Safety evaluation of newly expressed proteins
If substantial equivalence can be established except for a single or few specific traits of the genetically modified plant, further assessment focuses on the newly introduced trait itself (EU, 1997b). Demonstration of the lack of amino acid sequence homology to known protein toxins/allergens, and a rapid proteolytic degradation under simulated mammalian digestion conditions, was deemed to be sufficient to assume the safety of the new protein (FAO/WHO, 1996). However, there may be circumstances that require more extensive testing of the new protein, such as (i) the specificity and biological function/mode of action of the protein is partly known or unknown; (ii) the protein is implicated in mammalian toxicity; (iii) human and animal exposure to the protein is not documented; or (iv) modification of the primary structure of naturally occurring forms. Bacterial Bt proteins are an example of proteins that have been introduced into crop varieties by genetic modification.
Bt proteins (Cry proteins) from Bacillus thuringiensis strains have been introduced into genetically modified crop plants for their insecticidal properties in the larvae of target herbivoral insect species (Peferoen, 1997). The working mechanism is based on specific receptor binding in susceptible insect larvae in epithelial cells of the midgut, leading to pore formation, cell lysis, disintegration of the epithelium lining in their midgut, and eventually to death of the larvae due to starvation. This type of biological action of the newly introduced protein directs further toxicity testing in mammals. A general drawback is that newly expressed pesticidal proteins, such as Bt toxins and lectins, are often present in the genetically modified plant at levels too low for extensive testing. Therefore, sufficient amounts of the new proteins are obtained from cultures of overexpressing bacterial strains. This carries the potential hazard that toxic impurities can be present, and that protein processing, like glycosylation, may be different in plants and bacteria. Therefore it is important to demonstrate that production in an alternative host does not result in differences in toxicity. For these pesticidal proteins, the following properties must be comparatively investigated: (i) electrophoretic behaviour of full-length as well as trypsinated forms; (ii) immunoreactivity with poly- and/or monoclonal antibodies; (iii) identical patterns of post-translation modification; (iv) sequence similarity; and (v) functional characteristics to target insect species.
The safety of a number of newly inserted proteins has been tested on a case-by-case basis (Table 3). It should be noted that for transgenic viral proteins in crops approved in Canada and the USA, their consumption has been assumed to be safe based on the history of ingestion of the wild-type plant viruses contained within plant foods.
Table 3. Toxicity studies of proteins expressed in commercialized genetically modified crops a
|Acetolactate synthase (Arabidopsis thaliana)||1|| || || || || || || || |
|12 : 0 Acyl carrier protein thioesterase (Umbellularia californica)||2||2||2|| || || || || || |
|1-Aminocyclopropane-1-carboxylic acid deaminase (Pseudomonas chloroaphis)||3||3|| || || || || || || |
|Barnase (Bacillus amyloliquefaciens)||4|| || || || || || || || |
|Barstar (Bacillus amyloliquefaciens)||4|| || || || || || || || |
|Beta-glucuronidase (Escherichia coli K12)||5||5||5|| || || || || || |
|Bromoxynil nitrilase (Klebsiella pneumoniae var. ozaenae)||6||7|| || || || || || || |
|Coat protein (cucumber mosaic virus)||8|| || || || || || || || |
|Coat protein (potato virus Y)||9|| || || || || || || || |
|Coat protein (watermelon mosaic virus 2)||8|| || || || || || || || |
|Coat protein (zucchini yellows mosaic virus)||8|| || || || || || || || |
|Cry1Ab endotoxin (Bacillus thuringiensis var. kurstaki)||10||11||12||13||11|| || ||11||11|
|Cry1Ac endotoxin (Bacillus thuringiensis var. kurstaki)||14||12||12|| || || ||15|| ||16|
|Cry1F endotoxin (Bacillus thuringiensis var. aizawai)||17||17||17|| || || || || || |
|Cry3A endotoxin (Bacillus thuringiensis var. tenebrionis)||18||12||12|| || || || || || |
|Cry9C endotoxin (Bacillus thuringiensis var. tolworthi)||13||13||13||13||13||13|| || ||13|
|5-Enolpyruvylshikimate-3-phosphate synthase (Agrobacterium sp. CP4)||19||19||19|| || || || || || |
|5-Enolpyruvylshikimate-3-phosphate synthase (Zea mays)||20||20||20|| || || || || || |
|Glyphosate oxidoreductase (Ochromobactrum anthropii LBAA)||21||21||21|| || || || || || |
|Neomycin phosphotransferase II (Escherichia coli Tn5)||4||22||22|| || || || || || |
|Phosphinothricin acetyltransferase (Streptomyces hygroscopicus, bar gene)||4||23||14|| || || || || || |
|Phosphinothricin acetyltransferase (Streptomyces viridochromogenes, pat gene)||24||23||25|| || || || || || |
|Replicase (potato leaf roll virus)||26|| || || || || || || || |
In the case of the Cry1Ab5 and Cry9C proteins, various studies have been performed on binding to tissues of the gastro-intestinal tract of rodents and primates, including humans (EPA, 2000a; Noteborn et al., 1995). There is no evidence for the presence of specific receptors in mammalian tissues for these proteins, nor are there indications of an amino acid sequence homology to known protein toxins/food allergens. A number of toxicity tests have been performed with respect to:
digestibility and stability in in vitro simulated gastric and intestinal fluids and in vivo models;
acute oral toxicity in a rodent species;
subchronic toxicity (30-day repeated-dose feeding) with focus on a tier I immunotoxicity screening.
Experiments performed with single and repeated dosing of the Cry proteins Cry1Ab5 and Cry9C, at levels up to 10 000 times those produced in genetically modified plants, did not indicate toxic effects in the rat, and histopathological analysis did not show binding of the Cry proteins to the intestinal epithelium of rodents and tissues of other mammals. In contrast to Cry1Ab5, Cry9C showed resistance to proteolysis under simulated human gastric conditions (pH > 2.0) and denaturation at elevated temperatures. On the other hand, it was noted that Cry9C degraded completely upon pepsin treatment at pH <1.5 (human ’fasting’ values). However, the digestibility of protein preparations under simulated conditions is of limited value, as questions can be raised as to whether these assays do mimic the physiological state of human beings.
In cases of (i) a completely novel gene; (ii) novel proteins as anti-nutrients; (iii) novel proteins without a clear threshold (bacterial toxins); (iv) predicted high levels of intake of toxic proteins such as protease inhibitors; and (v) non-rapidly degradable proteins, more extensive toxicity testing with the pure protein at exaggerated doses may be required.
Safety evaluation of whole genetically modified foods
Examples of feeding studies with whole genetically modified foods are summarized in Table 4. In the case of the Bt tomato experiment, a semi-synthetic rodent diet was supplemented with 10% (w/w) of lyophilized genetically modified or control tomato powder, and fed during 91 days. The average daily intake was approximately 200 g tomato day−1 per rat, corresponding to a daily human consumption of 13 kg. No clinical, toxicological or histopathological abnormalities were observed. The 10% (w/w) tomato content of the diet was chosen because of the relatively high potassium content of tomato (40–60 g kg−1), while higher amounts could have caused renal toxicity (Noteborn and Kuiper, 1994).
Table 4. Toxicity studies performed with genetically modified food crops a
|Cottonseed||Bt endotoxin (Bacillus thuringiensis)||rat||28 days||body weight||Chen et al. (1996)|
| Maize|| Cry9C endotoxin (Bacillus thuringiensis var. tolworthi)|| human|| ||feed conversion histopathology of organs blood chemistry reactivity with sera from maize-allergic patients|| EPA (2000e)|
|Potato||lectin (Galanthus nivalis)||rat||10 days||histopathology of intestines||Ewen and Pusztai (1999)|
|Potato||Cry1 endotoxin||mouse||14 days||histopathology of intestines||Fares and El Sayed (1998)|
|(Bacillus thuringiensis var. kurstaki HD1)|| || || || |
|Potato||glycinin (soybean, Glycine max)||rat||28 days||feed consumption body weight blood chemistry blood count organ weights liver- and kidney- histopathology ||Hashimoto et al. (1999a) Hashimoto et al. (1999b)|
|Rice||glycinin (soybean, Glycine max)||rat||28 days||feed consumption body weight blood chemistry blood count organ weights liver- and kidney- histopathology ||Momma et al. (2000)|
|Riceb||phosphinothricin acetyltransferase (Streptomyces hygroscopicus)||mouse, rat||acute and 30 days||feed consumption body weight median lethal dose blood chemistry organ weight histopathology||Wang et al. (2000)|
|Soybean GTS 40-3-2||CP4 EPSPS (Agrobacterium)||rat, mouse||105 days||feed consumption body weight histopathology of intestines and immune system serum IgE and IgG levels||Teshima et al. (2000)|
|Soybean GTS 40-3-2 Soybean GTS 40-3-2 Soybean Tomato||CP4 EPSPS (Agrobacterium) CP4 EPSPS (Agrobacterium) 2S albumin (Brazil nut, Bertholetta excelsa) Cry1Ab endotoxin (Bacillus thuringiensis var. kurstaki)||human rat human rat|| 150 days 91 days||reactivity with sera from soybean-allergic patients blood chemistry urine composition hepatic enzyme activities reactivity with sera from Brazil nut-allergic patients feed consumption body weight organ weights blood chemistry histopathology||Burks and Fuchs (1995) Tutel'yan et al. (1999) Nordlee et al. (1996) Noteborn et al. (1995)|
|Tomato||antisense polygalacturonase||rat||28 days||feed consumption||Hattan (1996)|
|(tomato, Lycopersicon esculentum)|| || ||body weight organ weights blood chemistry histopathology || |
Fares and El Sayed (1998) reported that mice fed for 14 days on fresh potato immersed in a suspension of delta-endotoxin of B. thuringiensis var. kurstaki strain HD1 developed an increase of hyperplastic cells in their ileum. Feeding with fresh genetically modified potato expressing the cry1 gene caused mild adverse changes in the various ileac compartments, as compared to the control group on fresh potato. The occurrence of these effects in mice fed either ’spiked’ potato or genetically modified potato may have been due to the toxicity of the Cry1 protein; however, no details were given on the intake of Cry1 protein or on dietary composition, which limits interpretation of this study.
Following the short-term safety assessment of transgenic potato and rice with native and designed soybean glycinin (four additional methioninyl residues), Hashimoto et al. (1999a); Hashimoto et al. (1999b) and Momma et al. (2000) demonstrated that a daily administration of 2.0 g potato and 10 g rice kg−1 body weight to rats for 4 weeks indicated neither pathological nor histopathological abnormalities in liver and kidney.
The experiments reported by Ewen and Pusztai (1999) indicated, according to the authors, that rats fed genetically modified potato containing GNA lectin showed proliferative and antiproliferative effects in the gut. These effects are presumed to be due to alterations in the composition of the transgenic potato, rather than to the newly expressed gene product; however, various shortcomings of this study, such as the protein deficiency of the diets and the lack of control diets, make the results difficult to interpret (Kuiper et al., 1999). Similar criticisms have been made by the UK’s Royal Society (Royal Society, 1999).
Teshima et al. (2000) fed Brown Norway rats and B10A mice with either heat-treated genetically modified soybean meal containing the cp4-epsps gene, or control non-genetically modified soybean meal. These experimental animals were employed based on their immunosensitivity to oral challenges. The semi-synthetic animal diet was supplemented with 30% (w/w) heat-treated soybean meal, and fed over 105 days. Both treatments failed to cause immunotoxic activity or to cause the IgE levels to rise in the serum of rats and mice. Moreover, no significant abnormalities were observed histopathologically in the mucosa of the small intestine of animals fed either genetically modified or non-genetically modified soybean.
In addition to the feeding studies described above, studies have been performed on domestic animals fed genetically modified crops to establish performance (feed conversion; Table 5). It is apparent that no harmonized design exists yet for feeding trials in animals to test the safety of genetically modified foods.
Table 5. Performance studies on animals fed genetically modified crops a
|Canola GT73, meal||herbicide resistant||quail||weight increase feed consumption mortality ||5 days||ANZFA (2000a)|
|Canola GT73, meal||herbicide resistant||trout||weight increase||70 days||ANZFA (2000a)|
|Maize GA21, kernel||herbicide resistant||broiler chicken||weight increase feed consumption fat pads ||40 days||Sidhu et al. (2000)|
|Maize CBH351, kernel||insect resistant||broiler chicken||weight increase feed consumption breast muscle weight fat pads weight mortality ||42 days||EPA (2000f)|
|Maize, kernel||herbicide resistant||swine||feed conversion||8 days||Böhme and Aulrich (1999)|
|Maize Bt176, kernel||insect resistant||broiler chicken||weight increase feed consumption organ weights ||41 days||Brake and Vlachos (1998)|
|Maize Bt176, kernel||insect resistant||broiler chicken||feed consumption feed conversion ||35 days||Aulrich et al. (1999)|
|Maize Bt176, kernel||insect resistant||laying hen||feed consumption egg production feed conversion ||10 days||Aulrich et al. (1999)|
|Maize Bt176, silage||insect resistant||sheep||feed conversion||?||Aulrich et al. (1999)|
|Maize Bt176, silage||insect resistant||beef steer||weight increase feed conversion meat yield ||246 days||Aulrich et al. (1999)|
|Soybean||herbicide resistant||lactating cow||body weight||29 days||Hammond et al. (1996b)|
|GTS 40-30-2, raw|| || ||milk production milk composition dry matter digestibility ruminal fluid composition || || |
|Soybean||herbicide resistant||broiler chicken||weight increase||42 days||Hammond et al. (1996b)|
|GTS 40-30-2, meal|| || ||feed consumption breast muscle weight fat pads weight mortality || || |
|Soybean||herbicide resistant||channel catfish||weight increase||70 days||Hammond et al. (1996b)|
|GTS 40-30-2, meal|| || ||feed consumption filet composition || || |
|Soybean, meal||high oleic acid||swine||weight increase feed consumption ||17 days||ANZFA (2000d)|
|Soybean, meal||high oleic acid||broiler chicken||weight increase feed consumption ||18 days||ANZFA (2000d)|
|Sugar beet, beet||herbicide resistant||swine||feed conversion||8 days||Böhme and Aulrich (1999)|
The potential allergenicity of newly introduced proteins in genetically modified foods is a major safety concern. This is true in particular for genetic material obtained from sources with an unknown allergenic history, such as the soil bacterium B. thuringiensis. An illustrative case of a genetically modified food for which the allergenic risk has to be assessed is maize in which the truncated Cry9C protein (MW 68 kDa) is expressed, and which has been allowed as a transgene product in StarLink yellow maize for animal feed in the USA (EPA, 2000a). It should be noted that the protoxin Cry9C from B. thuringiensis var. tolworthi has been modified at residue 164 by substituting the arginine residue with lysine to increase serine protease resistance in the field (Lambert et al., 1996). Recent investigations have found traces of the Cry9C gene and/or protein in taco shells (CNN, 2000; EPA, 2000a). The Cry9C protein has also been detected in maize seeds of a non-StarLink variety or in maize from such seeds (FDA, 2000). This has raised the issue of potential allergenicity of the genetically modified maize for humans. Cry9C might be a potential allergen because the protein shows some characteristics of known food allergens: (i) an MW of 68 kDa; (ii) relative resistance to gastric proteolytic degradation and to heat and acid treatment; (iii) it is probably a glycoprotein; (iv) induces a positive IgE response in the Brown Norway rat, and is a high IgE responder on intraperitoneal and oral sensitization, in contrast to the related Cry1Ab5 protein; and (v) may be found intact in the bloodstream after oral feeding in a rat model. On the other hand, Cry9C has no amino acid sequence homology to any known allergen or protein toxin, and wild-type and StarLink maize protein extracts have been demonstrated to be indistinguishable in their reactivities towards sera of maize-allergic and major food-allergic patients. Furthermore, no immunogenic/toxic effects were observed in a 30-day repeated-dose study in mice with Cry9C (EPA, 2000b), and the bioavailability of the protein in the rat is relatively low (0.0002–0.0006%), which reduces the likelihood of sensitization.
Levels of Cry9C in maize-derived food products appear to be much less than the >1% level apparently characteristic of food allergens (10–80%). Post-harvest blending and mixing may have diluted the Cry9C protein in food products to the p.p.b. range for the harvest years 2000 and 1999. Maize in food channels is either wet-mill processed, which produces high-fructose corn syrup, glycose, dextrose, starch or oil; or dry-milled, which produces primarily cereals, flour and meal. A preliminary study using Cry9C ELISA well tests showed that there was no intact Cry9C protein in a limited number of starch samples (EPA, 2001). In this study no other wet-milling products were assayed, and the ELISA was not validated for detection of Cry9C in starch. In general, the protein fraction goes to feed use (FIFRA SAP, 2000b). Upon dry-milling, the Cry9C protein content is reduced by 40%. Additional processing, such as alkaline cooking (masa production), decreases the protein content to 0.1–0.2% of the original Cry9C protein (FIFRA SAP, 2000b). This suggests a further reduction in allergenic potency; however, protein denaturation by heat or partial proteolysis may uncover new allergenic epitopes (FIFRA SAP, 2000b; Hefle, 1996). It is therefore important to note the need for reproducible, validated methods for analysing Cry9C protein levels in processed foods and intermediates, as distinct from the PCR methods (CDC, 2001a; EPA, 2001). The estimated duration of exposure to Cry9C is uncertain, but may have been too short to promote sensitization and induce allergenic reactions.
After the media (CNN, 2000) reported the inadvertent introduction of StarLink maize into the food supply, some consumers reported adverse health effects consistent with allergic reactions after eating maize products, or from another cause (FIFRA SAP, 2000b). Subsequently the FDA, with the assistance of the Centers for Disease Control and Prevention (CDC), evaluated 28 consumer complaints linked to foods allegedly containing StarLink maize. Analysis by ELISA revealed, however, that the banked serum samples did not contain Cry9C-specific IgE antibody (CDC, 2001b).
Although reassuring, these follow-up studies of FDA/CDC's reported putative illnesses linked to StarLink maize are not conclusive as yet. The FDA's IgE-specific ELISA did not include the StarLink-derived Cry9C protein, but the recombinant Cry9C expressed in Escherichia coli as antigen. Consequently, it is possible that epitopes present on Cry9C in maize may not be present in the non-glycosylated E. coli-derived protein. It is also recognized that a specific goat antiserum against Cry9C was included in the ELISA, as there was no human serum available that contained the IgE antibody to Cry9C. The result is that the possibility of lack of specificity for human anti-Cry9C IgE cannot be entirely dismissed (CDC, 2001b; CDC, 2001c). The StarLink yellow maize case highlights the difficulty that there can be no final proof as to whether Cry9C is, or is not, a food allergen.
An example of a transgene from an allergenic source is that of the Brazil nut (Bertholetta excelsa) 2S albumin expressed in soybean. This protein is rich in methionine, and would therefore increase the nutritive value of soybean for animal feed. It was found, however, that the transgenic protein was reactive towards sera from patients who were allergic to Brazil nut, and the further development of this soybean was halted (Nordlee et al., 1996).
Detection and characterization of unintended effects
Upon random insertion of specific DNA sequences into the plant genome (intended effect), the disruption, modification or silencing of active genes or the activation of silent genes may occur, which may result in the formation of either new metabolites or altered levels of existing metabolites. Unintended effects may be partly predictable on the basis of knowledge of the place of the transgenic DNA insertion, the function of the inserted trait, or its involvement in metabolic pathways; while other effects are unpredictable due to the limited knowledge of gene regulation and gene–gene interactions (pleiotropic effects). It should be emphasized that the occurrence of unintended effects is not specific for genome modification through recDNA technology – it also occurs frequently in conventional breeding. Unintended effects may be identified by an analysis of the agronomical/morphological characteristics of the new plant and an extensive chemical analysis of key nutrients, anti-nutrients and toxicants typical for the plant. Limitations of this analytical, comparative approach are the possible occurrence of unknown toxicants and anti-nutrients, in particular in food plant species with no history of (safe) use; and the availability of adequate detection methods.
Examples of unexpected secondary effects due to either somaclonal variations, pleiotropic effects or genetic modification, which may be of biological or agronomic importance to the plant, are illustrated in Table 6. Some of these alterations would indicate that the experimental, genetically modified plant does not possess the appropriate properties to allow further development into a commercial crop plant. Others would be identified only through appropriate field trials (e.g. soybean; Gertz et al., 1999). In order to identify potential secondary effects of the genetic modification, which would result in alterations in the composition of genetically modified crops, different strategies may be applied, for example the targeted (compound-specific) approach, or the non-targeted (profiling/fingerprinting) approach.
Table 6. Unintended effects in genetic engineering breeding a
|Host plant||Trait||Unintended effect||Reference|
|Canola||overexpression of phytoene-synthase||multiple metabolic changes (tocopherol, chlorophyll, fatty acids, phytoene)||Shewmaker et al. (1999)|
|Potato||expression of yeast invertase||reduced glycoalkaloid content||Engel et al. (1998)|
|Potato||expression of soybean glycinin||increased glycoalkaloid content (+16–88%)||Hashimoto et al. (1999a); Hashimoto et al. (1999b)|
|Potato||expression of bacterial levansucrase||adverse tuber tissue perturbations;||Turk and Smeekens (1999);|
|impaired carbohydrate transport in the phloem||Dueck et al. (1998)|
|Rice||expression of soybean glycinin||increased vitamin B6-content||Momma et al. (1999)|
|Rice||expression of provitamin A biosynthetic pathway||formation of unexpected carotenoid derivatives (beta-carotene, lutein, zeaxanthin)||Ye et al. (2000)|
|Soybean||expression of glyphosphate (EPSPS) resistance||higher lignin content (20%) at normal soil temperatures (20°C); splitting stems and yield reduction (up to 40%) at high soil temperatures (45°C)||Gertz et al. (1999)|
|Wheat||expression of glucose oxidase||phytotoxicity||Murray et al. (1999)|
|Wheat||expression of phosphatidyl serine synthase||necrotic lesions||Delhaize et al. (1999)|
Targeted approach using single compound analysis
For any given transformation event, targeted studies should include baseline analyses of a number of key nutrients such as proteins, carbohydrates, fats, vitamins and other nutritional/anti-nutritional compounds which, if unintentionally modified, might affect nutritional value and safety. Selection of key nutrients and toxicants needs to take into account the target species, structure and function of the inserted gene(s), and possible interferences in metabolic pathways (Figure 3). Selection of compounds may be limited to a restricted number which represents essential biochemical/physiological pathways in the organism. It is plausible, but not proven, that expected changes in the metabolism as a possible result of the genetic modification will be identified by analysis of a great number of components, but unexpected changes are merely identified by chance. The targeted approach has severe limitations with respect to unknown anti-nutrients and natural toxins, especially in less well known crops.
Non-targeted approach using profiling methods
An alternative (non-targeted) approach for the detection of unintended effects is the use of so-called profiling techniques. New methods are being developed which allow for the screening of potential changes in the physiology of the modified host organism at different cellular integration levels: at the genome level; during gene expression and protein translation; and at the level of metabolic pathways. Many factors, such as genetic characteristics (cultivar, individual, isogenic lines, heterosis); agronomic factors (soil, fertilizers, plant protection products); environmental influences (location effect, weather, time of day, stress); plant–microbe interactions; maturity stage; and post-harvest effects determine the morphological, agronomic and physiological properties of a food crop. Screening for potential changes in these characteristics in genetically modified plants becomes more important as the newer genetic alterations changing agronomical or nutrition-related properties are more complex, involving insertion of large DNA fragments or clusters of genes.
Localization and characterization of the place(s) of insertion are the most direct approaches to predicting and identifying possible occurrence of (un-) intended effects due to transgene insertion in recipient-plant DNA. Data for transgene flanking regions will give leads for further analysis, in the case of a transgene insertion within or in the proximity of an endogenous gene. Transgene chromosomal location and structure can be detected by various methods such as genomic in situ hybridization (Iglesias et al., 1997) and fluorescence in situ hybridization (Pedersen et al., 1997), and by direct sequencing of flanking DNA (Spertini et al., 1999; Thomas et al., 1998). Knowledge of plant genomes is still limited, including the reliability of annotations in genomic databases, but the understanding of the genomic code and the regulation of gene expression in relation to the networks of metabolic activity is increasing. Therefore, the sequencing of the place of insertion(s) will become increasingly informative.
Gene expression analysis
The DNA microarray technology is a powerful tool to study gene expression. The study of gene expression using microarray technology is based on hybridization of mRNA to a high-density array of immobilized target sequences, each corresponding to a specific gene. mRNAs from samples to be analysed are labelled by incorporation of a fluorescent dye and subsequently hybridized to the array. The fluorescence at each spot on the array is a quantitative measure corresponding to the expression level of the particular gene. The major advantage of the DNA microarray technology over conventional gene profiling techniques is that it allows small-scale analysis of expression of a large number of genes at the same time, in a sensitive and quantitative manner (Schena et al., 1995, 1996). Furthermore, it allows comparison of gene-expression profiles under different conditions. The technology and the related field of bioinformatics are still in development, and further improvements can be anticipated (Van Hal et al., 2000).
The potential value of the application of technology for the safety assessment of genetically modified food plants is currently under investigation (E.J.K., unpublished results). The tomato is used as a model crop. To study differences in gene expression, two informative tomato expressed-sequence-tag (EST) libraries are obtained, one consisting of ESTs that are specific for the red stage of ripening, and the other for the green, unripe stage. Both EST libraries are spotted on the array and, in addition, selected functionally identified cDNAs, selected on the basis of their published sequence. The array is subsequently hybridized with mRNAs that are isolated from a number of different genetically modified varieties under investigation, as well as with the parent line and control lines. Preliminary results show that reproducible fluorescence patterns may reveal altered gene expression outside the ranges of natural variation, due to different stages of ripening (Figure 4). Prospects are that this method may effectively be used to screen for altered gene expression and, at the same time, provide initial information on the nature of detected alterations, whether the observed alteration(s) may affect the safety or nutritional value of the food crop under investigation.
Figure 4. The microarray technology is currently used to develop a non-biased system for the detection of altered gene expression in genetically modified crop plant varieties in comparison to the traditional parent line.
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Correlation between mRNA expression and protein levels is generally poor, as rates of degradation of individual mRNAs and proteins differ (Gygi et al., 1999). Therefore, understanding of the biological complexities in the plant cell can be expanded by exploiting proteomics, a technique that analyses many proteins simultaneously and will contribute to our understanding of gene function. Particularly, recent developments in mass spectrometry have increased the applicability of two-dimensional gel electrophoresis in the studies of complex protein mixtures. Proteomics can be divided into three main areas: (i) identification of proteins and their post-translational modifications; (ii) ’differential display’ proteomics for quantification of the variation in contents; and (iii) studies of protein–protein interactions.
The method most often used for analysing differences in protein pattern is sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), followed by excision of protein spots from the gel, digestion into fragments by specific proteases, and subsequently analysis by mass spectrometry (peptide mass fingerprinting). It allows the identification of proteins by comparing the mass of peptide fragments with data predicted by genetic or protein sequence information. Other much faster technologies, such as protein chip-based (microarray) approaches, are under development (MacBeath and Schreiber, 2000; Pandey and Mann, 2000). In addition, major technical hurdles remain to be overcome: proteins may constantly change in their secondary, tertiary and quaternary structures, depending on transfer and expression in different tissues and cellular compartments, which may profoundly influence their electrophoretic behaviour and molecular mass.
When searching for unintended changes by 2-D PAGE, the first step is to compare proteomes of the lines under investigation. If differences in protein profiles are detected, normal variations should be evaluated. If the profiles are outside normal variations, identification of the protein must be carried out, which may lead to further toxicological studies. Moreover, metabolic changes may be looked at if the identified protein has a known enzymatic activity.
There is one example of the use of proteomics to study alterations in the composition of a genetically modified plant, which illustrates that a targeted change in the level of a specific protein can result in other proteins being affected. The improvement in rice storage proteins by antisense technology resulting in low-glutelin genetically modified rice for commercial brewing of sake has been associated with an unintended increase in the levels of prolamins (FAO/WHO, 2000b). This would not have been detected by standard analyses such as total protein and amino acid profiling, but was observed only following SDS–PAGE.
Machuka and Okeola (2000) used 2-D PAGE for the identification of African yam bean seed proteins. Prominently resolved polypeptide bands showed sequence homology with a number of known anti-nutrient and inhibitory proteins, which may have implications for the safe use of these seeds as human food.
A multi-compositional analysis of biologically active compounds in plants – nutrients, anti-nutritional factors, toxicants and other relevant compounds (the so-called metabolome) – may indicate whether intended and/or unintended effects have taken place as a result of genetic modification. The three most important techniques that have emerged are gas chromatography (GC), high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). These methods are capable of detecting, resolving and quantifying a wide range of compounds in a single sample. For instance, metabolic profiling of isoprenoids by an HPLC method was described recently with applications to genetically modified tomato and Arabidopsis (Fraser et al., 2000). The potential of GC as a metabolic profiling method for plants was demonstrated some 10 years ago (Sauter et al., 1991), and GC/MS has been established as the most versatile and sensitive profiling method in the past 2 years following its systematic development by Roessner et al. (2000); Fiehn et al. (2000a); Fiehn et al. (2000b). Recently, it has been shown that the use of chemical fingerprinting techniques such as off-line LC-NMR may provide information on possible changes in plant matrices due to variations in environmental conditions (Lommen et al., 1998). Determination of a chemical fingerprint was based on the detection of alterations in 1H-NMR spectra obtained from different water and organic solvent extracts from genetically modified tomato varieties, such as the antisense RNA exogalactanase fruit, and from their non-modified counterpart(s) (Noteborn, 1998; Noteborn et al., 1998; Noteborn et al., 2000). Differences in concentration of low molecular weight components (MW < 10 kDa) could be traced by subtraction of the 1H-NMR spectra.
Application of these techniques will provide more detailed information on possible changes than can be obtained from single-compound analysis. Once differences have been identified, further safety evaluation of the observed differences may be needed by specific in vitro and/or in vivo testing. The design of such experiments will focus on the differences observed with the profiling methods. However, a number of problems must be addressed before such methods can be used on a routine basis: (i) standardization of sample collection, preparation and extraction procedures; (ii) standardization and validation of measurements; (iii) limited availability of data on profiles and natural variations; and (iv) lack of bioinformatic systems to treat large data sets.
Currently, different methods are tested for the detection and characterization of unintended effects as a result of genetic modification. Within an EU project, GMOCARE (QLK1-1999-00765; http://www.rikilt.wageningen-ur.nl/euprojects/euprojects.html), the above-mentioned approaches are exploited, including functional genomics, proteomics and metabolite profiling.
Assessment of marker genes
The most commonly used marker genes are those that code for resistance to herbicides or antibiotics (Table 7). The use of herbicide-resistant genes can be twofold: for selection purposes; and/or for altering the agronomic characteristics of a plant. In particular, the use of antibiotic-resistance genes is subject to controversy and intense debate, because of the risk of transfer and expression in bacteria which could compromise the clinical or veterinary use of certain antibiotics. Risk assessment of selectable marker genes with respect to the consumption by humans and animals of genetically modified foods or feed should focus, as with any new gene transfer, on micro-organisms residing in the gastro-intestinal tract of humans and animals, on the toxicity and allergenicity of newly expressed proteins, and on the impact of horizontal gene transfer. Health aspects of marker genes have been dealt with by, among others, WHO (1993); the Nordic Council (Karenlampi, 1996); FAO/WHO (1996); FAO/WHO (2000b). There is general agreement that transfer of antibiotic resistance genes from plants to micro-organisms residing in the human gastro-intestinal tract is unlikely to occur, given the complexity of steps required for gene transfer, expression, and impact on antibiotic efficacy (FAO/WHO, 1996). Under conditions of selective pressure (i.e. oral therapeutic use of the corresponding antibiotic), a selectable marker may provide selective advantage to the recipient micro-organism.
Table 7. Antibiotic- and herbicide-resistance genes commonly present in commercial- and field-tested genetically modified crops a
|Gene||Gene product||Antibiotic||Gene source|
|nptII||neomycin phosphotransferase II||kanamycin, neomycin, geneticin (G418), paromomycin, amikacin||Escherichia coli, transposon Tn5|
|bar||phosphinothricin acetyltransferase||glufosinate, l-phosphinothricin, bialaphos||Streptomyces hygroscopicus|
|pat||phosphinothricin acetyltransferase||glufosinate, l-phosphinothricin, bialaphos||Streptomyces viridochromogenes|
|bla||beta-lactamase||penicillin, ampicillin||Escherichia coli|
|aadA||aminoglycoside-3′-adenyltransferase||streptomycin, spectinomycin||Shigella flexneri|
|hpt||hygromycin phosphotransferase||hygromycin B||Escherichia coli|
|nptIII||neomycin phosphotransferase III||amikacin, kanamycin, neomycin, geneticin (G418), paromomycin||Streptococcus faecalis R plasmid|
|cp4 epsps||5-enoylpyruvate shikimate-3-phosphate synthase||glyphosate||Agrobacterium CP4|
|epsps||5-enoylpyruvate shikimate-3-phosphate synthase||glyphosate||Zea mays, Petunia hybrida, Arabidopsis thaliana|
|gox||glyphosate oxidoreductase||glyphosate||Achromobacter LBAA|
|bxn||bromoxynil nitrilase||bromoxynil||Klebsiella pneumoniae var. ozaenae|
|als||acetolactate synthase||sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidylbenzoates||Arabidopsis thaliana, Nicotiana tabacum, Brassica napus|
Transfer of plant DNA to microbial or mammalian cells would require the following steps (FAO/WHO, 2000b):
release of specific genes in the plant DNA;
survival of the gene(s) under gastro-intestinal conditions (plant, bacterial, mammalian nucleases);
competitive uptake of the gene(s);
recipient bacteria or mammalian cells must be competent for transformation, and gene(s) must survive restriction enzymes; insertion of the gene(s) into the host DNA by rare repair or recombination events.
There are no data available indicating that marker genes in genetically modified plants transfer to microbial or mammalian cells. Transfer and expression of plant genes in bacteria have been observed under laboratory conditions, and only when homologous recombination was possible (Nielsen et al., 1997). This would imply that an antibiotic resistance-marker gene is introduced from plants into bacteria only if the same gene or other genes with identical sequences were present in the bacteria. Model experiments with mice indicated the transfer of bacterially derived DNA fragments into mouse cells (Schubbert et al., 1998). These results have been criticized, along with others, regarding possible artefacts created during the analysis of foreign insertions in leukocyte DNA (Beever and Kemp, 2000). A relevant consideration for the assessment of horizontal gene transfer, if it occurs, is the consequences of the transfer. Information must be available on the role of the antibiotic in human and veterinary use, its specific therapeutic spectrum, existing resistance levels in the environment, and possible alternatives for treatment of diseases.
The 2000 FAO/WHO Consultation concluded: ‘For certain antibiotic resistance genes currently in use in genetically modified plants, available data suggest that consequences of horizontal gene transfer will be unlikely to pose a significant threat to the current therapeutic use of the respective drugs. With other genes that confer resistance to drugs that are important in specific medical use, or to drugs that have limited alternative therapies, the possibility of transfer and expression of these genes is a risk that warrants their avoidance in the genomes of widely disseminated GMOs and foods and food ingredients’ (FAO/WHO, 2000b). It then goes on: ‘In future developments, the Consultation encourages the use of alternative transformation technologies, if available and demonstrated to be safe, that do not result in antibiotic resistance genes in genetically modified foods. If further development of alternative technologies is required, additional research should be strongly encouraged’.
Non-antibiotic (alternative) marker genes such as tryptophane decarboxylase, β-glucuronidase and xylulose/phosphomannose isomerase should be evaluated according to the characteristics of the newly encoded proteins and metabolites formed as result of enzymatic reactions. Furthermore, the risks of the presence of multiple markers and of multiple copies of markers should be evaluated. In one example, the isopentenyl transferase (ipt) gene for plant hormone production (cytokines) allows modified cells to form shoots when cultured in dexamethasone-enriched media after the modification event (Kunkel et al., 1999). Another way is to use the xylA gene, which encodes xylose isomerase, enabling the genetically modified plant cell to grow in cultures with the sugar xylose added, which is normally toxic to the plant cells. Novartis, for example, has commercialized the manA gene as ‘Positech’, which encodes phosphomannose isomerase, that allows plant cells to be sustained in media containing mannose-6-phosphate (Joersbo et al., 1998).
In addition, methods have been developed to excise genes after successful introduction, such as the CreLox system in which Cre is an enzyme that removes the stretch of DNA flanked by the Lox sequences (Gleave et al., 1999). In a recent version of the CreLox system, both the antibiotic selection marker and the Cre recombinase gene were contained between the Lox sequences of the vector DNA that was introduced into plants. After successful transformation, expression of the recombinase gene was induced, and the marker and recombinase genes were subsequently removed by the recombinase (Zuo et al., 2001).
New models for safety testing, detection of unintended effects, gene transfer, detection and traceability of genetically modified foods are currently under development in the EU-funded research and technology development (RDT) projects SAFOTEST (QLK1-1999-00651); GMOCARE (QLK1-1999-00765); GMOBILITY (QLK1-1999-00527); and Qpcrgmofood (QLK1-1999-01301), clustered in the Thematic Network ENTRANSFOOD (http://www.entransfood.nl).