Omics in (Eco)toxicology: Case Studies and Risk Assessment February 2010, Málaga. Workshop Report No PDF

Omics in (Eco)toxicology: Case Studies and Risk Assessment February 2010, Málaga Workshop Report No. 19 EUROPEAN CENTRE FOR ECOTOXICOLOGY AND TOXICOLOGY OF CHEMICALS Omics in (Eco)toxicology: Case

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Omics in (Eco)toxicology: Case Studies and Risk Assessment February 2010, Málaga Workshop Report No. 19 EUROPEAN CENTRE FOR ECOTOXICOLOGY AND TOXICOLOGY OF CHEMICALS Omics in (Eco)toxicology: Case Studies and Risk Assessment February 2010, Málaga Workshop Report No. 19 Brussels, June 2010 ISSN (print) ISSN (online) ECETOC WORKSHOP REPORT No. 19 Copyright ECETOC AISBL European Centre for Ecotoxicology and Toxicology of Chemicals 4 Avenue E. Van Nieuwenhuyse (Bte 6), B-1160 Brussels, Belgium. All rights reserved. No part of this publication may be reproduced, copied, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the copyright holder. Applications to reproduce, store, copy or translate should be made to the Secretary General. ECETOC welcomes such applications. Reference to the document, its title and summary may be copied or abstracted in data retrieval systems without subsequent reference. The content of this document has been prepared and reviewed by experts on behalf of ECETOC with all possible care and from the available scientific information. It is provided for information only. ECETOC cannot accept any responsibility or liability and does not provide a warranty for any use or interpretation of the material contained in the publication. ECETOC WR No. 19 Omics in (Eco)toxicology: Case Studies and Risk Assessment CONTENTS 1. SUMMARY 1 2. WORKSHOP OVERVIEW Introduction Workshop structure Workshop aim and objectives 4 3. DEFINITIONS 5 4. BASELINE / NEW DESCRIPTORS / ADVERSE EFFECTS Sources of variation in baseline gene expression levels from toxicogenomics study control animals Toxicogenomics: The challenges and opportunities to identify biomarkers, signatures and thresholds to support mode of action New descriptors in toxicogenomics: Epigenetic studies and microrna IDENTIFICATION OF MODE OF ACTION AND APPLICATION TO RISK ASSESSMENT IN A QUALITATIVE WAY Linking transcriptomic profiles to population effects in Daphnia magna Profiling endocrine disruption potential using ToxCast and other relevant data CASE STUDIES Case 1: Using transcriptomic data to define non-cancer and cancer points of departure: A fivechemical case study Case 2: Protein biomarkers for in vitro testing of embryotoxicity Case 3: The identification of direct and indirect modes of action on the thyroid by means of metabolic profiling and its use for biology based chemicals grouping under REACH Case 4: Male reproductive health and endocrine toxicity: Application of toxicogenomic technologies to develop a mechanistic-based risk assessment Case 5: Liver toxicogenomics within the pharmaceutical industry. From in vivo, to slice, to permanent cell line Case 6: In vitro and in vivo transcriptomics for assessing toxicological effects of chemicals to aquatic organisms Case 7: A systems biology approach to small fish ecotoxicogenomics FUTURE PERSPECTIVES / SYSTEMS BIOLOGY / MODELLING Prediction in the face of uncertainty: A Monte Carlo strategy for systems biology of cancer treatment 25 ECETOC WR No. 19 8. REPORTS FROM THE SYNDICATE SESSIONS Syndicate 1a: MoA Syndicate 1b: MoA Syndicate 2: Quantitative risk assessment Syndicate 3: Linking from in vitro / in vivo and extrapolation across species: Mammals to humans, Daphnia to fish CONCLUSIONS AND RECOMMENDATIONS 37 ABBREVIATIONS 39 BIBLIOGRAPHY 40 APPENDIX A: LIST OF PARTICIPANTS 43 APPENDIX B: WORKSHOP PROGRAMME 44 APPENDIX C: ORGANISING COMMITTEE 47 ECETOC WR No. 19 1. SUMMARY This report presents recent progress made on the application of omics technologies in toxicological and ecotoxicological risk assessment as discussed at a workshop held in Malaga on 22 and 23 February Seven case studies were presented as well as sessions on the future perspective, system biology and modelling. This was followed by syndicate discussions on baseline, new descriptors, adverse effects, identification of mode of action and its qualitative application to risk assessment. The following conclusions were drawn in a final plenary session: Omics data are particularly valuable for understanding modes of action (MoA) via underlying molecular patterns and by exploring responses to model compounds in highly standardised systems. Novel patterns or biomarkers (e.g. gene signatures, metabolome profiles) can also be developed this way for screening chemical properties of novel compounds. Within the context of risk assessment omics data can already add value to risk assessment by improving mechanistic understanding and the identification of modes of action. To enhance the acceptance of omics data, for such risk assessment purposes, high quality data and a careful design of the biological experiment are essential. Mode of action recognition by fingerprints or biomarkers can be enhanced if the changes observed can be causally linked to the toxicological pathway. These technologies can potentially serve as a tool for the prioritisation of chemical testing and could help to provide a better (biology based) rationale for chemical grouping under the REACH legislation. To better assess the quantitative aspects of omics data, more information concerning the sensitivity of omics relative to classical toxicology testing is needed. It would seem that transcriptomic information may be more sensitive than classical toxicology, whereas metabolomics appears to be equally sensitive. In addition, there is a need for better standardisation of methods within the various activities in this dynamic field, particularly in the area of transcriptomics. The participants also agreed that in the near future, omics technologies could help to bridge in vitro testing to in vivo relevance. Guidance (communication of best practices), rather than guidelines will encourage improvements and adaptation to new technical developments. ECETOC WR No. 19 1 The workshop concluded that better standardisation, data interpretation and evaluation will build confidence in the value of omics technologies this being essential to increase their (regulatory) use. The workshop therefore called for an international effort to bring together scientists from academia, industry, agencies as well as the risk assessors themselves, to discuss and evaluate the necessary modifications that may be needed to enhance the use of omics data in risk assessment. 2 ECETOC WR No. 19 2. WORKSHOP OVERVIEW 2.1 Introduction In the 2007 ECETOC Workshop on the application of the omic technology in (eco)toxicology, it was agreed that toxicological relevance could only be ascribed to patterns of change indicative of a perturbation in a biochemical pathway whose relevance was understood (see Workshop Report No. 11). The significance of changes in single genes was considered unlikely to have toxicological significance due to the high likelihood of spurious and random variations. Three recommendations are therefore proposed: 1. In order to exemplify typical toxicological mechanisms, standardised studies are required using well-characterised reference chemicals. This will increase confidence in the interpretation of omic data. 2. As changes in biochemical pathways are accepted to be more relevant than changes in individual genes, it is necessary to obtain a common and agreed definition of what constitutes a toxicologically relevant biochemical pathway, based on well-studied and characterised examples. 3. In order to relate omics results to conventional toxicity, it is necessary to study the toxicity dose and time dependent transition in relevant biochemical pathways from normal variability, through adaptive response, to adverse effect. A benchmark dose approach might be the most suitable one for this exercise. To follow up and review the progress made on the application of omics technologies in toxicology and ecotoxicology, a second workshop Omics in (Eco)toxicology: Case Studies and Risk Assessment was organised as significant developments within the omics sciences have taken place over the last two years. The number of available case studies is far larger than two years ago, and some experiences concerning the regulatory use of omics data (e.g. to demonstrate a mode of action / toxicological mechanism) have been obtained by companies. It seems appropriate to address the question: Which of the recommendations from the 2007 Workshop were actually taken up by scientists and whether the recommendations need to be developed and progressed further. 2.2 Workshop structure The workshop was organised around case studies and syndicate discussion sessions where baseline, new descriptors, adverse effects and the identification of mode of action and the application of this to risk assessment in a qualitative way were discussed. Seven case studies were presented as well as a session on the future perspective, system biology and modelling. ECETOC WR No. 19 3 The discussions from the breakout groups were recapitulated in a final plenary session where several recommendations were made and conclusions drawn. A total of 35 scientific experts from industry, academia and governmental agencies participated in the workshop, which was held in Malaga on 22 and 23 February A list of participants is given in Appendix A, and the programme is detailed in Appendix B. The workshop was limited to participation by selected industry experts and invited external scientists. 2.3 Workshop aim and objectives The starting point for the second workshop was the recommendations of the first workshop The Application of Omics Technologies in Toxicology and Ecotoxicology: Case Studies and Risk Assessment the results of which were published in ECETOC Workshop Report no. 11. In short they were: Conduct studies in a more standardised form using reference chemicals. Obtain a common and agreed definition of what constitutes a toxicologically relevant biochemical pathway. Study the toxicity dose and time dependent transition on relevant biochemical pathways from normal variability through adaptive response, to adverse effect. Identify a relevant MoA from a risk assessment point of view. How can omics contribute to the above points? It was felt that gathering appropriate experts to help with the interpretation of data from omics studies was needed as well as the formation of working groups that would address specific topics. The aim of the 2010 ECETOC Workshop Omics in (Eco)toxicology: Case Studies and Risk Assessment was to bring together experts from industry, academia and the regulatory agencies to review the current state of the use of omics technology, the progress that has been made and to identify issues that need to be addressed in the (near) future. 4 ECETOC WR No. 19 3. DEFINITIONS Mode of Action (MoA) is a reference to the specific biochemical interaction through which a chemical substance / agent produces its influence on key processes in living organisms. It is a less detailed biochemical description of events than is meant by mechanism of action. However, specifically for risk assessment in toxicology a MoA should consist of a defined set of several key processes that all need to be fulfilled. Mode of action then, in the context of risk assessment, is defined as a biologically plausible series of key events leading to an adverse effect. Key events are those that are critical to the adverse outcome (i.e. necessary but not necessarily sufficient in their own right), measurable and repeatable. Mechanism of action: The mechanism of action is usually considered to include an identification of the specific molecular targets to which a chemical active substance binds or whose biochemical action it influences; it refers to the specific biochemical interaction through which a chemical substance produces a biochemical effect. Mechanism of action thus relates to understanding the molecular basis of adverse effects. While there is limited understanding of the mechanisms of toxicity for most adverse effects, identification of key events in an hypothesised mode of action, based on robust (but generally incomplete) mechanistic data provides important insight, which is critical to effective and efficient prediction and reduction of risk. Toxicity pathway: As a group of critical molecular events that, if perturbed by a toxic chemical, are expected to result in adverse effects, thus implying that changing such a pathway prevents the particular toxic endpoint of becoming manifest. Risk assessment: The process of determining the potential impact of risk by identification, evaluation and estimation of both the likelihood that effect will occur and, if it does occur, the impact it has on life, and then combining the results and comparing them against benchmarks or standards, to then determine an acceptable level of risk. ECETOC WR No. 19 5 4. BASELINE / NEW DESCRIPTORS / ADVERSE EFFECTS 4.1 Sources of variation in baseline gene expression levels from toxicogenomics study control animals Chris Corton Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, US EPA, USA The use of gene expression profiling to predict chemical mode of action would be enhanced by better characterisation of variance due to individual, environmental, and technical factors. Metaanalysis of microarray data from untreated or vehicle-treated animals within the control arm of toxicogenomics studies has yielded useful information on baseline fluctuations in gene expression. A dataset of control animal microarray expression data was assembled by a working group of the Health and Environmental Sciences Institute's Technical Committee on the Application of Genomics in Mechanism Based Risk Assessment in order to provide a public resource for assessments of variability in baseline gene expression. Data from over 500 Affymetrix microarrays from control rat liver and kidney were collected from 16 different institutions. Thirty-five biological and technical factors were obtained for each animal, describing a wide range of study characteristics, and a subset were evaluated in detail for their contribution to total variability using multivariate statistical and graphical techniques. The study factors that emerged as key sources of variability included gender, organ section, strain, and fasting state. These and other study factors were identified as key descriptors that should be included in the minimal information about a toxicogenomics study needed for interpretation of results by an independent source. Genes that are the most and least variable, gender-selective, or altered by fasting were also identified and functionally categorised. In other studies, examples of gene expression variability through mouse life stages were discussed. Better characterisation of gene expression variability in control animals will aid in the design of toxicogenomics studies and in the interpretation of their results. This abstract does not necessarily reflect US EPA policy. 6 ECETOC WR No. 19 4.2 Toxicogenomics: The challenges and opportunities to identify biomarkers, signatures and thresholds to support mode of action Richard A. Currie Syngenta, UK For over a decade toxicogenomics (TGx the application of transcript profiling and other massively parallel analytical techniques to toxicology) has been proposed to assist toxicologists and risk assessors in two ways. Firstly, by identifying patterns of gene-expression changes (signatures) that are surrogate markers, we might better predict hazard endpoints. Secondly by identifying the gene expression changes preceding toxicity, TGx can be used to investigate the underlying causes. The early challenges of technical reproducibility have been evaluated and addressed by both ILSI/HESI and the MAQC, which demonstrated that by the adoption of good practice, TGx can give reliable and reproducible information on gene expression changes in response to chemical treatment. Similarly the MAQC II is about to publish best practice on signature generation after conducting an extensive evaluation of numerous methods on existing large datasets. Two findings of particular note are the need for methods that control batch variability, and that the predictive power of a signature correlates with the intrinsic variability of the apical endpoint being predicted. Indeed this might create a fundamental challenge for the application of TGx in this way does this represent an upper limit on our ability to ever predict some endpoints? A traditional mode of action (MoA) is defined as the series of key events that ultimately result in the apical adverse endpoint of concern. Key events can either be casual or associative, which can thus act as surrogates for an underling key event. Obviously not every chemical-induced change need be a key event in the formation of toxicity and the observable key or incidental events may change with duration of chemical treatment. Clearly TGx can, and in practice does, identify the gene-expression changes underlying both key and incidental events. When considering the more investigative uses of TGx associated with MoA, there is therefore a clear need to adopt a suitable framework to aid in the analysis of the data. The application of the tests for causation used to build a more traditional argument is one such framework and its application to toxicogenomics data will also produce reasonable hypotheses linking altered pathways to phenotypic changes (as described in Figure 1). Challenges in interpretation still remain: Are all pathway changes equal; which are most important and can be plausibly considered as, or linked to, another key event? There are theoretical reasons why we might consider consistent alterations across genes in a metabolic pathway important, but similar changes in a signalling pathway may not alter information flow through that pathway. Only changes at the inputs, outputs or key integration nodes in a signalling transduction pathway may be considered relevant indicators for pathways of this type. Are some gene expression changes most often unrelated to chemical treatment? Can TGx data be used to separate two distinct MoAs (e.g. direct mitogenic stimulation and necrotic- ECETOC WR No. 19 7 damage induced regenerative proliferation)? How we perform interspecies comparisons is another major challenge for use in risk assessment: When can we use the same genes, or the same pathway. The use of the expert judgement of the toxicologist is still needed to identify those gene-expression changes that should be considered key events and those which should not. Do we know enough and are we adequately trained to make these judgments? Figure 1: Frameworks for interpretation of toxicity and toxicogenomics data IPCS framework for evaluating a MoA Postulated theory of cause the series of measurable events that are believed to be critical to the induction of the toxicity A body of evidence is then developed to support the association based on Sir Austin Bradford Hill type tests for causation Dose-response relationships are they parallel? Temporal relationship does the cause precede the effect? Consistent are the effects repeatable across studies? Specificity are the events unique or general? Are other modes of action also operating? Analogous to a previously proven causal relationship? Biologically plausible are they consistent with what is known about the underlying biology? Toxicogenomics data Gene expression changes are measurable (with suitable experimental protocols) and pathway tools inform us of the biological processes and pathway perturbations that may underlie existing key events Correct experimental design with suitable doses and time points plus replication Databases of TGx changes induced by a variety of compounds for comparison State of knowledge dependent that needs expert judgement and review of the weight of evidence in public literature One further opportunity for the application of TGx data is p
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