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GE - Review of GE risks report for Norwegian Government

We are grateful to Angela Ryan, molecular geneticist at the UK's Open
University, for giving permission to pass on the message below which may be
of considerable interest to others.

She reviews a recent report for the Norwegian Government which identifies
the skaky nature of the scientific foundations on which genetic engineering
is based.  Whislt the review deals with many specialist issues it also
includes details (towards the end) of existing practical examples of where
GMOs have already created hazards.

The Norwegian report questions whether the development of GMOs deserves the
label "technology" given the lack of predictability and control it provides.
The report itself includes 329 references.

NATURAL LAW PARTY WESSEX
nlpwessex@bigfoot.com
<http://www.btinternet.com/~nlpwessex>www.btinternet.com/~nlpwessex
----------------------------------------------------------------------------
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Dear NLP Wessex

I'm sending you this review for information exchange purposes.  And I would
like to thankyou for all the information you have been sending me. ( also
find as word file attached).
Best Wishes
Yours Faithfully
Angela Ryan

                    REVIEW


By Angela Ryan
Researcher in Molecular Genetics, Open University
Institute of Science in Society
42 Manor Road, High Barnet, London, EN5 2JJ
Tel/Fax 0181 441 6480
email.  A.Ryan@i-sis.dircon.co.uk


TOO EARLY MAYBE TOO LATE : Ecological risks associated with the use of naked
DNA as a biological tool for research, production and therapy.
Reported to the Directorate for Nature Management, Norway.


This report was commissioned by the Directorate for Nature Management for
the Norwegian Government in accordance with the Gene Technology Act which
stipulates that any genetically modified organism (GMO) must undergo an
environmental impact assessment before being released.

The report serves to highlight the lack of knowledge pertaining to the
ecological questions about the fate of naked DNA in the environment.
Horizontal gene transfer across species barriers is carefully considered as
is the uptake of naked DNA from the environment. It explains that most of
molecular biology and gene technology research takes place in agriculture,
industry and the biomedical sphere and that almost no environmental research
has been done.

The report acknowledges that there are indeed potential benefits with gene
technology but it stresses that the pertinent unanswered questions represent
flaws in basic insight, monitoring and risk assessments.  The importance of
the precautionary principle and the need for greater research comes over as
critical despite economic pressures and calls for a moratorium that lie at
the heart of the GMO debate within Europe.  Competition between the
pro-biotech sectors and their opponents for the attention of politicians,
experts, dealers, consumers and public opinion is considered to be an
important factor for the safety of GMOs and the report claims that the
absence of such public debate is in itself a risk.

The report refutes the idea that genetic modification is similar to
conventional breeding or cultivation.  Gene technology introduces new exotic
genes and creates unnatural recombination¹s whose genetic position within
the recipient cells is unpredictable and cannot yet be targeted.  This may
result in unpredictable effects on the metabolism, physiology and
biochemistry of the recipient.   The up-take, integration and expression of
naked DNA is reviewed in detail as are the potential hazards pertaining to
the regulation of endogenous gene expression.

Emphasis is placed on the fact that the vectors used in genetic manipulation
are made from genetic parasites (viruses, plasmids, mobile elements) which
are developed to express genes across species boundaries and ecological
barriers.  Many of them are able to invade and insert their DNA into the
chromosomes of any kind of cell and they are specially constructed to break
species barriers. They may in transit have the ability to pick up and
transfer genes from new host organisms or other genetic parasites therein.
Pathogenic viruses may result with potential to infect earlier refractory
hosts and during such potential relays, genetic rearrangements and mutations
may arise with unpredictable results.  Most vectors also carry antibiotic
resistance as markers for integration, these genes also have the potential
to leak.

The insertion of new promoters and enhancers that govern the transgene
expression also represent prominent uncertainties.  They may change the
nucleosome positioning of the host genome and or the methylation patterns on
the recipient chromosome(s) over long distances up and downstream from the
insertion site.  Promoters and enhancers often function in response to
signals received from the internal or external environment of the organism.
In GMOs unpredictability can be found with the expression level of the
transgene, the expression of a vast number of the organisms own genes, the
influence of geographical, climate, chemical (eg.xenobiotic) and ecological
changes in the environment and the transfer of vector sequences within the
chromosomes of the organism and vertical and or horizontal gene transfer to
other organisms.

The report suggests the major problem with biotechnology vectors is that
they contain genes that confer resistance to antibiotics, herbicides,
insecticides and other cytotoxic products and these can spread in the
environment and create ecological problems.  Plasmids used in gene
technology contain DNA sequences which ensure replication and genetic
expression in both procaryotic and eucaryotic cells.  Should they escape
they may multiply within and be spread by representatives of both kingdoms.

The report reviews in great detail the most commonly employed strategies of
gene transfer; direct injection, biolistics, electroporation, calcium
phosphate precipitation, liposomes, ligand/DNA conjugates, virus- retro,
adeno, adeno-assoc., vaccinia, herpes.  It concludes that none of these
methods can be carried out in ways which preclude the release of nucleic
acids.  The usual application of naked DNA involves the transfer of
double-stranded DNA but now rapid developments in the use of anti-sense
oligonucleotides and ribozymes and the direct introduction of RNA into cells
represents new potential problems for health and the environment.

The persistence of naked DNA in the environment in the form of both round
plasmids and strands and also its ability to remain biologically active is
discussed.  The distinction between phenotypic death and genetic death is
carefully explained and the report reviews the recent scientific evidence
suggesting that DNA belonging to dead organisms maintains an ability to be
biologically active for considerably longer than previously thought.
Knowledge as to what may happen to genetic material which is broken down by
microorganisms is largely non-existent.

The definition of horizontal gene transfer (HGT) and the scientific evidence
supporting it is well documented in this report.  Horizontal transfer takes
place for both genomic (usually non-mobile) sequences derived from
transposable genetic elements or mobile elements.  Documented cases exist of
genomic sequences being transferred from eucaryotes to procaryotes, from
procaryotes to eucaryotes, between procaryotes and between eucaryotes (
reviews in Heinemann, 1991; Kidwell, 1993;  Harding 1996;  Wostemayer et
al,.  1997 and Nielsen et al,. 1998).

Not only are there many examples of probable HGT events but the molecular
mechanisms which may contribute to such transfers have also been observed,
both physical means of transfer for DNA between cells and recombination
mechanisms which lead to the gene transfer becoming permanent.  Such
molecular mechanisms are reviewed; transduction, conjugation, transformation
/ transfection and transposition.

The biological and evolutionary important of HGT is not known but in order
for HGT to take place, genetic material has to overcome at least two types
of hypothetical barrier (Heinemann,. 1991),  an Œintroduction barrier¹ and
an Œestablishment barrier¹.

It is clear that introduction barriers are often broken down and that a
network of genetic exchange between organisms exists.  Oligo and
polynucleotides cannot diffuse through lipid membranes of living cells, it
has been shown that nucleic acids can be taken up by endocytosis which is
mediated by nucleic acid-specific receptors (Loke et al,. 1989; Vlassov et
al., 1994) and similar mechanisms may be active in bacteria also
(Dreiseikelmann, 1994; Lorenz & Wackernagel, 1994).

Following up take, in eucaryotes the nucleotides can escape from the
endosomes and reach nucleic acids located in the cytoplasm and the nucleus
(Vlassov et al., 1994).  Bacteria, remove foreign DNA using restriction
enzymes but this mechanism can clearly fail under certain circumstances
(Nielsen et al., 1998).  Many bacteria may be naturally competent at
transformation with DNA from any source whatsoever (Heinemann, 1991).
Conjugation transfer of DNA takes place within species but also across
species boundaries and even kingdoms e.g. agrobacteria transfer DNA into
their plant hosts and effective conjugation can take place between E.coli
and several species of yeast (Stachel & Zambryski, 1989).  This indicates
that establishment barriers are very effective and that they are necessary
for species to be able to remain distinct in a world of genetic promiscuity
(Heinemann, 1991).

The unanswered questions about HGT are further enhanced by the fact that we
are undertaking genetic modification and mutations which are intended to
make nucleic acids more effective in use and therefore more able to overcome
introduction and establishment barriers.  It has been shown that small
changes in a DNA sequence can change the host spectrum for a transferable
genetic element (Kipling & Kearsey, 1990).

The report begs the question whether our genetic constructs actually include
added dangers stemming from the fact that they are more able to overcome
genetic barriers.

The report also addresses transfer frequency and quotes Neilsen et al 1998,

³Transfer frequency should not be confounded with the likelihood of
environmental implications, since the frequency of HGT is probably only
marginally important compared with the selective force acting on the
outcome².



During the short time that GMOs (mostly plants) have been employed a number
of documented hazards and risks have emerged which are included in the
report:

… Genetically engineered Bovine Growth Hormone (BGH) was considered to be
substantially equivalent to its natural counterpart.  Independent research
demonstrated that epsilon-N-acethllysine was substituted for lysine in the
engineered hormone (Violand et al,. 1994)  Recent indications have been
published that suggest milk from cows treated with BGH contains an increased
concentration of IGF-1 which may lead to an enhanced risk of mammary cancer.
(Outwater et al ,. 1997 ; Gebauer et al,. 1998; Hawkinson et al,. 1998).

… GM cotton plants with inserted herbicide tolerance genes have shown two
types of malfunction.  In some cases the plants dropped their cotton balls
and in others the tolerance genes were not properly expressed so that the GM
plants were killed by the herbicide (Fox, 1997).  (The manufacturers blamed
extreme climate conditions and refuted claims of unpredictability put
forward by their opponents.  They did however agree to pay substantial out
of court settlements to all the farmers who pressed charges against them).

… GM Tobacco plants were engineered to produce gamma-linolenic acid.
Instead they produced a toxic product; octadecatetraenic acid which does not
exist in unmodified tobacco plants (Reddy & Thomas, 1996).

… GM yeast modified to obtain increased fermentation was found to accumulate
the metabolite methyl-glyoxal in toxic and mutagenic concentrations (Inose &
Murata, 1995)

… A brazil nut gene was inserted into Soya and unexpected strong allergic
reactions were recorded in nut-allergic persons whom had never had a problem
with normal Soya.  Also the inserted brazil nut gene did not code for any
known allergen (Nordlee et al,. 1996).

… A bacterium was engineered to produce increased levels of the amino acid
L-tryptophan which was harvested and sold as a nutritional supplement in a
tablet form extensively across health food stores.  Small amounts of a
toxic, tryptophan-related molecule was identified in the tablets (Sidransky
et al,. 1994).  This toxic tryptophan-related molecule may have been the
cause of EMS (easinophilia-myalgia syndrome) in persons whom consumed the
product and resulted in 37 deaths and 1500 cases of chronic neurological and
auto-immune symptoms.  However, this has never been clarified because the GM
stock of bacteria was not available for investigation (Australian Gen-Ethics
Network, 1994).

… Research at the Scottish Crop Research Institute in Dundee demonstrated
indirect ecological effects from GM potatoes expressing an inserted lecthin
gene to reduce aphid attacks.  Ladybirds predating the aphids had a
significant reduction in life expectancy and reproducibility.  Likewise,
researchers at the Swiss Federal Research Station for Agroecology in Zurich
demonstrated serious harm to lacewings foraging on aphids affected by the
insecticide Bt toxin produced by GM.  (The disappearance of predators of
crop-ruining insects via modern farming practices is already a major
worldwide problem for the maintenance of biodiversity, further acceleration
in this process would indeed be tragic, claims this report).

… Field trails in Denmark and Scotland have shown that GM oilseed rape
transferred its inserted transgene by cross-pollination of wild relatives
(Mikkelsen et al,. 1996).  In France transfer of resistance genes from rape
to radish have been documented (Chevre et al., 1997).  (Similar examples of
the spread of transgenes over long distances have been demonstrated for
other GM species and it is for this reason that organic farmers in European
countries have initiated legal actions for fear that their produce may
become deprived of the "organic" label).

… It was earlier thought that naked DNA introduced to an intact animal would
be very quickly broken down and would lack biological importance. This dogma
was removed (Wolff et al ,.1990) when it was discovered that naked DNA was
readily up-taken in muscle cells of living mice following direct injection.
The possibility for directed genetic expression using such strategies has
now been demonstrated in several species of animals including human beings.
Intravenous injection or local installation in the respiratory passages
achieved in vivo gene transfer to rabbit lungs (Canonico et al ,. 1994).
The report suggests that a careful study should be made to determine whether
genetic expression from liposome-plasmid complexes following installation in
the respiratory passages is a common phenomenon which happens to plasmids
that go astray and may lead to serious reactions in the respiratory
passages.

… The belief that DNA in food and forage cannot be up-taken from the
gastrointestinal tract is considered to be a dogma by this report.   Recent
research demonstrated that following ingestion by mice, DNA from the M13
bacteriophage could be detected as relatively long fragments in faeces,
peripheral leukocytes, spleen and liver cells in significant time intervals
after feeding.  In cells the ingested M13 DNA was found in a chromosome
integrated form (Doerfler et al,. 1997; Schubbert et al,.  1997). When such
DNA was fed to pregnant mice it was detected in various organs from foetuses
and newborn animals ( Doerfler and Schubbert 1998).


All these factors add up to a real possibility of genetic pollution via
cross-pollination, unplanned breeding and horizontal gene transfer.   The
level of naked DNA persistence in the environment is likely to increase the
chances of such pollution occurring and the report suggests that extensive
unpredictable health, environmental and socioeconomic problems may result.

This report questions whether the development of GMO deserves the label
"technology".  Technology is associated with predictability, control and
reproducibility yet the GM of cells and organisms means no possibility to
target specific genomic sites, no control over the changes in gene
expression patterns for the inserted gene and the endogenous genes of the
GMO and no control over the fate of the transgene or parts of the transgene
once in situ and once released into an ecosystem.

The report claims            that there has been a lack of competent,
independent expertise in many technological fields and goes on to document
examples of accidents and erroneous evaluations where the full extent of
ecological damage largely remains unknown. e.g. the mis-use of antibiotics
and the spread of antibiotic resistance,  the emergence of recombinant
viruses within transgenic plants engineered to be resistant to viruses
(Green & Allison, 1994), the laboratory escape of the hybrid "African killer
Bees" which resulted in the deaths of more than 1000 people and the BSE/
nvCJD episode in Britain.

The report demonstrates and promotes the importance of research into
molecular ecology.  It certainly does not lack confidence in existing
commercial and academic research groups and suggests a functional division
of labour and confidential co-operation in it¹s recommendations.  If this
should come into practice, academic and industrial gene technology and the
new molecular ecology will be able to mutually fortify one another both
intellectually and methodologically.  The overall objective is to
realistically utilize technology to the advantage of mankind without
compromising the biosphere. The use of gene technology may represent a
historical turning point for science.  Might it be the first example of
mankind¹s feeling of responsibility for life in the future being stronger
than the urge for short-term advantages?

Let us all hope so.


TO OBTAIN A REVISED VERSION IN ENGLISH CONTACT

DIRECTORATE FOR NATURE MANAGEMENT
TUNGASLETTA 2
7485 TRONDHEIM
NORWAY
TEL: +47 73 58 05 00
FAX: +47 73 58 05 01
ISSN 0804-1504
ISBN 82-7072-304-5
TE 803
<http://www.naturforvatning.no/>http://www.naturforvatning.no


References
  (There are a total of 329 references in the report)

Canonico, A.E. et al. 1994.  Aerosol and intravenous transfection of human
alpha 1 -antitrypsin gene to lungs of rabbits. Am. J. Repir. Cell. Mol.
Biol. 10:24-29

Chevre, A,M. et al. 1997.  Gene flow from transgene crops. Nature 389: 924

Doerfler, W. & Schubbert, R. 1998.  Uptake of foreign DNA from the
environment:  the gastrointestinal tract and the placenta as portals of
entry.  Wien Klin. Wochenschr. 110:40-4


Doerfler , W. et al. 1997.  Intergration of foreign DNA and it¹s
consequences in mammalian systems.  TIBTECH 1997; 312:401-6


Dreiseikelmann, B. 1994.  Translocation of DNA across bacterial membranes.
Microbiol. Rev. 58: 293-316

Gebauer, G. et al. 1998. mRNA expression of components of the insulin-like
growth factor system in breast cancer cell lines, tissues and metastatic
breast cancer cells.  Anti-cancer Res.  18:2A 1191-5

Green, A.E. & Allison, R.F. 1994.  Viruses and transgenic crops.  Science
263:1423-1424

Harding, K. 1996.  The potential for horizontal gene transfer within the
environment.  Agro Food Ind. Hi-Tech. 7:31-35

Hawkinson, S.E. et al. 1998. Circulating concentrations of insulin-like
growth factor 1 and risk of breast cancer. Lancet 352: 1393-6.

Heinemann, J. A 1991. Genetics of gene transfer between species.  Trends
Genet. 7:181-185.

Inose, T. & Murata, K. 1995.  Enhanced accumulation of tixic compound in
yeast cells having high glycolytic activity: a case study on the safety of
genetically engineered yeast.  Int. J. Food  Science Tech. 30:141-146

Kidwell, M.G. 1993.  Lateral transfer in natural populations of eukaryotes.
Annu. Rev. Genet. 27:235-256

Kipling, D. & Kearsey, S.E. 1990.  Reversion of autonomously replicating
sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic
replication origin with procaryotic vector DNA.  Mol. Cell.  Biol.
10:265-272

Loke, S.L. et al. 1989.  Characterization of oligonucleotide transport into
living cells.  Proc. Natl. Acad. Aci. USA 86: 4374-3478

Lorenz, M.G.  &  Wackernagel, W. 1994.  Bacterial gene transfer by natural
genetic transformation in the environment.  Microbiol. 156: 319-326

Mikkelsen, T.R. et al. 1996.  The risk of crop transgene spread. Nature
380:31

Nielsen, K.M. et al. 1998.  Horizontal gene transfer from transgenic plants
to terrestraial bacteria - a rare event?  FEMS Microbiological Reviews 22:
79-103.

Nordlee, J.A. et al. 1996.  Indentification of a Brazil-nut allergen in
transgenic soybeans.  New Engl. J. Med. 14: 688-728

Outwater, J.L. et al. 1997.  Dairy products and breast cancer:  the IGF-1,
estrogen and bGH hypothesis.  Med. Hypotheses 48: 453-61
.
Reddy, S.A. & Thomas, T.L. 1996.  Expression of a cyanobacteria delta
6-desaturase gene results in gamma-linolenic acid production in transgenic
plants.  Nature Biotechnol. 14: 639-42

Schubbert, R. et al. 1997.  Foreign (M13) DNA ingested by mice reaches
peripheral leukocytes, spleen and liver via intestinal wall mucosa and can
be covalently linked to mouse DNA.  Proc. Natl. Acad. Sci.  USA 94: 961-966

Sidransky, H. et al. 1994.  Studies with 1, 1¹-ethylidenebis (tryptophan), a
contaminant associated with L-tryptophan implicated in the
eosinophilia-myalgia syndrome.  Toxicol. Appl. Pharacol.  126: 108-13

Stachel, S.E.  & Zambryski, P.C. 1989.  Generic trans-kingdom sex?  Nature
340: 190-191

Violand, B.N. et al. 1994.  Isolation of Escherichia coli synthesized
recombinant eukaryotic proteins that contain epsilon-N-acetyllysine.
Protein Sci. 3: 1089-97

Vlassov, V.V. et al. 1994.  Transport of oligonucleotides across natural and
model membranes.  Biochim.Biophys. Acta 1197: 95:108

Wolff, J.A. et al. 1990.  Dirrect gene transfer into mouse muscle in vivo.
Science 247: 1465-1468.

Wostemayer, J. et al. 1997.  Horizontal gene transfer in the rhizophere: a
curiosity or a driving force in evolution?  Adv. Bot. Res. Incorp. Adv.
Plant Pathol.  24: 399-429