3
Unintended Effects from Breeding

This chapter explores the likelihood of unintended effects from diverse methods of genetic modification of plants and animals (see Operational Definitions in Chapter 1). Specifically, it discusses unexpected outcomes of breeding methods used to develop a food crop or strain and unexpected or unintended effects recorded in the scientific literature. It also includes analyses of methods intentionally used for modifying food sources and comparing the likelihood of unintended changes resulting from the use of genetic engineering versus other methods of genetic modification discussed in Chapter 2.

BACKGROUND

Novel gene combinations arising from the genetic manipulation of existing genes through conventional breeding techniques may introduce unintended and unexpected effects. However, through the breeder’s selection process, the genetic lines that express undesirable characteristics are eliminated from further consideration, and only the best lines—those that express desirable characteristics with no additional undesirable agronomic characteristics, such as increased disease susceptibility or poor grain quality—are maintained for possible commercial release. Although plants and animals produced from conventional breeding methods are routinely evaluated for changes in productivity, reproductive efficiency, reactions to disease, and quality characteristics, they are not routinely evaluated for unintended effects at the molecular level. New varieties of food crops, other than those produced using recombinant deoxyribonucleic acid (rDNA) technologies, are rarely subjected to toxicological or other safety assessments (WHO, 2000). Previous National Academies committees have addressed the question of



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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects 3 Unintended Effects from Breeding This chapter explores the likelihood of unintended effects from diverse methods of genetic modification of plants and animals (see Operational Definitions in Chapter 1). Specifically, it discusses unexpected outcomes of breeding methods used to develop a food crop or strain and unexpected or unintended effects recorded in the scientific literature. It also includes analyses of methods intentionally used for modifying food sources and comparing the likelihood of unintended changes resulting from the use of genetic engineering versus other methods of genetic modification discussed in Chapter 2. BACKGROUND Novel gene combinations arising from the genetic manipulation of existing genes through conventional breeding techniques may introduce unintended and unexpected effects. However, through the breeder’s selection process, the genetic lines that express undesirable characteristics are eliminated from further consideration, and only the best lines—those that express desirable characteristics with no additional undesirable agronomic characteristics, such as increased disease susceptibility or poor grain quality—are maintained for possible commercial release. Although plants and animals produced from conventional breeding methods are routinely evaluated for changes in productivity, reproductive efficiency, reactions to disease, and quality characteristics, they are not routinely evaluated for unintended effects at the molecular level. New varieties of food crops, other than those produced using recombinant deoxyribonucleic acid (rDNA) technologies, are rarely subjected to toxicological or other safety assessments (WHO, 2000). Previous National Academies committees have addressed the question of

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects whether unintended effects arising from the use of rDNA-based technologies in food production and the risks potentially associated with them differ in nature and frequency from those associated with non-rDNA-based breeding methods (NRC, 1987, 2000, 2002). There is a considerable amount of data compiled and available in the scientific literature addressing issues related to genetically modified (GM) and genetically engineered (GE) plants, including health and environmental impacts. In contrast, development of transgenic animals is a relatively new area of biotechnology, and so the amount of data collected and reported for GM animals is less than that for plants. However, the application of genetic modification techniques and the potential for unintended adverse effects are similar for both plants and animals, and much of the information obtained from plants can be applied to questions of concern in the genetic modification of animals. PLANT BREEDING Conventional Plant Breeding Conventional plant production occasionally generates foods with undesirable traits, some of which are potentially hazardous to human health. Most crops naturally produce allergens, toxins, or other antinutritional substances (see Chapter 5). Standard practice among plant breeders and agronomists includes monitoring the levels of potentially hazardous antinutritional substances relevant to the crop. For example, canola breeders monitor levels of glucosinolates in breeding lines under consideration for prospective commercial release, while potato breeders monitor for glycoalkaloid content. If a particular breeding line generates too much of an undesirable substance, that line is eliminated from consideration for commercial release. In the United States, the plant breeding community is largely self-monitored. Regulatory agencies do not evaluate conventional new crop varieties for health and environmental safety prior to commercial release. Some other countries require government agencies to conduct premarket evaluations for new crop varieties, both conventional and biotechnology-derived. Canada has a “merit system” for the commercial release of new varieties of major field crops, in which candidate varieties are grown in government-administered field trials. Performance data from these trials, related to agronomic factors, disease resistance, and food quality characteristics, are compiled for all candidate and standard commercial varieties. The data are collected from multiple locations over multiple years, as cereal chemists analyze the grain for chemical and nutritional composition and plant pathologists conduct tests to determine reactions to relevant diseases. These data are then evaluated by a team of experts from industry, government, and universities. The breeder of each candidate variety must convince these experts that it is competitive and worthy relative to other commercially available varieties of that

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects crop, based on the performance data from the trials. Only if the committee agrees is the candidate variety allowed to be registered as a new commercial variety. If the variety does not perform within prescribed parameters for all characteristics, it is not commercially released (CFIA, 2003). Unintended Effects of Conventional Plant Breeding Naturally Occurring Toxins All foods, whether or not they are genetically engineered, carry potentially hazardous substances or pathogenic microbes and must be properly and prudently assessed to ensure a reasonable degree of safety. Furthermore, all crop strains, including organic strains, potentially express traits generated by various forms of induced mutagenesis. (Under organic regulations, radiation breeding and induced mutagenesis are acceptable, but irradiation of the final food itself is not. For more information on organic regulations, see USDA-AMS, 2001.) History provides examples of traditional breeding that resulted in potentially hazardous foods (see Box 3-1). Solanaceous (tobacco family) crops, such as potato and tomato, naturally produce various steroidal glycoalkaloids. These substances are toxic not only to humans, but also to insects and pathogenic fungi. During the course of ordinary plant breeding assessments, breeding lines with increased levels of glycoalkaloids may be identified by the breeder as showing superior insect or disease resistance and retained for possible commercial release. The elevation of glycoalkaloid levels responsible for the pest tolerance may not be noted until people become ill from consuming the foods. Tomatine, a glycoalkaloid naturally present in tomatoes, can be produced in hazardous quantities in certain conventionally bred varieties. Ordinarily, alpha tomatine is present in immature tomato fruit, but is degraded as the fruit matures, so that by the time the fruit ripens to the preferred stage for human consumption, tomatine content is reduced to safe levels. Nevertheless, levels of naturally occurring tomatine in ordinary tomatoes bred using conventional methods can vary considerably, primarily based on maturity, type, and environmental growing conditions (Gilbert and Mohankumaran, 1969; Leonardi et al., 2000). In this respect, environment is more responsible for food hazards than genetic makeup or breeding method. Another example of a possible effect is the unintended elevation of glycoalkaloid content in potatoes. All potatoes produce the toxic glycoalkaloid solanine, but mature potatoes from most cultivars have amounts so small as to be nonhazardous. However, some varieties produce more than others, and certain environmental stimuli, such as growing or storage conditions, can cause potentially hazardous increases in solanine content, even within a usually safe cultivar (Concon, 1988). For example, potatoes exposed to sunlight turn green, making them particularly prone to high solanine content. Dark-skinned varieties are less

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects BOX 3-1 Kiwi: A New Food? Puffer fish, chile peppers, and mustard all are traditional foods that humans have learned to consume in moderation to minimize adverse health effects. But what about foods entirely new to the human digestive tract? Although some have designated (GE) foods as completely novel, the GE varieties currently approved and on the market are of the same composition as other foods. Corn oil, for example, is chemically identical regardless of the breeding method used to develop the corn variety. In recent history, the closest example of a new food might be kiwi fruit. Originally it was an edible but unpalatable plant producing small, hard berries in China. Breeders in New Zealand developed what we know now as kiwi fruit (Actinidia deliciosa) into a food during the twentieth century, and commercialized in the United States during the 1960s. There does not, however, appear to be any official record of a premarket safety analysis of the fruit. As a consequence, some humans who were not previously exposed to kiwi fruit developed allergic reactions. Recently, well after commercial release, the responsible allergenic protein (actinidin) was isolated and characterized (Pastorello et al., 1998). likely to turn green than light-skinned varieties, but in either case environment seems to be more important to solanine production than genetics. Certain potato lines have been found to express greater disease- or pest-resistance, and they have been selected as superior, not always with favorable or intended results. The most notorious such selection was the Lenape potato, which was developed using conventional breeding methods (Akeley et al., 1968). After a successful commercial launch, it was found to have dangerously elevated solanine content in the tubers and was removed from the market (Zitnak and Johnston, 1970). More recently, a similar high-solanine potato variety was detected and withdrawn from the market in Sweden (Hellanäs et al., 1995). In this case, the potato was a heritage variety, developed in the United Kingdom during the nineteenth century, but superseded by another variety due to its susceptibility to disease (Kuiper, 2003). Nevertheless, it became popular in Sweden under the name “Magnum Bonum,” until its predilection for overexpressing solanine resulted in its commercial demise (Hellanäs et al., 1995). In spite of occasional problems with the consumption of potato glycoalkaloids, conventional breeders continue to increase the glycoalkaloid content in the leaves to take advantage of its pest- and pathogen-deterrent properties. Consequently, the U.S. Department of Agriculture (USDA) recommends, but does not require, a limit for glycoalkaloid content in new potato varieties (Sinden and Webb, 1972).

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects Interestingly, the Lenape (also known as breeding line no. B5141-6) potato has been found to express some useful attributes, such as high solids content. Thus Lenape continues to be used successfully as a parent in conventional breeding programs, providing its genes to new commercial potato varieties, such as Atlantic and Denali. Progeny with genes providing the high solids content are selected or maintained, and the genes responsible for high solanine content in the tubers are selected against or rejected. Additionally, Lenape has been transformed with a genetic construct containing the solanidine glucose-adenosine diphosphate glucosyltransferase (SGT) transgene in the antisense direction, which is designed to interfere with the solanine biosynthesis (Moehs et al., 1997). In field trials, several transgenic Lenape-derived lines expressed substantially less solanine than the parent Lenape, apparently due to antisense expression, an rDNA method for “turning off” an undesirable gene (McCue et al., 2003). Several breeding programs are developing potatoes derived from conventional crosses between the ordinary potato Solanum tuberosum and relatives of other species, such as S. acaule (Kozukue et al., 1999) or S. chacoense (Sanford et al., 1998; Zimnoch-Guzowska et al., 2000). These are conventional breeding programs in which genes from two different species are exchanged. The intent of these conventional breeding programs is to generate potato varieties with new beneficial features, while minimizing deleterious traits from the foreign species. Breeders typically monitor levels of toxins in plants that are known to naturally contain them, even though such monitoring is only voluntary in the United States. However, an unexpected and unintended problem may result when combining different species because thousands of genes would be interacting, not just one or two genetic elements of interest. For example, hybrids of S. tuberosum and S. brevidens produced not only the usual glycoalkaloids, but also the toxin demissidine, which is not produced in either parent (Laurila et al., 1996). This singular result shows that non-genetic engineering breeding methods can have unintended effects and generate potentially hazardous new products. Any time genes are mutated or combined, as occurs in almost all breeding methods, the possibility of producing a new, potentially hazardous substance exists. Conceivably, similar outcomes could result from using rDNA to transfer specific genes from S. brevidans to S. tuberosum, giving rise to hybrids expressing the novel toxin demissidine. In either case, the hazard lies with the presence of the toxin, and not with the method of breeding. Genetic engineering could also be used to transfer only the beneficial genes from S. brevidans, leaving behind the genes responsible for the novel toxin. Another example of a toxic compound from traditional crops is psoralens in celery (see Box 3-2). Celery naturally produces these irritant chemicals that deter insects from feeding on the plant and also confer protection from some diseases (Beier and Oertli, 1983). Celery plants with an elevated expression of psoralens will suffer less damage from disease and insect predation and have more aesthetic appeal to consumers, who tend to reject insect- or disease-damaged produce.

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects BOX 3-2 Unintended Health Effects: A Conventionally-Bred Celery Cultivar Genetic engineering has raised the question of whether its products create unintended health effects for consumers, but how do conventionally bred products compare along the continuum of genetic modification? Celery provides an interesting case, as it contains naturally occurring toxic chemicals called psoralens, which are secondary metabolites found in a variety of fruits and vegetables, and members of the class of compounds called furanocoumarins (Diawara and Kulkosky, 2003). The psoralen in celery provides it with a biological defense mechanism of sorts—if it suffers disease or has been bruised, the celery plant can produce up to 100 times the level of psoralen that it typically contains, thus protecting itself from attacks by pests. The production of psoralens also depends on factors such as temperature, season and, particularly, availability of sunlight (Diawara et al., 1995). Psoralens temporarily sensitize human skin to longwave ultraviolet radiation. Consequently, they have been used for more than 25 years as a component in phototherapy—a means of treating acute skin diseases (New Zealand Dermatological Society, 2002). However, psoralen in celery has been implicated in cases of skin irri-tation, such as dermatitis, among farm workers and other handlers of these plants, and studies have suggested a potential correlation between psoralens and cancer in laboratory mice (Beier, 1990). Moreover, celery cultivars produced using conventional breeding methods—intended to enhance insect-resistance and aesthetic appeal to consumers through increased production of psoralen—have been associated with cases of dermatitis among grocery workers, as well as further cases of photosensitivity among farm workers handling these plants (Ames and Gold, 1999). This form of dermatitis, or “photodermatitis,” has been observed as far back as 1961 in field workers who had handled celery infected with the disease pink rot (Birmingham et al., 1961). The Centers for Disease Control and Prevention reports cases dating back to 1984 of severe skin rashes among laborers who handled celery on a regular basis. The initial onset of these cases spawned a study by the National Institute for Occupational Safety and Health, in which there appeared to be a relationship between the handling of celery and prolonged exposure to sunlight. In the study that was undertaken in 1984, a number of grocery workers who handled celery on a regular basis also had used a tanning salon, suggesting to researchers that the ultraviolet light from tanning had exacerbated the reactions of the workers’ skin in response to the celery they had handled (CDC, 1985). Exposure to elevated levels of psoralens remains a problem for laborers such as field workers, as constant exposure to sunlight—combined with the handling of vegetables such as celery—can induce reactions such as those occurring in the skin. Psoralens continue to be regarded as naturally occurring toxicants (Beier, 1990).

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects As a result, breeders may observe healthy, undamaged celery lines and select them for commercial release. Unfortunately, workers who harvest high psoralen-producing celery or pack it in grocery stores have, on occasion, developed severe photodermatitis, especially when they are exposed to bright sunlight and the celery is infected with pathogens (Berkley et al., 1986; Birmingham et al., 1961; Finkelstein et al., 1994). There are differences in psoralen content from one variety to another (Beier, 1990; Diawara et al., 1993), but environment seems to have the greatest influence on psoralen production (e.g., Diawara et al., 1995). Mutations Mutations, defined as any change in the base sequence of DNA, can either occur spontaneously or be induced, and both methods have produced new crop varieties. Most mutations are deleterious and therefore useless for breeding purposes. However, a mutation can result in desirable traits and may be selected for breeding. Spontaneous mutations, also called “sports” by horticulturists, are by definition not induced, so breeders wait a long time to see mutants arising from this process, and even longer to see useful ones. Most mutation breeders induce random changes in DNA by using ionizing radiation or mutagenic chemicals, such as ethyl methane sulfonate, to increase the rate and frequency of the mutation process. In spite of these intrusive methods, induced mutagenesis is considered a conventional breeding technique. Food derived from mutation breeding varieties is widely used and accepted. Organic farming systems permit food from mutated varieties to be sold as organic. In the United States many varieties have been developed using induced mutagenesis, such as lettuce, beans, grapefruit, rice, oats, and wheat. The Food and Agriculture Organization of the United Nations/ International Atomic Energy Agency Mutant Cultivar Database (FAO/IAEA, 2001) lists more than 2,200 varieties of various species worldwide that have been developed using induced mutagenesis agents, including ionizing irradiation and ethyl methane sulfonate. However, the database does not include spontaneous mutations, cell-selected mutants (Rowland et al., 1989), or somaclonal variants (Rowland et al., 2002). There is no mandated requirement to list new mutant varieties with the database. However, there do not appear to be outstanding examples of mutant varieties with documented unexpected effects beyond what the mutant was selected for, despite the expectation that mutant varieties may possess and generate more unexpected outcomes than ordinary crosses because of the unpredictable and uncontrollable nature of nontargeted mutations. Furthermore, there do not appear to be any examples in which mutant varieties were removed from the market due to unintended or unexpected adverse incidents.

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects Genetic Engineering in Plants As for conventional breeding techniques, the genetic engineering (GE) breeding and selection process is largely self-monitored and only new varieties of rDNA crops are subjected to assessment prior to commercialization. Evaluation of GE food products for other countries is described above, in the section Conventional Plant Breeding. Genetic engineering methods are considered by some to be more precise than conventional breeding methods because only known and precisely characterized genes are transferred. In contrast, conventional breeding involves transferring thousands of unknown genes with unknown function along with the desired genes. Similarly, in mutation breeding hundreds or thousands of random mutations are induced in each mutated line. Plant breeders take a variety of methods used to introduce desired traits into consideration during selection. The location where DNA expressing the desired traits is inserted into the host genome in rDNA technology may be immaterial—for example, if an insertion is made in an inappropriate place, the transformed plant is eliminated or selected against by the breeder, who will then select another plant with a preferable locus of insertion for continued evaluation and development as a new variety. The number of different lines developed by breeders varies according to the crop, the desired trait, and the choice of the breeder, but is it not unusual to start with evaluations of 2,000 “sister” lines from a cross of two parents to develop just one new variety. Thus 99 percent of sister lines are eliminated over the several years of evaluation, for various reasons. These include poor expression of the desired trait, poor yield performance, increased disease susceptibility, or even a lack of visual and tactile appeal, which is a largely subjective and arbitrary designation, but an important criterion nevertheless. Genetic engineering techniques require fewer lines or transformation events because the desired trait is known and identified early. Consequently, the evaluations—which still take several years—focus on eliminating any unstable lines or those with deleterious characteristics. In contrast to breeders using other techniques, genetic engineering breeders typically prefer to start with a small number of plants and then select only a few. For example, the two currently approved GE plant varieties from public institutions, papaya (Gonsalves, 1998; Swain and Powell, 2001) and flax (McHughen et al., 1997), started with only about 30 sister lines. Unintended Effects from Genetic Engineering Unexpected and unintended effects can be seen with all methods of breeding. Traditionally breeders observe such off-types regularly; they methodologically eliminate these individuals during the evaluation process, long before prepara-

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects tions are made for commercial release. An unexpected or unintended effect does not imply a health hazard, although clearly a plant expressing novel and unexpected characteristics warrants closer inspection prior to commercial release. A large body of transgenic plant material is developed by university and government laboratories investigating aspects of rDNA that are not related to commercial interests. The Organization for Economic Cooperation and Development (OECD) lists more than 10,000 field trials with GE plants that were conducted between 1986 and 1999. Most of these authorized trials were conducted on plants genetically engineered by public universities and government research institutions, not for commercial release but to test the plants for unexpected or unintended results (see OECD, 2000). Consequently, most of the information on unexpected or unintended results comes from these sources. Because GE crops are regulated to a greater degree than are conventionally bred, non-GE crops, it is more likely that traits with potentially hazardous characteristics will not pass early developmental phases. For the same reason, it is also more likely that unintentional, potentially hazardous changes will be noticed before commercialization either by the breeding institution or by governmental regulatory agencies. The following are among the examples of unexpected or unintended characteristics that are often cited in the scientific literature. Mycotoxin Content in Bt Corn Bt (Bacillus thuringiensis) corn was developed to help farmers protect corn crops from insect pests, particularly the European corn borer and, more recently, the corn root worm. An unexpected effect in this product was a substantial reduction in mycotoxins, which can adversely affect animal and human health. Researchers hypothesized that this is because mycotoxin production is stimulated by fungal spores that infect corn when worms bore into and injure the plant. In the absence of insect damage (as in the Bt corn plants), there is reduced opportunity for pathogenic fungal spores to infect the plant and therefore less opportunity to generate the associated and undesirable mycotoxins (Munkvold et al., 1997, 1999). Increased Lignin in Bt Corn Saxena and Stotzky (2001) reported that three Bt corn varieties increased their stem lignin content relative to their respective non-Bt isogenic parents. Since the increase was found in more than a single variety, this suggests that the lignin increase is directly associated with the inserted DNA and is not simply an isolated coincidence. However, increased lignin content has not been recorded in other Bt corn lines, so it is uncertain whether the reported increase was due to the rDNA insertion, the presence of Bt endotoxin, or some other mechanism. Al-

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects though lignin is a normal component of plants and of the human diet, and the increased lignin content in the stems of these corn plants is not so great as to present a novel health hazard, the authors do suggest that there might be an environmental consequence from a corn plant with lignin content that is significantly higher than normal. Petunias with Diminishing Color Over Generations In this example, a GE petunia line was noticed to have a diminished flower color pattern and intensity over the course of several generations. Molecular analysis showed that a mutation occurred in a gene that regulates flower pigmentation and methylation was responsible for inactivating the gene (Meyer et al., 1992). This phenomenon occasionally occurs with conventional breeding, so its occurrence in a GE petunia does not indicate a unique GE phenomenon. This mutation appears to have occurred in only one GE petunia line out of hundreds generated and tested, thus it is not a general or systematic event. Insertion of transfer DNA does not guarantee active expression of the associated gene. Inserted genes can be and are silenced or inactivated by the host plant by any of several mechanisms, including methylation (Dominguez et al., 2002). This is a well-known phenomenon and part of the reason genetic engineers generate as many different transgenic plants as possible. Other Examples Unexpected or unintentional effects, such as GE soybean stem splitting (Gertz et al., 1999) or Arabidopsis with unusual fertility or outcrossing characteristics (Bergelson et al., 1998), have been attributed to the rDNA breeding process. The GE soybean stem-splitting case documented that a high proportion of GE soybeans suffered split stems in dry, hot conditions. Unfortunately, the non-GE parent line was not measured for stem splitting under similar conditions, but other non-GE soybean varieties were, revealing that a high proportion also suffered split stems. They were not as prone to splitting, however, as the indicated GE line, but that line was within the general accepted range for soybeans. Whether the GE soybean lines tested were more prone to splitting than their parents remains unknown, and thus conclusions cannot be drawn as to whether the genetic engineering process contributed to this trait. In the Arabidopsis example, the authors compared the outcrossing of GE varieties with the outcrossing of a mutated strain that possibly had compromised fertility to begin with and was not an isogenic or near-isogenic line, necessary for properly conducting the type of experiment described. Other unexpected effects in GE plants have been documented. In GE high oleic soybean lines, metabolic analysis revealed trace amounts of an unintended metabolite, cis9,cis15-octadecadienoic acid, an isomer of the fatty acid linoleic

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects acid that is not usually present in nonhydrogenated soybean oil, but is present in hydrogenated soybean oil and in other food sources. Because it is a component of other foods, it was not considered a health threat. The developer of the lines, citing Kitamura (1995), argued that the fatty acid substitution was actually nutritionally advantageous. After consideration, the Food and Drug Administration (FDA) determined that the presence of the unexpected linoleic acid isomer and glycinin did not pose a health hazard (DHHS, 1996). USDA also evaluated the GE soybeans, as it does all GE plants, and approved it (APHIS, 1997). FDA did determine that the high oleic soybeans were not substantially equivalent to regular soybeans due to the intended effect of an elevated oleic acid content, thus the breeder had to distinguish them from standard commodity soybeans. The unexpected effects in these GE soybeans were small and required highly sensitive analytical tools to identify. Such small changes should be expected—although the exact metabolites and levels would remain unpredictable. Furthermore, because genetic changes result in protein changes, metabolic differences due to the presence or actions of new or differing levels of proteins cannot be unexpected. Such changes remain unintended, but they are common occurrences in new crop varieties, including new varieties from conventional breeding (although stringent and detailed analyses of conventional varieties are not conducted). An important consideration is how these examples of apparent unpredictability with rDNA compare with similar unpredicted effects from conventional breeding methods. Unfortunately, the comparisons typically are made only between these unusual rDNA examples and, if conventional plants are compared at all, it is not with isogenic lines but with mutant lines or with less closely related commercial varieties. The correct comparison should be between commercial cultivars of rDNA derivation and isogenic parental varieties. For example, the comparison between Arabidopsis and a herbicide-tolerant variety is misleading because the comparator was a mutant variety of uncertain genetic composition. In addition, as discussed earlier, undesirable, unexpected, and unintended traits are noted in conventional breeding lines of commercial crop species on rare occasions, and these lines are consequently discarded. ANIMAL BREEDING Conventional Animal Breeding Genetics is crucial for any livestock enterprise and requires breeders to evaluate production conditions and market objectives; the best breeds or lines of livestock for meeting market goals; selection of a breeding system and breed types; and selection of individual animals within the breed type. Generally, production conditions—so called gene-environment interactions—and market factors have the greatest influence on conventional animal breeding strategies. Breeders develop a breeding plan that may either be continuous if replace-

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects discussed earlier, unstable pigmentation in GE petunias provides an example of a systemic instability in particular GE plants (Meyer et al., 1992). Spontaneous mutations change DNA also, affecting everything from single base changes, known as “point mutations,” to entire genomes. Deletions of entire segments from a genome are predictably severe and deleterious, but point mutations or even more substantial mutations, such as those affecting large tracts of DNA in chromosomes, can be beneficial and adaptive. Because of the ubiquitous nature of spontaneous mutations, all plant varieties—whether conventional or GE—exhibit a small degree of genetic instability and generate mutants with some degree of frequency. If the frequency of spontaneous genetic instability is low, the typical loss-of-function mutants will probably not be noticed; if it is high, the variety cannot be commercialized. Natural types of recombination can also result in the same effects. Transposons, even Agrobacterium insertions, all interrupt any DNA sequence where they insert, regardless of whether they insert into homologous or nonhomologous DNA. Depending on the function of the interrupted DNA, there may or may not be phenotypic consequences from the insertion. Agrobacterium naturally inserts DNA into the host plant-cell genome, typically in a genetically stable manner. The substitution of desirable DNA by genetic engineering for the phyto-oncogenic DNA of the wild strains does not affect the mechanics of transfer. The genes responsible for Agrobacterium genetic transformation and recombination in the plant are physically and functionally separate from the T-DNA actually transferred and integrated. The mechanics of Agrobacterium appear the same whether the event is staged through a natural infection process by a crown gall-producing wild-type strain or by the same strain that has been disarmed by the removal of the gall-producing genes and subsequently replaced with known, desirable DNA. THE GENETIC MANIPULATION CONTINUUM Overview Predicting the likelihood of unintended hazards from compositional changes associated with genetic modifications does not fit a simple dichotomy comparing genetic engineering with non-genetic engineering breeding. This is because there are many mechanisms shared in common by both GE and non-GE methods, and also because there are techniques that slightly overlap each other. Furthermore, within the scope of genetic engineering (rDNA) technology, several mechanisms for genetically transforming plants are available as options to scientists, such as Agrobacterium-mediated gene transfer and the use of the particle gun (McHughen, 2000). These two examples of genetic engineering are as different mechanistically as the conventional methods of narrow crossing and wide crossing (discussed in Chapter 2). Consequently, it is unlikely that all methods of

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects either genetic engineering or conventional breeding will have equal probability of resulting in unintended changes. It is the final product of a given modification, rather than the modification method or process itself, that is more likely to result in an unintended adverse health effect. In Figure 3-1 some of the methods used to generate GM plants are shown to illustrate the full range of possibilities that might lead to unintended changes (these methods are described in Chapter 2). Analysis of the Continuum As noted earlier in this chapter, unintentional changes are possible with all conventional and biotechnological breeding methods for genetic modification. It is possible to represent the likelihood of such changes as a continuum, albeit only a partially understood one. Placement along this continuum has no bearing on risk of adverse outcomes, but only on the probability of unintended changes, which need not be hazardous. The potential for hazard resides in specific products of the modification regardless of whether the modification was intentional or unintentional. Again, unintended effects do not necessarily imply hazard. If a particular method were inherently hazardous, all products resulting from its use would be potentially harmful. However, it is known that each method can provide safe products, so the key for breeders and regulatory agencies, in their reviews of specific products, is to identify the relatively rare, potentially hazardous products resulting from any method. In breeding, the developer can generate literally thousands of breeding lines; each might be progeny of the same parents, thus bringing together the same set of genetic information, and yet possess subtle differences. As noted previously in this chapter, conventional breeders may scrutinize 2,000 of these “sister lines” of a single cross of two parent plants. Meiotic recombination allows sister lines to show a continuum of phenotypic expression. Over several years, the breeder eliminates most, or sometimes all, of those breeding lines because they are unsuitable. Plant breeding is often said to be a process not of selection, but of elimination. Any off-types, unstable lines, or lines showing characteristics such as significant differences in nutrient content, responses to environmental stresses, diseases, or the presence of other undesirable traits are discarded as soon as they are noticed. This winnowing takes place over several years, so the remaining lines identified for prospective commercial release are unlikely, but not guaranteed, to have any significant compositional changes other than those related to the desired trait. For this reason, regulatory scrutiny focuses most often on the new trait and its metabolic perturbations. Nevertheless, the appearance of subtle or obscure phenotypic changes can go unnoticed by breeders or regulators and may subsequently have to be recalled from the market, as in the case of the Lenape potato—

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects FIGURE 3-1 Relative likelihood of unintended genetic effects associated with various methods of plant genetic modification. The gray tails indicate the committee’s conclusions about the relative degree of the range of potential unintended changes; the dark bars indicate the relative degree of genetic disruption for each method. It is unlikely that all methods of either genetic engineering, genetic modification, or conventional breeding will have equal probability of resulting in unintended changes. Therefore, it is the final product of a given modification, rather than the modification method or process, that is more likely to result in an unintended adverse effect. For example, of the methods shown, a selection from a homogenous population is least likely to express unintended effects, and the range of those that do appear is quite limited. In contrast, induced mutagenesis is the most genetically disruptive and, consequently, most likely to display unintended effects from the widest potential range of phenotypic effects.

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects developed using non-genetic engineering methods—discussed previously in this chapter (Laurila et al., 1996). The products of a narrow cross-pollination, from a plant of a given species to another plant of the same species, or even the same subspecies, are less likely, but not guaranteed, to pose unintended risks. This is due to the expectation that pollination is inherently safer than other methods of gene transfer because the range of resulting products is typically limited to those products already present in the species or subspecies. However, even this likely “safe” method is subject to occasional spontaneous mutations or infelicitous genetic recombination events leading to novel and unexpected products, and these products may carry some degree of risk, such as disease susceptibility, leading to, for example, hazardous levels of fungal mycotoxins. Expanding the range of conventional breeding beyond the same species provides a greater opportunity to introduce novel genetic information and thereby novel sources for unexpected effects. Triticale is a man-made crop developed by breeding wheat with rye; hordecale is a similar product of barley and rye. Both examples bring together thousands of genes in a way that does not occur in nature and which conceivably could result in the production of undesirable toxicants. As discussed previously in this chapter, the conventional potato breeding program combining Solanum tuberosum and S. brevidens produced not only the expected toxicants, but also a new one, demissidine, which is not produced in either parent (Laurila et al., 1996). Although some products can be reasonably predicted to be hazardous, this does not imply that all products of rDNA—or other methods of bringing together genes—are necessarily hazardous. Agrobacterium and the particle gun are the only two rDNA methods represented on the continuum in Figure 3-1 because they provide almost all of the GE crops approved for commercial release. They are depicted separately because of the documented predilection of the particle gun to introduce multiple, broken, or rearranged DNA segments, in contrast with the usually singular and high-fidelity transfer typical of Agrobacterium.

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects The more disorganized particle gun mechanism increases the risk of insertion into crucial endogenous sequences. The degree of increased risk is as yet undetermined and may be minimal. However, in both cases sufficient numbers of events are generated and screened to make it possible to identify those with the most desirable insertion features, the best expression of desired genes, and the least likelihood of deleterious features, in much the same way that conventional breeders evaluate their breeding lines. Continuum Conclusions Unintended adverse health effects can be either predictable or unpredictable. All organisms undergo spontaneous mutations, giving rise to novel traits, which may carry some hazard. The incidence of such mutations is relatively rare and the type of hazard associated with them is generally not predictable. On the other hand, introduced mutations, such as by rDNA, theoretically allow a gene from any species to be inserted into and expressed by a food crop. Clearly, such a product has the potential to be hazardous if the inserted gene results in the production of a hazardous substance. Although the process of rDNA is itself not inherently hazardous, the resulting product of the process may be. DISCUSSION This chapter reviews examples of unintentional changes that resulted from the genetic modification of various organisms intended for food and the likelihood of unintentional changes arising from multiple methods of genetic modification. All forms of genetic modification, conventional and modern, may potentially lead to unintended changes in composition, some of which may have adverse health effects. The range of biotechnology methods, including genetic engineering, makes it possible to alter, add, or remove genes from conventional food organisms. This manipulation of genetic information may pose either risks or benefits to health or the environment. Examples abound in which a single gene can have a dramatic effect or no effect at all, depending on its features. Similarly, chromosomal changes can also have either a dramatic or no apparent effect, again depending on the features of the genes involved (NRC, 2002). Risks to human health from genetic changes in foods must, therefore, be placed in proper context. The introduction of allergenic proteins is a potential adverse health effect of concern that could arise from genetic modification, including genetic engineering, of food. These methods of breeding, however, are not the only way that potential allergens are introduced into the food supply (see Chapter 5). Although genetic modification techniques may introduce unpredicted adverse health effects, a given technique itself is not a determinant of many more com-

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Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects mon adverse effects, such as those that result from personal food intolerances or sensitivities; crops growing in soils containing toxicants; contamination from postharvest infection with pathogens; and adulterants inadvertently mixed into food. By comparison, genetic modifications to food account for a lesser likelihood for adverse health effects. Most of the novel features in commercially available GE products focus on single genes, such as herbicide tolerance or pest resistance in crops. Products developed using biotechnology express a wide range of new features, some of which may be benign, while others, such as industrial or pharmaceutical products, may pose a greater threat to food safety. Conventional breeding methods have also provided new crop varieties with enhanced herbicide tolerance, pest resistance, and other non-nutrient characteristics, as well as enhanced nutritional profiles, posing potential risks from ingestion that are similar to genetic engineering. Similar comparative examples can be found in animal breeding and in microbial strains, in which conventional methods have produced new products with unintended hazards similar to those that may result as a consequence of the application of rDNA technology. REFERENCES Akeley RV, Mills WR, Cunningham CE, Watts J. 1968. Lenape: A new potato variety high in solids and chipping quality. Am Potato J 45:142-145. Ames BN, Gold LS. 1999. Pollution, pesticides and cancer misconceptions. Pp. 18-39 in Fearing Food, J. Morris and R. Bate, eds. Oxford, UK: Butterworth Heinemann. Aoki S, Kawaoka A, Sekine M, Ichikawa T, Fujita T, Shinmyo A, Syono K. 1994. Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumors of N. glauca × N. langsdorfi. Mol Gen Genet 243:706–710. APHIS (Animal and Plant Health Inspection Service). 1997. DuPont Petition 97-008-01p for Determination of Nonregulated Status for Transgenic High Oleic Acid Soybean Sublines G94-1, G94-19, and G-168. Environmental Assessment and Finding of No Significant Impact. Online. U.S. Department of Agriculture. Online. Available at http://www.aphis.usda.gov/brs/dec_docs/9700801p_ea.HTM. Accessed December 12, 2002. Bauman DE. 1992. Bovine somatotropin: Review of an emerging animal technology. J Dairy Sci 75:3432–3451. Bauman DE. 1999. Bovine somatotropin and lactation: From basic science to commercial application. Domest Anim Endocrinol 17:101–116. Beier RC. 1990. Natural pesticides and bioactive components in foods. Rev Environ Contam Toxicol 113:47-137. Beier RC, Oertli EH. 1983. Psoralen and other linear furocoumarins as phytoalexins in celery. Phytochemistry 22:2595–2597. Bergelson J, Purrington CB, Wichmann G. 1998. Promiscuity in transgenic plants. Nature 395:25. Berkley SF, Hightower AW, Beier RC, Fleming DW, Brokopp CD, Ivie GW, Broome CV. 1986. Dermatitis in grocery workers associated with high natural concentrations of furanocoumarins in celery. Ann Intern Med 105:351-355. Birmingham, DJ, Key MM, Tubich GE, Perone VB. 1961. Phototoxic bullae among celery harvesters. Arch Dermatol 83:127-41.

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