POTENCY AND SPECIES SPECIFICITY OF ARYL HYDROCARBON RECEPTOR LIGANDS Richard J. Wall, BSc. MRes. Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy July 2012 Richard Wall Abstract The aryl hydrocarbon receptor (AhR) binds a wide range of structurally diverse compounds such as halogenated dibenzo-p-dioxins, dibenzofurans and biphenyls which are abundant in the environment. Activation of AhR leads to the regulation of a battery of xenobiotic enzymes including cytochrome P4501A1 (CYP1A1). The purely chlorinated compounds feature in the World Health Organisation’s (WHO) evaluation of dioxin-like compounds derived from a meta-analysis of previous potency data (toxic equivalency factors; TEFs), which is used to calculate the total toxic equivalence (TEQ). The first aim of this work was to fully characterise the three most environmentally abundant mono-ortho-substituted polychlorinated biphenyls (PCBs; PCB 105, 118 and 156) including a re-evaluation of their putative antagonistic effects on AhR. Secondly, the effects of mixed halogenated compounds, currently not included in the TEQ estimation, were investigated as AhR agonists based on their environmental exposure and potency. Quantitative real-time PCR (qRT-PCR) was used to measure the AhR mediated induction of CYP1A1 mRNA in rat H4IIE and human MCF-7 cells. The three mono-ortho-substituted PCBs were shown to be antagonists of rat and human AhRs, an effect which is not currently included in the TEQ calculation. 2-bromo-3,7,8-trichlorodibenzo-p-dioxin (2-B-3,7,8-TriCDD) was found to be an AhR agonist that was 2-fold more potent than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; considered one of the most potent in the environment). The majority of the other tested compounds were found to be within 10-fold less potent than TCDD and could therefore have a significant impact on the TEQ. A family of putative AhR agonists from AstraZencea were investigated and one of the compounds was shown to be a highly potent AhR agonist, 5-fold more potent than TCDD at inducing CYP1A1. i Richard Wall The results indicate approximately a 15-fold higher sensitivity of the rat cell line to the AhR agonists compared with the human cell line. It is not currently understood what confers these differences whether it is a difference in the mechanism of activation or purely as a result of differences in the AhR sequence. The mechanism of action is thought to be the same in both species and the associated proteins are both comparable. The amino acid sequences of the AhR, in both human and rat are quite similar but may play a significant role in the differences observed between species. Therefore in order to directly compare the rat and human AhRs, two novel cell line models were created using an inducible expression system to infect an AhR-deficient mouse cell line with a replication-defective virus containing either the rat or human AhR. The AhRs were activated with various compounds to induce mouse CYP1A1. The CYP1A1 mRNA was measured using qRT-PCR but showed that the two AhR genes were not expressed enough to produce a response detectable above the background CYP1A1 induction by the low levels of mouse AhR. This research has shown that these dioxin-like compounds can have very different potencies at AhRs in different species so it is not always possible to predict the potency in humans from in vitro or rat in vivo toxicity data. Furthermore, it has identified compounds, such as 5F-203, which are significantly more potent in human compared to rat. This thesis provides information on the AhR species differences between human and rat that can be applied to risk assessment. ii Richard Wall Acknowledgements Firstly, I would like to thank Dr. Ian Mellor for his advice, patience and humour, without that I’m not sure I would have survived. I’m sure Ian will be very sad that he no longer has to read through my manuscript drafts, hide in his office when he can see me coming (and at one point changing offices completely), answer simple questions and become an expert in AhR research purely to assist me with my work. For this I am eternally grateful. I would also like to thank Dr. David Bell for accepting me into this PhD and providing me with an interesting project. Despite being in another country, he has still taken the time to answer my questions, give me advice and make corrections. I would like to thank my FERA supervisors, Dr. Alwyn Fernandes and Dr. Martin Rose. They have been an excellent source of advice, and were very welcoming when I visited FERA or when I have attended conferences with them. Thanks also to Dr. J. Craig Rowlands for his advice and corrections for my manuscripts, and to the FSA for their financial and intellectual support. Thanks also to my two examiners, Dr. Nick Plant and Dr. Ian Duce, for actually allowing me to pass with only a few corrections! I would like to thank Declan Brady for his technical advice and conversation. He was always available if I had a problem (unless it was Friday night). I would like to thank all of the Bell Lab during my PhD, particularly Himanshu for his molecular biology knowledge, but also: Abeer, Ahmad, Cristina, Fikry, Hamad, Hao, Ning, Vroni, Wail, as well as Archana, Rakesh, Rosie who shared our office. I would also like to thank everyone at the School of Biology especially Mark, who was always around for a chat (or a moan) about life and has over the years, become a very good friend. I’d like to thank the Mellor/Duce Lab, especially young David, and for my office mate Charu, a lovely person and a great laugh (even if she was always late). I would also like to thank the many fantastic people I have met over the last 4 years; starting at Raleigh Park and finishing at Derby Hall. Without you all... well... I’d still of passed my PhD, probably even done better than I have... but I would not have enjoyed the last few years so much. Thank you for being such great distractions. Finally, as always, a huge thanks to my parents. They have always been there for me and I am very lucky to have such great family. They have supported me mentally (and sometimes financially) all the way through my earlier studies that allowed me to do this PhD. Thank you so much. iii Richard Wall Abbreviations 7-ER 7-Ethoxyresorufin 95% CI 95% Confidence Interval aā Amino acid residue AHH Aryl Hydrocarbon Hydroxylase (CYP1A1) AhR Aryl Hydrocarbon Receptor AIP Aryl hydrocarbon receptor interacting protein (see XAP2) α-NF Alpha-naphthoflavone Arnt Aryl Hydrocarbon Receptor Nuclear Translocator bHLH Basic Helix-Loop-Helix β-NF Beta-naphthoflavone BpRc1 Taoc1BPrc1 AhR-defective cell line cDMEM Complete Dulbecco’s modified Eagle’s medium cDNA Complementary DNA cMEM Complete minimum essential medium Chr 2-amino-isoflavones CYP1A1 Cytochrome P450 1A1 CYP1A2 Cytochrome P450 1A2 CYP1B1 Cytochrome P450 1B1 DMSO Dimethyl Sulfoxide Dox Doxycycline DR-CALUX Dioxin-Responsive-Chemical Activated LUciferase gene eXpression DRE Dioxin Response Element EC50 Concentration that gives 50% of maximal response EROD Ethoxyresorufin-O-deethylation GC/MS Gas chromatography/Mass spectrometry H4IIE Rat liver cell line HAH Halogenated aromatic hydrocarbon HepG2 Human hepatocellular carcinoma cell line Hepa1c1c7 Mouse hepatoma cell line Hsp90 90 kDa Heat shock protein IC50 Half maximal inhibitory concentration Kd Equilibrium dissociation constant Ki Equilibrium inhibition constant iv Richard Wall LBD Ligand binding domain Max Max Speed (centrifuge) MCF-7 Human breast carcinoma cell line mRNA Messenger RNA MMTV Mouse mammary tumour virus MOI Multiplicity of infection MoMuLV Moloney murine leukemia virus NIH/3T3 mouse embryonic fibroblast cell line p23 Prostaglandin E synthase 3 (Hsp90 accessory protein) PAH Polycyclic Aromatic Hydrocarbon PAS Per, Arnt, AhR, Sim PBS Dulbecco's phosphate buffered saline PCB Polychlorinated biphenyl PCDD Polychlorinated dibenzo-p-dioxin PCDF Polychlorinated dibenzofuran PeCDF 2,3,4,7,8- Pentachlorodibenzofuran Per Drosophila circadian rhythm protein PXB Polyhalogenated biphenyl PXDD Polyhalogenated dibenzo-p-dioxin PXDF Polyhalogenated dibenzofuran REP Relative potency value RevT Reverse Transcriptase RT Room temperature (20 – 25oC; centrifuge) qRT-PCR Quantitative Real-Time Polymerase Chain Reaction Sim Drosophila neurogenic protein SYBR SYBR green Tc Tetracycline TCDD 2,3,7,8-Tetracholordibenzo-p-dioxin TCDF 2,3,7,8-Tetrachlorodibenzofuran TEF Toxic equivalency factor TEQ Total toxic equivalency TRE Tet-response element WHO World Health Organisation XRE Xenobiotic Response Element XAP2 Immunophilin-like associated protein 2 (X-associated protein 2; see AIP) v Richard Wall Table of Contents Abstract ................................................................................................................................... i Acknowledgements ............................................................................................................... iii Abbreviations ........................................................................................................................ iv Table of Contents .................................................................................................................. vi List of Figures ....................................................................................................................... xi List of Tables ....................................................................................................................... xiii List of Equations ................................................................................................................