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

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

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

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

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

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

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

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

9. Anti-Histamines

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

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

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

13. 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

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

15. RAST basics

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

17. Results: SDS-PAGE IgE binding Total Proteins IgE binding

18. Results: Skin-Prick Test

19. 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.

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

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

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

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

24. Flour products tested

25. Food survey results

26. Western Blot

27. Testing Lab Supply SDS PAGE Western Blot

28. SDS PAGE Western Blot

29. Skin prick and IgE results

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

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

32. 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

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

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

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

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

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

38. Histamine Release for patient I Pretty similar!

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

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

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

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

43. 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

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

45. Results

46. Soybean Protein “Map”

47. Protein Analysis Wild typeP34 Silenced Missing P34 proteins and intermediates

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

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

50. 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)

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

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

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

54. 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.

55. 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

56. Questions??