Genetically Modified Crops : Concepts of Risk Assessment

1. Genetically Modified Crops : Concepts of Risk Assessment Dr. V.L. Maheshwari Professor & Director School of Life Sciences North Maharashtra university, Jalgaon-425001 (MS) India Norman E Borlaug Fellow BIGMAP Iowa State University, Ames, Iowa, USA

2. History of crop improvement By trial and error for almost 9900 years By scientific principles of breeding for last 100 years By chemical induced mutation for last 75 years By rDNA technology last 25 years

3. Green revolution Dr. MS Swaminathan Dr. NE Borlaug Irrigation facilities Improved/hybrid seeds Chemical fertilizers Pest management Farm credit Political will

4. CHALLENGES AHEAD -Population in 2050 AD: 1.5 B-Shrinking area of cultivated land-Diminishing water resources-Malnutrition and undernourishment-Deteriorationin soil quality -Climate change (global warming)

5. The Role of Biotechnology • Raise the yield ceiling and provide sustainable production systems Agriculture Resource BasedScience Based Industry Food Security ??

6. Genetically Modified (GM) Crops What is a GM crop? GM crops are genetically improved and contain a gene or genes from the same or a different species artificially inserted in its genome. Tissue Culture & Transformation – gives the maximum flexibility for moving genes within or between species.

7. CODING SEQUENCE PROMOTER poly A signal INTRON Plant Selectable Marker Gene Plasmid DNA Construct • bacterial genes • antibiotic marker • replication origin Building theTransgenes ON/OFF Switch Makes Protein stop sign Plant Transgene

8. Leaf Disk Method for A. t. Mediated Transformation Leaf Disk Preparation Co-cultivation with Agrobacterium Selection for Transformation Regeneration of Shoots

9. 22 countries, 117.7 M ha 21 crops, 107 events, 539 approvals, 29 countries

11. Data: Clive James 2006

12. Total plantation in India = 8.0 M Ha 0.80 M Ha 0.80 M Ha Others 1.6 M Ha 3.0 M Ha 1.40 M Ha

13. Bt- COTTON • Bt-cotton - First GM crop – 2002 • Second in global cotton production • Area – 8.0 million hectares – 2008 • Yield gain - 31% • Reduction in pesticide sprays – 39%

14. TARGET CROPS AND TRAITS IN THE ICAR NETWORK • Insect pest resistance • Stem borer • Rice • Sorghum • Maize • Pod Borer • Pigeon pea • Chick pea • Boll worm • Cotton • Fruit borer • Tomato • Brinjal • Aphid • Brassica • Virus resistance • Cotton • Soybean • Tomato • Potato • Banana • Papaya • Cassava • Drought stress • Brassica Transgenics • Fungal resistance • Rice • Banana • Delayed ripening • Tomato

15. NUTRITIONAL QUALITY “Golden Rice” *Expression of enzymes of β-carotene pathway in rice endosperm *Amelioration of Vitamin A deficiency

16. Bt Brinjal: Putting Science into Agriculture

17. Bt-Brinjal

18. GM FOOD CLASSIFICATION Food consisting of living organisms, e.g. soybean, maize Food derived from GMO e.g. soy oil, corn flour Foods containing ingredients produced by GMO, e.g. Vitamins or essential amino acids Foods containing ingredients processed by enzymes produced by GMO, e.g. high fructose corn syrup produced using recombinant glucose isomerase

19. The Risks….. Human Health Environment

20. Human Health… • Alteration in nutrition profile of the food • Introduction of toxins • Introduction of existing or new allergens

21. Environmental concerns… • Horizontal gene transfer • Effect on non target organisms • Development of resistance by pests

22. Societal concerns… • Unfamiliarity with the technology • Lack of reliable information • Negative media opinion • Opposition by activists group • Mistrust of the industry

23. Traditionally……. • Hardly anything what we eat today has been assessed for food safety • Even food known to be toxic or allergic or contain anti nutrients are being used based on our experience and history (Potatoes, tomatoes, eggs, milk, peanuts, fish, wheat etc)

24. Is this food safe ?

25. Is the food safe… ???

26. Some known allergenic food sources

27. What is safety ? • OECD defined it as “ the one which , as far as we know, and with the exception of some individual, who me be sensitive or allergic, when consumed in moderation over a period of time does not result in identifiable harm to the consumer” • Absolute safety is difficult to prove • One can show the absence of evidence of any harm at the most

28. Codex Alimentarius Commission TO PROVIDE A SUITABLE FRAMEWORK FOR UNDERTAKING RISK ANALYSIS ON THE SAFETY AND NUTRITIONAL ASPECTS OF FOOD DERIVED FROM MODERN BIOTECHNOLOGY

29. The Principles • Risk assessment : Identification of hazard Nature and Severity • Risk Management : Should be proportional to risk identified • Risk Communication : Should involve all stake holders, should be transparent, all stages documented Data can be obtained developer, literature, scientists, technical bulletins, regulatory agencies Intended and unintended effects New and altered hazards Changes in nutrients relevant to human health Data should be based on sound science, scientific peer review Food labeling, conditional marketing approvals, post-marketing monitoring

30. The Framework Core considerations • Gene (s) • Source (s) • Molecular characterization • Insert/copy no./integrity/ • stability • Food/Feed Composition • Proximate analysis • Key nutrients/anti nutrients • Animal performance • Protein • History of safe use & • Consumption • Function/specificity/ • mode of action • Levels • Toxicology & allergenicity Environmental

31. The Molecular Characterization

32. Molecular Characterization • Rigorous molecular characterization of each transgenic plant must be completed The following should be considered The transformation system (i) Agrobacterium mediated (ii) Microparticle bombardment Molecular characterization of the inserted DNA (i) Insert number (ii) Insert composition Genetic stability of the introduced trait (i) Segregation analysis (ii) Integron stability

33. Transformation system • A. tumefaciens mediated transformation is characterised by • Low transgene copy number • Limited molecular rearrangements in the insert • Higher transformation efficiency However it may show species specificity

34. Microparticle bombardment is characterised by Introduction of full length of transgene Transgene rearrangements Transgene copy number can vary between 1-20. Multiple copies within an insert generally co- segregate as a transgenic locus No species specificity

35. The information required… All the genetic elements (promoter, leader, terminator, marker etc) transferred along with citation Detailed map of plasmid used as a vector indicating location, orientation, size etc of genetic elements Relevant restriction enzyme sites, location of primers used in PCR, regions used as a probe

36. Allergenecity One of the components of overall risk analysis of GM Food. Potential or life threatening allergies are relatively rare It is important that a food allergen does not enter the food supply

37. Allergy : Some background • A specific adverse immune reaction to a protein ImmediateIgE mediated • Allergy DelayedCell Mediated • Most allergic reactions are caused by specific IgE antibodies • The mechanism involved is development of IgE antibodies which upon re-exposure bind to mast cells and release histamines • Occurrence ranges between 2-4% in adults and 4-8 % in children (US, Europe) • Peanuts, milk, wheat, eggs, fish, soybeans, crustacean, tree nuts together accounts for over 90% cases (EU adds celery roots, mustard and sesame seeds) • Disease management by avoidance

38. The two steps….

39. The causative agents • Food borne Peanut, tree nut, milk, eggs, crustaceans, (wheat, soybean), celery, sesame, kiwi, mustard • Air borne Pollen, weeds, molds, dust mites, latex • Other Bee and ant venom

40. Three Questions Is the novel protein an existing allergen ? Is the newly expressed protein going to cause allergic cross reactivity ? Is the new protein likely to sensitize and become an allergen ???

41. The Tests • Bioinformatics • Specific serum testing • Searchable specific allergen databases http://www.AllergenOnline.com (1313 in version 8.0 of known or putative allergens) NCBI (all sequences) • Review scientific literature for evidence of allergenicity

42. Guidelines for Allergenicity Assessment • International Food Biotechnology Council and International Life Sciences Institute (IFBC-ILSI), 1996 • Food and Agriculture Organization and World Health Organization (FAO/WHO), 2001 • Codex Alimetarius Commission (CAC), 2003

43. Databases Comprehensive databases like nrNCBI (GenPept, SwissProt, PIR, RPF, PDB) and exPASy Swiss Prot is a highly annotated database with a lot of valuable biological information Several specialized databases are also available

44. Some points to ponder… IFBS-ILSI and FAO/WHO guidelines follow a decision tree approach for evaluating risk of allergenicity whereas CAC follows a weight of evidence approach (Decision tree approach appears rigid as no single criteria is sufficiently predictive) IFBC-ILSI recommends in vivo clinical testing (SPT) and DBPCFC if a single 8 aa match is found but no cross reactivity in vitro with IgE FAO/WHO found in vivo clinical testing “impractical” or even “unethical”. Recommends 6 aa match rather than 8 aa match and targeted serum* and animal model testing (*even when the transgenic protein does not show sequence similarity and cross reactivity in specific serum test) CAC recommends a 35% identity over an 80 aa window to be a sufficient conservative prediction for potential cross reactivity.

45. Interpretation of results Evaluate the matches : E score is more useful than bit score or % identity A low E score with alignment over the entire sequence length is significant Review literature extensively

46. Issues…. ????? Animal Model tests : No validated models as yet Targeted serum IgE tests : most probably will lead to false positive results ??? Heat Stability ??? (CAC, 2003 guidelines and weight of evidence approach appears practical??)

47. GM Product Classification No significant sequence match with the aforementioned GM proteins as per data of major biotech companies around the world based on bio-informatics (no > 50% overall or > 35% identity in 80 aa match) Cry 1, 2, 3, CP4 EPSPS, NPT II and cry 1 F (except one 6 - mer match) Insert Codes for simple Functional/storage protein Does not code For protein Codes for functional enzyme Insecticidal (Cry1, cry3 etc.), ug/g Anti-fungal, ug/g Storage protein (high protein potato/high met corn), mg/g Herbicide tolerance (EPSPS roundup, PAT Soybean, rice or maize) Nutri. Enhancement (golden rice, high lys corn) Altered FA Synthesis Anti viral

48. Toxicological Studies • Food Ingredients Food additives, contaminants, pesticide residues etc. • Grains from GM crops Protein from the GM plant Whole grain

49. Acute Toxicity Protein Q’tative safety Characterization Toxicology Acute oral toxicity (mice) Limit dose (2000 mg/kg, OECD) Mortality, body wt., behavior, necropsy Source, HOSU, Mechanism of action Specificity Expression levels Bioinformatics Digestion/Heat Stability Toxic Yes No Equivalence SDS-PAGE, AA Composition, peptide finger Printing, N terminal sequencing, glycosylation, MALDI-TOF, Enzymatic/Biological activity ILSI Guidelines, 2008

50. Grains from GM crops • Codexapproach needs to be slightly modified • Foods are generally considered safe but absolute safety is difficult to establish • Take off point is ‘substantial equivalence’ • Objective is to establish that food from GM plant is ‘as safe as….’ the conventional counterpart