Endogenous allergen upregulation: Transgenic vs. traditionally bred crops

Food and Chemical Toxicology 49 (2011) 2667–2669

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Endogenous allergen upregulation: Transgenic vs. traditionally bred crops Rod A. Herman a,⇑, Gregory S. Ladics b a b

Dow AgroSciences LLC, 9330 Zionsville Rd., Indianapolis, IN 46268, USA Pioneer Hi-Bred International, Inc., DuPont Agricultural Biotechnology, PO Box 80353, Wilmington, DE 19880, USA

a r t i c l e

i n f o

Article history: Received 17 May 2011 Accepted 7 July 2011 Available online 19 July 2011 Keywords: Endogenous allergens Transgenic crops Regulation Safety

a b s t r a c t The safety assessment for transgenic food crops currently includes an evaluation of the endogenous allergy potential (via serum IgE screening) when the non-transgenic counterpart is a commonly allergenic food. The value of this analysis in the safety assessment of transgenic crops, especially with reference to recent requests to quantify individual allergen concentrations in raw commodities, is examined. We conclude that the likelihood of upregulating an endogenous allergen due to transgenesis is no greater than from traditional breeding which has a history of safety and is largely unregulated. The potential consequences of upregulating an endogenous allergen are also unclear. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction As part of the human health assessment for transgenic crops, an allergenicity assessment is performed (CODEX, 2009). This assessment looks at the allergenic potential of any novel food proteins produced as a result of the insertion of the transgene cassette, and, if the crop is considered a major allergenic source (e.g. soybean), any changes that occur in endogenous allergen concentrations within tissues from which food is derived (Thomas et al., 2008). This latter analysis is predicated on the premise that transgenesis is more likely to unintentionally upregulate endogenous-allergen concentrations compared with traditional breeding. The typical approach for comparing endogenous-allergen concentrations in a transgenic crop with non-transgenic comparators is to qualitatively measure the IgE antibody binding of sera or plasma from allergic patients to the extracts from the edible portion of the crop (Holzhauser et al., 2008). More recently, quantitation of known allergenic proteins in transgenic crops has been recommended (EFSA, 2010). In this article, we examine the utility of quantifying endogenous allergen concentrations in transgenic crops as part of the safety assessment. 2. Current situation Endogenous allergens are present in several commonly consumed foods, with 90% of allergy attributed to eight food groups (peanut, tree nuts, wheat, soybean, crustaceans, fish, cow’s milk, and chicken eggs) (Ladics, 2008). While the normal reaction of ⇑ Corresponding author. Tel.: +1 317 337 3551. E-mail address: [email protected] (R.A. Herman). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.07.018

humans to ingested proteins is oral tolerance, approximately 6% of children and up to 4% of adults in United States display the abnormal response of an allergic reaction (Burks et al., 2008). People are normally advised to avoid food to which they have an adverse reaction. Comparisons of food allergen concentrations across different cultivars of a crop have rarely been attempted because protein thresholds for eliciting an allergic reaction have typically not been determined and vary by individual (Blindslev-Jensen et al., 2002). However, where endogenous allergen concentrations have been characterized across crop cultivars, they have been found to vary considerably (Ahrazem et al., 2007; Ariyarathna et al., 2009; Houston et al., 2011; Kuppannan et al., 2010; Salekdeh and Komatsu, 2007; Zuidmeer et al., 2006). In addition to differences among cultivars, natural variability in allergen concentrations can occur in response to differing environmental conditions, harvest timing, or storage conditions (Doerrer et al., 2010; Sancho et al., 2006a,b). With this variability in mind, a couple of important questions can be asked: How much of an increase in allergen concentrations within a cultivar is a safety concern? How can scientists rationally set boundaries for factors where the impact is unknown and where the natural variability among cultivars is undetermined? Notably, clinical allergists do not advise allergic patients against eating certain cultivars of the offending food, nor are there data to indicate that allergic patients exhibit significantly altered clinical reactivity to different cultivars (Goodman et al., 2008). Since allergic individuals attempt to avoid offending foods completely, development of methods to quantify allergen concentrations in new cultivars produced by way of traditional breeding has not been considered necessary to protect public health. Thus, quantitative analytical methods designed to measure most allergens are absent. However, qualitative detection methods are commonly available

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for major allergenic foods to prevent contamination of non-allergic foods by allergenic food residues (e.g. peanuts) (Goodwin, 2004). 3. Risks of transgenesis Transgenesis is the process of inserting a genetic expression cassette, typically containing regulatory elements and a few genes from a sexually incompatible species, into the genome of another species. Risks have been postulated concerning upregulation of endogenous allergens via transgene insertion into a food crop known to cause allergic reactions in certain individuals. These risks include (1) insertional mutagenesis leading to enhancement of allergen-gene expression through promoter enhancement or read-through from the transgene promoter, (2) interaction of the transgenic protein with the biochemical synthetic pathway for the allergen, or (3) upregulation of the gene coding for the allergen (for modulating genes e.g. transcription factors and RNAi). In the following sections, we compare the likelihood of increasing the concentrations of endogenous allergens via transgenesis with that of traditional breeding. 3.1. Insertional mutagenesis While not widely recognized, insertional mutagenesis is a common and natural phenomenon in plants. Mobile genetic elements (transposons) are the origin of the majority of the crop genomes studied thus far, and transposons have been drivers for crop-plant evolution (Feschotte et al., 2002; Kazazian, 2004; Kidwell and Lisch, 1997; Parrott, 2010). In addition, natural and induced mutations have been selected to create thousands of commercial crop cultivars (Parrott, 2010). These non-transgenic cultivars have a history of being safely consumed and are thus largely unregulated. Insertion of a small number of well characterized transgenic cassettes is far less likely to upregulate an endogenous allergen due to insertional mutagenesis compared with the many random mutations and gene translocations that exist during non-transgenic breeding. This latter assertion is based on the large difference in the frequency of these events. For example, modifications in the rice transcriptome were found to be greater in mutagenized compared with transgenic rice plants (Batista et al., 2008). Furthermore, a recent literature review of profiling studies in a variety of transgenic and non-transgenic crops found that transgenesis had less impact on genome expression and concentrations of proteins or metabolites compared with conventional breeding or plant non-directed mutagenesis (Ricroch et al., 2011). Thus insertional mutagenesis due to transgenesis presents a negligible risk to safety. 3.2. Interaction with endogenous pathways It is possible for an exogenous protein to interact with endogenous biochemical pathways, including those involved with allergen synthesis. However, endogenous proteins that have coevolved with such synthetic pathways are far more likely to have experienced evolutionary pressure to coordinate with these pathways. A random interaction from an exogenous protein seems a very remote risk, unless the protein has been chosen to interact with that specific pathway. In this latter case, hypothesis-driven experiments are warranted to investigate such an interaction and its potential consequences. The vast array of uncharacterized gene products that are potentially brought into a crop genome from wild crop relatives (Hajjar and Hodgkin, 2007) are certainly more likely to interact with those in the related crop compared with genes from unrelated organisms, and natural and induced mutations are more likely to modify proteins within allergen pathways

compared with effects from exogenous proteins. Moreover, gene expression, protein distribution, and metabolite content were found to be affected to a greater extent by environmental factors (e.g. location, water, mineral nutrition, sampling time, or season) than by transgenesis (Baker et al., 2006; Barros et al., 2010; Zhou et al., 2009; Zolla et al., 2008). Thus, the risk of a transgenic protein interacting with an endogenous biochemical pathway to upregulate the expression of an allergen is lower than that of traditional breeding techniques that have been safely employed for centuries. 3.3. Regulation of genes Genes coding for transcription factors and interference RNA (RNAi) are being engineered into plants. These gene products are designed to modulate endogenous plant pathways to achieve desirable traits. Transcription factors and RNAi products are endogenous regulatory factors in plants (and animals) and are responsible for the natural regulation of gene-product expression. Phenotype enhancements within traditional breeding programs have often been accomplished based on selection for modifications in these factors (Parrott et al., 2010). Transcription factors or RNAi products may be coded for in transgenes to up- or down-regulate specific metabolic pathways. As such, these modulators are designed to elicit a desirable effect via a known mechanism. Genes (sometimes from wild crop relatives) and mutations selected to achieve a desirable trait within traditional breeding programs are rarely characterized. In addition, many uncharacterized genes and random mutations not related to the desirable trait are also integrated into the crop plant during traditional breeding. In spite of this lack of characterization, very few safety concerns have arisen over the long history of crop breeding. Unlike traditional breeding, the products of transgenes are well characterized (e.g. known mode of action and gene targets), so it is more likely that any undesirable effects would be predictable. Thus, the risk of a transgenic transcription factor or RNAi unintentionally upregulating an endogenous allergen is less than that of traditional breeding. 4. Consequences of higher allergen exposure The lack of analytical techniques to quantify allergens in crops and the resulting lack of knowledge often surrounding the natural variability of these allergens begs the question: why has this area received so little attention? The complete avoidance of offending foods by allergic individuals, along with the lack of identification of safe consumption levels, in part, explains this inattention. A further complication is related to the oral tolerance to allergens normally observed in the population. It is not clear that higher exposure to a protein in individuals results in a higher frequency of sensitization within a population. In fact, greater exposure at a young age to allergens can have the opposite epidemiology. For example, in countries where infant-appropriate peanut snacks are available, peanut allergy is rare (Burks et al., 2008; Du Toit et al., 2008). Similar relationships have been observed for certain milk, egg, and fish allergens where early exposure appears to reduce sensitization rates (Katz et al., 2010; Koplin et al., 2010; Wennergren, 2009). 5. Conclusions It seems sensible to look at traditionally-bred crops as a comparator for transgenic crops due to their history of safe consumption. Methods to determine allergen concentrations in nontransgenic cultivars are largely lacking, and where available, indi-

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cate a wide concentration range across cultivars and growing conditions (Houston et al., 2011; Kuppannan et al., 2010; Ricroch et al., 2011). Insertional mutagenesis, interaction with endogenous biochemical pathways, and gene modulation have all been posed as possible mechanisms whereby transgenesis could increase endogenous allergen concentrations. Here we have qualitatively compared the likelihood of these events occurring with transgenesis versus traditional breeding and concluded the likelihood to be lower for transgenesis. The same conclusion has been drawn from the empirical data on other endogenous compositional components of crops (Herman et al., 2009; Ricroch et al., 2011). Characterization of the endogenous allergen content within traditionally bred crop varieties has not been a priority because allergic individuals avoid the offending food regardless of the variety from which it comes, and because there is not a clear relationship between greater exposure to a protein by individuals and the frequency of allergy within populations (and the inverse relationship has actually been documented) (Burks et al., 2008; Du Toit et al., 2008; Katz et al., 2010; Koplin et al., 2010; Wennergren, 2009). Taken together, the value of quantifying the concentrations of endogenous allergens in transgenic crops is of negligible value in the safety assessment unless a plausible hypothesis for a particular transgene interacting with the synthetic pathway for an allergen is postulated. Development of analytical methods to quantify allergens for the express purpose of characterizing concentrations in transgenic crops, when this level of characterization is lacking for non-transgenic cultivars of the same crop, is not scientifically justified. Sufficient data exist to conclude that transgenesis presents less risk of upregulating endogenous allergens compared with traditional breeding (Ricroch et al., 2011), and that the safety consequences of such an alteration, should it occur, may actually be a decrease in the frequency of sensitization across a population. Conflict of Interest The authors are employed by companies that develop and market transgenic seed. Acknowledgements We thank Guomin Shan, Nicholas Storer, Ping Song, Kathryn Clayton, John Cuffe, and Brian Delaney for their review of a draft of this manuscript. References Ahrazem, O., Jimeno, L., López-Torrejón, G., Herrero, M., Espada, J.L., SánchezMonge, R., Duffort, O., Barber, D., Salcedo, G., 2007. Assessing allergen levels in peach and nectarine cultivars. Ann. Allergy Asthma Immunol. 99, 42–47. Ariyarathna, H., Pramod, S.N., Goodman, R.E., 2009. The abundance of lipid transfer protein (LTP), the major food allergen of corn, varies between hybrids and growth conditions. J. Allergy Clin. Immunol. 123, S28. Baker, J.M., Hawkins, N.D., Ward, J.L., Lovegrove, A., Napier, J.A., Shewry, P.R., Beale, M.H., 2006. A metabolomic study of substantial equivalence of field-grown genetically modified wheat. Plant Biotechnol. J. 4, 381–392. Barros, E., Lezar, S., Anttonen, M.J., van Dijk, J.P., Rohlig, R.M., Kok, E.J., Engel, K.H., 2010. Comparison of two GM maize varieties with a near-isogenic non-GM variety using transcriptomics, proteomics and metabolomics. Plant Biotechnol. J. 8, 436–451. Batista, R., Saibo, N., Lourenço, T., Oliveira, M.M., 2008. Microarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion. Proc. Natl. Acad. Sci. USA 105, 3640–3645. Blindslev-Jensen, C., Briggs, D., Osterballe, M., 2002. Can we determine a threshold level for allergenic foods by statistical analysis of published data in the literature? Allergy 57, 741–746. Burks, A.W., Laubach, S., Jones, S.M., 2008. Oral tolerance, food allergy, and immunotherapy: implications for future treatment. J. Allergy Clin. Immunol. 121, 1344–1350. CODEX, 2009. Foods Derived from Biotechnology, second ed. World Health Organization, Food and Agriculture Organization of the United Nations, Rome, pp. 7–34.

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