ToxSci Advance Access originally published online on December 15, 2007
Toxicological Sciences 2008 102(2):425-432; doi:10.1093/toxsci/kfm304
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A Gene-Shuffled Glyphosate Acetyltransferase Protein from Bacillus licheniformis (GAT4601) Shows No Evidence of Allergenicity or Toxicity
* Pioneer Hi-Bred International, Inc., Johnston, Iowa 50131
Charles River Laboratories, Redfield, Arkansas 72132
DuPont Haskell Laboratory, Newark, Delaware 19714
DuPont Agriculture and Nutrition, Wilmington, Delaware 19880
¶ Pioneer Hi-Bred International, Inc., Verdia Campus, Redwood City, California 94063
|| Department of Pediatrics and Immunobiology, Mount Sinai School of Medicine, New York, New York 10029
||| Sungkyunkwan University School of Medicine, Seoul, Korea
1 To whom correspondence should be addressed at Pioneer Hi-Bred International, Inc., 7250 NW 62nd Ave., P.O. Box 552, Johnston, IA 50131-0552. Fax: (515) 334-4478. E-mail: bryan.delaney{at}pioneer.com.
Received October 10, 2007; accepted December 11, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The glyphosate acetyltransferase (gat) gene from Bacillus licheniformis was subjected to multiple rounds of gene shuffling to optimize kinetics of corresponding GAT proteins to acetylate the herbicide active ingredient glyphosate. Genetically modified soybeans expressing the gat4601 gene (356043 soybeans) are tolerant to the application of glyphosate. The current manuscript reports the outcome of the allergenicity and toxicity assessment for the GAT4601 protein. Bioinformatic comparison of the amino acid sequence of GAT4601 did not identify similarities to known allergenic or toxic proteins. In vitro studies conducted with heterologously produced GAT4601 protein demonstrated that it was rapidly degraded in simulated gastric fluid containing pepsin (< 30 s) and in simulated intestinal fluid containing pancreatin (< 2 min) and completely inactivated at temperatures above 56°C. The GAT4601 protein expressed in planta is not glycosylated and similar protein profiles were observed in flour extracts from 356043 soybeans and nontransgenic near isoline comparator soybeans (Jack) using serum from soy allergic persons. No evidence of adverse effects was observed in mice following acute oral exposure to 2000 mg/kg of GAT4601 protein or in a repeated dose dietary exposure study at doses of 800–1000 mg/kg/day. This comprehensive assessment demonstrates that the GAT4601 protein does not present a risk for adverse effects in humans when used in the context of agricultural biotechnology.
Key Words: glyphosate acetyltransferase; bioinformatics; in vitro digestibility; allergenicity; toxicology; transgenic crop; gene shuffling.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Three alleles of a glyphosate acetyltransferase (gat) gene from the ubiquitous Bacillus licheniformis soil bacteria were isolated and subjected to numerous rounds of gene shuffling to optimize the ability to acetylate the herbicidal active ingredient glyphosate in the presence of acetyl-CoA (Castle et al., 2004
; Siehl et al., 2005). Following 7–11 rounds of shuffling, the efficiency of the GAT protein to acetylate glyphosate (kcat/KM) was increased approximately 5000 times compared with the native GAT enzyme. Expression of gene-shuffled variants of the GAT protein in field crops confers tolerance to glyphosate (Castle et al., 2004).
The gene from the seventh-round variant of GAT (gat4601) results in expression of a protein (GAT4601) with a high degree of similarity to the amino acid sequence of the native GAT protein. Twenty-one amino acid substitutions in GAT4601 were identified relative to the 146 amino acids of native GAT proteins. Only 4 of the 21 substitutions are in the active site of the enzyme, which is defined as lying within 5 Å of either substrate (acetyl-CoA or glyphosate; Siehl et al., 2007). None of the substitutions include amino acids identified through kinetic analysis of site-directed mutants as being involved with substrate binding or catalysis. The four substitutions in the active site (Y31F, V114A, I132T, and I135V) are to slightly smaller side-chains, which appear to accommodate glyphosate, a larger ligand, compared with the best known substrate for the native enzyme (D-2-amino-3-phosphonopropionate).
The gat4601 gene was transferred into the germplasm of soybeans to produce Optimum GAT soybeans that are tolerant to field applications of glyphosate. Although proteins as a general class of dietary substances are not associated with adverse effects, two safety concerns that have been raised are allergenicity and acute toxicity (Metcalfe et al., 1996; Sjoblad et al., 1992). Accordingly, a number of guidance documents have been published by scientific authorities describing processes to assess the potential allergenicity and toxicity of transgenic proteins used in the context of agricultural biotechnology (CODEX, 2003; FAO/WHO, 2001).
No single biochemical property can predict the allergenic potential of individual proteins and there are no validated animal models that accurately predict their allergenic potential. Consequently, a weight-of-evidence approach that takes into account a variety of relevant factors and experimental observations is used to derive an overall assessment of the allergenic potential of transgenic proteins such as those used on genetically modified (GM) crops (CODEX, 2003; Taylor, 2006). The allergenicity assessments for transgenic proteins are typically based on properties of known allergenic food proteins and include information about the history of exposure and safety of the gene source, amino acid sequence identity compared with known allergenic proteins, physicochemical properties such as glycosylation, stability to pepsin digestion in vitro or other digestive enzymes such as pepsin and pancreatin (Astwood and Fuchs, 1996; Ladics et al., 2006; Thomas et al., 2004). In some cases, the effect of processing (e.g., heating) on heterologously produced transgenic proteins may also be assessed. These studies are typically conducted using transgenic proteins obtained from heterologous (i.e., bacterial) expression systems following demonstration that they are equivalent to the protein expressed in planta.
Evaluation of the potential toxicity of transgenic proteins is similar to the allergenicity assessment of proteins because both rely on a multiple component strategy rather than emphasizing any one particular type of study. However, it differs in that it is based on a tiered approach rather than weight-of-evidence (Delaney et al., in press). The components of the first tier include an assessment of the history of use of the organism from which the gene was obtained especially when there is a documentable history of human exposure or consumption, bioinformatic comparison of the amino acid sequence of the transgenic protein for similarity to other proteins that are considered toxic, information about the mechanism of action of the protein, in vitro stability to digestive enzymes, and exposure assessment. Although the assessment of transgenic proteins using concepts in the first tier is comprehensive, a second tier was developed to be applied on a case-by-case basis depending on the nature of the particular protein. Specific studies within that tier include an assessment of the acute toxicity of the particular transgenic protein and hypothesis-based testing. Repeated dose toxicity studies with transgenic proteins may also be requested in some cases by regulatory authorities (European Commission, 2003).
This manuscript describes the comprehensive safety assessment process that was conducted to evaluate the potential allergenicity and toxicity of the gene-shuffled GAT4601 protein. The results demonstrate that the introduction of the gene-shuffled variant of the GAT4601 protein into 356043 soybeans (i.e., Optimum GAT soybeans) represents no risk for allergenicity or toxicity when used in the context of agricultural biotechnology.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Allergenicity assessment.
Similarity of the amino acid sequence of GAT4601 to known allergenic proteins was evaluated by FASTA34 bioinformatic comparison with the sequences of known allergenic proteins in the Allergen Database (Version 6.0 [January 2006]) at the Food Allergy Research and Research Program at the University of Nebraska (Lincoln, NE). Resistance of GAT4601 protein to digestion in simulated gastric fluid (SGF) containing pepsin containing pancreatin was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of pure heterologously produced GAT4601 protein following incubation for periods of time ranging from 0.5 to 60 min. Resistance to digestion in simulated intestinal fluid (SIF) was assessed by SDS-PAGE analysis and Western blot analysis of the GAT4601 protein following incubation for periods of time ranging from 0.5 to 60 min. The thermal stability of heterologously produced GAT4601 protein was assessed by determination of the enzymatic activity following exposure of the GAT4601 protein to temperatures ranging from 36°C to 60°C. The glycosylation of GAT4601 protein isolated from Optimum GAT soybeans (Event DP-356Ø43-5; 356043 soybeans) in comparison with a glycoprotein-positive control (horseradish peroxidase) and a glycoprotein-negative control (soybean trypsin inhibitor) was assessed using a GelCode Glycoprotein Staining kit (Pierce Biotechnology, Inc., Rockford, IL). The impact of genetic modification on expression of allergenic proteins in 356043 soybeans was assessed using one-dimensional IgE immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) inhibition analysis of protein extracts from 356043 and control (non-GM) soybeans using sera from patients with documented clinical sensitivity to soybeans or from nonatopic patients.
Toxicity assessment.
Similarity of the amino acid sequence of GAT4601 to the sequences of proteins in the National Center for Biotechnology Information Protein dataset was assessed by BLASTP bioinformatic analysis followed by manual inspection of sequence matches above a predetermined E score cutoff of 1.0. The acute oral toxicity of heterologously produced GAT4601 protein in mice was assessed per OECD 423 Guidelines by analysis of clinical signs and body weights following gavage at the limit dose of 2000 mg/kg (OECD, 2001). The repeated dose oral toxicity of GAT4601 protein was assessed in mice by blending heterologously produced GAT4601 protein into rodent diets (Purina Mills International, PMI LLC Certified Rodent LabDiet 5002, Richmond, IN) at concentrations corresponding to daily doses of approximately 10, 100, and 1000 mg/kg/day. Mice in the control groups consumed PMI 5002 rodent diets. Diets were prepared and administered on a weekly basis for a total of 27 days. The concentration, homogeneity, and stability of the GAT4601 protein were determined by antibody specific ELISA at Pioneer Hi-Bred International, Inc. (Johnston, IA). Assessment of the health of animals consuming the control and experimental diets was carried out, including body weights, feed consumption, clinical observations, ophthalmology, hematology, clinical chemistry, organ weights, and histopathology, per OECD 407 Guidelines (OECD, 1995).
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RESULTS |
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History of Use of B. licheniformis and Proteins from B. licheniformis in Foods
The gat4601 gene was derived from Bacillus licheniformis; a ubiquitous gram-positive soil bacteria. Bacillus licheniformis has a history of safe use as a host organism in the production of food enzymes (e.g., alpha-amylase, cyclodextrin glycosyltransferase, hemicellulase, protease, pullulanase), biocontrol agents, and as a dietary probiotic in the United States, Canada, and Europe (Alexopoulos et al., 2004a
,b; European Commission, 2000; Kritas et al., 2006; U.S. FDA, 2001). Bacillus licheniformis was determined by the U.S. Environmental Protection Agency (EPA) to present a low risk of adverse effects to human health and the environment (U.S. EPA, 1996). Although there have been reports of particular detergent enzymes from Bacillus species (including B. licheniformis) causing occupational asthma in detergent industry workers (Biagini et al., 1996; Hole et al., 2000; Sarlo et al., 1997), there are no documented reports of B. licheniformis or proteins derived from this organism causing allergic or toxic reactions in humans or other mammalian species following oral exposure.
Bioinformatic Analysis of the GAT4601 Protein Amino Acid Sequence
Although GAT4601 is a synthetic protein produced from shuffling of the native gat gene from B. licheniformis, it is 86% identical at the amino acid sequence level to the translated protein sequences of each of the three original gat alleles from which it was derived. It contains the definitive motif for the Gcn5-related N-acetyltransferase family of N-acetyltransferases; a superfamily of enzymes present in all organisms, including plants, mammals, fungi, algae, and bacteria (Dyda et al., 2000; Marchler-Bauer et al., 2005; Neuwald and Landsman, 1997; Vetting et al., 2005).
No matching sequences of eight or more contiguous amino acids and no significant alignments were observed between the amino acid sequence of GAT4601 when compared with the sequences of known allergenic proteins with a bioinformatic search. Similarly, no significant sequence similarity between the GAT4601 protein and proteins with known toxic or antinutritional qualities was identified using BLASTP bioinformatic analysis.
Isolation and Characterization of Heterologously Produced GAT4601 Protein
Recombinant GAT4601 protein for the studies described in subsequent sections was expressed in Escherichia coli strain BL21(DE3) and purified as a soluble protein. The microbial GAT4601 protein migrated as a single band with a molecular weight of approximately 17 kDa by SDS-PAGE (Supplemental Fig. S1). The purity of the GAT4601 protein was greater than 95% on a total protein basis. The concentration of GAT4601 protein on a mass basis in the lyophilized powder was determined to be 84% by amino acid composition analysis (data not presented). The GAT4601 protein demonstrated immunoreactivity in Western blot analysis using a GAT-specific antibody, which recognized a single protein band migrating at approximately 17 kDa (Supplemental Fig. S1). Additional analytical studies conducted to characterize the heterologously produced GAT4601 protein demonstrated that it was of high purity and enzymatically active (data not shown).
Digestion of GAT4601 in SGF and SIF
The resistance of heterologously produced GAT4601 to digestive enzymes was assessed by incubation in SGF (containing pepsin) or SIF (containing pancreatin) followed by SDS-PAGE analysis to evaluate apparent disappearance of protein bands. The GAT4601 protein was rapidly hydrolyzed (< 30 s) in SGF (Fig. 1) and in SIF (< 2 min; Fig. 2). Similarly, the GAT4601 protein band was only detectable in Western blot analysis following incubation in SIF for less than 2 min (Supplemental Fig. S2). These results demonstrated that the GAT4601 protein is not resistant to degradation by digestive enzymes.
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Thermal Stability of GAT4601
The thermal stability of GAT4601 was evaluated by determination of the percent residual enzyme activity following heat treatment compared with the activity of untreated GAT4601 protein. The enzymatic activity of GAT4601 was stable at incubation temperatures up to 49.5°C but decreased at higher temperatures. No enzymatic activity was observed at incubation temperatures above 56.1°C (Fig. 3).
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Glycosylation Analysis of the GAT4601 Protein
Although the presence of N-glycosylation in proteins does not necessarily indicate that they are allergenic, many allergenic proteins are glycosylated (Huby et al., 2000
). Therefore, the glycosylation of GAT4601 protein expressed in planta was assessed using protein isolated from 356043 soybean leaf tissue rather than heterologously produced protein. The results demonstrated that the GAT4601 protein expressed in 356043 soybean plants was not glycosylated (Fig. 4).
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Immunoblot and ELISA Inhibition Analysis of GAT4601 Protein in 356043 Soybeans
Expression of the GAT4601 protein in 356043 soybeans did not affect expression of soybean proteins as observed by comparison of the SDS-PAGE protein profiles for control (Jack) and 356043 soy flour extracts (Supplemental Fig. S3). Similarly, no differences were observed in the IgE binding profiles of control and 356043 soybean proteins using one-dimensional immunoblot analysis with sera from soy allergic humans (Fig. 5).
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ELISA inhibition analysis using sera from soy allergic humans showed similar inhibition patterns for control and 356043 soy flour extracts. IgE binding between control (Jack) and 356043 soybean extracts against 50 µg/ml Jack solid-phase with protein concentrations ranging from 50 to 500,000 ng/ml (Supplemental Fig. S4) did not exceed 2.7% standard deviation for 500 ng/ml, approaching 0.8% at the maximum concentration of inhibitor. Together, the SDS-PAGE, immunoblot, and ELISA inhibition analyses indicated that the protein and allergen profiles in flours from control and 356043 (Optimum GAT) soybeans were not different.
Acute Toxicity of GAT4601 Protein
The acute toxicity of the GAT4601 protein was assessed in mice by oral administration of 2000 mg/kg of the purified protein (corresponding to approximately 1680 mg/kg pure, full-length GAT4601 protein) via oral gavage. Control groups were administered either vehicle (water) alone or bovine serum albumin at 2000 mg/kg. All mice survived the duration of the study and no clinical signs of systemic toxicity were observed in any of the treatment groups (data not shown). Mice in all treatment groups gained weight relative to day 0 of dosing (Supplemental Table 1) and no gross lesions were present in any of the mice at necropsy indicating that the GAT4601 protein was not acutely toxic.
Repeated Dose Toxicity Assessment of GAT4601 Protein
To assess the potential for adverse effects from repeated exposure, GAT4601 protein was blended into rodent diets and fed to mice for 27 days at three different doses (10, 100, and 1000 mg/kg/day). No significant differences were observed in body weights over the course of the in-life phase of the study (Supplementary Figs. S5 and S6). Similarly, no differences were observed in feed consumption between control groups and those consuming diets containing the GAT4601 protein evaluated in this study (data not shown). Consumed dosages of GAT4601 protein were determined by ELISA analysis of dietary samples collected over the duration of the in-life phase of the study. In males, daily exposure to GAT4601 protein averaged 7.8, 76.7, and 783.1 mg/kg/day for the low, middle, and high dose groups, respectively. Consumed dosages for females averaged 9.2, 94.3, and 926.9 mg/kg/day for the low, middle, and high dose groups, respectively.
No deaths occurred and there were no abnormal clinical or ophthalmological observations in any of the treatment groups (data not shown). No adverse effects in clinical chemistry response (Tables 1 and 2) or hematology (Supplemental Tables 2 and 3) response variables or organ weights (Table 3) were observed in any of the GAT4601 treatment groups that were considered treatment related. Additionally, no gross or microscopic lesions in any tissues that were considered adverse or related to exposure to the GAT4601 protein in the experimental diets (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The guidelines and processes used to assess the safety of transgenic proteins used in the context of agricultural biotechnology differ somewhat from those used to assess the safety of other types of food ingredients (Delaney, 2007
). The assessments generally focus on evaluating the potential for particular proteins to cause allergenicity and acute toxicity. The potential for allergenicity is conducted using a multiple component weight-of-evidence approach that includes specific elements to compare the physical properties of the particular protein with those of known allergenic proteins (CODEX, 2003). Similarly, a multiple component approach has been applied to assess the toxicity of transgenic proteins (Delaney et al., in press). The studies described in this manuscript were conducted to assess the allergenicity and toxicity of the GAT4601 protein.
The gat4601 gene was produced from multiple rounds of gene shuffling of gat genes from B. licheniformis to increase the efficiency by which the protein acetylates the herbicidal ingredient glyphosate (Castle et al., 2004; Siehl et al., 2005). This gene was transferred into the germplasm of soybean seeds to produce Optimum GAT soybeans (356043) that are tolerant to field application of herbicides containing glyphosate.
The gat4601 gene was obtained from an organism (B. licheniformis) with a documented history of safe use in the human food supply, does not have amino acid sequence similarity to known allergenic proteins, is not resistant to in vitro gastric and intestinal digestion models, and is heat labile. Additionally, the GAT4601 protein expressed in 356043 soybeans was not glycosylated, a property that has been reported for many allergenic proteins (Lack, 2002; Taylor and Lehrer, 1996). When considered in the context of the weight-of-evidence approach, these results demonstrated that the GAT4601 protein bears no similarity to allergenic proteins and is therefore does not represent a risk for allergenicity (CODEX, 2003; Taylor, 2006).
Soybeans are one of the eight major foods known to cause allergic effects and a number of allergenic proteins have been identified (Cordle, 2004; Metcalfe et al., 1996; Ogawa et al., 1995, 2000). The serum of soy allergic patients contains IgE antibodies specific to one or more of these allergenic soy proteins. In the current report, serum from clinically reactive soy allergic humans was used to demonstrate that the genetic modification used to produce 356043 soybeans did not alter the allergen content of the seed crop compared with nontransgenic soybeans (see Goodman and Leach, 2004). From these studies it was concluded that no new allergenic proteins are present in 356043 soybeans.
Although the overwhelming majority of dietary proteins are degraded to constituent amino acids and small peptides that are absorbed for nutritive purposes in the mammalian gastrointestinal system, some proteins possess the potential to cause toxicity. Recommendations have recently been developed to assess the toxicity of transgenic proteins using a tiered approach (Delaney et al., in press). Application of the elements from the first tier to the GAT4601 protein in the current paper did not identify evidence for potential toxicity of the GAT4601 protein. It was obtained from a source with a history of safe use, does not have amino acid sequence similarity to proteins known to be toxic, and was readily degraded in the presence of digestive enzymes. The mechanism of action of the GAT4601 protein is enzymatic acetylation of glyphosate. Amino acid substitutions in GAT4601 relative to the native GAT protein to favor acetylation of glyphosate do not result in significant alterations in the structure or enzymatic mechanism of action. Analysis of the crystal structure demonstrated that only 4 of the 21 amino acid substitutions are in the active site, which is defined as lying within 5 Å of either the acetyl-CoA or glyphosate substrates (Siehl et al., 2007). The crystal structure, combined with kinetic characterization of site-directed mutants, defined structural elements that are involved in substrate binding and catalysis, all of which are retained in GAT4601, indicating that its catalytic mechanism is unchanged from the native GAT protein expressed in B. licheniformis.
Although there was no indication from the first tier of analysis that the GAT4601 presented any potential for adverse effects, additional studies were conducted with this protein because it is the first known gene-shuffled protein to be used in the context of agricultural biotechnology. With regard to the second tier of analysis, no evidence of toxicity was observed in mice following acute or repeated oral exposure to heterologously produced GAT4601. The overall nutritional contribution of the GAT4601 protein was not substantial as even the highest dose of GAT4601 administered in the diets accounted for less than 2% of total dietary protein intake. Accordingly, the No-Observed-Adverse-Effect-Level was determined to be greater than the high dose group (target of 1000 mg/kg/day) evaluated in this study. The actual exposure to pure GAT4601 protein was adjusted to 783 mg/kg/day for males and 927 mg/kg/day for females based on variability of body weights and feed consumption in the repeated dose study.
The doses of GAT4601 protein that were evaluated in the repeated dose toxicity study can be placed into context by comparison with the concentrations of this protein that have been detected in Optimum GAT soybeans using antibody specific ELISA (0.26 mg of GAT4601 protein/kg on a dry weight basis). Assuming an average body weight of 70 kg, humans would need to consume 54,810 mg/day of the GAT4601 protein in the diet to match the same exposure of the high dose male mice in the repeated dose study, which would require consumption of greater than 210,000 kg of raw soybeans per day. Even this is a very conservative estimate of human exposure to the GAT4601 protein because soybeans fractions that are consumed by humans, such as soybean oil, are processed to produce edible fractions. The processing of soybeans to edible fractions will further decrease protein activity because the GAT4601 protein is not stable to processing temperatures above 56°C. Additionally, the in vitro digestion studies conducted with the GAT4601 protein demonstrated that it is likely to be metabolized within the gut as occurs with most other dietary proteins and it therefore will not be absorbed intact.
In conclusion, the results in this paper describe the process that was followed to assess the potential allergenicity and toxicity of the GAT4601 protein expressed in Optimum GAT soybeans. Because GAT4601 was derived from a source that has no history of causing allergic effects in humans and did not present any similarity to known allergenic proteins using the step-wise, weight-of-evidence approach it was concluded that it was not allergenic. There was no evidence indicating that the GAT4601 protein is similar in sequence to known protein toxins and studies in laboratory animals did not identify any evidence of toxicity following single or repeated dose exposure. Further, potential human exposure to the GAT4601 protein is extremely low. These data support the food and feed safety of the GAT4601 protein and indicate that the GAT4601 protein expressed in Optimum GAT soybeans presents no risk for adverse effects to humans when used in the context of agricultural biotechnology.
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SUPPLEMENTARY DATA |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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FUNDING |
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Korea Research Foundation Grant funded by the Korean Government (MOEHRD, KRF-2006-214-C00099) to Y.H.; and Pioneer Hi-Bred, International, Inc.
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