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The Application of Transcriptomics in the Comparative Safety Assessment of (GMO-Derived) Plant Products

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The Application of Transcriptomics in the Comparative Safety Assessment of (GMO-Derived) Plant Products The application of transcriptomics in the comparative safety assessment of (GMO-derived) plant products E.J. Kok Promotoren Prof. dr. ir. I.M.C.M. Rietjens Hoogleraar Toxicologie, Wageningen Universiteit Prof. dr. M.R. Müller Hoogleraar Voeding, Metabolisme en Genomics, Wageningen Universiteit Co-promotoren Dr. ir. H.A. Kuiper Onderzoeker, RIKILT – Instituut voor Voedselveiligheid, Wageningen Prof. dr. ir. J. Keijer Groepshoofd, RIKILT – Instituut voor Voedselveiligheid, Wageningen Promotiecommissie Prof. dr. R.J. Bino (Wageningen Universiteit) Prof. dr. H.V. Davies (Scottish Crop Research Institute, Dundee, UK) Prof. dr. W.J. Stiekema (Wageningen Universiteit) Prof. dr. W.M. de Vos (Wageningen Universiteit) Dit onderzoek is uitgevoerd binnen de onderzoeksschool VLAG The application of transcriptomics in the comparative safety assessment of (GMO-derived) plant products Esther Jeannette (van Leeuwe-) Kok Proefschrift ter verkrijging van de graad van doctor op gezag van de Rector Magnificus van Wageningen Universiteit, Prof. dr. M.J. Kropff, in het openbaar te verdedigen op vrijdag 13 juni 2008 des morgens te elf uur in de Aula Esther Kok The application of transcriptomics in the comparative safety assessment of (GMO-derived) plant products PhD thesis Wageningen University and Research Centre, the Netherlands With summaries in English and Dutch. ISBN 978-90-8504-929-6 Aan mijn vader† en moeder, Sarina† en Carolien Abstract National and international organizations have discussed current approaches to the safety assessment of complex (plant) food products in general and the safety assessment of GMO- derived food products in particular. One of the recommendations of different expert meetings was that the new analytical techniques, in particular the ‘omics’ approaches, need to be explored for their potential to improve the analysis and thereby the toxicological and nutritional assessment of complex (GMO-derived) plant products. This thesis aims to further explore this approach in general and, more specifically, has evaluated the potential added value of transcriptomics to assess unintended side effects in a newly bred (genetically modified) plant variety. As one of the first initiatives in this area a small food safety-related tomato-array was developed with pathway-selected cDNAs on the basis of two subtractive cDNA libraries. This tomato array was used to hybridise mRNA derived from tomatoes in five subsequent ripening stages from green, via breaker, turning, and light red, to red, to obtain a background library of gene expression profiles of different ripening stages for future comparisons. At the same time these initial series of experiments were aimed to assess the potential of the approach with respect to its sensitivity and specificity. In addition the tomato array was used to hybridise mRNA derived from GM tomato transformant lines and the traditionally bred parent line and the results were analysed for the presence of differential expression patterns in both transformant lines, that have incorporated the same genetic construct, compared to the parent line. A similar study was performed in Arabidopsis to assess the extent of unintended effects in GM lines that have incorporated different numbers of the introduced genetic construct. The resulting data show that the methodology of transcriptomics has the potential to detect large as well as small differences in gene expression. The first was primarily shown in the comparative study on the developmental stage, the latter in the comparison of transcriptomics profiles of the two transformant lines vs the parent variety. It was also shown that, for direct comparison, plants to be sampled need to be grown under very similar conditions and the sampling needs to be performed in a structured way taking into account the developmental stage of the selected plant organs and/or tissues. All experiments illustrated the necessity to establish the bandwidth of natural variation for comparative purposes in order to determine the biological as well as toxicological and/or nutritional significance of differences detected in GM lines or in lines resulting from other breeding procedures. Finally, the results of the international debate on the assessment of complex (GMO-derived) plant products, the knowledge on current breeding strategies, and the results of the first publications on experiments that aim to detect unintended effects in plant breeding strategies using ‘omics’ technologies, including the experiments described here, are combined to review current approaches. A new overall approach for the safety evaluation of complex plant products, including GMO-derived products, is proposed. This approach applies currently available tools, including the ‘omics’ technologies, to assess food safety aspects of newly developed plant varieties already during the plant breeding process. Undesired effects of the breeding procedures for the plant’s physiology, can thus be traced at an early stage and this will help to further guarantee the safety of the final Theapplicationoftranscriptomicsinthecomparativesafetyassessmentof(GMO-derived)plantproducts plant-derived food products. Observed differences in the final plant product will form part of the comparative safety assessment. It can be envisioned on the basis of the data presented in this thesis as well as in other studies described in the scientific literature that in general few differences will be observed between GM lines and the WT counterparts that fall outside of the bandwidth of natural variation of commercial counterparts. The toxicological and nutritional evaluation of the final plant products will need to focus on the limited number of differences that are outside of this bandwidth and may affect the product’s food safety characteristics. To avoid inequalities in food safety assessment procedures that do not have a sound scientific basis, it is argued that these developments should have an impact on all novel plant varieties, not just on GMO-derived food plant products. Theapplicationoftranscriptomicsinthecomparativesafetyassessmentof(GMO-derived)plantproducts Table of contents Abstract 6 Introduction 10 Chapter 1. Comparative safety assessment for biotech crops 12 Chapter 2. The food safety risk assessment of GM animals 26 Chapter 3. Assessment of representational difference analysis (RDA) to construct informative cDNA microarrays for gene expression analysis of species with limited transcriptome information, using red and green tomatoes as a model 48 Chapter 4. Changes in gene and protein expression during tomato ripening - consequences for the safety assessment of new crop plant varieties 70 Chapter 5. Food safety analysis of genetically modified tomato lines using an ‘omics’ approach 98 Chapter 6. Evaluation of a non-targeted ‘omic’ approach in the safety assessment of genetically modified plants 118 Chapter 7. Comparative safety assessment of plant-derived foods 140 Chapter 8. Summary and conclusions 170 Chapter 9. Samenvatting en conclusies 180 About the author 190 Dankwoord 196 Theapplicationoftranscriptomicsinthecomparativesafetyassessmentof(GMO-derived)plantproducts Introduction Genetically modified food products, i.e. products derived from genetically modified organisms (GMOs), were firstly introduced onto the world market in 1994 in the United States. The first commercial product was the FLAVR SAVR tomato with delayed ripening characteristics. Already before this initial introduction the international debate had started on the most appropriate procedure to assess the safety of GMO-derived food and feed products. In the following years international consensus was reached as to the basic approach for GMO safety assessment with the application of the comparative safety assessment (CSA). This CSA entails a two-tiered procedure: 1) comparison of the new GMO variety with its nearest conventional counterpart on the market and 2) assessment of detected differences in terms of toxicological and nutritional relevance for the safety and nutritional status of consumers of the GMO-derived products. The basis for the CSA is an elaborate compositional analysis of known nutrients and anti-nutritional factors. At the same time it was recognized that this approach based on known constituents has its limitations in terms of detecting unintended side effects of the genetic modification. It was therefore proposed that unbiased methods such as the ‘omics’ technologies should be further explored for their applicability to detect unintended and undesired alterations in GM plant varieties. This thesis aims to evaluate the practical aspects of the application of one of the ‘omics’ technologies, transcriptomics, i.e. the generation of gene expression profiles using the microarray technology, to detect unintended effects in GMOs, as well as to assess the potential added value of transcriptomics in the comparative safety assessment of a newly bred (genetically modified) plant variety. Chapter 1 and 2 are introductory chapters and provide an overview of the state-of-the-art of the safety assessment procedures at the onset of the experiments. Chapter 1 introduces the concept of the Comparative Safety Assessment (CSA) as the basic principle in the safety assessment of GMOs and derived products, representing a tiered approach that would be an alternative for the similar and often cited Principle of Substantial Equivalence that is frequently misinterpreted as the endpoint of the safety assessment. Chapter 2 focuses on the safety assessment of GMO-derived
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