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- Materials and methods
Background: Assessing the allergenicity and toxicity of genetically modified (GM) crops is essential before they become a regular part of our food supply. The present study aimed to assess the allergenicity of Brassica juncea (mustard) expressing choline oxidase (codA) gene from Arthrobacter globiformis that provides resistance against abiotic stresses.
Methods: SDAP, Farrp, and Swiss-Prot databases were used to study allergenicity of choline oxidase. Digestibility of choline oxidase was assessed in simulated gastric fluid (SGF). Specific immunoglobulin E (IgE) reactivity of native and GM mustard was compared by using enzyme-linked immunosorbent assay (ELISA) and skin tests in respiratory-allergic patients. Allergenicity of GM and native mustard proteins was compared in Balb/c mice.
Results: Choline oxidase showed no significant homology with allergenic proteins in SDAP and Farrp databases. Cross-reactive epitope search showed a stretch similar to Hev b 6 having some antigenic properties. Purified choline oxidase showed complete degradation with SGF. Skin prick test of native and GM mustard extract on respiratory allergic patients showed significant correlation (P < 0.05). ELISA with 96 patients’ sera showed comparable IgE reactivity. Balb/c mice immunized with native and GM mustard proteins showed low IgE response. Presensitized mice on intravenous challenge with Brassica extract showed no anaphylactic symptoms unlike ovalbumin (OVA) sensitization that showed anaphylactic reaction in mice. Lung histology of OVA-sensitized mice showed narrowing of airway and large eosinophilic infiltration, whereas native and GM Brassica extract showed normal airway.
Conclusion: Genetically modified mustard with the codA gene possessed allergenicity similar to that of native mustard and no enhancement of IgE binding was observed due to genetic manipulation.
Agricultural biotechnology has potential to improve the food supplies required to feed the growing world population. The classical breeding method, where large amount of DNA is transferred, is replaced by the use of biotechnological approach and a specific trait is introduced into the host. Major traits being introduced in crops are improved tolerance to pests (1), herbicides (2, 3), drought (4), salt (5) and nutritional quality(6). Genetically modified (GM) crops can also be exploited for development of oral vaccines (7). However, critics have raised concern about the safety, allergenicity and toxicity of these crops. There are very few studies which provide information about allergenic potential of GM crops. FAO/WHO/OECD has proposed certain guidelines for safety and allergenicity evaluation of GM crops (8, 9).These guidelines recommends bioinformatics approach, digestibility studies, animal model, specific sera screening, etc. to study allergenicity of GM crops (10). However, the animal models are scarce to predict allergenic potential of GM crops.
The incidence of food allergy is 1–2% in adults and 6–8% in children (11). Food induced anaphylaxis is reported in 3/100 000 individual per year (12). Food allergens are mostly proteins which may be modified upon processing. GM foods likely to be introduced might have enhanced allergenicity due to the presence of cross-reactive epitopes (13). GM soybean containing Brazil nut allergen has improved protein content but retained its allergenicity (14). GM corn with cry9c gene has allergenic properties but no immunoglobulin E (IgE) was detected in patients’ sera (15). Therefore, extensive investigations are required for allergenicity assessment of GM food.
Mustard is a worldwide crop and leaves/seeds are consumed in various food forms. Mustard containing choline oxidase provides abiotic stress tolerance in plants by introducing glycinebetaine pathway and was expressed in leaves/seeds. Transgenic Brassica juncea expressing choline oxidase gene from Arthrobacter globiformis was evaluated for allergenicity using bioinformatic approach, in vitro digestibility, immuno screening and an ovalbumin (OVA) animal model.
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- Materials and methods
Genetically modified crops have reached the market and others are under development. This scenario necessitates the evaluation of safety and allergenicity of GM foods. GM food is assessed by genetic modification, safety analysis of new protein, occurrence and implication of unintended effects, gene transfer to gut micro flora, allergenicity of new protein, role of new food in the diet, influence of food processing, etc. (27). The present study was aimed to assess the allergenicity of transgenic mustard (B. juncea) expressing bacterial codA gene according to FAO/WHO guidelines. The codA gene has also been introduced in rice, tobacco, tomato, Arabidopsis (28–31), etc. but none of the plant products has been evaluated for allergenicity.
Bioinformatic analysis of choline oxidase sequence showed no homology with allergens listed in the SDAP and Farrp databases. Further studies identified cross-reactive epitope ‘VGGGSA’ similar to a latex allergen Hev b 6. This stretch is hydrophobic with less antigenicity in choline oxidase but more antigenicity in Hev b 6. Furthermore, the stretch was studied with ‘peptide cutter’ software showed three protease cleavable sites (http://us.expasy.org/tools/peptidecutter;accessed29December 2005). Thus the epitope seems to be susceptible to disruption by protease. Studies of structural biology of allergens have proposed that the α-helix region contains IgE-binding epitopes (32). The six amino acid stretch of choline oxidase falls in the β-sheet, indicating that its chances of being an epitopic region is low. Hev b 6 stretch falls under the β-coil region and this may not be epitopic region. An earlier study revealed that the six amino acid stretch analysis shows many random and irrelevant matches (33).
Food allergens are required to exhibit sufficient gastric stability to reach the intestinal mucosa where absorption and sensitization can occur. Many allergens such as milk β-lactoglobulin, peanut Ara h2, soybean β-conglycinin, etc. were stable to digestion with SGF (16). Purified choline oxidase protein was rapidly digested by SGF, thereby minimizing its potential for absorption by the intestinal mucosa and the chances of this to behave as an allergen are low.
Skin tests on allergic rhinitis and asthma patients with GM and native mustard extracts showed similar results (P < 0.05). Specific IgE values obtained with GM and native mustard in patients’ sera show no variation (P < 0.05). Batista et al. monitored IgE response to GM maize and soya and showed that none of the individuals reacted differently (34).
The intrinsic property of allergenic protein is to induce IgE antibody production. In our study, OVA induced high IgE antibody response both with the oral and the intraperitoneal route in mice models. IgE antibody response with GM and native mustard was low and showed no significant difference (P < 0.05). Thus, both the extracts have similar allergenic property and introduction of the codA gene did not enhance the IgE response of the GM mustard. Dearman et al. showed that intraperitoneal administration of OVA elicited allergenic response (35). Further, Knippels et al. demonstrated OVA allergenicity by oral administration (36).
Asthma is an immune inflammatory disease characterized by airway hyper-responsiveness. Inflammatory infiltrates are present in the bronchial walls containing eosinophils with elevated serum IgE levels (37). In the present study, presensitized mice with GM and native mustard on challenge with the same proteins showed no visible signs of anaphylaxis, but OVA induced severe anaphylaxis leading to mortality. Lungs histology further confirmed that exposure to GM and native mustard protein via the oral or the intraperitoneal route did not induce airway remodeling, whereas OVA increased eosinophils and caused airway constriction.
Avoidance of allergen(s) is the best therapeutic approach for food allergy. Therefore, testing of all new foods including GM crops should be made mandatory prior to release in the market. Researchers can think about hypoallergenic crops by silencing certain genes, epitope modification and/or alteration of secondary structure. The second-generation GM crops with reduced toxicity, allergenicity and no antibiotic resistance gene may be developed in the future to meet the growing food requirement.
In conclusion, both GM and native mustard showed same allergenicity with no enhancement in IgE binding due to genetic manipulation.