Unintended effects and their detection in genetically modified crops

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Abstract

The commercialisation of GM crops in Europe is practically non-existent at the present time. The European Commission has instigated changes to the regulatory process to address the concerns of consumers and member states and to pave the way for removing the current moratorium. With regard to the safety of GM crops and products, the current risk assessment process pays particular attention to potential adverse effects on human and animal health and the environment. This document deals with the concept of unintended effects in GM crops and products, i.e. effects that go beyond that of the original modification and that might impact primarily on health. The document first deals with the potential for unintended effects caused by the processes of transgene insertion (DNA rearrangements) and makes comparisons with genetic recombination events and DNA rearrangements in traditional breeding. The document then focuses on the potential value of evolving “profiling” or “omics” technologies as non-targeted, unbiased approaches, to detect unintended effects. These technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). The technologies are described, together with their current limitations. Importantly, the significance of unintended effects on consumer health are discussed and conclusions and recommendations presented on the various approaches outlined.

Introduction

The approaches used in the safety assessment of crops and foods derived from genetically modified organisms have been developed in collaborative work with international agencies such as the Organisation for Economic Co-ordination and Development (OECD, 1993) and the United Nations World Health Organisation/Food and Agricultural Organisation (FAO/WHO, 1991, FAO/WHO, 2000). The approach involves the concept of substantial equivalence, whereby the characteristics of the modified crop/food are compared to an existing food/crop with a history of safe use. This is most usually the parent crop from which the modifications were made. The process involves a targeted compositional analysis (profile of major nutrients and toxicants), and the expected intake and role in the diet of the novel food. This comparison provides the basis on which to focus further toxicological requirements for a safety assessment. Three scenarios may be considered (European Commission, 1997). Firstly, the novel food is equivalent to an accepted traditional food or ingredient, in which case no further testing is needed. Secondly, the novel food is equivalent to the traditional counterpart except for some well defined differences; safety assessments will be targeted to these differences. Finally, the novel food differs from the traditional counterpart in multiple and complex respects, or there are no traditional counterparts; such a novel food would require an extensive safety assessment. A stepwise procedure to carry out the safety assessment has been discussed by König et al. (2004).

Concerns have been raised that the current approach of using targeted analyses to compare the composition of GM crops to their traditional counterparts is biased (Millstone et al., 1999) and does not take into account the possibility of unintended effects and unexpected effects that could result directly or indirectly from the genetic modification. The potential occurrence of such “unintended effects” is currently one of the concerns being raised regarding the application of recombinant DNA techniques in the production of foods. In this report aspects related to the detection of unintended effects are discussed. As a basis for the paper, the following definitions have been adopted:

  • “Intended effects” of genetic engineering are those that are targeted to occur from the introduction of the gene(s) in question and that fulfil the original objectives of the genetic transformation process.

  • “Unintended effects” represent a statistically significant difference in the phenotype, response, or composition of the GM plant compared with the parent from which it is derived, but taking the expected effect of the target gene into account. Such comparisons should be made when GM and non-GM counterparts are grown under the same regimes and environments.

  • “Predictable unintended effects” are those unintended changes that go beyond the primary expected effect(s) of introducing the target gene(s), but that may be explicable in terms of our current knowledge of plant biology and metabolic pathway integration and interconnections.

  • “Unpredictable unintended effects” are those changes falling outside our present level of understanding.

Predictable and unpredictable unintended effects may or may not prove to have relevance in terms of product safety, but must be taken into account when assessing risk. In addition, there is little guidance for crop producers on which parameters should be measured for the comparison, which analytical methods should be used, and which sampling procedures should be followed to provide statistically sound analyses. With the development of new molecular techniques such as profiling techniques, it has been thought possible to address these concerns.

Safety assessments follow the well accepted paradigm, which includes hazard identification and characterisation, exposure assessments, and subsequent risk characterisation. The aim of the safety assessment of novel foods, including those produced by GM technologies, is to demonstrate that the novel food is as safe as its traditional counterpart (where one exists) and as such does not introduce any additional or new risks to the health of the consumer. The relevance of this issue with regard to consumer acceptance of GM crop-derived foods has been discussed by Frewer et al. (2004). Predictable and unpredictable unintended effects may or may not prove to have relevance in terms of product safety, but must be taken into account when assessing risk.

The present paper aims to critically discuss the detection of unintended effects in GM crops. However, prior to this, it is necessary to describe the mechanisms whereby unintended effects may arise during (GM) crop breeding and how this compares to natural DNA recombination in plants. The ways in which unintended effects are dealt with in conventional breeding programmes are outlined. General issues relating to analytical procedures to detect unintended effects are discussed and the present status of targeted approaches are reported. Current developments in non-targeted approaches, i.e., the profiling techniques of genomics, proteomics, and metabolomics are presented with a critical discussion of their potentials and limitations. This last point is closely linked to the relevance of searching for unintended effects with respect to safety assessments and raises the question as to whether the more information we have available would, in reality, reduce the uncertainties in the safety assessment.

Section snippets

Classical plant breeding

Plant breeding has always exploited genetic methods both by using natural genetic variation combined with artificial selection and by inducing new variability by artificial means. In this sense, plant breeding can also be defined as “applied plant genetics”. For plant breeders, genetic variability has not only been a matter of chance, but has also been induced, controlled, and exploited by artificial techniques. Plant population genetic structure has been widely changed by breeding practices (

Unintended effects in conventional breeding

As discussed previously, the occurrence of unintended effects is not a phenomenon specific to genetic engineering. In classical breeding programmes, extensive backcrossing procedures are applied in order to remove unintended effects. Fig. 1 indicates the steps taken to select lines in a traditional plant breeding programme using barley, an in-breeder, as an example.

This process would apply to most cereals. It is possible that with some parents the resulting F1 generation produces no lines with

Demonstration of “substantial equivalence” by investigation of defined constituents

The comparison of the chemical composition of the genetically modified plant to that of a traditionally obtained counterpart has been a key element in the safety assessment of genetically modified crops. Such a comparative approach will reveal similarities as well as differences between the transgenic crop and the selected comparator and will thus give information on the “status of equivalence” (König et al., 2004).

This concept has been applied in the pre-market assessment of the first

Safety assessment of unintended effects

Traditional plant sources of food with a long history of use have not been evaluated for safety in a systematic way. Typically, it was by trial and error where the plant was incorporated into the diet, often after some form of processing, e.g. cooking, to make it acceptable from both a taste and safety point of view. Traditional varieties of food crops are known to contain both beneficial components (nutrients and other compounds), as well as compounds with a toxic potential (natural plant

Overall conclusions

The aim of crop breeding is to apply selection aimed at specific characteristics, such as improving nutritional quality and yield. The major source of natural variation and of breeding programmes is the natural molecular mechanisms of DNA exchange and repair. These mechanisms are the same for all crops, irrespective of whether the DNA has been specifically modified by genetic engineering techniques or has been altered via conventional crossing of different varieties. In addition to the

References (198)

  • T.L Graham

    Flavonoid and flavonol glycoside metabolism in Arabidopsis

    Plant Physiology and Biochemistry

    (1998)
  • J.E Haber

    Recombinationa frank view of exchanges and vice versa

    Current Opinion in Cell Biology

    (2000)
  • G Hansen et al.

    Recent advances in the transformation of plants

    Trends in Plant Science

    (1999)
  • U Justesen et al.

    Quantitative analysis of flavonols, flavones and flavanones in fruits, vegetables and beverages by high-performance liquid chromatography with photo-diode array and mass spectrometric detection

    Journal of Chromatography A

    (1998)
  • B.S Kristal et al.

    Simultaneous analysis of the majority of low-molecular-weight, redox active compounds from mitochondria

    Analytical Biochemistry

    (1998)
  • J.C Lindon et al.

    Pattern recognition methods and applications in biomedical magnetic resonance

    Progress in NMR Spectroscopy

    (2001)
  • ACNFP, 1999. Annual Report 1999. Advisory Committee on Novel Foods and Processes, Ministry of Agriculture, Fisheries...
  • A Aharoni et al.

    Nontargeted metabolome analysis by use of Fourier Transform Ion Cyclotron Mass Spectrometry

    OMICS

    (2002)
  • R.W Allard

    History of plant population genetics

    Annual Review of Genetics

    (1999)
  • J.C Alwine et al.

    Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes

    Proceedings of the National Academy of Sciences U.S.A.

    (1977)
  • E Babiychuk et al.

    Efficient gene tagging in Arabidopsis thaliana using a gene trap approach

    Proceedings of the National Academy of Sciences U.S.A.

    (1997)
  • D.E Bassett et al.

    Gene expression informatics—it's all in your mine

    Nature Genetics

    (1999)
  • M.D Bauer et al.

    Sequencing of sulfonic acid derivatized peptides by electrospray mass spectrometry

    Rapid Communications in Mass Spectrometry

    (2000)
  • R.C Beier

    Natural pesticides and bioactive components in food

    Reviews of Environmental Contamination and Toxicology

    (1990)
  • R.G Birch

    Plant transformationProblems and strategies for practical application

    Annual Review of Plant Physiology and Plant Molecular Biology

    (1997)
  • A Bovy et al.

    High-flavonol tomatoes resulting from heterologous expression of the maize transcription factor genes LC and C1

    Plant Cell

    (2002)
  • A.B Britt

    DNA damage and repair in plants

    Annual Review of Plant Physiology and Plant Molecular Biology

    (1996)
  • P.O Brown et al.

    Exploring the new world of the genome with DNA microarrays

    Nature Genetics

    (1999)
  • R Camerini-Otero et al.

    Homologous recombination proteins in prokaryotes and eukaryotes

    Annual Review of Genetics

    (1995)
  • L.A Castle et al.

    Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

    Molecular and General Genetics

    (1993)
  • M.T Clegg et al.

    The evolution of plant nuclear genes

    Proceedings of the National Academy of Sciences U.S.A.

    (1997)
  • Codex Alimentarius Commission, 2003. Draft Guideline for the Conduct of Food Safety Assessment of Foods Derived from...
  • H.J Cooper et al.

    Electrospray ionization Fourier transform mass spectrometric analysis of wine

    Journal of Agricultural and Food Chemistry

    (2001)
  • S.J Cordwell et al.

    Subproteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional electrophoresis gels

    Electrophoresis

    (2000)
  • P Costa et al.

    Genetic analysis of needle proteins in maritime pine2

    Variation of protein accumulation. Silvae Genetica

    (1999)
  • P Costa et al.

    Separation and characterization of needle and xylem maritime pine proteins

    Electrophoresis

    (1999)
  • P Costa et al.

    A genetic map of Maritime pine based on AFLP, RAPD and protein markers

    Theoretical and Applied Genetics

    (2000)
  • F Coulston et al.

    Biotechnologies and foodassuring the safety of foods produced by genetic modification

    Regulatory Toxicology and Pharmacology

    (1990)
  • S De Buck et al.

    Molecular Breeding

    (2000)
  • Dueck, Th.A., van der Werf, A., Lotz, L.A.P., Jordi, W., 1998. Methodological Approach to a Risk Analysis for Polygene-...
  • Engel, K.H., Gerstner, G., Ross, A., 1998. Investigation of glycoalkaloids in potatoes as example for the principle of...
  • European Commission, 1997. 97/618/EC: Commission Recommendation of 29 July 1997 Concerning the Scientific Aspects and...
  • Strategies for Assessing the Safety of Foods Produced by Biotechnology, Report of a Joint FAO/WHO Consultation

    (1991)
  • FAO/WHO, 2000. Safety Aspects of Genetically Modified Foods of Plant Origin. Report of a Joint FAO/WHO Expert...
  • J.D Faris et al.

    Saturation mapping of a gene-rich recombination hot spot region in wheat

    Genetics

    (2000)
  • O Fiehn et al.

    Metabolite profiling for plant functional genomics

    Nature Biotechnology

    (2000)
  • O Fiehn et al.

    Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry

    Analytical Chemistry

    (2000)
  • O Fiehn

    Combining genomics, metabolome analysis, and biochemical modeling to understand metabolic networks

    Comparative and Functional Genomics

    (2001)
  • O Fiehn

    Metabolomics—the link between genotypes and phenotypes

    Plant Molecular Biology

    (2002)
  • M Fladung

    Gene stability in transgenic aspen (Populus) I. Flanking DNA sequences and T-DNA structure

    Molecular and General Genetics

    (1999)
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