GM food safty - Alexander Haslberger

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Development of concepts


WHO, Comparative assessment


Unintended effects, SAFOTEST


Allergenicity


HGT, GMOBILITY


Risks from the environment ?


Conclusions


Alexander Haslberger


7/04

Breeding: Irradiation

Irradiator at Institute of
Radiation Breeding

Ibaraki
-
ken, JAPAN
(
http://www.irb.affrc.go.jp/
)

Uninteded Changes in convent. breeding

Published examples of unintended changes in conventional crops that have

evaded pre
-
release evaluation include:



At least two potato varieties
withdrawn after commercial release

due to abnormally high levels of toxic glycoalkaloids in their tubers

(Zitnak & Johnson, 1970,
Am. Pot. J.
47: 256
-
260. Hellenas et al.,

1995,
J. Sci. Food Agric.
68: 249
-
255).



A pest
-
resistant celery variety
with abnormally high levels of

psoralens which caused light sensitive rashes and burns in pickers

(Ames & Gold, 1990;
Proc,. Natl. Acad. Sci. USA
87: 7777
-
7786).



A Spring barley variety,
Chariot, which was selected for high

malting quality in Cambridge, UK, but when grown in the northern

UK shows high levels of grain splitting and the resulted is a

reduction in quality (R. Ellis, SCRI
pers comm.
;
comments on the

UK recommended lists for cereals, 1999
-
2002).


Unintended Changes in GM plants

Nature Biotechnology

Haslberger, 2003



FAO/WHO expert consultation, 2003



Insertion of a transgene sometimes can affect expression of another
gene(s).



Expression of the transgene ideally should have no undesired effects
on the expression of other host genes or health of the host. Other
outcomes, however, have been observed.



The transgene can be silenced by methylation or through other
mechanisms.



Because expression of the transgene often is controlled by novel
regulatory elements outside of the host’s normal homeostatic
feedback mechanisms, expression of the transgene can have
pleiotropic effects, that is, effects upon multiple traits of the host.



The use of viral and transposon vectors poses the hazard that the
transgene might subsequently move within the genome


Existing traditional foods are considered to be
safe,
through their long history of use
, even though they may
contain anti
-
nutritional or toxic substances


The concept of
Substantial Equivalence embodies the
idea
that
conventional foods can serve as a basis for
the safety assessment of GM foods
, since most of these
foods are obtained from them


Concept of Substantial Equivalence

OECD, 1993

Criticism on the S.E.



Erik Millstone, Eric Brunner and Sue
Mayer


Nature,
October 7, 1999


Showing that a genetically modified food
is chemically similar to its natural
counterpart is not adequate evidence
that it is safe for human consumption


Range Controls:
7,3
-

71,2 meal

16,1
-
41,1 seed

Recommend: 30


Effects on environmental factors on

Glucosinolates in PGS
-

Rape

DROUGHT
-
STRESS ?


Novak WK, Haslberger
AG.
, Food Chem Toxicol
2000: 38


Canada and the S.E;



biotechnology
-
derived
products that are
considered to be the
same as their
conventional
counterparts should
not be exempted from
testing..

Canadian Food Inspection
Agency

Science Branch

Office of Biotechnology

An Overview of the Royal
Society Report on the Future of
Food Biotechnology










Hazard
-
, risk
-

, safety
-

assessments

Risk Analysis, in general

Risk Assessment

Risk Management


Risk Communication

Process Initiation



Science based



Policy based



Interactive exchange


of information and opinions

concerning risks

Uncertainty ?

Codex guideline principles

The key elements of the Principles are:




there should be a
pre
-
market food safety assessment,

on a case
-
by
-
case basis, for foods derived from biotechnology. The data and information
used in this assessment should be of a quality that would withstand scientific
peer review;




the food safety assessment is
based on a comparative analysis

with a
"conventional counterpart" to ensure that the resulting biotech food is no less
safe than the foods normally consumed by the population;




risk management measures

should be proportional to the risks identified in
the safety assessment and may include measures such as
labelling, post
-
market
monitoring and product tracing
;



The comparative safety assessment



The initial step is comprised of a thorough comparison with the closely
related conventional counterpart to identify any differences that may
have safety implications for the consumer.



This
comparison

includes both phenotypic characteristics as well as a
compositional analysis. The phenotypic analysis should also include
comparative health parameters.


The
compositional analysis

will focus on key substances in the animal
products under scrutiny and will be subject to changes according to
latest scientific state
-
of
-
the
-
art.


The second step of the CSA comprises the
toxicological and
nutritional evaluation

of the identified differences between the GMO
and its comparator.



As a result of this second step additional testing may be required and
can result in a
iterative process

in order to obtain all relevant
information for the final risk characterisation.

Comparative safety assessment, FAO/WHO, 2003,
Kok and Kuiper

CSA, II


Any differences found as a result of the CSA serve as comparable to the
hazard identification and hazard characterization steps in a traditional risk
assessment paradigm




The molecular characterisation

should comprise an analysis of the copy
number and a sequence analysis of the flanking regions of the place of
insertion



Food intake assessments will also include an estimate of the extent to which
current food products will be replaced by the GM



The limitations of standard toxicity testing applied to whole foods



Assessment of the replacement factor of important animal
-
derived sources
of micro
-

and macronutrients by GM animal products in the event of altered
composition with relation to these nutrients

Molecular characterisation,

RR Soya


US
-

EPA: FIFRA consultation 2004


Thus to more fully investigate the possibility that
T
-
DNA inserts are in active genes, Northern
hybridization blot hybridization to detect cognate
mRNA transcripts

should be done using 5’ and
3’ sequences that flank each T
-
DNA as probes.
Comparing the size of transcripts

detected by
Northern blot in fractionated RNA from non
-
transformed and transformed lines will assess
whether the T
-
DNA insertion physically disrupted
or significantly (2 SD from the norm) affected
expression of the cellular gene

Comparing Gene Expression

Control

Test

Isolate mRNA

& label cDNA

Isolate mRNA

& label cDNA

cDNA hybridised to microarray

Expressed only

in control

Expressed in

both conditions

Gene

induced

Not

expressed

cDNA binds to

corresponding gene

TOX: Difficulties Animal
Feeding Studies Whole
Foods









Small doses to be fed (bulk, satiety)

Nutritional imbalance of the diet

Many confounding factors

Small safety margins, if any

Insufficient sensitivity for specific endpoints



Microarrays/RIKILT

Metabolite profiling/ Engel

90 d rat tox for screening of
unintended effects

GM foods: allergenicity and
immune responses


FAO/WHO expert
consultations, 2001
-
2003



Distinguish the source of the
gene:


safe history as food,


no history as food,


Know allergen: discouraged
presently



Sequence homology


stability, digestibility



Models for sequence comparison


minimal epitope length, false positive


Additional vitro tests: APCs?


Sera testing


Animal tests?

Continuing discussions: allergenicity

FAO/WHO, 2003: It was recognized that animal models for
allergenicity testing, even those that are not yet validated, may
be of value to identify potential allergens.

It is recommended that additional efforts

should be directed to the further development and validation of
these models.


Post market Monitoring !


GMOBILITY


Safety evaluation of horizontal gene
transfer from genetically modified
organisms to the microflora of the food
chain and human gut




W
P
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WP1 Selection, construction and lab bench testing of donor
sequences and recipient strains for horizontal gene transfer

WP2 Horizontal gene transfer in food systems

WP3 Horizontal gene transfer in
in vitro

model systems

WP4 Horizontal gene transfer
in vivo

WP5 Quantitative risk assessment and evaluation of model
systems

HGT

Our Gut Flora Helps Prevent
Colonisation by Pathogens

Rapidly colonises gut after
birth

Comprises more than 10
14

organisms


More than 400 species


An individuals flora is
immunologically distinct


Symbiotic relationship with
host


HGT in Food

DNA digestion in porcine GI tract
material compared to TIM

Duodenum

Jejunum

Ileum

Pigle
t

TIM1

Piglet

TIM1

Pigle
t

TIM1

527
bp

<30”

<30”

< 2’

< 30”

<1’

30”
-

60’

1617
bp

<30”

<30”

<30”

<30”

<30”

<30”
-

5’

DNA
persistence
in vivo

Time

Stomach

Duodenum

Ileum

Caecum

Colon

3 hr

421

118

924

(+)

421

224

5 hr


421

118

421

924

(+)

924


Gnotobiotic rats (
B. subtilis)

receiving pDNA


Sacrificed after 3 or 5 hr


DNA extraction


PCR (
118
-
9
24

bp)


DNA can transiently persist during the passage of the GI tract

Marker rescue transformation of
B.
subtilis

LTH 5466 in milk and
chocolate milk


B
. subtilis

LTH 5466
developed competence
during growth (

⤠楮⁕䡔
milk (A) and chocolate milk
(B).


Marker rescue was observed
with
E. coli

DNA (10
µ
g/ml)
containing
npt
II gene


Transformation frequency
-
>
grey bars


Detection limit

-
>


white
bars.

A

B

In vivo
conjugation in
gnotobiotic rats


In vivo

experiment

10

ml

of

inoculum

(E
.

coli

pBHR
1
GFP)

overnight

at

37

°
C

in

BHI

Suspended

in

200

ml

of

physiological

solution

Administration

to

mice

for

28

days

2

times
-
week

Decimal dilutions

Plating on
specific media

Further analyses

PCR

Fluorescence

Microscope

FACS

FACS Analysis detected GFP
-

Donor E. coli


but no transkonjugants


M Co Isolates Co

GFP PCR 800bp


Isolates Co Co

GFP protein expression

GFP detection in bacteria from faeces

99 % similarity with Bacteroides

Summary HGT: GMOBILITY


Small amounts of plasmid, plant DNA in all parts of the
GI tract



in all parts of the GI tract development of competence
and transformation ( low rates ) possible in vitro



No transformation seen from marker gene fragments
from plants in vivo models using sensitive models,
gnotobiotic rats / marker rescue assays



Conjugation in the GI tract

FAO/WHO expert consultation, 2003



The DNA construct used to change the genetic make
-
up of the animal
should be considered within an assessment, especially if the gene or
its promoter is derived from a
viral source




There is potential for horizontal transfer of the gene construct:
food
-
ingested foreign DNA may not be completely degraded in
the gastrointestinal tract




For the food safety assessment, it is prudent to assume that DNA
fragments may survive the human gastrointestinal tract and be
absorbed by either the gut microflora or somatic cells lining the
intestinal tract.



In general, the Consultation advocated avoiding the use of any
unnecessary DNA sequences including marker genes in the genetic
construct.



Outcrossing, Herbicides, Pesticides




BT
-

cotton : local
factors decide on
pesticide
reductions and
benefits

Local factors
and agro
-

ecological base
lines

..
analyze technologies, e.g. Organic Farming, Integrative Pest
management, precision agriculture, marker directed breeding,
GM foods …. for their effects on bio
-
diversity …as the basis of
ecosystem services to humans … ( e.g. sustainable food
production …)

Before agriculture

Modern plant breeding (ca 1900)

Onset of domestication

Wild populations

Landraces

Modern lines

Ex situ

collections

Genetic diversity

FAO: concern
because of crop
diversity

WHO : GM food aspects need to
be seen “holistic”



Interaction environment, human health


Socio
-

economic aspects, patenting


Globalised trading but regional
consequences


Ethic aspects ( consultation 2003 )


FAO, Food ethics, 2003


While risk assessment is based on science,
scientific
evidence and analysis cannot always provide immediate
answers

to questions posed. Much scientific evidence is
tentative, as the established processes of science
include checking and re checking outcomes in order to
obtain the required level of confidence.


Decisions usually are defended as based on “science,”

and sometimes on economic costs and benefits as well,
which offer seemingly objective, verifiable evidence that
the policy choice is “correct.”


Decisions explicitly based on ethical principles and value
preferences can be just as defensible
, if the society
agrees broadly on the ethical assumptions used to make
policy. The emphasis on science and the exclusion of
ethical argument as the basis for decisions may polarize
the scientific debate.

Four principles have
been established as
fundamental in the
biomedical field:

respect for autonomy,
beneficence, non
-
maleficence and justice
(Beauchamp and
Childress, 2001).

FAO/WHO consultation GM food
safety, 2003 includes Ethics

Leiden conference, Sept 2004:


Global Code of Ethics for the introduction of Modern Food

Biotechnology in global markets

Gesche Astrid H., Entsua
-
Mensah Mamaa and Haslberger G. Alexander

QUO VADIS?


GM food safety debate has resulted in a
high level of safety of present products



New products (stacking of different traits,
foods with intended changes of nutrients )
will need improved health and
environmental assessments


Quo vadis II

EPA
-

FIFRA consultation, 2004:

site directed insertion

Quo vadis: III