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

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

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

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

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

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

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

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

Anti-Histamines

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

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

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

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

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

RAST basics

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

Results: SDS-PAGE IgE binding Total Proteins IgE binding

Results: Skin-Prick Test

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.

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

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

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

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

Flour products tested

Food survey results

Western Blot

Testing Lab Supply SDS PAGE Western Blot

SDS PAGE Western Blot

Skin prick and IgE results

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

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

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

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

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

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

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

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

Histamine Release for patient I Pretty similar!

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

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

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

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

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

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

Results

Soybean Protein “Map”

Protein Analysis Wild typeP34 Silenced Missing P34 proteins and intermediates

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

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

Who’s in charge? Coordinated Framework for the Regulation of Biotechnology regulatory bodies of genetically modified foods: (Animal and Plant Health Inspection Service)

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

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

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

Additional Works Consulted USDA Website. “Biotechnology FAQs.” Accessed 11/21/ d=AGRICULTURE&contentid=BiotechnologyFAQs.xml d=AGRICULTURE&contentid=BiotechnologyFAQs.xml Singer, S., Raven, D., Johnson G., Losos, J Biology 7 th Edition. McGraw Hill. New York, NY.

Picture References statistihttp://tharwacommunity.typepad.com/tharwa_review/images/2008/03/12/gm_foods.jpg Y5iFz4Ef69JQNJKYzZ5lyynC5e9rpsiR7KJHFqW*CGRvzuPN6AianENPQ159UhHB680/pha0155l.jpg

Questions??