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Review of Norwegian Report to the Directorate of Nature
Management, Norway
By Angela Ryan
Researcher in Molecular Genetics, Open University
Director, Institute of Science in Society
42 Manor Road, High Barnet, London, EN5 2JJ
Tel/Fax 0181 441 6480
email. aryan35@onetel.net.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 recombination's 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 constructed 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 'introduction
barrier' and an 'establishment barrier'.
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 raises 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,
"Transfer 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
outcome".
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 taken up 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 it's 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
mankind's feeling of responsibility for life in the future being stronger
than the urge for short-term advantages?
Let us all hope so.
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http://www.naturforvatning.no
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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 it's
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Dreiseikelmann, B. 1994. Translocation of DNA across bacterial membranes.
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Gebauer, G. et al. 1998. mRNA expression of components of the
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Green, A.E. & Allison, R.F. 1994. Viruses and transgenic crops.
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Harding, K. 1996. The potential for horizontal gene transfer within the
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Inose, T. & Murata, K. 1995. Enhanced accumulation of tixic compound
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Nordlee, J.A. et al. 1996. Indentification of a Brazil-nut allergen in
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Schubbert, R. et al. 1997. Foreign (M13) DNA ingested by mice reaches
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Sidransky, H. et al. 1994. Studies with 1, 1'-ethylidenebis (tryptophan),
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