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.

Review by Angela Ryan, ISIS

The above 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 these 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 denied claims of unpredictability put forward by their opponents. They did however concede 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. Prior to the more stringent tests conducted by Nordlee et al,. 1996, the inserted brazil nut gene, methionine-rich 2S albumin, was thought not to code for any known allergen.

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

Angela Ryan is
Researcher in Molecular Genetics, Open University
Science Monitor, Institute of Science in Society