Perr, Hilary A.
LIMITED RESOURCES TO MEET THE NEEDS OF A GROWING POPULATION
The global population is expected to increase from 6 billion to 9 to 10 billion by the mid 21st century (1). Even now, 800 million people are malnourished (2), including half the world's children (3). Globally, the most profound micronutrient deficiencies include vitamin A, iron, iodine, and vitamin E (4). The earth's fertile lands are nearly fully cultivated and the water supply is finite, yet the world will need 80% more cereal crops than were produced in 1990 within 25 years (5). The increased demand for nutrients worldwide must be accomplished while simultaneously conserving the environment.
Over the millennia, various forms of biotechnology have been employed to cultivate crops with desired traits and increased yield. The difference now is that biotechnology encompasses not only traditional crossbreeding but also genetic engineering. Genetic engineering uses recombinant DNA technology to transfer genes by nonsexual procedures from one organism to another. Genes can be taken from one organism (e.g., bacteria) and transferred into a completely different organism, such as a plant. Such technology has already been used in microorganisms to make human insulin. Gene transfer is accomplished by several means. One method of gene transfer involves coating microscopic metal particles with the desired DNA and accelerating the particles directly into plant cells with a particle gun. An alternative method involves inserting the gene of interest into the DNA of a bacterial or yeast plasmid which serves as a molecular vehicle to then infect plant cells. These plasmid-bearing bacteria can be used to transfer recombinant DNA into host plant cells which then inserts into the plant genome. As in traditional crossbreeding, gene insertion is random. Transfer of the recombinant DNA into the plant cell by either coated particles or by plasmid insertion is followed by plant cell division eventually yielding a plant with the transferred trait.
Genetically engineered plants differ from crossbred plants in several ways. Genes for desired traits can be obtained not only from related species, but from unrelated species, thus allowing the expression of novel proteins or changes in the levels of endogenous proteins. Bioengineered plants are developed more efficiently. The development of a new and successful plant variety by traditional plant breeding techniques may take 5 to 7 years (6). For example, the development of canola oil required nearly 15 years. In contrast, the development of new, bioengineered oilseed crops took less than half that time (7). Bioengineering is much more precise than cross breeding. Instead of transferring many thousands of genes, a highly selected few can be transferred by bioengineering techniques. Bioengineered plants can be designed to contain several new and desirable properties at once. Species may be developed which are not only less susceptible to climatic variation, but also grow in poor soil and resist pests. Genetically engineered plants which can grow without fertilizers or pesticides result in a reduction in the generation of greenhouse gases which accompany fertilizer uses and less environmental pollution associated pesticides (1).
The first genetically modified crops were introduced in the early 1990s. Some of the common genetically modified food crops are soybeans, corn, cotton, canola, potatoes, and tomatoes. Transgenic crops have rapidly become prevalent. Worldwide, one half of the soybean crops and one third of the corn crops are transgenic species (8). In the USA, 30 to 50% planted soybeans and corn are transgenic while about two thirds of processed food contain transgenic ingredients. Early crops were developed to resist insects and disease, and to tolerate climate and herbicides. Recently traits that confer consumer benefit such as improved taste, longer shelf life, increased nutritional value, and reduced toxicity are being selected.
Genetically modified foods are currently being developed to both improve nutrition and prevent disease in children. Examples of genetically modified foods that could impact children's health include: 1) vitamin and iron fortified rice which reduce common deficiency states, 2) designer oils from oilseed crops which decrease disease risk, and 3) fruits which deliver edible vaccines. Concerns have been expressed regarding potential allergenicity, increased anti-nutrient levels, and potential gene transfer from modified plants to bacteria or humans. The ensuing overview focuses on potential applications and risks to children's health of bioengineered foods.
RICE TO PREVENT AND TREAT VITAMIN A AND IRON DEFICIENCIES
Deficiencies of Vitamin A and Iron Cause Global Morbidity and Mortality
Deficiencies of vitamin A and iron remain among the most serious nutritional disorders worldwide, despite intensive efforts since the 1990 World Summit for Children. Infants, children, and women of reproductive age are the most vulnerable. Vitamin A is necessary for vision, reproductive function, bone growth, epithelial cell integrity, and differentiation. Worldwide, inadequate vitamin A causes blindness in 250 million children each year among whom 500,000 cases are irreversible (9). The severity of ocular lesions such as keratomalacia is inversely proportional to age (10). Some 3.7 billion people have iron deficiency, half of whom are anemic (11). In developing countries, 30 to 60% of women and children are anemic. (An excellent review of public health approaches to iron deficiency is found in reference 12). Fifty percent of the world's one-year-old children are iron deficient, either with frank anemia or with biochemical evidence of deficiency (13). In the United States, iron deficiency affects 5% young children, and 5 to 10% women of childbearing age (14). The resulting low–birth-weight infants of iron-deficient mothers are at increased risk of hemorrhage, sepsis, and death during childbirth. Postnatal iron deficiency anemia impairs subsequent cognitive development and immune function.
Local Food Sources Do Not Provide Adequate Dietary Vitamin A or Iron
Most provitamin A rich crops are seasonal and thus of limited availability. Even in season, such crops can be compromised by environmental stresses, such as temperature and water supply (15). Once harvested, the nutritional value of such crops drops further with storage, home processing, and cooking (16). Neither medicinal nor fortified food vitamin A supplementation has proven sustainable. Such programs have been undermined by irregular supplies, difficulty of administering proper doses, difficulty determining frequency and timing of supplementation, identification of appropriate foods to fortify, high cost, and difficulty reaching high-risk individuals (15). The bioavailability and utilization of vitamin A is also compromised by low fat and protein intake, and concomitant deficiencies of vitamin E and zinc.
Iron absorption varies from less than 1% to greater than 20% depending on the food source (12). Iron is better absorbed from meat than from dairy products or vegetable sources (17). However, meat intake in developing countries is low. Iron bio-availabity is further diminished by inhibitors of iron uptake present in local plant-based diets containing phytate, polyphenols, and fiber. Lack of compliance has undermined the effectiveness of iron supplementation (18). In Romania, a UNICEF infant supplementation program only reduced the prevalence of anemia from 69% to 45%. In the United States, one third of low-income women are anemic in the third trimester despite supplement availability (19).
Food fortification has been used for other nutritional deficiencies when supplementation has failed. Pellagra is rare since grains have been fortified with niacin. Water fluoridation has reduced dental caries. In the U.S., dairy products are fortified with vitamins A and D. Iodine deficiency has been ameliorated with iodinated salt where it can be distributed. These measures have been most successful where foods are commercially distributed by centralized industry. Unfortunately, food fortification is less practical in developing nations where the diet is usually locally produced (20).
Rice Was Genetically Modified to Contain More Vitamin A and Iron
For much of the world, rice is a food staple and so is a viable food vehicle. As currently grown, rice is low in vitamin A and iron. Bioengineering can be used to improve the quality of locally produced rice, by genetically enhancing its vitamin A and iron content. The technique requires careful selection of vehicle plant as well as the metabolic form of the desired nutrient.
Dietary vitamin A occurs in two forms: retinol (preformed vitamin A) and carotenoids (provitamin A). Retinol is a 20-carbon compound found in organ meats, fish oils, flesh meats, cream, milk, cheese, egg, butter, and margarine. Carotenoids are 40-carbon compounds which, when cleaved and reduced, yield vitamin A. β-carotene is one of the most potent carotenoids from the standpoint of vitamin A conversion. While present in animal tissues such as liver, animal fat, and egg yolk, the primary dietary sources are yellow, orange and green plants. Enhancement of the vitamin A content of foods is limited by the toxicity of its metabolites. Retinol can be toxic at only 5-fold RDA levels of intake. Retinol is teratogenic and acutely results in vomiting, exfoliative dermatitis, and increased intracranial pressure which can progress to seizures, coma, and respiratory failure. Chronic toxicity alters the integrity of hair, skin, and nails as well as inducing hepatic infiltration and ascites. The maximum safe intake of β-carotene is 20 times that of retinol (or 100 times the RDA of vitamin A). Massive doses of β-carotene are not converted to vitamin A rapidly enough to induce vitamin A toxicity. Therefore, genetic manipulation of β-carotene content rather than retinol content was chosen as a means of providing increased dietary vitamin A. Four genes were introduced into the rice genome to encode proteins necessary for synthesis of β-carotene; two from the daffodil plant and two from the bacterium Erwinia uredovora (21–23). The enhanced β-carotene content of the transgenic grain produces rice with yellow tint, hence the moniker, “Golden Rice”. Some rice strains contain enough β-carotene such that 300 g of cooked rice provides the RDA of vitamin A (24).
The strategy for genetically enhancing dietary iron in rice involved optimizing iron uptake on three levels: increased iron content, improved bioavailability, and decreased inhibition of iron absorption. Ferritin is a storage protein that can concentrate iron in cells to levels far above that of the free metal ion (25). A gene encoding cellular ferritin was introduced into the rice genome from the French bean (26). To decrease inhibition of iron absorption by decreasing endogenous phytates contained in rice, a gene encoding phytase was introduced from Aspergillus fumigatus (26). Another gene was introduced from basmati rice which encodes a cysteine-rich protein (26). Cysteine improves iron resorption in the human digestive tract. The β-carotene-producing rice was subsequently crossed with the iron-enriched strain. The resulting transgenic rice plant contains seven foreign genes which offer the potential to improve intakes of vitamin A and iron. These genes are in the process of being transferred to a commercial variety of rice. Studies are in progress by Robert Russell from the USDA laboratory in Boston to determine the bioavailability and bioefficacy of β-carotene in Golden Rice. As the β-carotene is stored in lipid membranes and in a tissue which is extremely digestible, one of the developers of Golden Rice postulates that this delivery of provitamin A will be advantageous over other sources (personal communication with Ingo Potrykus, 6/30/02). Once possible risks to human health and the environment are assessed, the rice will be distributed free of charge to farmers in developing countries (27).
DESIGNER OILS FROM OILSEED CROPS TO DECREASE DISEASE RISK
Fat Intake During Childhood Increases Risk of Adult Disease
While some regions of the world suffer from too little food and a lack of specific nutrients, other regions suffer from too much food and an inappropriate balance of nutrients. In the United States, children consume 35 to 36% of their total calories as fat in contrast to the recommended 20 to 30% of total calories (28,29). Children consume 14% of total calories as saturated fat in contrast to the recommended less than 10% (28,30). Between 1990 and 1991, approximately 15% children in the United States consumed greater than 40% of total calories as fat and only 15% of children ages 6 to 19 years old consumed the recommended 30% of total calories as fat (29).
Inappropriate dietary fat intake is not without consequences. The risk of adult diseases including cancer, myocardial infarction, hypertension, stroke, and atherosclerosis is increased directly by early childhood patterns of fat consumption and indirectly by childhood obesity. Obesity affects one in five children in the United States, making it the most prevalent nutritional disease of children and adolescents in the country. In childhood, obesity is linked to accelerated sexual maturation, glucose intolerance, hepatic steatosis, cholelithiasis, sleep apnea, pseudotumor cerebri, and orthopedic complications (There is an excellent review of this subject in reference 31). However, it is the associated childhood hypertension, altered lipid profile, and high body mass index (kg/m2) that are associated with later cardiovascular disease (31,32). Thirteen million Americans have coronary heart disease, which causes 1.5 million myocardial infarctions and 450,000 deaths annually (33).
The Structure of Specific Fatty Acids Determines Effects on Health
Both the type and amount of fat are important determinants of disease risk. Serum cholesterol increases the risk of coronary heart disease. Several lipoproteins transport cholesterol in the blood; low-density lipoprotein (LDL) increases coronary heart disease risk, while high-density lipoprotein (HDL) decreases coronary heart disease risk. The structure determines whether a fat possesses deleterious or beneficial effects on health (32).
Fats are categorized by the number of double bonds and the orientation of hydrogens on either side of the double bonds. Trans fatty acids, present in margarines and spreads, display a straight carbon chain configuration because the attached hydrogen moieties are on opposite sides of the double bond. Cis fatty isomers, contained in vegetable oils, are bent because the hydrogen moieties occur on the same side of the double bond. Saturated fats contain no double bonds and possess chain lengths of 12 to 18 carbons. In descending order of intake, dietary saturated fatty acids include palmitic (16:0), stearic (18:0), myristic (14:0), and lauric (12:0) acids. Myristic and palmitic acids increase plasma cholesterol concentrations (33) whereas stearic acid has little impact (34,35). The lack of effect on plasma cholesterol may reflect the rapid metabolism of stearic acid to the monounsaturated fatty acid, oleic acid (cis 18:1n–9) (36). Saturated fatty acids increase LDL-cholesterol concentrations (37,38). Elevations in LDL-cholesterol result in cholesterol deposition within arterial wall macrophages and smooth muscle cells, eventually leading to atherosclerosis.
Monounsaturated fats contain one double bond. Oleic acid is the major monounsaturated fat and contains a double bond at the ninth carbon. Serum cholesterol can be lowered if monounsaturated fats are substituted for saturated fats. However, dietary monounsaturated fats are largely derived from the same food sources as saturated fats. Therefore, simply consuming foods high in monounsaturated fats also increases saturated fat intake. Some monounsaturated fats pose greater risk of CHD depending on the configuration. Trans fatty acids elevate LDL–cholesterol and reduce HDL–cholesterol, and increase the concentrations of lipoprotein(a), an independent risk factor for CHD. Therefore, it is recommended that intake of hydrogenated and trans fatty acids be kept to a minimum (32).
The Structure of Specific Fatty Acids Influences Use in Food Preparation and Consumption
Altering dietary fat composition must take into consideration not only nutritional value, but the knowledge that individual fatty acid structures possess different physicochemical properties important to cooking and storage (7). For instance, more double bonds lower the melting point and the oxidative stability, and therefore decrease the usefulness of these fats in baking and processing food. Hydrogenation of unsaturated fatty acids is required to make oil semisolid for spreads and to increase its oxidative stability during storage or frying (39). Unfortunately, hydrogenation produces trans fatty acids, compounds superior for baking and shelf life but with increased health risks when ingested in large quantities. Frequently, such conflicts exist between nutritional, cooking, and storage properties of various fats and their health risks.
Children's Food Preferences Further Complicate Dietary Compliance
Children's food preferences are physiologically, genetically, and cognitively complex. An excellent review is provided by Birch and Fisher (40). Offering a healthy selection of food alone does not insure healthy intake. Food purchase and preparation is frequently unsupervised (3). In controlled settings, some children still select and consume a 42% fat diet despite provision of alternatives with lower fat (41). High fat foods are more palatable. The desire and predilection to eat more fat, as well as phenotype, may be heritable (42). Suboptimal food selection is reinforced by food advertisements which accompany children's television programming. Eighty percent of child-directed food advertisements feature foods high in fat, sugar, and salt (43). Yet, lower fat intake alone does not guarantee a healthier diet. One study of 10-year-old children showed that lower fat intake was associated with higher intake of simple sugars (44). Birch and Fisher note, “Continuing Survey of Food Intakes by Individuals data from 1989 to 1991 revealed that only one in five children and adolescents consumed five or more servings of fruit and vegetables per day and that 25% of the vegetables were French fries” (40,45).
Commercial Oilseed Crops Provide a Significant Source of Dietary Fat in Baked and Cooked Food
In developed countries, 25% of energy intake is derived from plant fatty acids (46). In the United States in 1993, vegetable oils comprised more than 90% of total fat intake (47). The predominant oilseed crops—soybean, oil palm, rapeseed and sunflower—generate 65% of the world's vegetable oil production (48). In the United States, 80% of all dietary vegetable oils come from soybean (32).
Bioengineered Oilseed Crops May Offer a Means to Improve the Quality of Fat Intake in the Context of Cultural Dietary Practices
Oilseed crops are already being genetically modified to contain increased concentrations of healthy fatty acids and decreased concentrations of the deleterious fatty acids. Genetic engineering has also been used to produce oilseed crops with more stabile oils and those with a reduced need for hydrogenation (49). The amount of stearic acid has been increased as much as 40% in oilseeds to produce a semisolid which requires no hydrogenation (50–53). Stearic acid-enriched semisolid oils can substitute for cocoa butter, margarines, and shortening. Soybeans have been engineered to contain a healthier lipid composition while providing oxidatively stable liquid oil useful for high-temperature frying (50). These soybeans contain up to 86% oleic acid compared to 25% in wild type soybeans. Saturated fat in these beans has been reduced to less than 10%. Therefore, the technology already exists to modify the fat composition of oils commonly used in food preparation and processing.
Other candidates for genetic modification include plants with enhanced phytosterol content. Phytosterols are plant-derived compounds which are consumed at low levels in the American diet, but can inhibit cholesterol absorption. Plants expressing polyunsaturated fatty acids (PUFAs), usually derived from fish oils, may be considered as well. Specifc PUFAs modulate immunologic and inflammatory processes present in cardiovascular disease and inflammatory disease. Potential applications of modified fat include use in infant formulas, intravenous nutrition, and athletic supplements. It is even conceivable that plants could be modified to provide products with fat composition beneficial to select patient populations such as those with cystic fibrosis, chronic liver disease, pancreatic disorders, or short gut syndrome.
Hepatitis and Diarrhea Cause Morbidity and Mortality
Hepatitis and diarrhea contribute to morbidity and mortality of children worldwide. Over 2 billion individuals are infected with hepatitis B virus (HBV) with 1 million deaths annually (54). Carriers incur increased risk of chronic liver disease and hepatocellular carcinoma. Carriers assure the continued infection of children with HBV through lateral and vertical transmission. In some countries, children experience 7 to 10 episodes of diarrhea per year by age 2 years (55,56). Diarrheal diseases kill three million infants each year, especially in poor or undeveloped areas. Developed nations are increasingly vulnerable under specific circumstances. For example, infectious exposure is enhanced among children in daycare (57) particularly that caused by rotavirus and Shigella. Environmental disaster and political/social destabilization can compromise sanitation leading to disease outbreaks. International trade, travel, and adoption bring carriers into contact with individuals of low immunity.
Vaccinations Are One of the Most Effective Means to Prevent Disease
The high costs of production, packaging, and delivery undermine the feasibility of using current vaccines in developing nations. Injectable vaccines are not only expensive, but require refrigeration, trained personnel, and appropriate facilities for needle and syringe sterilization and disposal. The Children's Vaccine Initiative has encouraged new technologies to facilitate vaccine availability (58). Ease of administration and patient acceptance make oral vaccines attractive candidates. Additionally, mucosal immunity may be more effectively stimulated by oral than by injectable vaccines.
Luminal Presentation of Antigens Stimulate Mucosal Immunity
Luminal presentation of an antigen, either in food or vaccine, is an important means of stimulating mucosal immunity (59,60). The antigen is taken up by specialized M cells within the intestinal lining and transferred to macrophages and B cells. Portions of the antigen displayed on macrophage membranes stimulate helper T cells, and activate B cells to produce neutralizing antibodies. Later ingestion of an intact pathogen elicits memory helper T cells to produce cytotoxic T cells, which attack infected cells. Memory helper T cells also induce a brisk secretion of antibodies by stimulated B cells. Therefore, oral vaccines may be a particularly effective first line of defense against many of the ingested pathogens responsible for gastrointestinal disease.
Edible Recombinant Vaccines
Recombinant vaccines contained in food have been in development for over a decade. (An excellent review of this topic can be found in reference 61). Recombinant vaccines do not contain intact pathogens and thus may be safer. Edible plants containing vaccines may be fed directly to individuals and do not require purification. Transformations have already been reported in a variety of food crops (62–64). The first recombinant plant vaccines utilized tobacco plants for their ease of genetic manipulation, rapid growth, and ready regeneration. Endogenous alkaloids and nicotine, and a lack of palatability detract from tobacco's feasibility as a vehicle for commercial vaccines (65). Potatoes appeared more practical. From a molecular standpoint, potatoes allow efficient genetic transformation, clonal propagation, and have tissue-specific promoters. As a food, they are edible, and can be stored for prolonged periods. However, potatoes are low in protein content, making adequate production of a foreign protein challenging. Cooking may further denature recombinant antigen integrity. Tomatoes can be eaten raw and present similar molecular advantages. But, like the potato, the total protein content is low.
Bananas are currently favored as a possible vehicle for vaccine delivery. A popular snack in the West, bananas provide 25% of all food calories in Western and Central Africa and feed tens of millions in Central America and Asia (66). They are grown locally, thus avoiding the cost of foreign production and transport. Bananas are often eaten raw thereby avoiding denaturation of recombinant protein by cooking. Bananas can be consumed by infants. Originally, bananas were genetically modified to provide more food. Transgenic bananas were also designed to resist fungi and increase crop yield. However, banana trees require up to three years to produce mature fruit because they reproduce vegetatively, i.e., without pollination. Therefore, other plant models are being employed to determine how best to maximize expression of a vaccine antigen in plant tissue before specifically developing a vaccine in bananas. Ripening bananas have already been found to contain several upregulated genes which may later prove useful for expression of edible vaccines (67). Potentially, a single banana could yield up to 10 vaccine doses, reducing the cost of one dose to less than one cent (Charles Arntzen, PhD, president of Cornell University's Boyce Thompson Institute for Plant Research in (68)). In contrast, one dose rHBsAg now costs $0.90, which is more than the daily income of nearly one billion people (69).
An excellent review of candidate vaccines is discussed by Richter and Kipp (61). Traditionally, vaccines employ one of three strategies; 1) killed pathogens, e.g., Salk polio vaccine, 2) attenuated live pathogens, e.g.,Sabin polio vaccine, 3) pathogenic strains which induce disease in host different from the species being vaccinated, e.g., Jenner's cowpox vaccine. Alternatively, a subunit vaccine may be used. This strategy induces a host immune response to proteins or components, rather than intact pathogens. For instance, Hepatitis B surface antigen (HBSAg) uses the S protein of the viral capsid which self-assembles into virus-like particles (VLPs). Recombinant VLPs include Hepatitis B (70,71); Hepatitis E (72); Norwalk virus (73); Rotavirus (74). The first model of a vaccine grown in plants used Hepatitis B virus which was also the first recombinant vaccine and which, conveniently, contained only one transgene. HBsAg has been expressed in tobacco and potato plants with subsequent formation of VLPs (71,75). Studies are in progress to increase the expression of particles in tobacco plant tissue. The Norwalk virus capsid protein (NVCP) has been similarly studied and expressed in tobacco and potato plants (73). Oral immunogenicity has been demonstrated in mice fed tubers containing recombinant NVCP (76). Thirty two of forty three mice demonstrated significant rises in serum antibody titers, but fecal IgA titers were only noted in 12 of 41. Current studies are determining the ideal oral dose.
Edible Vaccines are Feasible and May Protect Against Multiple Pathogens
Preliminary studies of NVCP in mice demonstrate that oral vaccines can survive gastric protease digestion and stimulate a gut immune response. Phase I trials in human volunteers fed tubers containing heat-labile enterotoxin from enterotoxigenic E. coli demonstrated successful delivery of recombinant antigens via plant ingestion by humans (77). Plant cell walls and membranes encapsulate candidate vaccines thus protecting against denaturation. Antigen dosing must be adequate and predictable. Current plant models produce only small amounts of vaccine. Adjuvants may serve to enhance uptake of plant vaccines and stimulate the immune response. The B subunit of Vibrio cholera toxin binds well to M cells. When coupled with other antigens, it can stimulate protection against multiple diseases simultaneously (78). Therefore, the concept of an edible vaccine is feasible.
Oral Tolerance and Autoimmunity
Before edible vaccination can be practicable, oral tolerance must be regulated. Oral tolerance occurs when oral administration of a protein antigen suppresses, rather than stimulates, systemic humoral and cell-mediated immune response (60). It is postulated that oral tolerance may be the underlying mechanism that prevents immune response to commensal bacteria and to food antigens in the gut. Oral tolerance may be desirable or undesirable. Obviously, this phenomenon would be counterproductive in an edible plant vaccine intended to provide immunity. However, oral tolerance may provide a means to suppress autoimmunity. For example, plant-based vaccines containing insulin or glutamic acid decarboxylase, proteins linked to type I diabetes, delay or inhibit autoimmune disease in diabetes-prone mice (60). Ongoing studies are investigating the technical aspects of creating transgenic plants which express adequate autoantigens to produce vaccines against autoimmune human disease, as well as insuring that immunity is stimulated by oral plant-based vaccines intended to combat infectious disease.
Future Edible Vaccines
Future prospects for edible vaccines include: 1) food vaccines ingested by mothers to passively immunize the fetus via the placenta or via breast milk, 2) oral delivery of “autoantigen” to suppress autoimmune diseases, and 3) oral vaccines for H. pylori (60).
GENETICALLY MODIFIED FOOD AND SAFE USE IN CHILDREN
The ability to treat nutritional disorders and prevent infectious diseases on such a massive scale is unquestionably beneficial, but do bioengineered foods pose risks for children? Particular concern has been expressed regarding allergic potential, altered levels of antinutritionals, and risks of gene transfer.
Children and Food Safety
The safety of novel foods, bioengineered or not, involves both specific safety considerations in children and food safety in general (79). The Acceptable Daily Intake recommendations established for adults may not apply. Children may react to novel foods in novel ways because children differ physiologically and behaviorally from adults. The effects or significance of a food may change throughout development and growth. Children consume more food relative to body weight, so the dose of a harmful food component may be relatively higher in children. The composition of the diet changes, altering the context in which a particular food ingredient may be ingested and interact. Hepatic detoxification and metabolism may be immature in children. Therefore, susceptibility to food borne toxicity may change with age. Though difficult to achieve in practice, novel foods ideally should be evaluated for both their short- and long-term effects on reproduction, endocrine function, neurological development, and immunotoxicity.
Regulating and Defining Safety
Safety assessment strategies are devised by a complex network of national and international regulatory and industrial agencies including the International Food Biotechnology Council (IFBC), the International Life Sciences Institute (ILSI), the Organization for Economic Cooperation and Development (OECD), the Food and Agricultural Organization (FAO), and the World Health Organization (WHO) (summarized in ). Three federal agencies are responsible for regulating food safety, including bioengineered foods, in the United States. The Food and Drug Administration (FDA) is responsible for labeling and safety pertaining to crops used for human consumption and animal feed. The Environmental Protection Agency (EPA) regulates pesticides, including plants engineered to produce their own pesticides. The United States Department of Agriculture (USDA) ensures the environmental safety of planting and field testing (81).
Safety determinations depend on the end-product crop, not the method that created it. This approach is supported by the American Society for Microbiology (82). Novel foods are compared to existing foods, which is the basis of the principle “substantial equivalence”. To achieve this standard, bioengineered food plants must contain the desired trait without significantly increasing deleterious allergens, antinutritionals, toxic plant metabolites, and or diminishing endogenous nutrients relative to the pre-existing conventional crop.
There have been no confirmed allergic reactions due to genetically modified food. In fact, any food plant may have allergic potential regardless of production method. Nonetheless, particular concern has been placed on the allergenicity of bioengineered crops. This sentiment may reflect recognition that the incidence of food allergies and anaphylactic shock secondary to food is rising (79) and that bioengineering involves the introduction of a foreign protein into a plant.
The much publicized Starlink corn exemplifies some of the issues related to the development, assessment and distribution of potentially allergenic foods (83). Corn–borers are insect larvae which cost farmers nearly one billion dollars annually in destroyed corn crops. Starlink corn contains a foreign gene derived from the soil bacterium, Bacillus thuringiensis. The gene encodes a Bt toxin called cryc9c, which confers insect resistance. Bt toxin naturally exists in hundreds of forms with varying insecticidal properties. Bt toxin is harmless to humans and has been used in sprays and powders for decades. Safety assessment of cryc9c did not reveal allergenicity of the gene source and there was no homology with known allergens. However, the protein was resistant to digestion. While characteristic of some allergens, digestive stability is not fully predictive of allergenicity. Nonetheless, approval was restricted to use in animals and delayed for human consumption. Limited planting ensued from 1998 to 2000. Faulty segregation of harvested corn led to the presence of Starlink corn in human food products such as taco shells, which were subsequently recalled. Lay media exaggerated and misreported the allergic risk. In fact, consumer exposure to cryc9c was slight. Taylor and Hefle emphasize that Starlink comprised only 0.05% of all harvested corn and cryc9c constituted only 0.0129% of a kernel. So, processed food made from corn contained only minute quantities of cryc9c protein that “likely has been insufficient to elicit allergic sensitization” (83). Not one case of the tested individuals who reported symptoms, such as itchy eyes, was confirmed as an allergic reaction due to Bt corn. However, the Starlink corn incident demonstrated the failure of the crop harvesting, food processing and food distribution system to effectively segregate crops of different planting technology and intended for different uses.
The IFBC and the Allergy and Immunology Institute of the ILSI convened a task force in 1996 (84) to develop strategies to prevent bioengineered crops from introducing allergens into the diet. Their initial recommendations underwent more stringent modifications at the January 2001 consultation of the FAO and WHO. There is no single test to determine allergenicity of a novel protein. The latest consensus recommended several levels of assessment including; the source of the gene, its sequence homology to known allergens, the expression level of the novel protein in the modified crop, the functional classification of the novel protein, the reactivity of the novel protein with serum IgE derived from individuals known to be allergic to the gene source, and various physicochemical properties. More comprehensive reviews are provided by Metcalfe et al. (84), Kimber et al. (85), Taylor and Hefle (83) and Taylor (86).
These recommendations are largely based on whether the foreign protein is related to a known allergen. Most food allergies are attributed to peanuts, soybeans, tree nuts, wheat, milk, eggs, fish, and crustaceans. There is also cross-reactivity between some food allergens and pollen allergens (87). A gene derived from such a source can be evaluated to see if it encodes a known environmental or food allergen. Further determinations of short sequence homologies can be performed. For risk of allergenicity, it is thought that there must be at least 6 contiguous, identical amino acids or an overall homology greater than 35% (88). The choice of six contiguous, identical amino acids is based on studies of Ara h 1 and Ara h 2 epitopes in peanut hypersensitivity which requires at least 6 contiguous amino acids to bind IgE (89,90). Protein amino acid sequence databanks include Genbank, PIRprotein, EMBL, and Swissport (91).
The digestive stability of the novel protein is another criterion. Most allergens resist degradation by heat, acid, and peptidase activity; a property that permits an intact protein to reach the intestine and thus provoke an immune response. The recommended assessment uses much higher concentrations of the novel protein than those present in the transgenic plant. The FAO/WHO suggests a specific standardized protocol for a pepsin resistance test (88).
The level of protein expression is not currently assessed but, may be important (84). There appears to be a threshold dose below which an allergen will not provoke an adverse reaction. For instance, some studies estimate the threshold dose for peanut protein at 1 to 20 mg (92). Therefore, as Taylor and Hefle state, “if an allergenic protein were expressed in a food produced through agricultural biotechnology at levels well below 1 mg per serving, the hazard for allergic consumers would be minimal” (83). Determination of the appropriate threshold dose still remains (93). Other studies have detected symptoms in individuals exposed to as low as 100 mcg of peanut (94). A future opportunity in the field of bioengineered crops is the development of food crops which lack established allergens (95).
ALTERED GENE EXPRESSION IN PLANTS, BACTERIA, AND HUMANS
Safety assessment criteria and procedures are continually being modified to identify unintentional consequences of biotechnological advancements.
At the molecular level, there are three basic questions with regard to human health:
Does insertion of novel gene(s) into plants alter the expression of other endogenous proteins which increase toxicity or decrease nutritional value?
Can edible plants transfer genes to the bacteria in soil, animal rumens, or the human gut which might lead to enhanced pathogenicity?
Can humans absorb intact DNA in food which either directly causes disease or can be incorporated into the human genome?
Antinutrient Levels in Food Plants
The uncertainty of where the foreign gene will integrate into the host plant genome raises the possibility that genetic modifications might produce unintentional perturbations in other plant constituents. Theoretical mechanisms underlying such altered gene expression include gene silencing or gene activation (96,97). Attention is primarily focused on endogenous antinutritionals. Antinutritionals are variably defined, but are basically acknowledged as compounds that result in suboptimal nutrient utilization. These compounds are present in many conventionally grown food crops. Toxicity may not occur until a threshold concentration is achieved (96,98,99) or may be obviated by heating or soaking. Respective examples include the toxic oxalates contained in spinach and rhubarb, and soaking kidney beans to reduce lectin toxicity. Some antinutritionals confer other advantageous antibacterial or anticarginogenic properties (96,100).
Novak and Haselberger compared the composition of genetically modified plants and their parent plants: rape plants and canola for glucosinolates and phytate; maize for phytate; tomatoes for tomatine, solanine, chaconine, lectins and oxalate; potatoes for glykoalkaloids, protease-inhibitors and phenols; and soybeans for protease-inhibitors, lectins, isoflavones, and phytate (96). They found that the composition of most genetically modified plants was within the range of the parent plant. Depending on the crop and compound studied, the genetically modified plant might contain lower or higher levels than the parent plant. In some instances, the location of the crop and local environmental stresses led to more variation than the genetic transformation.
Integration of Foreign DNA into Bacteria and Antibiotic Resistance
The transfer and functional integration of ingested plant DNA into gut microflora or human cells appears unlikely, but is still under investigation. The reader is referred to the comprehensive review and position paper of the Novel Foods Task Force ILSI Europe Workshop on Safety Considerations of DNA in Food held June 26–28, 2000 (101), as well as discussions by Droge (102), and Nielson (103).
The primary concerns in bacteria are the acquisition of antibiotic resistance and the emergence of untreatable pathogens (104). Antibiotic resistance genes have been routinely used as selection markers to document successful incorporation of desired foreign genes into plants and other organisms. Examples include nptII gene encoding resistance to kanamycin and neomycin, the aaa gene which confers resistance to streptomycin and spectinomycin, the nptIII gene which provides resistance to amikacin, and the bla TEM gene which leads to cephalosporin resistance (104,105). Some antibiotics have greater medical significance than others. Kanamycin and neomycin are less frequently used due to their inherent toxicity, but spectinomycin may be the only option for a pregnant woman with N. gonorrhea (105).
The spread of bacterial resistance may not remain an issue for several reasons. Jonas et al., in the Executive Summary Statement of the ILSI state, “The [bacterial] cells would need to have the capacity to take up DNA. Then the DNA would need to be integrated into the genome as linear fragment, would require extensive sequence homology, or would need to form an independent replicon. In addition, for a gene on the integrated DNA to be expressed, it would need to be associated with appropriate regulatory sequences. A new trait may be maintained without selection, but for the transformed bacterium to become a large part of the population there would need to be selection for the trait. Each of these events is rare and they would need to happen sequentially” (101). Nielson et al. cite the low frequency of gene transfer from plants to bacteria throughout evolution, but recommend more study of selection processes in natural environments (103). In the interim, the threat of acquired bacterial resistance may be eliminated by the alternative use of other selection markers (106) or marker elimination from transgenic plants (107).
Incorporation of Novel Genes into Humans
Review of currently available data on human ingestion of genetically modified plants does not identify any increased risk of human disease or risk of incorporation of novel genes into the human genome (101). A comprehensive review of the structure and function of nucleic acids, occurrence of nucleic acids in food, stability of nucleic acids, safety of DNA, and food safety consequences is beyond the scope of this paper, but appears in the position paper of the Novel Foods Task Force ILSI Europe Workshop on Safety Considerations of DNA in Food held June 26–28, 2000 (101). The consensus of the expert panel is based in part on comparison to DNA already present in the human diet and is as follows:
* “All DNA including recDNA [recombinant DNA] is composed of the same four nucleotides.
* In view of the variability of dietary intake of DNA, consumption of foods derived from GMOs [genetically modified organisms] does not measurably change the overall amount of DNA ingested through the diet.
* Taking into account the natural variations of DNA sequences, the present use of recombinant techniques in the food chain does not introduce changes in the chemical characteristics of the DNA.
* There is no difference in the susceptibility of recDNA and other DNA to degradation by chemical or enzymatic hydrolysis.
* The metabolic fate of DNA digestion products is not influenced by the origin of the DNA.
* DNA is not toxic at levels usually ingested. Where there is potential for adverse effects, e.g., gout, this is due to excessive intake, not the origin of the DNA.
* There is no indication that ingested DNA has allergenic or other immunogenic properties that would be of relevance for consumption of foods derived from GMOs.
* In vivo uptake of DNA fragments by mammalian cells after oral administration has been observed. However, there are effective mechanisms to avoid genomic insertion of foreign DNA. There is no evidence that DNA from dietary sources has ever been incorporated into the mammalian genome.”
There is a need for more and better food worldwide. The use of genetically modified food plants is already pervasive and represents an efficient means to provide more food, more nutritious food, or to possibly to eradicate some diseases. No human hazard has yet been identified. The allergic and toxic potential of plants exists regardless of breeding technique. More important is the need to identify and monitor beneficial and deleterious proteins in novel food plants, regardless of technology. Children stand to accrue the greatest potential benefits of this technology through the eradication of various nutritional and infectious disorders. However, because children will have the longest potential exposure one must consider potential long-term effects which have not been identified and would be difficult to assess. Pediatric gastroenterologists have much to contribute regarding how food bioengineering is best used and to help insure that it is done in a safe manner.
The author would like to thank T. Gradman, Ph.D. for his support and encouragement and I.N. Perr, M.D., J.D., whose ability to analyze and synthesize material within and across academic disciplines I hope to emulate.
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