellet air-dried for

10 min. The pellet was then resuspended in DEPC-treated water (50-100 uL), and incubated at 55°C for 20 min. To remove any possible genomic DNA contamination, all

RNA samples were treated with DNase I (20 U; GE Healthcare Bio-Sciences, Baie d'Urfe, QC) for 25 min at 37°C, followed by a 10 min incubation at 55°C to inactivate the enzyme. RNA yield and purity were determined by measuring the A260/A280 ratio, with a ratio of > 1.7 considered acceptable. The RNA integrity was assessed by comparing the relative intensities of the 28S and 18S ribosomoal RNA (rRNA) bands as visualized on ethidium bromide-stained 1.5% agarose gels (Fig. 2.2). RNA was

109 visualized on an UV light box and integrity was confirmed by the presence of two

distinct bands representing the 28S and 18S rRNAs, and a greater intensity of the 28S

band. RNA samples were stored at -70°C until use.

28S

18S

Figure 2.2. Visualization of 28S and 18S rRNA in total RNA samples prepared by the acid guanidinium thiocyanate-phenol-chloroform extraction method. RNA was isolated from (A) 5L (lanes 1, 2) and BP8 (lanes 3 ,4) hepatoma cells or (B) livers of rats receiving hydrodynamics-based plasmid injections. Representative RNA samples (2.5 ng) were separated on 1.5% ethidium bromide-stained agarose gels and visualized on an UV light box.

110 2.4.2 Reverse-transcription reactions

Reverse-transcription (RT) was performed on extracted RNA samples. For

primer annealing, RNA (1 \ig), oligo(dT)i5 (2 ug; Roche Diagnostics), and DEPC-treated

water (4 uL) were incubated at 60°C for 5 min. For primer extension, samples were

incubated in a final volume of 40 uL with Moloney murine leukemia virus (MMLV)- reverse transcriptase (400 U; Invitrogen Corporation), 10 mM dithiothreitol, IX RT buffer (50 mM Tris; 75 mM KC1; 3 mM MgCl2), 1 mM of each dNTP and RNA Guard

(60 U; GE Healthcare Bio-Sciences). Reactions proceeded for 60 min at 37°C, followed by incubation at 70°C for 10 min. RT products were stored at -20°C until use.

2.4.3 Detection of mRNA by RT-PCR

CYP2C11, a-tubulin, AHR and P-actin mRNA levels were analyzed in rat hepatoma 5L and BP8 cells by reverse transcription coupled polymerase chain reaction (RT-PCR). CYP1A1 and P-actin mRNA levels were analyzed in RNA samples isolated from rat liver tissue. PCR primer sequences and cycling conditions are shown in

Table 2.2. The 50-uL PCR reactions contained input cDNA derived from 50 ng of RNA,

Taq DNA polymerase (2.5 U; Invitrogen Corporation), IX PCR buffer (20 mM Tris; 50 mM KC1; 3 mM MgCl2), an optimized primer concentration (0.5 uM for CYP2C11; 0.4 uM for a-tubulin; 0.2 uM for AHR,CYP1A1, or p-actin), and 0.2-0.4 mM of each dNTP.

CYP1A1 and P-actin cDNA were amplified in separate reactions; AHR/ P-actin assays and CYP2C11/a-tubulin assays were run as duplex PCR reactions. All PCR reactions began with a hot start phase, typically 5 min at 95°C, and ended with a final extension at

72°C for 7 min.

Ill TABLE 2.2. Primer sequences and thermal cycling conditions used for analysis of steady-state mRNA levels by RT-PCR.

Target Forward and reverse PCR primer sequences PCR product size Thermal cycling parameters Reference (denature; anneal; extend) x cycles

CYP2C11 5'-GTATCGCTGTCATCCATAC (FP) 412-bp (94°/30 s; 56°/60 s; 72760 s) x 34 Pampori and Shapiro 2000

5'-GGAAATGGGGATATGTG (RP)

a-tubulin 5'-TGCTGCCATTGCCACCATCA (FP) 350-bp (94730 s; 56760 s; 72760 s) x 34 El-Husseini et al. 1995

5'-CTCACCCTCACCCTCCACCG (RP)

AhR 5'-AGGGAGGTTAAAGTATCTTCATGGAC(FP) 917-bp (94720 s; 54720 s; 72740 s) x 28 Franc et al. 2001

5'-TCCCTAGGTTTCTCATGATGCTATAC (RP)

p-actin 5'-ACCGTGAAAAGATGACCCAG (FP) 688-bp (94720 s; 52720 s; 72740 s) x 17 or x 28 Franc etal. 2001

5'-GAGCCACCAATCCACACAG (RP)

CYP1A1 5'-ACGTTATGACCACGATGACC (FP) 672-bp (94720 s; 52720 s; 72740 s) x 17 Franc etal. 2001

5'-AGGCCGGAACTCGTTTG (RP)

FP, forward primer; RP, reverse primer

112 PCR products were separated on 6% non-denaturing polyacrylamide gels. Each

gel contained 6.5 mL water, 9 mL 5X TBE (4.5 M Tris-borate, 0.01 M EDTA, pH 8.3),

9 mL acrylamide/BIS (30% T, 2.7% C, where % T represents the total percentage [w/v]

of acrylamide monomer and N,N'-methylenebisacrylamide (BIS) crosslinker, and % C

represents the amount of BIS crosslinker expressed as a percentage of the sum of

acrylamide monomer and BIS), 0.45 mL 10% ammonium persulfate and 22.5 fiL

N,N,N\N'-tetramethylethylenediamine (TEMED). Gels were placed in a Gibco BRL

V-16-2 Vertical Gel Electrophoresis System and PCR products fractionated at 130 V for

1.5 h in IX TBE buffer. Gels were stained with Vistra Green (1:10,000 dilution in IX

TBE; GE Healthcare Bio-Sciences), by gentle shaking for 30 min at room temperature.

Each gel was imaged using the STORM Phosphorimager (Molecular Dynamics,

Sunnyvale, CA). The presence of CYP1 Al mRNA in in vivo studies was used as a

qualitative positive control for MC treatment and not subjected to quantitative analysis.

Similarly, CYP2C11 and AHR mRNA signals in 5L and BP8 cells were not subject to

quantitative analysis.

2.5 Transient transfection in continuous cell lines

2.5.1 Optimization of transfection efficiency

For each cell line, in situ P-galactosidase staining was used to determine the

optimal quantities of plasmid DNA and Superfect transfection reagent (Qiagen Inc.) needed to yield maximal transfection efficiency. HepG2, Hepa-1, 5L and BP8 cells were grown in 6-well plates (3x10 cells/well). Twenty-four hours after plating, cells were transfected with 1.5-6 ug of pSV-P-galactosidase plasmid in combination with 4-12 pL of Superfect transfection reagent in all possible permutations. After 24 h, cells were

113 rinsed with PBS (1 mL per well), and fixed with 3% formaldehyde in PBS. Fixed cells were incubated for 15 min at room temperature. Formaldehyde was removed and cells rinsed with PBS. Cells were then treated with 500 uL of X-gal (5-bromo-4-chloro-3- indolyl-P-galactopyranoside) solution (0.4 ug/uL X-gal; 4 mM MgCk; 4 mM potassium ferrocyanate; 4 mM potassium ferricyanate in PBS), and incubated at room temperature.

After 3 h, X-gal solution was removed and 70% glycerol (1 mL) was added to each well.

Since P-galactosidase converts X-gal into a blue compound, cells expressing the pSV-0- galactosidase plasmid turned blue following treatment with the X-gal solution. The transfection efficiency was determined by the number of blue cells (those expressing the pSV-P-galactosidase plasmid) as a percentage of the total number of cells counted under the highest magnification of a light microscope.

2.5.2 Transient transfection

Cells were seeded in 12-well plates (1.5 x 105 cells/well) and cultured for 24 h to

-40% confluency. For transfections, firefly luciferase constructs (1.3 ug per well for

HepG2, Hepa-1 and 5L cells, and 2.8 ug per well for BP8 cells), the pRL-TK Renilla plasmid (0.1 ug per well for HepG2, Hepa-1 and 5L cells, and 0.2 ug per well for BP8 cells), and the appropriate volume of Superfect were incubated in sterile microfuge tubes for 10 min in serum-free medium (75 uL). Medium containing 10% FBS (400 uL) was then added to the plasmid DNA/Superfect mixture. Cells were washed with PBS and incubated with the transfection solution for 3 h under normal growing conditions. The transfection solution was then aspirated, cells were gently washed with PBS, and medium containing 10% FBS was added to each well (0.5 mL).

114 2.5.3 Chemical treatment of continuous cell lines

Twenty-four h after transfections, cells were treated with vehicle [0.1% dimethyl sulfoxide (DMSO)], 10 nM TCDD (Wellington Laboratories Inc., Guelph, Ontario,

Canada), or 1 uM MC (Aldrich Chemical Company, Milwaukee, WI; 98% purity). In the

AHR antagonist studies, cells were treated with vehicle (0.1% DMSO), TCDD (1 nM or

10 nM), cc-NF (1 uM; Sigma Chemical Company), or a combination of TCDD and CX-NF.

2.5.4 Isolation of cell extracts

Cells were harvested after 24 h of treatment. Medium was aspirated and each well rinsed twice with PBS (1 mL). Cell extracts were prepared in IX Passive Lysis

Buffer (250 uL per well; Promega), and incubated by gentle shaking for 20 min at room temperature. Cell lysates were then transferred to microfuge tubes and cell debris removed by centrifugation at 12,000 rpm for 2 min. Dual luciferase assays were performed on the supernatant as described in Section 2.10.1.

2.5.5 Bradford protein assay

The method of Bradford (1976) was used to determine the protein concentration of cell lysates and microsomal protein. This method is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Using 0 ug, 10 ug, 20 jug,

30 ug, 40 ug and 50 ug of BSA, a standard curve was generated and used as a reference for protein determinations. Cell lysates (45 uL) and the BSA standards were diluted with

2.5 mL of vacuum-filtered Bio-Rad reagent (1 part Bio-Rad dye protein reagent concentrate diluted with 4 parts water; Bio-Rad Laboratories Inc., Hercules, CA), while microsomal protein (3 pJL) was diluted with 5 mL Bio-Rad reagent. Samples were

115 incubated for 14 min. Using a background reference of 465 nm, the absorbance at 595 nm was read on a Beckman DU-65 spectrophotometer. The Quant II Quadratic Soft-Pac

Module was used to fit a standard curve via non-linear regression.

2.6 Animals and treatment

Male Fischer 344 rats (7-9 weeks of age; 150-200 g) were purchased from

Charles River Canada (St.-Constant, Quebec, Canada). Rats were fed Harlan Teklad rodent laboratory chow and water ad libitum. All animal experimentation was approved by the University of Toronto Animal Care Committee and rats were cared for in accordance with guidelines of the Canadian Council on Animal Care. Rats were housed two animals per cage, and exposed to a 12 h-light cycle followed by a 12 h-dark cycle.

Animals were acclimatized to living conditions in the Division of Comparative Medicine at the University of Toronto for 7 days before experimental procedures were begun.

Rats were placed under a heat lamp for -20 min prior to high-volume injections to facilitate the procedure by dilating the tail vein. Prior to hydrodynamics-based injections, rats were anesthetized by isoflurane inhalation (5% isoflurane in oxygen for induction;

2% isoflurane in oxygen for maintenance). Fig. 2.3 shows a rat from my study receiving the hydrodynamics-based injection. Rats received a single i.p. injection of either MC

(80 mg/kg) or an equivalent volume of vehicle (sterile Mazola corn oil). Rats were then injected via the tail vein with a firefly luciferase plasmid and the pRL-TK Renilla luciferase plasmid, dissolved in sterile lactated Ringer's solution (130 mM Na; 4 mM K;

1.4 mM Ca; 109 mM CI; 28 mM lactate; Baxter Co., Mississauga, Ontario, Canada) at a volume equivalent to 8% of the rat's body weight. Injection time never exceeded 10 s.

The Renilla luciferase construct was used to normalize for hepatic DNA transfection

116 efficiency. Tail vein injections were performed using a 22-guage, 1 inch long catheter connected to a 25 rnl-capacity syringe. With exceptions noted below, each rat received

20 ug of firefly luciferase plasmid and 1.75 |ig of Renilla luciferase plasmid DNA per 1 ml of Ringer's solution injected. Rats injected with the (-5.6-2C11) plasmid (both 24 h and 72 h time-points), or the (-10.1-2C11) plasmid (6 h time-point only), received 5 ug of firefly luciferase plasmid and 0.5 ug of Renilla luciferase plasmid DNA per 1 mL of

Ringer's solution. These plasmid quantities fall within optimal conditions required for high-level reporter gene expression in the liver (Liu et al., 1999; Maruyama et al., 2002).

Figure 2.3. Co-administration of firefly and Renilla luciferase plasmid DNA for hydrodynamics-based hepatocyte transfection. Anesthetized rats were injected via the tail vein with firefly luciferase and Renilla luciferase DNA constructs dissolved in sterile lactated Ringer's solution. The volume of the DNA-containing solution was equivalent to 8% of the rat's body weight and injected in <10 s. pRL-TK {Renilla luciferase) was used to normalize for transfection efficiency into the liver. Rats were euthanized by decapitation 6 h, 24 h or 72 h post-injection.

117 Rats were euthanized by decapitation at 6 h, 24 h or 72 h following high-volume injections. The liver of each rat was perfused in situ with ice-cold 1.15% KC1. Livers were excised and weighed. Small pieces of individual livers (~0.1 g) were frozen in liquid nitrogen and stored at -70°C until RNA isolations were performed. Fresh liver

(1.5 g) was isolated from the left-lobe of the liver and used for subsequent in vivo dual luciferase assays as discussed in Section 2.10.2.

The remaining liver (~8 g) was homogenized in 4 volumes of cold phosphate- buffered KC1 (1.15% KC1; 10 mM potassium phosphate, pH 7.4) with a Potter-Elvehjem apparatus. Microsomes were isolated by differential centrifugation (McCluskey et al.,

1986). Homogenate samples were centrifuged in a pre-cooled JA-17 rotor at 9,000 x g for 22 min at 4°C. The supernatant was collected and centrifuged at 106,000 x g for 60 min at 4°C. Microsomal pellets were resuspended in phosphate-buffered KC1 (5 mL), and re-centrifuged at 106,000 x g for 60 min at 4°C. The resulting microsomal pellets were re-suspended in storage buffer [10 mM Tris (pH 7.4); 20% glycerol; 1 mM EDTA], frozen in liquid nitrogen and stored at -70°C. Microsomal protein concentrations were determined by the method of Bradford (1976) as described in Section 2.5.5.

2.7 In vivo AHR antagonist studies

To determine whether the AHR is involved in the in vivo suppression of the

CYP2C1 i-luciferase constructs, I attempted to antagonize the receptor in vivo using cc-

NF. Male Fischer 344 rats (7-9 weeks of age) were weighed and dosed daily with one or more of the following treatments: vehicle corn oil (3 or 4 days, i.p.), MC (50 mg/kg or

80 mg/kg; single i.p. injection), or a-NF (80 mg/kg; 3 or 4 days, i.p). I implemented two study designs in attempts to antagonize the AHR in vivo (Tables 2.3,2.4). Doses of

118 a-NF and study time-lines were based on prior papers in which this chemical was used to antagonize the AHR in vivo and estrogen-related endpoints were measured (Mattison and

Thorgeirsson, 1979; Thompson et al., 2005). Rats were injected with a-NF 24 h prior to treatment with vehicle or MC, in attempts to antagonize the AHR before aromatic hydrocarbon treatment was administered (Mattison and Thorgeirsson, 1979). Rats were euthanized by decapitation 24 h following the last i.p. injection, which coincides with

48 h or 72 h following MC or vehicle treatment.

CYP1A1 protein and mRNA levels were assessed qualitatively to determine the antagonistic ability of a-NF on MC-induced in vivo AHR activation.

2.8 Analysis of CYP2C11 and 0-actin mRNA by real time RT-PCR

Primers and probes used for real-time analyses were designed using the Primer 3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). For each gene product amplified, the probe and one primer were designed to span an intron of the gene to ensure that any contaminating genomic DNA would not be amplified. Primer specificity and in silico PCR analyses were assessed using the PUNS database (Boutros and Okey, 2004). The probability of primers and probes forming secondary structures such as hairpin loops, homo-dimers and hetero-dimers was calculated using the online

SciTools option provided by Integrated DNA Technologies. All primers and probes were synthesized by Integrated DNA Technologies Inc., and these sequences are listed in

Table 2.5. Molecular probes were labeled with the reporter fluorescent dye 6- carboxyfluorescein at the 5'-end, and the quencher fluorescent dye Iowa Black at the 3'- end. Optimization studies were employed to determine the concentration of primers and probes that would yield the highest level of fluorescence at the lowest cycle in the real-

119 TABLE 2.3. Study design 1: In vivo AHR antagonist time-line.

-24 h Oh 24 h 48 h Group 1 (n=4) Vehicle Vehicle Vehicle Euthanized

Group 2 in—~~r) Vehicle MC Vehicle Euthanized

Group 3 (n=4) cc-NF cc-NF cc-NF Euthanized

Group 4 (n=4) oc-NF cc-NF + MC cc-NF Euthanized

Rats received either 80 mg/kg MC, 80 mg/kg cc-NF, the combination of both MC and cc-NF, or the equivalent volume of vehicle corn oil.

TABLE 2.4. Study design 2: In vivo AHR antagonist time-line.

-24 h Oh 24 h 48 h 72 h Group 1 (n=l) Vehicle Vehicle Vehicle Vehicle Euthanized

Group 2 (n=3) Vehicle MC Vehicle Vehicle Euthanized

Group 3 (n=3) oc-NF oc-NF + MC oc-NF cc-NF Euthanized

Rats received either 50 mg/kg MC, the combination of both MC and 80 mg/kg cc-NF, or the equivalent volume of vehicle corn oil.

120 TABLE 2.5. Primer and probe sequences used for measurements of steady-state mRNA levels in rat liver by real-time quantitative RT-PCR.

CYP2C11 p-actin

Forward Primer 5'-1219-TTTGACCCTGGTCACTTTCT-1238-3' 5'-357-GACCCAGATCATGTTTGAGACCTTC-381-3'

Reverse Primer 5'-1318-GGGCTTCTCCTGCACATATC-1299-3' 5'-465-GGAGTCCATCACAATGCCAGTG-444-3'

Probe 5'-1272-CTCTTTCCTGCTGAGAATGGCATAAAG-1298-3' 5'-441-ACGACCAGAGGCATACAGGGACAACACAG-413-3'

Product Size 10O-bp 109-bp

Reference New design Tijet et al., 2006

121 time RT-PCR reaction for each gene product. Primer and probe sets were tested at final concentrations of 0.2 pJVl, 0.3 |iM and 0.4 |iM in separate reactions.

Prior to real-time RT-PCR assays, CYP2C11 and P-actin cDNAs were amplified in conventional PCR reactions using the primers designed for use in real-time RT-PCR assays. The specificity of these primers was confirmed by the observation of a single band of predicted size when PCR products were run on a 6% non-denaturing polyacrylamide gel, stained with Vistra Green and imaged using the STORM

Phosphorimager (Fig. 2.4).

RNA was reverse-transcribed as described in Section 2.5.2. Real-time PCR was performed on the 3V1X4000 real-time PCR system (Stratagene). Each 25-uL reaction contained a final concentration of 0.3 uM (CYP2C11) or 0.4 uM (P-actin) primers,

0.2 uM (CYP2C11) or 0.3 uM (P-actin) probe, input cDNA derived from 50 ng of RNA and the 2X Brilliant QPCR Master Mix (Stratagene). A standard curve consisting of a

10-fold dilution series derived from pooling cDNA from several rats was generated with each real-time RT-PCR run to determine the efficiency. Each dilution used in the standard curve was performed in triplicate. Representative standard curves for CYP2C11 and P-actin are displayed in Fig. 2.5. The efficiency (E) for each standard curve was calculated using the following equation: E=(10-1 slope-i). The efficiency of the reaction was considered to be acceptable if it was in the range of 90-110%. Efficiency can also be expressed as E=10"1/slope, which would generate the numerical value 2, in the case of

100% efficiency, which translates as a doubling of the amplicon at each cycle. Cycling conditions consisted of an initial 10 min denaturation at 95°C, followed by 40 cycles of:

95°C/ 30 s; 55°C/ 60 s; 72°C/ 30 s.

122 200-bp

100-bp

CYP2C11 p-actin 100-bp 109-bp

Figure 2.4. CYP2C11 and p-actin amplicons from RT-PCR amplification using real-time RT-PCR primers specific for each gene. RT-PCR analysis of CYP2C11 mRNA (lane 1) and the normalization control p-actin mRNA (lane 2) using total RNA isolated from a vehicle-treated male Sprague Dawley rat as a template for reverse transcription followed by conventional PCR amplification. PCR products were amplified for 35 cycles and separated on a 6% polyacrylamide gel, stained with Vistra Green.

123 3

1 S 5

U B P-actin Standard curve

Figure 2.5. Representative CYP2C11 and p-actin standard curves generated by real-time RT-PCR. Standard curves consisting of 10-fold dilution series were generated for (A) CYP2C11 and (B) the normalization control p-actin, from pooled cDNA samples, performed in triplicate replicates for each dilution. The efficiency of amplification was determined using the equation: E=(10"1/slope-l). The efficiency of the reaction was considered to be acceptable if it was in the range of 90-110%. These two representative standard curves were obtained from the Stratagene MX4000 real-time quantitative detection software.

125 Normalized expression (NE) was determined for each sample using the calculation: t^tf* &***»/&* (Pmcii)^ where E is the efficiency of the PCR

amplification as calculated by the equation E=10"1/Slope, determined from the standard curve for each gene product, and C, is the cycle at which threshold fluorescence is detected (Simon, 2003).

2.9 Immunoblot anlaysis

Microsomal protein samples from each rat were thawed on ice. Each sample was prepared at a final protein concentration of 0.5 |ig/uL by diluting the microsomal protein in IX sample loading buffer [62.5 mM Tris-HCl (pH 6.8); 10% glycerol; 2% sodium dodecyl sulfate (SDS); 5% P-mercaptoethanol; 0.001% bromophenol blue dye] in a final volume of 200 jxL. Samples were placed in a 100°C heat block for 4 min, then allowed to cool to room temperature and stored at -20°C until use. Microsomal protein (5 jig for

CYP1A1; 3.5 |ig for CYP2C11 analyses) from each liver was resolved by SDS- polyacrylamide gel electrophoresis. The 10% polyacrylamide separating gel contained:

2.66% BIS crosslinker; 375 mM Tris-HCl (pH 8.8); 0.1% SDS; 0.05% ammonium persulphate; and 0.1% TEMED. The 4% polyacrylamide stacking gel was compromised of: 2.66% BIS crosslinker; 125 mM Tris-HCl (pH 6.8); 0.1% SDS; 0.05% ammonium persulphate; and 0.1% TEMED.

Gels were run in a Bio-Rad Mini-Protein II gel apparatus electrophoresis tank

(Bio-Rad) filled with IX running buffer [25 mM Tris-HCl (pH 8.3); 192 mM glycine;

0.1% SDS]. Microsomal protein was resolved at 150 V for ~1 h, then transferred to nitrocellulose membranes (Hybond-ECL; GE Healthcare Bio-Sciences). The transfer was performed in the Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell containing

126 IX transfer buffer [25 mM Tris-HCl (pH 8.3); 192 mM glycine; 20% methanol] run at

100 V for ~1 h. Following the transfer, membranes were stained with Ponceau-S (Sigma-

Aldrich) to observe the transfer efficiency. To remove the stain, membranes were rinsed

with IX TNT [20 mM Tris (pH 7.6); 137 mM NaCl; 0.1% Tween-20] for -15 min. Blots

were incubated overnight at 4°C in 5% skim milk powder dissolved in IX TNT to block

non-specific binding sites. The next day, membranes were rinsed three times for 15 min

with IX TNT. They were then incubated for 1 h with primary antibody. For CYP1A1

detection, mouse monoclonal antibody 1-31-2 (Dr. H. V. Gelboin, National Cancer

Institute, Bethesda, MD) was used at a dilution of 1:5000 in IX TNT containing 5% skim milk powder. For CYP2C11 protein detection, a rabbit anti-CYP2Cl 1 polyclonal antibody [Dr. E. T. Morgan, Emory University, Atlanta, GA; (Morgan et al., 1985b)] was used at a dilution of 1:5000 in IX TNT containing 5% skim milk. Membranes were rinsed for 15 min, three times in IX TNT, then incubated for 1 h with secondary antibody dissolved in IX TNT containing 5% skim milk powder. The secondary antibody used for

CYP1A1 detection was a sheep anti-mouse Ig-horseradish peroxidase conjugate (GE

Healthcare Bio-Sciences) at a dilution of 1:5000. The secondary antibody used for

CYP2C11 detection was a donkey anti-rabbit Ig-horseradish peroxidase conjugate (GE

Healthcare Bio-Sciences) at a 1:5000 dilution. Following the 1 h incubation, membranes were rinsed for 15 min, three times in IX TNT. An enhanced chemiluminescence system

(ECL; GE Healthcare Bio-Sciences) was used for protein detection by incubating the membranes for 1 min in the ECL solution. The films were developed using the

SRX101A developer (Konica Minolta, Wayne, NJ). Films were scanned on an HP

Scanjet 3970 scanner (Hewlitt-Packard Company, Palo Alto, CA). Relative quantitation

127 was performed using IPLabGel software (Signal Analytics, Vienna, VA). CYP1A1

immunoblots were used as a qualitative positive control for MC treatment and not

subjected to quantitative analyses. CYP2C11 quantitative analyses were performed

under conditions that yielded a linear relationship between amount of microsomal protein

and immunoreactive signal intensity (Fig. 3.11).

2.10 Reporter gene assays

2.10.1 Dual luciferase reporter assays for in vitro transfections

The dual luciferase reporter assay system (Promega) was used to measure the

luminescent signal from both luciferase reporter enzymes (firefly and Renilla) present in

cell lysates. Light is emitted by the oxidation of beetle luciferin in a reaction that

requires oxygen, magnesium and ATP. This reaction is catalyzed by firefly luciferase.

Renilla luciferase catalyzes the oxidation of the substrate coelenterazine to produce

coelenteramide and light. Luciferase Assay Reagent II (LAR II) was prepared by adding

Luciferase Assay Buffer II (10 mL) to lyophilized Luciferase Assay substrate (which contains luciferin). Cell lysates (10 uL; corresponding to -0.01 mg of protein) were mixed with LAR II (50 uL) at room temperature. Firefly Luciferase activity was measured. Quenching of firefly luciferase activity and concurrent activation of Renilla luciferase activity was accomplished by the addition of Stop & Glo reagent (50 uL; 1 volume of 50X Stop & Glo substrate diluted in 50 volumes of Stop & Glo buffer). The sample was vortexed and Renilla luciferase activity measured. Firefly luciferase activity was normalized to Renilla luciferase activity. Luminescence was read using a TD-20/20 luminometer (Turner Designs, Inc., Sunnyvale, CA).

128 2.10.2 Dual luciferase reporter assays for in vivo transfections

The left-lobe of the liver (1.5 g) was homogenized in IX Passive Lysis Buffer

(1 mL). Homogenates were centrifuged at 4°C for 25 min at 14,000 rpm (Liu et al.,

2001). The supernatant was stored at -70°C until dual luciferase assays were performed.

Thawed supernatant (20 uL) was mixed with 100 uL LARII. Firefly luciferase activity was measured using a TD-20/20 luminometer. Stop & Glo reagent (100 uL) was added, the sample vortexed and Renilla luciferase activity measured. Firefly luciferase activity was normalized to Renilla luciferase activity.

2.11 Statistical analysis

2.11.1 Statistical analysis for in vitro studies

Data are presented in arbitrary normalized luciferase units as mean ± S.D. of three determinations. All statistical analyses were performed on the raw data and not on the percentage control data presented in the figures. Data were analyzed initially using a randomized design two-way ANOVA to identify significant drug and plasmid effects. If a significant drug effect was identified, the following analyses were performed. In experiments involving more than two drug treatments, a randomized design one-way

ANOVA followed by post hoc Newman-Keuls test was performed to identify the drug treatments producing effects that differed from vehicle control. In experiments involving two drug treatments, unpaired Student's r-tests (two-tailed) were performed to determine significant differences from vehicle control. In all cases, a result was considered to be statistically significant if/? < 0.05.

129 2.11.1 Statistical analysis for in vivo studies

Data are presented as a percentage of the mean for the vehicle-treated controls

and each bar represents the mean + S.D. of determinations from three or four rats. All

statistical analyses were performed on the raw data rather than the percent control data presented in the figures. Student's /-tests were performed to determine whether the mean value for MC-treated rats differed from the mean value for the corresponding vehicle control rats at each time-point. Statistical significance of linear correlation data was determined by the Pearson correlation test. In all cases, a result was considered to be statistically significant ifp < 0.05.

130 3.0 RESULTS

3.1 Cloning, sequencing and bioinformatics

3.1.1 Bioinformatic analysis of putative TF binding sites located in the CYP2C11 5'- flanking region

Bioinformatic analysis of the proximal 20-kb stretch of the CYP2C11 5'-flanking region revealed that the 10.1-kb section closest to the start site of transcription is particularly enriched for DRE-like motifs (also known as AHRE-I elements). The distribution of putative sites for TFs of interest within the proximal 10.1-kb region of the

CYP2C11 5'-flank is shown in Fig. 3.1. This large region also contains one perfect match to the AHRE-II consensus sequence (Boutros et al., 2004; Sogawa et al., 2004), a novel site first identified in the CYP1A2 gene, that recruits the AHR/ARNT complex as a co-activator. Also included in this 10.1-kb region are putative sites for TFs involved in

GH control of this gene (STAT5, HNF-3; Park and Waxman, 2001; Timsit and Riddick,

2002), and suppression of this gene by inflammatory cytokines (NF-KB; Iber et al., 2000).

The proximal 10.1-kb section of the CYP2C11 5'-flanking region also contains five perfect matches to the AP-1 consensus sequence. Although the role of AP-1 in CYP2C11 regulation is unclear, AHR agonists can increase the expression of AP-1 components (c- fos, c-jun, junB, junD) and increase binding of AP-1 to DNA (Hoffer et al., 1996; Puga et al., 2000a). As well, the AHR can influence gene transcription by interacting with AP-1 components at a variety of response elements (Vasiliou et al, 1995; Gillesby et al.,

1997).

131 •AHRE-I (or DRE) HNF-KB HNF-3 AHRE-II STAT-5

Distance upstream of CYP2C11 transcription start site (bp)

Figure 3.1. Location of putative transcription factor binding sites in the proximal 10.1-kb of the CYP2C11 5'-flanking region. Consensus sequences for the listed transcription factor binding sites of interest were defined as follows: AHRE-I (DRE): must have the invariant core CGTG plus S2 mismatches from the AHR binding consensus [5'- GCGTGNN(A/T)NNN(C/G)-3'] defined by Yao and Denison (1992). NF-KB: SI mismatch from consensus [5'-GGGANT(C/T)(C/T)CC-3']. HNF-3:

132 3.1.2 Luciferase reporter constructs: confirmation by diagnostic restriction digestion

The identity of the novel luciferase constructs was first confirmed by analyses of restriction digestion fragments separated on agarose gels. The observation of DNA fragments with mobility consistent with predicted sizes suggests that CYP2C11 genomic sequence was correctly ligated into luciferase reporter plasmids (Fig. 3.2A-E). The tables below the gels in Fig. 3.2 show the expected sized fragments following digestion with the indicated restriction endonucleases for each particular CYP2C1 i-luciferase construct.

3.1.3 Luciferase reporter constructs: confirmation by DNA sequencing

The identity of the novel luciferase constructs was further confirmed by DNA sequencing. The complete [in the case of (-10.1-2C11)] or partial (all other constructs) sequence of PCR product inserts was aligned against the known CYP2C11 5'-flanking genome sequence from the UCSC database using a Blast-like alignment tool (BLAT;

Kent, 2002). In all cases, a 98-100% match to the database genomic sequence was confirmed.

133 12 3 4 5 6 7

5,000-bp 3,000-bp

1,500-bp 1,000-bp

500-bp

Lane Restriction Predicted endonucleases fragment sizes < hi) >

l.(-2.4-2Cll) MluVNcol 2,411 + 4,747

2. (-1.3-2C11) MluVNcoI 1,323 + 4,747

3. (-0.2-2C11) MluVNcol 217 + 4,747

4. (-2.4to-1.3-2Cll)-0.2 MluV SacI 1,042 + 4,960

5. (-2.4 to -1.3-2C1 l)-Promoter MluV BgM 1,042 + 4,989

6. (-2.4to-1.8-2Cll)-0.2 MluV SacI 532 + 4,960

7. (DRE-2Cll)-0.2 MluV SacI 525 + 4,960

134 B

7,126-bp

5,090-bp

4,072-bp

Lane Restriction endonucleases Predicted fragment sizes (bp)

l.(-5.6-2Cll) MluVNcol 4,747 + 5,633

2.(-7.1-2Cll) MM 4,818 + 7,084

3. (-10.1-2C11) MluV Sad 4,814+10,081

135 (-5.6-2C11)

7,126-bp 5,090-bp

3,054-bp

1,636-bp

Lane Restriction endonucleases Approximate predicted fragment sizes (kb) 1. NcolIMM 4.7 + 5.6

2. Ncol 10.3

3. Hindill 8.0+1.8+0.5

4. Bglll 10.2 + 0.1

136 D (-7.1-2C11) 1 2

10,000 bp 7,000 bp 5,000 bp 3,000 bp

1,500 bp

Lane Restriction endonucleases Approximate predicted fragment sizes (kb) 1. MM 4.8 + 7.1

2. Bsal 6.3 + 4.3 + 1.3

137 E (-10.1-2C11) 1 2 3

23,130-bp 9,416-bp 7,126-bp 6,557-bp 5,090-bp

3,054-bp

2,036-bp

Lane Restriction endonucleases Approximate predicted fragment sizes (kb) 1. Mlull Sad 10.1+4.8

2. uncut (supercoiled) 14.9

3. MM 14.9

4. Sad 14.9

5. Bsal 6.6 + 4.3 + 2.7+1.3

Figure 3.2. Diagnostic restriction digestion analysis of CYP2C77-luciferase reporter constructs. Reactions (20 uL) contained 0.5-1 ug plasmid DNA, 2 uL of 10X NE Buffer 1-4 (as recommended for each enzyme used), restriction endonucleases (10-20 U) and water. Reactions were incubated for 1-1.5 h at 37°C and separated on 0.8-1.5 % agarose gels. The restriction enzymes and predicted fragment sizes following restriction digestion are shown in a table below each gel. The sizes of the DNA ladder fragments are shown beside each gel. (A) Digestion of (-2.4-2C11), (-1.3-2C11), (-0.2-2C11), (-2.4 to -1.3- 2Cll)-0.2, (-2.4 to 1.3-2Cll)-Promoter, (-2.4 to -1.8-2Cll)-0.2 and (DRE-2Cll)-0.2; (B) Digestion of the three longest CYP2C11 constructs containing extended stretches of 5'-flanking sequence: (-10.1-2C11), (-7.1-2C11) and (-5.6-2C11); (C) Digestion of (-5.6-2C11); (D) Digestion of (-7.1-2C11); and (E) Digestion of (-10.1-2C11).

138 3.2 In vitro studies

3.2.1 Optimization of transfection efficiency

For HepG2, Hepa-1 and 5L cells, the combination of Superfect transfection

reagent and quantity of pSV-P-galactosidase plasmid DNA yielding the highest

transfection efficiencies was found to be 4 uL and 1.5 (Xg, respectively. In BP8 cells,

8 uL of Superfect reagent and 3.0 (ig of pS V-p-galactosidase plasmid yielded optimal

transfection efficiency. High transfection efficiencies were obtained with human HepG2

cells (23%) and mouse Hepa-1 cells (26%); however, the rat 5L and BP8 hepatoma cells proved more difficult to transfect with efficiencies between 3.5-4%.

3.2.2 CYP2C11 mRNA levels in rat hepatoma 5L and BP8 cells

Using RNA isolated from the 5L rat hepatoma cell line as well as the AHR- deficient BP8 variant (Weiss et al., 1996), RT-PCR assays were run as duplex reactions with both CYP2C11 and a-tubulin cDNA being amplified simultaneously in a single reaction tube. Endogenous CYP2C11 mRNA was not detected in 5L and BP8 cells following 35 cycles of PCR amplification (Fig. 3.3). This is consistent with the known loss of expression of several constitutive P450s in continuous hepatoma cell lines. As a positive control for the presence of CYP2C11 mRNA, the PCR was performed on reverse-transcribed RNA isolated from a corn oil-treated male Sprague-Dawley rat. A

PCR product displaying mobility consistent with the predicted size of 412-bp was observed. All samples displayed the presence of a PCR product corresponding to the expected size of the a-tubulin amplicon (350-bp).

3.2.3 AHR mRNA levels in rat hepatoma 5L and BP8 cells

RT-PCR assays were run as duplex reactions containing both AHR and P-actin

139 500-bp CYP2C11 400-bp 412-bp

a-tubulin 300-bp 350-bp

Figure 3.3. Endogenous CYP2C11 mRNA is undetectable in BP8 and 5L cells. RT-PCR analysis of CYP2C11 mRNA and the internal control a-tubulin using RNA isolated from BP8 (lane 1) and 5L (lane 2) cells. PCR products were amplified for 35 cycles and separated on a 6% polyacrylamide gel, stained with Vistra Green. Male Sprague-Dawley rat cDNA was used as a positive control for the presence of CYP2C11 mRNA (lane 3). Lanes 4-6 are negative controls corresponding to the PCR reaction performed with no cDNA input (lane 4); no MMLV reverse transcriptase added to the RT reaction (lane 5); and no RNA added to the RT reaction (lane 6).

140 primers. This analysis confirmed the lack of endogenous AHR mRNA in BP8 cells, and

its presence in 5L cells (Fig. 3.4A, inset) following 29 cycles of amplification. An AHR

amplicon displaying mobility consistent with the predicted size of 917-bp was observed.

A PCR product corresponding in size to the expected P-actin amplicon (688-bp) was

detected in all samples.

3.2.4 Transient transfections in 5L, BP8 and Hepa-1 cells

Rat 5L and BP8 hepatoma cells were transfected with my newly generated

CYP2C11 constructs and control plasmids. The basal luciferase activity of vehicle- treated 5L cells transfected with the various plasmids was as follows, expressed in arbitrary normalized light units as mean + SD: (-5.6-2C11), 0.02 ± 0.004; (-7.1-2C11),

0.07 ± 0.01; (-10.1-2C11), 0.06 ± 0.01; pGudlucl.l, 0.08 + 0.01; pGL3-Basic, 0.56 ±

0.04. CFP2C7i-luciferase activity was not altered significantly by TCDD or MC in 5L cells (Fig. 3.4A). Luciferase activity of pGudlucl.l was induced 25-fold by TCDD and

15-fold by MC. Since pGudlucl.l is comprised of the pGL2-Basic vector backbone and luciferase is driven by the MMTV promoter, the extent of induction from this construct cannot be directly compared to that of CYP2C11 -luciferase constructs, which utilize the pGL3-Basic vector backbone. Induction of pGudlucl.l serves as a positive control marker for AHR activation. The pGL3-Basic vector was unaffected by TCDD or MC treatment. The CYP2C11 plasmids showed no response to TCDD or MC in BP8 cells

(Fig. 3.4B), and induction of pGudlucl.l activity was absent in these cells. The basal luciferase activity of vehicle-treated BP8 cells transfected with the various plasmids was as follows, expressed in arbitrary normalized light units as mean ± SD: (-5.6-2C11), 0.01

± 0.005; (-7.1-2C11), 0.006 ± 0.001; (-10.1-2C11), 0.066 ± 0.008; pGudlucl.l, 0.05 ±

141 w > Relative luciferase activity Relative luciferase activity (% of vehicle control) (% of vehicle control) _». _Jk r«o fO Ol o en o en o o o o o _l__.o _ o o o• o

01 01 • p- • ^U

03 , I T) ^^ CD •

-TJ; > ct i I w JO to 3 ^—»v 3 <0 00 00 O* cr H X*w3- c

95004

B 8 TO ot * o •DMSO JD u •TCDD j> .y IMC o a> >

A-4, . &.©-*•

Figure 3.4. Effects of TCDD and MC on CYP2C11 reporter gene activity in transiently transfected cells. 5L cells (A), BP8 cells (B), or Hepa-1 cells (C), were transiently transfected with a specified firefly luciferase construct and pRL-TK. After 24 h, cells were exposed to vehicle (0.1% DMSO), 10 nM TCDD or 1 |nM MC and incubated for a further 24 h. Firefly luciferase activity was normalized to Renilla luciferase activity. Arbitrary normalized luciferase data are expressed as a percentage of the mean for the vehicle controls for each plasmid. Data represent the mean ± S.D. of three determinations. Data were analyzed initially using a randomized design two-way ANOVA to identify significant drug and plasmid effects; for clarity of presentation, only significant drug effects are shown. *, significantly different (p < 0.05) from DMSO control; **, significantly different (p < 0.01) from DMSO control; ***, significantly different (p < 0.001) from DMSO control, based on a randomized design one-way ANOVA followed by a post hoc Newman-Keuls test. (A), inset; RT-PCR analysis of AHR and p-actin mRNA levels in 5L and BP8 cells. An ethidium bromide-stained agarose gel depicting relative AHR and P-actin mRNA levels is shown. Representative results are shown for a single RNA isolation.

143 0.02; pGL3-Basic, 0.4 ± 0.07.

Mouse Hepa-1 hepatoma cells were also used as recipients for transfection. The basal luciferase activity of vehicle-treated Hepa-1 cells transfected with the various plasmids was as follows, expressed in arbitrary normalized light units as mean ± SD:

(-2.4-2C11), 0.177 ± 0.01; (-5.6-2C11), 0.224 ± 0.01; (-7.1-2C11), 0.228 ± 0.01;

(-10.1-2C11), 0.142 ± 0.002; pGudlucl.l, 0.141 + 0.015; pGL3-Basic, 0.971 + 0.05.

Luciferase activity from all three of the novel CYP2C11 constructs: (-10.1-2C11),

(-7.1-2C11), (-5.6-2C11), as well as an additional luciferase plasmid containing 2.4-kb of the 5'-flanking region (-2.4-2C11), was increased 2- to 3-fold upon treatment of transfected mouse Hepa-1 cells with TCDD or MC, compared to vehicle-treated cells

(Fig. 3.4C). Induction of these CYP2C11 reporter constructs following aromatic hydrocarbon treatment was comparable to the 1.5-fold induction of the empty pGL3-

Basic vector, suggesting minimal response of CYP2C11 -luciferase constructs in Hepa-1 cells. As indicated above, basal luciferase activity of the CYP2C11 constructs was less than that of the promoterless pGL3-Basic vector in Hepa-1, 5L and BP8 cells. This suggests that these hepatoma cells may lack TFs and/or co-activators needed to drive high basal transcription from the CYP2C11 promoter. As a positive control for AHR activation, pGudlucl.l luciferase expression was increased 96-fold by TCDD and 67-fold byMC.

3.2.5 Transient transfections and analyses of "transcriptionally active" region within the CYP2C11 5'-flank in HepG2 cells

The final cell model examined was the human HepG2 hepatocellular carcinoma cell line. Cells were transfected with six plasmids containing various lengths of the

144 CYP2C11 5'-flank: (-10.1-2C11), (-7.1-2C11), (-5.6-2C11), (-2.4-2C11), (-1.3-2C11) and

(-0.2-2C11) (Fig. 3.5A). The basal luciferase activity of vehicle-treated HepG2 cells

transfected with the various plasmids was as follows, expressed in arbitrary normalized

light units as mean + SD: (-0.2-2C11), 0.154 + 0.01; (-1.3-2C11), 0.537 + 0.4;

(-2.4-2C11), 0.412 + 0.07; (-5.6-2C11), 0.12 + 0.17; (-7.1-2C11), 0.207 + 0.01;

(-10.1-2C11), 0.197 + 0.03; pGudlucl.l, 0.09 + 0.01; pGL3-Basic, 0.069 + 0.003; pGL3-

Promoter, 3.693 + 0.27. CKP2C7/-luciferase plasmids displayed higher basal luciferase

activity than the promoterless pGL3-Basic vector in this cell line, suggesting that HepG2 cells contain a collection of TFs and/or co-activators capable of driving high basal expression from the CYP2C11 promoter. Luciferase activity of the two shortest constructs, (-0.2-2C11) and (-1.3-2C11), was increased between 2- to 3-fold after TCDD treatment and did not respond to MC. The empty pGL3-Basic vector was slightly induced by aromatic hydrocarbon treatment to an extent comparable to that observed for

(-0.2-2C11) and (-1.3-2C11). The four longer CYP2C11 constructs displayed a pronounced 5- to 8-fold induction by TCDD and a trend for increased activity following

MC treatment. This unexpected and pronounced paradoxical induction by TCDD in

HepG2 cells was a novel observation that we followed up mechanistically.

I wanted to determine whether the region between the four longer constructs and the two shortest constructs, -2.4 to -1.3-kb, behaves as a conventional "enhancer" sequence by augmenting transcription from a heterologous promoter. PGL3-Promoter contains the heterologous SV40 promoter ligated into the pGL3-Basic vector backbone.

When the region between -2.4 to -1.3-kb was cloned upstream of the SV40 promoter, the luciferase activity of (-2.4 to -1.3-2Cll)-Promoter was significantly increased ~2.5-fold

145 Relative luciferase activity (% of vehicle control) Relative luciferase activity O w (% of vehicle control) Relative luciferase activity _* -* M (% of vehicle control) Oi O tn O K> N> W W3 OOO 01 00 0

-J

•s ID O 3p. 52 1Is D off 3 52 o o a o D

% % c AA ^e&* ,%o^ ((v^ ^ <***

Figure 3.5. Effects of TCDD and MC on CYP2C11 deletion constructs in transiently transfected HepG2 cells. Cells were transiently transfected with a specified firefly luciferase construct and pRL-TK as follows: (A) the full range of CYP2C11 deletion constructs, (B) a plasmid containing a putative "transcriptional stimulatory" region in the context of a heterologous SV40 promoter, (C) a plasmid containing a putative "transcriptional stimulatory" region in the context of the CYP2C11 promoter and (D) plasmids that differ in the presence or absence of the AHR-binding CYP2C17-DRE3 element. After 24 h, cells were exposed to vehicle (0.1% DMSO), 10 nM TCDD or 1 uM MC and incubated for a further 24 h. Firefly luciferase activity was normalized to Renilla luciferase activity. Arbitrary normalized luciferase data are expressed as a percentage of the mean for the vehicle controls for each plasmid. Data represent the mean ± S.D. of three determinations. Data were analyzed initially using a randomized design two-way ANOVA to identify significant drug and plasmid effects; for clarity of presentation, only significant drug effects are shown. *, significantly different (p < 0.05) from DMSO control; **, significantly different (p < 0.01) from DMSO control; ***, significantly different (p < 0.001) from DMSO control, based on a randomized design one-way ANOVA followed by a post hoc Newman-Keuls test.

148 by TCDD (Fig. 3.5B). The empty pGL3~Promoter vector was increased 1.8-fold by

TCDD but this response did not achieve statistical significance. When this region of the

CYP2C11 5'-flank was placed proximal to its natural promoter in the plasmid (-2.4 to

-1.3-2C11)-0.2, it displayed a 3.6-fold induction by TCDD treatment, while (-0.2-2C11) was only induced 1.3-fold (Fig. 3.5C). Since the region between -2.4 to -1.3-kb confers greater transcriptional stimulation when placed upstream of its natural promoter compared to a heterologous promoter, I referred to this region as a "transcriptionally active" sequence rather than a classical enhancer sequence.

My attention became focused on the CYP2C11-DRE3 site located at positions

-1546 to -1533-bp. This site was shown previously by our laboratory to bind the activated AHR (Bhathena et al., 2002), but this element has never been characterized for transcriptional stimulatory activity. The (DRE-2C11)-0.2 plasmid encompasses the

CYP2CU-DRE3 site and this construct was induced ~5-fold by TCDD (Fig. 3.5D). The

(-2.4 to -1.8-2Cll)-0.2 plasmid lacks CYP2C17-DRE3 and this construct was induced

2.6-fold by TCDD, similar to the response shown by the (-0.2-2C11) construct. The

CYP2C11 genomic sequence contained in (DRE-2C11)-0.2 and corresponding to positions -1827 to -1303-bp of the 5'-flanking region is shown in Fig. 1.7.

3.2.6 Effect of site-directed mutagenesis of CYP2C11-DRE3 on TCDD-induced luciferase activity in HepG2 cells

To further define the functional significance of the CFP2C7i-DRE3 located at positions -1546 to -1533-bp of the CYP2C11 5'-flanking region, three of the four core nucleotides required for AHR/ARNT binding (Yao and Denison, 1992) were mutated using site-directed mutagenesis. In HepG2 cells, TCDD increased luciferase activity of

149 (-10.1-2C11), (-2.4-2C11) and (-2.4 to -1.3-2Cll)-0.2 by ~3-fold compared to vehicle, whereas (DRE-2C11)-0.2 luciferase activity was increased 2-fold by TCDD (Fig. 3.6).

Mutation of the CYP2C11-DKE3 within these four constructs eliminated the luciferase induction by TCDD.

3.2.7 Effect of the AHR antagonist a-NF on TCDD-induced luciferase activity in

HepG2 cells

I next asked whether a-NF, an AHR antagonist/partial agonist, could modulate induction of CYP2C11 -luciferase constructs by TCDD in HepG2 cells. a-NF (1 uM) was ineffective in blocking pGudlucl.l induction caused by a 10 nM concentration of

TCDD (Fig. 3.7A). When the TCDD concentration was reduced from 10 nM to 1 nM, the antagonistic effect of 1 |J,M a-NF became more apparent (Fig. 3.7B). TCDD (1 nM) induced pGudlucl.l activity by ~50-fold, and co-treatment with a-NF (1 |J,M) reduced this effect to 17-fold. TCDD (1 nM) induced (-10.1-2C11) activity by ~3.5-fold, while co-treatment with a-NF (1 U.M) blocked this response. At this lower concentration of

TCDD, the (DRE-2Cll)-0.2 plasmid was induced only 1.7-fold and this small TCDD response was prevented by a-NF.

150 ***

•DMSO •TCDD

v; .•«£>

Figure 3.6. Site-directed mutagenesis reveals that induction of CYP2C11- lucfierase activity by TCDD in HepG2 cells is at least partially mediated by the CYP2C11-DRE3 element. Cells were transiently transfected with a specified firefly luciferase construct and pRL-TK. After 24 h, cells were exposed to vehicle (0.1% DMSO) or 10 nM TCDD and incubated for a further 24 h. Firefly luciferase activity was normalized to Renilla luciferase activity. Arbitrary normalized luciferase data are expressed as a percentage of the mean for the vehicle controls for each plasmid. Data represent the mean ± S.D. of three determinations. Data were analyzed initially using a randomized design two-way ANOVA to identify significant drug and plasmid effects; for clarity of presentation, only significant drug effects are shown. **, significantly different (p < 0.01) from DMSO control; ***, significantly different (p < 0.001) from DMSO control, based on unpaired Student's ?-test (two-tailed).

151 [TCDD]= 10 nM

p act ; S3 o o m o 2 3> <& "a 38> 5 > 5 o J3 0s- "3 rt

DMSO TCDD TCDD + a -NF a-NF

152 B [TCDD]= 1 nM

7000'

.-£* 300CH • DMSO 1 8CO f §8K •TCDD o> 8 IHTCDD + a-NF •a-NF

Figure 3.7. Effects of a-NF on TCDD-induced luciferase activity in HepG2 cells. Cells were transiently transfected with a specified firefly luciferase construct and pRL-TK. After 24 h, cells were exposed to vehicle (0.1 % DMSO), (A) 10 nM TCDD or (B) 1 nM TCDD, 1 uM a-NF, or both TCDD and a-NF, and incubated for a further 24 h. Firefly luciferase activity was normalized to the Renilla luciferase activity. Arbitrary normalized luciferase data are expressed as a percentage of the mean for the vehicle controls for each plasmid. Data represent the mean ± S.D. of three determinations. Data in (B) were analyzed initially using a randomized design two-way ANOVA to identify significant drug and plasmid effects; for clarity of presentation, only significant drug effects are shown. *, significantly different (p < 0.05) from DMSO control; **, significantly different (p < 0.01) from DMSO control; ***, significantly different (p < 0.001) from DMSO control; $, significantly different (p < 0.01) from TCDD + a-NF, based on a randomized one-way ANOVA followed by a post hoc Newman-Keuls test.

153 3.3 In vivo studies

3.3.1 Hepatic luciferase activity in rats receiving hydrodynamics-based injections

Using the hydrodynamics-based technique, rats received a tail vein injection

consisting of one of five firefly luciferase plasmids [pGudlucl. 1, pGL3-Basic,

(-10.1-2C11), (-5.6-2C11) or (-2.4-2C11)] and the Renilla luciferase plasmid (pRL-TK)

to normalize for in vivo transfection efficiency. MC or vehicle corn oil (CO) was

administered to rats immediately prior to the high-volume injection and rats were

euthanized at 6 h, 24 h or 72 h post-treatment.

Hepatic luciferase activity of the positive control plasmid pGudlucl.l was

increased -2000-fold by 24 h and 3000-fold by 72 h following MC administration

(Fig. 3.8A). This confirmed that the dose of MC (80 mg/kg) used in this study increased the transcription of a reporter gene delivered to the liver by the hydrodynamics-based approach. The large standard deviation in the MC-treated group at 72 h results from one rat, which displayed considerably lower normalized luciferase activity compared to the other three rats in this group.

Under in vivo conditions in which MC activates the AHR to induce pGudlucl.l reporter gene transcriptional activity, I examined the MC-responsiveness of the four other firefly luciferase reporter plasmids: pGL3-Basic and three CYP2C11 constructs containing 10.1-kb, 5.6-kb and 2.4-kb of 5'-flanking region (Fig. 3.8B). While Fig. 3.8 presents normalized luciferase activity as a percentage of vehicle control levels, Table

3.1 shows the raw values of firefly luciferase activity normalized to Renilla luciferase activity, as measured in hepatic lysates. pGL3-Basic is a luciferase reporter plasmid devoid of an eukaryotic promoter, thus serving as a negative control for aromatic

154 eso.oooi

£, 575,000

I I 500,0004

g © 425,000.

J g 350,000.

•| £ 275,000 13 w 200.000*

24h 72 h Time after MC exposure B

6h •P MC-24h § 8 MC-72h s *>

"5 ^? «i

pGL3-Basic (-10.1-2C11) (-5.6-2C1I) (-2.4-2C11) Plasmid received by hydrodynamics-based injection

Figure 3.8. Effect of MC on hepatic reporter gene activity in rats receiving hydrodynamics- based injections. Rats received one of five firefly luciferase plasmids: (A) pGudlucl.l, or (B) pGL3-Basic, (-10.1-2C11), (-5.6-2C11), (-2.4-2C11), as well as the pRL-TK Renilla construct, and were given i.p. injections of vehicle corn oil (CO) or 80 mg/kg MC. Rats were euthanized 6 h, 24 h or 72 h later as indicated. Hepatic lysates were prepared from each rat and firefly luciferase activity was measured and normalized to Renilla luciferase activity. Results for MC-treated rats are expressed as a percentage of the mean for the vehicle-treated controls at each time point. Each bar represents the mean ± S.D. of determinations from three or four rats. Asterisks indicate significant differences from vehicle-treated rats at a given time point (*pS 0.05; ** p £ 0.01; *** p S 0.001); based on a two-tailed Student's /-test for pGL3-Basic luciferase data, and a one- tailed Student's /-test for all CYP2C11 constructs and pGudlucl.l luciferase data.

155 hydrocarbon responsiveness. MC induced pGL3-Basic luciferase activity slightly at both

time-points studied, but this response was not statistically significant. At 24 h post-

treatment, all CYP2C11 constructs were down-regulated by MC compared to vehicle-

treated rats. The most pronounced down-regulation was observed with (-5.6-2C11),

which was suppressed to 36% of vehicle levels. (-2.4-2C11) showed the smallest

magnitude of suppression 24 h post-treatment, being decreased to 63% of vehicle levels.

At 72 h, luciferase activity of (-10.1-2C11) and (-5.6-2C11) plasmids was strikingly

down-regulated in response to MC treatment by 69% and 76%, respectively. Suppression

of (-5.6-2C11) did not achieve statistical significance at 72 h since vehicle-treated rats

displayed large variation in basal luciferase activity, whereas variations at 24 h were

minimal (Table 3.1). MC up-regulated (-2.4-2C11) luciferase activity by 53% at 72 h;

however, this was not statistically significant. Since all plasmids displayed a decline in

luciferase activity by MC at 24 h, we examined an earlier time-point to gain a clearer

understanding of the time course involved in CYP2C11 reporter suppression. Rats were

euthanized 6 h following MC or vehicle treatment and hydrodynamics-based injections of

(-10.1-2C11). Regardless of chemical treatment, large variations in luciferase activity

were observed at this early time-point. There was a 43% decline in luciferase activity 6 h

following MC administration, an effect that was not statistically significant (Fig. 3.8B).

For CYP2C11 constructs that were suppressed by MC at both 24 h and 72 h

[(-10.1-2C11) and (-5.6-2C11)], basal luciferase activity in vehicle-treated rats was

always higher at 72 h compared to 24 h following injections (Table 3.1). The opposite

trend was observed for pGudlucl.l and pGL3-Basic luciferase activity, which displayed high levels of activity at 24 h, but tapered off by 72 h. All Renilla luciferase levels were

156 relatively similar within a single plasmid group at the same time-point, indicating

consistent transfection efficiencies between rats. Renilla luciferase values were

considerably higher in all rats when examined 24 h post-treatment compared to 72 h.

3.3.2 Endogenous CYP1A1 levels in rats receiving hydrodynamics-based injections

The effectiveness of MC (80 mg/kg) was also confirmed by monitoring the

induction of endogenous CYP1A1 protein/mRNA in rats receiving hydrodynamics-based

injections as a qualitative positive control response for AHR activation. MC induced

CYP1A1 protein (Fig. 3.9A) and mRNA (Fig. 3.10A) levels at all time-points, although

the extent of induction varied between individual rats. No observable signs of toxicity

were associated with this dose of MC.

3.3.3 Endogenous CYP2C11 levels in rats receiving hydrodynamics-based injections

The effects of MC administration on endogenous hepatic CYP2C11 expression in

rats receiving hydrodynamics-based injections were examined at the protein level (Fig.

3.9B,C). Before conducting relative quantitation of endogenous CYP2C11 apoprotein

levels, optimization studies were performed using samples from a vehicle-treated male

rat. Microsomal samples containing 0-10 ng of protein were used to generate a standard

curve (Fig. 3.11). The amount of microsomal protein chosen for future studies was

3.5 jig, since this amount lies within the linear range of the standard curve. Suppression

of CYP2C11 apoprotein levels by MC was detected at 24 h (62-77% of vehicle levels); however, statistical significance was achieved only in rats receiving pGudlucl.l,

(-10.1-2C11) and (-2.4-2C11). Dramatic down-regulation of CYP2C11 protein was

observed at 72 h in MC-treated rats receiving pGL3-Basic, pGudlucl.l and (-5.6- 2C11) with levels falling to 24-54% of vehicle levels. Rats receiving (-2.4-2C11) showed no

157 TABLE 3.1. Effect of MC on hepatic reporter gene activity in rats receiving hydrodynamics-based injections.

6h 24h 72h Plasmid delivered Vehicle MC Vehicle MC Vehicle MC

pGL3-Basic N.D. N.D. 0.60 + 0.07 0.71+0.08 0.21+0.01 0.28 ±0.10

pGudlucl.l N.D. N.D. 0.15 ±0.02 305.95 ± 62.22 0.10 + 0.03 335.05 ± 256.84

(-10.1-2C11) 5.29 + 3.62 3.00+1.79 153.72+ 19.73 74.84 ±50.13 4225.88 ± 2386.38 1330.27 ±485.98

(-5.6-2C11) N.D. N.D. 1.95 + 0.25 0.70 + 0.36 49.65 + 38.53 11.79 ±1.74

(-2.4-2C11) N.D. N.D. 3.50 + 0.42 2.22 + 0.51 1.48 + 0.83 2.28 ±1.32

All data are expressed in arbitrary light units (normalized for Renilla activity) as mean + S.D. of determinations from hepatic lysates prepared from three or four rats. Statistically significant effects are summarized in Fig. 3.8. N.D., not determined.

158 A CYP1A1 (~55kDa>

B CYP2C11 (-50 kDa)

pGL3-Basic pGudfucl.1 (-10.1-2C11) (-5.6-2C11) (-2.4-2C11)

160, 140-

3 £ 120- b O 100--

c IE 80- 60- 40 20-

-Basvc cU .2CU") acW) rfiV* pGu^ ^Q>*^ C-5.6 t-aA Plasmid received by hydrodynamics-based injection Figure 3.9. Immunoblot analysis of endogenous hepatic CYP1A1 and CYP2C11 protein levels following vehicle or MC treatment in rats receiving hydrodynamics- based injections. Rats received one of five firefly luciferase plasmids: pGL3-Basic, pGudlucl.l, (-10.1-2C11), (-5.6-2C11) or (-2.4-2C11), as well as the pRL-TK Renilla construct, and were given i.p. injections of vehicle (V) or MC (M). Rats were euthanized 6 h, 24 h or 72 h later as indicated. (A) Immunoblot analysis of hepatic microsomal protein (5 ug) using a CYP1A1 monoclonal antibody. (B) Immunoblot analysis of hepatic microsomal protein (3.5 jig) using a C YP2C11 polyclonal antibody. Representative immunoblots are shown for one rat sample selected from the three or four rats within each treatment group. (C) Relative quantitation of CYP2C11 apoprotein levels. Results for MC-treated rats are presented as a percentage of the mean for the vehicle-treated control rats at each time point. Data are expressed as mean + S.D. of determinations from microsomal samples prepared from three or four rats. Similar results were obtained in two or three independent immunoblot analyses. Asterisks indicate significant differences from vehicle-treated rats at a given time point (* p < 0.05; ** p < 0.01); based on a one-tailed Student's f-test

159 A CYP1A1 (672 bp)l «-jp;; CMI r - £4 ^ imi B P-actin (688 bp) Aw*l ir»t W*i im4 -1—1—1—r- —r—1—1—1— -1—r-i—r- "1 I III 1- 1—I—I—r V M V M V M V M V M V M V M V M V M V M V M 24 h 72 h 24 h 72 h 6 h 24 h 72 h 24h 72 h 24 h 72 h • I • I • I I • pGL3-Basic pGudlucl.l (-I0.1-2C11) (-S.6-2C11) (-2.4-2CI1)

MC-6H MC- 24 h IMC- 72 h

.Cr* V* 4*>»* ^^. ^ & &

Plasmid received by hydrodynamics-based injection

Figure 3.10. Effect of MC administration on endogenous hepatic CYP1A1 and CYP2C11 mRNA levels in rats receiving hydrodynamics-based injections. Rats received one of five fireflyluciferas e plasmids: pGL3-Basic, pGudlucl.l, (-10.1-2C11), (-5.6-2C11) or (-2.4-2C11), as well as the pRL-TK Renilla construct, and were given i.p. injections of vehicle (V) or MC (M). Rats were euthanized 6 h, 24 h or 72 h later as indicated. RT-PCR analysis of (A) CYP1A1 and (B) (3-actin mRNA as visualized on Vistra Green-stained polyacrylamide gels. Representative results are shown for one rat sample selected from the three or four rats within each treatment group. (C) Relative quantitation of CYP2C11 mRNA levels as determined by real-time RT-PCR using molecular beacons. CYP2C11 signal was normalized to (3-actin. Results for MC-treated rats are presented as a percentage of the mean for the vehicle-treated control rats at each time point. Data are expressed as mean ± S.D. of determinations from hepatic RNA samples prepared from three or four rats. Asterisks indicate significant differences from vehicle-treated rats at a given time point (* p £ 0.05; **p£ 0.01); based on a one-tailed Student's Mest.

160 overall changes in CYP2C11 apoprotein levels between vehicle- and MC-treatment at

72 h. These rats displayed high levels of variation in CYP2C11 protein, with standard

deviations of 45-54% of the mean (Fig.3.9C and Table 3.2). No differences in

CYP2C11 protein expression were detected between MC- and vehicle-treated rats at 6 h

post-treatment.

I used real-time quantitative RT-PCR to determine whether endogenous hepatic

CYP2C11 mRNA expression was down-regulated by MC in rats receiving

hydrodynamics-based injections (Fig. 3.10C). At 24 h, modest trends for CYP2C11

mRNA suppression were observed in MC-treated rats receiving high-volume injections

(67-76% of vehicle levels), but statistical significance was only detected in rats receiving

(-5.6-2C11). Suppression of endogenous CYP2C11 mRNA levels was detected at 72 h in

all rats (52-69% of vehicle control) except rats receiving pGL3-Basic. Statistical

significance at 72 h was achieved in rats receiving pGudlucl.l and (-10.1-2C11), since

large variations in CYP2C11 mRNA expression were observed in vehicle-treated rats

receiving (-5.6-2C11) and (-2.4-2C11) (Table 3.2). At 6 h post-treatment, no

suppression of CYP2C11 mRNA levels following MC treatment was observed.

3.3.4 Correlation studies of endogenous CYP2C11 protein and mRNA levels in rats

receiving hydrodynamics-based injections

In general, CYP2C11 protein levels paralleled CYP2C11 mRNA levels

(Fig. 3.12). A significant positive correlation was observed between endogenous

CYP2C11 protein and mRNA expression in rats receiving all plasmids by hydrodynamics-based injections, except for rats in the (-2.4-2C11) plasmid group. Of particular interest, CYP2C11 mRNA levels in rats receiving (-2.4-2C11) extend over a

161 14- y=1.G45x-2.532 r2= 0.9799 ^y* . .'/"•<. 12-

•&•*' 10-

s s ••* J>****^ * 1? 8- — « 6- 4- Ba n arb i • .w 2"

n- i i i i

Microsomal protein (fig)

Figure 3.11. Optimization of CYP2C11 immunoblot assay. A standard curve was generated showing a linear relationship between immunoreactive band intensity and amount of microsomal protein loaded. The immunoblot corresponding to the amount of microsomal protein loaded is shown beneath the x-axis of the graph. The equation of the line of best fit was generated by least-squares linear regression analysis. A value of 3.5 u.g microsomal protein was chosen for future CYP2C11 immunoblotting studies.

162 TABLE 3.2. Variation in endogenous CYP2C11 protein and mRNA levels for vehicle-treated rats receiving hydrodynamics- based injections.

S.D. for CYP2C11 protein (% of mean) S.D. for CYP2C11 mRNA (% of mean) Plasmid delivered 6h 24 h 72 h 6h 24 h 72 h

pGL3-Basic N.D. 24.5 27.1 N.D. 33.6 17.0

pGudlucl.l N.D. 8.4 53.4 N.D. 53.5 29.7

(-10.1-2C11) 19.7 12.8 54.8 6.42 31.6 23.6

(-5.6-2C11) N.D. 17.7 45.1 N.D. 21.3 48.0

(-2.4-2C11) N.D. 14.7 45.3 N.D. 23.0 39.0

S.D. data are expressed as a percentage of the mean, based on determinations from microsomal or RNA samples prepared from three or four vehicle-treated rats. The variation in the corresponding MC-treated groups of rats and statistically significant effects are summarized in Fig.3.9C and Fig.3.10C. N.D., not determined.

163 greater range compared to measurements from rats within the other plasmid groups (Fig.

3.12C). There are a few noticeable outliers among the rats receiving (-2.4-2C11), which display strikingly high CYP2C11 mRNA levels but relatively normal protein levels compared to the majority of rats in this group. Since immunoblotting and real-time RT-

PCR studies were only performed at the same time within a single plasmid group, I could not analyze the overall correlation between endogenous CYP2C11 protein and mRNA levels in all rats receiving hydrodynamics-based injections. A single analysis including all rats would likely generate a strong positive correlation between endogenous

CYP2C11 protein and mRNA levels.

3.3.5 Correlation studies of endogenous CYP2C11 mRNA levels and Iuciferase activity in rats receiving hydrodynamics-based injections

For CYP2C11 constructs strongly suppressed by MC [(-10.1-2C11) and (-5.6-

2C11)], Iuciferase activity was positively correlated with endogenous CYP2C11 mRNA levels (Fig. 3.13A,B)- pGudlucl.l Iuciferase activity displayed a slight trend for negative correlation with endogenous CYP2C11 mRNA levels; however, this was not statistically significant (Fig. 3.13D). For (-10.1-2C11), (-5.6-2C11) and pGudlucl.l, I also graphed the correlation between endogenous CYP2C11 mRNA levels and Iuciferase activity on a logio-transformed x-axis scale [Fig. 3.13A(ii), B(ii), D(ii)]. This was done because the

Iuciferase activity in rats receiving these plasmids extended over a large range. MC treatment markedly reduced Iuciferase activity in rats receiving (-10.1-2C11) and

(-5.6-2C11) compared to vehicle-treated levels. As a result, values corresponding to the

Iuciferase activity from MC-treated rats are very low and scattered close to the y-axis,

164 (-2.4-2C11) {(-10.1-2C11) B (-5.6-2C11) | y=-2.248x + 6.546; r= -03071; P=0.266 y=4 S49x -1.668, r= 0.577;P e • ••" —i—i—i—i—i i—i—i 0.0 ia as i.a 15 0.00 025 0.50 9.75 1.110 US 1.50 1,75 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.(

CYP2C11 protein (arbitraryunits) CYP2C11 protein (arbitrary units) CYP2CI1 protein (arbitrary Hits)

D pGL3-Basic j E pGudlud 1 y=0,407x + 1.61; r= 0.693; P<0.01 y=t^I6x + 0.92l4;r=0.73;P<0.01 6" < 9 5- 3 1e 1x •s & 4- Z g 3- • • • 1 £u _^rf - 8 2- *i • II • I 0.0 9.5 U> 1.5 2.0 2.5 3.0 J.5

CYP2C1I protein (arbitrary units) CYP2C11 protein (arbitrary units)

Figure 3.12. Correlation between endogenous hepatic CYP2C11 protein and mRNA levels in rats following hydrodynamics-based injections. Rats received one of the following luciferase constructs: (A) (-10.1-2C11), (B) (-5.6-2C11), (C) (-2.4-2C11), (D) pGL3-Basic or (E) pGudlucl, and endogenous CYP2C11 protein and mRNA levels were determined following treatment with corn oil or MC. The equations of the lines of best fit were generated by least-squares linear regression analysis. Statistical significance was determined using the two-tailed Pearson correlation test with a significance level set at p ^ 0.05.

165 CO

CYP2Cll/p-actin mRNA CYP2Cll/p-actin mRNA (normalized expression) (normalized expression)

• NW*«0>-JI

ON ON 5. 0 e -4 b _J WJ ^^^^^^^^ • (T« r e 9 ia • f oa e> *• - H' ~ 3 • ?J * 5 s s £ 3 • a- e. ST B. i- .. 3 8 • <4 2. ••* 1 n o K — K r ffa 0 «• t iS" n i1 r» K s. • 1| 6.8 \ s!•• a 4J, » 8 ~ ft) 1 J3l T\ •*« T • jS. 25s. •3 (-• 1* •V- "< M *vT N |(-2.4-2Cll)j y=-1.6x+8.06;r=0.521O4; P<0.01 isn

II iH

H >- g

Normalized luciferase activity (aribtrary light units') pGudhicI.lj D(i) y--0.002x +2.71 8: r- -0.264; P-0.324 (") 6-j 6-

% 3 4 1 • • 3-i 2 1 !! i i i I I 1 I -T- i 100 200 300 400 500 600 700 0.01 0.1 100 1000 10 Normalized luciferase activity Logu Normalized luciferase activity (arbitrary light units) (arbitrary-light units)

Figure 3.13. Correlation between hepatic luciferase activity and endogenous CYP2C11 mRNA levels in rats receiving hydrodynamics-based injections. Rats received one of the following luciferase plasmids: (A) (-10.1-2C11), (B) (-5.6-2C11), (C) (-2.4-2C11) or (D) pGudlucl, and endogenous CYP2C11 mRNA levels and hepatic luciferase activity were determined following treatment with corn oil or MC. Figures (A)ii, (B)ii, and (D)ii show the same data represented on a log-transformed x axis. The equations of the lines of best fit were generated by least-squares linear regression analysis. Statistical significance was determined using the two-tailed Pearson correlation test with a significance level set atp ^ 0.05.

167 while values from vehicle controls are dispersed along the entire length of the x-axis

[Fig. 3.13A(i), B(i)]. The opposite scenario occurs in rats receiving pGudlucl.l, since measurements recorded from vehicle-treated rats are in the low range of luciferase activity, while values from rats administered MC are much higher, spanning the entire length of the x-axis [Fig. 3.13D(i)]. All statistical analyses were performed on the original untransformed data.

Luciferase activity of (-2.4-2C11) was down-regulated at 24 h following MC treatment but showed a trend for increased activity at 72 h, resulting in significant negative correlation between luciferase activity and CYP2C11 mRNA expression

(Fig. 3.13C). There was no significant relationship between pGL3-Basic luciferase activity and endogenous CYP2C11 mRNA (data not shown).

3.3.6 Effect of a-NF on MC-induced endogenous CYP1A1 levels in vivo

It was of interest to determine whether a-NF, an AHR antagonist/partial agonist, could block the in vivo effects of MC on CFP2C/i-luciferase activity. However, a-NF was not effective in preventing the induction of CYP1A1 protein and mRNA expression by MC under the conditions used in study designs 1 and 2 (Fig. 3.14A,B). Since there were no apparent differences (by visual inspection) in CYP1A1 induction between rats administered both a-NF and MC compared with MC alone, I did not proceed to optimize analytical conditions that would permit quantitation of these data.

168 Vehicle MC oc-NF MC+a-NF A

CYP1A1 mRNA 672-bp

CYP1A1 protein 55kDa

B Vehicle MC MC + a-NF

^^^^^

—* t 700-bp *«*»* i. Vv>, J W\ «.* CYP1A1 mRNA 600-bp —* i—* 672-bp

flpsfflWffflff 700-bp —» tti&xU <,>. " '. M ' • a P-actin mRNA 600-bp MM* 688-bp

CYP1A1 protein 55kDa

Figure 3.14. CYP1A1 induction by MC is not affected by a-NF in vivo. Rats received vehicle corn oil, MC, a-NF or both MC and a-NF as described in Table 2.3 [(A) Study design 1] and Table 2.4 [(B) Study design 2]. RT-PCR was performed on RNA samples prepared from rat livers. Products were separated on a 6% polyacrylamide gel and stained with Vistra Green. Liver microsomal protein (5 jug) was resolved by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and probed with a CYP1 Al monoclonal antibody. Each lane represents RNA or microsomal protein isolated from a representative rat within each of the treatment groups indicated above the lane.

169 4.0 DISCUSSION

4.1 Luciferase reporter assays in continuous cell lines

To date, luciferase reporter plasmids encompassing the proximal 2390-bp of the

CYP2C11 5'-flanking region have not been down-regulated by TCDD in liver-derived

cell lines and primary rat hepatocytes (Bhathena et al, 2002). My in vitro studies

focused on the responses to TCDD and MC in cultured hepatoma cells of novel

C7P2Cii-luciferase constructs encompassing up to 10.1-kb of the 5'-flanking region.

4.1.1 Lack of suppression of CYP2C11 reporter constructs in continuous cell lines

The first major finding of my in vitro work is that reporter constructs containing

extended stretches of the CYP2C11 5'-flanking region are not down-regulated by TCDD

and MC in hepatoma cells of rat, mouse and human origin. The suppression of CYP2C11

gene expression by aromatic hydrocarbons observed in vivo cannot be reproduced in reporter gene assays conducted in continuous hepatoma cells that do not express endogenous CYP2C11 mRNA. It may be that the collection of TFs, co-activators and co- repressors in hepatoma cells do not permit the CYP2Cii-luciferase constructs to respond to aromatic hydrocarbons in the same manner that the endogenous gene responds in rat liver (Wiwi and Waxman, 2004). Hormonal factors (in particular, GH), which are absent in hepatoma cells, play a major role in regulating the in vivo expression of CYP2C11 and other constitutive P450s, and seem to be important targets for disruption by aromatic hydrocarbons. This will be discussed further in Section 4.2.5.2.

4.1.2 Role of the AHR in paradoxical induction of CYP2C11 -luciferase constructs in

HepG2 cells

170 The second major finding is that CYP2C1 i-luciferase plasmids display a

paradoxical induction by aromatic hydrocarbons that is evident in both Hepa-1 and

HepG2 hepatoma cell lines, but is particularly pronounced in human HepG2 cells. In

Hepa-1 cells, constructs containing between 2.4-kb to 10.1-kb of the CYP2C11 5'-

flanking region were induced approximately 2- to 2.5-fold by aromatic hydrocarbon

treatment, whereas these constructs displayed a strikingly greater 3- to 8-fold magnitude

of induction in HepG2 cells. As recipients for transfection of luciferase constructs, we used rat 5L and BP8 cells, mouse Hepa-1 cells and human HepG2 cells. A functional

AHR pathway is present in 5L (Weiss et al., 1996), Hepa-1 (Riddick et al, 1994), and

HepG2 cells (Roberts et al., 1990); these lines have been characterized for their gene expression patterns and have been used extensively as model systems that reflect the in vivo induction of CYP1A enzymes by AHR agonists (Li et al., 1998; Puga et al., 2000b;

Wei et al., 2004; Fong et al., 2005). In addition, the constitutive expression of several

P450s has been documented at the mRNA level in HepG2 cells, and CYP3A subfamily members are induced in this cell line by dexamethasone and rifampicin (Maruyama et al.,

2007). Plasmids containing >2.4-kb of the CYP2C11 5'-flanking region showed an unexpected 3- to 8-fold induction in response to TCDD in HepG2 cells. Since the AHR is present and functional in rat 5L cells, mouse Hepa-1 cells and human HepG2 cells, the preferential induction response observed in HepG2 cells cannot be explained simply on the basis of AHR expression and function. I hypothesize that human HepG2 cells may contain a repertoire of TFs, co-activators and co-repressors that allow luciferase constructs driven by the rat CYP2C11 5'-flank and promoter to be markedly induced by aromatic hydrocarbons under my culture conditions. A previous study examining the

171 response of CYP2C11 reporter constructs to treatment with peroxisome proliferators showed negative regulation of constructs in HepG2, but not H4HE or CV-1 continuous cells lines (Ripp et al., 2003). This study also suggests that the collection of TFs and signaling pathways present in HepG2 cells, but absent in the other cell models, permit the response of CYP2C11 constructs in this particular cell line.

Rat 5L cells and the AHR-deficient BP8 derivative are useful models to examine the AHR's role in transcriptional responses; however, since the CYP2C11 constructs did not respond to aromatic hydrocarbon treatment in the 5L cells, no conclusions regarding the role of the AHR in in vitro CYP2C11 regulation could be drawn from this cell model.

In Hepa-1 and HepG2 cells, pGL3-Basic and pGL3-Promoter were slightly induced by

TCDD. This has also been reported in other studies, and is thought to represent a response to chemical treatment of cryptic elements in the vector backbone (Annicotte et al., 2001; Bhathena et al., 2002; Nishihashi et al., 2006).

The third major in vitro finding is that the paradoxical induction of CYP2C11- luciferase constructs in HepG2 cells is AHR-dependent and DRE-mediated. The deletion studies suggest the presence of a transcriptional stimulatory region located between -2.4 and -1.3-kb of the CYP2C11 5'-flank. This region does not behave as a conventional enhancer since it seems to confer greater transcriptional stimulation in the context of the

CYP2C11 promoter compared to the heterlogous SV40 promoter. A more narrowly defined region between -1.8 and -1.3-kb possesses most of the transcriptional stimulatory activity; thus, my focus was centered on the CYP2C11-DRE3 element located at positions -1546 to -1533-bp, previously shown to be a high affinity AHR-binding site

(Bhathena et al., 2002). Site-directed mutagenesis confirmed that this DRE is critical for

172 the paradoxical induction response. It is likely that the AHR/ARNT complex at least

partially mediates induction of the ClT2C/i-luciferase constructs in HepG2 cells by

interacting with this site. This does not rule out the possibility that other cis-acting

element(s) may contribute to the induction.

The role of the AHR in the suppression of CYP2C11 by aromatic hydrocarbons

is supported by SAR data (Yoshihara et al., 1982; Dannan et al., 1983; Safa et al., 1997).

My results with the AHR antagonist/partial agonist a-NF now support a central role for

the AHR in the paradoxical induction of CYP2Cii-luciferase constructs in HepG2 cells.

Under conditions in which the induction of pGudlucl.l by TCDD was partially blocked

by a-NF, the paradoxical induction of CYP2C11 constructs containing the full 10.1-kb

region and the more narrowly defined region between -1.8 and -1.3-kb was prevented by

a-NF.

To take into consideration the effects of both PAHs and HAHs on the response of the CYP2C11 constructs, I studied both TCDD and MC in the in vitro work. Throughout this study, the transcriptional responses of pGudlucl.l and the C7P2Cii-luciferase constructs to MC (1 uM) were considerably weaker than responses to TCDD (10 nM).

This is expected for AHR-mediated responses after 24 h of cellular exposure since MC is less persistent due to rapid metabolism (Riddick et al, 1994). In HepG2 cells, MC undergoes extensive metabolism within the first day of treatment with concentrations up to 10 |iM (Swedenborg et al., 2008). A recent study compared the gene expression profiles following treatment of HepG2 and MCF-7 cells with B[a]P and TCDD (Hockley et al., 2007). Not only did this investigation discern differences in gene expression changes between the two cell lines, they also report marked differences between the two

173 chemical treatments within each individual cell type. This suggests that HepG2 and

MCF-7 cells differ in their respective signaling pathways and responses to the same

chemical treatment, but also that TCDD, which is resistant to metabolism and is present

for a longer time period in the cell, induces gene expression changes within the same cell

line that are distinct from B[a]P, a readily metabolized PAH.

When removed from the natural environment of the rat liver and placed in the

specific cellular context of HepG2 cells, CYP2Cii-luciferase constructs respond to

aromatic hydrocarbons with a paradoxical induction that is AHR-dependent and DRE- mediated. On the other hand, the endogenous CYP2C11 gene is down-regulated by aromatic hydrocarbons in rat liver via an unknown mechanism. It was essential to follow-up these in vitro transfection studies with an examination of the behaviour of the

CYP2 Cii-luciferase plasmids in the liver of live rats to determine if the in vitro paradoxical induction response relates in any way to mechanisms of in vivo gene suppression. One possibility arising from the in vitro work was that CYP2C11-DRE3, now implicated in the paradoxical induction in HepG2 cells, may represent an AHR binding site that plays a role in the in vivo suppression of CYP2C11 in its native chromosomal context by disrupting the binding and/or function of key GH-dependent

TFs that normally stimulate the expression of this gene. Wolff et al. (2001) found that

(transforming growth factor-P) TGF-p mediates the suppression of an AHR reporter gene in A549 cells, while this construct is induced by TFG-P treatment in HepG2 cells. The opposite responses are attributed to the balance of the expression of co-activators and co- repressors in each cell line. The position of CYP2C11-DRE3 in naked plasmid DNA in the HepG2 cellular context seems to preferentially unmask an induction response not

174 observed in rat liver. This cell-specific in vitro paradoxical induction is potentially

misleading and serves as a reminder that findings from reporter gene studies obtained in

cell lines may not reliably predict in vivo responses of the endogenous gene to xenobiotic

treatment.

4.2 Luciferase reporter assays in vivo

4.2.1 Considerations when using the hydrodynamics-based approach

CyP2Cii-luciferase constructs containing extended stretches of the 5'-flanking

region were hydrodynamically introduced into rat hepatocytes in vivo. Rats were

anesthetized for -10 min to facilitate hydrodynamics-based injections. Isoflurane was

chosen since it is fairly resistant to metabolism and therefore less likely to interfere with

our findings. This anesthetic is fast-acting and molecularly stable, leading to minimal

metabolic activation and potential toxicity. To the best of my knowledge, there are no

existing reports of altered P450 levels following acute isoflurane exposure. A recent

study showed that exposure of rats to 4.5% isoflurane in air leads to some clinical

pathology parameters indicative of toxicity such as alterations in haemoglobin levels,

blood ion composition, and reduced serum T3, T4 and prolactin levels (Deckardt et al.,

2007). Overall, this study concludes that isoflurane is preferred to other inhalation

anesthetics such as ether and methoxyflurane.

Following HVTV injections, the rats in my study recovered quickly and within

minutes, exhibited normal behavior. Some rats experience apnea and "wet" breathing

immediately after the injection, but recovery can be facilitated by gentle thoracic massage. The survival rate of these rats following high-volume injections exceeded 90%.

Histology reports on rat liver slices identified traces of macrophage and monocyte cells,

175 and mild focal hepatic necrosis at 48 h to 72 h following injection. Overall, histology

confirmed good hepatocyte viability and no major signs of inflammation. No clinical

signs of toxicity were evident.

Although the hydrodynamics-based approach of gene delivery has many

advantages, there are some concerns associated with this technique. Rats may experience

stress following high-volume injections, which could alter the physiological state of the

liver. This in turn may influence the response of the transgene to chemical challenge.

The sheer injection volume may adversely affect heart function and venous pressure.

Electrocardiograms of mice undergoing HVTV injections show transient cardiac rhythm disruptions that return to normal in 60 s (Zhang et al., 2004a). Apoptotic or necrotic cells

are found in 20-30% of livers in mice receiving high-volume injections, while cell death is apparent in 2% of hepatocytes, all of which express plasmid DNA (Budker et al.,

2006). Hydrodynamics-based injections do not affect growth rates in mice (Liu et al.,

1999), although immediately following injections there is a transient 2 h increase in body weight which returns to baseline by 12 h (Kameda et al., 2003). This is likely due to the large volume of liquid injected which causes the liver to swell to twice its normal size

(Budker et al., 2006). Serum ALT and aspartate aminotransferase (AST) levels increase transiently following high-volume injections but return to normal within 2-3 days (Liu et al, 1999; Maruyama et al., 2002; Kameda et al., 2003; Budker et al., 2006). Other biochemical parameters such as major ion concentrations, albumin, alkaline phosphatase and total bilirubin levels are within normal ranges in mice following hydrodynamics- based injections (Liu et al., 1999). In general, this suggests that high-volume injections

176 may lead to transient adverse outcomes that return to baseline shortly afterwards. Liver

morphology at 24 h post-injection appears normal (Budker et al., 2006).

High in vivo liver transfection efficiency will only result if the full volume of the

injection is administered rapidly through the tail vein. Consequently, a small number of rats in my study were not included in analyses because they did not receive the full volume of their injection in the time required. Such rats exhibited extremely low hepatic transfection efficiency as revealed by both firefly and Renilla luciferase activities. When studying reporter plasmids in vivo, large quantities of plasmid DNA are required for adequate detection of luciferase activity in the liver. Most of the rats in my study received 20 ug firefly luciferase and 1.75 fig Renilla luciferase plasmid DNA for every

1 ml of Ringer's solution injected. Rats studied at 24 h and 72 h following injection with

(-5.6-2C11) and rats studied at 6 h following injection with (-10.1-2C11), received 5 ug firefly luciferase and 0.5 |ig Renilla luciferase plasmid DNA for every 1 mL of Ringer's solution injected. These amounts still fall within optimal ranges required for high-level reporter expression in the liver (Maruyama et al., 2002), but were lowered to conserve plasmid. I injected rats with plasmid DNA according to the mass of DNA rather than the number of DNA molecules. This may be a limitation in my in vivo studies as the number of plasmid DNA constructs present within hepatocytes may affect the magnitude and time course of response following MC treatment.

4.2.2 Transgene expression

Following hydrodynamics-based injections, transgene expression may or may not mimic the behavior of the endogenous gene. In many situations, the behavior of the reporter gene is in fact similar to that of the endogenous or homologous gene. For

177 example, the induction of the human CYP3A4 transgene in mice correlates with the

behavior of endogenous CYP3A11 mRNA expression in response to xenobiotic treatment

(Zhang et al., 2003b). The induction of endogenous CYP2C29 mRNA is consistent with that observed for the Cyp2c29 reporter gene hydrodynarnically-transfected in mice

(Jackson et al., 2004). The human PXR in transgenic mice responds strongly to inducers of the human CYP3A4 gene as measured by the induction of endogenous CYP3A11 mRNA levels in these mice (Xie et al., 2000), indicating that regulation of genes encoding drug metabolizing enzymes can occur normally in a foreign host.

On the other hand, transgene expression may respond differently than the endogenous gene. A group studying CYP2B regulation found that induction of the gene transfected in situ is lower in magnitude than that observed for the endogenous gene

(Honkakoski et al., 1992). Discrepancies between transgene and endogenous gene expression can also arise in cell culture. Transient transfection studies of a SOCS2 reporter gene reveals induction of reporter gene activity by TCDD in Hepa-1 cells despite no corresponding elevation in the native message (Boverhof et al., 2004). The human

CYP1B1 reporter gene is induced by TCDD in HepG2 cells, while endogenous mRNA levels are unaffected (Shehin et al., 2000). Such discrepancies between the responses of the reporter and endogeneous genes may be due to the absence of chromatin in naked plasmid DNA (Eltom et al., 1999). In the absence of chromatin structure, which protects and conceals DNA sequences from the collection of activators and repressors within the cell, TFs within proximity may bind to regulatory sequences that are normally concealed by nucleosomes or histones. The paradoxical induction of CYP2C11 reporter gene activity by TCDD in HepG2 cells may be explained by the accessibility of AHR/ARNT

178 to DREs located in the CYP2C11 5'-flanking region due to the absence of chromatin

structure that normally conceals these binding sites (Boverhof et al., 2004). AHR/ARNT

accessibility to CYP2Cll-DREs in constructs introduced to living rats may be of lesser

significance since the host environment contains an extensive repertoire of TFs, co-

activators, co-repressors, signaling pathways and hormones that support endogenous

CYP2C11 down-regulation by MC.

Interestingly, CFP2C7i-luciferase plasmids strongly suppressed by MC [(-5.6-

2C11) and (-10.1-2C11)], displayed a positive correlation between luciferase activity and endogenous CYP2C11 mRNA levels, indicating that luciferase activity paralleled the response of the endogenous gene. Luciferase activity of (-2.4-2C11), which was weakly suppressed by MC at 24 h, was negatively correlated with CYP2C11 mRNA expression.

This correlation may be skewed by the unexpected trend for induction of this plasmid at

72 h while endogenous CYP2C11 mRNA levels showed a tendency for down-regulation.

Hydrodynamics-based delivery allows for the study of reporter genes in a living organism, which likely produces findings that are more physiologically relevant than reporter gene studies conducted in isolated culture systems. As a result, in vitro reporter assays may not reliably predict in vivo responses of the endogenous gene to xenobiotic treatment. Studying CFP2Cii-luciferase constructs in HepG2 cells revealed paradoxical induction of the reporter gene that is AHR-dependent and DRE-mediated. Conversely, I found that these same reporter plasmids were suppressed by MC treatment when studied in the living rat, a response that is analogous to the behavior of the endogenous CYP2C11 gene. Similarly, Sasaki et al. (1999) found that studying GH regulation of rat CYP2C12 reporter constructs in living rats was more mechanistically accurate than studying these

179 plasmids in HepG2 cells. Transfection studies in HepG2 cells reveal that STAT5b

represses CYP2C12 reporter activity induced by HNF-6 (Delesque-Touchard et al.,

2000). When CYP2C12 constructs were introduced in situ to living rats, STAT5, HNF-4

and HNF-6 co-operatively activated reporter gene transcription (Sasaki et al., 1999). In

vivo analysis is critical when examining genes whose transcription is modulated by GH

(Gardmo and Mode, 2006). Recently, mechanisms involving regulation of the rat A1BG

(oclB-glycoprotein) gene by the female pattern of GH secretion were studied using

hydrodynamics-based injections to introduce reporter constructs to living rats (Gardmo

and Mode, 2006).

Furthermore, one must be cautious when interpreting reporter gene findings if

adequate lengths of DNA regulatory sequences are not included in the plasmid. For

example, (-2.4-2C11) behaves differently than (-5.6-2C11) and (-10.1-2C11); whereas the latter two plasmids, which contain more CYP2C11 regulatory DNA sequence, are more uniform in their in vivo responses to MC.

4.2.3 Effects of MC on endogenous CYP2C11 expression

Treatment of rats with 20 mg/kg (Dannan et al., 1983) and 50 mg/kg (Yeowell et al., 1987; Jones and Riddick, 1996) MC can simultaneously induce CYP1A1 and suppress CYP2C11 expression. To enhance the likelihood of observing endogenous

CYP2C11 and reporter gene suppression, I injected rats with 80 mg/kg MC, a dose which has been used in mice to characterize the suppression of murine P450s without any adverse effects (Lee et al., 2006). Corn oil was used as the vehicle for MC administration in my studies. Previous data show that rat hepatic P450 levels are not affected by corn oil injections compared to untreated intact rats (Thomas et al., 1981).

180 When examining endogenous CYP2C11 protein and mRNA levels in rats

receiving high-volume injections, there was not always statistically significant

suppression in MC-treated rats compared to vehicle controls. Unlike gene induction,

suppression is more difficult to measure reproducibly because it is often a relatively small

magnitude decrease from basal levels, compared to nearly unlimited fold-inductions that can be observed if a gene is up-regulated from low basal levels. It is possible that the high-volume injection itself could produce hepatotoxicity and/or stimulate the release of inflammatory cytokines that are known to suppress endogenous CYP2C11 levels. As a result, rats receiving a hydrodynamics-based injection may have partially suppressed

CYP2C11 levels, regardless of MC treatment. This may explain why MC-treated rats did not always have a statistically significant lowering of CYP2C11 expression compared to vehicle controls. Alternatively, large-scale plasmid preparation may result in abnormally high endotoxin levels due to insufficient removal during plasmid purification. Injection of plasmids containing high endotoxin concentrations may suppress endogenous

CYP2C11 (Cheng et al., 2003) in both vehicle- and MC-treated rats. Had we measured cytokine production, endotoxin levels or serum liver enzymes, we would have a better understanding of the damage elicited by the high-volume injection and its effect on endogenous CYP2C11 expression.

CYP2C11 mRNA levels were quite variable in both vehicle- and chemical- treated male rats. This may result in large standard deviations that make it difficult to detect statistical significance between the two groups (Caron et al., 2005). CYP2C11 variation may be a result of differences in GH pulsatile release between rats (Caron et al.,

2005), although a study which measured CYP2C11 mRNA at several time-points over

181 24 h report that levels do not fluctuate with the time of day (Emi and Omura, 1988). I found that the variability in CYP2C11 expression between rats also coincided with variability in the degree of CYP1A1 induction in MC-treated rats. Overall, there was a positive correlation between CYP2C11 protein and mRNA levels in collections of individual rats, which reached significance in all but one of the five plasmid treatment groups. This further supports a pre-translational mechanism for the down-regulation of

CYP2C11 protein by MC. In some plasmid treatment groups, a decrease in endogenous

CYP2C11 protein levels was detected at an earlier time-point than significant mRNA

suppression was detected. Such findings suggest the possibility that in addition to transcriptional mechanisms, increased protein degradation or decreased protein synthesis may contribute to CYP2C11 suppression by MC.

Greater endogenous CYP2C11 suppression may have been observed if TCDD was used instead of MC in my in vivo studies. These two chemicals differ greatly in their half-lives, which corresponds to differences in their duration of action and observed in vivo potency (Poland and Glover, 1974). In rats, the half-life of TCDD is 12-31 days

(Rose et al., 1976), while only 16 h for MC (Aitio, 1974). In rat liver, TCDD is 30,000- fold more potent than MC at inducing AHH activity (Poland and Glover, 1974). While both compounds produce the same maximal response, MC does so at a much higher dose.

In Hepa-1 cells, Riddick et al. (1994) reported that MC is a 1000-fold less potent inducer of AHH activity compared to TCDD; however, competitive binding assays revealed that

TCDD displays only a 3- to 4-fold higher AHR binding affinity than MC. This study concluded that differences in the ability of these two aromatic hydrocarbons to induce

AHH activity was attributed to the more rapid metabolism of MC and/or differences in

182 the nuclear signaling events required for CYP1A1 induction. Furthermore, there is a difference in the duration of action between the non-metabolizable TCDD and the readily metabolizable MC. AHH activity in rats treated with a single dose of either chemical is similar for the first 4 days, after which AHH activity rapidly declines in MC-treated rats yet remains elevated in TCDD-treated rats (Poland and Glover, 1974). In Hepa-1 cells,

TCDD and MC display similar potencies for CYP1A1 mRNA induction at 2 h but TCDD is -1000-fold more potent at 16 h, suggesting that time-dependent metabolism of MC is responsible for the reduced potency of this chemical (Riddick et al., 1994). While numerous groups use TCDD to characterize aromatic hydrocarbon-responsive genes in vitro, most use lower potency ligands such as MC to characterize aromatic hydrocarbon- responsive genes in vivo (Thomas et al., 1981; Kimura et al, 1989; Jones and Riddick,

1996; Kurose et al., 1998; Lee and Riddick, 2000; Timsit and Riddick, 2000; Nukaya et al., 2004; Arpiainen et al., 2005; Jenkins et al., 2006; Lee et al., 2006). Nearly all in vivo rat studies use MC to characterize the regulation of CYP2C11 by aromatic hydrocarbons.

As a result, I was confident with respect to in vivo MC doses and time-points expected to produce CYP1A1 induction and CYP2C11 suppression. On the downside, the use of a less potent AHR ligand such as MC, may diminish the magnitude of endpoint responses including both endogenous CYP2C11 and reporter suppression.

The use of a PAH, such as MC which undergoes metabolism, may initiate secondary AHR-independent pathways that could contribute to suppression of endogenous CYP2C11 and CFP2Ci7-luciferase constructs. B[a]P exposure leads to the initiation of AHR-independent pathways including changes in the expression of genes involved in the DNA-damage response and cell cycle regulation (Hockley et al., 2007).

183 Hockley's study aimed to identify DNA-damage pathways that are induced by PAH metabolites by comparing gene expression profiles following treatment of HepG2 and

MCF-7 cells with TCDD and B[a]P. Genes that responded only to B[a]P or its major metabolite, but not TCDD, were predominantly involved in DNA repair, cell cycle control and apoptosis. Other AHR-independent pathways activated by PAH exposure may be initiated following the generation of reactive oxygen species as a byproduct of metabolism. The production of reactive oxygen species may induce numerous signaling pathways (Morel et al., 1999; Radjendirane and Jaiswal, 1999) that could potentially contribute to CYP2C11 suppression. Microarray analysis of liver samples from MC- treated rats shows that this chemical triggers the up-regulation of acute phase genes associated with the inflammatory response (Kondraganti et al., 2005). Biotransformation of the PAH benzo[&]fluoranthene results in the production of metabolites that are active in inducing CYP1A1 reporter gene activity and endogenous CYP1A1/CYP1B1 enzymatic activity in T-47D human breast carcinoma cells, stressing the importance of PAH metabolites in the modulation of P450 levels (Spink et al., 2008).

A role for MC in the disruption of hormonal pathways has been established and continues to be of major interest. The estrogen pathway is a known target of MC.

Although TCDD is also known to interfere with estrogen signaling, these two aromatic hydrocarbons differ in their effect on this pathway. A recent study demonstrates that MC directly activates the AHR and also causes ERcc/p activation, although induction of estrogenic signaling is primarily mediated by MC metabolites (Swedenborg et al., 2008).

Other studies show that MC can directly activate ERa, as shown by competitive binding assays, fluorescence resonance energy transfer and the ability of MC to induce estrogen-

184 responsive reporter gene activity in the absence of the AHR (Abdelrahim et al., 2006).

Unlike MC, TCDD cannot activate estrogen-dependent reporter gene activity (Shipley and Waxman, 2006) and TCDD is primarily associated with AHR-dependent anti estrogenic effects (Safe, 1995; Ruegg et al., 2008). MC can induce ER-dependent gene transcription in both 5L and BP8 cells, indicating that MC or its metabolite(s) may directly activate ERa regardless of AHR status (Shipley and Waxman, 2006). Although

MC can induce ERa-dependent transcription, it does so at higher concentrations than required for AHR-dependent gene transcription. Estrogenic effects of PAHs may also be due to an AHR-dependent "hijacking" of ERa in the absence of estrogen (Ohtake et al.,

2003). In female rats, MC can increase the length of the estrus cycle and suppress plasma testosterone levels (Yeowell et al., 1987; Konstandi et al., 1997). MC has also been suggested to interfere with normal pheromonal signaling pathways in mice (Shiraiwa et al., 2007). The use of MC- a compound whose metabolites may activate numerous signaling pathways, might lead to indirect transcriptional suppression of endogenous

CYP2C11 and CyP2C77-luciferase constructs. Although most TCDD effects are AHR- mediated (Tijet et al., 2006), this chemical may also function by activating other signaling pathways such as Src kinases (Dunlap et al., 2002).

4.2.4 Time-course of CYP2C11 reporter suppression

When examining suppression by a xenobiotic, CYP2C11 -luciferase activity should ideally be expressed at a relatively stable steady-state level. However, a detailed time-course of luciferase activity following hydrodynamics-based injection of CYP2C11 reporter constructs was not possible due to cost. Since 40 mg/kg MC can partially suppress the rate of CYP2C11 gene transcription within 6 h (Lee and Riddick, 2000) and

185 blockage of transcription is not complete due to ongoing MC metabolism, I included time-points in the in vivo study that would provide a realistic window for detecting suppression of endogenous CYP2C11 and reporter plasmids: 6 h, 24 h and 72 h. The half-life of CYP2C11 mRNA in primary rat hepatocytes ranges from 9.8 h (Iber et al.,

2000) to 16 h (Bhathena et al., 2002). A prior study in our laboratory found that maximal suppression of endogenous CYP2C11 protein and mRNA levels occurs between 72 h to

120 h following in vivo MC treatment (Jones and Riddick, 1996). At 6 h post-treatment,

I did not detect down-regulation of CYP2C11 protein, mRNA or luciferase activity of a construct containing 10.1-kb of flanking region. At 24 h and 72 h after MC treatment, there were overall trends for suppression of endogenous CYP2C11 levels and CYP2C11- luciferase activity, with several of these specific responses achieving statistical significance.

A study investigating the time course involved in TCDD-mediated gene expression changes reports that gene induction through AHR activation is evident by 6 h

(Hanlon et al., 2005). Hanlon et al. (2005) suggest that an increase in gene expression observed after 12 h is likely an outcome of secondary TCDD-related effects and not direct AHR/ARNT binding. In accordance, another study reports that 5 h post-treatment is an adequate time to observe gene induction; however, the authors caution that such an early time-point may not be sufficient to observe a decline in gene expression (Patel et al., 2007). CYP2C11 suppression and down-regulation of other genes show a delayed time-course because of the need to allow for decay of pre-existing mRNA and/or protein.

Since mRNA suppression occurs at 24 h or later in our studies, this may be indicative of an indirect mechanism of suppression caused by the secondary effects of MC (Hanlon et

186 al., 2005). However, a previous study was able to detect CYP2C11 mRNA suppression as early as 3 h following MC treatment, which supports a direct transcriptional mechanism (Emi and Omura, 1988). A combination of both direct and indirect mechanisms may be involved in the suppression of CYP2C11 by MC and further investigations are needed to clarify this.

4.2.5 Possible mechanisms mediating CYP2C11 suppression by MC in vivo

All of the CKP2Ci7-luciferase constructs were suppressed 24 h post-treatment, suggesting that TF binding sites located in the first 2.4-kb of this gene's 5'-flanking region are required for transient suppression by MC. By 72 h post-treatment, only (-10.1-

2C11) and (-5.6-2C11) luciferase activity remain down-regulated. This indicates that negative regulatory elements located between -5.6 and -2.4-kb on the CYP2C11 5'-flank mediate persistent suppression by MC. Although the precise mechanism(s) of CYP2C11 suppression remains to be elucidated, possible mechanisms can be postulated based on the presence of TF binding sites located between -5.6 and -2.4-kb (Fig. 4.1). While the functionality of these binding elements has yet to be determined, they include: six DRE- like sites, one AHRE-II site, five NF-KB sites, one STAT5 site and eight HNF-3 sites. I envision three possible mechanisms by which MC suppresses CYP2C11 expression: direct AHR-mediated suppression via DREs and/or AHRE-II; indirect suppression through interference with GH signaling needed to maintain CYP2C11 transcription; and/or indirect suppression involving inflammatory cytokines activated by MC.

4.2.5.1 Role of the AHR

SAR studies first introduced the notion that CYP2C11 suppression coincided with the stimulation of AHR-mediated responses (Dannan et al., 1983; Safa et al., 1997). Our

187 laboratory has demonstrated that the transformed AHR binds with high affinity to a DRE- like sequence at positions -1546 to -1533-bp of the CYP2C11 5'-flanking region

(Bhathena et al., 2002). My in vitro studies show that this DRE is functional in the paradoxical induction of CFjP2Cii-luciferase activity by TCDD in HepG2 cells, as determined by site-directed mutagenesis. Multiple DRE-like elements and/or the AHRE-

II motif located between -5.6 and -2.4-kb on the CYP2C11 5'-flank, may function as negative regulatory elements. Such motifs have been shown in other genes to bind the activated AHR, yet interfere with the binding of positive trans-acting factors to nearby enhancer elements resulting in the down-regulation of gene expression [reviewed in:

(Riddick et al., 2003)]. I was unable to verify the role of the AHR in CYP2C11- luciferase suppression since in vivo conditions were not identified in which oc-NF alleviated CYP1A1 induction by MC.

4.2.5.2 Interference with GH signaling pathways by MC

The second mechanistic possibility involves the interference by MC with hormonal signaling pathways required to drive basal CYP2C11 transcription. The primary physiological regulator of hepatic CYP2C11 expression is the pulsatile pattern of pituitary GH secretion and STAT5b seems to be a key intracellular messenger that is at least partially responsible for this process (Park and Waxman, 2001; Timsit and Riddick,

2002; Verma et al, 2005). MC interferes with the ability of GH to stimulate hepatic

CYP2C11 expression in the liver of hypx male rats (Timsit and Riddick, 2000). In rat models, MC does not seem to disrupt GH-stimulated STAT5b signaling (Timsit and

Riddick, 2002), although several components of the GHR/JAK2/STAT5b pathway are targets for disruption by MC in mice (Nukaya et al., 2004; Lee et al., 2006).

188 -10.1-kb ||- 24 h 72 h

V7 V7

24 h 72 h -5.6-kb

AHRE-I (6) V V AHRE-II (1) NF-KB (5) -2.4-kb 24 h STAT-5 (1) HNF-3 (8) V

Figure 4.1. Possible transcription factor binding sites mediating in vivo CYP2C11 reporter suppression by MC. In vivo reporter gene studies suggest that transcription factor binding sites located in the proximal 2.4-kb of the CYP2C11 5'-flanking region may mediate transient (24 h) suppression by MC. Regulatory elements) located between -5.6 and -2.4-kb on the CYP2C11 5'-f]ank may mediate a more persistent and greater magnitude reporter suppression by MC. Potential transcription factor binding sites and their frequency of occurrence between -5.6 and -2.4-kb as identified by bioinformatics analyses are shown. Arrows do not necessarily reflect the magnitude or statistical significance of the reporter suppression by MC.

189 Ethylbenzene, a simple aromatic hydrocarbon, can decrease CYP2C11 protein only in intact but not in hypx rats (Serron et al., 2001), suggesting that CYP2C11 down- regulation by both simple and polycyclic aromatic hydrocarbons may occur by a mechanism that disrupts the ability of GH to maintain constitutive CYP2C11 expression.

MC could modulate other parts of the GH signaling pathway such as the SOCS and CIS family of inhibitory proteins whose expression is increased in male rats following the GH pulse. The SOCS/CIS proteins inhibit JAK2/STAT5b signaling

(Thangavel and Shapiro, 2007), and their modulation could impact the maintenance of

CYP2C11 transcription. Aromatic hydrocarbon treatment may increase the expression of these inhibitory proteins as shown by a previous study reporting that treatment of murine

B cell lymphoma cells with TCDD leads to a 3-fold elevation of SOCS2 mRNA levels

(Boverhof et al., 2004). MC could be targeting other pathways downstream of the GHR such as MAPK signaling (Wauthier et al., 2006a; Dhir et al., 2007), which may modulate

JAK2/STAT5b activity (Dhir et al., 2007). STAT5b may function as a repressor of GH- dependent genes by interfering with nuclear receptor signaling (Su et al., 2005; Mode and

Gustafsson, 2006; Ahmed et al., 2007), inducing transcriptional repressors (Holloway et al., 2007), or inducing micro-RNA expression leading to mRNA degradation (Ono et al.,

2007). It is possible that one or more of these mechanisms contributes to CYP2C11 reporter suppression by MC through interference with GH-activated signaling.

4.2.5.3 Involvement of inflammatory cytokines

A third possible mechanism mediating CYP2C11 suppression by MC involves inflammatory cytokines. There are five potential NF-KB binding sites located between

-5.6 and -2.4-kb on the CYP2C11 5'-flank. MC could activate inflammatory cytokines

190 which may suppress the reporter constructs by: initiating NF-KB binding to regions on the

CYP2C11 5'-flank (Iber et al., 2000), interfering with GH-dependent pathways (Ahmed et al., 2007; Chen et al, 2007; Dhir et al., 2007), or disrupting AHR signaling (Zhao and

Ramos, 1998; Kim et al., 2000).

As discussed in Section 1.6.5, aromatic hydrocarbons may activate cytokines directly or via the production of reactive oxygen species. Interactions between the AHR and p65 subunits of NF-KB may also mediate the down-regulation of CYP2C11. Since interaction with the AHR sequesters p65 subunits in the cytoplasm, it is possible that MC treatment enables p50 subunits, typically implicated in transcriptional repression, to enter the nucleus and suppress CYP2C11 by binding to NF-KB consensus sequences.

Recent studies show inflammatory cytokines can interfere with various components of GH-activated signaling pathways including JAK2/STAT5b and

SOCS/CIS proteins (Ahmed et al., 2007; Chen et al., 2007; Dhir et al, 2007). A recent study reports IL-6 can attenuate GH-inducible STAT5 DNA binding activity (Ahmed et al., 2007). Endotoxin treatment inhibits GH-induced hepatic JAK2/STAT phosphorylation, STAT5 nuclear translocation and DNA binding while increasing inflammatory cytokine levels in the liver (Chen et al., 2007). In turn, SOCS and CIS levels increase in response to inflammatory cytokines, which may contribute to impaired

GH-mediated signaling. LPS can down-regulate GHR mRNA expression and reporter gene activity, which would also impair GH-activated pathways (Dejkhamron et al.,

2007). Furthermore, high levels of NF-KB in the male rat may attenuate the activity of the JAK2/STAT5 pathway and thus decrease CYP2C11 transcription (Dhir et al., 2007).

191 A "triple-null" mouse was generated that lacks two tumor necrosis factor (TNF) receptors and the IL-1 receptor (Pande et al., 2005). These mice are protected from some aspects of liver inflammation caused by TCDD compared to wild-type mice, confirming the attenuation of TCDD-induced IL-1 and TNF signaling. It would be interesting to observe whether CYP2C1 i-luciferase constructs introduced to the "triple-null" mouse would still be down-regulated by MC in the absence of these three cytokine receptors.

4.3 Overall summary of findings

This thesis work has contributed novel insights into the areas of cytochrome P450 regulation, molecular toxicology and the study of environmental contaminants such as dioxins and PAHs. Modulation of gene expression is thought to be the primary mechanism by which TCDD and related chemicals elicit toxic and adaptive responses

(Okey et al., 1994). Alterations in hepatic P450 expression following exposure to aromatic hydrocarbons are of considerable interest since P450s play a central role in the metabolism of numerous exogenous and endogenous substances. AHR-dependent induction of P450s by aromatic hydrocarbons is at least partly understood, whereas mechanisms by which these chemicals down-regulate the expression of constitutive

P450s (Kurose et al., 1998; Riddick et al., 2004; Vezina et al, 2004; Lee et al, 2006;

Ovando et al., 2006) and other genes are poorly characterized. Data from our laboratory

(Jones and Riddick, 1996), in agreement with earlier studies [reviewed in: (Riddick et al.,

2003)] as well as recent studies (Shaban et al, 2005; Caron et al., 2005), report that exposure of male rats to aromatic hydrocarbons results in the suppression of hepatic

CYP2C11 catalytic activity, protein and mRNA levels via an unknown pre-translational mechanism.

192 My thesis research provides key mechanistic insights into the regulation of

CYP2C11 by aromatic hydrocarbons both in vitro and in vivo by studying extended lengths of this gene's 5'-flanking region that until now, have never been cloned or characterized. The in vitro studies presented in this thesis, as well as previous studies in our laboratory (Bhathena et al., 2002), show that TCDD and MC do not down-regulate expression of CYP2C11 -luciferase reporter constructs containing up to 10.1-kb of the

CYP2C11 5'-flanking region in transient transfection assays in cell culture. In human

HepG2 cells, I discovered a surprising 6- to 8-fold paradoxical induction of the 10.1-kb construct by TCDD, which was blocked by a-NF. A DRE located between -1546 to

-1533-bp was found to participate in the paradoxical induction as shown by site-directed mutagenesis. CyP2Cii-luciferase constructs display a potentially misleading paradoxical in vitro induction in HepG2 cells that is AHR-dependent and DRE-mediated.

Lack of suppression of CYP2C11 -luciferase constructs by aromatic hydrocarbons in cell culture studies suggests this gene should be studied in vivo where the full complement of

TFs, co-activators, co-repressors and endocrine factors necessary for physiological

CYP2C11 regulation are present. By utilizing the hydrodynamics-based approach to deliver naked plasmid DNA to the liver of live rats, I have conducted the first and only study of CYP2C11 reporter constructs in vivo.

My in vivo findings indicate that negative regulatory element(s) responsible for

CYP2C11 reporter suppression by MC exist within the first 2.4-kb of the 5'-flanking region and promoter of this gene. Additional upstream sequences located between -5.6 and -2.4-kb mediate a more persistent suppression of CFP2Cii-driven luciferase activity.

Modulation of reporter gene activity by MC was accompanied by induction of

193 endogenous CYP1A1 and varying degrees of endogenous CYP2C11 suppression in rats

receiving hydrodynamics-based injections. This is the first observed down-regulation of

CYP2C11 reporter gene activity by aromatic hydrocarbons in any model system, and my results suggest that CYP2C11 suppression in vivo occurs by a transcriptional mechanism

mediated by the 5'-flanking region and promoter of this gene.

4.4 Future studies

4.4.1 Additional deletion and mutant constructs introduced to rats by hydrodynamics-based injections

To more precicely define the region of the CYP2C11 5'-flank that is mediating

suppression of reporter constructs in vivo, additional deletion and mutant constructs must be generated. Of particular interest are deletion constructs that allow further analysis of the region between -5.6 and -2.4-kb, which was found to confer at least a portion of the

suppression response. Bioinformatics search results for putative TF binding sites of interest (Fig. 4.1) can help direct generation of novel mutant constructs that would be defective in the binding of these TFs to their consensus sequences within the CYP2C11- luciferase constructs. This would clearly identify key trans- and cis- acting elements involved in the suppression of reporter gene activity following MC exposure.

4.4.2 Studying CFP2CI/-luciferase constructs in hypophysectomized rats

Studying CYP2C1 i-luciferase constructs in the hypx rat model will allow a determination of whether MC interferes with pituitary factors that maintain constitutive

CYP2C11 transcription. Exogenous GH administration to hypx rats could determine if

CYP2C17-luciferase constructs are positively regulated by GH and if MC interferes with the ability of GH to stimulate CIT2Cii-luciferase expression in vivo. Recently, a novel

194 GHR antagonist known as BVT-A has been characterized in rats and this compound is functional in vivo (Rosengren et al., 2007). It would be interesting to determine the MC- responsiveness of Cyj°2C7/-luciferase constructs in GH-treated hypx rats that have been administered BVT-A. Alternatively, the response of the CYP2C11 constructs to aromatic hydrocarbons could be studied following their transfection into primary rat hepatocytes that are responsive to GH pulses (Thangavel et al., 2006). Until this published report appeared in 2006, it was not possible to induce male-specific P450s in isolated hepatocytes by mimicking a pulsatile pattern of GH secretion.

One must be cautious when studying rats that have undergone endocrine organ ablation surgeries. The removal of endocrine organs may alter the expression of the

AHR as observed in hypx rats (Timsit et al., 2002); such changes may affect the response of genes to aromatic hydrocarbon treatment. Dwarf rats or MSG-treated rats, which display a more selective GH deficiency (Waxman et al, 1990; Shimada et al., 1997), may also be used to determine whether GH status influences suppression of CYP2C11- luciferase activity by aromatic hydrocarbons. These two rat models do not have altered levels of other pituitary hormones which are affected in hypx rats and therefore can be used to determine GH's role with greater precision.

4.4.3 Additional in vivo AHR antagonists

Although my attempts to antagonize the AHR in vivo were not successful, there are several alternative approaches to achieve this goal. Several promising in vivo AHR antagonists will need to be investigated to determine their selectivity and potency in inhibiting AHR-mediated events in the rat. Although most AHR antagonists are actually partial agonists displaying varying degrees of agonist-like activity, there are several

195 compounds that may prove successful if studied under the correct conditions. These candidates include: MCDF (Astroff et al., 1988; Bannister et al., 1989); CH-223191 (Kim et al., 2006); and 3'M4'NF (Nazarenko et al., 2001). These antagonists have been studied in living rodents and the latter two chemicals appear to be the most promising in vivo AHR antagonists to date. Ultimately, one must be cautious of the non-specific actions associated with antagonists including anti-estrogenic signaling as observed with

MCDF (Merchant et al., 1993). As an aside, I was not able to obtain sufficient quantities of CH-223191 or 3'M4'NF for my proposed in vivo rat studies; custom synthesis of these compounds may be required.

Another technique that can be used to examine the role of the AHR in CYP2C11 reporter suppression is the in vivo employment of siRNA targeting this receptor. It will be interesting to determine whether the introduction of siRNA molecules to rats by hydrodynamics-based injections can down-regulate AHR expression. Once the time course and extent of AHR down-regulation is established, CYP2C11 reporter constructs can be introduced to rats by high-volume injections and the effects of MC administration can be determined.

4.4.4 Chromatin immunoprecipitation assays to study the binding of TFs of interest to specific regions of the CYP2C11 5'-flank in a chromosomal context

ChIP assays will be useful to determine whether specific TFs interact with their target sites on the CYP2C11 5'-flanking region when the gene is in its native nucleosomal configuration. This is critical since the regulation of transcription in eukaryotic cells is strongly influenced by chromatin structure. Crosslinking using formaldehyde covalently

"freezes" proteins bound to chromatin at a specific time-point and antibodies can be used

196 to immunoprecipitate candidate TFs of suspected importance (eg. AHR, STAT5b, NF-

KB). Together with hydrodynamics-based in vivo reporter transfections, these ChIP studies will permit definitive characterization of the key protein-DNA interactions involved in the suppression of CYP2C11 by aromatic hydrocarbons in vivo. This approach will also facilitate construction of models of how changes in protein binding at distal DNA elements can alter promoter occupancy.

4.4.5 Related studies in a mouse model

4.4.5.1 Mouse P450s down-regulated by aromatic hydrocarbons

Some mouse hepatic P450s from the CYP2 and CYP3 families are also subject to sex-dependent GH regulation (Udy et al., 1997; Jarukamjorn et al., 2006). Aromatic hydrocarbons suppress the expression of mouse CYP3A13 (Ovando et al., 2006), mouse

CYP2D9 (Jenkins et al., 2006; Lee et al., 2006), and mouse CYP3A11 (Jenkins et al.,

2006; Lee et al., 2006) by unknown mechanisms. It will be interesting to study the mechanism(s) involved in the suppression of mouse P450s by aromatic hydrocarbons.

Such studies could contribute to our understanding of the mechanisms involved in

CYP2C11 suppression since specific mouse P450s (e.g. CYP2D9) seem to display similar

GH regulatory pathways. If down-regulation of these genes is found to occur at the transcriptional level, in vivo reporter gene studies in mice can be implemented with the goal of defining key sequences mediating reporter gene suppression.

4.4.5.2 Regulation of CYP2Cll-luciferase constructs by aromatic hydrocarbons in wild-type and Ahr-nutt mice

Another approach to investigate the AHR's role in CYP2C11 suppression will be the use of mice as a heterologous species to study this gene's down-regulation.

197 Numerous studies have examined transgene regulation in foreign species. Human P450 reporter genes have been transfected and their responses characterized in rats or mice

(Schuetz et al., 2002; Zhang et al., 2003b; Fisher et al., 2007). Likewise, rat P450s reporters have been studied in mice (Schuetz et al., 2002). Promoters from various

species introduced to mice by HVTV injections are all transcriptionally active, suggesting mice harbor the necessary transcriptional machinery needed to drive activity from diverse promoters (Al-Dosari et al., 2006). An interesting future direction would be to study the aromatic hydrocarbon-responsiveness of C7P2C7i-luciferase constructs in both wild- type and A/ir-null mice (Schmidt et al., 1996). Such studies may provide a clearer understanding of the AHR's involvement in CYP2C11 reporter suppression by aromatic hydrocarbon treatment.

4.5 Physiological and global relevance of studying CYP2C11 down-regulation

Although it is difficult to speculate on the physiological significance of CYP2C11 down-regulation, it has been proposed that this is either a pathophysiological response to stress signals (e.g. toxicants, inflammation) or an adaptive response which minimizes the production of reactive oxygen species, nitric oxide, or arachidonic acid metabolites that can be generated during P450 reactions (Morgan, 2001; Riddick et al., 2004). An important aspect of P450 evolution is thought to be "animal-plant warfare", in which plants produced toxic chemicals for the purpose of self-protection and animals adapted by detoxifying such chemicals via P450-catalyzed reactions (Yang et al., 1992). CYP2C11 suppression could be a protective mechanism in male rats such that it prevents the toxicity of ingested plant toxins by decreasing their bioactivation (Yamada et al., 1998).

On the other hand, suppression of this P450 could impair the ability of the organism to

198 respond to foreign chemicals and metabolize endogenous substrates.

Studying mechanisms of CYP2C11 suppression has useful implications from both clinical and molecular viewpoints. P450 suppression is of clinical significance as shown by altered drug toxicity in patients during inflammatory conditions (Ueyama et al., 2004;

Morgan et al., 2008). Inflammation results in reduced P450 drug-metabolizing activity, which may cause adverse effects in humans due to increased plasma drug concentrations and lowered drug clearance. In humans injected with a small dose of LPS, the clearance of three probe drugs used as markers for P450 activity is reduced, suggesting decreased drug metabolism in humans following an inflammatory response (Shedlofsky et al.,

1994). The physiological significance of CYP2C11 suppression following inflammation has been questioned. Morgan (2001) speculates that this may be a mechanism allowing the liver to deal with inflammation by transcribing acute-phase proteins (Morgan, 2001).

Studying CYP2C11 regulation may improve our understanding of human pathology. For example, CYP2C11 mRNA suppression in a rat model of hemorrhage suggests ways in which drug-metabolizing capacity may be impacted in patients suffering from hemorrhage or sepsis (Higuchi et al., 2007). CYP2C11 has also been used as a model to study the effects of fasting on drug metabolism (Shimizu et al., 2003).

The CYP2C subfamily constitutes 10-20% of the total P450 content in the human liver. CYP2C9 and CYP2C19 are the most abundant members of the human CYP2C subfamily (Shimada et al., 1994). No direct CYP2C11 orthologue in the human exists and none of the CYP2C isoforms show orthologous relationships between humans and rodents (Uno et al., 2006). Many CYP2C members metabolize common substrates, but each isoform acts in a regio- and stereo-selective manner (Miyazawa et al., 2003; Ohhira

199 et al., 2006). CYP2C members epoxidize arachidonic acid, yet each isoform produces a unique spectrum of metabolites (Barbosa-Sicard et al., 2005). This subfamily contributes to tetrahydrocannabinol metabolism in rats, mice and humans (Yamamoto et al., 1995).

Populations exposed to PAHs are at an increased risk of developing certain types of cancers (Pavanello et al., 1999; Lee and Shim, 2007), and B[a]P is classified as a known human carcinogen by the IARC. Occupational exposure to PAHs has been reported to suppress human P450s (Lemm et al., 2004), yet little is known about mechanisms involved in this suppression. Although P450 down-regulation in response to

aromatic hydrocarbon treatment occurs in several animal species, the effects of these chemicals on the expression of human P450s remains largely unexplored. An important recent study showed that CYP2C8 mRNA levels are decreased by MC in human hepatocytes (Ning et al., 2008). CYP2C11 serves as a valuable model system to understand how HAHs and PAHs decrease the transcription of other hormonally- regulated genes.

Although the sex difference in hepatic P450 expression is not as obvious in humans as it is in rats, it does exist and appears to be under hormonal control (Shapiro et al., 1995; Wiesener et al., 2001; Wolbold et al., 2003; Cheung et al., 2006; Dhir et al.,

2006; Laz et al., 2007). Human P450s such as CYP3A4, CYP1A2 and CYP2D6 are responsive to the sexually dimorphic patterns of GH secretion as observed in human hepatocyte cultures treated with recombinant human GH (Dhir et al., 2006). Transgenic male mice harboring the human CYP3A4 and CYP3A7 genes show induced hepatic

CYP3A4 expression following exposure to the feminine pattern of continuous GH infusion (Cheung et al., 2006). This shows that human CYP3A4 is responsive to

200 hormonal signals, even in a heterologous species. Furthermore, human P450 activity is also modulated by growth hormone (Jurgens et al., 2002).

My thesis research focused on CYP2C11, a GH-regulated rat hepatic P450 that is down-regulated in vivo by aromatic hydrocarbons. Disruption of GH signaling pathways is a novel and relatively unexplored aspect of endocrine disruption by HAHs and PAHs.

Animal models are essential in studying regulation of hormone-dependent genes by toxicants. Consequently, studying the molecular mechanisms involved in the control of rat P450s may potentially reveal fundamental mechanistic pathways that are conserved in other species.

201 5.0 REFERENCES

Abbott BD, Held GA, Wood CR, Buckalew AR, Brown JG and Schmid J (1999a) AhR, ARNT, and CYP1A1 mRNA quantitation in cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in culture. Toxicol Sci 47:62-75. Abbott BD, Schmid JE, Pitt JA, Buckalew AR, Wood CR, Held GA and Diliberto JJ (1999b) Adverse reproductive outcomes in the transgenic Ah receptor-deficient mouse. Toxicol Appl Pharmacol 155:62-70. Abdelrahim M, Ariazi E, Kim K, Khan S, Barhoumi R, Burghardt R, Liu S, Hill D, Finnell R, Wlodarczyk B, Jordan VC and Safe S (2006) 3-Methylcholanthrene and other aryl hydrocarbon receptor agonists directly activate estrogen receptor-a. Cancer Res 66:2459-2467. Adachi J, Mori Y, Matsui S and Matsuda T (2004) Comparison of gene expression patterns between 2,3,7,8-tetrachlorodibenzo-/?-dioxin and a natural aryl hydrocarbon receptor ligand, indirubin. Toxicol Sci 80:161-169. Agrawal AK and Shapiro BH (2000) Differential expression of gender-dependent hepatic isoforms of cytochrome P450 by pulse signals in the circulating masculine episodic growth hormone profile of the rat. J Pharmacol Exp Ther 292:228-237. Agrawal AK and Shapiro BH (2001) Intrinsic signals in the sexually dimorphic circulating growth hormone profiles of the rat. Mol Cell Endocrinol 173:167-181. Ahluwalia A, Clodfelter KH and Waxman DJ (2004) Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray analysis. Mol Endocrinol 18:747-760. Ahmed TA, Buzzelli MD, Lang CH, Capen JB, Shumate ML, Navaratnarajah M, Nagarajan M and Cooney RN (2007) Interleukin-6 inhibits growth hormone- mediated gene expression in hepatocytes. Am J Physiol Gastrointest Liver Physiol 292:G1793-1803. Aitio A (1974) Different elimination and effect on mixed function oxidase of 20-methylcholanthrene after intragastric and intraperitoneal administration. Res Commun Chem Pathol Pharmacol 9:701-710. Aitken RJ, Koopman P and Lewis SE (2004) Seeds of concern. Nature 432:48-52. Akiyama TE and Gonzalez FJ (2003) Regulation of P450 genes by liver-enriched transcription factors and nuclear receptors. Bba-Gen Subjects 1619:223-234. Al-Dosari M, Zhang G, Knapp JE and Liu D (2006) Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver. Biochem Biophys Res Commun 339:673-678. Amakura Y, Tsutsumi T, Nakamura M, Kitagawa H, Fujino J, Sasaki K, Yoshida T and Toyoda M (2002) Preliminary screening of the inhibitory effect of food extracts on activation of the aryl hydrocarbon receptor induced by 2,3,7,8- tetrachlorodibenzo-/?-dioxin. Biol Pharm Bull 25:272-274. Anderson MD, Bandiera SM, Chang TKH and Bellward GD (1998) Effect of androgen administration during puberty on hepatic CYP2C11, CYP3A, and CYP2A1 expression in adult female rats. Drug Metab Dispos 26:1031-1038.

202 Andreola F, Calvisi DF, Elizondo G, Jakowlew SB, Mariano J, Gonzalez FJ and DeLuca LM (2004) Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion. Hepatology 39:157-166. Annicotte JS, Schoonjans K, Haby C and Auwerx J (2001) An E-box in pGL3 reporter vectors precludes their use for the study of sterol regulatory element-binding proteins. Biotechniques 31:993-994,196. Anyanwu EC, El-Saeid MH, Akpan AI and Saled MA (2003) Evaluation of the most current and effective methods in the analysis of chlorinated dioxins in ground beef. ScientificWorld Journal 3:913-921. Arpiainen S, Lamsa V, Pelkonen O, Yim SH, Gonzalez FJ and Hakkola J (2007) Aryl hydrocarbon receptor nuclear translocator and upstream stimulatory factor regulate Cytochrome P450 2A5 transcription through a common E-box site. J Mol Biol 369:640-652. Arpiainen S, Raffalli-Mathieu F, Lang MA, Pelkonen O and Hakkola J (2005) Regulation of the Cyp2a5 gene involves an aryl hydrocarbon receptor-dependent pathway. Mol Pharmacol 67:1325-1333. Astroff B and Safe S (1988) Comparative antiestrogenic activities of 2,3,7,8- tetrachlorodibenzo-/?-dioxin and 6-methyl-l,3,8-trichlorodibenzofuran in the female rat. Toxicol Appl Pharmacol 95:435-443. Astroff B, Zacharewski T, Safe S, Arlotto MP, Parkinson A, Thomas P and Levin W (1988) 6-Methyl-l,3,8-trichlorodibenzofuran as a 2,3,7,8-tetrachlorodibenzo-/>- dioxin antagonist: inhibition of the induction of rat cytochrome P450 isozymes and related monooxygenase activities. Mol Pharmacol 33:231-236. Baccarelli A, Pesatori AC, Consonni D, Mocarelli P, Patterson DG, Jr., Caporaso NE, Bertazzi PA and Landi MT (2005) Health status and plasma dioxin levels in chloracne cases 20 years after the Seveso, Italy accident. Br J Dermatol 152:459- 465. Baccarelli A, Pesatori AC, Masten SA, Patterson DG, Needham LL, Mocarelli P, Caporaso NE, Consonni D, Grassman JA, Bertazzi PA and Landi MT (2004) Aryl-hydrocarbon receptor-dependent pathway and toxic effects of TCDD in humans: a population-based study in Seveso, Italy. Toxicol Lett 149:287-293. Backes WL, Sequeira DJ, Cawley GF and Eyer CS (1993) Relationship between hydrocarbon structure and induction of P450: effects on protein levels and enzyme activities. Xenobiotica 23:1353-1366. Bacsi SG, Reiszporszasz S and Hankinson O (1995) Orientation of the heterodimeric aryl hydrocarbon (dioxin) receptor complex on its asymmetric DNA recognition sequence. Mol Pharmacol 47:432-438. Badger TM, Ronis MJJ, Frank SJ, Chen Y and He L (2003) Effects of chronic ethanol on hepatic and renal CYP2C11 in the male rat: Interactions with the Janus-kinase 2- signal transducer and activators of transcription proteins 5b pathway. Endocrinology 144:3969-3976. Bannister R, Biegel L, Davis D, Astroff B and Safe S (1989) 6-Methyl-l,3,8- trichlorodibenzofuran (MCDF) as a 2,3,7,8-tetrachlorodibenzo-/?-dioxin antagonist in C57BL/6 mice. Toxicology 54:139-150.

203 Barbosa-Sicard E, Markovic M, Honeck H, Christ B, Muller DN and Schunck WH (2005) Eicosapentaenoic acid metabolism by cytochrome P450 enzymes of the CYP2C subfamily. Biochem Biophys Res Commun 329:1275-1281. Beischlag TV and Perdew GH (2005) ERa-AHR-ARNT protein-protein interactions mediate estradiol-dependent transrepression of dioxin-inducible gene transcription. J Biol Chem 280:21607-21611. Benedict WF, Considine N and Nebert DW (1973) Genetic differences in aryl hydrocarbon hydroxylase induction and benzo[a]pyrene-produced tumorigenesis in the mouse. Mol Pharmacol 9:266-277. Bergeron RM, Serron SC, Rinehart JJ, Cawley GF and Backes WL (1998) Pituitary component of the aromatic hydrocarbon-mediated expression of CYP2B and CYP2C11. Xenobiotica 28:303-312. Bertazzi PA, Zocchetti C, Guercilena S, Consonni D, Tironi A, Landi MT and Pesatori AC (1997) Dioxin exposure and cancer risk: A 15-year mortality study after the "Seveso accident". Epidemiology 8:646-652. Bhathena A, Lee C and Riddick DS (2002) Suppression of cytochrome P450 2C11 by aromatic hydrocarbons: mechanistic insights from studies of the 5 '-flanking region of the CYP2C11 gene. Drug Metab Dispos 30:1385-1392. Biagini C and Celier C (1996) cDNA-directed expression of two allelic variants of cytochrome P450 2C11 using COS1 and SF21 insect cells. Arch Biochem Biophys 326:298-305. Birnbaum LS (1994) The mechanism of dioxin toxicity: Relationship to risk assessment. Environ Health Perspect 102:157-167. Birnbaum LS and Cummings AM (2002) Dioxins and endometriosis: A plausible hypothesis. Environ Health Perspect 110:15-21. Black VH and Quattrochi LC (2004) Molecular cloning of the guinea pig CYP1A2 gene 5'-flanking region: Identification of functional aromatic hydrocarbon response element and characterization of CYP1A2 expression in GPC16 cells. Drug Metab Dispos 32:595-602. Bock KW and Kohle C (2006) Ah receptor: dioxin-mediated toxic responses as hints to deregulated physiologic functions. Biochem Pharmacol 72:393-404. Bodner KM, Collins JJ, Bloemen LJ and Carson ML (2003) Cancer risk for chemical workers exposed to 2,3,7,8-tetrachlorodibenzo-/?-dioxin. Occup Environ Medicine 60:672-675. Boffetta P, Jourenkova N and Gustavsson P (1997) Cancer risk from occupational and environmental exposure to polycyclic aromatic hydrocarbons. Cancer Causes Control 8:444-472. Boutros PC, Moffat JD, Franc MA, Tijet N, Tuomisto J, Pohjanvirta R and Okey AB (2004) Dioxin-responsive AHRE-II gene battery: identification by phylogenetic footprinting. Biochem Biophys Res Commun 321:707-715. Boutros PC and Okey AB (2004) PUNS: transcriptomic- and genomic- in silico PCR for enhanced primer design. Bioinformatics 20:2399-2400. Boverhof DR, Burgoon LD, Williams KJ and Zacharewski TR (2008) Inhibition of estrogen-mediated uterine gene expression responses by dioxin. Mol Pharmacol 73:82-93.

204 Boverhof DR, Tam E, Harney AS, Crawford RB, Kaminski NE and Zacharewski TR (2004) 2,3,7,8-Tetrachlorodibenzo-/?-dioxin induces suppressor of cytokine signaling 2 in murine B cells. Mol Pharmacol 66:1662-1670. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254. Brunner LJ, Bennett WM and Koop DR (1998) Cyclosporine suppresses rat hepatic cytochrome P450 in a time-dependent manner. Kidney Int 54:216-223. Budker V, Budker T, Zhang G, Subbotin V, Loomis A and Wolff JA (2000) Hypothesis: naked plasmid DNA is taken up by cells in vivo by a receptor-mediated process. J Gene Med 2:76-88. Budker VG, Subbotin VM, Budker T, Sebestyen MG, Zhang G and Wolff JA (2006) Mechanism of plasmid delivery by hydrodynamic tail vein injection. II. Morphological studies. J Gene Med 8:874-888. Bunger MK, Moran SM, Glover E, Thomae TL, Lahvis GP, Lin BC and Bradfield CA (2003) Resistance to 2,3,7,8-tetrachlorodibenzo-/>-dioxin toxicity and abnormal liver development in mice carrying a mutation in the nuclear localization sequence of the aryl hydrocarbon receptor. J Biol Chem 278:17767-17774. Buters JTM, Sakai S, Richter T, Pineau T, Alexander DL, Savas U, Doehmer J, Ward JM, Jefcoate CR and Gonzalez FJ (1999) Cytochrome P450 1B1 determines susceptibility to 7,12-dimethylbenz[a]anthracene-induced lymphomas. ProcNat Acad Sci USA 96:1977-1982. Caron E, Rioux N, Nicolas O, Lebel-Talbot H and Hamelin BA (2005) Quantification of the expression and inducibility of 12 rat cytochrome P450 isoforms by quantitative RT-PCR. J Biochem Mol Toxicol 19:368-378. Carver LA and Bradfield CA (1997) Ligand-dependent interaction of the aryl hydrocarbon receptor with a novel immunophilin homolog in vivo. J Biol Chem 272:11452-11456. Casper RF, Quesne M, Rogers IM, Shirota T, Jolivet A, Milgrom E and Savouret JF (1999) Resveratrol has antagonist activity on the aryl hydrocarbon receptor: Implications for prevention of dioxin toxicity. Mol Pharmacol 56:784-790. Cauchi S, Stucker I, Solas C, Laurent-Puig P, Cenee S, Hemon D, Jacquet M, Kremers P, Beaune P and Massaad-Massade L (2001) Polymorphisms of human aryl hydrocarbon receptor (AhR) gene in a French population: relationship with CYP1A1 inducibility and lung cancer. Carcinogenesis 22:1819-1824. Celier C and Cresteil T (1989) Induction of drag-metabolizing enzymes in Gunn rat liver. Effect of polycyclic aromatic hydrocarbons on cytochrome P450 regulation. Biochem Pharmacol 38:2825-2832. Chang TKH and Bellward GD (1996) Peripubertal androgen imprinting of rat hepatic cytochrome P450 2C11 and steroid 5a-reductase: pre-translational regulation and impact on microsomal drug activation. J Pharmacol Exp Ther 278:1383-1391. Chen JQ, Strom A, Gustafsson J-A and Morgan ET (1995a) Suppression of the constitutive expression of cytochrome P450 2C11 by cytokines and interferons in primary cultures of rat hepatocytes: Comparison with induction of acute-phase genes and demonstration that CYP2C11 promoter sequences are involved in the suppressive response to intevleukins 1 and 6. Mol Pharmacol 47:940-947.

205 Chen JZ, Murray M, Liddle C, Jiang XM and Farrell GC (1995b) Downregulation of male-specific cytochrome P450s 2C11 and 3A2 in bile duct-ligated male rats: importance to reduced hepatic content of cytochrome P450 in cholestasis. Hepatology 22:580-587. Chen Y, Sun D, Krishnamurthy VM and Rabkin R (2007) Endotoxin attenuates growth hormone-induced hepatic insulin-like growth factor I expression by inhibiting JAK2/STAT5 signal transduction and STAT5b DNA binding. Am J Physiol Endocrinol Metab 292:E 1856-1862. Chen YH, Riby J, Srivastava P, Bartholomew J, Denison M and Bjeldanes L (1995c) Regulation of CYP1A1 by indolo[3,2-&]carbazole in murine hepatoma cells. J Biol Chem 270:22548-22555. Cheung C, Yu AM, Chen CS, Krausz KW, Byrd LG, Feigenbaum L, Edwards RJ, Waxman DJ and Gonzalez FJ (2006) Growth hormone determines sexual dimorphism of hepatic cytochrome P450 3A4 expression in transgenic mice. J Pharmacol Exp Ther 316:1328-1334. Choi HK and Waxman DJ (1999) Growth hormone, but not prolactin, maintains low- level activation of STAT5a and STAT5b in female rat liver. Endocrinology 140:5126-5135. Choi HK and Waxman DJ (2000) Plasma growth hormone pulse activation of hepatic JAK-STAT5 signaling: Developmental regulation and role in male-specific liver gene expression. Endocrinology 141:3245-3255. Clodfelter KH, Holloway MG, Hodor P, Park SH, Ray WJ and Waxman DJ (2006) Sex- dependent liver gene expression is extensive and largely dependent upon signal transducer and activator of transcription 5b (STAT5b): STAT5b-dependent activation of male genes and repression of female genes revealed by microarray analysis. Mol Endocrinol 20:1333-1351. Cole P, Trichopoulos D, Pastides H, Starr T and Mandel JS (2003) Dioxin and cancer: a critical review. Regul Toxicol Pharmacol 38:378-388. Corchero J, Martin-Partido G, Dallas SL and Fernandez-Salguero PM (2004) Liver portal fibrosis in dioxin receptor-null mice that overexpress the latent transforming growth factor-P-binding protein-1. Int J Exp Pathol 85:295-302. Corton JC, Fan LQ, Brown S, Anderson SP, Bocos C, Cattley RC, Mode A and Gustafsson J-A (1998) Down-regulation of cytochrome P450 2C family members and positive acute-phase response gene expression by peroxisome proliferator chemicals. Mol Pharmacol 54:463-473. Courey AJ and Jia S (2001) Transcriptional repression: the long and the short of it. Genes Dev 15:2786-2796. Cox MB and Miller CA (2004) Cooperation of heat shock protein 90 and p23 in aryl hydrocarbon receptor signaling. Cell Stress Chaperones 9:4-20. Crespo A, Peydro A, Dasi F, Benet M, Calvete JJ, Revert F and Alino SF (2005) Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles. Gene Ther 12:927-935. Dannan GA, Guengerich FP, Kaminsky LS and Aust SD (1983) Regulation of cytochrome P-450. Immunochemical quantitation of eight isozymes in liver microsomes of rats treated with polybrominated biphenyl congeners. J Biol Chem 258:1282-1288.

206 Dannan GA, Guengerich FP and Waxman DJ (1986) Hormonal regulation of rat liver microsomal enzymes. Role of gonadal steroids in programming, maintenance and suppression of D4-steroid 5a-reductase, flavin-containing monooxygenase, and sex-specific cytochromes P450. J Biol Chem 261:10728-10735. Davey HW, McLachlan MJ, Wilkins RJ, Hilton DJ and Adams TE (1999) STAT5b mediates the GH-induced expression of SOCS-2 and SOCS-3 mRNA in the liver. Mol Cell Endocrinol 158:111-116. Deckardt K, Weber I, Kaspers U, Hellwig J, Tennekes H and van Ravenzwaay B (2007) The effects of inhalation anaesthetics on common clinical pathology parameters in laboratory rats. Food Chem Toxicol 45:1709-1718. Dejkhamron P, Thimmarayappa J, Kotlyarevska K, Sun J, Lu C, Bonkowski EL, Denson LA and Menon RK (2007) Lipopolysaccharide (LPS) directly suppresses growth hormone receptor (GHR) expression through MyD88-dependent and -independent Toll-like receptor-4/MD2 complex signaling pathways. Mol Cell Endocrinol 274:35-42. Delescluse C, Lemaire G, de Sousa G and Rahmani R (2000) Is CYP1A1 induction always related to AHR signaling pathway? Toxicology 153:73-82. Delesque-Touchard N, Park SH and Waxman DJ (2000) Synergistic action of hepatocyte nuclear factors 3 and 6 on CYP2C12 gene expression and suppression by growth hormone-activated-STAT5b proposed model for female-specific expression of CYP2C12 in adult rat liver. J Biol Chem 275:34173-34182. Denis M, Cuthill S, Wikstrom A-C, Poellinger L and Gustafsson J-A (1988) Association of the dioxin receptor with the Mr 90,000 heat shock protein: a structural kinship with the glucocorticoid receptor. Biochem Biophys Res Commun 155:801-807. Denison MS, Fisher JM and Whitlock JP Jr. (1988) The DNA recognition site for the dioxin-Ah receptor complex. Nucleotide sequence and functional analysis. J Biol Chem 263:17221-17224. Denison MS, Pandini A, Nagy SR, Baldwin EP and Bonati L (2002) Ligand binding and activation of the Ah receptor. Chem Biol Inter 141:3-24. Dertinger SD, Lantum HBM, Silverstone AE and Gasiewicz TA (2000) Effect of 3 '- methoxy-4 '-nitroflavone on benzo[a]pyrene toxicity - Aryl hydrocarbon receptor- dependent and -independent mechanisms. Biochem Pharmacol 60:189-196. Devito MJ and Birnbaum LS (1994) Toxicology of dioxins and related chemicals, in: Dioxins and Health (Schecter A ed), pp 139-162, Plenum Press Div Plenum Publishing Corp, 233 Spring St, New York, NY 10013. Dhir RN, Dworakowski W, Thangavel C and Shapiro BH (2006) Sexually dimorphic regulation of hepatic isoforms of human cytochrome P450 by growth hormone. J Pharmacol Exp Ther 316:87-94. Dhir RN and Shapiro BH (2003) Interpulse growth hormone secretion in the episodic plasma profile causes the sex reversal of cytochrome P450s in senescent male rats. Proc Nat Acad Sci Usa 100:15224-15228. Dhir RN, Thangavel C and Shapiro BH (2007) Attenuated expression of episodic growth hormone-induced CYP2C11 in female rats associated with suboptimal activation of the JAK2/STAT5b and other modulating signaling pathways. Drug Me tab Dispos 11:2102-2110.

207 Donahue BS, Skottnerlundin A and Morgan ET (1991) Growth hormone-dependent and hormone-independent regulation of cytochrome P450 esozyme expression in streptozotocin-diabetic rats. Endocrinology 128:2065-2076. Duan R, Porter W, Samudio I, Vyhlidal C, Kladde M and Safe S (1999) Transcriptional activation of c-fos protooncogene by 17p-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition. Mol Endocrinol 13:1511-1521. Dunlap DY, Dceda I, Nagashima H, Vogel CFA and Matsumura F (2002) Effects of src- deficiency on the expression of in vivo toxicity of TCDD in a strain of c-src knockout mice procured through six generations of backcrossings to C57BL/6 mice. Toxicology 172:125-141. Dunn RT, Ruh TS and Ruh MF (1993) Binding of the Ah receptor to receptor binding factors in chromatin. Biochem Pharmacol 45:1121-1128. Eguchi H, Ikuta T, Tachibana T, Yoneda Y and Kawajiri K (1997) A nuclear localization signal of human aryl hydrocarbon receptor nuclear translocator hypoxia-inducible factor ip is a novel bipartite type recognized by the two components of nuclear pore-targeting complex. J Biol Chem 272:17640-17647. El-Husseini AE, Bladen C and Vincent SR (1995) Molecular characterization of a type II cyclic GMP-dependent protein kinase expressed in the rat brain. J Neurochem 64:2814-2817. Eltom SE, Zhang LY and Jefcoate CR (1999) Regulation of cytochrome P450 (CYP) 1B1 in mouse Hepa-1 variant cell lines: A possible role for aryl hydrocarbon receptor nuclear translocator (ARNT) as a suppressor of CYP1B1 gene expression. Mol Pharmacol 55:594-604. Ema M, Ohe N, Suzuki M, Mimura J, Sogawa K, Ikawa S and Fujii-Kuriyama Y (1994) Dioxin binding activities of polymorphic forms of mouse and human aryl hydrocarbon receptors. J Biol Chem 269:27337-27343. Emi Y and Omura T (1988) Synthesis of sex-specific forms of cytochrome P450 in rat liver is transiently suppressed by hepatic monooxygenase inducers. J Biochem 104:40-43. Enan E and Matsumura F (1995) Evidence for a second pathway in the action mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)- significance of Ah receptor mediated activation of protein kinase under cell-free conditions. Biochem Pharmacol 49:249-261. Endo M, Takahashi Y, Sasaki Y, Saito T and Kamataki T (2005) Novel gender-related regulation of CYP2C12 gene expression in rats. Mol Endocrinol 19:1181-1190. Estabrook RW (2003) A passion for P450s (Remembrances of the early history of research on cytochrome P450). Drug Metab Dispos 31:1461-1473. Estabrook RW, Hildebrandt AG, Baron J, Netter KJ and Leibman K (1971) A new spectral intermediate associated with cytochrome P450 function in liver microsomes. Biochem Biophys Res Commun 42:132-139. Evans BR, Karchner SI, Allan LL, Pollenz RS, Tanguay RL, Jenny MJ, Sherr DH and Halm ME (2008) Repression of aryl hydrocarbon receptor (AHR) signaling by AHR repressor: role of DNA binding and competition for AHR nuclear translocator. Mol Pharmacol 73:387-398. Evans BR, Karchner SI, Franks DG and Hahn ME (2005) Duplicate aryl hydrocarbon receptor repressor genes (ahrrl and ahrr2) in the zebrafish Danio rerio: structure,

208 function, evolution, and AHR-dependent regulation in vivo. Arch Biochem Biophys 441:151-167. Feng DM, He CX, Miao CY, Lu B, Wu WJ, Ding YF and Xue JL (2004) Conditions affecting hydrodynamics-based gene delivery into mouse liver in vivo. Clin Exp Pharmacol Physiol 31:850-855. Fernandez-Salguero P, Pineau T, Hilbert DM, Mcphail T, Lee SST, Kimura S, Nebert DW, Rudikoff S, Ward JM and Gonzalez FJ (1995) Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268:722-726. Fetissov SO, Huang P, Zhang Q, Mimura J, Fujii-Kuriyama Y, Rannug A, Hokfelt T and Ceccatelli S (2004) Expression of hypothalamic neuropeptides after acute TCDD treatment and distribution of Ah receptor repressor. Regul Peptides 119:113-124. Fisher CD, Augustine LM, Maher JM, Nelson DM, Slitt AL, Klaassen CD, Lehman- McKeeman LD and Cherrington NJ (2007) Induction of drug-metabolizing enzymes by garlic and allyl sulfide compounds via activation of constitutive androstane receptor and nuclear factor E2-related factor 2. Drug Metab Dispos 35:995-1000. Fong CJ, Burgoon LD and Zacharewski TR (2005) Comparative microarray analysis of basal gene expression in mouse Hepa-lclc7 wild-type and mutant cell lines. Toxicol Sci 86:342-353. Franc MA, Pohjanvirta R, Tuomisto J and Okey AB (2001) Persistent, low-dose 2,3,7,8- tetrachlorodibenzo-p-dioxin exposure: Effect on aryl hydrocarbon receptor expression in a dioxin-resistance model. Toxicol Appl Pharmacol 175:43-53. Franzen B, Haaparanta T, Gustafsson J-A and Toftgard R (1988) TCDD receptor ligands present in extracts of urban air particulate matter induce aryl hydrocarbon hydroxylase activity and cytochrome P450c gene expression in rat hepatoma cells. Carcinogenesis 9:111-115. Frumkin H (2003) Agent Orange and cancer: an overview for clinicians. CA Cancer J Clin 53:245-255. Fujii-Kuriyama Y and Mimura J (2005) Molecular mechanisms of AhR functions in the regulation of cytochrome P450 genes. Biochem Biophys Res Commun 338:311- 317. Fujisawa-Sehara A, Sogawa K, Yamane M and Fujii-Kuriyama Y (1987) Characterization of xenobiotic responsive elements upstream from the drug- metabolizing cytochrome P-450c gene: a similarity to glucocorticoid regulatory elements. Nucleic Acids Res 15:4179-4191. Fujita I, Sindhu RK and Kikkawa Y (1995) Hepatic cytochrome P450 enzyme imprinting in adult rat by neonatal benzo[a]pyrene administration. Pediatr Res 37:646-651. Fukuda I, Mukai R, Kawase M, Yoshida K and Ashida H (2007) Interaction between the aryl hydrocarbon receptor and its antagonists, flavonoids. Biochem Biophys Res Commun 359:822-827. Fukuda I, Sakane 1, Yabushita Y, Kodoi R, Nishiumi S, Kakuda T, Sawamura SI, Kanazawa K and Ashida H (2004) Pigments in green tea leaves (Camellia sinensis) suppress transformation of the aryl hydrocarbon receptor induced by dioxin. JAgr Food Chem 52:2499-2506.

209 Gabriel SM, Roncancio JR and Ruiz NS (1992) Growth hormone pulsatility and the endocrine milieu during sexual maturation in male and female rats. Neuroendocrinology 56:619-625. Gardmo C and Mode A (2006) In vivo transfection of rat liver discloses binding sites conveying GH-dependent and female-specific gene expression. J Mol Endocrinol 37:433-441. Garfinkel D (1958) Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions. Arch Biochem Biophys 77:493- 509. Garrison PM, Tullis K, Aarts JMMJG, Brouwer A, Giesy JP and Denison MS (1996) Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. FundAppl Toxicol 30:194- 203. Gasiewicz TA and Rucci G (1991) a-Naphthoflavone acts as an antagonist of 2,3,7,8- tetrachlorodibenzo-/?-dioxin by forming an inactive complex with the Ah- receptor. Mol Pharmacol 40:607-612. Gebert CA, Park SH and Waxman DJ (1999) Termination of growth hormone pulse- induced STAT5b signaling. Mol Endocrinol 13:38-56. Geusau A, Tschachler E, Meixner M, Sandermann S, Papke O, Wolf C, Valic E, Stingl G and McLachlan M (1999) Olestra increases faecal excretion of 2,3,7,8- tetrachlorodibenzo-p-dioxin. Lancet 354:1266-1267. Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, Scherer S, Scott G, Steffen D, Worley KC, Burch PE, Okwuonu G, Hines S, Lewis L, DeRamo C, Delgado O, DuganRocha S, Miner G, Morgan M, Hawes A, Gill R, Holt RA, Adams MD, Amanatides PG, BadenTillson H, Barnstead M, Chin S, Evans CA, Ferriera S, Fosler C, Glodek A, Gu ZP, Jennings D, Kraft CL, Nguyen T, Pfannkoch CM, Sitter C, Sutton GG, Venter JC, Woodage T, Smith D, Lee HM, Gustafson E, Cahill P, Kana A, DoucetteStamm L, Weinstock K, Fechtel K, Weiss RB, Dunn DM et al. (2004) Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428:493-521. Gillesby BE, Santostefano M, Wu ZF, Safe S and Zacharewski TR (1995) Identification of an inhibitory dioxin response element (iDRE) required for TCDD-mediated inhibition of pS2. Proc Am Assoc Cancer Res 36:257. Gillesby BE, Stanostefano M, Porter W, Safe S, Wu ZF and Zacharewski TR (1997) Identification of a motif within the 5' regulatory region of pS2 which is responsible for AP-1 binding and TCDD-mediated suppression. Biochemistry 36:6080-6089. Gokhale MS, Bunton TE, Zurlo J and Yager JD (1997) Cytochrome P450 isoenzyme activities in cultured rat and mouse liver slices. Xenobiotica 27:341-355. Gradin K, Mcguire J, Wenger RH, Kvietikova I, Whitelaw ML, Toftgard R, Tora L, Gassmann M and Poellinger L (1996) Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor. Mol Cell Biol 16:5221-5231. Grassman JA, Masten SA, Walker NJ and Lucier GW (1998) Animal models of human response to dioxins. Environ Health Perspect 106:761-775.

210 Green NJ, Hassanin A, Johnston AE and Jones KC (2004) Observations on historical, contemporary, and natural PCDD/Fs. Environ Sci Technol 38:715-723. Gronemeyer H, Gustafsson J-A and Laudet V (2004) Principles for modulation of the nuclear receptor superfamily. Nat Rev Drug Discov 3:950-964. Guengerich FP (1993) Cytochrome P450 enzymes. Am Sci 81:440-447. Guengerich FP (2004) Cytochrome P450: What have we learned and what are the future issues? Drug Metab Rev 36:159-197. Guengerich FP, Dannan GA, Wright ST, Martin MV and Kaminsky KS (1982) Purification and characterization of liver microsomal cytochromes P450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or |3- naphthoflavone. Biochemistry 21:6019-6030. Guengerich FP, Martin MV, McCormick WA, Nguyen LP, Glover E and Bradfield CA (2004) Aryl hydrocarbon receptor response to indigoids in vitro and in vivo. Arch Biochem Biophys 423:309-316. Gunton JE, Kulkarni RN, Yim S, Okada T, Hawthorne WJ, Tseng YH, Roberson RS, Ricordi C, O'Connell PJ, Gonzalez FJ and Kahn CR (2005) Loss of ARNT/HEFip mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes. Cell 122:337-349. Gustafsson J-A and Ingelman-Sundberg M (1979) Changes in steroid hormone metabolism in rat liver microsomes following administration of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). Biochem Pharmacol 28:497-499. Haarmann-Stemmann T, Bothe H, Kohli A, Sydlik U, Abel J and Fritsche E (2007) Analysis of the transcriptional regulation and molecular function of the aryl hydrocarbon receptor repressor in human cell lines. Drug Metab Dispos 35:2262- 2269. Hahn ME and Karchner SI (1995) Evolutionary conservation of the vertebrate Ah (dioxin) receptor: amplification and sequencing of the PAS domain of a teleost Ah receptor cDNA. Biochem J 310:383-387. Hahn ME, Poland A, Glover E and Stegeman JJ (1994) Photoaffinity labeling of the Ah receptor- phylogenetic survey of diverse vertebrate and invertebrate species. Arch Biochem Biophys 310:218-228. Hankinson O (2005) Role of coactivators in transcriptional activation by the aryl hydrocarbon receptor. Arch Biochem Biophys 433:379-386. Hanlon PR, Zheng W, Ko AY and Jefcoate CR (2005) Identification of novel TCDD- regulated genes by microarray analysis. Toxicol Appl Pharmacol 202:215-228. Harper PA, Riddick DS and Okey AB (2006) Regulating the regulator: factors that control levels and activity of the aryl hydrocarbon receptor. Biochem Pharmacol 72:267-279. Harper PA, Wong JMY, Lam MSM and Okey AB (2002) Polymorphisms in the human AH receptor. Chem Biol Inter 141:161-187. Hassoun EA, AlGhafri M and Abushaban A (2003) The role of antioxidant enzymes in TCDD-induced oxidative stress in various brain regions of rats after subchronic exposure. Free Radical Biol Med 35:1028-1036.

211 Heid SE, Pollenz RS and Swanson HI (2000) Role of heat shock protein 90 dissociation in mediating agonist-induced activation of the aryl hydrocarbon receptor. Mol Pharmacol 57:82-92. Henley DY, Bellone CJ, Williams DA, Ruh TS and Ruh MF (2004) Aryl hydrocarbon receptor-mediated post-transcriptional regulation of IL-ip. Arch Biochem Biophys 422:42-51. Higuchi N, Maruyama H, Kuroda T, Kameda S, lino N, Kawachi H, Nishikawa Y, Hanawa H, Tahara H, Miyazaki J and Gejyo F (2003) Hydrodynamics-based delivery of the viral interleukin-10 gene suppresses experimental crescentic glomerulonephritis in Wistar-Kyoto rats. Gene Ther 10:1297-1310. Higuchi S, Wu R, Zhou M, Ravikumar TS and Wang P (2007) Downregulation of hepatic cytochrome P450 isoforms and PPAR-y: their role in hepatic injury and proinflammatory responses in a double-hit model of hemorrhage and sepsis. J Surg Res 137:46-52. Hirasawa F, Kawagoe M, Arany S, Koizumi Y, Ueno Y and Sugiyama T (2005) Styrene monomer primarily induces CYP2B1 mRNA in rat liver. Xenobiotica 35:1089- 1099. Hockley SL, Arlt VM, Brewer D, Te Poele R, Workman P, Giddings I and Phillips DH (2007) AHR- and DNA-damage-mediated gene expression responses induced by benzo[a]pyrene in human cell lines. Chem Res Toxicol 20:1797-1810. Hodges BL and Scheule RK (2003) Hydrodynamic delivery of DNA. Expert Opin Biol Ther 3:911-918. Hoffer A, Chang CY and Puga A (1996) Dioxin induces transcription of fos and jun genes by Ah receptor-dependent and -independent pathways. Toxicol Appl Pharmacol 141:238-247. Holloway MG, Cui Y, Laz EV, Hosui A, Hennighausen L and Waxman DJ (2007) Loss of sexually dimorphic liver gene expression upon hepatocyte-specific deletion of Stat5a-Stat5b locus. Endocrinology 148:1977-1986. Holloway MG, Laz EV and Waxman DJ (2006) Codependence of growth hormone- responsive, sexually dimorphic hepatic gene expression on signal transducer and activator of transcription 5b and hepatic nuclear factor 4a. Mol Endocrinol 20:647-660. Hong J, Pan J, Gonzalez FJ, Gelboin HV and Yang CS (1987) The induction of a specific form of cytochrome P450 (P450j) by fasting. Biochem Biophys Res Commun 142:1077-1083. Honkakoski P, Kojo A and Lang MA (1992) Regulation of the mouse liver Cytochrome- P450 2B subfamily by sex hormones and phenobarbital. Biochem 7285:979-983. Hook GER, Haseman JK and Lucier GW (1975) Induction and suppression of hepatic and extrahepatic microsomal foreign-compound-metabolizing enzyme systems by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Chem-Biol Interactions 10:199-214. Hosoya T, Harada N, Mimura J, Motohashi H, Takahashi S, Nakajima O, Morita M, Kawauchi S, Yamamoto M and Fujii-Kuriyama Y (2008) Inducibility of cytochrome P450 1A1 and chemical carcinogenesis by benzo[a]pyrene in AhR repressor-deficient mice. Biochem Biophys Res Commun 365:562-567.

212 Hu SW, Chen CC, Kuo CY, Lin WH and Lin P (2005) Increased cytochrome P450 1B1 gene expression in peripheral leukocytes of municipal waste incinerator workers. Toxicol Lett 160:112-120. Iber H, Chen Q, Cheng PY and Morgan ET (2000) Suppression of CYP2C11 gene transcription by interleukin-1 mediated by NF-KB binding at the transcription start site. Arch Biochem Biophys 377:187-194. Iber H, Chen Q, Sewer M and Morgan ET (1997) Regulation of hepatic cytochrome P450 2C11 by glucocorticoids. Arch Biochem Biophys 345:305-310. Iber H and Morgan ET (1998) Regulation of hepatic cytochrome P450 2C11 by transforming growth factor-P, hepatocyte growth factor, and interleukin-11. Drug Metab Dispos 26:1042-1044. Iber H, Tong LM, Qi CH, Sheng Y and Morgan ET (2001) Regulation of hepatic cytochrome P4502C11 via cAMP: Implications for down-regulation in diabetes, fasting, and inflammation. J Pharmacol Exp Ther 297:174-180. Ickenstein LM, Holsmer SL and Bandiera SM (2004) Tamoxifen alters hepatic cytochrome P450 enzyme expression and circulating growth hormone levels in intact male rats. Pharmaceut Res 21:1631-1636. Iseki M, Acuta T, Kobayashi T and Kawajiri K (2005) Growth suppression of Leydig TM3 cells mediated by aryl hydrocarbon receptor. Biochem Biophys Res Commun 331:902-908. Ishizuka M, Yonemoto J, Zaha H, Tohyama C and Sone H (2003) Perinatal exposure to low doses of 2,3,7,8-tetrachlorodibenzo-/?-dioxin alters sex-dependent expression of hepatic CYP2C11. J Biochem Mol Toxicol 17:278-285. Ito T, Tsukumo S, Suzuki N, Motohashi H, Yamamoto M, Fujii-Kuriyama Y, Mimura J, Lin TM, Peterson RE, Tohyama C and Nohara K (2004) A constitutively active aryl hydrocarbon receptor induces growth inhibition of Jurkat T cells through changes in the expression of genes related to apoptosis and cell cycle arrest. J Biol Chem 279:25204-25210. Jackson JP, Ferguson SS, Moore R, Negishi M and Goldstein JA (2004) The constitutive active/androstane receptor regulates phenytoin induction of Cyp2c29. Mol Pharmacol 65:1397-1404. Jackson ME (1991) Negative regulation of eukaryotic transcription. J Cell Sci 100 (Pt l):l-7. Jarukamjorn K, Sakuma T, Jaruchotikamol A, Ishino Y, Oguro M and Nemoto N (2006) Modified expression of cytochrome P450 mRNAs by growth hormone in mouse liver. Toxicology 219:97-105. Jenkins RE, Kitteringham NR, Hunter CL, Webb S, Hunt TJ, Elsby R, Watson RB, Williams D, Pennington SR and Park BK (2006) Relative and absolute quantitative expression profiling of cytochromes P450 using isotope-coded affinity tags. Proteomics 6:1934-1947. Jeuken A, Keser BJG, Khan E, Brouwer A, Koeman J and Denison MS (2003) Activation of the Ah receptor by extracts of dietary herbal supplements, vegetables, and fruits. JAgr Food Chem 51:5478-5487. Jones EJ and Riddick DS (1996) Regulation of constitutive rat hepatic cytochromes P450 by 3-methylcholanthrene. Xenobiotica 26:995-1012.

213 Jurgens G, Lange KHW, Reuther LO, Rasmussen BB, Brosen K and Christensen HR (2002) Effect of growth hormone on hepatic cytochrome P450 activity in healthy elderly men. Clin Pharmacol Ther 71:162-168. Kamataki T, Maeda K, Shimada M and Kato R (1986) Effects of phenobarbital, 3- methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P450 in liver microsomes of rats. J Biochem 99:841-845. Kameda S, Maruyama H, Higuchi N, Nakamura G, lino N, Nishikawa Y, Miyazaki J and Gejyo F (2003) Hydrodynamics-based transfer of PCR-amplified DNA fragments into rat liver. Biochem Biophys Res Commun 309:929-936. Kapitulnik J and Gonzalez FJ (1993) Marked endogenous activation of the CYP1A1 and CYP1A2 genes in the congenitally jaundiced Gunn rat. Mol Pharmacol 43:722- 725. Karolchik D, Baertsch R, Diekhans M, Furey TS, Hinrichs A, Lu YT, Roskin KM, Schwartz M, Sugnet CW, Thomas DJ, Weber RJ, Haussler D and Kent WJ (2003) The UCSC Genome browser database. Nucleic Acids Res 31:51-54. Kato R and Yamazoe Y (1993) Hormonal regulation of cytochrome P450 in rat liver, in: Cytochrome P450 (Schenkman JB and Greim H eds), pp 447-459, Springer- Verlag, Berlin. Kato S, Sato T, Watanabe T, Takemasa S, Masuhiro Y, Ohtake F and Matsumoto T (2005) Function of nuclear sex hormone receptors in gene regulation. Cancer Chemother Pharmacol 56 Suppl 1:4-9. Kawabata K, Takakura Y and Hashida M (1995) The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake. Pharm Res 12:825-830. Kawai M, Bandiera SM, Chang TKH, Poulet FM, Vancutsem PM and Bellward GD (1999) Modulation of hepatic CYP2A1, CYP2C11, and CYP3A9 expression in adult rats by neonatal administration of tamoxifen. Drug Metab Dispos 27:1392- 1398. Kawajiri K, Watanabe J, Eguchi H, Nakachi K, Kiyohara C and Hayashi S (1995) Polymorphisms of human Ah receptor gene are not involved in lung cancer. Pharmacogenetics 5:151-158. Kazerouni N, Sinha R, Hsu CH, Greenberg A and Rothman N (2001) Analysis of 200 food items for benzo[a]pyrene and estimation of its intake in an epidemiologic study. Food Chem Toxicol 39:423-436. Kazlauskas A, Poellinger L and Pongratz I (1999) Evidence that the co-chaperone p23 regulates ligand responsiveness of the dioxin (aryl hydrocarbon) receptor. J Biol Chem 21'4:13519-13524. Kent WJ (2002) BLAT-the BLAST-like alignment tool. Genome Res 12:656-664. Kewley RJ, Whitelaw ML and Chapman-Smith A (2004) The mammalian basic helix- loop-helix/PAS family of transcriptional regulators. Int J Biochem Cell Biol 36:189-204. Kim DW, Gazourian L, Quadri SA, Romieu-Mourez R, Sherr DH and Sonenshein GE (2000) The RelA NF-KB subunit and the aryl hydrocarbon receptor (AhR) cooperate to transactivate the c-myc promoter in mammary cells. Oncogene 19:5498-5506.

214 Kim SH, Henry EC, Kim DK, Kim YH, Shin KJ, Han MS, Lee TG, Kang JK, Gasiewicz TA, Ryu SH and Suh PG (2006) Novel compound 2-methyl-2H-pyrazole-3- carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191) prevents 2,3,7,8-TCDD-induced toxicity by antagonizing the aryl hydrocarbon receptor. Mol Pharmacol 69:1871-1878. Kimura S, Kozak CA and Gonzalez FJ (1989) Identification of a novel P450 expressed in rat lung: cDNA cloning and sequence, chromosome mapping, and induction by 3- methylcholanthrene. Biochemistry 28:3798-3803. Klingenberg M (1958) Pigments of rat liver microsomes. Arch Biochem Biophys 75:376- 386. Ko HP, Okino ST, Ma Q and Whitlock JP (1997) Transactivation domains facilitate promoter occupancy for the dioxin-inducible CYP1A1 gene in vivo. Mol Cell Biol 17:3497-3507. Kondraganti SR, Muthiah K, Jiang W, Barrios R and Moorthy B (2005) Effects of 3- methylcholanthrene on gene expression profiling in the rat using cDNA microarray analyses. Chem Res Toxicol 18:1634-1641. Konstandi M, Pappas P, Johnson E, Lecklin A, Karageorgou M and Marselos M (1997) Modification of reproductive function in the rat by 3-methylcholanthrene. Pharmacol Res 35:107-111. Korkalainen M, Tuomisto J and Pohjanvirta R (2004) Primary structure and inducibility by 2,3,7,8-tetrachlorodibenzo-/?-dioxin (TCDD) of aryl hydrocarbon receptor repressor in a TCDD-sensitive and a TCDD-resistant rat strain. Biochem Biophys Res Commun 315:123-131. Kot M and Daniel WA (2007) Effect of cytochrome P450 (CYP) inducers on caffeine metabolism in the rat. Pharmacol Rep 59:296-305. Kouri RE (1976) Relationship between levels of aryl hydrocarbon hydroxylase activity and susceptibility to 3-methylcholanthrene and benzo[a]pyrene-induced cancers in inbred strains of mice. In: Poynuclear Aromatic Hydrocarbons: Chemistry, Metabolism and Carcinogenesis (R.I. Freudenthal and P.W. Jones, Eds).ppl39- 151. Kozak KR, Abbott B and Hankinson O (1997) ARNT-deficient mice and placental differentiation. Dev Biol 191:297-305. Kraner JC, Morgan ET and Halpert JR (1994) Selective suppression of rat hepatic cytochrome P450 2C11 by chloramphenicol. J Pharmacol Exp Ther 270:1367- 1372. Kransler KM, McGarrigle BP and Olson JR (2007) Comparative developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the hamster, rat and guinea pig. Toxicology 229:214-225. Kriek E, Rojas M, Alexandrov K and Bartsch H (1998) Polycyclic aromatic hydrocarbon- DNA adducts in humans: relevance as biomarkers for exposure and cancer risk. MutatRes 400:215-231. Krishnan V, Porter W, Santostefano M, Wang XH and Safe S (1995) Molecular mechanism of inhibition of estrogen-induced cathepsin D gene expression by 2,3,7,8-tetrachlorodibenzo-/?-dioxin (TCDD) in MCF-7 cells. Mol Cell Biol 15:6710-6719.

215 Kurose K, Tohkin M and Fukuhara M (1999) A novel positive regulatory element that enhances hamster CYP2A8 gene expression mediated by xenobiotic responsive element. Mol Pharmacol 55:279-287. Kurose K, Tohkin N, Ushio F and Fukuhara M (1998) Cloning and characterization of Syrian hamster testosterone 7 alpha-hydroxylase, CYP2A9. Arch Biochem Biophys 351:60-65. Lahvis GP, Pyzalski RW, Glover E, Pitot HC, McElwee MK and Bradfield CA (2005) The aryl hydrocarbon receptor is required for developmental closure of the ductus venosus in the neonatal mouse. Mol Pharmacol 67:714-720. Lai KP, Li W, Xu Y, Wong MH and Wong CK (2004) Dioxin-like components in human breast milk collected from Hong Kong and Guangzhou. Environ Res 96:88-94. Lai ZW, Pineau T and Esser C (1996) Identification of dioxin-responsive elements (DREs) in the 5' regions of putative dioxin-inducible genes. Chem Biol Interact 100:97-112. Landers JP and Bunce NJ (1991) The Ah receptor and the mechanism of dioxin toxicity. Biochem 7276:273-287. Landrigan P, Garg A and Droller DBJ (2003) Assessing the effects of endocrine disruptors in the national children's study. Environ Health Perspect 111:1678- 1682. Laz EV, Holloway MG, Chen CS and Waxman DJ (2007) Characterization of three growth hormone-responsive transcription factors preferentially expressed in adult female liver. Endocrinology 148:3327-3337. LeBlanc GA and Waxman DJ (1988) Feminization of rat hepatic P450 expression by cisplatin. Evidence for perturbations in the hormonal regulation of steroid- metabolizing enzymes. J Biol Chem 263:15732-15739. LeBlanc GA and Waxman DJ (1990) Mechanisms of cyclophosphamide action on hepatic P450 expression. Cancer Res 50:5720-5726. Lee BM and Shim GA (2007) Dietary exposure estimation of benzo[a]pyrene and cancer risk assessment. J Toxicol Environ Health A 70:1391-1394. Lee C, Hutson JR, Tzau VK and Riddick DS (2006) Regulation of constitutive mouse hepatic cytochromes P450 and growth hormone signaling components by 3- methylcholanthrene. Drug Metab Dispos 34:1530-1538. Lee C and Riddick DS (2000) Transcriptional suppression of cytochrome P4502C11 gene expression by 3-methylcholanthrene. Biochem Pharmacol 59:1417-1423. Lee JE and Safe S (2000) 3 ',4 '-Dimethoxyflavone as an aryl hydrocarbon receptor antagonist in human breast cancer cells. Toxicol Sci 58:235-242. Legraverend C, Mode A, Westin S, Strom A, Eguchi H, Zaphiropoulos PG and Gustafsson J-A (1992) Transcriptional regulation of rat P450 2C gene subfamily members by the sexually dimorphic pattern of growth hormone secretion. Mol Endocrinol 6:259-266. Lemm F, Wilhelm M and Roos PH (2004) Occupational exposure to polycyclic aromatic hydrocarbons suppresses constitutive expression of CYP1B1 on the transcript level in human leukocytes. Int J Hyg Environ Health 207:325-335. Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ and Wood WI (1987) Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537-543.

216 Levine M and Manley JL (1989) Transcriptional repression of eukaryotic promoters. Cell 59:405-408. Li JX, Chen HB, Ke QD, Feng ZH, Tang MS, Liu BC, Amin S, Costa M and Huang CS (2004) Differential effects of polycyclic aromatic hydrocarbons on transactivation of AP-1 and NF-KB in mouse epidermal C141 cells. Mol Carcinogen 40:104-115. Li W, Harper PA, Tang BK and Okey AB (1998) Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells- CYP1A2 expression in the LSI80 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin or 3-methylcholanthrene. Biochem Pharmacol 56:599-612. Li X and Stark GR (2002) NFKB-dependent signaling pathways. Exp Hematol 30:285- 296. Li XL, Eckard J, Shah R, Malluck C and Frenkel K (2002) Interleukin-la up-regulation in vivo by a potent carcinogen 7,12-dimethylbenz(A)anthracene (DMBA) and control of DMBA-induced inflammatory responses. Cancer Res 62:417-423. Liddle C, Mode A, Legraverend C and Gustafsson J-A (1992) Constitutive expression and hormonal regulation of male sexually differentiated cytochromes P450 in primary cultured rat hepatocytes. Arch Biochem Biophys 298:159-166. Liu F, Song Y and Liu D (1999) Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 6:1258-1266. Liu JR, Callahan SM and Brunner LJ (2003) Effect of sodium alterations on hepatic cytochrome P450 3A2 and 2C11 and renal function in rats. Drug Develop Ind Pharm 29:767-775. Liu M and Alkayed NJ (2005) Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) la-linked induction of P450 2C11 epoxygenase in astrocytes. J Cereb Blood Flow Metab 25:939-948. Liu SQ, Rivera-Rivera I, Bredemeyer AJ and Kemper B (2001) Functional analysis of the phenobarbital-responsive unit in rat CYP2B2. Biochem Pharmacol 62:21-28. Lu SK, Callahan SA, Jin RY and Brunner LJ (2004a) Cyclosporine and bromocriptine- induced suppressions of CYP3A1/2 and CYP2C11 are not mediated by prolactin. Eur J Pharmacol 501:215-224. Lu SK, Callahan SM and Brunner LJ (2003) Suppression of hepatic CYP3A1/2 and CYP2C11 by cyclosporine is not mediated by altering growth hormone levels. J Pharmacol Exp Ther 305:331-337. Lu ZJ, Lee EY, Robertson LW, Glauert HP and Spear BT (2004b) Effect of 2,2',4,4',5,5'- hexachlorobiphenyl (PCB-153) on hepatocyte proliferation and apoptosis in mice deficient in the p50 subunit of the transcription factor NF-KB. Toxicol Sci 81:35- 42. Lund AK, Goens MB, Kanagy NL and Walker MK (2003) Cardiac hypertrophy in Aryl hydrocarbon receptor null mice is correlated with elevated angiotensin II, endothelin-1, and mean arterial blood pressure. Toxicol Appl Pharmacol 193:177- 187. Lusska A, Shen E and Whitlock JP (1993) Protein-DNA interactions at a dioxin- responsive enhancer- Analysis of 6 bona-fide DNA-binding sites for the liganded Ah receptor. J Biol Chem 268:6575-6580. Ma JX, Zhang KL, Liu X, Ma YL, Pei LN, Zhu YF, Zhou L, Chen XY, Kong QY, Li H and Liu J (2006) Concurrent expression of aryl hydrocarbon receptor and

217 CYP1A1 but not CYP1A1 Mspl polymorphism is correlated with gastric cancers raised in Dalian, China. Cancer Lett 240:253-260. Ma Q and Lu AY (2007) CYP1A induction and human risk assessment: an evolving tale of in vitro and in vivo studies. Drug Metab Dispos 35:1009-1016. Ma Q and Whitlock JP (1997) A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Biol Chem 272:8878-8884. Mahadevan B, Parsons H, Musafia T, Sharma AK, Amin S, Pereira C and Baird WM (2004) Effect of artificial mixtures of environmental polycyclic aromatic hydrocarbons present in coal tar, urban dust, and diesel exhaust particulates on MCF-7 cells in culture. Environ Mol Mutagen 44:99-107. Marlowe JL, Knudsen ES, Schwemberger S and Puga A (2004) The aryl hydrocarbon receptor displaces p300 from E2F-dependent promoters and represses S phase- specific gene expression. J Biol Chem 279:29013-29022. Marlowe JL and Puga A (2005) Aryl hydrocarbon receptor, cell cycle regulation, toxicity, and tumorigenesis. J Cell Biochem 96:1174-1184. Martini R and Murray M (1994) Suppression of the constitutive microsomal cytochrome P450 2C11 in male rat liver during dietary vitamin A deficiency. Biochem Pharmacol 48:1305-1309. Maruyama H, Higuchi N, Kameda S, Miyazaki J and Gejyo F (2004) Rat liver-targeted naked plasmid DNA transfer by tail vein injection. Mol Biotechnol 26:165-172. Maruyama H, Higuchi N, Nishikawa Y, Kameda S, lino N, Kazama J J, Takahashi N, Sugawa M, Hanawa H, Tada N, Miyazaki J and Gejyo F (2002) High-level expression of naked DNA delivered to rat liver via tail vein injection. / Gene Med 4:333-341. Maruyama M, Matsunaga T, Harada E and Ohmori S (2007) Comparison of basal gene expression and induction of CYP3As in HepG2 and human fetal liver cells. Biol Pharm Bull 30:2091-2097. Matikainen T, Perez GI, Jurisicova A, Pru JK, Schlezinger JJ, Ryu HY, Laine J, Sakai T, Korsmeyer SJ, Casper RF, Sherr DH and Tilly JL (2001) Aromatic hydrocarbon receptor-driven Box gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals. Nat Genet 28:355-360. Matsuda T, Imaoka S, Funae Y, Otori K and Fukushima S (1995) Induction of CYP isoenzymes in various organs of rats by 3-methylcholanthrene or P- naphthoflavone. Cancer Lett 97:137-143. Matsumoto A, Masuhara M, Mitsui K, Yokouchi M, Ohtsubo M, Misawa H, Miyajima A and Yoshimura A (1997) CIS, a cytokine inducible SH2 protein, is a target of the JAK-STAT5 pathway and modulates STAT5 activation. Blood 89:3148-3154. Matsumura F (2003) On the significance of the role of cellular stress response reactions in the toxic actions of dioxin. Biochem Pharmacol 66:527-540. Matsushita N, Sogawa K, Ema M, Yoshida A and Fujii-Kuriyama Y (1993) A factor binding to the xenobiotic responsive element (XRE) of P450 1A1 gene consists of at least two helix-loop-helix proteins, Ah receptor and Arnt. J Biol Chem 268:21002-21006. Matthews J, Wihlen B, Heldring N, MacPherson L, Helguero L, Treuter E, Haldosen LA and Gustafsson J-A (2007) Co-planar 3,3',4,4',5-pentachlorinated biphenyl and

218 non-co-planar 2,2',4,6,6'-pentachlorinated biphenyl differentially induce recruitment of oestrogen receptor a to aryl hydrocarbon receptor target genes. Biochem J 406:343-353. Matthews J, Wihlen B, Thomsen J and Gustafsson J-A (2005) Aryl hydrocarbon receptor-mediated transcription: ligand-dependent recruitment of estrogen receptor a to 2,3,7,8-tetrachlorodibenzo-/>-dioxin-responsive promoters. Mol Cell Biol 25:5317-5328. Mattison DR and Thorgeirsson SS (1979) Ovarian aryl hydrocarbon hydroxylase activity and primordial oocyte toxicity of polycyclic aromatic hydrocarbons in mice. Cancer Res 39:3471-3475. McCluskey SA, Racz WJ and Marks GS (1986) Properties of 17- to 19-day-old chick embryo liver microsomes. Induction of cytochrome P450, effect of storage at low temperature, and resistance to lipid peroxidation. J Pharmacol Methods 16:111- 123. McGuire J, Whitelaw ML, Pongratz I, Gustafsson J-A and Poellinger L (1994) A cellular factor stimulates ligand-dependent release of hsp90 from the basic helix-loop- helix dioxin receptor. Mol Cell Biol 14:2438-2446. McMillan BJ and Bradfield CA (2007) The aryl hydrocarbon receptor is activated by modified low-density lipoprotein. Proc Natl Acad Sci USA 104: 1412-1417. Merchant M, Krishnan V and Safe S (1993) Mechanism of action of cc-naphthoflavone as an Ah receptor antagonist in MCF-7 human breast cancer cells. Toxicol Appl Pharmacol 120:179-185. Merchant M, Morrison V, Santostefano M and Safe S (1992) Mechanism of action of Aryl hydrocarbon receptor antagonists- inhibition of 2,3,7,8-tetrachlorodibenzo-/?- dioxin-induced CYP1A1 gene expression. Arch Biochem Biophys 298:389-394. Merrill AH, Nikolova-Karakashian M, Schmelz EM, Morgan ET and Stewart J (1999) Regulation of cytochrome P450 expression by sphingolipids. Chem Phys Lipids 102:131-139. Meyer BK, Pray-Grant MG, Vanden Heuvel JP and Perdew GH (1998) Hepatitis B virus X-associated protein 2 is a subunit of the unliganded aryl hydrocarbon receptor core complex and exhibits transcriptional enhancer activity. Mol Cell Biol 18:978-988. Mimura J, Ema M, Sogawa K and Fujii-Kuriyama Y (1999) Identification of a novel mechanism of regulation of Ah (dioxin) receptor function. Gene Develop 13:20- 25. Mimura J, Yamashita K, Nakamura K, Morita M, Takagi TN, Nakao K, Ema M, Sogawa K, Yasuda M, Katsuki M and Fujii-Kuriyama Y (1997) Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-/?-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes to Cells 2:645-654. Miyamoto K (2004) Effects of dioxin on gene expression in female reproductive system in the rat. Environ Sci 11:47-55. Miyazawa M, Sugie A and Shimada T (2003) Roles of human CYP2A6 and 2B6 and rat CYP2C11 and 2B1 in the 10-hydroxylation of (-)-verbenone by liver microsomes. Drug Metab Dispos 31:1049-1053. Mode A and Gustafsson J-A (2006) Sex and the liver- a journey through five decades. Drug Metab Rev 38:197-207.

219 Moennikes O, Loeppen S, Buchmann A, Andersson P, Ittrich C, Poellinger L and Schwarz M (2004) A constitutively active dioxin/aryl hydrocarbon receptor promotes hepatocarcinogenesis in mice. Cancer Res 64:4707-4710. Moffat ID, Boutros PC, Celius T, Linden J, Pohjanvirta R and Okey AB (2007) microRNAs in adult rodent liver are refractory to dioxin treatment. Toxicol Sci 99:470-487. Monshouwer M and Hoebe KHN (2003) Hepatic (dys-)function during inflammation. Toxicol Vitro 17:681-686. Morel Y, Mermod N and Barouki R (1999) An autoregulatory loop controlling CYP1A1 gene expression: Role of H202 and NFL Mol Cell Biol 19:6825-6832. Morgan ET (2001) Regulation of cytochrome P450 by inflammatory mediators: Why and how? Drug Metab Dispos 29:207-212. Morgan ET, Goralski KB, Piquette-Miller M, Renton KW, Robertson GR, Chaluvadi MR, Charles KA, Clarke SJ, Kacevska M, Liddle C, Richardson TA, Sharma R and Sinai CJ (2008) Regulation of drug-metabolizing enzymes and transporters in infection, inflammation, and cancer. Drug Metab Dispos 36:205-216. Morgan ET, MacGeoch C and Gustafsson J-A (1985a) Hormonal and developmental regulation of expression of the hepatic microsomal steroid 16a-hydroxylase cytochrome P450 apoprotein in the rat. J Biol Chem 260:11895-11898. Morgan ET, MacGeoch C and Gustafsson J-A (1985b) Sexual differentiation of cytochrome P450 in rat liver: evidence for a constitutive isozyme as the male- specific 16a-hydroxylase. Mol Pharmacol 27:471-479. Morishima N, Yoshioka H, Higashi Y, Sogawa K and Fujii-Kuriyama Y (1987) Gene structure of cytochrome P-450 (M-l) specifically expressed in male rat liver. Biochemistry 26:8279-8285. Murray M, Butler AM, Fiala-Beer E and Su GM (2005) Phospho-STAT5 accumulation in nuclear fractions from vitamin A-deficient rat liver. FEBS Lett 579:3669-3673. Myers SR, Blake JW and Flesher JW (1989) Metabolism of 3-methylcholanthrene in rat liver cytosol. Chem-Biol Interactions 71:393-401. Myers SR and Flesher JW (1990) Metabolism of the carcinogen 3-methylcholanthrene in human bone marrow preparations. Drug Metab Dispos 18:664-669. Nagai H, Takei T, Tohyama C, Kubo M, Abe R and Nohara K (2005) Search for the target genes involved in the suppression of antibody production by TCDD in C57BL/6 mice. Int Immunopharmacol 5:331-343. Nair SC, Toran EJ, Rimerman RA, Hjermstad S, Smithgall TE and Smith DF (1996) A pathway of multi-chaperone interactions common to diverse regulatory proteins: Estrogen receptor, Fes tyrosine kinase, heat shock transcription factor Hsfl, and the aryl hydrocarbon receptor. Cell Stress Chaperones 1:237-250. Naka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono A, Nishimoto N, Kajita T, Taga T, Yoshizaki K, Akira S and Kishimoto T (1997) Structure and function of a new STAT-induced STAT inhibitor. Nature 387:924-929. Nakajima T and Wang RS (1994) Induction of cytochrome P450 by toluene. Int J Biochem 26:1333-1340. Nakayama K, Sudo Y, Sasaki Y, Iwata H, Takahashi M and Kamataki T (2001) Studies on transcriptional regulation of Cyp3al6 gene in mouse livers by application of direct DNA injection method. Biochem Biophys Res Commun 287:820-824.

220 Natsume Y, Satsu H, Kitamura K, Okamoto N and Shimizu M (2004) Assessment system for dioxin absorption in the small intestine and prevention of its absorption by food factors. Biofactors 21:375-377. Nazarenko DA, Dertinger SD and Gasiewicz TA (2001) In vivo antagonism of AhR- mediated gene induction by 3 '-methoxy-4'-nitroflavone in TCDD-responsive lacZ mice. Toxicol Sci 61:256-264. Nebert DW, Dalton TP, Okey AB and Gonzalez FJ (2004) Role of aryl hydrocarbon receptor-mediated induction of the CYP1 enzymes in environmental toxicity and cancer. J Biol Chem 279:23847-23850. Nebert DW and Gonzalez FJ (1987) P450 genes: structure, evolution, and regulation. Annu Rev Biochem 56:945-993. Nebert DW, Goujon FM and Gielen JE (1972) Aryl hydrocarbon hydroxylase induction by polycyclic hydrocarbons: simple autosomal dominant trait in the mouse. Nat New Biol 236:107-110. Nebert DW, Roe AL, Dieter MZ, Solis WA, Yang Y and Dalton TP (2000) Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis. Biochem Pharmacol 59:65-85. Nelson DR (2003) Comparison of P450s from human and fugu: 420 million years of vertebrate P450 evolution. Arch Biochem Biophys 409:18-24. Nelson DR, Zeldin DC, Hoffman SMG, Maltais LJ, Wain HM and Nebert DW (2004) Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics 14:1-18. Nguyen LP and Bradfield CA (2008) The search for endogenous activators of the aryl hydrocarbon receptor. Chem Res Toxicol 21:102-116. Nicholas JS and Barron HD (1932) The use of sodium amytal in the production of anesthesia in the rat. J Pharmacol Exp Ther 46:125-129. Nichols KD and Kirby GM (2008) Microarray analysis of hepatic gene expression in pyrazole-mediated hepatotoxicity: Identification of potential stimuli of Cyp2a5 induction. Biochem Pharmacol 75:538-551. Ning B, Dial S, Sun Y, Wang J, Yang J and Guo L (2008) Systematic and simultaneous gene profiling of 84 drug-metabolizing genes in primary human hepatocytes. J Biomol Screen 13:194-201. Nishihashi H, Kanno Y, Tomuro K, Nakahama T and Inouye Y (2006) Primary structure and organ-specific expression of the rat aryl hydrocarbon receptor repressor gene. Biol Pharm Bull 29:640-647. Nukaya M, Takahashi Y, Gonzalez FJ and Kamataki T (2004) Aryl hydrocarbon receptor-mediated suppression of GH receptor and Janus kinase 2 expression in mice. Febs Lett 558:96-100. Ohhira S, Enomoto M and Matsui H (2006) Sex difference in the principal cytochrome P450 for tributyltin metabolism in rats. Toxicol Appl Pharmacol 210:32-38. Ohtake F, Takeyama K, Matsumoto T, Kitagawa H, Yamamoto Y, Nohara K, Tohyama C, Krust A, Mimura J, Chambon P, Yanagisawa J, Fujii-Kuriyama Y and Kato S (2003) Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature 423:545-550.

221 Okey AB (2007) An aryl hydrocarbon receptor odyssey to the shores of toxicology: the Deichmann Lecture, International Congress of Toxicology-XL Toxicol Sci 98:5- 38. Okey AB, Franc MA, Moffat ID, Tijet N, Boutros PC, Korkalainen M, Tuomisto J and Pohjanvirta R (2005) Toxicological implications of polymorphisms in receptors for xenobiotic chemicals: The case of the aryl hydrocarbon receptor. Toxicol Appl Pharmacol 207:43-51. Okey AB, Riddick DS and Harper PA (1994) The Ah receptor- mediator of the toxicity of 2,3,7,8-tetrachlorodibenzo-/>-dioxin (TCDD) and related compounds. Toxicol Lett 70:1-22. Okey AB and Vella LM (1982) Binding of 3-methylcholanthrene and 2,3,7,8- tetrachlorodibenzo-/?-dioxin to a common Ah receptor site in mouse and rat hepatic cytosols. EurJBiochem 127:39-47. Okey AB, Vella LM and Harper PA (1989) Detection and characterization of a low affinity form of cytosolic Ah receptor in livers of mice nonresponsive to induction of cytochrome Pl-450 by 3-methylcholanthrene. Mol Pharmacol 35:823-830. Okino ST and Whitlock JP (1995) Dioxin induces localized, graded changes in chromatin structure: implications for Cyplal gene transcription. Mol Cell Biol 15:3714- 3721. Omura T and Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370-2378. Ono M, Chia DJ, Merino-Martinez R, Flores-Morales A, Unterman TG and Rotwein P (2007) Signal transducer and activator of transcription (Stat) 5b-mediated inhibition of insulin-like growth factor binding protein-1 gene transcription: a mechanism for repression of gene expression by growth hormone. Mol Endocrinol 21:1443-1457. Oropeza-Hernandez LF, Lopez-Romero R and Albores A (2003) Hepatic CYP1A, 2B, 2C, 2E and 3A regulation by methoxychlor in male and female rats. Toxicol Lett 144:93-103. Oshima M, Mimura J, Yamamoto M and Fujii-Kuriyama Y (2007) Molecular mechanism of transcriptional repression of AhR repressor involving ANKRA2, HDAC4, and HDAC5. Biochem Biophys Res Commun 364:276-282. Otto DME, Henderson CJ, Carrie D, Davey M, Gundersen TE, Blomhoff R, Adams RH, Tickle C and Wolf CR (2003) Identification of novel roles of the cytochrome P450 system in early embryogenesis: Effects on vasculogenesis and retinoic acid homeostasis. Mol Cell Biol 23:6103-6116. Ovando BJ, Vezina CM, McGarrigle BP and Olson JR (2006) Hepatic gene downregulation following acute and subchronic exposure to 2,3,7,8- tetrachlorodibenzo-/?-dioxin. Toxicol Sci 94:428-438. Oyama T, Sugio K, Uramoto H, Iwata T, Onitsuka T, Isse T, Nozoe T, Kagawa N, Yasumoto K and Kawamoto T (2007) Increased cytochrome P450 and aryl hydrocarbon receptor in bronchial epithelium of heavy smokers with non-small cell lung carcinoma carries a poor prognosis. Front Biosci 12:4497-4503. Paajarvi G, Viluksela M, Pohjanvirta R, Stenius U and Hogberg J (2005) TCDD activates Mdm2 and attenuates the p53 response to DNA damaging agents. Carcinogenesis 26:201-208.

222 Paleologo M, van Schooten FJ, Pavanello S, Kriek E, Zordan M, Clonfero E, Bezze C and Levis AG (1992) Detection of benzo[a]pyrene-diol-epoxide-DNA adducts in white blood cells of psoriatic patients treated with coal tar. Mutat Res 281:11-16. Pampori NA and Shapiro BH (2000) Nominal growth hormone pulses in otherwise normal masculine plasma profiles induce intron retention of overexpressed hepatic CYP2C11 with associated nuclear splicing deficiency. Endocrinology 141:4100-4106. Pande K, Moran SM and Bradfield CA (2005) Aspects of dioxin toxicity are mediated by interleukin 1-like cytokines. Mol Pharmacol 67:1393-1398. Park SH and Waxman DJ (2001) Inhibitory cross-talk between STAT5b and liver nuclear factor HNF3P- Impact on the regulation of growth hormone pulse-stimulated, male-specific liver cytochrome P450 gene expression. J Biol Chem 276:43031- 43039. Park YK, Li H and Kemper B (1996) Phenobarbital induction mediated by a distal CYP2B2 sequence in rat liver transiently transfected in situ. J Biol Chem 271:23725-23728. Pascussi JM, Gerbal-Chaloin S, Drocourt L, Assenat E, Larrey D, Pichard-Garcia L, Vilarem MJ and Maurel P (2004) Cross-talk between xenobiotic detoxication and other signalling pathways: clinical and toxicological consequences. Xenobiotica 34:633-664. Pastorelli R, Carpi D, Campagna R, Airoldi L, Pohjanvirta R, Viluksela M, Hakansson H, Boutros PC, Moffat ID, Okey AB and Fanelli R (2006) Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses. Mol Cell Proteomics 5:882-894. Patel RD, Hollingshead BD, Omiecinski CJ and Perdew GH (2007) Aryl hydrocarbon receptor activation regulates constitutive androstane receptor levels in murine and human liver. Hepatology 46:209-218. Pavanello S, Favretto D, Brugnone F, Mastrangelo G, Dal Pra G and Clonfero E (1999) HPLC/fluorescence determination of anti-BPDE-DNA adducts in mononuclear white blood cells from PAH-exposed humans. Carcinogenesis 20:431-435. Perdew GH (1988) Association of the Ah receptor with the 90-kDa heat shock protein. J Biol Chem 263:13802-13805. Perissi V and Rosenfeld MG (2005) Controlling nuclear receptors: the circular logic of cofactor cycles. Nat Rev Mol Cell Biol 6:542-554. Petrick JS and Klaassen CD (2007) Importance of hepatic induction of constitutive androstane receptor and other transcription factors that regulate xenobiotic metabolism and transport. Drug Metab Dispos 35:1806-1815. Pirkle JL, Wolfe WH, Patterson DG, Needham LL, Michalek JE, Miner JC, Peterson MR and Phillips DL (1989) Estimates of the half-life of 2,3,7,8-tetrachlorodibenzo-p- dioxin in Vietnam veterans of Operation Ranch Hand. J Toxicol Environ Health 27:165-171. Pocar P, Fischer B, Klonisch T and Hombach-Klonisch S (2005) Molecular interactions of the aryl hydrocarbon receptor and its biological and toxicological relevance for reproduction. Reproduction 129:379-389. Pohjanvirta R, Wong JMY, Li W, Harper PA, Tuomisto J and Okey AB (1998) Point mutation in intron sequence causes altered carboxyl-terminal structure in the aryl

223 hydrocarbon receptor of the most 2,3,7,8-tetrachlorodibenzo-/?-dioxin-resistant rat strain. Mol Pharmacol 54:86-93. Poiger H and Schlatter C (1979) Biological degradation of TCDD in rats. Nature 281:706-707. Poland A and Glover E (1974) Comparison of 2,3,7,8-tetrachlorodibenzo-/?-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene. Mol Pharmacol 10:349-359. Poland A and Glover E (1975) Genetic expression of aryl hydrocarbon hydroxylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin: evidence for a receptor mutation in genetically non-responsive mice. Mol Pharmacol 11:389-398. Poland A, Glover E and Kende AS (1976) Stereospecific, high affinity binding of 2,3,7,8- tetrachlorodibenzo-/?-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. J Biol Chem 251:4936- 4946. Poland A and Knutson JC (1982) 2,3,7,8-Tetrachlorodibenzo-/?-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Ann Rev Pharmacol Toxicol 22:517-554. Poland AP, Glover E, Robinson JR and Nebert DW (1974) Genetic expression of aryl hydrocarbon hydroxylase activity. Induction of monooxygenase activities and cytochrome Pl-450 formation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice genetically "non-responsive" to other aromatic hydrocarbons. J Biol Chem 249:5599-5606. Puga A, Barnes SJ, Chang CY, Zhu H, Nephew KP, Khan SA and Shertzer HG (2000a) Activation of transcription factors activator protein-1 and nuclear factor-KB by 2,3,7,8-tetrachlorodibenzo-jP-dioxin. Biochem Pharmacol 59:997-1005. Puga A, Maier A and Medvedovic M (2000b) The transcriptional signature of dioxin in human hepatoma HepG2 cells. Biochem Pharmacol 60:1129-1142. Qin HT and Powell-Coffman JA (2004) The Caenorhabditis elegans aryl hydrocarbon receptor, AHR-1, regulates neuronal development. Develop Biol 270:64-75. Racky J, Schmitz HJ, Kauffmann HM and Schrenk D (2004) Single nucleotide polymorphism analysis and functional characterization of the human Ah receptor (AhR) gene promoter. Arch Biochem Biophys 421:91-98. Radjendirane V and Jaiswal AK (1999) Antioxidant response element-mediated 2,3,7,8- tetrachlorodibenzo-/?-dioxin (TCDD) induction of human NADPH: quinone oxidoreductase 1 gene expression. Biochem Pharmacol 58:1649-1655. Rahmaniyan M, Patrick K and Bell NH (2005) Characterization of recombinant CYP2C11: a vitamin D 25-hydroxylase and 24-hydroxylase. Am J Physiol Endocrinol Metab 288:E753-760. Ramadoss P and Perdew GH (2005) The transactivation domain of the Ah receptor is a key determinant of cellular localization and ligand-independent nucleocytoplasmic shuttling properties. Biochemistry 44:11148-11159. Ramos KS, Steffen MC, Falahatpisheh MH and Nanez A (2007) From genomics to mechanistic insight: a global perspective on molecular deficits induced by environmental agents. Environ Mol Mutagen 48:395-399.

224 Ramsden R, Beck NB, Sommer KM and Omiecinski CJ (1999) Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice. Gene 228:169-179. Ray SS and Swanson HI (2004) Dioxin-induced immortalization of normal human keratinocytes and silencing of p53 and pl6(INK4a). J Biol Chem 279:27187- 27193. Reinik M, Tamme T, Roasto M, Juhkam K, Tenno T and Kiis A (2007) Polycyclic aromatic hydrocarbons (PAHs) in meat products and estimated PAH intake by children and the general population in Estonia. FoodAddit Contain 24:429-437. Reisdorph R and Lindahl R (2007) Constitutive and 3-methylcholanthrene-induced rat ALDH3A1 expression is mediated by multiple xenobiotic response elements. Drug Metab Dispos 35:386-393. Renkawitz R (1990) Transcriptional repression in eukaryotes. Trends Genet 6:192-197. Riddick DS, Huang Y, Harper PA and Okey AB (1994) 2,3,7,8-Tetrachlorodibenzo-p- dioxin versus 3-mefhylcholanthrene- comparative studies of Ah receptor binding, transformation, and induction of Cyplal. J Biol Chem 269:12118-12128. Riddick DS, Lee C, Bhathena A and Timsit YE (2003) The 2001 Veylien Henderson Award of the Society of Toxicology of Canada. Positive and negative transcriptional regulation of cytochromes P450 by polycyclic aromatic hydrocarbons. Can J Physiol Pharmacol 81:59-77. Riddick DS, Lee C, Bhathena A, Timsit YE, Cheng PY, Morgan ET, Prough RA, Ripp SL, Miller KKM, Jahan A and Chiang JYL (2004) Transcriptional suppression of cytochrome P450 genes by endogenous and exogenous chemicals. Drug Metab Dispos 32:367-375. Rier S and Foster WG (2002) Environmental dioxins and endometriosis. Toxicol Sci 70:161-170. Ripp SL, Falkner KC, Pendleton ML, Tamasi V and Prough RA (2003) Regulation of CYP2C11 by dehydroepiandrosterone and peroxisome proliferators: Identification of the negative regulatory region of the gene. Mol Pharmacol 64:113-122. Rivera SP, Saarikoski ST and Hankinson O (2002) Identification of a novel dioxin- inducible cytochrome P450. Mol Pharmacol 61:255-259. Rivera SP, Wang F, Saarikoski ST, Taylor RT, Chapman B, Zhang R and Hankinson O (2007) A novel promoter element containing multiple overlapping xenobiotic and hypoxia response elements mediates induction of cytochrome P450 2S1 by both dioxin and hypoxia. J Biol Chem 282:10881-10893. Rivera-Rivera I, Kim J and Kemper B (2003) Transcriptional analysis in vivo of the hepatic genes, Cyp2b9 and Cyp2bl0, by intravenous administration of plasmid DNA in mice. Bba-Gen Subjects 1619:254-262. Roberts EA, Johnson KC, Harper PA and Okey AB (1990) Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line HepG2. Arch Biochem Biophys 276:442-450. Roberts SG and Green MR (1995) Transcription. Dichotomous regulators. Nature 375:105-106. Robertson GR, Field J, Goodwin B, Bierach S, Tran M, Lehnert A and Liddle C (2003) Transgenic mouse models of human CYP3A4 gene regulation. Mol Pharmacol 64:42-50.

225 Roblin S, Okey AB and Harper PA (2004) AH receptor antagonist inhibits constitutive CYP1A1 and CYP1B1 expression in rat BP8 cells. Biochem Biophys Res Commun 317:142-148. Rodriguez-Sosa M, Elizondo G, Lopez-Duran RM, Rivera I, Gonzalez FJ and Vega L (2005) Over-production of DFN-y and IL-12 in AhR-null mice. FEBS Lett 579:6403-6410. Rose JQ, Ramsey JC, Wentzler TH, Hummel RA and Gehring PJ (1976) The fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat. Toxicol Appl Pharmacol 36:209-226. Rosengren L, Parrow V, Chmielewska J, Mode A and Fholenhag K (2007) In vivo evaluation of a novel, orally bioavailable, small molecule growth hormone receptor antagonist. Growth Horm IGF Res 17:47-53. Ross G (2004) The public health implications of polychlorinated biphenyls (PCBs) in the environment. Ecotoxicol Environ Safety 59:275-291. Ruegg J, Swedenborg E, Wahlstrom D, Escande A, Balaguer P, Pettersson K and Pongratz I (2008) The transcription factor aryl hydrocarbon receptor nuclear translocator functions as an Estrogen receptor p-selective coactivator, and its recruitment to alternative pathways mediates anti-estrogenic effects of dioxin. Mol Endocrinol 22:304-316. Ryan DE, Thomas PE, Levin W, Maines SL, Bandiera S and Reik LM (1993) Monoclonal antibodies of differentiating specificities as probes of Cytochrome- P450h (2C11). Arch Biochem Biophys 301:282-293. Saeki K, Matsuda T, Kato T, Yamada K, Mizutani T, Matsui S, Fukuhara K and Miyata N (2003) Activation of the human Ah receptor by aza-polycyclic aromatic hydrocarbons and their halogenated derivatives. Biol Pharm Bull 26:448-452. Safa B, Lee C and Riddick DS (1997) Role of the aromatic hydrocarbon receptor in the suppression of cytochrome P450 2C11 by polycyclic aromatic hydrocarbons. Toxicol Lett 90:163-175. Safe S (1990) Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and related compounds: environmental and mechanistic considerations which support the development of toxic equivalency factors (TEFs). Critical Rev Toxicol 21:51-88. Safe S, Bandiera S, Sawyer T, Zmudzka B, Mason G, Romkes M, Denomme MA, Sparling J, Okey AB and Fujita T (1985) Effects of structure on binding to the 2,3,7,8-TCDD receptor protein and AHH induction-halogenated biphenyls. Environ Hlth Perspect 61:21-33. Safe SH (1995) Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-/>-dioxin and related compounds. Pharmacol Ther 67:247-281. Santostefano M, Merchant M, Arellano L, Morrison V, Denison MS and Safe S (1993) a- Naphthoflavone-induced CYP1A1 gene expression and cytosolic aryl hydrocarbon receptor transformation. Mol Pharmacol 43:200-206. Santostefano M and Safe S (1996) Characterization of the molecular and structural properties of the transformed and nuclear aryl hydrocarbon (Ah) receptor complexes by proteolytic digestion. Chem Biol Interact 100:221-240.

226 Sasaki Y, Takahashi Y, Nakayama K and Kamataki T (1999) Cooperative regulation of CYP2C12 gene expression by STAT5 and liver-specific factors in female rats. J Biol Chem 274:37117-37124. Savouret JF, Antenos M, Quesne M, Xu J, Milgrom E and Casper RF (2001) 7- ketocholesterol is an endogenous modulator for the aryl hydrocarbon receptor. J Biol Chem 276:3054-3059. Schecter A, Birnbaum L, Ryan JJ and Constable JD (2006) Dioxins: an overview. Environ Res 101:419-428. Schmidt CW (2004) Diet & dioxins- The need to cut back. Environ Health Perspect 112:A40-A43. Schmidt JV, Su GHT, Reddy JK, Simon MC and Bradfield CA (1996) Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc Natl Acad Sci USA 93:6731-6736. Schuetz E, Lan L, Yasuda K, Kim R, Kocarek TA, Schuetz J and Strom S (2002) Development of a real-time in vivo transcription assay: application reveals pregnane X receptor-mediated induction of CYP3A4 by cancer chemotherapeutic agents. Mol Pharmacol 62:439-445. Sebestyen MG, Budker VG, Budker T, Subbotin VM, Zhang G, Monahan SD, Lewis DL, Wong SC, Hagstrom JE and Wolff JA (2006) Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules. J Gene Med 8:852-873. Semenza GL (1998) Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Curr Opin Genet Develop 8:588-594. Serron SC, Zhang SX, Bergeron RM and Backes WL (2001) Effect of hypophysectomy and growth hormone replacement on the modulation of P450 expression after treatment with the aromatic hydrocarbon ethylbenzene. Toxicol Appl Pharmacol 172:163-171. Sewer MB and Morgan ET (1997) Nitric oxide-independent suppression of P450 2C11 expression by interleukin-ip and endotoxin in primary rat hepatocytes. Biochem Pharmacol 54:729-737. Sewer MB and Morgan ET (1998) Down-regulation of the expression of three major rat liver cytochrome P450s by endotoxin in vivo occurs independently of nitric oxide production. J Pharmacol Exp Titer 287:352-358. Shaban Z, El-Shazly S, Abdelhady S, Fattouh I, Muzandu K, Ishizuka M, Kimura K, Kazusaka A and Fujita S (2004) Down regulation of hepatic PPARa function by AhR ligand. J Vet Med Sci 66:1377-1386. Shaban Z, Soliman M, El-Shazly S, El-Bohi K, Abdelazeez A, Kehelo K, Kim HS, Muzandu K, Ishizuka M, Kazusaka A and Fujita S (2005) AhR and PPARa: antagonistic effects on CYP2B and CYP3A, and additive inhibitory effects on CYP2C11. Xenobiotica 35:51-68. Shapiro BH, Agrawal AK and Pampori NA (1995) Gender differences in drug metabolism regulated by growth hormone. Int J Biochem Cell Biol 27:9-20. Shedlofsky SI, Israel BC, Mcclain CJ, Hill DB and Blouin RA (1994) Endotoxin administration to humans inhibits hepatic cytochrome P450-mediated drug metabolism. J Clin Invest 94:2209-2214.

227 Shehin SE, Stephenson RO and Greenlee WF (2000) Transcriptional regulation of the human CYP1B1 gene- Evidence for involvement of an aryl hydrocarbon receptor response element in constitutive expression. J Biol Chem 275:6770-6776. Shelnutt SR, Cornett LE and Owens SM (1997) Phencyclidine continuous dosing produces a treatment time-dependent regulation of rat CYP2C11 function, protein expression and mRNA levels. J Pharmacol Exp Ther 281:574-581. Shen AL, OLeary KA and Kasper CB (2002) Association of multiple developmental defects and embryonic lethality with loss of microsomal NADPH-cytochrome P450 oxidoreductase. J Biol Chem 277:6536-6541. Shen ES and Whitlock JP (1992) Protein-DNA Interactions at a dioxin-responsive enhancer- mutational analysis of the DNA-binding site for the liganded Ah receptor. J Biol Chem 267:6815-6819. Shertzer HG, Clay CD, Genter MB, Chames MC, Schneider SN, Oakley GG, Nebert DW and Dalton TP (2004) Uncoupling-mediated generation of reactive oxygen by halogenated aromatic hydrocarbons in mouse liver microsomes. Free Radical Biol Med 36:618-631. Shertzer HG, Nebert DW, Puga A, Ary M, Sonntag D, Dixon K, Robinson LJ, Cianciolo E and Dalton TP (1998) Dioxin causes a sustained oxidative stress response in the mouse. Biochem Biophys Res Commun 253:44-48. Shimada M, Murayama N, Nagata K, Hashimoto H, Ishikawa H and Yamazoe Y (1997) A specific loss of growth hormone abolished sex-dependent expression of hepatic cytochrome P450 in dwarf rats: Reversal of the profiles by growth hormone- treatment. Arch Biochem Biophys 337:34-42. Shimada M, Murayama N, Yamauchi K, Yamazoe Y and Kato R (1989) Suppression in the expression of a male-specific cytochrome P450, P450-male: difference in the effect of chemical inducers on P450-male mRNA and protein in rat livers. Arch Biochem Biophys 270:578-587. Shimada T and Fujii-Kuriyama Y (2004) Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes P450 1A1 and 1B1. Cancer Sci 95:1-6. Shimada T, Yamazaki H, Mimura M, Inui Y and Guengerich FP (1994) Interindividual variations in human liver cytochrome P450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414-423. Shimba S, Hayashi M, Sone H, Yonemoto J and Tezuka M (2000) 2,3,7,8- Tetrachlorodibenzo-/j-dioxin (TCDD) induces binding of a 50 kDa protein on the 3' untranslated region of urokinase-type plasminogen activator mRNA. Biochem Biophys Res Commun 272:441-448. Shimizu M, Matsushita R, Matsumoto Y and Fukuoka M (2003) 4'-hydroxylation of flurbiprofen by rat liver microsomes in fasting and feeding conditions. Biol Pharm Bull 26:1448-1454. Shimizu Y, Nakatsuru Y, Ichinose M, Takahashi Y, Kume H, Mimura J, Fujii-Kuriyama Y and Ishikawa T (2000) Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor. Proc Nat Acad Sci Usa 97:779-782.

228 Shipley JM and Waxman DJ (2003) Down-regulation of STAT5b transcriptional activity by ligand-activated peroxisome proliferator-activated receptor (PPAR)a and PPAR y. Mol Pharmacol 64:355-364. Shipley JM and Waxman DJ (2006) Aryl hydrocarbon receptor-independent activation of estrogen receptor-dependent transcription by 3-methylcholanthrene. Toxicol Appl Pharmacol 213:87-97. Shiraiwa T, Kamiyama N and Kashiwayanagi M (2007) Decreases in urinary pheromonal activities in male mice after exposure to 3-methylchoranthrene. Toxicol Lett 169:137-144. Shiraki H and Guengerich FP (1984) Turnover of membrane proteins: kinetics of induction and degradation of seven forms of rat liver microsomal cytochrome P450, NADPH-cytochrome P450 reductase, and epoxide hydrolase. Arch Biochem Biophys 235:86-96. Shou MG and Yang SK (1996) 3-Hydroxy-methylcholanthrene: metabolic formation from 3-methlylcholanthrene and stereoselective metabolism by rat liver microsomes. Drug Metab Dispos 24:595-601. Silkworm JB, Koganti A, Elouz K, Possolo A, Zhao M and Hamilton SB (2005) Comparison of TCDD and PCB CYP1A induction sensitivities in fresh hepatocytes from human donors, Sprague-Dawley rats, and Rhesus monkeys and HepG2 cells. Toxicol Sci 87:508-519. Simon P (2003) Q-Gene: processing quantitative real-time RT-PCR data. Bioinformatics 19:1439-1440. Sinai CJ and Bend JR (1997) Aryl hydrocarbon receptor-dependent induction of Cyplal by bilirubin in mouse hepatoma hepa lclc7 cells. Mol Pharmacol 52:590-599. Smart J and Daly AK (2000) Variation in induced CYP1A1 levels: relationship to CYPlAl,Ah receptor and GSTM1 polymorphisms. Pharmacogenetics 10:11-24. Smith AG, Clothier B, Carthew P, Childs NL, Sinclair PR, Nebert DW and Dalton TP (2001) Protection of the Cypla2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Appl Pharmacol 173:89-98. Sogawa K, Numayama-Tsuruta K, Takahashi T, Matsushita N, Miura C, Nikawa J, Gotoh O, Kikuchi Y and Fujii-Kuriyama Y (2004) A novel induction mechanism of the rat CYP1A2 gene mediated by Ah receptor-Arnt heterodimer. Biochem Biophys Res Commun 318:746-755. Song JS, ClagettDame M, Peterson RE, Hahn ME, Westler WM, Sicinski RR and DeLuca HF (2002) A ligand for the aryl hydrocarbon receptor isolated from lung. Proc Nat Acad Sci Usa 99:14694-14699. Spink DC, Wu SJ, Spink BC, Hussain MM, Vakharia DD, Pentecost BT and Kaminsky LS (2008) Induction of CYP1A1 and CYP1B1 by benzofflfluoranthene and benzo[a]pyrene in T-47D human breast cancer cells: Roles of PAH interactions and PAH metabolites. Toxicol Appl Pharmacol 226:213-224. Starr TB (2003) Significant issues raised by meta-analyses of cancer mortality and dioxin exposure. Environ Health Per sped 111:1443-1447. Steenland K, Bertazzi P, Baccarelli A and Kogevinas M (2004) Dioxin revisited: Developments since the 1997 IARC classification of dioxin as a human carcinogen. Environ Health Perspect 112:1265-1268.

229 Strom A, Eguchi H, Mode A, Legraverend C, Toilet P, Stromstedt PE and Gustafsson J-A (1994) Characterization of the proximal promoter and two silencer elements in the CYP2C11 gene expressed in rat liver. DNA Cell Biol 13:805-819. Stupans I, Richards DA and Mcclure MT (1995) Effects of 5-fluorouracil treatment on rat liver microsomal enzymes. Xenobiotica 25:1-8. Su GM, Fiala-Beer E, Weber J, Jahn D, Robertson GR and Murray M (2005) Pre- translational upregulation of microsomal CYP4A in rat liver by intake of a high- sucrose, lipid-devoid diet containing orotic acid. Biochem Pharmacol 69:709-717. Su GM, Sefton RM and Murray M (1999) Down-regulation of rat hepatic microsomal cytochromes P450 in microvesicular steatosis induced by orotic acid. J Pharmacol Exp Ther 291:953-959. Sugihara K, Kitamura S, Yamada T, Okayama T, Ohta S, Yamashita K, Yasuda M, Fujii- Kuriyama Y, Saeki K, Matsui S and Matsuda T (2004) Aryl hydrocarbon receptor-mediated induction of microsomal drug-metabolizing enzyme activity by indirubin and indigo. Biochem Biophys Res Commun 318:571-578. Sun YV, Boverhof DR, Burgoon LD, Fielden MR and Zacharewski TR (2004) Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences. Nucl Acid Res 32:4512-4523. Sundseth SS, Alberta JA and Waxman DJ (1992) Sex-Specific, growth hormone- regulated transcription of the cytochrome P450 2C11 and 2C12 Genes. J Biol Chem 267:3907-3914. Sunouchi M, Ishimi Y, Mizokami K, Fukuhara M, Takanaka A, Hanaoka F and Yamada M (1988) Preparation and characterization of monoclonal antibodies against a form of hamster liver cytochrome P450 highly specific to aflatoxin Bl. Biochem Biophys Res Commun 153:441-447. Swedenborg E, Ruegg J, Hillenweck A, Rehnmark S, Faulds MH, Zalko D, Pongratz I and Pettersson K (2008) 3-Methylcholanthrene displays dual effects on estrogen receptor (ER)oc and ERfi signaling in a cell-type specific fashion. Mol Pharmacol 73:575-586. Tanabe S, Watanabe M, Minh TB, Kunisue T, Nakanishi S, Ono H and Tanaka H (2004) PCDDs, PCDFs, and coplanar PCBs in albatross from the North Pacific and Southern Oceans: levels, patterns, and toxicological implications. Environ Sci Technol 38:403-413. Tanaka J, Yonemoto J, Zaha H, Kiyama R and Sone H (2007) Estrogen-responsive genes newly found to be modified by TCDD exposure in human cell lines and mouse systems. Mol Cell Endocrinol 272:38-49. Thangavel C, Dhir RN, Volgin DV and Shapiro BH (2007) Sex-dependent expression of CYP2C11 in spleen, thymus and bone marrow regulated by growth hormone. Biochem Pharmacol 74:1476-1484. Thangavel C, Dworakowski W and Shapiro BH (2006) Inducibility of male-specific isoforms of cytochrome P450 by sex-dependent growth hormone profiles in hepatocyte cultures from male but not female rats. Drug Metab Dispos 34:410- 419. Thangavel C and Shapiro BH (2007) A molecular basis for the sexually dimorphic response to growth hormone. Endocrinology 148:2894-2903.

230 Thomae TL, Glover E and Bradfield CA (2004) A maternal Ahr null genotype sensitizes embryos to chemical teratogenesis. J Biol Chem 279:30189-30194. Thomae TL, Stevens EA, Liss AL, Drinkwater NR and Bradfield CA (2006) The teratogenic sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin is modified by a locus on mouse chromosome 3. Mol Pharmacol 69:770-775. Thomas PE, Kouri RE and Hutton JJ (1972) The genetics of aryl hydrocarbon hydroxylase induction in mice: a single gene difference between C57BL/6J and DBA/2J. Biochem Genet 6:157-168. Thomas PE, Reik LM, Ryan DE and Levin W (1981) Regulation of three forms of cytochrome P450 and epoxide hydrolase in rat liver microsomes. Effects of age, sex and induction. J Biol Chem 256:1044-1052. Thompson KE, Bourguet SM, Christian PJ, Benedict JC, Sipes IG, Flaws JA and Hoyer PB (2005) Differences between rats and mice in the involvement of the aryl hydrocarbon receptor in 4-vinylcyclohexene diepoxide-induced ovarian follicle loss. Toxicol Appl Pharmacol 203:114-123. Tian Y, Ke S, Denison MS, Rabson AB and Gallo MA (1999) Ah receptor and NF-KB interactions, a potential mechanism for dioxin toxicity. J Biol Chem 274:510-515. Tijet N, Boutros PC, Moffat ID, Okey AB, Tuomisto J and Pohjanvirta R (2006) Aryl hydrocarbon receptor regulates distinct dioxin-dependent and dioxin-independent gene batteries. Mol Pharmacol 69:140-153. Timsit YE, Chia FSC, Bhathena A and Riddick DS (2002) Aromatic hydrocarbon receptor expression and function in liver of hypophysectomized male rats. Toxicol Appl Pharmacol 185:136-145. Timsit YE and Riddick DS (2000) Interference with growth hormone stimulation of hepatic cytochrome P450 2C11 expression in hypophysectomized male rats by 3- methylcholanthrene. Toxicol Appl Pharmacol 163:105-114. Timsit YE and Riddick DS (2002) Stimulation of hepatic signal transducer and activator of transcription 5b by GH is not altered by 3-methylcholanthrene. Endocrinology 143:3284-3294. Tomita S, Jiang HB, Ueno T, Takagi S, Tohi K, Maekawa S, Miyatake A, Furukawa A, Gonzalez FJ, Takeda J, Ichikawa Y and Takahama Y (2003) T cell-specific disruption of aryl hydrocarbon receptor nuclear translocator (Arni) gene causes resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced thymic involution. J Immunol 171:4113-4120. Tsuchiya Y, Nakajima M, Itoh S, Iwanari M and Yokoi T (2003) Expression of aryl hydrocarbon receptor repressor in normal human tissues and inducibility by polycyclic aromatic hydrocarbons in human tumor-derived cell lines. Toxicol Sci 72:253-259. Udy GB, Towers RP, Snell RG, Wilkins RJ, Park SH, Ram PA, Waxman DJ and Davey HW (1997) Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc Natl Acad Sci USA 94:7239-7244. Ueda R, Iketaki H, Nagata K, Kimura S, Gonzalez FJ, Kusano K, Yoshimura T and Yamazoe Y (2006) A common regulatory region functions bidirectionally in transcriptional activation of the human CYP1A1 and CYP1A2 genes. Mol Pharmacol 69:1924-1930.

231 Ueyama J, Kitaichi K, Nadai M, Iwase M, Tomyo N, Kanazawa H, Suzuki R, Takagi K and Hasegawa T (2004) Effect of pioglitazone on endotoxin-induced decreases in hepatic drug-metabolizing enzyme activity and expression of CYP3A2 and CYP2C11. Eur J Pharmacol 498:257-265. Uno S, Dalton TP, Derkenne S, Curran CP, Miller ML, Shertzer HG and Nebert DW (2004a) Oral exposure to benzo[a]pyrene in the mouse: detoxication by inducible cytochrome P450 is more important than metabolic activation. Mol Pharmacol 65:1225-1237. Uno S, Dalton TP, Sinclair PR, Gorman N, Wang B, Smith AG, Miller ML, Shertzer HG and Nebert DW (2004b) Cyplal(-/-) male mice: protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria. Toxicol Appl Pharmacol 196:410-421. Uno Y, Fujino H, Kito G, Kamataki T and Nagata R (2006) CYP2C76, a novel cytochrome P450 in cynomolgus monkey, is a major CYP2C in liver, metabolizing tolbutamide and testosterone. Mol Pharmacol 70:477-486. Vasiliou V, Puga A, Chang CY, Tabor MW and Nebert DW (1995) Interaction between the Ah receptor and proteins binding to the AP-1-like electrophile response element (EpRE) during murine phase II [Ah] battery gene expression. Biochem Pharmacol 50:2057-2068. Vecchini F, Lenoirviale MC, Cathelineau C, Magdalou J, Bernard BA and Shroot B (1994) Presence of a retinoid responsive element in the promoter region of the human cytochrome P4501A1 gene. Biochem Biophys Res Commun 201:1205- 1212. Verma AS, Dhir RN and Shapiro BH (2005) Inadequacy of the Janus kinase 2/signal transducer and activator of transcription signal transduction pathway to mediate episodic growth hormone-dependent regulation of hepatic CYP2C11. Mol Pharmacol 67:891-901. Vezina CM, Walker NJ and Olson JR (2004) Subchronic exposure to TCDD, PeCDF, PCB126, and PCB153: effect on hepatic gene expression. Environ Health Perspect 112:1636-1644. Villard PH, Caverni S, Baanannou A, Khalil A, Martin PG, Penel C, Pineau T, Seree E and Barra Y (2007) PPARot transcriptionally induces AhR expression in Caco-2, but represses AhR pro-inflammatory effects. Biochem Biophys Res Commun 364:896-901. Vlassov VV, Balakireva LA and Yakubov LA (1994) Transport of oligonucleotides across natural and model membranes. Biochim Biophys Acta 1197:95-108. Walisser JA, Bunger MK, Glover E and Bradfield CA (2004a) Gestational exposure of Ahr and Arnt hypomorphs to dioxin rescues vascular development. Proc Natl AcadSci U S A 101:16677-16682. Walisser JA, Bunger MK, Glover E, Harstad EB and Bradfield CA (2004b) Patent ductus venosus and dioxin resistance in mice harboring a hypomorphic Arnt allele. J Biol Chem 279:16326-16331. Walker NJ (2007) Unraveling the complexities of the mechanism of action of dioxins. Toxicol Sci 95:297-299.

232 Wang F, Samudio I and Safe S (2001) Transcriptional activation of cathepsin D gene expression by 17P-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition. Mol Cell Endocrinol 172:91-103. Wang H, Chen W, Zheng H, Guo L, Liang H, Yang X, Bai Y, Sun J, Su Y, Chen Y, Yuan J, Bi Y, Wei Q and Wu T (2007) Association between plasma BPDE-Alb adduct concentrations and DNA damage of peripheral blood lymphocytes among coke oven workers. Occup Environ Med 64:753-758. Wang RS, Nakajirna T, Tsuruta H and Honma T (1996) Effect of exposure to four organic solvents on hepatic cytochrome P450 isozymes in rat. Chem Biol Interact 99:239-252. Wang S, Ge K, Roeder RG and Hankinson O (2004) Role of mediator in transcriptional activation by the aryl hydrocarbon receptor. J Biol Chem 279:13593-13600. Warner M, Samuels S, Mocarelli P, Gerthoux PM, Needham L, Patterson DG and Eskenazi B (2004) Serum dioxin concentrations and age at menarche. Environ Health Perspect 112:1289-1292. Watanabe H, Suzuki A, Goto M, Ohsako S, Tohyama C, Handa H and Iguchi T (2004) Comparative uterine gene expression analysis after dioxin and estradiol administration. J Mol Endocrinol 33:763-771. Wauthier V, Dubois P, Verbeeck RK and Calderon PB (2006a) Induction of CYP2C12 expression in senescent male rats is well correlated to an increase of HNF3|3 expression, while the decline of CYP2C11 expression is unlikely due to a decrease of STAT5 activation. Biochem Pharmacol. Wauthier V, Schenten V, Verbeeck RK and Calderon PB (2006b) Ageing is associated with increased expression but decreased activity of CYP2E1 in male Wistar rats. LifeSci 79:1913-1920. Wauthier V, Verbeeck RK and Calderon PB (2007) The effect of ageing on cytochrome P450 enzymes: consequences for drug biotransformation in the elderly. CurrMed Chem 14:745-757. Waxman DJ (1988) Interactions of hepatic cytochromes P450 with steroid hormones. Regioselectivity and stereospecificity of steroid metabolism and hormonal regulation of rat P450 enzyme expression. Biochem Pharmacol 37:71-84. Waxman DJ, Dannan GA and Guengerich FP (1985) Regulation of rat hepatic cytochrome P450: age-dependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific isoenzymes. Biochemistry 24:4409-4417. Waxman DJ, Morrissey JJ, Maclxod JN and Shapiro BH (1990) Depletion of serum growth hormone in adult female rats by neonatal monosodium glutamate treatment without loss of female-specific hepatic enzymes P450 2d (IIC12) and steroid 5a-reductase. Endocrinology 126:712-720. Waxman DJ and O'Connor C (2006) Growth hormone regulation of sex-dependent liver gene expression. Mol Endocrinol 20:2613-2629. Waxman DJ, Pampori NA, Ram PA, Agrawal AK and Shapiro BH (1991) Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450. Proc Natl Acad Sci USA 88:6868-6872. Wei YD, Tepperman K, Huang MY, Sartor MA and Puga A (2004) Chromium inhibits transcription from polycyclic aromatic hydrocarbon-inducible promoters by

233 blocking the release of histone deacetylase and preventing the binding of p300 to chromatin. J Biol Chem 279:4110-4119. Weiss C, Faust D, Durk H, Kolluri SK, Pelzer A, Schneider S, Dietrich C, Oesch F and Gottlicher M (2005) TCDD induces c-jun expression via a novel Ah (dioxin) receptor-mediated p38-MAPK-dependent pathway. Oncogene 24:4975-4983. Weiss C, Kolluri SK, Kiefer F and Gottlicher M (1996) Complementation of Ah receptor deficiency in hepatoma cells: negative feedback regulation and cell cycle control by the ah receptor. Exp Cell Res 226:154-163. Wen X and Walle T (2007) Cytochrome P450 1B1, a novel chemopreventive target for benzo[a]pyrene-initiated human esophageal cancer. Cancer Lett 246:109-114. Weng MW, Hsiao YM, Chen CJ, Wang JP, Chen WC and Ko JL (2004) Benzo[a]pyrene diol epoxide up-regulates COX-2 expression through NF-KB in rat astrocytes. Toxicol Lett 151:345-355. Westwell AD (2004) The therapeutic potential of aryl hydrocarbon receptor (AhR) agonists in anticancer drug development. Drug Future 29:479-491. Wiesener MS, Munchenhagen PM, Berger I, Morgan NV, Roigas J, Schwiertz A, Jurgensen JS, Graber G, Maxwell PH, Loning SA, Frei U, Maher ER, Grone HJ and Eckardt KU (2001) Constitutive activation of hypoxia-inducible genes related to over-expression of hypoxia-inducible factor-la in clear cell renal carcinomas. Cancer Res 61:5215-5222. Wilhelmsson A, Cufhill S, Denis M, Wikstrom AC, Gustafsson J-A and Poellinger L (1990) The specific DNA binding activity of the dioxin receptor is modulated by the 90 kDa heat shock protein. Embo J 9:69-76. Willey JJ, Stripp BR, Baggs RB and Gasiewicz TA (1998) Aryl hydrocarbon receptor activation in genital tubercle, palate, and other embryonic tissues in 2,3,7, 8- tetrachlorodibenzo-/?-dioxin-responsive lacZ mice. Toxicol Appl Pharmacol 151:33-44. Williamson MA, Gasiewicz TA and Opanashuk LA (2005) Aryl hydrocarbon receptor expression and activity in cerebellar granule neuroblasts: implications for development and dioxin neurotoxicity. Toxicol Sci 83:340-348. Wiwi CA, Gupte M and Waxman DJ (2004) Sexually dimorphic P450 gene expression in liver-specific hepatocyte nuclear factor 4 a-deficient mice. Mol Endocrinol 18:1975-1987. Wiwi CA and Waxman DJ (2004) Role of hepatocyte nuclear factors in growth hormone- regulated, sexually dimorphic expression of liver cytochromes P450. Growth Factors 22:79-88. Wiwi CA and Waxman DJ (2005) Role of hepatocyte nuclear factors in transcriptional regulation of male-specific CYP2A2. J Biol Chem 280:3259-3268. Wolbold R, Klein K, Burk O, Nussler AK, Neuhaus P, Eichelbaum M, Schwab M and Zanger UM (2003) Sex is a major determinant of CYP3A4 expression in human liver. Hepatology 38:978-988. Wolff JA and Budker V (2005) The mechanism of naked DNA uptake and expression. Adv Genet 54:3-20. Wolff MS, Britton JA and Russo JC (2005) TCDD and puberty in girls. Environ Health Perspect 113:A17; author reply A18.

234 Wolff S, Harper PA, Wong JMY, Mostert V, Wang YP and Abel J (2001) Cell-specific regulation of human aryl hydrocarbon receptor expression by transforming growth factor-P(l). Mol Pharmacol 59:716-724. Wong JMY, Okey AB and Harper PA (2001) Human aryl hydrocarbon receptor polymorphisms that result in loss of CYP1A1 induction. Biochem Biophys Res Commun 288:990-996. Wu Q, Ohsako S, Ishimura R, Suzuki JS and Tohyama C (2004) Exposure of mouse preimplantation embryos to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the methylation status of imprinted genes H19 and Igf2. Biol Reprod 70:1790- 1797. Xie W, Barwick JL, Downes M, Blumberg B, Simon CM, Nelson MC, Neuschwander- Tetri BA, Bruntk EM, Guzelian PS and Evans RM (2000) Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406:435-439. Xu DX, Wei W, Sun MF, Wu CY, Wang JP, Wei LZ and Zhou CF (2004) Kupffer cells and reactive oxygen species partially mediate lipopolysaccharide-induced downregulation of nuclear receptor pregnane X receptor and its target gene Cyp3a in mouse liver. Free Radical Biol Med 37:10-22. Xu H, Rajesan R, Harper P, Kim RB, Lonnerdal B, Yang M, Uematsu S, Hutson J, Watson-MacDonell J and Ito S (2005a) Induction of cytochrome P450 1A by cow milk-based formula: a comparative study between human milk and formula. Br J Pharmacol 146:296-305. Xu M, Nelson GB, Moore JE, McCoy TP, Dai J, Manderville RA, Ross JA and Miller MS (2005b) Induction of Cyplal and Cyplbl and formation of DNA adducts in C57BL/6, Balb/c, and Fl mice following in utero exposure to 3- methylcholanthrene. Toxicol Appl Pharmacol 209:28-38. Xu ZM, Kawai M, Bandiera SM and Chang TKH (2001) Influence of dietary zinc deficiency during development on hepatic CYP2C11, CYP2C12, CYP3A2, CYP3A9, and CYP3A18 expression in postpubertal male rats. Biochem Pharmacol 62:1283-1291. Yamada H, Nakamura T and Oguri K (1998) Induction of rat hepatic cytochromes P450 by toxic ingredients in plants: lack of correlation between toxicity and inductive activity. J Toxicol Sci 23:395-402. Yamamoto I, Watanabe K, Narimatsu S and Yoshimura H (1995) Recent advances in the metabolism of cannabinoids. Int J Biochem Cell Biol 27:741-746. Yamazaki H, Shimizu M, Nagashima T, Minoshima M and Murayama N (2006) Rat cytochrome P450 2C11 in liver microsomes involved in oxidation of anesthetic agent propofol and deactivated by prior treatment with propofol. Drug Metab Dispos 34:1803-1805. Yamazoe Y, Shimada M, Kamataki T and Kato R (1986) Effects of hypophysectomy and growth hormone treatment on sex-specific forms of cytochrome P450 in relation to drug and steroid metabolisms in rat liver microsomes. Jpn J Pharmacol 42:371-382. Yang CS, Brady JF and Hong JY (1992) Dietary effects on cytochromes P450, xenobiotic metabolism, and toxicity. FASEB J 6:737-744.

235 Yang X, Liu D, Murray TJ, Mitchell GC, Hesterman EV, Karchner SI, Merson RR, Hahn ME and Sherr DH (2005) The aryl hydrocarbon receptor constitutively represses c-myc transcription in human mammary tumor cells. Oncogene 24:7869-7881. Yao EF and Denison MS (1992) DNA sequence determinants for binding of transformed Ah receptor to a dioxin-responsive enhancer. Biochemistry 31:5060-5067. Yeikilis R, Gal S, Kopeiko N, Paizi M, Pines M, Braet F and Spira G (2006) Hydrodynamics based transfection in normal and fibrotic rats. World J Gastroenterol 12:6149-6155. Yeowell HN, Waxman DJ, Wadhera A and Goldstein JA (1987) Suppression of the constitutive, male-specific rat hepatic cytochrome P-450 2c and its mRNA by 3,4,5,3',4',5'-hexachlorobiphenyl and 3-methylcholanthrene. Mol Pharmacol 32:340-347. Yoshida K and Nakanishi J (2003) Estimation of dioxin risk to Japanese from the past to the future. Chemosphere 53:427-436. Yoshihara S, Nagata K, Wada I, Yoshimura H, Kuroki H and Masuda Y (1982) A unique change of steroid metabolism in rat liver microsomes induced with highly toxic polychlorinated biphenyl (PCB) and polychlorinated dibenzofuran (PCDF). J Pharm Dyn 5:994-1004. Yoshinari K, Sueyoshi T, Moore R and Negishi M (2001) Nuclear receptor CAR as a regulatory factor for the sexually dimorphic induction of CYP2B1 gene by phenobarbital in rat livers. Mol Pharmacol 59:278-284. Yoshioka H, Morohashi K-I, Sogawa K, Miyata T, Kawajiri K, Hirose T, Inayama S, Fujii-Kuriyama Y and Omura T (1987) Structural analysis and specific expression of microsomal cytochrome P-450(M-1) mRNA in male rat livers. J Biol Chem 262:1706-1711. Yu Z, Ng VY, Su P, Engler MM, Engler MB, Huang Y, Lin E and Kroetz DL (2006) Induction of renal cytochrome P450 arachidonic acid epoxygenase activity by dietary y-Linolenic Acid. J Pharmacol Exp Ther 317:732-738. Yuan W, White TB, White JW, Strobel HW and Backes WL (1995) Relationship between hydrocarbon structure and induction of P450: Effect on RNA levels. Xenobiotica 25:9-16. Zatloukalova J, Svihalkova-Sindlerova L, Kozubik A, Krcmar P, Machala M and Vondracek J (2007) P-Naphthoflavone and 3'-methoxy-4'-nitroflavone exert ambiguous effects on Ah receptor-dependent cell proliferation and gene expression in rat liver 'stem-like' cells. Biochem Pharmacol 73:1622-1634. Zhang G, Budker V and Wolff JA (1999) High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther 10:1735-1737. Zhang G, Gao X, Song YK, Vollmer R, Stolz DB, Gasiorowski JZ, Dean DA and Liu D (2004a) Hydroporation as the mechanism of hydrodynamic delivery. Gene Ther 11:675-682. Zhang LY, Savas U, Alexander DL and Jefcoate CR (1998) Characterization of the mouse Cyplbl gene- Identification of an enhancer region that directs aryl hydrocarbon receptor-mediated constitutive and induced expression. J Biol Chem 273:5174-5183.

236 Zhang LY, Zheng WC and Jefcoate CR (2003a) Ah receptor regulation of mouse Cyplbl is additionally modulated by a second novel complex that forms at two AhR response elements. Toxicol Appl Pharmacol 192:174-190. Zhang WS, Moorthy B, Chen M, Muthiah K, Coffee R, Purchio AF and West DB (2004b) A Cypia2-luciferase transgenic CD-I mouse model: Responses to aryl hydrocarbons similar to the humanized AhR mice. Toxicol Sci 82:297-307. Zhang WS, Purchio A, Chen K, Burns SM, Contag CH and Contag PR (2003b) In vivo activation of the human CYP3A4 promoter in mouse liver and regulation by pregnane X receptors. Biochem Pharmacol 65:1889-1896. Zhang WS, Purchio AF, Chen K, Wu JM, Lu L, Coffee R, Contag PR and West DB (2003c) A transgenic mouse model with a luciferase reporter for studying in vivo transcriptional regulation of the human CYP3A4 gene. Drug Metab Dispos 31:1054-1064. Zhang WS, Purchio AF, Coffee R and West DB (2004c) Differential regulation of the human CYP3A4 promoter in transgenic mice and rats. Drug Metab Dispos 32:163-167. Zhang Y, Bressler JP, Neal J, Lai B, Bhang HE, Laterra J and Pomper MG (2007) ABCG2/BCRP expression modulates D-luciferin based bioluminescence imaging. Cancer Res 67:9389-9397. Zhao W and Ramos KS (1998) Cytotoxic response profiles of cultured rat hepatocytes to selected aromatic hydrocarbons. Toxicol In Vitro 12:175-182. Zhou J and Gasiewicz TA (2003) 3'-methoxy-4'-nitroflavone, a reported aryl hydrocarbon receptor antagonist, enhances Cyplal transcription by a dioxin responsive element-dependent mechanism. Arch Biochem Biophys 416:68-80. Zhou M, Maitra SR and Wang P (2008) The potential role of transcription factor aryl hydrocarbon receptor in downregulation of hepatic cytochrome P450 during sepsis. IntJMol Med 21:423-428. Zudaire E, Cuesta N, Murty V, Woodson K, Adams L, Gonzalez N, Martinez A, Narayan G, Kirsch I, Franklin W, Hirsch F, Birrer M and Cuttitta F (2008) The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers. J Clin Invest 118:640-650.

237 6.0 LIST OF PUBLICATIONS AND ABSTRACTS

Refereed Scholarly Publications • Sawaya RM and Riddick DS. 2008. Cytochrome P450 2C11 5'-flanking region and promoter: regulation by aromatic hydrocarbons in vitro. Toxicology, 248:104-112. • Sawaya RM and Riddick DS. 2008. Cyrochrome P450 2C11 5'-flanking region and promoter mediate in vivo suppression by 3-methylcholanthrene. Drug Metab Dispos, 36:1803-1811.

Refereed Conference Abstracts • Sawaya RM and Riddick DS. 2007. CYP2C11 5'-flanking region and promoter mediates in vivo suppression by aromatic hydrocarbons. Proceedings of the 11' International Congress of Toxicology [Montreal, Quebec; July/07] poster • Sawaya RM and Riddick DS. 2006. CYP2C11 5'-flanking region and promoter mediates in vivo suppression by aromatic hydrocarbons. Proceedings of the 39th Annual Society of Toxicology of Canada Symposium [Montreal, Quebec; December /06] poster • Sawaya RM and Riddick DS. 2006. Cytochrome P450 2C11 5'-flanking region and promoter: regulation by aromatic hydrocarbons in vitro and in vivo. FASEB Journal 20: A260-A261. [Experimental Biology '06; San Francisco, California; April /06] poster • Sawaya RM and Riddick DS. 2005. Cytochrome P450 2C11 5' -flanking region and promoter: regulation by aromatic hydrocarbons in vitro and in vivo. Proceedings of the 38th Annual Society of Toxicology of Canada Symposium; [Montreal, Quebec; December /05] poster • Sawaya RM and Riddick DS. 2005. Cloning and characterization of the extended 5'- flanking region of the cytochrome P450 2C11 gene. FASEB Journal 19: A1567. [Experimental Biology '05; San Diego, California; April /05] poster • Sawaya RM and Riddick DS. 2004. Cloning and characterization of the extended 5'- flanking region of the cytochrome P450 2C11 gene. Proceedings of the 37th Annual Society of Toxicology of Canada Symposium [Montreal, Quebec; December /04] poster

Non-refereed Conference Abstracts • Sawaya RM and Riddick DS. 2007. CYP2C11 5'-flanking region and promoter mediates in vivo suppression by aromatic hydrocarbons. Visions in Pharmacology, p.39. [Toronto, Ontario; May /07] poster • Sawaya RM and Riddick DS. 2006. Cytochrome P450 2C11 5'-flanking region and promoter: regulation by aromatic hydrocarbons in vitro and in vivo. Visions in Pharmacology, p.44. [Toronto, Ontario; May /06] poster • Sawaya RM and Riddick DS. 2005. Cloning and characterization of the extended 5'- flanking region of the cytochrome P450 2C11 gene. Visions in Pharmacology, p.45. [Toronto, Ontario; May /05] poster • Sawaya RM and Riddick DS. 2004. Cytochrome P450 2C11 5'-flanking region and promoter: regulation by aromatic hydrocarbons in rat liver. Visions in Pharmacology, p.45. [Toronto, Ontario; May /04] poster

238