GMO risks and hazards: Absence of evidence is not evidence of absence of risk
As more genetically modified foods are being produced and marketed and as many different types of genetically modified organisms (GMOs) continue to be released into the environment, the debate on the safety of genetic engineering has intensified. In this article, Professor Terje Traavik argues that given the unpredictable consequences of genetic technology and the fact that any adverse consequences will only be evident in the long run, this is a proper case for the application of the 'precautionary principle'.
A HIGH number of plant, and some animal, species have been genetically modified by recombinant DNA techniques, methods included in the collective term 'gene technology'. In the USA ca. 40 genetically modified plant species have been approved for commercial use. After having been introduced in 1996, herbicide-resistant soybeans now represent 27% of the total, while GM maize is assumed to represent ca. 25% (Williams, 1998). Herbicide-tolerance (54%), insect- (37%) and virus-resistance (14%) make up the vast majority of gene modifications, while quality improvements with regard to growth and nutrient compositions represent less than 1% (James, 1997). This is caused by the wish of transnational manufacturing companies to offer packages of the company's own pesticide and GM plants which tolerate the pesticide (Ho, 1998).
Production, marketing and consumption of GM food are highly controversial. The controversy mainly concerns whether the first-generation GM organisms should at all be commercialised and also to what extent, and how, GM food should be labeled. In the EU, serious conflicts of interest and opinion are seen within and between member countries. The UK, Austria, Luxembourg, France and Greece have at present some sort of moratoria on GM plants, and the Environmental Advice Committee of the European Parliament has recently called for a limited moratorium. Organic plant breeders in various EU countries have brought governmental and commercial institutions to court, because scientifically based risk assessments cannot exclude cross-pollinations from GM crops from taking place. In the USA commercialisation of GM plants has met little opposition from government or consumers (Williams, 1998), and in Scandinavia public debate is nearly absent, in itself a risk factor.
Experience with large-scale production and consumption of GM organisms is, of course, very limited. Proponents of early GM plant commercialisation focus on the hypothetical advantages, especially in the context of the critical global nutritional situation (Brown, 1997), and a suppressive effect on chemical pollution in all forms of primary production. The opponents bring forward lack of knowledge, uncertainty and potential hazard to public health and the environment, and insist that the 'precautionary principle' should be invoked (Rissler and Mellon, 1996; Ho, 1998). Both parties are fighting for the attention of politicians, 'experts', dealers, consumers and the general opinion.
Risk factors and hazards
In order to carry out a genetic modification, a recombinant, genetic vector has to be constructed. The vector is intended to carry the cloned gene safely into the chosen organism and order the gene to become expressed, i.e. produce the protein that it codes for.A vector will, in addition to the chosen gene, be composed of a number of other DNA elements. Typically, it needs a control element (promoter/enhancer) necessary for expression of the gene, and an extra gene coding for resistance to an antibiotic or another cytotoxic substance. An alternative to the highly controversial antibiotic resistance genes for GM plants, is a gene (csr-1) providing resistance to the herbicide chlorsulfon (Bergelson et al; 1998).
Vector DNA molecules are transferred across cell membranes aided by so-called biolistics ('gene-cannon'), chemical treatment or by exposing cells to an electric field. Thereafter vector molecules are transported to the cell nucleus to become inserted (integrated) into the chromosome(s) of the recipient cell. Integration takes place at unpredictable locations in the chromosomes.
Vector transfer techniques are inefficient. Only a fraction of the treated cells take up and express the chosen gene. In order to select the vector-containing cell population, an antibiotic is added. Cells not expressing the vector resistance gene are then killed. Surviving cells are the basis for development of GMOs, in which all the cells of the organism contain integrated vector DNA and express the wanted gene.
Genetic modification is different from traditional cultivation and breeding
It is often argued that genetic modification represents a more precise, but not fundamentally different, kind of breeding or cultivation. This must be rejected because unnatural recombinations are created. Genetic material is recombined between species for which there is no, or very low, probability of natural progeny.
New, exotic genes or DNA sequences are introduced into unpredictable chromosomal locations. Conventional breeding shuffles around aberrant versions (alleles) of the same genes, while these are fixed in the chromosomal locations they have been given by evolution. Gene technology introduces new, exotic genes. Their location within the recipient cell DNA is unpredictable and with no possibility of targeting. This may result in unpredictable effects on the metabolism, physiology and biochemistry of the recipient, transgenic organism, effects not detected with traditional methods of control.
The vectors used are efficient genetic parasites. They are gene shuttles developed to move and express genes across species borders and ecological barriers:
i) They are mosaics of genetic elements and sequences derived from the most efficient genetic parasites (viruses/plasmids/mobile elements). Many of them are able to invade and insert their DNA into the chromosomes of any kind of cell, with possible genetic or metabolic harm in consequence.
ii) They are specifically constructed to break species barriers. In transit they may pick up and transfer genes from new host organisms or from the genetic parasites of these. Such newly created genetic mosaics may then become transferred to new species, or recombine between them to result in pathogenic viruses with the potential to infect earlier refractory hosts etc. During such relays genetic rearrangements and mutations may arise at any given time, with unpredictable results.
iii) They carry resistance genes that in themselves may represent new, or enhance existing, public health and environmental problems (i.e. antibiotic resistance in pathogenic bacteria or herbicide-tolerant 'super-weeds').
Changes in GMOs or their products
The most serious scientifically based arguments against large-scale, commercial use of the first-generation GMOs are based on the unpredictability with regard to where in the recipient cell chromosomes insertion of vector DNA takes place. The consequences of insertion may vary considerably according to the precise insertional location (Doerfler et al; 1997). This is valid for the expression of the inserted transgene as well as for changes in the recipient organism's own genes and their expression levels.
Some of the most prominent uncertainties are related to the fact that the recipient organism has received a new promoter/enhancer. These elements govern the gene expression levels of their attached transgenes, but after insertion they may also change the gene expression and methylation patterns in the recipient chromosome(s) over long distances up- and downstream from the insertion site. Promoters/enhancers function in response to signals received from the internal or external environment of the organism. For a GMO this results in unpredictability with regard to:
* The expression level of the inserted foreign gene(s).
* Expression of a vast number of the organism's own genes.
* Influence of geographical, climatic, chemical (i.e. xenobiotics) and ecological changes in the environment.
* Transfer of vector sequences within the chromosomes of the organism and vertical and/or horizontal gene transfer to other organisms.
Changes in ecosystems and the environment in a broad sense
Genetic pollution from GMOs is a real possibility. This can be exerted by cross-pollination, unplanned breeding and horizontal gene transfer (reviews: Kidwell, 1994; Nielsen et al., 1998; Traavik, 1999). Such events may result in extensive and unpredictable health, environmental and socioeconomic problems. Environmental persistence and transfer of nucleic acids are extensively discussed in the latter two references. The issue has assumed added urgency after demonstrations by a highly respected research group that ingested DNA under some circumstances may be taken up from the intestines of mice, inserted into chromosomes and vertically transmitted to offspring (Doerfler et al., 1997; Schubbert et al., 1997; Doerfler and Schubbert, 1998).
Risk and assessment of risk
The term 'risk' is very often confused with 'probability', and hence used erroneously. Risk is defined as the probability that a certain event will take place multiplied by the consequences arising if it takes place. The atomic bomb makes a good basis for conceiving the contents of the term. With regard to development and commercialisation of GMOs, we often are able to define neither the probability of unintended events nor the consequences of them. Hence, the present state of ignorance makes scientifically based risk assessments impossible. This calls for invoking the 'precautionary principle'.
The precautionary principle
This principle is now established in international declarations and agreements. It was introduced as an ethical road sign. The principle implies that responsibility for future generations and the environment is to be combined with the anthropocentric needs of the present.
In the context of gene technology and use of GMOs, a general definition might be: 'In order to obtain sustainable development, politics should be based on the precautionary principle. Environmental and health policies must be aimed at predicting, preventing and attacking the causes of environmental or health hazards. When there is reason to suspect threats of serious, irreversible damage, lack of scientific evidence should not be used as a basis for postponement of preventive measures' (revised after Cameron and Abouchar, 1991).
Documented hazards and risks
During the short time period that GMOs (mostly plants) have been employed, a number of warning signals have already emerged (for reviews: Ho, 1998; Ho et al., 1998; Nielsen et al., 1998; Traavik, 1999).
Changes in the GMO or its products
For a long while the manufacturers of genetically engineered bovine growth hormone (BGH), injected into cows in order to increase milk production, claimed that it was identical with its natural counterpart. Later on it was demonstrated by independent research that epsilon-N-acethyllysine was substituted for lysine in the engineered hormone (Violand et al; 1994). Such aminoacid substitutions may have unpredictable consequences for the conformation and functions of proteins. Recently indications have been published that milk from BGH-treated cows may contribute to increased mammary cancer risk by enhancing the IGF-1 concentration in milk (Outwater et al., 1997; Hawkinson et al., 1998; Gebauer et al; 1998).
Tobacco plants were genetically engineered to produce gamma-linolenic acid. Instead the plants mainly produced the toxic product octadecatetraenic acid. Unmodified tobacco plants do not contain this substance (Reddy and Thomas, 1996).
When yeast was genetically modified to obtain increased fermentation, it was unexpectedly discovered that the metabolite methyl-glyoxal accumulated in toxic and mutagenic concentrations (Inose and Murata, 1995).
When a gene from Brazil nut was inserted in soybean plants, unexpected strong allergic reactions were recorded in nut-allergic persons who had never had any problems with soybean products. The inserted gene did not code for any known allergen (Nordlee et al., 1996).
A bacterium (Bacillus amyloliquefaciens) was genetically engineered to produce increased levels of the aminoacid L-tryptophan, which has widespread application in tablets as a nutritional supplement. In the tablets small amounts of a toxic, tryptophan-related molecule were identified (Sidransky et al., 1994). Whether this was the cause of EMS (easinophilia-myalgia syndrome) which resulted in 37 deaths and 1,500 cases of chronic neurologic and autoimmune symptoms has never been clarified, mainly because the genetically modified stock of bacteria was not available for investigation (Australian Gen-Ethics Network, 1994).
Researchers at the Scottish Crop Research Institute in Dundee have demonstrated indirect ecological effects of GM potato plants. The plants expressed an inserted lecthin gene in order to reduce aphid attacks. Ladybirds predating lecthin-containing aphids had their lifetime expectancies and reproducibility significantly reduced. Likewise, researchers at the Swiss Federal Research station for Agroecology in Zurich have demonstrated serious harm to lacewings foraging on aphids affected by the insecticide Bt toxin produced by GM maize (Williams, 1998). It is already a major worldwide agricultural problem that natural predators of crop-ruining insects are disappearing. An acceleration of this process would be tragic.
Field trials in Denmark and Scotland have shown that GM oilseed rape may transfer their inserted transgene by cross-pollination of wild relatives (Mikkelsen et al., 1996), while experiments in France have demonstrated transfer of resistance genes from rape to radish (Chevre et al., 1997). Similar examples, with spread of transgenes over long distances, have been demonstrated for other GM plant species. Organic plant farmers in European countries have initiated legal actions on this ground. When their farms are situated in the vicinity of GM crop fields, their products may be deprived of the 'organic' labelling.
Recently it was demonstrated that self-pollinating GM plants may have a forced, augmented capability to cross-pollinate other plants, with a resulting transfer of inserted transgenes (Bergelson et al., 1998). The unpredictability was demonstrated by the fact that inbred, identical plants genetically modified in separate experiments had differing abilities to cross-pollinate other plants. Although the experiments were carried out on a single plant species, Arabidopsis thaliana, these results have general interest, also because the inserted gene (csr-1) has been introduced in various plant species as an alternative selection marker to replace antibiotic resistance genes.
GM cotton plants with inserted herbicide tolerance genes have demonstrated two types of malfunctions. In some cases the plants dropped their cotton bolls, in others the tolerance genes were not properly expressed, so that the GM plants were killed by herbicide (Fox, 1997). The manufacturers blamed extreme climatic conditions, indirectly assessing claims of general unpredictability from opponents. A number of cotton farmers pressed charges. The manufacturers offered economic settlements out of court.
We can move genes - but do we have a 'technology'?
'Technology' is derived from the Greek term 'tekhne' which is connected to handicraft or arts. Our associations with the word include predictability, control and reproducibility. The parts of genetic engineering that concern construction of vectors are truly technology. But present techniques for moving new genes into cells and organisms mean:
* No possibility of targeting the vector/transgene to specific sites within the recipient genomes. In practical terms this means that modifications performed with identical recipients and vector gene constructs under the same standardised conditions may result in vastly different GMOs depending on where the transgenes become inserted.
* No control of changes in gene expression patterns for the inserted or the endogenous genes of the GMO.
* No control of whether the inserted transgene(s), or parts thereof, move within or from the recipient genome, or where transferred DNA sequences end up in the ecosystems.
Poor track record
Technology is developed to achieve benefits and there are many tragic examples of how people, elated over these, have both overlooked and neglected to adequately investigate the possibilities for dramatic disadvantages, which have therefore only first been acknowledged much later.
Frightening examples from the last half of the 20th century are the application of organochlorines and other chemicals to fight plant pests, and the peaceful exploitation of nuclear power. We are now aware that the environment on the Earth has been seriously damaged by these senseless encroachments on the ecosystems, but it will still be a long time before we are able to recognise how serious the damage is.
In both these cases, sectors of informed public opinion in many countries posed serious questions concerning the safety and possible side-effects of their use. The research communities on the other hand, with a few brave exceptions, made themselves available for a nave, optimistic development, and were unanimous in their view that there were no real risks of undesirable effects for health and the environment. The same experts and research milieus that had participated in developing the new technology were employed as advisors by political authorities in connection with the pre-assessment of risks and the setting-up of systems to record damage.
Researchers are people like everyone else. The ability for critical and objective evaluation of risks associated with a person's own lifework is not a predominant part of human nature. There was, and still is, a lack of competent, independent expertise in many technological fields.
Recent years have witnessed many examples of unforeseen side effects from 'safe technology' that have led to health risks and threatened to disturb the ecological balance. What the following examples of accidents and erroneous evaluations have in common is that the consequences are still developing and we do not know the full extent of the damage.
Antibiotic resistance and horizontal gene transfer
Development of bacteria that are resistant to antibiotics now represents a brewing catastrophe. Multiresistant bacteria do not remain in hospitals but have now been spread to the 'healthy' community and, moreover, to large numbers of freely living, naturally occurring species of bacteria (Davies 1994; Kruse, 1994: Kruse and S¿rum, 1994; Thomson et al., 1994). Antibiotics have saved numerous human lives, prevented suffering and preserved food resources and valuable resources in animal husbandry and aquaculture. However, senseless use of antibiotics has at the same time resulted in microbes now being on the warpath. Strains of increasing numbers of microbe species that are important for medicine and veterinary medicine are being found to be resistant to all relevant antibiotics. 'Old' infectious diseases, such as tuberculosis, are returning, and freely living bacteria in the ecosystems have acquired resistance to antibiotics. During the last few decades, confidence in antibiotics has, moreover, led to the stagnation of research and testing of alternative strategies for preventing and treating infectious diseases. These fields of research now have to be re-awakened, because no one, including the pharmaceutical industry, believes that the constant development of new antibiotics is the right path to take. Horizontal gene transfer of antibiotic resistance genes lies at the root of the problem.
Recombinant plant viruses
In the last decade, researchers have been eager to make plants resistant to viral infections by inserting virus genes in the plant genome. If, for example, the gene that codes for the coat protein for the cowpea chlorotic mottle virus (CCMV) is inserted in plants, the plants become resistant to both CCMV and several other related viruses. It has now been shown that when such transgenic plants are infected with other viruses, new, recombinant viruses can arise which have had their host specificity and other biological properties changed (Greene and Allison, 1994). Critically inclined scientists had pointed out this possibility, and the necessity of investigating it, for many years, but their protests had been drowned out by representatives of both the biotechnological industry and the research community optimistically eager to develop the technique. Even after the publication of Greene and Allison's results, such experts attempted to undermine the significance of the discoveries without having alternative results of their own to point to (Falk and Bruening, 1994). Incidentally, the story did not end here. In further work Greene and Allison demonstrated that a targeted trimming of the viral transgene seemed to eliminate development of viral recom-binants (Greene and Allison, 1996). This illustrates the importance of invoking the 'precautionary principle', to gain time for identification of risk-imposing mechanisms and look for means to prevent them.
Dogmas concerning absence of hazards have often been proven wrong (e.g. the Titanic). A relevant example is the belief that DNA in food and forage cannot be taken up from the gastrointestinal tract. Some experimental studies, and the whole evolutionary history as well as our daily intake of vast amounts of DNA from various sources supported this belief. Absolute biological and ecological truths are, however, very rare, and rare phenomena may have important consequences when they take place.
Recently this was illustrated by the demonstration that following ingestion by mice, DNA from the M13 bacteriophage could be detected as relatively long fragments in faeces, peripheral leukocytes, and spleen and liver cells in significant time intervals after feeding. In the cells the ingested M13 DNA was found in a chromosome-integrated form (Schubbert et al., 1997; Doerfler et al., 1997). When such DNA was fed to pregnant mice, the test DNA was detected in various organs from foetuses and new-born animals (Doerfler and Schubbert, 1998).
The experimental conditions strongly indicated that the DNA had been transferred across the placenta. The authors concluded that the consequences of foreign-DNA uptake in the context of mutagenesis and oncogenesis should be subject to controlled experiments. Such experiments are still absent. Another unclarified issue is connected with the detection of long M13 DNA fragments in the faeces (Schubbert et al., 1997). If such fragments are taken up by enteric bacteria, unwanted establishment of sequences from transgenes, i.e. antibiotic resistance genes, may take place in pathogenic or opportunistic bacteria.
Another striking example is represented by the BSE (Bovine spongiform encephalopathia) story. Against the explicit conclusions of experts, the BSE prions crossed the hypothesised 'species' barrier and initiated new variant Creutzfeldt-Jakob disease (nv CJD) in human beings. Recently it has been demonstrated that a vast number of BSE prion-carrying, symptom-free cattle may have been consumed, and at the moment the extent of nv CJD is impossible to forecast.
In these cases, and many others, the experts were wrong. To the extent that any prior investigations of damaging effects had been undertaken, methods and approaches had been used that were only capable of disclosing short-term effects, whereas in ecological contexts it is the long-term impacts that are the most important and most serious (Colburn et al., 1994; Leaning, 1994). Long-term impacts in these contexts, and also in connection with the possible damaging effects of the dispersal of naked DNA, are assessed in terms of not months or years, but at least ten to hundreds of years.
We will soon be putting behind us the bloodiest century in the history of mankind. In our technological fervour, in our inquisitiveness and in our pleasure at being able to rub magic lamps, we have made enormous, and still unpredictable, erroneous assessments of our relationship to our surroundings and the, relatively speaking, ever smaller vessel in which all of us are sailing.
Is the use of genetic engineering to be a historical turning point, the first example of mankind's feeling of responsibility for life in the future being stronger than the need for short-term advantages? May the precautionary principle be invoked in this area? (Third World Resurgence No. 104/105, April/May 1999)
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Dr Terje Traavik is Professor of Virology at the University of Troms, Norway and Scientific Director of the Norwegian Institute of Gene Ecology.
The above article is an edited version of the first chapter of his research report for the Directorate for Nature Management, Norway entitled "Too early may be too late".