Unintended effects and their detection in genetically modified crops☆
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)
- et al.
Functional genomics by mass spectrometry
FEBS Letters
(2000) - et al.
The distribution of T-DNA in the genomes of transgenic Arabidopsis and rice
FEBS Letters
(2000) - et al.
Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of SI endonuclease-digested hybrids
Cell
(1977) - et al.
Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionisation tandem mass spectrometry
Analytical Biochemistry
(2001) - et al.
Genetic engineering of crops as potential source of genetic hazard in the human diet
Mutation Research
(1999) - et al.
Cloning and expression of a wheat (Triticum aestivum) phosphatidylserine synthase cDNA. Overexpression in plants alters the composition of phospholipids
Journal of Biological Chemistry
(1999) Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures
Progress in Nuclear Magnetic Resonance Spectroscopy
(1996)- et al.
Factors influencing cDNA microarray hybridisation on silylated glass slides
Analytical Biochemistry
(2002) The introduction and expression of transgenes in plants
Current Opinion in Biotechnology
(1998)- et al.
How plants make ends meetDNA double-strand break repair
Trends in Plant Science
(1999)
Flavonoid and flavonol glycoside metabolism in Arabidopsis
Plant Physiology and Biochemistry
Recombinationa frank view of exchanges and vice versa
Current Opinion in Cell Biology
Recent advances in the transformation of plants
Trends in Plant Science
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
Simultaneous analysis of the majority of low-molecular-weight, redox active compounds from mitochondria
Analytical Biochemistry
Pattern recognition methods and applications in biomedical magnetic resonance
Progress in NMR Spectroscopy
Nontargeted metabolome analysis by use of Fourier Transform Ion Cyclotron Mass Spectrometry
OMICS
History of plant population genetics
Annual Review of Genetics
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.
Efficient gene tagging in Arabidopsis thaliana using a gene trap approach
Proceedings of the National Academy of Sciences U.S.A.
Gene expression informatics—it's all in your mine
Nature Genetics
Sequencing of sulfonic acid derivatized peptides by electrospray mass spectrometry
Rapid Communications in Mass Spectrometry
Natural pesticides and bioactive components in food
Reviews of Environmental Contamination and Toxicology
Plant transformationProblems and strategies for practical application
Annual Review of Plant Physiology and Plant Molecular Biology
High-flavonol tomatoes resulting from heterologous expression of the maize transcription factor genes LC and C1
Plant Cell
DNA damage and repair in plants
Annual Review of Plant Physiology and Plant Molecular Biology
Exploring the new world of the genome with DNA microarrays
Nature Genetics
Homologous recombination proteins in prokaryotes and eukaryotes
Annual Review of Genetics
Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis
Molecular and General Genetics
The evolution of plant nuclear genes
Proceedings of the National Academy of Sciences U.S.A.
Electrospray ionization Fourier transform mass spectrometric analysis of wine
Journal of Agricultural and Food Chemistry
Subproteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional electrophoresis gels
Electrophoresis
Genetic analysis of needle proteins in maritime pine2
Variation of protein accumulation. Silvae Genetica
Separation and characterization of needle and xylem maritime pine proteins
Electrophoresis
A genetic map of Maritime pine based on AFLP, RAPD and protein markers
Theoretical and Applied Genetics
Biotechnologies and foodassuring the safety of foods produced by genetic modification
Regulatory Toxicology and Pharmacology
Molecular Breeding
Strategies for Assessing the Safety of Foods Produced by Biotechnology, Report of a Joint FAO/WHO Consultation
Saturation mapping of a gene-rich recombination hot spot region in wheat
Genetics
Metabolite profiling for plant functional genomics
Nature Biotechnology
Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry
Analytical Chemistry
Combining genomics, metabolome analysis, and biochemical modeling to understand metabolic networks
Comparative and Functional Genomics
Metabolomics—the link between genotypes and phenotypes
Plant Molecular Biology
Gene stability in transgenic aspen (Populus) I. Flanking DNA sequences and T-DNA structure
Molecular and General Genetics
Cited by (0)
- ☆
All authors contributed equally to the paper