Genetically Modified Soybeans: Equal Allergenicity as Natural Counterparts?

Genetically Modified Soybeans: Equal Allergenicity as their Wild Type Counterparts?Katie Van Den EindeNovember 24, 2009Advisor: Dr. Chastain1OverviewIntroduction: GM foods, allergies, controversyPaper 1Paper 2Paper 3Paper 4Current regulationsConclusions

2ImportanceGM foods:SoybeansCornTomatoesRiceCanolaPotatoesSugar beetsSugar cane

3ModificationsHerbicide resistanceInsect resistanceDisease resistanceAddition of proteins/vitamins

2003 84% of US soybean acreage was glyphosate tolerant (Roundup ready)

4Basics of Genetic ModificationProcedures1. Plasmid insertion2. Gene guns3. Protoplasts

5AllergiesMajority of allergic reactions are immunoglobulin E (IgE) mediated.IgE allergies affect about 1-2% of adults2-8% of children6Symptoms:Itchy, watery eyesRash CongestionItchinessDifficulty breathingAnaphylactic shock (Can be life threatening)

7Basics of allergic reactions1-Allergen2-IgE antibodies3-Mast cells4-Histamine release

8Anti-Histamines

9GM ControversyEthicsGene flowResistanceHarm to other organismsAllergens???

10OverviewIntroduction: GM foods, allergies, controversyPaper 1Paper 2Paper 3Paper 4Current regulationsConclusions

11Paper 1:Identification of a Brazil-nut allergen in transgenic soybeansNew England Journal of Medicine 1996-2S albumin to add methionine to soybeans because they dont have it12Purpose:To assess ability of proteins from

1)soybeans (Glycine max)2)transgenic soybeans3)Brazil nuts (Bertholletia excelsa)4)purified 2S albumin to bind to IgE serum13Methods:Radio allergosorbent test (RAST) 4 serumsSodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) 9 serumsSkin Prick Tests

Serum from those allergice to brazil nuts14RAST basics

15Results: RAST

More inhibition of IgE binding = more allergic.Triangles= WTSquares= GM soybeanCircles= Brazil nutAdded human serum first, which bound if allergic. The more that bound, the more the labeled IgE binding was inhibited16Results: SDS-PAGE

IgE bindingTotal ProteinsIgE bindingStandardsWT SBTG SBBrazil Nut2SWT SBTG SBBrazil Nut2S2S in TG SBBrazil Nut17Results: Skin-Prick Test

Also on three control people with no allergies only had positive response to the histamine prick18Main 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.19OverviewIntroduction: GM foods, allergies, controversyPaper 1Paper 2Paper 3Paper 4Current regulationsConclusions

20Paper 2Lack of detectable allergenicity of transgenic maize and soya samplesJournal of Allergy and Clinical Immunology 200521Purpose:Monitor 5 GM products whose transgenes came from sources with no allergenic history

Such as bacteria22Methods:Food SurveyPrevious exposure?Skin Prick Tests27 kids with food allergies50 patients with asthma rhinitisSDS-PAGE23Flour products tested

24Food survey results

25Western Blot

26Testing Lab Supply

SDS PAGEWestern Blot27

SDS PAGEWestern Blot28Skin prick and IgE results

29Main Point:No detectable difference in IgE reactivity between wild type and GM soybeans or corn.

30OverviewIntroduction: GM foods, allergies, controversyPaper 1Paper 2Paper 3Paper 4Current regulationsConclusions

31Paper 3A comparative study of the allergenic potency of wild-type and glyphosate-tolerant gene-modified soybean cultivarsActa pathologica, microbiologica et immunologica Scandinavica 2003

32Purpose:To compare allergenicity of 8 wild type and 10 GM soybeans varieties (all for CP4 EPSPS)EPSPS: 5-Enolpyruvylshikimate-3-phosphate synthase33Methods:RAST (serum from 10 patients)SDS-PAGEHistamine Release testSkin prick tests34RAST results

More inhibition of labeled IgE binding = more original serum bound first.35RAST results

Concentration of extract needed for 50% inhibition of IgE binding (variety #12)If GM was more allergenic, the dots wouldve been way father down- hardly any needed, and the WT wouldve been really high up lots needed for the IgE binding36Histamine Release results

Notice lack of any major differences no where to point an arrow!Skin Prick TestHistamine Release(0=negative,6=lots)Also did statistical analysis of these found differences between the patients, but no significant difference between the two groups of soybeans37Histamine Release for patient I

Pretty similar!38Main Points:Difference between patients response, but no statistical difference between WT and TG soybeans.Addition of CP4 EPSPS gene higher allergenicity

39OverviewIntroduction: GM foods, allergies, controversyPaper 1Paper 2Paper 3Paper 4Current regulationsConclusions

40Paper 4Genetic modification removes an immunodominant allergen from soybeanPlant Physiology 200341Purpose:To silence the Gly m Bd 30K (P34) gene transgenically

42P34A major soybean allergenMore than 65% of soy-sensitive patients react only to the P34 proteinLess than 1% of total proteinPigs, calves and salmon also allergic

43Methods:Created a P34 silencing vector (plasmid pKS73)Grew these into homozyous strainsUsed SDS-PAGE for presence of P34 protein

44Results

Monoclonal antibodies45Soybean Protein Map

46Protein Analysis

Wild typeP34 SilencedMissing P34proteins and intermediates47Main Points:TG and WT were indistinguishable in size, shape, protein and oil content

P34 gene silencing was successful

48OverviewIntroduction: GM foods, allergies, controversyPaper 1Paper 2Paper 3Paper 4Current regulationsConclusions

49Whos in charge?Coordinated Framework for the Regulation of Biotechnology - 19863 regulatory bodiesof genetically modified foods:

(Animal and Plant Health Inspection Service)50Considerations:Effect on environment (animals, insects)Transferable to wild typeDigestive stabilityToxicityWeediness

51FDAFood additivesManufacturers responsible for checkingVoluntary consultation process - but all on U.S. market have undergone

52ConclusionsAllergens can be addedMostly, there is no differenceCan also remove allergensContinue studiesContinue monitoring

53Additional Works ConsultedUSDA Website. Biotechnology FAQs. Accessed 11/21/2009. http://www.usda.gov/wps/portal/!ut/p/_s.7_0_A/7_0_1OB?contentidonly=true&navid=AGRICULTURE&contentid=BiotechnologyFAQs.xml

Singer, S., Raven, D., Johnson G., Losos, J. 2005. Biology 7th Edition. McGraw Hill. New York, NY. 54Picture Referenceshttp://agriculture.sc.gov/UserFiles/Image/soybeans7.jpgstatistihttp://tharwacommunity.typepad.com/tharwa_review/images/2008/03/12/gm_foods.jpghttp://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-20/2032.jpghttp://repairstemcell.files.wordpress.com/2009/03/genetically-modified-food-fda.jpghttp://animalid.aphis.usda.gov/nais/wp_images/extension/high_res/usda-logo.jpghttp://blogs.venturacountystar.com/motorhead/epa.jpghttp://www.marlerblog.com/uploads/image/fda-logo.jpghttp://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/gel.jpghttp://www.life.umd.edu/classroom/bsci423/songhttp://media.photobucket.com/image/Ige%20allergy/belldandy_84/Allergies.jpg/F03-44.jpghttp://api.ning.com/files/f7sw9nvb2lvWKi0Z-603fV67e5PN0http://www.flourallergy.com/images/allergy-test.jpg Y5iFz4Ef69JQNJKYzZ5lyynC5e9rpsiR7KJHFqW*CGRvzuPN6AianENPQ159UhHB680/pha0155l.jpghttp://www.worldcommunitycookbook.org/season/guide/photos/corn.jpghttp://e-internetbusiness.com/wp-content/uploads/2009/06/benadryl.png http://www.ars.usda.gov/is/graphics/photos/sep02/k9975-3i.jpghttp://intmedweb.wfubmc.edu/grand_rounds/1999/Image15.jpg55 Questions??

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