- Email: [email protected]
Genetically Engineered Foods
432 Genetically Engineered Foods Miura T, Patierno SR, Sahuramoto T, and Landolph JR (1989) Morphological and neoplastic transformation of C3H/10T1/2 Cl 8 mouse embryo cells by insoluble carcinogenic nickel compounds. Environmental and Molecular Mutagenesis 14: 65–78. O’Neill JP and Hsie AW (1979) The CHO/HGPRT mutation assay experimental procedure. In: Hsie AW, O’Neill JP, and McElhenny VK (eds.) Mammalian Cell Mutagenesis: The Maturation of Test Systems, Banbury Report No. 2, pp. 55–70, 311–318, 407–420. Cold Spring Harbor, NY: Cold Spring Harbor Press. Patierno SR, Banh D, and Landolph JR (1988) Transformation of C3H/10T1/2 mouse embryo cells to focus formation and anchorage independence by insoluble lead
chromate but not soluble calcium chromate: Relationship to mutagenesis and internalization of lead chromate particles. Cancer Research 48: 5280–5288. Preston RJ and Hoffmann GR (2001) Genetic toxicology. In: Klaassen CD (ed.) Casarett and Doull’s Toxicology, The Basic Science of Poisons, 6th edn., pp. 321–350. New York: McGraw-Hill Medical Publishing Division. Verma R, Ramnath J, Clemens F, Kaspin LC, and Landolph JR (2004) Molecular biology of nickel carcinogenesis: Identification of differentially expressed genes in morphologically transformed C3H/10T1/2 Cl 8 mouse embryo fibroblast cell lines induced by specific insoluble nickel compounds. Molecular and Cellular Biochemistry 255: 203–216.
Genetically Engineered Foods William Frez
Current Technologies
& 2005 Elsevier Inc. All rights reserved.
In its simplest terms, the production of GMOs is derived from manipulation of the subject genome in order to achieve some desired trait or end result. Therefore, traditional agricultural practices intended to manipulate breeding or reproduction to select for desired traits over a somewhat protracted period of time can be thought of as genetic modification practiced over thousands of years. While drawing on these long used natural selection methods, current technologies have now been developed to achieve rapid and more dramatic results such as crop resistance to insects, production of novel pharmaceuticals, and increased animal growth and milk production. Thus, a brief outline of the essential gene manipulation technologies is fundamental to further understanding of potential global health concerns.
Introduction Genetically engineered foods are a subset of genetically modified organisms (GMOs). The definition of a GMO may vary depending on the source of the information. However, the World Health Organization (WHO) specifically defines GMOs as ‘‘yorganisms in which the genetic material (DNA) has been altered in a way that does not occur naturally.’’ Farmers began using GMOs for the growth of soy-beans, potatoes, and corn in the mid-1990s finding benefits ranging from reduced operating cost to enhanced crop production. However, the use of GMO technology had not been without controversy then and is still debated now. Concern over the use of GMOs became heightened by the inadvertent introduction of a genetically modified corn (StarLinkTM) into the human food supply. Ostensibly registered by the US Environmental Protection Agency for use as an insect resistant plant pesticide and animal feed, the StarLinkTM controversy propelled its producer to voluntarily cancel its registration and cease production. This episode in the evolution of GMO production has since then prompted citizen’s watch groups, nongovernmental organizations (NGOs), and governmental agencies worldwide to address concerns regarding the use of GMOs ranging from specific toxicity to humans, plants, fish, and livestock to fear of large scale ecosystem disruption.
Transgenic Manipulation
Transgenic manipulation refers to the insertion, removal, and modification of the plant genome for placement into an individual of the same species, or across species to achieve the desired results in a relatively rapid period of time. Two widely used techniques developed to achieve these ends are termed in vitro and vector-based techniques. In vitro techniques mechanically insert or inject a specific protein, gene, or genetic sequence into the subject organism. Within that general category, three methods are often defined; microinjection, particle or microprojectile bombardment, and DNA uptake directly into organism. Alternatively, vector-assisted techniques use live viral or bacterial carriers (i.e., vectors) to facilitate transfer of genetic material into the subject organism.
Genetically Engineered Foods 433
In Vitro Methods Microinjection in vitro methods use a fine needle and microscopic manipulation to directly inject the gene material into protoplasts of the subject organism. The difficulty and cost of the microinjection process may be circumvented by DNA transfer via protoplast mixing. In this method, the ‘protoplast-associated’ DNA of one individual is allowed contact with that of another individual in a polyethylene glycol environment that is favorable to DNA exchange. Subsequent replication processes of the genetic material facilitate incorporation of the desired gene into the subject organism for an increase in copy number and favorable trait selection. Temporary disruption of the cell membrane via electrical stimulation (i.e., electroporation) can be used to enhance the transfer efficiency of this process. Another often-used in vitro technique is ‘particle bombardment’. In this method, microparticles of tungsten or gold are coated with the transgenic material and are literally ‘shot’ into the plant cell with compressed helium gas or an electrical discharge. Recombination and replication of the DNA material transpires within the subject organism DNA prior to manipulations to increase copy number and trait selection. Vector-Facilitated Methods Vector-facilitated methods depend on the use of nonpathogenic viruses, or sections of bacterial DNA to transfer portions of the transgene or the transgene in its entirety using the biological invasive capabilities of the vector. Essentially, the vector DNA is modified and subsequently allowed to ‘infect’ the subject DNA to facilitate DNA transfer to the host. These vectors are frequently engineered to eliminate their virulence yet retain the DNA transfer capability of the original pathogen.
Commonly Produced GMOs GMO technologies discussed above and others have been used to create a variety of modified crop, fish, and animal species for crop prey resistance, pharmaceutical development, and increased production from livestock. It is these applications that have attracted public and scientific interest over the years. As will be noted below, several organisms are currently used, though it should be anticipated that additional organisms will be employed in the future. Genetically Modified Crops
The use of genetically modified corn and cotton has increased over 10-fold from 1992 to 1999 and as of 2002, 50 crop species have been evaluated for uses by the US Food and Drug Administration (FDA). In the development of transgenic crops, genes isolated from several varieties of the bacterium, Bacillus thuringienses (Bt) are probably the best known and most often cited example of GMO development. The use of this bacterial species has been deemed to be ideally suited for GMO use due to possession of toxins known as delta endotoxins. The structure of the delta endotoxin, is complex, containing three major regions or sections that each connote differential characteristics to the ultimate toxin (Figure 1).
Marker Gene Incorporation The mere delivery of the transgene into the host species or individual does not guarantee that the production of a GMO will be successful. This is due to the relative inefficient transfer process and the somewhat random selection process that must subsequently be used to enhance the number of individual copies of the transgene for further trait development. In order to address this limitation, genes or DNA segments coding for known and readily observable phenotypes (i.e., marker genes) are ‘coinserted’ with the transgene to better detect successful transformations. Such marker genes are now commonly used to detect color expressions or other visible attributes for better identification and subsequent selection and enhancement of the desired trait.
Figure 1 Molecular structure of Bacillus thuringienses delta endotoxin. (Reproduced from Li J, Carroll J, and Ellar DJ (1991) Nature 353: 815–821, with permission of the Nature Publishing Group.)
434 Genetically Engineered Foods
The domains of the molecule have markedly different conformations that control key aspects of ‘Bt’ toxicity to target insects. The a-helical bundles of the domain I region can be inserted into the gut cell membrane of the target species, thus, facilitating ion leakage. The domain II region consists of three b-sheet conformations that are structurally similar to immunoglobulin antigen binding regions, suggesting that the key characteristic section resides in the gut area. Domain III is a densely packed structure that is believed to protect the exposed end of the toxin from cleavage by digestive proteases; thus helping to ensure structural integrity and subsequent toxicity to the target species. By incorporating these characteristics of the Bt toxin into valued crop species, crop resistance to insects feeding on crop species may be enhanced helping to increase vitality, size, and acreage yields. Coded genes for the Bt delta endotoxin production have been isolated for insertion into a variety of vegetables including potatoes, field corn, sweet corn, and soybeans. Genetically Modified Fish
No genetically modified fish have been approved for entry into the US human food supply as of this writing although growing pressure for such use is anticipated. However, as with crop species, the use of GMO technologies in fish has been deemed useful for obtaining an increase in production, size, disease resistance, optimal food consumption, and pharmaceutical development. Common species selected for research into the application of transgenic manipulations include salmon, tilapia, channel catfish, and medaka. Alternatively, very active research in the use of transgenic fish as research models has been ongoing for several years with ever increasing focus on their use in the investigation of mutagenicity and environmental toxicology. Typical organisms for such applications tend to be small and easily cultured species such as medaka, mummichog, zebra fish, and others displaying favorable genetic attributes. Transgenic fish were noted as being first introduced into the research community in 1985. Since that time, the use of transgenic fish to refine methods
of transgene manipulation and application as potential analytical tools has grown substantially in the assessment of mutation frequencies. Several mutation assays using bacteriophage and plasmid vectors have been developed. Examples are shown in Table 1. As can be noted, detection methods may be based on modifications in enzyme systems that become evident in subsequent growth of bacterial species on selective growth media. In concert with the mutation assays that are shown in Table 1, the potential use of transgenic fish in the assessment of environmental health risk assessment offers opportunities to further assess the effects of water- and sediment-associated contaminants in the environment. Genetically Modified Livestock
The genetic modification of livestock stems from a desire to enhance growth, increase production of high protein milk and cheese, facilitate biomedical research, as well as potentially protect against incidental toxicity via exposure to pesticides that may be associated with food crops. Animal genomes that have been successfully modified include sheep, pigs, cows, rabbits, and chickens. Two key research areas applied to livestock are discussed below. Enhanced Production Growth, as a primary metric of production, has been and is the subject of much research. Of substantial interest is the potential for use of transgenes in stimulating ‘overproduction’ of growth hormones to enhance animal growth. Several test protocols have been evaluated with moderate success in various livestock models. For instance, blood levels of zinc-induced growth hormones have been increased in pigs and sheep. However, such activity was minimal in pigs and negligible via attempted phosphoenolpyruvate carboxykinase induction. Enhanced growth, via increased production of growth hormones also has been used to modify the qualitative nature of the animal such as lean muscle mass. However, such manipulations were not without negative effects such as renal failure and gastric ulcers of the subject animal.
Table 1 Various transgenic fish mutation assays Assay
Vector/fish species
Detection method
lacI Mutation assay cII Mutation assay rpsL Mutation assay lacZ Mutation assay
Bacteriophage l LIZ vector/medaka Bacteriophage l/medaka pML4 Plasmid vector/zebra fish Plasmid pUR288 vector/medaka and mummichog
a-Galactosidase functionality Plaque formation Kanamycin and streptomycin resistance Galactose-sensitivity
Genetically Engineered Foods 435
Pharmaceutical Development As of 2001, three companies were engaged in the manufacture of B30 pharmaceuticals to produce proteins and antibodies derived from transgenic procedures. The transgenic-mediated production of pharmaceuticals is primarily achieved by facilitating construction of proteins that can then be expressed in milk of organisms are often termed ‘bioreactors’. Examples of biologically active proteins that have been produced include antitrypsin, tissue plasmogen activator, and human blood clotting factor.
Human Health Risk Although potentially beneficial, the development of GMOs is fraught with concern over the potential for adverse effects on human health. While human health effects of popular concern consist of increased toxicity, decreased nutritional value of food, and increased antibiotic resistance, possibly the most widely recognized concern centers on the potential increase in allergic reactions of individuals consuming transgenically modified food. Approximately 1–2% of the population and 5–8% of children experience food allergies that are the result of natural selection processes that have occurred over thousands of years. Elevations in these frequencies may suggest allergic reactions to GMOs. Such allergic reactions are of two general types, immuoglobulin E (IgE)-mediated and non-IgE mediated reactions. IgE-mediated allergenicity requires that individuals must first be exposed to an allergen in a sensitization dose prior to showing overt signs of the allergy. In this reaction, antigen specific binding of IgE to mast cells and basophils, followed by release of pharmacologically active chemicals such as histamines, cytokines, chemokines, and arachidonic metabolites are ultimately responsible for the classic rapid allergic reaction often termed ‘immediate hypersensitivity reactions’. Toxic responses may range from, dermatitis, urticaria, and itching, to fatal anaphylactic shock and may be induced by proteins associated with a variety of foods including peanuts, grains, and fish. Non-IgE mediated allergic reactions do not require a sensitization exposure and may occur especially in infants and children consuming milk protein and grains, and are characterized by a delayed onset of symptoms after exposure to the food. Food-induced enterocolitic syndromes caused by milk protein ingestion may result in vomiting, diarrhea, and general deterioration of the individual. Celiac disease is a specific example of such a wasting syndrome stemming from a reaction to cereal or grain (i.e., wheat, rye, barley, oats, and spelt) ‘glutens’. Individuals
experiencing this disease may show weight loss, diarrhea, abdominal cramps, gas and bloating, general weakness, oily stool, and stunted growth in children. Though significant in terms of effects to an afflicted individual, only B1 in 300–3000 individuals in a population seem to be affected by celiac disease. To date, no confirmed cases of increased allergic reactions to GMOs have been documented and thus the extent to which genetically modified food contributes to significant allergic reactions in the population is not accurately known. The only reported incident of potential GMO allergenicity occurred after production of soybeans modified with Brazil nut protein. Allergenicity to the genetically modified soybean was detected and the product was not marketed, precluding exposure and toxicity. The previously discussed StarLinkTM episode is perhaps the most widely evaluated incidence potentially leading to potential health effects associated with GMOs. Using enzyme-linked immunosorbent assay (ELISA) in a retrospective study, the US FDA hypothesized that exposure to the Bt Cry9c protein could be cause for allergic responses. However, the results of the FDA research concluded that there was no evidence of GMO-mediated allergenicity subsequent to potential exposure to the StarLinkTM corn. Because the lack of evidence of health effects cannot conclusively show that GMOs are not a human health concern, current global efforts are focused on the development of protocols to proactively and systemically detect and assess potential adverse effects.
Environmental Health Risk The American Medical Association (AMA) has estimated that in 1999, 200 million acres of land had been planted worldwide with transgenic crops. The AMA further indicated that over 25 000 field trials for environmental effects of GMOs had been performed in 45 countries without noted adverse environmental consequences. Despite these conclusions, the limited geographical size and comprehensiveness of such trials confounds definitive conclusions regarding the potential for adverse effects such as enhanced crop pest resistance, out crossing with weedy relatives of crops, reduced biodiversity, nutritional deficiency of food sources, and toxicity to nontarget species. Concerns over the safety of transgene introduction into environment was sensitized early in the GMO debate with significant focus on the potential toxicity of Bt endotoxins to monarch butterfly (Danaus plexippus) larvae exposed to transgenic pollen. Early data suggested that Bt corn pollen could result in potentially significant reactions in the monarch gut.
436 Genetically Engineered Foods
However, at that time (mid-1990s), the general regulatory consensus in the United States suggested that the adverse effects of Bt toxins were negligible when actual exposure conditions in the field were considered. In 1999, laboratory tests were conducted to determine if Bt transformed corn pollen consumed by monarch larvae at environmentally significant concentrations could result in adverse effects under well-controlled conditions. The widely published data resulting from this testing, indeed showed that larvae consuming Bt corn pollen experienced a decreased growth rate, expressed as the concentration of protein (0.76 ng ml 1) required to result in growth reduction of 50% of the population (EC50). Mortality, expressed as the LD50, was determined to occur at 3.3 ng protein ml 1 diet. Estimated pollen densities of B10 and 50–100 grains cm 2 were deemed to potentially result in these adverse effects. As a potentially significant environmental and ecological risk, such Bt pollen toxicity notably stirred the interest and emotions of the scientific and public interest communities. In 2001, a collaborative research effort of Canadian and US scientists developed a weight of evidence risk assessment to address the concern. The risk assessment results suggested that the likelihood of an adverse effect on the monarch population was less than 1 in 10 000 (or less than 1 10 4) when the toxicity information and exposure estimates were integrated into a risk analysis. While these data support the conclusion that an adverse effect on the monarch receptor was unlikely, it should be noted that the absence of data suggesting risk under given assumptions and circumstances should be interpreted carefully as a general conclusion of safety in all instances. The concern for adverse ecological effects is not limited to plant or insect communities and direct toxicity. For instance, experimental data collected using fish suggests that the incorporation of transgenes may actually benefit the transgenic individual. For instance, mathematical models have been developed and evaluated to assess population effects of the transgenic fish of wild populations. Using the same data that show that the transgene may benefit the individual, models have predicted that the inadvertent release of the transgenically modified fish into the environment may result in significant adverse environmental effects such as invasion by genetically modified fish resulting in increased competition with and displacement of native species, reduced hardiness of the modified fish offspring in the wild, and potential extinction of the naturally occurring population. Although such effects have not been conclusively supported by field evaluations or verifying laboratory data, the concerns for long-term adverse
ecosystem effects remains a subject of continued research. As noted earlier in this article, transgenically modified growth hormones have been administered to cattle to increase milk production. However, it has been noted that the administration of growth hormones can result in adverse effects in the exposed animal including, fertility reduction, changes in bone growth, increase chance of mastitis, and reductions in endocrine function. The long-term effects of inadvertent exposures to the transgene-modified growth hormone on nontreated cattle herds, livestock, or breeding stock is poorly understood.
Safety Evaluation Methodology The complexity of GMO introduction to the human food chain as well as uncertainties regarding potential ecosystem effects has prompted governmental agencies and NGOs to recommend highly structured and systematic safety evaluation protocols prior to GMO release. While several protocols have been proposed, developed, and are being adopted worldwide, those of the European Union and the WHO provide reasonable and illustrative examples for reference. Substantial Equivalence
A fundamental concept essential to the currently defined approaches for determining GMO safety is that of ‘substantial equivalence’ (SE). Used as the basis for establishing a comparative benchmark, SE depends on two key concepts. First, existing traditional foods are assumed to be safe as evidenced by their longterm use. Second, the response to traditional foods can be used as a basis for comparison to transgenically modified foods which are often derived from traditional foods. Predicated on the definition of substantial equivalence, a decision process for further evaluation of the GMO can be defined according to the general rules shown in Table 2. While the decision matrix shown in Table 2 suggests a rather simple testing approach, in reality, adherence to the SE concept requires that it be applied to a multitude of characteristic variables including chemical SE, biological SE, SE of potential exposure routes, and the SE of possible overall safe use. The WHO Decision Tree for Assessment of Potential Allergenicity
Allergenicity is the current overriding concern regarding potential human exposures to GMOs. Thus, the
Genetically Engineered Foods 437 Table 2 Simplified decision matrix for evaluation of GMO safety according to the concept of substantial equivalence If the GMO demonstrates
Then
Substantial equivalence to traditional food Substantial equivalence to traditional food but also demonstrates evidence of a specific new trait No substantial equivalence to traditional food
No further testing or evaluation is necessary The assessment must focus on the potential effects of the new trait or gene Comprehensive toxicological and nutritional testing is required
Is gene source allergic? Yes
No
DNA sequence homolgous to traditional food?
DNA sequence homolgous to traditional food?
Yes
Possible Allergen
No
Yes
Is specific serum screen positive?
No
Yes
Is targeted serum screen positive?
No No Yes Yes Demonstrable animal model allergenicity
No Yes
Yes
High
Intermediate
Yes No
Demonstrable pepsin resistance
Low No
No
Figure 2 Decision tree for the evaluation of allegenic potential associated with GMO. (Adapted from FAO/WHO (2000) Safety Aspects of Genetically Modified Foods of Plant Origin. Geneva, Switzerland: World Health Organization.)
WHO has developed a decision tree approach to determine the human health risk from exposure to GMOs. The decision tree and associated rationale and documentation is best reviewed in WHO records. However, Figure 2 shows the general approach (with modifications for presentation purposes) suggested by WHO to determine if a GMO is a potential allergen. The decision tree is not a prescriptive and mandated method with substantial recommended technical protocols but rather is a conceptual model to help the evaluator determine the potential that the GMO
may trigger allergic reactions in greater proportion than the unexposed populations. As noted by examination of Figure 2, the incorporation of comparisons with traditional foods coupled with an allowance for graded responses ensures that the risks of allergic reactions are assessed in the context of exposures and the substantial equivalence concept.
Concluding Remarks The development of GMOs, and genetically modified foods in particular, is, in large part, a direct response to ever increasing global food demands. Current
438 Genomics, Toxicogenomics
information suggests that GMOs are unlikely to have substantial adverse near term effects on human health and the environment. However, the assessment of the potential for longer-term adverse effects poses a greater challenge. As a result of perceived needs, the technology inspired over the last 20 years and continues to develop; both in methodology and application. As a result of increased concerns over health effects, such activities will be scrutinized with growing vigilance on the part of citizens, scientists, regulatory bodies, and advisory commissions; ultimately to assure nearand long-term protection of global human health and the environment.
See also: Food and Drug Administration, US; Toxicity Testing, Mutagenicity; Transgenic Animals.
Corn. A Report to the U.S. Food and Drug Administration from the Centers for Disease Control and Prevention. Cockburn A (2002) Assuring the safety of genetically modified (GM) foods: The importance of an holistic, integrative approach. Journal of Biotechnology 98(1): 79–106. Konig A, Cockburn A, Crevel RW, et al. (2004) Assessment of the safety of foods derived from genetically modified (GM) crops. Food and Chemical Toxicology 42(7): 1047–1088. Kuiper HA, Konig A, Kleter GA, Hammes WP, and Knudsen I (2004) Safety assessment, detection and traceability, and societal aspects of genetically modified foods. European Network on Safety Assessment of Genetically Modified Food Crops (ENTRANSFOOD). Concluding remarks. Food and Chemical Toxicology 42(7):1195– 1202 (review). World Health Organization, Food and Agriculture Organization (2000) Safety Aspects of Genetically Modified Foods of Plant Origin. Geneva, Switzerland: World Health Organization.
Further Reading Bakshi A (2003) Potential adverse health effects of genetically modified crops. Journal of Toxicology and Environmental Health B Critical Reviews 6(3): 211–225. CDC (2001) Investigation of Human Health Effects Associated with Potential Exposure to Genetically Modified
Genetically Modified Organisms
Relevant Website http://www.pbs.org – Tyson P (2001), NOVA Public Broadcasting System, ‘Should We Grow GM Crops’ Documentary aired April 24, 2001.
See Genetically Engineered Foods.
Genomics, Toxicogenomics Kartik Shankar and Harihara M Mehendale & 2005 Elsevier Inc. All rights reserved.
Toxicogenomics refers to the application transcriptonomic (high-throughput analyses of gene expression) techniques to the field of toxicology. Classically, toxicologists examine potential adverse outcomes and putative mechanisms due to xenobiotic exposure by biochemical and histopathological markers of toxicity. In the current healthcare and regulatory environment, chemicals suspected to have potential for significant adverse health effects are selected to undergo subsequent testing for carcinogenicity and chronic toxicity. However, long-term studies are typically labor intensive and time consuming and can cost $2–4 million. As of 2002, the National Toxicology Program had tested or is currently testing 505 chemicals in long-term studies, 66 in short-term studies, and only a single chemical in a subchronic study. Given that almost 70 000–85 000 chemicals
are used in commerce today, it is evident that alternative high-throughput methods for screening the toxic potential of chemicals is needed. This would allow for prioritization of untested chemicals in the classical approach of toxicity testing. Concurrent with rapid advances in bioinformatic tools and classification algorithms toxicogenomic analyses is fast becoming a viable option in high-throughput screening of potentially hazardous chemicals.
Applications of Toxicogenomics Predictive Toxicology
The underlying premise of toxicogenomics is that toxicity is associated with changes in the global gene expression. Since toxicity by itself is resultant due to some form of cellular dysfunction or cell death, it will either be preceded or followed by some level of
chromate but not soluble calcium chromate: Relationship to mutagenesis and internalization of lead chromate particles. Cancer Research 48: 5280–5288. Preston RJ and Hoffmann GR (2001) Genetic toxicology. In: Klaassen CD (ed.) Casarett and Doull’s Toxicology, The Basic Science of Poisons, 6th edn., pp. 321–350. New York: McGraw-Hill Medical Publishing Division. Verma R, Ramnath J, Clemens F, Kaspin LC, and Landolph JR (2004) Molecular biology of nickel carcinogenesis: Identification of differentially expressed genes in morphologically transformed C3H/10T1/2 Cl 8 mouse embryo fibroblast cell lines induced by specific insoluble nickel compounds. Molecular and Cellular Biochemistry 255: 203–216.
Genetically Engineered Foods William Frez
Current Technologies
& 2005 Elsevier Inc. All rights reserved.
In its simplest terms, the production of GMOs is derived from manipulation of the subject genome in order to achieve some desired trait or end result. Therefore, traditional agricultural practices intended to manipulate breeding or reproduction to select for desired traits over a somewhat protracted period of time can be thought of as genetic modification practiced over thousands of years. While drawing on these long used natural selection methods, current technologies have now been developed to achieve rapid and more dramatic results such as crop resistance to insects, production of novel pharmaceuticals, and increased animal growth and milk production. Thus, a brief outline of the essential gene manipulation technologies is fundamental to further understanding of potential global health concerns.
Introduction Genetically engineered foods are a subset of genetically modified organisms (GMOs). The definition of a GMO may vary depending on the source of the information. However, the World Health Organization (WHO) specifically defines GMOs as ‘‘yorganisms in which the genetic material (DNA) has been altered in a way that does not occur naturally.’’ Farmers began using GMOs for the growth of soy-beans, potatoes, and corn in the mid-1990s finding benefits ranging from reduced operating cost to enhanced crop production. However, the use of GMO technology had not been without controversy then and is still debated now. Concern over the use of GMOs became heightened by the inadvertent introduction of a genetically modified corn (StarLinkTM) into the human food supply. Ostensibly registered by the US Environmental Protection Agency for use as an insect resistant plant pesticide and animal feed, the StarLinkTM controversy propelled its producer to voluntarily cancel its registration and cease production. This episode in the evolution of GMO production has since then prompted citizen’s watch groups, nongovernmental organizations (NGOs), and governmental agencies worldwide to address concerns regarding the use of GMOs ranging from specific toxicity to humans, plants, fish, and livestock to fear of large scale ecosystem disruption.
Transgenic Manipulation
Transgenic manipulation refers to the insertion, removal, and modification of the plant genome for placement into an individual of the same species, or across species to achieve the desired results in a relatively rapid period of time. Two widely used techniques developed to achieve these ends are termed in vitro and vector-based techniques. In vitro techniques mechanically insert or inject a specific protein, gene, or genetic sequence into the subject organism. Within that general category, three methods are often defined; microinjection, particle or microprojectile bombardment, and DNA uptake directly into organism. Alternatively, vector-assisted techniques use live viral or bacterial carriers (i.e., vectors) to facilitate transfer of genetic material into the subject organism.
Genetically Engineered Foods 433
In Vitro Methods Microinjection in vitro methods use a fine needle and microscopic manipulation to directly inject the gene material into protoplasts of the subject organism. The difficulty and cost of the microinjection process may be circumvented by DNA transfer via protoplast mixing. In this method, the ‘protoplast-associated’ DNA of one individual is allowed contact with that of another individual in a polyethylene glycol environment that is favorable to DNA exchange. Subsequent replication processes of the genetic material facilitate incorporation of the desired gene into the subject organism for an increase in copy number and favorable trait selection. Temporary disruption of the cell membrane via electrical stimulation (i.e., electroporation) can be used to enhance the transfer efficiency of this process. Another often-used in vitro technique is ‘particle bombardment’. In this method, microparticles of tungsten or gold are coated with the transgenic material and are literally ‘shot’ into the plant cell with compressed helium gas or an electrical discharge. Recombination and replication of the DNA material transpires within the subject organism DNA prior to manipulations to increase copy number and trait selection. Vector-Facilitated Methods Vector-facilitated methods depend on the use of nonpathogenic viruses, or sections of bacterial DNA to transfer portions of the transgene or the transgene in its entirety using the biological invasive capabilities of the vector. Essentially, the vector DNA is modified and subsequently allowed to ‘infect’ the subject DNA to facilitate DNA transfer to the host. These vectors are frequently engineered to eliminate their virulence yet retain the DNA transfer capability of the original pathogen.
Commonly Produced GMOs GMO technologies discussed above and others have been used to create a variety of modified crop, fish, and animal species for crop prey resistance, pharmaceutical development, and increased production from livestock. It is these applications that have attracted public and scientific interest over the years. As will be noted below, several organisms are currently used, though it should be anticipated that additional organisms will be employed in the future. Genetically Modified Crops
The use of genetically modified corn and cotton has increased over 10-fold from 1992 to 1999 and as of 2002, 50 crop species have been evaluated for uses by the US Food and Drug Administration (FDA). In the development of transgenic crops, genes isolated from several varieties of the bacterium, Bacillus thuringienses (Bt) are probably the best known and most often cited example of GMO development. The use of this bacterial species has been deemed to be ideally suited for GMO use due to possession of toxins known as delta endotoxins. The structure of the delta endotoxin, is complex, containing three major regions or sections that each connote differential characteristics to the ultimate toxin (Figure 1).
Marker Gene Incorporation The mere delivery of the transgene into the host species or individual does not guarantee that the production of a GMO will be successful. This is due to the relative inefficient transfer process and the somewhat random selection process that must subsequently be used to enhance the number of individual copies of the transgene for further trait development. In order to address this limitation, genes or DNA segments coding for known and readily observable phenotypes (i.e., marker genes) are ‘coinserted’ with the transgene to better detect successful transformations. Such marker genes are now commonly used to detect color expressions or other visible attributes for better identification and subsequent selection and enhancement of the desired trait.
Figure 1 Molecular structure of Bacillus thuringienses delta endotoxin. (Reproduced from Li J, Carroll J, and Ellar DJ (1991) Nature 353: 815–821, with permission of the Nature Publishing Group.)
434 Genetically Engineered Foods
The domains of the molecule have markedly different conformations that control key aspects of ‘Bt’ toxicity to target insects. The a-helical bundles of the domain I region can be inserted into the gut cell membrane of the target species, thus, facilitating ion leakage. The domain II region consists of three b-sheet conformations that are structurally similar to immunoglobulin antigen binding regions, suggesting that the key characteristic section resides in the gut area. Domain III is a densely packed structure that is believed to protect the exposed end of the toxin from cleavage by digestive proteases; thus helping to ensure structural integrity and subsequent toxicity to the target species. By incorporating these characteristics of the Bt toxin into valued crop species, crop resistance to insects feeding on crop species may be enhanced helping to increase vitality, size, and acreage yields. Coded genes for the Bt delta endotoxin production have been isolated for insertion into a variety of vegetables including potatoes, field corn, sweet corn, and soybeans. Genetically Modified Fish
No genetically modified fish have been approved for entry into the US human food supply as of this writing although growing pressure for such use is anticipated. However, as with crop species, the use of GMO technologies in fish has been deemed useful for obtaining an increase in production, size, disease resistance, optimal food consumption, and pharmaceutical development. Common species selected for research into the application of transgenic manipulations include salmon, tilapia, channel catfish, and medaka. Alternatively, very active research in the use of transgenic fish as research models has been ongoing for several years with ever increasing focus on their use in the investigation of mutagenicity and environmental toxicology. Typical organisms for such applications tend to be small and easily cultured species such as medaka, mummichog, zebra fish, and others displaying favorable genetic attributes. Transgenic fish were noted as being first introduced into the research community in 1985. Since that time, the use of transgenic fish to refine methods
of transgene manipulation and application as potential analytical tools has grown substantially in the assessment of mutation frequencies. Several mutation assays using bacteriophage and plasmid vectors have been developed. Examples are shown in Table 1. As can be noted, detection methods may be based on modifications in enzyme systems that become evident in subsequent growth of bacterial species on selective growth media. In concert with the mutation assays that are shown in Table 1, the potential use of transgenic fish in the assessment of environmental health risk assessment offers opportunities to further assess the effects of water- and sediment-associated contaminants in the environment. Genetically Modified Livestock
The genetic modification of livestock stems from a desire to enhance growth, increase production of high protein milk and cheese, facilitate biomedical research, as well as potentially protect against incidental toxicity via exposure to pesticides that may be associated with food crops. Animal genomes that have been successfully modified include sheep, pigs, cows, rabbits, and chickens. Two key research areas applied to livestock are discussed below. Enhanced Production Growth, as a primary metric of production, has been and is the subject of much research. Of substantial interest is the potential for use of transgenes in stimulating ‘overproduction’ of growth hormones to enhance animal growth. Several test protocols have been evaluated with moderate success in various livestock models. For instance, blood levels of zinc-induced growth hormones have been increased in pigs and sheep. However, such activity was minimal in pigs and negligible via attempted phosphoenolpyruvate carboxykinase induction. Enhanced growth, via increased production of growth hormones also has been used to modify the qualitative nature of the animal such as lean muscle mass. However, such manipulations were not without negative effects such as renal failure and gastric ulcers of the subject animal.
Table 1 Various transgenic fish mutation assays Assay
Vector/fish species
Detection method
lacI Mutation assay cII Mutation assay rpsL Mutation assay lacZ Mutation assay
Bacteriophage l LIZ vector/medaka Bacteriophage l/medaka pML4 Plasmid vector/zebra fish Plasmid pUR288 vector/medaka and mummichog
a-Galactosidase functionality Plaque formation Kanamycin and streptomycin resistance Galactose-sensitivity
Genetically Engineered Foods 435
Pharmaceutical Development As of 2001, three companies were engaged in the manufacture of B30 pharmaceuticals to produce proteins and antibodies derived from transgenic procedures. The transgenic-mediated production of pharmaceuticals is primarily achieved by facilitating construction of proteins that can then be expressed in milk of organisms are often termed ‘bioreactors’. Examples of biologically active proteins that have been produced include antitrypsin, tissue plasmogen activator, and human blood clotting factor.
Human Health Risk Although potentially beneficial, the development of GMOs is fraught with concern over the potential for adverse effects on human health. While human health effects of popular concern consist of increased toxicity, decreased nutritional value of food, and increased antibiotic resistance, possibly the most widely recognized concern centers on the potential increase in allergic reactions of individuals consuming transgenically modified food. Approximately 1–2% of the population and 5–8% of children experience food allergies that are the result of natural selection processes that have occurred over thousands of years. Elevations in these frequencies may suggest allergic reactions to GMOs. Such allergic reactions are of two general types, immuoglobulin E (IgE)-mediated and non-IgE mediated reactions. IgE-mediated allergenicity requires that individuals must first be exposed to an allergen in a sensitization dose prior to showing overt signs of the allergy. In this reaction, antigen specific binding of IgE to mast cells and basophils, followed by release of pharmacologically active chemicals such as histamines, cytokines, chemokines, and arachidonic metabolites are ultimately responsible for the classic rapid allergic reaction often termed ‘immediate hypersensitivity reactions’. Toxic responses may range from, dermatitis, urticaria, and itching, to fatal anaphylactic shock and may be induced by proteins associated with a variety of foods including peanuts, grains, and fish. Non-IgE mediated allergic reactions do not require a sensitization exposure and may occur especially in infants and children consuming milk protein and grains, and are characterized by a delayed onset of symptoms after exposure to the food. Food-induced enterocolitic syndromes caused by milk protein ingestion may result in vomiting, diarrhea, and general deterioration of the individual. Celiac disease is a specific example of such a wasting syndrome stemming from a reaction to cereal or grain (i.e., wheat, rye, barley, oats, and spelt) ‘glutens’. Individuals
experiencing this disease may show weight loss, diarrhea, abdominal cramps, gas and bloating, general weakness, oily stool, and stunted growth in children. Though significant in terms of effects to an afflicted individual, only B1 in 300–3000 individuals in a population seem to be affected by celiac disease. To date, no confirmed cases of increased allergic reactions to GMOs have been documented and thus the extent to which genetically modified food contributes to significant allergic reactions in the population is not accurately known. The only reported incident of potential GMO allergenicity occurred after production of soybeans modified with Brazil nut protein. Allergenicity to the genetically modified soybean was detected and the product was not marketed, precluding exposure and toxicity. The previously discussed StarLinkTM episode is perhaps the most widely evaluated incidence potentially leading to potential health effects associated with GMOs. Using enzyme-linked immunosorbent assay (ELISA) in a retrospective study, the US FDA hypothesized that exposure to the Bt Cry9c protein could be cause for allergic responses. However, the results of the FDA research concluded that there was no evidence of GMO-mediated allergenicity subsequent to potential exposure to the StarLinkTM corn. Because the lack of evidence of health effects cannot conclusively show that GMOs are not a human health concern, current global efforts are focused on the development of protocols to proactively and systemically detect and assess potential adverse effects.
Environmental Health Risk The American Medical Association (AMA) has estimated that in 1999, 200 million acres of land had been planted worldwide with transgenic crops. The AMA further indicated that over 25 000 field trials for environmental effects of GMOs had been performed in 45 countries without noted adverse environmental consequences. Despite these conclusions, the limited geographical size and comprehensiveness of such trials confounds definitive conclusions regarding the potential for adverse effects such as enhanced crop pest resistance, out crossing with weedy relatives of crops, reduced biodiversity, nutritional deficiency of food sources, and toxicity to nontarget species. Concerns over the safety of transgene introduction into environment was sensitized early in the GMO debate with significant focus on the potential toxicity of Bt endotoxins to monarch butterfly (Danaus plexippus) larvae exposed to transgenic pollen. Early data suggested that Bt corn pollen could result in potentially significant reactions in the monarch gut.
436 Genetically Engineered Foods
However, at that time (mid-1990s), the general regulatory consensus in the United States suggested that the adverse effects of Bt toxins were negligible when actual exposure conditions in the field were considered. In 1999, laboratory tests were conducted to determine if Bt transformed corn pollen consumed by monarch larvae at environmentally significant concentrations could result in adverse effects under well-controlled conditions. The widely published data resulting from this testing, indeed showed that larvae consuming Bt corn pollen experienced a decreased growth rate, expressed as the concentration of protein (0.76 ng ml 1) required to result in growth reduction of 50% of the population (EC50). Mortality, expressed as the LD50, was determined to occur at 3.3 ng protein ml 1 diet. Estimated pollen densities of B10 and 50–100 grains cm 2 were deemed to potentially result in these adverse effects. As a potentially significant environmental and ecological risk, such Bt pollen toxicity notably stirred the interest and emotions of the scientific and public interest communities. In 2001, a collaborative research effort of Canadian and US scientists developed a weight of evidence risk assessment to address the concern. The risk assessment results suggested that the likelihood of an adverse effect on the monarch population was less than 1 in 10 000 (or less than 1 10 4) when the toxicity information and exposure estimates were integrated into a risk analysis. While these data support the conclusion that an adverse effect on the monarch receptor was unlikely, it should be noted that the absence of data suggesting risk under given assumptions and circumstances should be interpreted carefully as a general conclusion of safety in all instances. The concern for adverse ecological effects is not limited to plant or insect communities and direct toxicity. For instance, experimental data collected using fish suggests that the incorporation of transgenes may actually benefit the transgenic individual. For instance, mathematical models have been developed and evaluated to assess population effects of the transgenic fish of wild populations. Using the same data that show that the transgene may benefit the individual, models have predicted that the inadvertent release of the transgenically modified fish into the environment may result in significant adverse environmental effects such as invasion by genetically modified fish resulting in increased competition with and displacement of native species, reduced hardiness of the modified fish offspring in the wild, and potential extinction of the naturally occurring population. Although such effects have not been conclusively supported by field evaluations or verifying laboratory data, the concerns for long-term adverse
ecosystem effects remains a subject of continued research. As noted earlier in this article, transgenically modified growth hormones have been administered to cattle to increase milk production. However, it has been noted that the administration of growth hormones can result in adverse effects in the exposed animal including, fertility reduction, changes in bone growth, increase chance of mastitis, and reductions in endocrine function. The long-term effects of inadvertent exposures to the transgene-modified growth hormone on nontreated cattle herds, livestock, or breeding stock is poorly understood.
Safety Evaluation Methodology The complexity of GMO introduction to the human food chain as well as uncertainties regarding potential ecosystem effects has prompted governmental agencies and NGOs to recommend highly structured and systematic safety evaluation protocols prior to GMO release. While several protocols have been proposed, developed, and are being adopted worldwide, those of the European Union and the WHO provide reasonable and illustrative examples for reference. Substantial Equivalence
A fundamental concept essential to the currently defined approaches for determining GMO safety is that of ‘substantial equivalence’ (SE). Used as the basis for establishing a comparative benchmark, SE depends on two key concepts. First, existing traditional foods are assumed to be safe as evidenced by their longterm use. Second, the response to traditional foods can be used as a basis for comparison to transgenically modified foods which are often derived from traditional foods. Predicated on the definition of substantial equivalence, a decision process for further evaluation of the GMO can be defined according to the general rules shown in Table 2. While the decision matrix shown in Table 2 suggests a rather simple testing approach, in reality, adherence to the SE concept requires that it be applied to a multitude of characteristic variables including chemical SE, biological SE, SE of potential exposure routes, and the SE of possible overall safe use. The WHO Decision Tree for Assessment of Potential Allergenicity
Allergenicity is the current overriding concern regarding potential human exposures to GMOs. Thus, the
Genetically Engineered Foods 437 Table 2 Simplified decision matrix for evaluation of GMO safety according to the concept of substantial equivalence If the GMO demonstrates
Then
Substantial equivalence to traditional food Substantial equivalence to traditional food but also demonstrates evidence of a specific new trait No substantial equivalence to traditional food
No further testing or evaluation is necessary The assessment must focus on the potential effects of the new trait or gene Comprehensive toxicological and nutritional testing is required
Is gene source allergic? Yes
No
DNA sequence homolgous to traditional food?
DNA sequence homolgous to traditional food?
Yes
Possible Allergen
No
Yes
Is specific serum screen positive?
No
Yes
Is targeted serum screen positive?
No No Yes Yes Demonstrable animal model allergenicity
No Yes
Yes
High
Intermediate
Yes No
Demonstrable pepsin resistance
Low No
No
Figure 2 Decision tree for the evaluation of allegenic potential associated with GMO. (Adapted from FAO/WHO (2000) Safety Aspects of Genetically Modified Foods of Plant Origin. Geneva, Switzerland: World Health Organization.)
WHO has developed a decision tree approach to determine the human health risk from exposure to GMOs. The decision tree and associated rationale and documentation is best reviewed in WHO records. However, Figure 2 shows the general approach (with modifications for presentation purposes) suggested by WHO to determine if a GMO is a potential allergen. The decision tree is not a prescriptive and mandated method with substantial recommended technical protocols but rather is a conceptual model to help the evaluator determine the potential that the GMO
may trigger allergic reactions in greater proportion than the unexposed populations. As noted by examination of Figure 2, the incorporation of comparisons with traditional foods coupled with an allowance for graded responses ensures that the risks of allergic reactions are assessed in the context of exposures and the substantial equivalence concept.
Concluding Remarks The development of GMOs, and genetically modified foods in particular, is, in large part, a direct response to ever increasing global food demands. Current
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information suggests that GMOs are unlikely to have substantial adverse near term effects on human health and the environment. However, the assessment of the potential for longer-term adverse effects poses a greater challenge. As a result of perceived needs, the technology inspired over the last 20 years and continues to develop; both in methodology and application. As a result of increased concerns over health effects, such activities will be scrutinized with growing vigilance on the part of citizens, scientists, regulatory bodies, and advisory commissions; ultimately to assure nearand long-term protection of global human health and the environment.
See also: Food and Drug Administration, US; Toxicity Testing, Mutagenicity; Transgenic Animals.
Corn. A Report to the U.S. Food and Drug Administration from the Centers for Disease Control and Prevention. Cockburn A (2002) Assuring the safety of genetically modified (GM) foods: The importance of an holistic, integrative approach. Journal of Biotechnology 98(1): 79–106. Konig A, Cockburn A, Crevel RW, et al. (2004) Assessment of the safety of foods derived from genetically modified (GM) crops. Food and Chemical Toxicology 42(7): 1047–1088. Kuiper HA, Konig A, Kleter GA, Hammes WP, and Knudsen I (2004) Safety assessment, detection and traceability, and societal aspects of genetically modified foods. European Network on Safety Assessment of Genetically Modified Food Crops (ENTRANSFOOD). Concluding remarks. Food and Chemical Toxicology 42(7):1195– 1202 (review). World Health Organization, Food and Agriculture Organization (2000) Safety Aspects of Genetically Modified Foods of Plant Origin. Geneva, Switzerland: World Health Organization.
Further Reading Bakshi A (2003) Potential adverse health effects of genetically modified crops. Journal of Toxicology and Environmental Health B Critical Reviews 6(3): 211–225. CDC (2001) Investigation of Human Health Effects Associated with Potential Exposure to Genetically Modified
Genetically Modified Organisms
Relevant Website http://www.pbs.org – Tyson P (2001), NOVA Public Broadcasting System, ‘Should We Grow GM Crops’ Documentary aired April 24, 2001.
See Genetically Engineered Foods.
Genomics, Toxicogenomics Kartik Shankar and Harihara M Mehendale & 2005 Elsevier Inc. All rights reserved.
Toxicogenomics refers to the application transcriptonomic (high-throughput analyses of gene expression) techniques to the field of toxicology. Classically, toxicologists examine potential adverse outcomes and putative mechanisms due to xenobiotic exposure by biochemical and histopathological markers of toxicity. In the current healthcare and regulatory environment, chemicals suspected to have potential for significant adverse health effects are selected to undergo subsequent testing for carcinogenicity and chronic toxicity. However, long-term studies are typically labor intensive and time consuming and can cost $2–4 million. As of 2002, the National Toxicology Program had tested or is currently testing 505 chemicals in long-term studies, 66 in short-term studies, and only a single chemical in a subchronic study. Given that almost 70 000–85 000 chemicals
are used in commerce today, it is evident that alternative high-throughput methods for screening the toxic potential of chemicals is needed. This would allow for prioritization of untested chemicals in the classical approach of toxicity testing. Concurrent with rapid advances in bioinformatic tools and classification algorithms toxicogenomic analyses is fast becoming a viable option in high-throughput screening of potentially hazardous chemicals.
Applications of Toxicogenomics Predictive Toxicology
The underlying premise of toxicogenomics is that toxicity is associated with changes in the global gene expression. Since toxicity by itself is resultant due to some form of cellular dysfunction or cell death, it will either be preceded or followed by some level of