Genetically Modified Soybeans Equal Allergenicity as their Wild

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 (Ig. E) mediated. �Ig. E 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 -Ig. E 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 2 S albumin to bind to Ig. E serum

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

RAST basics

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

Results: SDS-PAGE Total Proteins 2 S Standards WT SBTG SB Brazil Nut Ig. E binding 2 S WT SB TG SB Brazil Nut Ig. E binding 2 S Brazil Nut in TG SB

Results: Skin-Prick Test

Main Points: �GM soybean protein successfully competed with Brazil nut protein. �Ig. E from 8/9 allergic to Brazil nut bound to introduced 2 S 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 Ig. E results

Main Point: �No detectable difference in Ig. E 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 glyphosatetolerant 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 CP 4 EPSPS)

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

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

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

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

Histamine Release for patient I Pretty similar!

Main Points: �Difference between patients’ response, but no statistical difference between WT and TG soybeans. �Addition of CP 4 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 30 K (P 34) gene transgenically

P 34 �A major soybean allergen �More than 65% of soy-sensitive patients react only to the P 34 protein �Less than 1% of total protein �Pigs, calves and salmon also allergic

Methods: �Created a P 34 silencing vector (plasmid p. KS 73) �Grew these into homozyous strains �Used SDS-PAGE for presence of P 34 protein

Results Monoclonal antibodies

Soybean Protein “Map”

Protein Analysis Missing P 34 proteins and intermediates Wild type P 34 Silenced

Main Points: �TG and WT were indistinguishable in size, shape, protein and oil content �P 34 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 - 1986 � 3 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 11/21/2009. http: //www. usda. gov/wps/portal/!ut/p/_s. 7_0_A/7_0_1 OB? contentidonly=true &navid=AGRICULTURE&contentid=Biotechnology. FAQs. xml � Singer, S. , Raven, D. , Johnson G. , Losos, J. 2005. Biology 7 th Edition. Mc. Graw Hill. New York, NY.

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Questions? ?