PART III PROCESS SAFETY
1. Genetically Modified Organisms
Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms defines GMO as an organism, with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination.
Within the terms of this definition techniques of genetic modification are:
i. recombinant nucleic acid techniques involving the formation of new combi- nations of genetic material by the insertion of nucleic acid molecules produced by whatever means outside an organism, into any virus, bacterial plasmid or other vector system and their incorporation into a host organism in which they do not occur but in which they are capable of continued propagation
ii. methods involving the direct introduction into an organism of heritable mate- rial prepared outside the organism including micro-injection, macro-injection and micro-encapsulation
iii. cell fusion (including protoplast fusion) or hybridisation techniques where live cells with new combinations of heritable genetic material are formed through the fusion of two or more cells by means of methods that do not occur naturally.
In the Cartagena Protocol on Biosafety to the Convention on Biological Diversity instead of GMO (genetically modified organism) the term Living Modified Organism (LMO) was introduced. This is defined as any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology. The modern biotechnology means the application of:
1Dezider Tóth, PhD., DSc, Slovak Agricultural University of Nitra, Tr.A.Hlinku 2, SK- 94976 NITRA, Slovak Republic, E-mail: [email protected]
i. in vitro nucleic acid techniques, including recombinant DNA and direct injec- tion of nucleic acid into cells or organelles, or
ii. fusion of cells beyond the taxonomic family, that overcome natural physio- logical reproductive or recombinant barriers and that are not techniques used in traditional breeding and selection.
Both definitions are fairly similar in meaning, even though the approach is dif- ferent. To consider the potential impact of genetically modified organisms on environment and human health, a sound understanding of the biology of an organ- ism is required, as well as its relationship and interactions with the environment and other organisms. The most important information focused on those biologi- cal attributes used for evaluation of GMOs safety are compiled by OECD in the so called Consensus Documents on the biology of several crop plant species, trees and microorganisms (such as OECD 2002). These documents deal with many important issues in assessing safety, including genetic characteristics, reproduc- tive biology, data concerning the origin and biodiversity, characteristics on pests, diseases and ecology.
Genetically modified organisms have already entered the food stream in many parts of the world (FAO, 2005). Various species of microorganisms, mostly bacteria and fungi, have been modified for increased production of pro- teins, amino acids and commercial chemicals (Tourte, 2003). Early work in this area relied primarily on discovery of naturally occurring or by mutagenesis- induced variant microbial strains. Often these variant genotypes were blocked in specific metabolic pathways, or they expressed higher levels of a key rate- limiting enzyme, with the result that their metabolic output was being chan- nelled into the desired product. Such mutants provided valuable biological tools for researchers, and for the fermentation industry they also represented a key commercial asset.
However, the current generation of GMOs consists mostly of plants modified for a limited number of traits. With the expected increase in the availability of genomic information for many species in the next years, the floodgates of genetic modifications could open and release an unprecedented variety of genetically modified products onto the market. In parallel with this rapid market penetration, there is increasing concern about the use of genetic engineering for food produc- tion, particularly about possible deleterious effects on human health and about the possible impacts of the widespread deployment of GMOs in the environment.
Plants play a critical role in global life as they are the base of the food chain.
Most animals consume plant material as food, although some animals are carni- vores, dependent on plant-eating animals. Microorganisms can also utilize either living or decaying plants as source of essential substrates. Pathogenic microor- ganisms may induce a hypersensitive reaction in plants or a systematically acquired resistance to pathogens. Along with complex interactions inside of plant kingdom (competition for light, space and nutrients) there are additional interac- tive relations between plants and pests, herbivores, pollinating insects or seed- dispersing animals.
It is generally accepted that the plants play a number of roles in the environ- ment and may affect other organisms through different direct and indirect mech- anisms. The risk/safety assessor, in considering the safety of transgenic plants, should evaluate the potential for disruption of these interactions. Applications of crop genetic engineering could be subdivided into several groups:
i. Transformation for insect resistance – This is important in field and horticul- tural crops as well as plantation trees. The most common modification involves use of genes for protein toxins from the soil bacterium Bacillus thuringiensis (Bt). The Bt plants are insecticidal to a limited range of specific caterpillars and beetles like European corn borer (Bt maize), bollworm and budworm (Bt cotton) and Colorado potato beetles (Bt potatoes). Another example involves the transformation of peas with a kidney bean amylase inhibitor gene to develop a source of resistance to pea weevils. A cowpea gene has also been used in strawberries to confer weevil resistance.
ii. Transformation with plant quality genes – This is important for both food and feed production. The first quality-enhanced product on the market was tomatoes with a reduced level of the polygalacturonase gene product. The lower activity of this enzyme is responsible for slowing down the long pectin chains cleaving in the cell walls of the fruit so that the ripe fruit does not soften so quickly and remains for firm for a longer time. Another case is canola with a thio-esterase transgene from a bay laurel tree prevents the synthesis of longer chain fatty acids and thus enabling formation of high lauric acid in the oil (an useful raw material in detergent manufacture). The third example that applies to a range of horticul- tural crops (potatoes, apple, lettuce, bananas, grapes, lettuce, pineapple), is pre- vention of damage-induced browning by blocking polyphenoloxidase genes.
iii. Transformation for disease resistance –The transformation for virus resist- ance is well developed. Transgenic plants containing various parts of a viral genome can be protected against a virus. For example, the expression of viral coat protein genes in the target plant, as in tomato and tobacco plants resist- ant to mosaic virus, potatoes resistant to potato leaf roll virus (PLRV) and white clover resistant to alfalfa mosaic virus.
iv. Transformation for herbicide tolerance – The development of crop resistance to a herbicide is an important economical and environmental achievement of weed control. Commercially-used transgenic herbicide-resistant crops are maize, soy- beans, cotton and canola. These are resistant to the broad-spectrum herbicides such as glyphosate (Roundup®) and glufosinate (Basta®). Herbicide-tolerant pasture crops, such as subterranean clover, enable farmers to control broadleaf pasture weeds earlier in the growing season with low levels of herbicide.
v. Transformation for nutritional purposes – Examples of other transformations include enhanced lysine content in maize or enhanced vitamin A content in canola. Edible vaccines, for viral and diarrhoeal diseases, using proteins expressed in transgenic plants is extremely important for developing coun- tries. The vaccine is expressed in the fruit or vegetable and even multiple vac- cines could be produced in one plant.
1.1. GMO: new biotechnology products
Biotechnology, broadly defined, includes any technique that uses living organisms, or their parts, to make or modify products, to improve plant or animals, or to develop microorganisms for specific use. It ranges from traditional biotechnology to the most advanced modern biotechnology (Doyle and Persley, 1996).
Commercial biotechnology consists of an expanding range of interrelated tech- niques, procedures and processes for practical applications in the health care, agri- cultural and industrial sectors. The background of biotechnology is formed by a mixture of scientific disciplines – biochemistry, cell biology, embryology, genet- ics, microbiology and molecular biology combined with practical disciplines such as chemical engineering, information technology and robotics. From the synergy of these building blocks are born the desired results – new thoughts, new products.
Biotechnology should be seen as an integration of new techniques emerging from modern biotechnology with well established approaches of traditional biotechnol- ogy, such as plant and animal breeding, food production, fermentation process and production of pharmaceuticals, biopesticides and fertilizers.
Taking into account the historical progressive aspects, biotechnology consists of a gradient of technologies, ranging from the long-established and widely used techniques and traditional processes like fermentation to novel techniques of modern biotechnology. As shown by Persley (1990), these technologies started with the oldest microbial fermentations; these were then followed by plant tissue cultures, embryo transfer in animals, monoclonal antibody production up to recombinant DNA technology used for genetic engineering of microorganisms, plants and animals.
One of the major issues relating to the role and application of biotechnology in agriculture is the safety of organisms with novel traits and the appropriate regu- latory measures for research and development, field testing, marketing of organ- isms with novel traits and their use as food or feed. There is fear that uncontrolled introduction of genetically modified organisms might cause undesirable effects on ecological or genetic relationships in some communities or potential harm to some consumers in the food chain. Therefore organisms with novel traits should be carefully designed and their introduction into the environment or the mar- ket allowed only after a proper testing.
Both the traditional and the modern plant breeding have the potential to alter the nutritional value of plants and their products or lead to unexpected or unintended changes in concentration of various natural toxicants or antinutrients (Table.1).
It is important to evaluate all new varieties in order to reduce the likelihood that unexpected changes will produce adverse health effects. Unintended changes in levels of nutrients can theoretically arise in several ways:
i. Insertion of genetic material could disrupt or alter the expression of normally expressed plant genes.
ii. Expression of the introduced gene resulting in a protein synthesis might reduce the availability of amino acids used for synthesis of normal plant compounds.
iii. Production of normal plant compounds might also be affected if the expressed protein diverted substrates from other important metabolic path- ways.
iv. Finally, either the expressed protein or altered levels of other proteins might have antinutritional effects. These possible concerns are related to the random- ness of DNA insertion. However, changes in gene expression can also occur when traditional breeding method are used; such changes may be less frequent in transgenic plants since only a limited number of genes are transferred during the genetic modification.
Food safety should consider the potential for any change in nutritional compo- sition, especially in key elements that have a significant impact on the diet, as well as the potential for any change in the bioavailability of key nutritional com- ponents. Where additional assurance of safety is needed, analytical methods tra- ditionally applied in the evaluation of food constituents such as total protein, fat, ash, fibre and micronutrients may be supplemented with additional analyses to identify unexpected effects.
Because of the potential for broad changes in nutrient levels and interactions with other nutrients and unexpected effects, it may be necessary in certain instances to undertake feeding studies with animals to determine outcomes that result from changes in nutrient profiles and nutrient bioavailability. Nutritional modifications which are within the normal range of nutrient variation might require a less extensive evaluation than those outside normal ranges.
In GM plants that were not developed to have intentionally altered nutritional value, the aim_ of the nutritional evaluation is to demonstrate that there have been no unintentional changes in the levels of key nutrients, natural toxicants or anti- nutrients, or in the bioavailability of the nutrients. In this case, food substitution TABLE1. Plant toxins and antinutrients in some common crop plants
Crop Toxin/antinutrient
Rape (Brassica napus, B. rapa) Glucosinonales
Erucic acid Phytate
Maize (Zea mays) Phytate
Tomato (Lycopersicon esculentum) Alpha-tomatine
Solanine Chaconine Lectins Oxalate
Potato (Solanum ruberosum) Solanine
Chaconine Protease inhibitors Phenols
Soybean (Glycine max) Protease inhibitors
Lectins Isoflavones Phytate
using products from the genetically engineered plant should not adversely affect the health or nutritional status of the consumer. Implications for the population as a whole and for specific subgroups like children and/or the elderly should be considered.