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Genetically Modified Soybeans: Equal Allergenicity as their Wild Type Counterparts? Katie Van Den Einde November 24, 2009 Advisor: Dr. Chastain

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Overview Introduction: GM foods, allergies, controversy Paper 1 Paper 2 Paper 3 Paper 4 Current regulations Conclusions

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Importance GM foods: Soybeans Corn Tomatoes Rice Canola Potatoes Sugar beets Sugar cane

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Modifications Herbicide resistance Insect resistance Disease resistance Addition of proteins/vitamins 2003 84% of US soybean acreage was glyphosate tolerant (Roundup ready)

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Basics of Genetic Modification Procedures 1. Plasmid insertion 2. Gene guns 3. Protoplasts

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Allergies Majority of allergic reactions are immunoglobulin E (IgE) mediated. IgE allergies affect about 1-2% of adults 2-8% of children

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Symptoms: Itchy, watery eyes Rash Congestion Itchiness Difficulty breathing Anaphylactic shock (Can be life threatening)

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Basics of allergic reactions 1-Allergen 2-IgE antibodies 3-Mast cells 4-Histamine release

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Anti-Histamines

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GM Controversy Ethics Gene flow Resistance Harm to other organisms Allergens???

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Overview Introduction: GM foods, allergies, controversy Paper 1 Paper 2 Paper 3 Paper 4 Current regulations Conclusions

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Paper 1: Identification of a Brazil-nut allergen in transgenic soybeans New England Journal of Medicine 1996

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Purpose: To assess ability of proteins from 1)soybeans (Glycine max) 2)transgenic soybeans 3)Brazil nuts (Bertholletia excelsa) 4)purified 2S albumin to bind to IgE serum

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Methods: Radio allergosorbent test (RAST) 4 serums Sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) 9 serums Skin Prick Tests

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RAST basics

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Results: RAST More inhibition of IgE binding = more allergic. Triangles= WT Squares= GM soybean Circles= Brazil nut

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Results: SDS-PAGE IgE binding Total Proteins IgE binding

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Results: Skin-Prick Test

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Main Points: GM soybean protein successfully competed with Brazil nut protein. IgE from 8/9 allergic to Brazil nut bound to introduced 2S albumin in GM soybeans.

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Overview Introduction: GM foods, allergies, controversy Paper 1 Paper 2 Paper 3 Paper 4 Current regulations Conclusions

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Paper 2 Lack of detectable allergenicity of transgenic maize and soya samples Journal of Allergy and Clinical Immunology 2005

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Purpose: Monitor 5 GM products whose transgenes came from sources with no allergenic history

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Methods: Food Survey Previous exposure? Skin Prick Tests 27 kids with food allergies 50 patients with asthma rhinitis SDS-PAGE

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Flour products tested

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Food survey results

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Western Blot

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Testing Lab Supply SDS PAGE Western Blot

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SDS PAGE Western Blot

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Skin prick and IgE results

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Main Point: No detectable difference in IgE reactivity between wild type and GM soybeans or corn.

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Overview Introduction: GM foods, allergies, controversy Paper 1 Paper 2 Paper 3 Paper 4 Current regulations Conclusions

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Paper 3 A comparative study of the allergenic potency of wild-type and glyphosate- tolerant gene-modified soybean cultivars Acta pathologica, microbiologica et immunologica Scandinavica 2003

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Purpose: To compare allergenicity of 8 wild type and 10 GM soybeans varieties (all for CP4 EPSPS)

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Methods: RAST (serum from 10 patients) SDS-PAGE Histamine Release test Skin prick tests

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RAST results More inhibition of labeled IgE binding = more original serum bound first.

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RAST results Concentration of extract needed for 50% inhibition of IgE binding (variety #12)

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Histamine Release results Notice lack of any major differences no where to point an arrow! Skin Prick Test Histamine Release (0=negative, 6=lots)

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Histamine Release for patient I Pretty similar!

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Main Points: Difference between patients response, but no statistical difference between WT and TG soybeans. Addition of CP4 EPSPS gene higher allergenicity

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Overview Introduction: GM foods, allergies, controversy Paper 1 Paper 2 Paper 3 Paper 4 Current regulations Conclusions

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Paper 4 Genetic modification removes an immunodominant allergen from soybean Plant Physiology 2003

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Purpose: To silence the Gly m Bd 30K (P34) gene transgenically

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P34 A major soybean allergen More than 65% of soy-sensitive patients react only to the P34 protein Less than 1% of total protein Pigs, calves and salmon also allergic

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Methods: Created a P34 silencing vector (plasmid pKS73) Grew these into homozyous strains Used SDS-PAGE for presence of P34 protein

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Results

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Soybean Protein Map

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Protein Analysis Wild typeP34 Silenced Missing P34 proteins and intermediates

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Main Points: TG and WT were indistinguishable in size, shape, protein and oil content P34 gene silencing was successful

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Overview Introduction: GM foods, allergies, controversy Paper 1 Paper 2 Paper 3 Paper 4 Current regulations Conclusions

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Whos in charge? Coordinated Framework for the Regulation of Biotechnology - 1986 3 regulatory bodies of genetically modified foods: (Animal and Plant Health Inspection Service)

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Considerations: Effect on environment (animals, insects) Transferable to wild type Digestive stability Toxicity Weediness

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FDA Food additives Manufacturers responsible for checking Voluntary consultation process - but all on U.S. market have undergone

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Conclusions Allergens can be added Mostly, there is no difference Can also remove allergens Continue studies Continue monitoring

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Additional Works Consulted USDA Website. Biotechnology FAQs. Accessed 11/21/2009. http://www.usda.gov/wps/portal/!ut/p/_s.7_0_A/7_0_1OB?contentidonly=true&navi d=AGRICULTURE&contentid=BiotechnologyFAQs.xml http://www.usda.gov/wps/portal/!ut/p/_s.7_0_A/7_0_1OB?contentidonly=true&navi d=AGRICULTURE&contentid=BiotechnologyFAQs.xml Singer, S., Raven, D., Johnson G., Losos, J. 2005. Biology 7 th Edition. McGraw Hill. New York, NY.

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Picture References http://agriculture.sc.gov/UserFiles/Image/soybeans7.jpg statistihttp://tharwacommunity.typepad.com/tharwa_review/images/2008/03/12/gm_foods.jpg http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-20/2032.jpg http://repairstemcell.files.wordpress.com/2009/03/genetically-modified-food-fda.jpg http://animalid.aphis.usda.gov/nais/wp_images/extension/high_res/usda-logo.jpg http://blogs.venturacountystar.com/motorhead/epa.jpg http://www.marlerblog.com/uploads/image/fda-logo.jpg http://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/gel.jpg http://www.life.umd.edu/classroom/bsci423/song http://media.photobucket.com/image/Ige%20allergy/belldandy_84/Allergies.jpg/F03-44.jpg http://api.ning.com/files/f7sw9nvb2lvWKi0Z-603fV67e5PN0 http://www.flourallergy.com/images/allergy-test.jpg Y5iFz4Ef69JQNJKYzZ5lyynC5e9rpsiR7KJHFqW*CGRvzuPN6AianENPQ159UhHB680/pha0155l.jpg http://www.worldcommunitycookbook.org/season/guide/photos/corn.jpg http://e-internetbusiness.com/wp-content/uploads/2009/06/benadryl.png http://www.ars.usda.gov/is/graphics/photos/sep02/k9975-3i.jpg http://e-internetbusiness.com/wp-content/uploads/2009/06/benadryl.png http://www.ars.usda.gov/is/graphics/photos/sep02/k9975-3i.jpg http://intmedweb.wfubmc.edu/grand_rounds/1999/Image15.jpg

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Questions??