Corinne Herouet-Guicheney

Evaluation of protein safety in the context of agricultural biotechnology

One component of the safety assessment of agricultural products produced through biotechnology is evaluation of the safety of newly expressed proteins. The ILSI International Food Biotechnology Committee has developed a scientifically based two-tiered, weight-of-evidence strategy to assess the safety of novel proteins used in the context of agricultural biotechnology. Recommendations draw upon knowledge of the biological and chemical characteristics of proteins and testing methods for evaluating potential intrinsic hazards of chemicals. Tier I (potential hazard identification) includes an assessment of the biological function or mode of action and intended application of the protein, history of safe use, comparison of the amino acid sequence of the protein to other proteins, as well as the biochemical and physico-chemical properties of the proteins. Studies outlined in Tier II (hazard characterization) are conducted when the results from Tier I are not sufficient to allow a determination of safety (reasonable certainty of no harm) on a case-by-case basis. These studies may include acute and repeated dose toxicology studies and hypothesis-based testing. The application of these guidelines is presented using examples of transgenic proteins applied for agricultural input and output traits in genetically modified crops along with recommendations for future research considerations related to protein safety assessment.

Quantitation of soybean allergens using tandem mass spectrometry.

Soybean (Glycine max) seed contain some proteins that are allergenic to humans and animals. However, the concentration of these allergens and their expression variability among germplasms is presently unknown. To address this problem, 10 allergens were quantified from 20 nongenetically modified commercial soybean varieties using parallel, label-free mass spectrometry approaches. Relative quantitation was performed by spectral counting and absolute quantitation was performed using multiple reaction monitoring (MRM) with synthetic, isotope-labeled peptides as internal standards. During relative quantitation analysis, 10 target allergens were identified, and five of these allergens showed expression levels higher than technical variation observed for bovine serum albumin (BSA) internal standard (∼11%), suggesting expression differences among the varieties. To confirm this observation, absolute quantitation of these allergens from each variety was performed using MRM. Eight of the 10 allergens were quantified for their concentration in seed and ranged from approximately 0.5 to 5.7 μg/mg of soy protein. MRM analysis reduced technical variance of BSA internal standards to approximately 7%, and confirmed differential expression for four allergens across the 20 varieties. This is the first quantitative assessment of all major soybean allergens. The results show the total quantity of allergens measured among the 20 soy varieties was mostly similar.

Toxicological evaluation of proteins introduced into food crops.

Abstract This manuscript focuses on the toxicological evaluation of proteins introduced into GM crops to impart desired traits. In many cases, introduced proteins can be shown to have a history of safe use. Where modifications have been made to proteins, experience has shown that it is highly unlikely that modification of amino acid sequences can make a non-toxic protein toxic. Moreover, if the modified protein still retains its biological function, and this function is found in related proteins that have a history of safe use (HOSU) in food, and the exposure level is similar to functionally related proteins, then the modified protein could also be considered to be “as-safe-as” those that have a HOSU. Within nature, there can be considerable evolutionary changes in the amino acid sequence of proteins within the same family, yet these proteins share the same biological function. In general, food crops such as maize, soy, rice, canola etc. are subjected to a variety of processing conditions to generate different food products. Processing conditions such as cooking, modification of pH conditions, and mechanical shearing can often denature proteins in these crops resulting in a loss of functional activity. These same processing conditions can also markedly lower human dietary exposure to (functionally active) proteins. Safety testing of an introduced protein could be indicated if its biological function was not adequately characterized and/or it was shown to be structurally/functionally related to proteins that are known to be toxic to mammals.

Review of animal models designed to predict the potential allergenicity of novel proteins in genetically modified crops

The safety assessment of genetically modified crops involves the evaluation of the potential allergenicity of novel proteins by using several in silico and in vitro endpoints. In this publication, the variables and questions associated with the development of in vivo models are examined and several unpublished results are presented. Both rodent and non-rodent (dog and pig) models have been investigated using various routes of administration with purified proteins or food extracts, with or without the use of an adjuvant. The ideal model should be simple, reproducible across laboratories over time, specific and sensitive enough for distinguishing a threshold beyond which relevant allergenicity would be predicted and, for ranking proteins correlated with the allergic responses in humans, and acceptable under animal care. Preliminary data suggest that a few appear promising; however, further evaluation of these models is required. In particular, more extensive validation testing with additional allergenic and non-allergenic material should be performed before using them in the safety assessment of genetically modified crops.

Murine models for evaluating the allergenicity of novel proteins and foods.

Genetically modified crops convey many benefits to world population. However, a rigorous safety assessment procedure, including an evaluation of the allergenic potential, is fundamental before their release into the food chain. As an integral part of the safety assessment process, regulatory authorities worldwide strongly recommend the use of tests that can predict the allergenic potential of the novel proteins. All guidance documents are based on an array of tests that have been proposed in 2003 by the Codex Alimentarius. Although the animal model is not a requirement of the Codex Alimentarius weight of evidence approach, allergenic hazard of novel proteins could only be evaluated by an in vivo model that can potentially identify and distinguish commonly allergenic proteins from rarely allergenic proteins. Therefore, food allergy experts encourage its development. During the 2007 International Life Science Institute (ILSI) workshop (Nice, France), worldwide experts shared their latest research results on rodent models to evaluate the allergenic potential of proteins and foods. This review presents the most promising rodent models for assessing food protein allergenicity that were evaluated during this ILSI workshop.

Investigation of endogenous soybean food allergens by using a 2-dimensional gel electrophoresis approach

As part of the safety assessment of genetically modified (GM) soybean, 2-dimensional gel electrophoresis analyses were performed with the isoxaflutole and glyphosate tolerant soybean FG72, its non-GM near-isogenic counterpart (Jack) and three commercial non-GM soybean lines. The objective was to compare the known endogenous human food allergens in seeds in the five different soybean lines in order to evaluate any potential unintended effect(s) of the genetic modification. In total, 37 protein spots representing five well known soybean food allergen groups were quantified in each genotype. Qualitatively, all the allergenic proteins were detected in the different genetic backgrounds. Quantitatively, among 37 protein spots, the levels of accumulation of three allergens were slightly lower in the GM soybean than in the non-GM counterparts. Specifically, while the levels of two of these three allergens fell within the normal range of variation observed in the four non-GM varieties, the level of the third allergen was slightly below the normal range. Overall, there was no significant increase in the level of allergens in FG72 soybean seeds. Therefore, the FG72 soybean can be considered as safe as its non-GM counterpart with regards to endogenous allergenicity. Additional research is needed to evaluate the biological variability in the levels of endogenous soybean allergens and the correlation between level of allergens and allergenic potential in order to improve the interpretation of these data in the safety assessment of GM soybean context.

Safety evaluation of the double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) from maize that confers tolerance to glyphosate herbicide in transgenic plants

Glyphosate tolerance can be conferred by decreasing the herbicides ability to inhibit the enzyme 5-enol pyruvylshikimate-3-phosphate synthase, which is essential for the biosynthesis of aromatic amino acids in all plants, fungi, and bacteria. Glyphosate tolerance is based upon the expression of the double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) protein. The 2mEPSPS protein, with a lower binding affinity for glyphosate, is highly resistant to the inhibition by glyphosate and thus allows sufficient enzyme activity for the plants to grow in the presence of herbicides that contain glyphosate. Based on both a review of published literature and experimental studies, the potential safety concerns related to the transgenic 2mEPSPS protein were assessed. The safety evaluation supports that the expressed protein is innocuous. The 2mEPSPS enzyme does not possess any of the properties associated with known toxins or allergens, including a lack of amino acid sequence similarity to known toxins and allergens, a rapid degradation in simulated gastric and intestinal fluids, and no adverse effects in mice after intravenous or oral administration (at 10 or 2000 mg/kg body weight, respectively). In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the 2mEPSPS protein in human food or in animal feed.

Allergic sensitization: screening methods

Experimental in silico, in vitro, and rodent models for screening and predicting protein sensitizing potential are discussed, including whether there is evidence of new sensitizations and allergies since the introduction of genetically modified crops in 1996, the importance of linear versus conformational epitopes, and protein families that become allergens. Some common challenges for predicting protein sensitization are addressed: (a) exposure routes; (b) frequency and dose of exposure; (c) dose-response relationships; (d) role of digestion, food processing, and the food matrix; (e) role of infection; (f) role of the gut microbiota; (g) influence of the structure and physicochemical properties of the protein; and (h) the genetic background and physiology of consumers. The consensus view is that sensitization screening models are not yet validated to definitively predict the de novo sensitizing potential of a novel protein. However, they would be extremely useful in the discovery and research phases of understanding the mechanisms of food allergy development, and may prove fruitful to provide information regarding potential allergenicity risk assessment of future products on a case by case basis. These data and findings were presented at a 2012 international symposium in Prague organized by the Protein Allergenicity Technical Committee of the International Life Sciences Institute’s Health and Environmental Sciences Institute.

Current and future methods for evaluating the allergenic potential of proteins: International workshop report 23-25 October 2007

The International Life Science Institutes Health and Environmental Sciences Institutes Protein Allergenicity Technical Committee hosted an international workshop October 23-25, 2007, in Nice, France, to review and discuss existing and emerging methods and techniques for improving the current weight-of-evidence approach for evaluating the potential allergenicity of novel proteins. The workshop included over 40 international experts from government, industry, and academia. Their expertise represented a range of disciplines including immunology, chemistry, molecular biology, bioinformatics, and toxicology. Among participants, there was consensus that (1) current bioinformatic approaches are highly conservative; (2) advances in bioinformatics using structural comparisons of proteins may be helpful as the availability of structural data increases; (3) proteomics may prove useful for monitoring the natural variability in a plants proteome and assessing the impact of biotechnology transformations on endogenous levels of allergens, but only when analytical techniques have been standardized and additional data are available on the natural variation of protein expression in non-transgenic bred plants; (4) basophil response assays are promising techniques, but need additional evaluation around specificity, sensitivity, and reproducibility; (5) additional research is required to develop and validate an animal model for the purpose of predicting protein allergenicity.

Measurement of endogenous allergens in genetically modified soybeans--short communication.

The measurement of endogenous allergens is required by the European Commission (EC) as part of the compositional analysis for GM products from host plants that are common causes of food allergy, such as soybean (EC Implementing Regulation No. 503/2013). In each case, the EC Implementing Regulation indicates that analysis be conducted on identified allergens as specified in the Organization of Economic Cooperation and Development (OECD) consensus documents on compositional considerations for new plant varieties. This communication discusses the methods available to measure endogenous allergens as well as the endogenous soybean allergens that should be analyzed. It is suggested herein that in conjunction with the 2012 OECD consensus document on soybean, any list of soybean allergens should be based on clinically relevant data among publicly available allergen databases and peer-reviewed scientific publications, and the ability to measure the identified allergen. Based on a detailed analysis of the scientific literature, the following key points are recommended: (1) the acceptance of serum-free, quantitative analytical method data as an alternative to traditional IgE reactivity qualitative or semi-quantitative data for evaluation of endogenous soybean allergen content; (2) eight of the 15 potential allergens listed in the OECD soybean consensus document (Gly m 3, Gly m 4, Gly m Bd28K, Gly m Bd30K, Gly m 5, Gly m 6, Gly m 8, and Kunitz trypsin inhibitor) have both appropriate supporting clinical data and sufficient sequence information to be evaluated in comparative endogenous soybean allergen studies; and (3) the remaining seven proteins (Gly m 1, Gly m 2, unknown 50kDa protein, unknown 39kDa protein, P-22-25, lipoxygenase and lectin) lack sufficient data for clear classification as confirmed allergens and/or available sequence information and should not be currently included in the measurement of endogenous soybean allergens in the compositional analysis for the EU.