UC San Diego UC San Diego Electronic Theses and Dissertations Title Xenobiotic regulation of Phase I and Phase II metabolism enzymes : beyond the Ah receptor paradigm Permalink https://escholarship.org/uc/item/5nz70459 Author Bonzo, Jessica A. Publication Date 2007 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Xenobiotic Regulation of Phase I and Phase II Metabolism Enzymes: Beyond the Ah Receptor Paradigm A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Jessica A. Bonzo Committee in charge: Professor Robert H. Tukey, Chair Professor Daniel J. Donoghue Professor Ronald M. Evans Professor Michael Karin Professor Julian I. Schroeder 2007 Copyright Jessica A. Bonzo, 2007 All rights reserved. The Dissertation of Jessica A. Bonzo is approved, and it is acceptable in quality and form for publication on microfilm: Chair University of California, San Diego 2007 iii Dedication To my parents, Roy T. and Flora E. A. Bonzo. iv Table of Contents Signature Page …………………………………………………. iii Dedication ……….……………………………………………... iv Table of Contents …………………………………………….... v List of Abbreviations .………………………………………...... vii List of Figures ………………..………………………………… xi List of Tables ............................................................................... xiv Acknowledgements ..................................................................... xv Vita .............................................................................................. xix Abstract of the Dissertation ......................................................... xxiii Chapter I Introduction Historical Perspective on Phase I and Phase II Enzymes................ 1 Xenobiotics and CYP and UGT Induction...................................... 11 The Ah Receptor Regulates Cytochrome P450 1A1....................... 16 Structure and Signal Transduction Pathway of the Ah Receptor.... 18 Regulation of Ah Receptor Function............................................... 22 Objectives of the Dissertation......................................................... 25 Chapter II Materials and Methods.................................................................... 29 Chapter III Results Effects of As3+ on Apoptosis and Cell Cycle.................................. 48 Inhibition of CYP1A1 Induction During Cell Cycle Arrest............ 52 As3+ Treatment Blocks TCDD Induction of CYP1A1.................... 54 The Actions of As3+ on TCDD-Induced Transcriptional control of CYP1A1........................................................................................... 58 Gene and Ligand Dependence of As3+ Inhibition of Ah Receptor Responsive Genes............................................................................ 64 Chrysin Regulates the UGT1A1 Gene Through a XRE.................. 68 Effects of Chrysin on TCDD Binding to the Ah Receptor.............. 71 The Role of the Ah Receptor in Chrysin Induction of UGT1A1..... 75 v Chrysin Activation of the UGT1A1 Promoter Occurs Through the MAP kinase Pathway...................................................................... 85 Actions of Oral Chrysin Treatment in Transgenic UGT1 (Tg- UGT1) Mice..................................................................................... 87 Regulation of UGT1A6 by the Xenobiotic-Sensing Nuclear Receptors PXR and CAR................................................................ 91 Regulation of UGT1A6 by PPARα................................................. 97 UGT1As and the High Fat Diet Model of Type 2 Diabetes............ 101 High Fat Diet Influences on UGT1A Isoform Exression................ 108 Chapter IV Discussion Summary.......................................................................................... 112 As3+ and CYP1A1........................................................................... 113 Chrysin and UGT1A1...................................................................... 120 Nuclear Receptor Control of UGT1A6 Expression......................... 125 Implications for UGT1A Enzymes In Metabolic Syndrome........... 132 Conclusions and Future Work......................................................... 142 References................................................................................................... 147 vi List of Abbreviations Ah aryl hydrocarbon AHRR aryl hydrocarbon receptor repressor Arnt Aryl hydrocarbon nuclear translocator As3+ arsenite, inorganic trivalent arsenic B[a]P benzo[a]pyrene, 3,4-benzpyrene bHLH basic helix loop helix CAR constitutive androstane receptor ChIP chromatin immunoprecipitation CN-I, -II Crigler-Najjar Syndrome Type I or Type II CYP cytochrome P450 DMEM Dulbecco’s modified Eagle medium DMSO dimethyl sulfoxide DRE dioxin response element DTT dithiothreitol ELISA enzyme-linked immunosorbent assay EMSA electrophorectic mobility shift assay ERK1/2 extracellular signal-regulated kinase 1 or 2, p44/42 EROD ethoxyresorufin-O-deethylase FA fatty acid FACS fluorescence-activated cell sorting FFA free fatty acid GAPDH glyceraldehydes-3-phosphate dehydrogenase vii GI tract gastrointestinal tract GPR40 G-coupled protein receptor 40 GRDBD glucocorticoid receptor DNA binding domain GRE glucocorticoid response element GTT glucose tolerance test HAH halogenated aromatic hydrocarbon HDAC histone deacetylase HF high-fat diet HNF hepatic nuclear factor hnRNA heteronuclear RNA Hsp90 Heat shock protein 90 IL-1β interleukin 1β ITT insulin tolerance test JNK1/2 c-Jun N-terminal kinase LXRα liver x receptor α MAPK mitogen activated protein kinase MEK MAPK ERK kinase MEF mouse embryonic fibroblast MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium NEFA non-esterified free fatty acid NF-κB nuclear factor κB NQO1 NAD(P)H:quinone oxidoreductase 1 NSAID nonsteroidal anti-inflammatory drug viii PAH polycyclic aromatic hydrocarbon PARP poly(ADP-ribose)polymerase PAS Per-Arnt-Sim PCR polymerase chain reaction PD98059 2'-amino-3'-methoxyflavone PKC protein kinase C polII RNA polymerase II PMSF phenylmethylsulfonyl fluoride PPARα, γ, δ peroxisome proliferator-activated receptor α, γ, δ PPRE PPAR response element PXR pregnane x receptor RT-PCR reverse transcriptase-polymerase chain reaction SB203580 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H- imidazole SNP single nucleotide polymorphism SP600125 1,9-pyrazoloanthrone 2,4,5-T 2,4,5-trichlorophenoxyacetic acid TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin TCDF 2,3,7,8-tetrachlorodibenzo-furan TNFα tumor necrosis factor α UDP-GA UDP-glucuronic acid UGT/UDPGT UDP-glucuronosyltransferase UO126 1,2-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol] butadiene ix WT wild type WY-14643 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid XAP2 hepatitis B virus X-associated protein 2 XRE xenobiotic response element x List of Figures Chapter I Figure 1: Organization of the human UGT1 locus………………… 7 Figure 2: Ah receptor signal transduction pathway……………….. 19 Chapter III Figure 3: Cell viability, apoptosis, and cell cycle arrest after As3+ exposure…………………………………………………. 51 Figure 4: G2/M arrest inhibits CYP1A1 mRNA expression but not transcriptional activation………………………………... 53 Figure 5: CYP1A1 EROD activity, protein, and mRNA expression as a function of As3+ treatment………………………….. 56 Figure 6: As3+ inhibition of CYP1A1 induction in transgenic CYP1A1N+/- primary hepatocytes……………………….. 58 Figure 7: Activation of Ah receptor is unaffected by the presence of As3+................................................................................ 59 Figure 8: Ah receptor nuclear translocation and DNA binding activity is unchanged by As3+ treatment………………… 60 Figure 9: Transcriptional control of CYP1A1……………………... 63 Figure 10: Identification of As3+ inhibition region on CYP1A1 promoter…………………………………………………. 64 Figure 11: Inhibition of chrysin-mediated CYP1A1 induction by As3+……………………………………………………… 65 Figure 12: Induction of NQO1 by TCDD and As+3………………… 66 Figure 13: Differential regulation of UGT1A1 in response to TCDD and chrysin mixtures with As3+…………………………. 68 Figure 14: Chrysin induction of TCDD responsive genes………….. 70 Figure 15: Identification of the chrysin responsive region within the UGT1A1 promoter………………………………………. 71 xi Figure 16: Chrysin alters TCDD binding affinity to the Ah receptor. 73 Figure 17: Differential effects of TCDD and chrysin mixtures…….. 75 Figure 18: Confirmation of Ah receptor/Arnt-binding complex on CYP1A1 and UGT1A1 XREs……………………………. 76 Figure 19: Effects of chrysin on Ah receptor activation……………. 80 Figure 20: Transactivation of the Ah receptor by chrysin and TCDD 82 Figure 21: siRNA knockdown of Ah receptor……………………… 84 Figure 22: U0126 activates the Ah receptor………………………… 85 Figure 23: Involvement of MAP kinase pathway in chrysin induction of UGT1A1…………………………………… 87 Figure 24: The actions of oral chrysin treatment…………………… 90 Figure 25: Induction of UGT1A6 in Tg-UGT1 primary hepatocytes. 93 Figure 26: UGT1A6 promoter constructs and previously characterized response elements………………………… 94 Figure 27: Preliminary identification of PXR and CAR responsive regions on the UGT1A6 promoter……………………….. 95 Figure 28: Identification of the PXR responsive region in the UGT1A6 promoter………………………………………. 97 Figure 29: PPARα is functional in the presence of UGT1A6 DR1 sequence…………………………………………………. 100 Figure 30: Increased weight in response to a high fat diet………….. 104 Figure 31: Increased adiposity in high fat diet mice………………..