Risk%20Assessment%20of%20genetically%20modified%20food

1
Biological and Environmental aspects of GM crop
usage
Prof. Parthadeb Ghosh
UGC Emeritus Fellow Plant Biotechnology
Research Unit Department of Botany University of
Kalyani
2
History of crop improvement

• By trial and error for almost 9900 years
• By scientific principles of breeding for last 110
  years
• By chemical induced mutation for last 85 years
• By rDNA technology last 34 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-Dete
rioration in soil quality -Climate change (global
warming)
5
The Role of Biotechnology

• Raise the yield ceiling and provide
  sustainable production systems
• Agriculture
• Resource Based Science 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
Building the Transgenes
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
10
Biotech crop countries and mega-countries
11
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
12
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

13
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

14
NUTRITIONAL QUALITY
Golden Rice
Expression of enzymes of ß-carotene pathway in
rice endosperm Amelioration of Vitamin A
deficiency
15
Bt Brinjal Putting Science into Agriculture
16
Bt-Brinjal
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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
18
The Risks..

• Human Health
• Environment

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Human Health

• Alteration in nutrition profile of the food
• Introduction of toxins
• Introduction of existing or new allergens

20
Environmental concerns

• Horizontal gene transfer
• Effect on non target organisms
• Development of resistance by pests

21
Societal concerns

• Unfamiliarity with the technology
• Lack of reliable information
• Negative media opinion
• Opposition by activists group
• Mistrust of the industry

22
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)

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Is this food safe ?
24
Is the food safe ???
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Some known allergenic food sources
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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

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Codex Alimentarius Commission
TO PROVIDE A SUITABLE FRAMEWORK FOR UNDERTAKING
RISK ANALYSIS ON THE SAFETY AND NUTRITIONAL
ASPECTS OF FOOD DERIVED FROM MODERN BIOTECHNOLOGY
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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
29
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
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The Molecular Characterization
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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

32
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

33
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

34
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

35
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

36
Allergy Some background

• A specific adverse immune reaction to a protein
• Immediate IgE mediated
• Allergy
• Delayed Cell 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

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The two steps.
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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

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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 ???

40
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

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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

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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

43
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.

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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

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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??)

46
GM Product Classification
Insert
Codes for simple Functional/storage protein
Does not code For protein
Codes for functional enzyme

• No significant sequence match with the
  aforementioned GM proteins as per data of major
  biotech companies around the world based on
  bio-informatics (no gt 50 overall or gt 35
  identity in 80 aa match)
• Cry 1, 2, 3, CP4 EPSPS, NPT II and cry 1 F
  (except one 6 - mer match)

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
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Toxicological Studies

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

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Acute Toxicity
Protein
Qtative safety
Characterization
Toxicology
Acute oral toxicity (mice) Limit dose (2000
mg/kg, OECD) Mortality, body wt., behavior,
necropsy
Source, HOSU, Mechanism of action Specificity Expr
ession 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
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Grains from GM crops

• Codex approach 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

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Food from GM plant
Food
Nutritional Eq
Characterization
Toxicology
Qlitative Ction
HOSU Comparison with non GM isogenic parental
line
Subchronic rodent dietary feeding studies (rats,
90 days)
Broiler chicken (42 days)
GM Food As Safe As
Rapid growing sp Sensitive to changed nutrition
Biochemistry, Haematology Histopathology Organ
wt etc
Compositional Analyses Agronomic characters
Yes
No
Reference non GM Tolerance limit
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Animal tests may not be warranted.

• Source not known to synthesize toxin protein (s)
• The protein has a history of safe use
• Amino acid sequence analysis lacks identity with
  known toxins
• Protein is easily digested/degraded
• Protein is unstable to heat and other processing

52
The choice should be based on objective science
based decision
53
Indian Scenario
The regulatory framework for transgenic crop
in India comprises following rules and
guidelines Rule 1989 under EPA of 1986
rDNA 1990 Seed policy of 2002 Res. in
transgenic crop 1998 Ministry of Forests
and Environment, GoI and Dept. of Biotechnology
are implementing Agencies
Approvals
Rules Policies
Guidelines
rDNA Advisory Committee (RDAC) Rev Com on Gen
Manipulations (RCGM) Gen. Engg. Approval
Committee (GEAC) Institutional Biosafety
Committee (IBSC) State Biosafety coord committee
(SBCC) District Level Committee (DLC)
IBSC RCGM (DBT) GEAC (MoEF)
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Biotechnology Policy of India, 2005Some
excerpts

• A comprehensive and integrated view should be
  developed of r-DNA and non r-DNA based
  applications of biotechnology with other
  technological components required for agriculture
  as a whole
• Regulatory requirement in compliance with
  Cartagena Protocol, and other international
  treaties and protocol for biosafety, germplasm
  exchange and access and the guiding principles of
  Codex Alimentarius will be implemented through
  inter ministerial consultative process
• availability, access, release and efficient
  system for biosafety assessment of GMOs and
  products thereof safe use of approved
  technologies and prevention of unauthorized ones
  building public trust
• India a signatory and has ratified, along with
  138 countries, the Cartagena Protocol which
  provide guidelines for safe handling and
  trans-boundary movement of LMOs

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• Rules, 1989 The Ministry of Environment
  Forests, GoI notified the rules and procedures
  under EPA of 1986 covering areas of research as
  well as large scale applications of GMOs and
  products made there from throughout India
• rDNA Guidelines Formulated by DBT, revised in
  1994, cover R D activities on GMOs, transgenic
  crops, large scale production, deliberate release
  of GMOS in environment, shipment and import for
  lab. research
• Guidelines for research in transgenic plants
  Separate guidelines formulated by DBT in 1998.
  Covers R D in plants, development of
  transgenics, their growth in soil for molecular
  and field evaluation, also include guidelines for
  toxicity and allergenicity evaluation of
  transgenic seeds, plants or plant parts
• Seed Policy Issued by Ministry of Agriculture
  in 2002 contains a separate section (no. 6) on
  transgenic plants. It states that all
  transgenics will be tested for environment and
  biosafety before commercial release, agronomic
  traits to evaluated for at least 2 seasons under
  the all India coordinated project trials (AICPT)
  by ICAR. Seeds to be registered as per the
  provisions of seed act. Post market monitoring
  for 3-5 years by MoA.
• Prevention of Food Adulteration Act Issued by
  Ministry of health Family Welfare, GoI for
  assuring quality and safety of food and encourage
  fair trade practices.

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The Depts., Ministries and Institutions

• MoEF Holds GEAC, apex body that gives approval
  for manufacture, sale, import and export of all
  GMOs and products thereof
• DBT Holds RCGM, approves research and small
  scale trials etc of GMOs
• MoHFW Regulates PFA Act
• ICMR Advisory body for MoHFW
• MoA Nodal Ministry for Agriculture growth.
  Implements Seed Policy, 2002
• Ministry of Commerce Industry Formulates
  EXIM policy
• Ministry of Food Processing Industries
• National Institute of Nutrition, Hyderabad
• Central Food Technology Research Institute,
  Mysore
• Defense Food Research Laboratory, Mysore
• Industrial Toxicology Research Institute, Lucknow
• National Bureau of Plant Genetic Resources, New
  Delhi
• Centre for DNA Finger Printing and Diagnostics,
  Hyderabad

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Indian Regulatory Framework
Source of gene, cloning strategy, Sequence
details of inserted gene Vector and method of
tformation Genetic analysis and segregation
stability Biochemistry of expressed gene
product by chemical, immunological methods

• Rationale and benefits
• Biology of plant system
• Molecular biology and
• transformation method

Part A
Competitive toxicant analysis Potential for
weediness Risk during processing/handling Gene
and pollen transfer Effect of diseases pests

• Field trial plans
• Phenotype of plant,
• fruits/seeds

Part B
Consequence to the environment
Part C
Nutritional studies, acute and chronic
toxicity Allergenecity testing Classical animal
feeding trials Immunotoxicological studies Gut
toxicological studies Effect of transgene
product on gene integration, regulation
expression
Part D
Food Safety Evaluations
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Bioinformatics.

• Learnt and practiced FASTA and BLASTP local
  sequence alignment tools for matching sequences
  of interest with online databases
• Result analyses based on E score, identity,
  similarity and literature review
• E-score is inversely related to similarity of two
  proteins depends on overall length of joined
  sequence alignments, quality (similarity,
  identity) of overlap database size.
• Codex (2003) recommendations for bioinformatics
  search using FASTA/BLASTP algorithms for
  allergenecity assessment
• 35 identity over 80 aa sliding window
• 50 identity over entire sequence length

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Purpose

• To have an in depth understanding of the
  framework for biosafety analysis of GM food
• Molecular characterization
• Toxicology studies
• Allergenicity assessment
• Bioinformatics

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The outcome

• Pretty good understanding of different frameworks
  for analysis (ILSI, FAO/WHO, Codex)
• Understanding from risk assessor, academician and
  developers points of view
• Personal interactions for studies wrt toxicology,
  allergenecity, genetic characterization and
  bioinformatics
• Industrial visits and interactions
• A well informed University Professor
• Transfer to students of Masters Program (MAP)
• Participation in capacity building programs (HRD
  T)
• Strengthening technical capacity to assess,
  manage and monitor risks associated with GMO ?
• Assist the regulatory agency in the home country
  ??

62
Biosafety
Protecting human and animal health and
environment from possible side effects of the
products of modern biotechnology such as
genetically modified plants
63

• Many food considered safe based on history of
  safe consumption
• Hazards associated are analyzed as per guidelines
  of CAC and CWPRA
• Being used for long time for chemical, microbial
  and nutritional factors
• May need suitable modification for whole food

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Limitations.

• Disproportionate attention relative to
  information it imparts in terms of food safety
• In the absence of phenotypic data, unlikely to
  predict unforeseen effects on nutrient profile
• No correlation between copy number and safety
• May provide information on positional and
  pleiotropic effects and gene silencing
• Ensures appropriate characterization of the
  genetic modification

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The Mechanism ..
From Nagai et al, 2006
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Choice of Algorithm

• It is based on desired comparison type,
  computational resources available and goal
• FASTA, BLAST P and Ssearch are the algorithm of
  choice for protein sequence alignments
• Low specificity filter help avoid false positive
  statistically significant scores
• Scoring matrix and Gap penalties use default

67
Amino acid sequence comparison

• Overall FASTA vs AllergenOnline
• (gt50 overall identity or E score lt1 e-7 )
• (Most predictive, allergic cross reactivity most
  likely)
• FASTA scanning over 80 aa window
• (gt35 identity indicates some chances of
  reactivity)
• Scanning 6 or 8 aa segments ???
• If identity detected above the specified limit,
  go for specific serum testing

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Serum IgE test

• Serum donors must have relevant and proven
  allergy
• The test must be specific and validated (should
  be able to detect binding to conformational or
  linear epitopes)
• Must include positive (who react to gene source
  or sequence matched allergen) and negative
  control allergic sera and proteins/extracts
• Use protein free from N linked glycosylation, if
  possible
• No established claims of allergenecity of new
  proteins in GM crops (upto Dec., 2007)

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Some more considerations

• Many potent food allergen are stable in pepsin
• Many are abundant protein, up to 1 of total
  proteins in food
• If the transgene is transferred to known allergen
  source, then changes in endogenous allergenicity
  needs to be monitored
• However, it is necessary to establish natural
  variability of allergenicity among the non GM
  varieties.
• Special attention must be given when a
  transcription activator is transferred or
  transgene is transferred in the coding region for
  an allergen

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Transgenic crop under development and field
trials in India

• Crop Organization Gene
• Brinjal IARI, New
  Delhi cry1Ab, cry1Ac
• MAHYCO, Mumbai
• Cauliflower MAHYCO, Mumbai cry1Ac
• Sungrow Seeds Ltd., New Delhi
• Cabbage Sungrow Seeds Ltd., New Delhi cry1Ac
• Chickpea ICRISAT, Hyderabad cry1Ac, cry1Ab
• Groundnut ICRISAT, Hyderabad IPCVcp, IPCV
  replicase,
• Maize Monsanto, Mumbia CP4 EPSPS
• Mustard IARI, New Delhi CodA, Osmotin,
• NRCWS, Jabalpur bar, barnase, barstar
• TERI, New Delhi Ssu-maize, Psy, Ssu-tpCrtI
• UDSC, New Delhi bar, barnase, barstar
• Okra MAHYCO, Mumbai cry1Ac
• Pigeonpea ICRISAT, Hyderabad cry1Ab SBTI
• MAHYCO, Mumbai cry1Ac
• Potato CPRI, Simla cry1Ab
• NCPGR, New Delhi Ama-1
• Rice Directorate of Rice Research, Bacterial
  blight res, Xa-21,

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Thank You