Evaluation of in Silico and in Vitro Screening Methods for EDC Hazard Characterisation
Total Page：16
File Type：pdf, Size：1020Kb
` CRANFIELD UNIVERSITY Louise Claire Youngs EVALUATION OF IN SILICO AND IN VITRO SCREENING METHODS FOR CHARACTERISING ENDOCRINE DISRUPTING CHEMICAL HAZARDS SCHOOL OF ENERGY, ENVIRONMENT AND AGRIFOOD PhD Thesis Doctor of Philosophy Academic Year: 2014 Supervisor: Dr Ruth Bevan Nov 2014 CRANFIELD UNIVERSITY SCHOOL OF ENERGY, ENVIRONMENT AND AGRIFOOD Institute for Environment, Health, Risk and Futures PhD Thesis Academic Year 2014 Louise Claire Youngs EVALUATION OF IN SILICO AND IN VITRO SCREENING METHODS FOR CHARACTERISING ENDOCRINE DISRUPTING CHEMICAL HAZARDS Supervisor: Dr Ruth Bevan Nov 2014 © Cranfield University 2014. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner. ABSTRACT Anthropogenic activities have drastically altered chemical exposure, with traces of synthetic chemicals detected ubiquitously in the environment. Many of these chemicals are thought to perturb endocrine function, leading to declines in reproductive health and fertility, and increases in the incidence of cancer, metabolic disorders and diabetes. There are over 90 million unique chemicals registered under the Chemical Abstracts Service (CAS), of which only 308,000 were subject to inventory and/or regulation, in September 2013. However, as a specific aim of the EU REACH regulations, the UK is obliged to reduce the chemical safety initiatives reliance on in vivo apical endpoints, promoting the development and validation of alternative mechanistic methods. The human health cost of endocrine disrupting chemical (EDC) exposure in the EU, has been estimated at €31 billion per annum. In light of the EU incentives, this study aims to evaluate current in silico and in vitro tools for EDC screening and hazard characterisation; testing the hypothesis that in silico virtual screening accurately predicts in vitro mechanistic assays. Nuclear receptor binding interactions are the current focus of in silico and in vitro tools to predict EDC mechanisms. To the author’s knowledge, no single study has quantitatively assessed the relationship between in silico nuclear receptor binding and in vitro mechanistic assays, in a comprehensive manner. Tripos ® SYBYL software was used to develop 3D-molecular models of nuclear receptor binding domains. The ligand binding pockets of estrogen (ERα and ERβ), androgen (AR), progesterone (PR) and peroxisome proliferator activated (PPARγ) receptors were successfully modelled from X-ray crystal structures. A database of putative-EDC ligands (n= 378), were computationally ‘docked’ to the pseudo-molecular targets, as a virtual screen for nuclear receptor activity. Relative to in vitro assays, the in silico screen demonstrated a sensitivity of 94.5%. The SYBYL Surflex-Dock method surpassed the OECD Toolbox ER-Profiler, DfW and binary classification models, in correctly identifying endocrine active substances (EAS). Aiming to evaluate the current in vitro tools for endocrine MoA, standardised ERα transactivation (HeLa9903), stably transfected AR transactivation (HeLa4-11) assays in addition to novel transiently transfected reporter gene assays, predicted the mechanism and potency of test substances prioritised from the in silico results (n = 10 potential-EDCs and 10 hormone controls). In conclusion, in silico SYBYL molecular modelling and Surflex-Dock virtual screening sensitively predicted the binding of ERα/β, AR, PR and PPARγ potential EDCs, and was identified as a potentially useful regulatory tool, to support EAS hazard identification. Keywords: Endocrine Disrupt*, Test Methods, Prioritisation, Regulation, SYBYL Surflex-Dock Page i ACKNOWLEDGEMENTS “From the world we actually live in, the world that is given by our senses, our intuitions of beauty and goodness, our emotions and impulses, our moods and sentiments, the man of science abstracts a simplified and private universe of things possessing only those qualities which used to be called “primary”. Arbitrarily, because it happens to be convenient, because his methods do not allow him to deal with the immense complexity of reality, he selects from the whole of experience only those elements which can be weighed, measured, numbered, or which lend themselves in any other way to mathematical treatment. By using this technique of simplification and abstraction, the scientist has succeeded to an astounding degree in understanding and dominating the physical environment. The success was intoxicating and, with an illogicality which, in the circumstances, was doubtless pardonable, many scientists and philosophers came to imagine that this useful abstraction from reality was reality itself” Aldous Huxley. Firstly, I would like to thank all those who have aided my plight for scientific abstraction, and in particular Ruth Bevan, Mike Roberts and Joe Lunec, who supported the project from the beginning and throughout its duration. Thanks also goes to: the ol’ IEH team (Paul Harrison, Terry Brown, Derrick Crump, Lini Ashdown and Christina Tam); Kal Karim; Phil Holmes; Taisen Iguchi; and Miyagawa Shinichi for their support. However, the largest thanks’ goes to those that reminded me life is not just facts, error and statistical significance, but the beauty of anomalies. My home girls - Milner, HG, Pols, Digby, Dale, Bibby, Root and Cobbie – you have filled my life with love and laughter for over 15 years. Always accepting me, from the red hair and nose ring to PhD, you have given me the strength to be whatever it is I am. To the friends that opened my doors of perception at Exeter, I am eternally grateful and constantly inspired by you - Sammy, Els, Sweeney, Shells and Ellie, I love and thank you. Thanks also goes to the friends I’ve met along the way; Zineb, Natalia, Rob, Stefan, Thierry, Dave, Gail, Paul and the countless others who have helped me cope with the social and psychological isolation of Cranfield. Last, but by no means least, I would like to thank my family. Always feeling free, supported and loved, I grew up naïve to how existence is for some. As an adult I know how lucky I was to feel this freedom and see these opportunities. Mumges, Dadge and Sedge - without you, none of this would have been possible. Page iii TABLE OF CONTENTS ABSTRACT ...................................................................................................................... i ACKNOWLEDGEMENTS ............................................................................................ iii LIST OF FIGURES ......................................................................................................... ix LIST OF TABLES .......................................................................................................... xi LIST OF ABBREVIATIONS ....................................................................................... xiii 1 INTRODUCTION ......................................................................................................... 1 1.1 The Scientific Context ............................................................................................ 1 1.1.1 A Human Health Concern? ............................................................................. 2 1.1.2 Mechanism of Action ...................................................................................... 3 1.2 A Regulatory Conundrum ...................................................................................... 6 1.2.1 Chemical Safety Testing Strategies ................................................................. 6 1.3 The EDC Problem .................................................................................................. 8 1.4 Research Aims and Objectives ............................................................................... 9 2 BACKGROUND INFORMATION ............................................................................ 13 2.1 The ‘Start’ of the Science ..................................................................................... 13 2.1.1 Endocrinology ............................................................................................... 13 2.1.2 The Rise of Environmentalism ...................................................................... 15 2.1.3 Wildlife Effects ............................................................................................. 16 2.1.4 Summary ........................................................................................................ 20 2.2 Evidence for Endocrine Disruption in Humans: A Human Health Concern? ...... 21 2.2.1 Reproductive Health ...................................................................................... 21 2.2.2 Hormonal Cancers ......................................................................................... 25 2.2.3 Metabolism and Development ....................................................................... 26 2.2.4 Summary ........................................................................................................ 27 2.3 Chemical Regulation and Testing ......................................................................... 28 2.3.1 Legislation & Regulation .............................................................................. 28 2.3.2 A Defined Mode-of-Action? ......................................................................... 29 2.3.3 Chemical Safety Test Guidelines .................................................................. 30 2.3.4 Alternative (Non-Animal) Methods .............................................................. 33 2.3.5 Exposure