1110 VOLUME 111 | NUMBER 8 | June 2003 • Environmental Health Perspectives

Evaluating the AllergenicPotential of GeneticallyModified FoodsThe application of biotechnology to foodproduction offers great promise in increasingcrop production through development ofplants that have an increased yield throughresistance to changes of temperature anddrought and the expression of natural pesti-cides, lessening the need for the application ofexogenous pesticides (Astwood et al. 1997;Mann 1997). Plants may also be developed inwhich toxin content is downregulated, immu-nizing proteins are expressed, fat/protein ratiois altered, palatability is increased, and appear-ance is more appealing (Arntzen 1998). Cropsmay be developed that naturally express vita-mins or that are deficient in specific allergensknown to cause problems upon ingestion byindividuals sensitive to the allergens within thenative plant (Friedrich 1999).

It is inevitable that application of thistechnology has raised a number of fundamen-tal concerns, including the consequences ofreporter genes, the spread of resistance genesto surrounding plants, the use of suicidegenes to prohibit reuse of seed from engi-neered plants, and finally, whether thesealtered plants may be allergenic (Lehrer et al.1996; Miller 1993; Taylor 1997; Van Damand de Vriend 1999; Wal and Pascal 1998).It is this last question that was the subject ofthe conference “Assessment of the AllergenicPotential of Genetically Modified Foods”held 10–12 December 2001 in ResearchTriangle Park, North Carolina. Specifically,how do we determine if a genetically modifiedfood is likely to be allergenic, given that peanutand tree nut allergies alone are observed in1.1% of the general U.S. population (Sichereret al. 1999)?

Overview of Allergic Reactionsto Foods

To address this question, it is first necessary toappreciate that a number of defined clinicalpathologic entities fall within the general pub-lic perception of what defines food allergy(Table 1) (Burks et al. 1988; Lake 1997; Minand Metcalfe 1997; Smith and Munoz-Furlong 1997; Sollid et al. 1989). Indeed, themajority of reactions to foods are classic aller-gic reactions (Jansen et al. 1994). These reac-tions occur within minutes after ingestion of afood and are manifested by urticaria, angio-edema, rhinoconjunctivitis, nausea, vomiting,diarrhea, and asthma. These reactions dependon the synthesis of antigen-specific immuno-globin E (IgE) by B cells in allergic individualswith an inherited tendency for T-helper type 2cell–like reactions, in which the T cells, whenactivated, secrete cytokines such as interleukin(IL)-4 and IL-5. This antigen-specific IgEbecomes fixed on the surfaces of mast cells andbasophils within that sensitized individual.Basophils circulate through the blood, andmast cells are found with greater frequency onsurfaces of the body that interface the externalenvironment. When that individual is reex-posed to the same antigen, it then cross-linksIgE on the surfaces of basophils and mast cells.This leads to basophil and mast cell activation,with the release of histamine, generation ofarachidonic metabolites, and the release andgeneration of potent cytokines and chemo-kines. These chemicals then interact with tar-get-sensitive tissues and generate the allergicresponse. For instance, if mast cells are acti-vated within the gastrointestinal tract, this thenleads to nausea, vomiting, diarrhea, and theingress of antigen into the systemic circulationwhere it may degranulate basophils within thevascular compartment and mast cells within

target tissues. If such a reaction is severe, itmay result in profound hypotension andbe life threatening. This latter reaction iscalled anaphylaxis.

However, not all reactions to foods on animmunologic basis are IgE mediated. Thereare non–IgE-mediated delayed reactions, par-ticularly in infants and children, to such sub-stances as milk protein. These reactions maylead to vomiting, diarrhea, and failure tothrive. Such reactions are sometimes termed“food-induced enterocolitic syndromes”(Lake 1997). The food components thatinduce such reactions and the mechanismsbehind these reactions, which includeimmune complex formation and activation oflymphocytes, have yet to be fully defined.Similarly, celiac disease (Sollid et al. 1989) isa specific form of food allergy in which thebody reacts to components of specific cerealgrains, termed “glutens,” which leads to aspecific pathologic picture.

Efforts to date to address issues of aller-genicity in engineered foods have concentratedon IgE-mediated immediate reactions. Notonly are these reactions the most frequent, butthey are easily the most potentially life threat-ening. Further, the specific antigens withinfood that lead to such reactions and the effec-tor cells and mediators involved have been rea-sonably well characterized. The same cannotbe said for delayed reactions to food compo-nents, seen primarily in infants and children.Because gluten-sensitive entropy, or celiac dis-ease, is specifically caused by glutens, any con-sideration of allergenicity in a new food shouldspecifically address whether genes coding forglutens have been transferred, and thus createproducts that will be a problem for those withceliac disease. This disease aside, however, themajority of current efforts remain directed atIgE-mediated allergic reactions.

The tools to aid in the diagnosis of foodallergy are limited (Bock et al. 1988; Jansenet al. 1994; Pastorello et al. 1989). The most

Introduction: What Are the Issues in Addressing the Allergenic Potential of Genetically Modified Foods?

Dean D. Metcalfe

Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,Maryland, USA

This article is part of the mini-monograph“Assessment of the Allergenic Potential ofGenetically Modified Foods.”

Address correspondence to D.D. Metcalfe,Laboratory of Allergic Diseases, NIAID, NIH,Bldg. 10, Rm. 11C205, MSC 1881, 10 Center Dr.,Bethesda, MD 20892-1881 USA. Telephone:(301) 496-2165. Fax: (301) 480-8384. E-mail:dean_metcalfe@nih.gov

The author declares he has no conflict of interest.Received 31 May 2002; accepted 27 September 2002.

There is growing concern among the general public and the scientific community regarding thepotential toxicity of genetically modified organisms (GMOs). The use of biotechnology toenhance pest resistance or nutritional value has raised a number of fundamental questions includ-ing the consequences of insertion of reporter genes, the spread of resistance genes to surroundingplants, and the use of suicide genes to prohibit reuse of seed from engineered plants. Of particularinterest is the ability of proteins from GMOs to elicit potentially harmful immunologic responses,including allergic hypersensitivity. The lack of information of the potential toxicity of these prod-ucts suggests a need to identify the critical issues and research needs regarding these materials andto develop testing strategies to examine the allergenicity of these compounds. Key words: biotech-nology, decision tree, food allergy, genetically modified crops, IgE, immunology, sensitization.Environ Health Perspect 111:1110–1113 (2003). doi:10.1289/ehp.5810 available viahttp://dx.doi.org/ [Online 19 December 2002]

Genetically Modified Foods Mini-Monograph

critical feature in the diagnosis of food allergyis a carefully obtained clinical history of spe-cific reactions to foods. What foods wereingested, in what quantities, and in what con-text? A careful history often reveals the foodthat induced the allergic response. The his-tory may be supplemented by careful use ofdiet diaries and elimination diets (when safefor a given individual). The suspicion that aspecific food induced an allergic response maybe reinforced by the demonstration of IgEspecific to the food in question. Testing usu-ally takes one of several forms. The mostcommon test employed is the use of water-soluble extracts applied to the skin. The skinis then “tented” through the extract. If a per-son is sensitive to allergens within the food inquestion, a local, small allergic reaction con-sisting of erythema, edema, and itching willoccur. Alternatively, blood obtained frompatients with suspected food allergies can besubjected to examination using a radioallergo-sorbent test (RAST) or enzyme-linkedimmunosorbent assay (ELISA), which identi-fies the presence of food-specific IgE withinserum. If history, cutaneous testing, andRAST/ELISA do not demonstrate sensitivity,it may on some occasions be necessary tochallenge the patient in a double-blind fash-ion with suspected foods, masked either incapsules or in another food (Jansen et al.1994). This latter testing strategy should notbe used if the person is anaphylactically sensi-tive to the suspected food. Further, if suchtesting is performed, it should be done onlyby a physician skilled in application of thisprocedure, in the context of the equipmentavailable for intubation and the treatment of asevere allergic reaction, and with the patient’sinformed consent.

Much of the need to address the safety ofmodified foods is because currently there isno available means to cure a given individualof an immediate reaction to food. The strat-egy for protection of a sensitive individualinvolves instruction on dietary avoidance ofthe foods in question. Patients also areinstructed on how to self-treat in the case that

inadvertent exposure occurs. Immediatetreatment for systemic reactions is the intra-muscular injection of epinephrine followed byacquisition of medical help. The reliance ondietary avoidance by patients with food aller-gies is the reason it is so important that newallergenic foods not be created in the processof the application of biotechnology.

Strategies to Determine If aModified Food Is AllergenicIt would seem, on first pass, that the strategyto prevent the creation of new allergenic foodsshould be straightforward. The strategy thathas been proposed is simply to avoid transfer-ring genes or genetic material that code forknown allergens or potential allergens, basedon their structure, and to screen products ofupregulated genes to determine if they codefor proteins with allergenic potential. The dif-ficulty in this strategy is that the characteristicsof a protein with known allergenicity thatwould distinguish this protein from a proteinunlikely to be allergenic are not known. Thisin turn dictates that strategies to screen forallergenicity must rely on what is known orwhat can be determined about proteins codedfor by transferred genes or upregulated withinthe target plant. To address these concerns, aseries of questions can be asked similar tothose in Table 2. Does the gene transferredfrom a source material to the altered plantcode for a known allergen? Two approaches inthis regard have been widely applied. First, isthere sequence and structural similaritybetween the transferred gene product andknown allergens? Second, does the transferredgene code for a protein known to be aller-genic? This is particularly relevant when thegene is derived from a known allergenicsource. In this latter case, serum from individ-uals sensitive to the source of the transferredgene can be used to screen for the presence ofIgE reactive to the transferred protein asexpressed in the modified food. Clearly, sim-ply examining sequence similarity does notaccount for discontinuous and conformationalepitopes. However, with crystallization ofknown allergens, it is possible to foresee a daywhen such epitopes may be identified.

A more difficult question is whether thetransferred gene codes for a protein that may

be allergenic, when the source of the trans-ferred protein is not known for its allergenicity.Again, efforts can be made to determinesequence similarity of the transferred gene toknown genes that code for allergens. Here,resistance to degradation by proteases and acidhas been suggested as a relative screeningmethodology (Astwood et al. 1996). Resistanceof a transferred protein to degradation is partlybased on the theory that resistance to degrada-tion protects a protein from digestion and thusallows for greater absorption of the protein.Assessment of transferred proteins for potentialallergenicity has also generated interest in thepossible use of animal models to assess proteinallergenicity. This leads to another question.Has the genetic manipulation of the modifiedfood upregulated endogenous substances thatmay be allergenic? In the possible instancewhere such upregulated proteins may not havebeen identified, screening for allergenicityagain through the use of an animal model hasbeen attractive.

Decision Tree Approaches

The first organized attempt to synthesize aplan in the form of a decision tree to addressissues of the allergenicity of engineered foodswas sponsored by the International FoodBiotechnology Counsel and the InternationalLife Sciences Allergy and ImmunologyInstitute. This decision tree and accompany-ing articles were published in a supplement ofCritical Reviews in Food Science andNutrition (Metcalfe et al. 1996) after a num-ber of other similar initiatives (FAO/WHO1995, 1996; U.S. FDA 1992). This decisiontree envisioned two primary scenarios. Thefirst scenario is that the source of the trans-ferred gene was allergenic. In this case, serumfrom individuals allergic to the source of thegene could be used to screen extracts of themodified food to determine if an allergenicprotein had been transferred. The initialscreening is done using an in vitro measure-ment of allergen-specific IgE, possibly fol-lowed by skin testing of extracts of themodified plant in individuals known to besensitive to the source material, and finally, bythe rare possibility of employing blinded foodchallenge procedures in sensitive individuals.In the second scenario, where the source of

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Table 1. Clinical patterns of immune-mediatedadverse reaction to foods.

IgE mediated (immediate)Urticaria/angioedemaRhinoconjunctivitis, asthmaOral allergy syndromeNausea, colic, vomiting, diarrheaAnaphylaxis

Non-IgE mediated (delayed)Food-induced enterocolitic syndromesCeliac disease

OverlapAtopic dermatitisAllergic eosinophilic gastroenteritis

ControversialMigraine headachesIrritable bowel syndrome, etc.

Table 2. Examples of technical approaches to address questions concerning the possible allergenicity ofmodified foods.

Sequence Reaction to Stability to Allergenicity inQuestions similarity specific IgE digestion an animal model

Does the gene transferred code X X Xfor a known allergen? (Source is acommon allergenic food.)Does the gene transferred code X X Xfor a protein that may be allergenic?Does the genetic manipulation lead to the Xexpression or upregulation of a new allergen?

the gene is not known to be allergenic, thedecision tree recommends examining thetransferred gene for sequence similarity toknown allergens and for stability to digestion.This decision tree has been widely discussedand critiqued.

This initial decision tree was published in1996 at a time when a number of modifiedfoods were in the process of being approved.However, with the increasing use of geneticallymodified plants in the late 1990s came a signif-icant rise in public concern about the safety ofgenetically modified foods (Enserink 1999;Ferber 1999; Gaskell et al. 1999). Among themany issues of public concern was the issue ofallergenicity. In part, public attention to theseissues was heightened by the circumstances sur-rounding the approval of StarLink corn. In1998, the U.S. Environmental ProtectionAgency (U.S. EPA) decided to limit the use ofthis corn for animal feed because the Cry9Cprotein engineered into this corn to providepesticide resistance was resistant to degrada-tion. Subsequently, in September 2000,Cry9C DNA was detected in taco shells, andthe U.S. EPA and U.S. FDA became involvedin an assessment of whether the StarLink cornwas indeed allergenic (U.S. EPA 2001).

Over the same period, a number of criti-cisms had been voiced about the 1996International Life Sciences Institute/Inter-national Food Biotechnology Consortium(ILSI/IFBC) decision tree approach in assess-ing allergenicity. In some cases, the use ofskin prick tests and double-blind placebo-controlled food challenge were felt to beinappropriate because of ethical concerns inthe use of normal volunteers for the purposesof safety assessment. There was also signifi-cant concern over exactly how many contigu-ous amino acids defined sequence similarity.There was increasing evidence that stability todigestion was a poor screen for allergenicity.Further, there was no screen for cross-reactingallergens. Finally, individuals interested inanimal models of allergenicity were makingthe case that such models would help todefine allergenicity for safety assessment.

To address these concerns, the FAO/WHO convened a special panel in 2001 toupdate the ILSI/IFBC decision tree. Thisrevised decision tree most noticeably differsfrom the ILSI/IFBC decision tree through theinsertion of screens for cross-reacting allergenssuch as those that exist between ragweed andmelons, and through its advocacy of the use ofanimal models in a relative scale of allergenic-ity (FAO/WHO 2001). The FAO/WHOpanel also recommended that such approachesto assess allergenicity must constantly take intoaccount new and evolving information onparameters that define allergenicity and on theevolving use of specific animal models. Theyalso noted that use of human in vivo methods

to evaluate the allergenicity of food derivedfrom biotechnology in many circumstancesraised ethical issues, and their use would haveto be considered on a case-by-case basis and asrelates to the value of premarketing evaluation.This was particularly important in the assess-ment of foods claimed to be hypoallergenicthrough the downregulation of genes thatcode for known allergens.

Questions in Risk Assessment

If it were possible to list with certainty thecharacteristics that allow a specific protein toinduce specific IgE in a susceptible individual,there would have been no need for this con-ference. The reality is, however, that we haveonly imperfect and relative measures of aller-genic potential at present (Table 2). Giventhe current situation, what issues now need tobe addressed?

It may be argued that the first set of issuescenters around the understanding of patho-physiologic mechanisms of allergenicity. Howdo we define susceptible populations? Whatare the thresholds for sensitization? What arethe thresholds for elicitation of a reaction?What do we know about dose–responserelationships between the amount of foodingested and the final reaction? And finally,what available biomarkers are there ofexposure and effect?

A second series of issues related to themechanisms of allergenicity is directed toallergenic structure. What are the moleculardeterminants of allergenicity? Is there a rela-tionship between allergenicity and function?And finally, is it possible to use animalmodels to predict allergenicity?

Assuming that any effort to identifygenetically modified foods of potential aller-genicity using current technology will not beperfect, what then is the role of postmarketsurveillance? Is it practical to label geneticallymodified organism (GMO) foods, consider-ing that many of these foods will beprocessed? And what about the issue of post-marketing surveillance in situations wherefoods are sold in restaurants and throughstreet vendors and in other situations wheremonitoring of labeling becomes difficult?

It was thus the charge of this meeting toexamine the issues surrounding the potentialallergenicity of GMO foods. What is the truevalue and role of an assessment of new proteinsin GMO foods in terms of similarity to knownallergens? How many contiguous amino acidsdefine similarity? What are the limitations andstrengths of tests to examine stability of pro-teins? Is there sufficient information to sup-port the use of animal models in evaluatingallergens to allow their rational use in safetyassessment? To what degree can safety assess-ment rely on testing of GMO foods using serafrom those known to be allergic to source

materials, pollens, and other substances in theenvironment? In the end, decisions made as tohow to apply existing knowledge and databasesin the assessment of GMO foods for potentialallergenicity will be only as successful as theyare creditable to research scientists, industry,and to the public at large.

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