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In this article we will discuss about the production of BD foods with allergenic properties.
Current techniques of developing organisms used in the production of BD foods typically involve the transfer to the host of the desired gene or genes in combination with a promoter and a gene for a selectable marker trait that allows the efficient isolation of cells or organisms that have been transformed from those that have not.
Common selectable markers in plants have included resistance to antibiotics or herbicides. Several key issues have been raised with respect to the potential toxicity associated with BD foods, including the inherent toxicity of the transgenes and their products, and unintended effects resulting from the insertion of the new genetic material into the host genome.
Unintended effects of gene insertion might include an over-expression by the host of inherently toxic or pharmacologically-active substances, silencing of normal host genes, or alterations in host metabolic pathways.
It is important to recognise that, with the exception of the introduction of marker genes, the process of genetic engineering does not, in itself, create new types of risk. Most of the hazards listed above are also inherent in conventional breeding methods.
The guiding principle in the evaluation of BD foods by regulatory agencies in Europe and the USA is that their human and environmental safety is most effectively considered relative to comparable products and processes currently in use. From this arises the concept of “substantial equivalence.”
If a new food is found to be substantially equivalent in composition and nutritional characteristics to an existing food, it can be regarded as being as safe as the conventional food and does not require extensive safety testing.
Evaluation of substantial equivalence includes consideration of the characteristics of the transgene and its likely effects within the host, and measurements of protein, fat and starch content, amino acid composition and vitamin and mineral equivalency together with levels of known allergens and other potentially toxic components.
BD foods can either be substantially equivalent to an existing counterpart, substantially equivalent except for certain defined differences, or be non-equivalent, which would mean that more extensive safety testing might be necessary.
The examination of substantial equivalence therefore may only be the starting point of the safety assessment. It provides a valuable guide to the definition of potential hazards from BD foods and illuminates necessary areas for further study.
While there is some concern relative to the meaning of “substantial” and how equivalency should be established, and debate over its use continues, the concept appears to be logical and robust in assessing the safety of foods derived from both genetically-modified plants and microorganisms.
If it can be established with reasonable certainty that a BD food is no less safe than its conventional counterpoint, it provides a standard likely to be satisfactorily protective of public health. It is also an approach that has the flexibility to evolve in concert with the field of transgenic technology.
Humans typically consume a minimum of 0.1 to 1 gram of DNA in their diet each day. Therefore, the transgene in a genetically engineered plant is not a new type of material to our digestive systems and it is present in extremely small amounts. In transgenic corn, the transgenes represent about 0.0001% of the total DNA. Decades of research indicate that dietary DNA has no direct toxicity itself.
On the contrary, exogenous nucleotides have been shown to play important beneficial roles in gut function and the immune system. Likewise, there is no compelling evidence for the incorporation and expression of plant-derived DNA, whether a transgene or not, into the genomes of consuming organisms.
Defense processes have evolved, including extensive hydrolytic breakdown of the DNA during digestion, excision of integrated foreign DNA from the host genome, and silencing of foreign gene expression by targeted DNA methylation that prevent the incorporation or expression of foreign DNA.
Although much remains to be learned about the fate of dietary DNA in mammalian systems, the possibility of adverse effects arising from the presence of transgenic DNA in foods either by direct toxicity or gene transfer is minimal.
The potential toxicity of the transgene product must be considered on a case-by-case basis. Particular attention must be paid if the transgene produces a known toxin (such as the Bacillus thuringiensis (Bt) endotoxins) or a protein with allergenic properties.
The level of risk of these gene products to consumers and those involved in food production can be and is evaluated by standard toxicological methods. The toxicology testing for the Bt endotoxins typifies this approach and has been described in detail by USEPA. The safety of most Bt toxins is assured by their easy digestibility as well as by their lack of intrinsic activity in mammalian systems.
In this case, the good understanding of the mechanism of action of Bt toxins, and the selective nature of their biochemical effects on insect systems, increases the degree of certainty of the safety evaluations. However, each new transgenic product must be considered individually based on exposure levels and its potency in causing any toxic effects, as is typical of current risk assessment paradigms for chemical agents.
Allergenicity is one of the major concerns about food derived from transgenic crops. However, it is important to keep in mind that eating conventional food is not risk- free; allergies occur with many known and even new conventional foods. For example, the kiwi fruit was introduced into the U.S. and the European market in the I960′ s with no known human allergies; however, today there are people allergic to this fruit.
The issues that have to be addressed regarding the potential allergenicity of BD foods are:
i. Do the products of novel genes have’ the ability to elicit allergic reactions in individuals who are already sensitized to the same, or a structurally similar, protein?
ii. Will transgenic techniques alter the level of expression of existing protein allergens in the host crop plant?
iii. Do the products of novel genes engineered into food plants have the ability to induce de novo sensitization among susceptible individuals?
Considerable scientific resources are being committed to determine the most appropriate and accurate approaches for identifying and characterizing potentially allergenic proteins.
The first systematic approach to allergenicity assessment was developed by the International Life Sciences Institute (ILSI) in collaboration with the International Food Biotechnology Council and published in 1996. The hierarchical approach described therein has been reviewed and revised by the World Health Organisation (WHO) and the Food and Agriculture Organisation of the United Nations (FAO).
The main approaches currently used in the evaluation of allergenicity are:
1. Determinations of structural similarity, sequence homology and serological identity:
The objective is to determine whether, and to what extent, the novel protein of interest resembles other proteins that are known to cause allergy among human populations. There are essentially three generic approaches. The first is to examine the overall structural similarity between the protein of interest and known allergens.
The second is to determine, using appropriate databases, whether the novel protein is similar to known allergens with respect to either overall amino acid homology, or with respect to discrete areas of the molecule where complete sequence identity with a known allergen may indicate the presence of shared epitopes.
The third approach is to determine whether specific IgE antibodies in serum drawn from sensitized subjects are able to recognise the protein of interest.
2. Assessment of proteolytic stability:
There exists a good, but incomplete, correlation between the resistance of proteins to proteolytic digestion and their allergenic potential, the theory being that relative resistance to digestion will facilitate induction of allergic responses provided the protein possesses allergenic properties.
One approach therefore is to characterize the susceptibility of the protein of interest to digestion by pepsin or in a simulated gastric fluid. However, this approach alone may not be sufficient to identify cross- reactive proteins with the potential to elicit allergic responses in food- or latex-sensitized individuals as in the case of oral allergy syndrome or latex-fruit syndrome.
Nor are considerations of stability to digestion necessarily relevant for allergens that act through dermal or inhalation exposure and that may have significance for worker health. In these cases, other approaches such as structural homology searches and the use of animal models may be effective in identifying potential new allergens.
3. Use of animal models:
Currently there are available no widely accepted or thoroughly evaluated animal models for the identification of protein allergens. Nevertheless, progress is being made and methods based on the characterisation of allergic responses or allergic reactions in rodents and other species have been described.
Although testing strategies for allergens are still evolving and no single test is fully predictive of human responses, when used in combination, allow scientists to address questions of potential allergenicity and these will increase in precision and certainty with time.
Considerations of this type led the US federal agencies to deny approval of Star-Link corn for human consumption because of the possibility that its Bt protein, Cry9C, may be a human allergen.
This protein had been modified to slow its digestion and prolong its effect in the insect gut and this change rendered the protein less digestible in the human gut as well. After the accidental introduction of Star-Link corn into the human food chain, a limited number of illnesses among consumers were reported.
These were investigated by the centres for Disease Control who found no evidence that the corn products were responsible. The only documented case where a human allergen was introduced into a food component by genetic engineering occurred when attempts were made to improve the nutritional quality of soybeans using a Brazil nut protein, the methionine-rich 2S albumin.
Allergies to the Brazil nut have been documented, and while still in pre-commercial development, testing of these new soybeans for allergenicity was conducted in university and industrial laboratories. It was found that serum from people allergic to Brazil nuts also reacted to the new soybean. Once this was discovered, further development of the new soybean variety was halted and it was never marketed.
Concern has been expressed about the randomness with which genes are inserted into the host by current genetic engineering processes. This could, and does, result in pleiotropic and insertional mutagenic effects.
The former term refers to the situation where a single gene causes multiple changes in the host phenotype and the latter to the situation where the insertion of the new gene induces changes in the expression of other genes.
Such changes due to random insertion might cause the silencing of genes, changes in their level of expression or, potentially, the turning on of existing genes that were not previously being expressed. Pleiotropic effects could be manifested as unexpected new metabolic reactions arising from the activity of the inserted gene product on existing substrates or as changes in flow rates through normal metabolic pathways.
Unexpected and potentially undesirable pleiotropic or mutagenic changes in the genome of the host do occur, but these would likely be revealed by their effects on the development, growth or fertility of the host, or by the extensive testing of its chemical composition compared with isogenic untransformed plants which is a necessary part of any safety evaluation of transgenic crops.
In the US, since 1987, the USDA Animal and Plant Health Inspection Service has completed over 5000 field trials with more than 70 different transgenic plant species. The only unexpected result was a mutation in a colour gene and gene silencing through changes in the methylation status of these genes that led to unexpected colour patterns in petunia flowers.
Both of these effects are also seen in conventional plant breeding. While the possibility of an undetected increase in a toxic component in a new food cannot be entirely eliminated, the current safeguards make this unlikely and no toxicologically or nutritionally significant changes of this type are evident in the transgenic plants so far marketed for food production.
Substantial public concern about the safety of BD products was raised in 1989 when a number of cases of eosinophilia-myalgia syndrome (EMS) were reported among users of the amino acid tryptophan as a dietary supplement. By mid-1993, 37 deaths had been attributed to this outbreak.
The development of the syndrome appeared among users of some batches of the supplement after a change in the manufacturing process that included the use of a new genetically modified microorganism in the fermentation. However, concomitant with this change were additional alterations in certain filtration and purification steps used previously in the manufacturing process.
The exact cause of the outbreak and the nature of the toxic impurity have not been established with certainty. Thus, it is not possible to determine whether the change in purification, the genetic engineering of the organism, or some other factor or factors were to blame.
A subsequent investigation revealed that cases of EMS occurred among consumers of tryptophan before the GM organism was introduced into the manufacturing process, although at a lower incidence.
The genetic modifications might have caused an increase in the level of the agent which was responsible for tryptophan-associated EMS, but it did not create a novel toxicant. This event is troubling in that the tryptophan would be regarded as highly purified and no adequate animal model has been found to replicate EMS, a probable autoimmune disease.
This illustrates that toxicology has limits in its ability to explain and predict adverse effects in humans. These examples indicate that careful analysis of the changes in BD organisms is necessary to ensure against unexpected alterations in the levels of toxins, allergens and essential nutrients.
This will be particularly critical if, as seems likely, engineering of the synthetic pathways of secondary metabolites is undertaken in plants e.g. to increase their resistance to insects and pathogens or produce compounds of pharmaceutical value.
Such changes might create new and unanticipated secondary compounds with unknown toxic properties. New approaches to profiling changes in metabolites, proteins, and gene expression may be helpful in such cases.
The development of antibiotic resistance among pathogenic bacteria is a significant human health issue. However, no contribution to antibiotic resistance in gut bacteria arising from antibiotic resistance markers in BD foods has been documented.
For several reasons, including the efficient destruction of the resistance gene in the human gut and the very low intrinsic rate of plant-microbe gene transfer, any contribution from this source is expected to be extremely small.
Genes for resistance to kanamycin and related antibiotics already occur quite commonly in the environment including the flora of the human gut which naturally contains about 1 trillion kanamycin- or neomycin-resistant bacteria. Even if the occasional transfer of resistance from plant to bacterium did occur, the practical impact would be negligible.
However, since any increase in antibiotic resistance is recognised as undesirable, and the technology is now available to omit the use of such marker genes, future genetically-modified organisms are unlikely to contain them. Thus concerns related to their use are likely to diminish.
In the USA, the FDA is entrusted with assuring that the nutritional composition of BD foods is substantially equivalent to that of the non-modified food. Studies are performed to determine whether nutrients, vitamins and minerals in the new food occur at the same level as in the conventionally-bred food sources. A typical example is the case of Roundup Ready soybeans.
In this case, the protein, oil, fibre, ash, carbohydrates and moisture content and the amino acid and fatty acid composition in seeds and toasted soybean meal were compared with conventional soybeans.
Fatty acid compositions and protein or amino acid levels of soybean oil were compared and special attention was given to checking the levels of anti-nutrients typically found in soybeans, e.g., trypsin inhibitors, lectins and iso-flavones.
One difference between the conventional and non- conventional soybeans was detected in defatted, non- toasted soybean meal, the starting material for commercially utilised soybean protein which is not itself consumed. In this material, trypsin inhibitor levels were 11-26% higher in the transgenic soybeans.
The levels of the trypsin inhibitors were similar in all lines in the seeds and in defatted, toasted soybean meal, the form used in foods. Except for this difference in trypsin inhibitor levels, all other nutritional aspects were equivalent between the transgenic line and the conventional soybean cultivars.
Feeding studies demonstrated that there were no evident differences in nutritional value between the conventional and transgenic soybeans in rats, chickens, catfish and dairy cattle. Domestic animal feeding studies with a number of other transgenic crops have similarly shown no significant adverse changes in nutritional value.
In addition to the general concerns addressed that relate to food safety, additional attention is needed when the gene product is pesticidal or otherwise may be toxic to non-target organisms that consume it.
The effects of each transgene product that is designed for pesticidal effects must be evaluated on a case-by-case basis against target and non-target organisms under specific field growth conditions for each transgenic crop.
The foremost current example of this is the incorporation of Bt genes into crop plants for insect control. The toxic properties of Bt endotoxins to both target and non-target species of many kinds are well known. They show a narrow range of toxicity limited to specific groups of insects, primarily Lepidoptera, Coleoptera or Diptera, depending on the Bt strain.
Nevertheless, Bt-producing plants have been tested broadly to determine whether any alteration in this limited spectrum of toxicity has occurred, without the discovery of any unexpected results. Exotoxins and enterotoxins, which are much more broadly toxic than the endotoxins, are also produced by some Bt strains, but these are not present in the transformed plant because their genes are not transferred into the crop.
In plants transformed with Bt genes to control lepidopterans, toxicity to non-target lepidopterans would be expected if exposure occurs by feeding on the transformed crop. Particular concern has been expressed over the potential toxicity of the Bt toxin in corn pollen to the Monarch butterfly after initial laboratory studies showed increased mortality in larvae fed on leaves dusted with transgenic pollen.
However, most transgenic corn pollen contains much lower, non-lethal levels of Bt toxins than the strain used and there is only a limited synchrony between the feeding period of the most sensitive younger larvae and the period when corn pollen is shed. Also, corn pollen does not typically move far beyond the borders of the field, leaving significant amounts of milkweed uncontaminated in many locations.
It is unlikely that a substantial risk to these butterflies exists in the field since only a negligible portion of the population is exposed to toxic levels of Bt. Beyond the question of the potential toxicity of Bt corn to such valued insects, it is also important to recollect that the common alternative is to spray corn with synthetic insecticides, which are not as selective as the Bt toxin.
In a sweet corn field containing milkweed plants and treated with a synthetic pyrethroid for insect control, 91-100% of the monarch butterfly larvae placed on the milkweed leaves after spraying were killed. In plots where Bt sweet corn was planted and the pollen fell naturally on the milkweed leaves, larval death rates were much lower (7-20%) and indistinguishable from those in untreated non-Bt corn plots.