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[Genetically modified organisms--problems and legislation].

Genetically modified organisms are defined by law as entities capable of replication and/or transmission of hereditary material that had been altered by the insertion or removal of a DNA fragment. By the EU legal regulation as well as by the Czech law, such organisms are considered risky whereas other products of breeding, though obtained by, e.g., induced mutagenesis, are claimed as safe. Organisms transferred from other ecosystems are also considered safe. The Czech law on the use of genetically modified organisms is based on registers of users and organisms for specific use. Application for the registration that is valid as an approval should be submitted to the Ministry of Environment. The applicant is obliged to present the risk assessment of the particular use of genetically modified organisms. Genetically modified organisms are connected with certain risk to ecology, however health risks are brought about almost exclusively by microorganisms. Modified organisms used for food production are thoroughly tested for substantial equivalency with standard crops and with respect to health parameters of the protein(s) newly introduced due to genetic modification. Detail tests as well as their cost are close to the testing of new drugs. European as well as Czech rules for food labelling are motivated by the psychology of consumers rather than by health impact. They result to absurdities but do not meet the task of public psychology. This is why the EU authorities are looking for measures to change the present situation that other wise would bring Europe well behind the developed countries.

Czech Republic↗

[Examination of DNA extract from kernels and processed foods using silica-base resin].

A rapid and simple DNA extraction method is needed to detect genetically modified recombinant DNA in soybean kernels and processed foods. However, since various kernels and processed foods differ greatly in form, a uniform DNA extraction method has proved elusive. The silica-base resin DNA extraction method does not use any organic solvent, and the operation is simple and the cost per extraction is low, although the frequency of its use is very low and few domestic reports exist. We therefore studied suitable conditions for a silica-base resin method. We also developed the method to get more pure DNA from soybean kernels. The silica-base resin method was found to be adequate for extracting DNA from various processed foods for PCR amplification with endogenous gene primers. In the case of DNA extraction from soybean kernels, pure DNA could be efficiently extracted after pre-heating the soybean suspension in TNE buffer. The extracted DNA showed higher ratios of absorption at 260 nm/280 nm and 260 nm/230 nm than those for samples obtained with previous methods. Moreover, our observations suggested that the extraction time could be reduced to within 30 min for processed foods such as tofu.

DNA, Recombinant↗

Quantitation of transgenic Bt event-176 maize using double quantitative competitive polymerase chain reaction and capillary gel electrophoresis laser-induced fluorescence.

In this work, a new procedure useful to quantitatively analyze genetically modified organisms (GMOs) in foods is described and applied to analyze transgenic Bt Event-176 maize. The method developed consists of coamplifications of specific DNA maize sequences with internal standards using quantitative competitive PCR (QC-PCR). The QC-PCR products are quantitatively analyzed using a capillary gel electrophoresis (CGE) with laser-induced fluorescence detection (LIF) method developed at our laboratory that utilizes a physically adsorbed coating. The CGE-LIF procedure allows the use of internal standards differing by only 10 bp from the original target fragments, to our knowledge, the smallest size difference that can be found in the bibliography for QC-PCR of GMOs. A spectrofluorometric procedure using ROX reference dye is proposed to solve calibration problems of input DNA concentration. It is demonstrated that the use of ROX drastically enhances the accuracy of the quantitative analysis by QC-PCR. Reproducibility of analysis times and corrected peak areas (measured as target/competitor PCR products ratio) for the CGE-LIF separations are determined to be better than 0.91 and 1.93% (RSD, n = 15) respectively, for three different days. It is shown that CGE-LIF provides better resolution and a signal/noise ratio improvement of approximately 700-fold compared to slab gel electrophoresis. The good possibilities in terms of quantitative analysis of GMOs provided by this new method are confirmed by determining the Bt Event-176 maize content in certified reference maize powder and food samples of known composition. This procedure opens the possibility for accurate quantitation of multiple GMOs in a single run.

Bacterial Proteins↗

A rapeseed-specific gene, acetyl-CoA carboxylase, can be used as a reference for qualitative and real-time quantitative PCR detection of transgenes from mixed food samples.

Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA.

Acetyl-CoA Carboxylase↗

Effect of food components and processing parameters on DNA degradation in food.

The effect of food components on degradation of DNA by DNase I (EC 3.1.21.1) was monitored by electrotransformation of Escherichia coil, making it possible to determine the number of plasmid molecules capable of giving rise to transformed cells. The transformation frequency increased linearly with the plasmid number within the range of 2 x 10(6) to 2 x 10(10). DNA degradation was reduced by one order of magnitude in the presence of 0.05% (w.v(-1)) maltol or 1 mM putrescine. Complete inhibition of degradation was observed with > or = 0.2% (w.v(-1)) maltol, > or = 0.01% (w.v(-1)) octyl gallate or > or = 0.5 mM of spermine. To monitor degradation of plant DNA during food processing, a real-time PCR system was established. The ratio of copy numbers of a potato gbss DNA fragment of 325 bp and a nested 96 bp fragment was determined. The latter served as internal reference for normalization. The system made it possible to exclude process-dependent changes of DNA concentration in the food matrix. Processing of genetically modified potatoes to dried potato sticks, crisps or flakes was studied and drying steps were shown to exert the strongest effect on DNA degradation, resulting in a drop of the ratio from 0.73 to 0.16.

DNA↗

Labeling to manage marketing of GM foods.

Biotechnology has the potential to introduce new food safety risks, liabilities and benefits, and although privately managed supply chains (involving proactive management of the production of branded products) are effective at providing, managing and communicating adequate information about products with well understood risks, products with uncertain risks pose a greater challenge. The demand for increased product information regarding genetically modified content, in particular, places new constraints on food supply chains, frequently resulting in communication failures. Here we assess and reject mandatory labeling as an appropriate response.

Australia↗

[The safety and usefulness of transgenic plants].

Transgenic crop plants, used in food and feed production, carry different beneficial transgenes, mostly for resistance to pests, herbicides and diseases. All new transgenic plant varieties, the genes they carry and their products have been thoroughly tested before released for agriculture and even more for marketing. Genetically modified organisms carry the same risk as any other organism. Food derived from genetically modified organisms due to legal regulation is most controlled and therefore most safe food ever placed on the market. In future, transgenic plants offer many new possibilities also for medical use, like plant vaccines, antibiotics and rare proteins of clinical importance produced by plants.

Consumer Product Safety↗

[Animal models for assessment of GMO allergenicity: advantages and limitations].

Incidence of IgE-mediated allergic reactions to foods is increasing as well as the severity of associated symptoms and numerous foods are now incriminated, probably in relation with modifications of dietary habits and increased exposure to new or modified food ingredients. Therefore, the introduction on the market of food composed of or derived from genetically modified organisms (GMOs) raised the question of their potential allergenicity. Particularly with regards to the allergenicity of a newly expressed protein, it is necessary to obtain, from several steps in the risk assessment process, a cumulative body of evidence which minimises any uncertainty. This may include the use of animal model despite no fully reliable validated model is available yet. Such animal models should allow to address 3 major issues: Is the novel protein a sensitizer, i.e. does it possess intrinsic properties that allow to sensitize a predisposed individual? Is the protein an elicitor i.e. is it able to elicit an allergic reaction in a sensitised individual? And is the protein an adjuvant, i.e. can it facilitate or enhance the sensitisation to an other protein? Animal models under investigation currently include mice, rats and guinea pigs but models such as dogs and swine also appeared a few years ago. The aim is to mimic the mechanism and characteristics of the sensitisation phase and/or the elicitation phase of the allergic reaction as it occurs in atopic humans. They are necessary because sensitisation studies can obviously not be done in human and because in vitro tests cannot reproduce the complexity of the immune system. We propose a mouse model which mimics both phases of the allergic reaction. It has permitted to evidence that biochemical and clinical manifestations occuring during the active phases of the allergic reaction differ according to the structure of the allergen used for the challenge. This may allow to compare the allergenic potential of a genetically modified protein with that of the conventional one and to identify possible unintended effects. However, pathogenesis of food allergy in human is very complex and multifactorial, including individual differences in susceptibility, environmental factors, conditions of exposure, ... No animal model can take into account all these factors and allow a reliable prediction of the prevalence and severity of allergic reactions which would result from the exposure to a (novel) protein. Nevertheless, point by point analysis using the different models available may provide useful informations on the potential allergenicity of a novel protein.

Adjuvants, Immunologic↗

Assessment of the allergenic potential of foods derived from genetically engineered crop plants.

This article provides a science-based, decision tree approach to assess the allergenic concerns associated with the introduction of gene products into new plant varieties. The assessment focuses on the source from which the transferred gene was derived. Sources fall into three general categories: common allergenic food proteins; less common allergenic foods or other known allergen sources; and sources with no history of allergenicity. Information concerning the amino acid sequence identity to known allergenic proteins, in vitro and/or in vivo immunologic assays, and assessment of key physiochemical properties are included in reaching a recommendation on whether food derived from the genetically modified plant variety should be labeled as to the source of the transferred gene. In the end, a balanced judgement of all the available data generated during allergenicity assessment will assure the safety of foods derived from genetically engineered crops. Using the approaches described here, new plant varieties generated by genetic modification should be introduced into the marketplace with the same confidence that new plant varieties developed by traditional breeding have been introduced for decades.

Allergens↗

Risk assessment of meat and milk from cloned animals.

Research on, and commercialization of, cloned cattle has been conducted for more than 20 years. Early techniques relied on the physical splitting of embryos or using embryo cells for nuclear transfer to generate cloned animals. Milk and meat from these animals entered into the human food market with no evidence of problems. With the advent of nuclear transfer, which enables the direct transference and preservation of high-value meat- and milk-producing genotypes to offspring, concerns have been raised about whether the products from somatic cell nuclear transfer-produced animals are safe for human consumption. Studies on the biochemical properties of food products from cloned and noncloned animals have thus far not detected any differences. All data to date indicate no significant differences in the measured parameters between animals created by nuclear transfer and normally bred animals. Public acceptance of cloned animal products depends upon forthcoming US Food and Drug Administration approval along with convincing safety data.

Animals↗

Genetic technologies. Bioengineered food--safety and labeling.

The safety and labeling of genetically engineered foods are two areas that have elicited considerable public concern and debate. This Policy Forum provides a legal analysis of these issues in the context of two bills that have been recently proposed in The U.S. Congress, the Genetically Engineered Food Safety Act and the Genetically Engineered Food Right to Know Act. Most transgenic components of foods currently on the market are plant-incorporated protectants or their inert ingredients. Therefore, they have been evaluated for safety by the Environmental Protection Agency (as well as the Food and Drug Administration), and their disclosure in labeling should not be required. If plant-incorporated protectants are considered safer than chemical pesticides, and chemical pesticides do not have to be disclosed in labels, then bioengineered foods should not be subject to stricter regulation, nor should they be required to be labeled. The two bills are inconsistent, in many respects, with well-established principles of food regulation.

Animals↗

Real-time quantitative PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods.

A fast and quantitative method was developed to detect transgenic "Maximizer" maize "event 176" (Novartis) and "Roundup Ready" soybean (Monsanto) in food by real-time quantitative PCR. The use of the ABI Prism 7700 sequence detection system allowed the determination of the amplified product accumulation through a fluorogenic probe (TaqMan). Fluorescent dyes were chosen in such a way as to coamplify total and transgenic DNA in the same tube. Using real-time quantitative PCR, 2 pg of transgenic or total DNA per gram of starting sample was detected in 3 h after DNA extraction and the relative amounts of "Maximizer" maize and "Roundup Ready" soybean in some representative food products were quantified.

DNA↗

Random amplified polymorphic DNA analysis of genetically modified organisms.

Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products.

Food Analysis↗

Mechanisms of food allergy.

The prevalence of food allergy continues to rise, particularly in 'westernized' societies; it has been linked to the 'hygiene hypothesis' and the increased diversity of food consumption worldwide. The pathogenic mechanisms and Th1/Th2 paradigm are being closely examined with respect to the occurrence of inflammatory and injury/repair responses at different mucosal sites. Genetically modified plants as potential food sources and allergenicity are current topics of controversy.

Animals↗

Biosafety of E. coli beta-glucuronidase (GUS) in plants.

The beta-glucuronidase (GUS) gene is to date the most frequently used reporter gene in plants. Marketing of crops containing this gene requires prior evaluation of their biosafety. To aid such evaluations of the GUS gene, irrespective of the plant into which the gene has been introduced, the ecological and toxicological aspects of the gene and gene product have been examined. GUS activity is found in many bacterial species, is common in all tissues of vertebrates and is also present in organisms of various invertebrate taxa. The transgenic GUS originates from the enterobacterial species Escherichia coli that is widespread in the vertebrate intestine, and in soil and water ecosystems. Any GUS activity added to the ecosystem through genetically modified plants will be of no or minor influence. Selective advantages to genetically modified plants that posses and express the E. coli GUS transgene are unlikely. No increase of weediness of E. coli GUS expressing crop plants, or wild relatives that might have received the transgene through outcrossing, is expected. Since E. coli GUS naturally occurs ubiquitously in the digestive tract of consumers, its presence in food and feed from genetically modified plants is unlikely to cause any harm. E. coli GUS in genetically modified plants and their products can be regarded as safe for the environment and consumers.

Ecology↗