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GMO the true form of Conspiracy
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Horizontal Gene Transfer from GMOs Does Happen
Genetic engineering, horizontal gene transfer, and the emergence of infectious diseases
Genetic engineering creates vast arrays of transgenic DNA that could spread, not only through cross-pollination with the same or related species, but also through the direct uptake of the transgenic DNA by cells of unrelated species, a process called horizontal gene transfer. We have been alerting regulators to Horizontal Gene Transfer - The Hidden Hazards of Genetic Engineering [1] on many occasions since the late 1990s [2-4] (Genetic Engineering Dream or Nightmare, ISIS Publication) when the regulators and their scientific advisors had denied vehemently that horizontal gene transfer could happen, and assumed mistakenly that transgenic DNA, like all DNA, would be rapidly degraded once out of the cell.
In a review published in 1998 [5], we presented extensive evidence that DNA persists in all environments and can indeed be taken up by cells of many species throughout the living world. We called for a public enquiry on the extent to which the poorly regulated discharge of transgenic organisms and transgenic nucleic acids into the environment could have been responsible for the increased emergence of new viral and bacterial diseases and antibiotic and drug resistance since genetic engineering began in the mid 1970s. Horizontal gene transfer and recombination is the main route for generating new pathogens and spreading antibiotic and drug resistance, and genetic engineering is nothing if not greatly facilitated horizontal gene transfer and recombination.
Transgenic DNA is different from natural DNA and more likely to spread
Regulators frequently dismiss our concerns with remarks such as "The safety of nucleic acids is widely accepted. Both RNA and DNA are part of all food products that we consume. " [6] (USDA FONSI for Transgenic Poplars Absurd & Dangerous, SiS 38). The implication is that transgenic DNA (or RNA) is no different from natural nucleic acids, and hence no more likely to spread by horizontal gene transfer.
There is no doubt that transgenic DNA is different from natural DNA; not only does it contain new combinations of genes, but also new synthetic genes that have never existed in billions of years of evolution: new coding sequences, promoters and other non-coding regulatory sequences that boost gene expression to abnormally high levels.
Furthermore, there are indeed reasons to suspect transgenic DNA is more likely to transfer horizontally and recombine than natural DNA (see Box adapted from [7] Living with the Fluid Genome, ISIS publication), and this has been borne out by accumulating evidence, even though dedicated research is still extremely rare.
Transgenic DNA more likely to spread horizontally
1. Transgenic DNA is designed to jump into genomes, often through viral or bacterial plasmid vectors that can integrate into genomes.
2. Transgenic DNA tends to be structurally unstable and hence prone to break and rejoin, giving rise to numerous deletions, duplications, and other rearrangements during the transformation process, which spread into the host genome; and this is in part responsible for the instability of transgenic varieties [8, 9] (see Transgenic Lines Unstable hence Illegal and Ineligible for Protection and MON810 Genome Rearranged Again, Stability of All Transgenic Lines in Doubt, SiS 38).
3. The mechanisms that enable transgenic constructs to jump into the genome enable them to jump out again and reinsert at another site or into another genome.
4. The borders of the most commonly used vector for transgenic plants, the T-DNA of Agrobacterium, are recombination hotspots (sites that tend to break and join). In addition, a recombination hotspot is also associated with the cauliflower mosaic virus (CaMV) promoter and many transcription terminators, which means that the whole or parts of the integrated DNA will have an increased propensity for secondary horizontal gene transfer and recombination (see main text).
5. The Agrobacterium vector remaining in transgenic plants may be a vehicle for gene escape and can transfer genes widely to many bacteria as well as into human cells (see main text).
6. Transgenic constructs tend to integrate at recombination hotspots in the genome, which again, would tend to increase the chances that they will disintegrate and transfer horizontally [8].
7. Transgenic DNA often has other genetic signals, such as the origin of replication left over from the plasmid vector. These are also recombination hotspots, and in addition, can enable the transgenic DNA to be replicated independently as a plasmid that is readily transferred horizontally among bacteria and other cells.
8. The metabolic stress on the host organism due to the continuous over-expression of transgenes linked to aggressive promoters such as the CaMV 35S will also increase the instability of the transgenic DNA, thereby facilitating horizontal gene transfer
9. Transgenic DNA is typically a mosaic of DNA sequences copied from many different species and their genetic parasites; these homologies mean that it will be more prone to recombine with, and successfully transfer to the genomes of many species and their genetic parasites. Homologous recombination typically occurs at one thousand to one million times the frequency of non-homologous recombination, and short homologous sequences could act as anchors for acquiring non-homologous sequences (see main text).
CaMV 35S promoter active in all species including human cells
In 1999-2000, we alerted our regulators to the CaMV 35S promoter that is in practically every commercial transgenic crop commercialised. A promoter is a signal necessary for a gene to be expressed, and the CaMV 35S promoter is used with many transgenes. It has a recombination (fragmentation) hotspot that would enhance horizontal transfer of transgenic DNA, making transgenic DNA and transgenic lines unstable [10, 11]. Furthermore, contrary to the then common assumption that the CaMV 35S promoter was only active in plants and plant-like organisms, it is in fact active in species across the living world, animal and human cells included [12]. Consequently, it has the potential to activate dormant viruses and trigger cancer, if it happens to land next to certain cancer-related 'proto-oncogenes'. Since then, the CaMV 35S promoter was demonstrated to be active in human enterocyte-like cells [13]. And evidence of transgenic instability has also emerged, with the CaMV 35S promoter representing a major breakpoint [14, 15] (Transgenic Lines Proven Unstable, SiS 20) precisely as we had predicted.
Agrobacterium vector a vehicle for gene escape
We have also provided evidence strongly suggesting that the most common method of creating transgenic plants may also serve as a ready route for horizontal gene transfer [16, 17].
Agrobacterium tumefaciens, the soil bacterium that causes crown gall disease, has been developed as a major gene transfer vector for making transgenic plants. Foreign genes are typically spliced into the T-DNA - part of a plasmid of A. tumefaciens called Ti (tumour-inducing) – which ends up integrated into the genome of the plant cell that subsequently develops into a tumour.
But further investigations revealed that the process whereby Agrobacterium injects T-DNA into plant cells strongly resembles conjugation, the mating process between bacterial cells.
Conjugation, mediated by certain bacterial plasmids requires a sequence called the origin of transfer (oriT) on the DNA that's transferred. All the other functions can be supplied from unlinked sources, referred to as 'trans-acting functions' (or tra). Thus, 'disabled' plasmids, with no trans-acting functions, can nevertheless be transferred by 'helper' plasmids that carry genes coding for the trans-acting functions. And that's the basis of a complicated vector system devised, involving Agrobacterium T-DNA, which has been used for creating numerous transgenic plants.
It soon transpired that the left and right borders of the T-DNA are similar to oriT, and can be replaced by it. Furthermore, the disarmed T-DNA, lacking the trans-acting functions (virulence genes that contribute to disease), can be helped by similar genes belonging to many other pathogenic bacteria. It seems that the trans-kingdom gene transfer of Agrobacterium and the conjugative systems of bacteria are both involved in transporting macromolecules, not just DNA but also protein.
That means transgenic plants created by the T-DNA vector system have a ready route for horizontal gene escape, via Agrobacterium, helped by the ordinary conjugative mechanisms of many other bacteria that cause diseases, which are present in the environment.
In fact, the possibility that Agrobacterium can serve as a vehicle for horizontal gene escape was first raised in 1997 in a study sponsored by the UK Government [18, 19], which found it extremely difficult to get rid of the Agrobacterium in the vector system after transformation. Treatment with an armoury of antibiotics and repeated subculture of the transgenic plants over 13 months failed to get rid of the bacterium. Furthermore, 12.5 percent of the Agrobacterium remaining still contained the binary vector (T-DNA and helper plasmid), and were hence fully capable of transforming other plants.
Agrobacterium not only transfers genes into plant cells; there is possibility for retrotransfer of DNA from the plant cell to Agrobacterium [20]. High rates of gene transfer are associated with the plant root system and the germinating seed, where conjugation is most likely [21]. There, Agrobacterium could multiply and transfer transgenic DNA to other bacteria, as well as to the next crop to be planted. These possibilities have yet to be investigated empirically.
Finally, Agrobacterium attaches to and genetically transforms several human cell lines [22]. In stably transformed HeLa cells (a human cell line derived originally from a cancer patient), the integration of T-DNA occurred at the right border, exactly as would happen when it is transferred into a plant cell genome. This suggests that Agrobacterium transforms human cells by a mechanism similar to that which it uses for transforming plants cells.
The possibility that Agrobacterium is a vehicle for horizontal transfer of transgenic DNA remains unresolved to this day.
Evidence of horizontal transgene transfer to bacteria denied and dismissed
By 1999, there was already evidence that horizontal transfer of transgenic DNA could occur, not only in the laboratory but also in the field [23]. Unfortunately, the researchers were far too cautious as scientists, and ended up denying the prima facie evidence that the transgenic DNA had transferred horizontally from plant to soil bacteria [24]; whereas, a proper application of the precautionary principle would have resulted in the researchers stressing the possibility that it had occurred could not be dismissed.
High frequencies of horizontal transfer of transgenic plant DNA were demonstrated for soil bacteria, Pseudomonas stutzeri and Acinetobacter sp. when the transgenic plant DNA contained sequence homologies to the bacteria [25]. Again, the authors stressed that the transfer "strictly depends on homologous sequences", which could give the uninformed a false sense of assurance, forgetting that transgenic constructs contain homologies to many different species of bacteria and viruses, and are therefore capable of engaging in high frequencies of horizontal gene transfer and recombination with all of them [24].
We drew attention to further evidence of the enhanced horizontal transfer of transgenic DNA in our submissions [26, 27] (Molecular Pharming by Chloroplast Transformation, GM Pharmaceuticals from Common Green Alga, SiS 27) to the regulatory authorities in Hawaii objecting to an intended outdoor large-scale facility for transgenic strains of the alga, Chlamydomonas reinhardtii producing a range of pharmaceutical proteins in chloroplast integrated transgenes, which would greatly increase copies of transgenic DNA per cell. We pointed that DNA not only persists in all environments, but also that transformation by direct uptake of DNA is a major route of horizontal gene transfer among bacteria [25]. The close similarities (homologies) between the transformed chloroplasts in transgenic C. reinhardtii and bacterial genomes is expected to further enhance the frequency of horizontal gene transfer, by up to a billion-fold.
In fact, researchers at the University of Oldenburg in Germany demonstrated that the horizontal transfer of non-homologous DNA also occurs at relatively high frequencies when a homologous DNA 'anchor sequence' is present, which can be as short as 99bp [28]. In a review published in 2004, the researchers listed at least 87 species of naturally transformable bacteria [29], which represent 2 percent of all known species. And transgenic DNA can spread not only via the roots and plant debris, but also via pollen drift into fields that had never cultivated transgenic crops. The authors had even developed a bio-monitoring technique for detecting transgenic DNA based on transformation of a competent strain of bacteria that depends on double cross-over event between the transgenic DNA and the bacterial chromosome, a theoretically much rarer event than a single cross-over. Nevertheless, the bio-monitoring technique is at least as sensitive as a routine polymerase chain reaction (PCR) for detecting minute amounts of DNA, indicating that horizontal transfer of transgenic DNA is not exactly a rare event. This conflicts with their conclusion in the same review that, "each of the many step involved from the release of intact DNA from a plant cell to integration into a prokaryotic genome has such a low probability that a successful transfer event [is] extremely rare."
Researchers at Cardiff University in the UK have confirmed that horizontal transfer of transgenic DNA occurs at detectable levels using a similar system [30]. Transgene sequences kanamycin resistance (nptII) and green flourescent protein (gfp) were driven by their own bacterial promoters. Recipient bacteria carried a copy of these two genes with deletions in their 3' ends abolishing marker activity. Successful recombination between the plant transgene and the bacterial genome resulted in restoration of the markers, allowing detection through antibiotic selection and fluorescence. Measurable transformation frequencies were obtained in increasingly complex conditions approaching field conditions. In sterile soil microcosms, transformation was detected using pure plant DNA at 3.6 x 10-8 and in ground leaves at 2.5 x 10-11 transformants per recipient; for non-sterile soil using pure plant DNA, the frequency was 5.5 x 10-11 transformants per recipient.
Evidence has continued to accumulate [31] Horizontal Gene Transfer Happens - II, ISIS Report) indicating that transgenic DNA in food and feed can transfer into animal and human cells [32] (DNA in GM Food & Feed, SiS 23). Several studies have documented the survival of DNA in food/feed throughout the intestinal tract in mice and pigs [33, 34 and references therein], in the rumen of sheep [35], and in the rumen and duodenum of cattle [36], with varying degrees of sensitivities in PCR methods.
In the only feeding trial in human volunteers [37], a single meal containing GM soya with about 3 x 1012 copies of the soya genome, the complete 2 266 bp of the epsps transgene was recovered from the colostomy bag in six out of seven ileostomy subjects, though at highly variable levels, ranging from 1011 copies (3.7 percent) in one subject to 105 copies in another. This is a strong indication that DNA is not rapidly broken down in the gastrointestinal tract, confirming earlier results from the same research group. In three of the seven ileostomy subjects, about 1 to 3 per million bacteria cultured from the contents of the colostomy bag were positive for the GM soya transgene, indicating that horizontal transfer of transgenic DNA had occurred, either before the single meal was taken, as claimed, or else as the result of the single GM soya meal, a possibility that cannot be ruled out [32]. Interestingly, no bacteria were found to have taken up non-transgenic soya DNA, suggesting that transgenic DNA may be more successfully transferred for reasons given above.
No transgenic DNA was found in the faeces of any of 12 healthy volunteers tested. Either the remaining DNA has completely broken down by then as claimed by the researchers, or else detectable fragments have all passed into the blood stream from the intestine [31]. The researchers had not checked for the presence of transgenic DNA in the bloodstream. It is already known that food material can reach lymphocytes entering the intestinal wall directly, through Peyer's patches. And fragments of plant DNA were detected in cow's peripheral blood lymphocytes [38]. From the blood, the DNA can be transported to and taken up by tissue cells, and this has been known from experiments since the late 1990s. Transgenic DNA and viral DNA fed to mice ended up in cells of several tissues [39], and when fed to pregnant mice, the DNA crossed the placenta and entered the cells of the foetus and the newborn [40]. DNA from ingested food plants were also taken up into tissue cells [41].
In summary, the evidence shows that horizontal transfer of transgenic DNA does happen and has happened both in the soil and in the gastrointestinal tract, though many scientists are unable or unwilling to acknowledge this, or else dismiss it by saying it has a "low probability" and is "extremely rare". But recent evidence shows it has been greatly underestimated.
Evidence that horizontal transfer of transgenic DNA has been greatly underestimated
A team of researchers led by Aurora Rizzi at the University of Milan, Italy, developed a strategy for detecting transformation in the soil bacterium Acinetobacter baylyi BD413 in situ by the expression of green fluorescent protein (gfp) [42]. The transformed bacteria growing on plant tissues can be seen and counted directly by fluorescence microscopy. Using this method, the researchers showed that conventional methods based on cultivating and selecting the transformants on agar-plates containing antibiotics underestimates the frequencies of transformation by at least a hundred-fold.
The agar-plating step destroys the original material, so no information can be obtained on the specific location of the gene transfer events or the interaction between the bacteria and the transforming material, including the donor DNA. So it is not possible to locate the hotspots of transformation in a complex environment such as the soil, or within the plant. Furthermore, conventional plating techniques will only isolate cells that can be cultured (which is probably less than one percent of soil microbes).
The reporter strain is engineered with a green fluorescent protein (gfp) that is not expressed due to a deletion that includes its promoter, and which is present in the transgenic DNA. So when the required piece of transgenic DNA is transferred into the correct position – which it will do because the deletion is flanked by sequences homologous to the transgenic DNA – the gfp will be expressed and lights up the cells under the fluorescence microscope.
Using this technique the researchers located actual transformation events on a membrane filter, and in decaying tobacco leaves and roots. The transformation frequencies measured were at least two orders of magnitude higher than previously recorded by cultivation-based plating.
In decaying leaves, transformed cells were located in the interstices between epidermal cells on the surface of leaves and close to the stoma (pores for gas exchange) at the border with other epidermal cells. Transformed bacteria were also found on root surfaces.
Horizontal transfer to plant and animal genomes may occur at even higher frequencies
While researchers on biosafety have been focussing on horizontal gene transfer from plants to bacteria, evidence is emerging suggesting that genomes of higher plants and animals may be even softer targets for horizontal gene transfer. Transgenic DNA may well be taken up by the cells of other plants, and by animals including humans feeding on the plants. We have been warning of this possibility at least since 2001, when experiments in 'gene therapy'- making transgenic human cells - were demonstrating how easy it was for transgenic constructs to be taken up by human and animal cells [43] (SLIPPING THROUGH THE REGULATORY NET: 'Naked' and 'free' nucleic acids)
Now, information from transgenic Arabidopsis and rice, with sequenced genomes, and the huge amounts of relic viruses, transposons, retroelements, and chloroplast and mitochondrial DNAs found in these and other sequenced genomes are persuading geneticists that [44] "nuclear genomes of plants, like those of other eukaryotes, are promiscuous in integrating nonhomologous DNA." "Illegitimate recombination", a rarity in prokaryotes, is the main route for transgene integration in eukaryotes. In other words, eukaryotic genomes, including the human genome, integrate foreign DNA much more readily than bacterial genomes. We have spelt out what such consequences could be [43]: insertion mutations including cancer, activation of dormant viruses, and recombination with viral sequences in the genome to generate new viruses.
Eesearch in recent years has also uncovered substantial amounts of DNA and RNA circulating in peripheral blood, which are actively secreted by living cells, and fully capable of transforming other cells [45, 46]. The nucleic acids appear to play a role in disease progression and metastasis of cancers. In plants too, foreign and endogenous nucleic acids circulate [47], apparently acting as intercellular messengers. There is a distinct possibility, therefore, that transgenic DNA could be vectored between plants through insects as well as soil bacteria; and transgenic DNA from food could end up in peripheral blood and gain entry into cells [45].
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Recent Evidence Confirms Risks of Horizontal Gene Transfer
Horizontal gene transfer is one of the most serious, if not the most serious hazard of transgenic technology. I have been drawing our regulators' attention to it at least since 1996 [1], when there was already sufficient evidence to suggest that transgenic DNA in GM crops and products can spread by being taken up directly by viruses and bacteria as well as plant and animals cells.
The oft-repeated refrain that "transgenic DNA is just like ordinary DNA" is false. Transgenic DNA is in many respects optimised for horizontal gene transfer. It is designed to cross species barriers and to jump into genomes, and it has homologies to the DNA of many species and their genetic parasites (plasmids, transposons and viruses), thereby enhancing recombination with all of them [2]. Transgenic constructs contain new combinations of genes that have never existed, and they also amplify gene products that have never been part of our food chain [3].
The health risks of horizontal gene transfer include:
1. Antibiotic resistance genes spreading to pathogenic bacteria.
2. Disease-associated genes spreading and recombining to create new viruses and bacteria that cause diseases.
3. Transgenic DNA inserting into human cells, triggering cancer.
The risk of cancer is highlighted by the recent report that gene therapy - genetic modification of human cells - claimed its first cancer victim [4]. The procedure, in which bone marrow cells are genetically modified outside the body and re-implanted, was previously thought to avoid creating infectious viruses and causing cancer, both recognized major hazards of gene therapy.
The transgenic constructs used in genetic modification are basically the same whether it is of human cells or of other animals and plants. An aggressive promoter from a virus is often used to boost the expression of the transgene, in animal and human cells, from the cytomegalovirus that infects mammalian cells, and in plants, the 35S promoter from the cauliflower mosaic virus (CaMV) that infects Cruciferae plants.
Unfortunately, although the CaMV virus is specific for plants, its 35S promoter is active in species across the living world, human cells included, as we discovered in the scientific literature dating back to 1989. Plant geneticists who have incorporated the promoter into practically all GM crops now grown commercially are apparently unaware of this crucial information [5].
In 1999, another problem with the CaMV 35S promoter was identified: it has a 'recombination hotspot' where it tends to break and join up with other DNA [6]. Since then, we have continued to warn our regulators that the CaMV 35S promoter will be extra prone to spread by horizontal gene transfer and recombination [7-9]. The recent controversy over the transgenic contamination of the Mexican landraces [10] hinges on observations suggesting that the transgenic DNA with the CaMV 35S promoter is "fragmenting and promiscuously scattering throughout the genome" of the landraces, observations that would be consistent with our expectations [11].
Similarly, I was not surprised by the research results released earlier this year by the Food Standards Agency [12], indicating that transgenic DNA from GM soya flour, eaten in a single hamburger and milk shake meal, was found transferred to the bacteria in the gut contents from the colostomy bags of human volunteers.
What I found unacceptable was the way the Agency dismissed the findings and downplayed the risks. The comments, "it is extremely unlikely that genes from genetically modified (GM) food can end up in bacteria in the gut of people who eat them", and "the findings had been assessed by several Government experts who had ruled that humans were not at risk", are seriously misleading.
First, the experimental design stacked the odds heavily against finding a positive result. For example, the probe for transgenic DNA covered only a tiny fraction of the entire construct. So, only a correspondingly tiny fraction of the actual transfers would ever be detected, especially given the well-known tendency of transgenic constructs to fragment and rearrange.
Second, the scope of the investigation was intentionally restricted. There was no attempt to check for transgenic DNA in the blood and blood cells, even though scientific reports dating back to the early 1990s had already indicated transgenic DNA could pass through the intestine and the placenta, and become incorporated into the blood cells, liver and spleen cells and cells of the foetus and newborn [13].
The observation in the FSA report [12] that no transgenic DNA was found in the faeces of the 'healthy volunteers', far from being reassuring, raises the worrying possibility that the transgenic DNA has all been taken up into the intestinal cells and/or passed into the bloodstream.
Third, no attempt was made to address the limitations of the detection method and the scope of the investigation, which grossly underestimated the extent and frequency of horizontal gene transfer, and hence failed completely in assessing the real risks. On the contrary, false assurances were made that "humans were not at risk".
Another research project on horizontal gene transfer commissioned by the Ministry of Agriculture, Fisheries and Food (MAFF), the predecessor to the Food Standards Agency, concerns Agrobacterium tumefaciens, the soil bacterium that causes crown gall disease, which has been developed as a major gene transfer vector for making transgenic plants. Foreign genes are typically spliced into T-DNA - part of a plasmid called Ti (tumour-inducing) – that's integrated into plant genome.
It turns out that Agrobacterium injects T-DNA into plant cells in a process that strongly resembles conjugation, ie, mating between bacterial cells; and all the necessary signals and genes involved are interchangeable with those for conjugation [14].
That means transgenic plants created by T-DNA vector system have a ready route for horizontal gene escape, via Agrobacterium, helped by the ordinary conjugative mechanisms of many other bacteria that cause diseases [15].
A report submitted to MAFF in 1997 had indeed raised the possibility that Agrobacterium tumefaciens could be a vector for gene escape [16, 17].
The researchers found that it was extremely difficult to get rid of the Agrobacterium used in the vector system after transformation.
High rates of gene transfer are known to be associated with the plant root system and the germinating seed [18]. There, Agrobacterium could multiply and transfer transgenic DNA to other bacteria, as well as to the next crop plant.
Agrobacterium was also found to transfer genes into several types of human cells [19], and in a manner similar to that which it uses to transform plant cells.
We have submitted two relevant ISIS reports together with some specific questions to the ACNFP for consideration at the November 13 Open Meeting [20].
All the risks of horizontal gene transfer described above are real, and far outweigh any potential benefits that GM crops can offer. There is no case for allowing any commercial release of GM crops and food products.
The following experiments and tests should be done to address the risks of horizontal gene transfer.
1. Feeding experiments similar to those carried out by Dr. Arpad Pusztai's team should be done, using well-characterized transgenic soya and/or maize meal feed, with full, adequate, monitoring for transgenic DNA in the faeces, blood and blood cells, and post-mortem histological examinations that include tracking transfer of transgenic DNA into the genome of cells. As an added control, nontransgenic DNA from the same GM feed sample should also be monitored.
2. Feeding trials on human volunteers should be carried out using well-characterized transgenic soya and/or maize meal feed, with full, adequate monitoring for transgenic DNA in the faeces, blood and blood cells. Also as an added control, nontransgenic DNA from the same GM feed sample should also be monitored.
3. The stability of transgenic plants in successive generations should be systematically investigated, especially for those containing CaMV 35S promoter, using adequate quantitative molecular techniques.
4. Full molecular characterisation of all transgenic lines must be carried out to establish uniformity and genetic stability of the insert(s).
5. All transgenic plants created by the Agrobacterium T-DNA vector system should be tested for the persistence of the bacteria and vectors. The soil in which they have been grown should also be monitored for gene escape to soil bacteria. And the potential for horizontal gene transfer to the next crop via the germinating seed and root system should be carefully monitored.
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Common Plant Vector Injects Genes into Human Cells
The genetic engineering community has assumed that Agrobacterium, a commonly used gene transfer vector for plants, does not infect animal cells, and certainly would not transfer genes into them. But this has been proved wrong. Prof. Joe Cummins warns of hazards to laboratory and farm workers.
Agrobacterium tumefaciens is a bacterium that causes tumours to appear on the stems of infected plants. The bacterium causes the tumours by transferring genes to the cells of the infected plant cells from a tumour inducing plasmid (Ti). The Ti plasmid has virulence genes that determine attachment to cells and transfer of a segment of the plasmid, T-DNA, to the plant cell. The transferred DNA is integrated essentially randomly (no apparent sequence bias at the site of insertion) into the plant chromosomes and normally add bacterial genes that stimulate plant tumour cell growth.
In crop genetic manipulation (GM), the growth-stimulating genes that give rise to tumours are replaced by GM constructs which include genes for antibiotic resistance, plant viral promoters and genes for desired crop traits such as herbicide tolerance.
Until quite recently, the genetic engineering community has assumed that Agrobacterium does not infect animal cells, and certainly would not transfer genes into them. But this has been proved wrong.
A paper published earlier this year reports that T-DNA can be transferred to the chromosomes of human cancer cells [1]. In fact, Agrobacterium attaches to and genetically transforms several types of human cells. The researchers found that in stably transformed HeLa cells, the integration event occurred at the right border of the Ti plasmid's T-DNA, exactly as would happen when it is being transferred into a plant cell genome. This suggests that Agrobacterium transforms human cells by a mechanism similar to that which it uses for transformation of plants cells.
The paper shows that human cancer cells along with neuron and kidney cells were transformed with the Agrobacterium T-DNA. Such observations should raise alarm for those who use Agrobacterium in the laboratory.
The integrated T-DNA will almost certainly act as a mutagen as it integrates into human chromosomes. Cancer can be triggered by activation of oncogenes (ie, cancer genes) or inactivation of cancer suppressing genes. Furthermore, the sequences carried within the T-DNA in the transforming bacterium can be expressed in the transformed cells (the viral promoter CaMV has been found to be active in HeLa cells [2]) and constructions currently being tested include pharmaceutically active human genes such as the interleukins [3].
It is clear that little has been done to prevent environmental escape of the transforming bacteria or to quantify such releases. In conclusion, a study of cancer incidence among those exposed to Agrobacterium tumefaciens in the laboratory and
in the field is needed. It would be worthwhile to screen workers for T-DNA sequences.
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Horizontal Gene Transfer – The Hidden Hazards of Genetic Engineering
Genetic engineering involves designing artificial constructs to cross species barriers and to invade genomes. In other words, it enhances horizontal gene transfer – the direct transfer of genetic material to unrelated species. The artificial constructs or transgenic DNA typically contain genetic material from bacteria, viruses and other genetic parasites that cause diseases as well as antibiotic resistance genes that make infectious diseases untreatable. Horizontal transfer of transgenic DNA has the potential, among other things, to create new viruses and bacteria that cause diseases and spread drug and antibiotic resistance genes among pathogens. There is an urgent need to establish effective regulatory oversight to prevent the escape and release of these dangerous constructs into the environment, and to consider whether some of the most dangerous experiments should be allowed to continue at all.
Key words: antibiotic resistance genes, dormant viruses, CaMV promoter, cancer, naked DNA, transgenic DNA,
Transgenic pollen and baby bees
Prof. Hans-Hinrich Kaatz from the University of Jena, is reported to have new evidence, as yet unpublished, that genes engineered into transgenic plants have transferred via pollen to bacteria and yeasts living in the gut of bee larvae(1).
If Prof. Kaatz' claim can be substantiated, it indicates that the new genes and gene-constructs introduced into transgenic crops and other transgenic organisms can spread, not just by ordinary cross-pollination or cross-breeding to closely related species, but by the genes and gene-constructs invading the genomes (the totality of the organisms' own genetic material) of completely unrelated species, including the microorganisms living in the gut of animals eating transgenic material.
This finding is not unexpected. Some scientists have been drawing attention to this possibility recently(2), but the warnings actually date back to the mid-1970s when genetic engineering began. Hundreds of scientists around the world are now demanding a moratorium on all environmental releases of transgenic organisms on grounds of safety(3), and horizontal gene transfer is one of the major considerations.
Some of us have argued that the hazards of 'horizontal' gene transfer to unrelated species are inherent to genetic engineering(4). The genes and gene-constructs created in genetic engineering have never existed in billions of years of evolution. They consist of genetic material originating from bacteria, viruses and other genetic parasites that cause diseases and spread drug and antibiotic resistance genes. They are designed to cross all species barriers and to invade genomes. The spread of such genes and gene-constructs have the potential to make infectious diseases untreatable and to create new viruses and bacteria that cause diseases.
Horizontal gene transfer may spread transgenes to the entire biosphere
Horizontal gene transfer is the transfer of genetic material between cells or genomes belonging to unrelated species, by processes other than usual reproduction. In the usual process of reproduction, genes are transferred vertically from parent to offspring; and such a process can occur only within a species or between closely related species.
Bacteria have been known to exchange genes across species barriers in nature. There are three ways in which this is accomplished. In conjugation, genetic material is passed between cells in contact; in transduction, genetic material is carried from one cell to another by infectious viruses; and in transformation, the genetic material is taken up directly by the cell from its environment. For horizontal gene transfer to be successful, the foreign genetic material must become integrated into the cell's genome, or become stably maintained in the recipient cell in some other form. In most cases, foreign genetic material that enters a cell by accident, especially if it is from another species, will be broken down before it can incorporate into the genome. Under certain ecological conditions which are still poorly understood, foreign genetic material escapes being broken down and become incorporated in the genome. For example, heat shock and pollutants such as heavy metals can favor horizontal gene transfer; and the presence of antibiotics can increase the frequency of horizontal gene transfer 10 to 10 000 fold(5).
While horizontal gene transfer is well-known among bacteria, it is only within the past 10 years that its occurrence has become recognized among higher plants and animals(6). The scope for horizontal gene transfer is essentially the entire biosphere, with bacteria and viruses serving both as intermediaries for gene trafficking and as reservoirs for gene multiplication and recombination (the process of making new combinations of genetic material (7)).
There are many potential routes for horizontal gene transfer to plants and animals. Transduction is expected to be a main route as there are many viruses which infect plants and animals. Recent research in gene therapy indicates that transformation is potentially very important for cells of mammals including human beings. A great variety of 'naked' genetic material are readily taken up by all kinds of cells, simply as the result of being applied in solution to the eye, or rubbed into the skin, injected, inhaled or swallowed. In many cases, the foreign gene constructs become incorporated into the genome(8).
Direct transformation may not be as important for plant cells, which generally have a protective cell wall. But soil bacteria belonging to the genus Agrobacterium are able to transfer the T (tumour) segment of its Tumour-inducing (Ti) plasmid (see below) into plant cells in a process resembling conjugation. This T-DNA is widely exploited as a gene transfer vehicle in plant genetic engineering (see below). Foreign genetic material can also be introduced into plant and animal cells by insects and arthropods with sharp mouthparts. In addition, bacterial pathogens which enter plant and animal cells may take up foreign genetic material and carry it into the cells, thus serving vectors for horizontal gene transfer(9). There are almost no barriers preventing the entry of foreign genetic material into the cells of probably any species on earth. The most important barriers to horizontal gene transfer operate after the foreign genetic material has entered the cell(10).
Most foreign genetic material, such as those present in ordinary food, will be broken down to generate energy and building-blocks for growth and repair. There are many enzymes which break down foreign genetic material; and in the event that the foreign genetic material is incorporated into the genome, chemical modification can still put it out of action and eliminate it.
However, viruses and other genetic parasites such as plasmids and transposons, have special genetic signals and probably overall structure to escape being broken down. A virus consists of genetic material generally wrapped in a protein coat. It sheds its overcoat on entering a cell and can either hi-jack the cell to make many more copies of itself, or it can jump directly into the cell's genome. Plasmids are pieces of 'free', usually circular, genetic material that can be indefinitely maintained in the cell separately from the cell's genome. Transposons, or 'jumping genes', are blocks of genetic material which have the ability to jump in and out of genomes, with or without multiplying themselves in the process. They can also land in plasmids and be propagated there. Genes hitch-hiking in genetic parasites, ie, viruses, plasmids and transposons, therefore, have a greater probability of being successfully transferred into cells and genomes. Genetic parasites are vectors for horizontal gene transfer.
Natural genetic parasites are limited by species barriers, so for example, pig viruses will infect pigs, but not human beings, and cauliflower viruses will not attack tomatoes. It is the protein coat of the virus that determines host specificity, which is why naked viral genomes (the genetic material stripped of the coat) have generally been found to have a wider host range than the intact virus(11). Similarly, the signals for propagating different plasmids and transposons are usually specific to a limited range of host species, although there are exceptions.
As more and more genomes have been sequenced, it is becoming apparent that gene trafficking or horizontal gene transfer has played an important role in the evolution of all species(12). However, it is also clear that horizontal gene trafficking is regulated by internal constraints in the organisms in response to ecological conditions(13).
Genetic engineering is unregulated horizontal gene transfer
Genetic engineering is a collection of laboratory techniques used to isolate and combine the genetic material of any species, and then to multiply the constructs in convenient cultures of bacteria and viruses in the laboratory. Most of all, the techniques allow genetic material to be transferred between species that would never interbreed in nature. That is how human genes can be transferred into pig, sheep, fish and bacteria; and spider silk genes end up in goats. Completely new, exotic genes are also being introduced into food and other crops.
In order to overcome natural species barriers limiting gene transfer and maintenance, genetic engineers have made a huge variety of artificial vectors (carriers of genes) by combining parts of the most infectious natural vectors – viruses, plasmids and transposons - from different sources. These artificial vectors generally have their disease-causing functions removed or disabled, but are designed to cross wide species barriers, so the same vector may now transfer, say, human genes spliced into the vector, to the genomes of all other mammals, or of plants. Artificial vectors greatly enhance horizontal gene transfer (see Box 1).(14)
Box 1
Artificial vectors enhance horizontal gene transfer
* They are derived from natural genetic parasites that mediate horizontal gene transfer most effectively.
* Their highly chimaeric nature means that they have sequence homologies (similarities) to DNA from viral pathogens, plasmids and transposons of multiple species across Kingdoms. This will facilitate widespread horizontal gene transfer and recombination.
* They routinely contain antibiotic resistance marker genes which enhance their successful horizontal transfer in the presence of antibiotics, either intentionally applied, or present as xenobiotic in the environment. Antibiotics are known to enhance horizontal gene transfer between 10 to 10 000 fold.
* They often have 'origins of replication' and 'transfer sequences', signals that facilitate horizontal gene transfer and maintenance in cells to which they are transferred.
* Chimaeric vectors are well-known to be structurally unstable, ie, they have a tendency to break and join up incorrectly or with other DNA, and this will increase the propensity for horizontal gene transfer and recombination.
* They are designed to invade genomes, to overcome mechanisms that breakdown or disable foreign DNA and hence will increase the probability of horizontal transfer.
Although different classes of vectors are distinguishable on the basis of the main-frame genetic material, practically every one of them is chimaeric, being composed of genetic material originating from the genetic parasites of many different species of bacteria, animals and plants. Important chimaeric 'shuttle' vectors enable genes to be multiplied in the bacterium E. coli and transferred into species in every other Kingdom of plants and animals. Simply by creating such a vast variety of promiscuous gene transfer vectors, genetic engineering biotechnology has effectively opened up highways for horizontal gene transfer and recombination, where previously the process was tightly regulated, with restricted access through narrow, tortuous footpaths. These gene transfer highways connect species in every Domain and Kingdom with the microbial populations via the universal mixing vessel used in genetic engineering, E. coli. What makes it worse is that there is currently still no legislation in any country to prevent the escape and release of most artificial vectors and other artificial constructs into the environment (15).
What are the hazards of horizontal gene transfer?
Most artificial vectors are either derived from viruses or have viral genes in them, and are designed to cross species barriers and invade genomes. They have the potential to recombine with the genetic material of other viruses to generate new infectious viruses that cross species barriers. Such viruses have been appearing at alarming frequencies. The antibiotic resistance genes carried by artificial vectors can also spread to bacterial pathogens. Has the growth of commercial-scale genetic engineering biotechnology contributed to the resurgence of drug and antibiotic infectious diseases within the past 25 years (16)? There is already overwhelming evidence that horizontal gene transfer and recombination have been responsible for creating new viral and bacterial pathogens and for spreading drug and antibiotic resistance among the pathogens. One way that new viral pathogens may be created is through recombination with dormant, inactive or inactivated viral genetic material that are in all genomes, plants and animals without exception. Recombination between external and resident, dormant viruses have been implicated in many animal cancers (17).
As stated earlier, the cells of all species including our own can take up foreign genetic material. Artificial constructs designed to invade genomes may well invade our own. These insertions may lead to inappropriate inactivation or activation of genes (insertion mutagenesis), some of which may lead to cancer (insertion carcinogenesis)(18). The hazards of horizontal gene transfer are summarized in Box 2.
Box 2
Potential hazards of horizontal gene transfer from genetic engineering
* Generation of new cross-species viruses that cause disease
* Generation of new bacteria that cause diseases
* Spreading drug and antibiotic resistance genes among the viral and bacterial pathogens, making infections untreatable
* Random insertion into genomes of cells resulting in harmful effects including cancer
* Reactivation of dormant viruses, present in all cells and genomes, which may cause diseases
* Spreading new genes and gene constructs that have never existed
* Multiplication of ecological impacts due to all of the above.
Transgenic DNA may be more likely to transfer horizontally than non-transgenic DNA
Both the artificial vectors used in genetic engineering and the genes transferred to make transgenic organisms are predominantly from viruses and bacteria associated with diseases, and these are being brought together in combinations that have never existed in billions of years of evolution.
Genes are never transferred alone. They are transferred in unit-constructs, known as an 'expression cassettes'. Each gene has to be accompanied by a special piece of genetic material, the promoter, which signals the cell to turn the gene on, ie, to transcribe the DNA gene sequence into RNA. At the end of the gene there has to be another signal, a terminator, to end the transcription and to mark the RNA, so it can be further processed and translated into protein. The simplest expression cassette looks like this:
Promoter gene terminator
Typically, each bit of the construct: promoter, gene and terminator, is from a different source. The gene itself may also be a composite of bits from different sources. Several expression cassettes are usually linked in series, or 'stacked' in the final construct. At least one of the expression cassettes will be that of an antibiotic resistance marker gene to enable cells that have taken up the foreign construct to be selected with antibiotics. The antibiotic resistance gene cassette will often remain in the transgenic organism.
The most commonly used promoters are from viruses associated with serious diseases. The reason is that such viral promoters give continuous over-expression of genes placed under their control. The same basic construct is used in all applications of genetic engineering, whether in agriculture or in medicine, and the same hazards are involved. There are reasons to believe that transgenic DNA is much more likely to spread horizontal than the organisms' own DNA (see Box 3) (19).
Box 3
Reasons to suspect that transgenic DNA may be more likely to spread horizontally than non-transgenic DNA
* Artificial constructs and vectors are designed to be invasive to foreign genomes and overcome species barriers.
* All artificial gene-constructs are structurally unstable (20), and hence prone to recombine and transfer horizontally.
* The mechanisms enabling foreign genes to insert into the genome also enable them to jump out again, to re-insert at another site, or to another genome.
* The integration sites of most commonly used artificial vectors for transferring
* genes are 'recombination hotspots', and so have an increased propensity to transfer horizontally.
* Viral promoters, such as that from the cauliflower mosaic virus, widely used to make transgenes over-express, contain recombination hotspots (21), and will therefore further enhance horizontal gene transfer.
* The metabolic stress on the host organism due to the continuous over expression of transgenes may also contribute to the instability of the insert (22).
* The foreign gene-constructs and the vectors into which they are spliced, are typically mosaics of DNA sequences from numerous species and their genetic parasites; that means they will have sequence homologies with the genetic material of many species and their genetic parasites, thus facilitating wide-ranging horizontal gene transfer and recombination.
Additional hazards from viral promoters
We have recently drawn attention to additional hazards associated with the promoter of the cauliflower mosaic virus (CaMV) most widely used in agriculture (23). It is in practically all transgenic plants already commercialized or undergoing field trials, as well as a high proportion of transgenic plants under development, including the much acclaimed 'golden rice' (24).
CaMV is closely related to human hepatitis B virus, and less so, to retroviruses such as the AIDS virus (25). Although the intact virus itself is infectious only for cruciferae plants, its promoter is promiscuous in function, and is active in all higher plants, in algae, yeast, and E. coli (26), as well as frog and human cell systems (27). Like all promoters of viruses and of cellular genes, it has a modular structure, with parts common to, and interchangeable with promoters of other plant and animal viruses. It has a recombination hotspot, flanked by multiple motifs involved in recombination, similar to other recombination hotspots including the borders of the Agrobacterium T DNA vector most frequently used in making transgenic plants. The suspected mechanism of recombination requires little or no DNA sequence homologies. Finally, viral genes incorporated into transgenic plants have been found to recombine with infecting viruses to generate new viruses (28). In some cases, the recombinant viruses are more infectious than the original.
Proviral sequences – generally inactive copies of viral genomes - are present in all plant and animal genomes, and as all viral promoters are modular, and have at least one module – the TATA box - in common, if not more. It is not inconceivable that the CaMV 35S promoter in transgenic constructs can reactivate dormant viruses or generate new viruses by recombination. The CaMV 35S promoter has been joined artificially to copies of a wide range of viral genomes, and infectious viruses produced in the laboratory (29). There is also evidence that proviral sequence in the genome can be reactivated (30).
These considerations are especially relevant in the light of recent findings that certain transgenic potatoes - containing the CaMV 35S promoter and transformed with Agrobacterium T-DNA - may be unsafe for young rats, and that a significant part of the effects may be due to "the construct or the genetic transformation (or both) (31)" The authors also report an increase in lymphocytes in the intestinal wall, which is a non-specific sign of viral infection (32).
Evidence for horizontal transfer of transgenic DNA
It is often argued that transgenic DNA, once incorporated into the transgenic organism, will be just as stable as the organism's own DNA. But there is both direct and indirect evidence against this supposition. Transgenic DNA is more likely to spread, and has been found to spread by horizontal gene transfer.
Transgenic lines are notoriously unstable and often do not breed true (33). There is a paucity of molecular data documenting the structural stability of the transgenic DNA, both in terms of its site of insertion in the genome and its arrangement of genes, in successive generations. Instead, transgenes may be silenced in subsequent generations or lost altogether (34).
A herbicide-tolerance gene, introduced into Arabidopsis by means of a vector, was found to be up to 30 times more likely to escape and spread than the same gene obtained by mutagenesis (35). One way this may happen is by secondary horizontal gene transfer via insects visiting the plants for pollen and nectar (36). The reported finding that pollen can transfer transgenic DNA to bacteria in the gut of bee larvae is relevant here.
Secondary horizontal transfer of transgenes and antibiotic resistant marker genes from genetically engineered crop-plants into soil bacteria and fungi have been documented in the laboratory. Transfer to fungi was achieved simply by co-cultivation (37), while transfer to bacteria has been achieved by both re-isolated transgenic DNA or total transgenic plant DNA (38). Successful transfers of a kanamycin resistance marker gene to the soil bacterium Acinetobacter were obtained using total DNA extracted from homogenized plant leaf from a range of transgenic plants: Solanum tuberosum (potato), Nicotiana tabacum (tobacco), Beta vulgaris (sugar beet), Brassica napus (oil-seed rape) and Lycopersicon esculentum (tomato) (39). It is estimated that about 2500 copies of the kanamycin resistance genes (from the same number of plant cells) is sufficient to successfully transform one bacterium, despite the fact that there is six million-fold excess of plant DNA present. A single plant with say, 2.5 trillion cells, would be sufficient to transform one billion bacteria.
Despite the misleading title in one of the publications,(40) a high gene transfer frequency of 5.8 x 10-2 per recipient bacterium was demonstrated under optimum conditions. But the authors then proceeded to calculate an extremely low gene transfer frequency of 2.0 x 10-17 under extrapolated "natural conditions", assuming that different factors acted independently. The natural conditions, however, are largely unknown and unpredictable, and even by the authors' own admission, synergistic effects cannot be ruled out. Free transgenic DNA is bound to be readily available in the rhizosphere around the plant roots, which is also an 'environmental hotspot' for gene transfer (41). Other workers have found evidence of horizontal transfer of kanamycin resistance from transgenic DNA to Acinetobactor, and positive results were obtained using just 100ml of plant-leaf homogenate (42).
Defenders of the biotech industry still insist that just because horizontal gene transfer occurs in the laboratory does not mean it can occur in nature. However, there is already evidence suggesting it can occur in nature. First of all, genetic material released from dead and live cells, is now found to persist in all environments; and not rapidly broken down as previously supposed. It sticks to clay, sand and humic acid particles and retains the ability to infect (transform) a range of micro-organisms in the soil (43). The transformation of bacteria in the soil by DNA adsorbed to clay sand and humic acid has been confirmed in microcosm experiments (44).
Reseachers in Germany began a series of experiments in 1993 to monitor field releases of transgenic rizomania-resistant sugar beet (Beta vulgaris), containing the marker gene for kanamycin resistance, for persistence of transgenic DNA and of horizontal gene transfer of transgenic DNA into soil bacteria (45). It is the first such experiment to be carried out; after tens of thousands of field releases and tens of millions of hectares have been planted with transgenic crops. It will be useful to review their findings in detail.
Transgenic DNA was found to persist in the soil for up to two years after the transgenic crop was planted. Though they did not comment on it, the data showed that the proportion of kanamycin resistant bacteria in the soil increased significantly between 1.5 and 2 years. Could it be due to horizontal transfer of antibiotic resistance marker gene in the transgenic DNA? Although none of 4000 colonies of soil bacteria isolated – a rather small number - was found to have taken up transgenic DNA by the probes available, two out of seven samples of total bacterial DNA yielded positive results after 18 months. This suggests that horizontal gene transfer may have taken place, but the specific bacteria which have taken up the transgenic DNA cannot be isolated as colonies. That is not surprising as less than 1% of all the bacteria in the soil are culturable. The authors were careful not to rule out transgenic DNA being adsorbed to the surface of bacteria rather than being tranferred into the bacteria.
The researchers also carried out microcosm experiments to which total transgenic sugar-beet DNA was added to non-sterile soil with its natural complement of microorganisms. The intensity of the signal for transgenic DNA decreased during the first days and subsequently increased. This may be interpreted as a sign that the transgenic DNA has been taken up by bacteria and become amplified as a result.
In parallel, soil samples were plated and the total bacterial lawn allowed to grow for 4 days, after which DNA was extracted. Several positive signals were found, "which might indicate uptake of transgenic DNA by competent bacteria."
The authors were cautious not to claim conclusive results simply because the specific bacteria carrying the transgenic DNA sequences were not isolated. The results do show, however, that horizontal gene transfer may have taken place both in the field and in the soil microcosm.
DNA is not broken down sufficiently rapidly in the gut either, which is why transfer of transgenic DNA to microorganisms in the gut of bee larvae would not be surprising. A genetically engineered plasmid was found to have a 6 to 25% survival after 60 min. of exposure to human saliva. The partially degraded plasmid DNA was capable of transforming Streptococcus gordonii, one of the bacteria that normally live in the human mouth and pharynx. The frequency of transformation dropped exponentially with time of exposure to saliva, but it was still detectable after 10 minutes. Human saliva actually contains factors that promote competence of resident bacteria to become transformed by DNA (46).
Viral DNA fed to mice is found to reach white blood cells, spleen and liver cells via the intestinal wall, to become incorporated into the mouse cell genome (47). When fed to pregnant mice, the viral DNA ends up in cells of the fetuses and the new born animals, suggesting that it has gone through the placenta as well (48). The authors remark that "The consequences of foreign DNA uptake for mutagenesis and oncogenesis have not yet been investigated (49)." As already mentioned, recent experiments in gene therapy leave little doubt that naked nucleic acid constructs can readily enter mammalian cells and in many cases become incorporated into the cell's genome.
Horizontal gene transfer is an established phenomenon. It has taken place in our evolutionary past and is continuing today. All the signs are that natural horizontal gene transfer is a regulated process, limited by species barriers and by mechanisms that break down and inactivate foreign genetic material. Unfortunately, genetic engineering has created a huge variety of artificial constructs designed to cross all species barriers and to invade essentially all genomes. Although the basic constructs are the same for all applications, some of the most dangerous may be coming from the waste disposal of contained users of transgenic organisms(50). These will include constructs containing cancer genes from viruses and cells from laboratories researching and developing cancer and cancer drugs, virulence genes from bacteria and viruses in pathology labs. In short, the biosphere is being exposed to all kinds of novel constructs and gene combinations that did not previously exist in nature, and may never have come into being but for genetic engineering.
There is an urgent need to establish effective regulatory oversight, in the first instance, to prevent the escape and release of these dangerous constructs into the environment, and then to consider whether some of the most dangerous experiments should be allowed to continue at all.
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Survival of free DNA encoding antibiotic resistance from transgenic maize and the transformation activity of DNA in ovine saliva, ovine rumen fluid and silage effluent. Duggan PS, Chambers PA, Heritage J and Forbes JM. FEMS Microbiology Letters 2000, 191, 71-7.
Insect-resistant maize line CG00526-176 contains three bacterial genes: the cry1A(b) specific to lepidopterans, the bar gene conferring tolerance to glufosinate, and a bla gene encoding TEM-1 b-lactamase (ampicillin resistance). The bla gene originates from the cloning vector PUC18 and is not expressed in maize, but has bacterial regulatory sequences that would allow it to become functional were it to be transferred back into bacteria. There are at least two copies of crylA(b) and bla genes integrated into the DNA of maize line CG00526-176.
The researchers investigated the survival of DNA from transgenic maize and the transfer of the antibiotic resistance bla gene to bacteria in the presence of saliva, rumen fluid and silage effluent, which are relevant to horizontal gene transfer in the oral cavity, the rumen, and in silage.
E. coli strain DH5a was the test micro-organism for horizontal gene transfer. Degradation of DNA was followed by gel electrophoresis as well as by polymerase chain reaction (PCR). Both pUC18 plasmid and transgenic maize DNA were used in the experiments.
On gel electrophoresis, plasmid DNA and maize DNA were shown to be degraded rapidly by rumen fluid or silage effluent within one minute, but both were incompletely degraded after at least l h exposure to saliva.
On PCR analysis, large fragments of the bla gene (>350bp) were still found in rumen fluid up to 30 mins for the plasmid and up to1 min for maize DNA. Even larger fragments (>350 and >684 bp) from plasmid and maize DNA were found up to 30 min incubation in silage effluent, and up to 24h and 2 h respectively in saliva.
PCR analysis also showed that fragments of the cryl1A(b) (>1914bp) in maize DNA could be found up to 1 min with rumen fluid, 5 min with silage effluent, and 60 min with saliva.
Plasmid DNA exposed to saliva for 24h was still capable of transforming E. coli to ampicillin resistance, but at low efficiency: 20 cfu (colony forming units) per ml compared with 1.6 x103 cfu per ml after 24h in sterile water. Previous exposure to rumen fluid for 30s reduced transformation 5-fold. No transformants were obtained after the plasmid DNA was exposed to silage eflluent or rumen fluid for longer than 1 min.
However, when E. coli and plasmid were simultaneously added to filter-sterilized silage effluent or rumen fluid, 4.75x103 cfu per ml transformants were recovered after 4.5h in rumen fluid and 11cfu per ml were recovered after 3h in silage effluent.
In summary, horizontal gene transfer can occur before the DNA is completely broken down, even when the breakdown is rapid, as in the rumen or in silage. DNA breakdown is extremely slow in saliva, and hence the oral cavity will be a very important site for horizontal gene transfer.
The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly depends on homologous sequences in the recipient cells. DeVries J, Meier P and Wackernagel W. FEMS Microbiology Letters 2001, 195, 211-5.
The nptII gene in transgenic potato plants coding for kanamycin resistance, transforms naturally competent cells of the soil bacteria Pseudomonas stutzeri and Acinetobacter BD413 (both harboring a plasmid with an nptII gene containing a small deletion (hence nonfunctional) with the same high efficiency as nptII genes on plasmid DNA ( 3x10-5 -1x10-4) despite the presence of a more than 106 fold excess of plant DNA. However, in the absence of homologous sequences in the recipient cells the transformation dropped by at least about 108 fold in P. stutzeri and 109 fold in Acinetobacter, below the detection limit.
More than 60 bacterial species have been shown to take up and incorporate DNA (undergo transformation). Many bacteria like Bacillus subtilis and Acinetobacter sp. strain BD413, apparently take up DNA of any source into the cytoplasm. Stable maintenance and expression depends on integration into the genome by genetic recombination.
The authors state, "This indicates a very low probability of non-homologous DNA fragments to be integrated by illegitimate recombination events during transformation". Should we be reassured? Not at all.
The high frequencies of homologous recombination obtained are relevant to GM constructs released in large concentrations into the environment in GM crops and transgenic wastes. GM constructs contain homologies to many different species of bacteria and viruses, and are therefore capable of engaging in high frequencies of recombination with a wide variety of bacteria and viruses.
Illegitimate recombination events may occur at lower frequencies, but they become substantial as GM constructs are released on massive scales. In particular, recombination hotspots associated with many GM constructs (such as those containing CaMV 35S promoter) may increase the frequency of illegitimate recombination.
Effect of genomic location on horizontal transfer of a recombinant gene cassette between Pseudomonas strains in the rhizosphere and spermosphere of barley seedlings. Sengelov G, Kristensen KJ, Sorensen AH, Kroer N, and Sorensen SJ. Current Microbiology 2001, 42, 160-7.
The rhizosphere - surfaces around the plant roots - and the spermosphere - surfaces around the germinated seeds – are recognised hotspots for horizontal gene transfer between bacteria. But the frequency of horizontal transfer also depends on the location of the genes; whether in the bacterial chromosome, or in a mobilizable plasmid, ie, a plasmid that can be transferred with helper functions supplied by other plasmids, or in a conjugative plasmid, ie, a plasmid that has its own functions for transfer during conjugation (mating between bacterial cells). Not surprisingly, researchers found the highest frequencies of horizontal gene transfer in both the rhizosphere and spermosphere when the GM cassette was in a conjugative plasmid, somewhat lower when it was in a mobilizable plasmid, but could not be detected when it was inserted into the bacterial chromosome.
However, that does not mean GM constructs located on bacterial chromosomes do not transfer. The authors were careful to point out that the main mode of horizontal gene transfer in both the rhizosphere and the spermosphere is conjugation. Elsewhere, transformation (by direct uptake of DNA) will be more important, and there is evidence that chromosomal constructs are more efficient in transformation.
The authors warn: "On the basis of these experiments, we cannot rule out the possibility that horizontal gene transfer by transformation occurs at low frequencies in soil and that this process might have significant effect at field scale, which is an especially important point as regards risk assessment. Such rare events cannot be studied in microcosm experiments, but must be addressed in retrospective field studies."
The only retrospective study carried out has indeed found evidence of horizontal gene transfer from transgenic plants to soil bacteria (see "Horizontal gene transfer happens", ISIS News 5). An investigation yet to be done is horizontal transfer from GM plants to bacteria in the rhizosphere.
Evidence for recent invasion of the medaka fish genome by the Tol2 transposable element. Koga A, Shimada A, Shima, A, Sakaizumi, M, Tachida H and Hori H. Genetics 2000, 155, 273-81.
Transposable elements are genetic units that can move from one chromosome to another, with or without multiplying themselves. Tol2 is a 4.7 kbp element found in the genome of the medaka fish Oryzias latipes. It has terminal inverted repeats and contains four genes similar to a group of transposable elements found in fruitflly, maize and snapdragon. There are some 10 to 30 copies of Tol2 in the medaka genome that are highly homogeneous in structure, and no variation in base sequence was found when 5 random clones were examined. This is unusual, as transposable elements are typically heterogeneous, with many defective copies being present in the same genome.
The genus Oryzias contains more than 10 species. The authors examined 10 species but Tol2 was found in only 2 of them: O. curvinotus and O. latipes. The structure of the Tol2 is homogenous and identical both within each species and between the two species, which are not closely related and do not crossbreed in nature.
These results suggest very recent horizontal gene transfer. The two species overlap in distribution probably somewhere in Southern China. Tol2 could have transferred from one species to another, or both species could have acquired it from the same source. They also illustrate the dangers of using transposable elements as gene transfer vectors.
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Unstable Transgenic Lines Illegal
Further evidence that most if not all commercially approved transgenic lines are genetically unstable and non-uniform has come to light. The transgenic lines fail to satisfy the current EU Directive requirements for proof of genetic stability and uniformity, and are hence illegal. Dr. Mae-Wan Ho reports.
In a recent study [1] on five commercially approved transgenic lines carried out by two French laboratories [2], all five transgenic inserts were found to have rearranged, not just from the construct used in transformation, but also from the original structure reported by the company. This was clear evidence that all the lines were genetically unstable.
Further evidence has come to light since. The Service of Biosafety and Biotechnology (SBB) of the Scientific Institute of Public Health (IPH) in Brussels has published on its website ( [link to biosafety.ihe.be] reports on the molecular characterisation of the genetic map of six transgenic lines, four of which overlap with those analysed by the French laboratories: Bt 176 maize (Syngenta), Mon 810 maize (Monsanto), T25 maize (Bayer CropScience) and GTS 40-3-2 soybean (Monsanto).
The IPH is a Scientific Institute of the State, linked to the Belgian Federal Ministry of Social Affairs, Public Health and the Environment.
The Brussels reports are an overview of data presented at a meeting of the Belgian Biosafety Advisory Council. The data come from different scientific institutions, the applicants and from published papers. The reports found evidence of genetic instability similar to those described in the French study.
However, there are small and large discrepancies when the two sets of data are compared. In one case, Bt 176, there may even have been a misreporting or misidentification of the Bt transgene present, which the company claimed to be crylAb. Comparison with the public database revealed that the transgene has only 65% homology with the native crylAb, but 94% homology with crylAc. Bt toxins are potential allergens and immunogens; crylAc, in particular, was identified as a potent systemic and mucosal immunogen, as potent as cholera toxin [3].
The studies also revealed a discrepancy in regulatory practice. UK's Advisory Committee on Novel Foods and Processes (ACNFP) and the Belgian authority both appear to have allowed Monsanto to submit new molecular data on Roundup Ready soybean when independent analysis revealed its insert had rearranged.
Most of the discrepancies involve the structure of the insert, the number of insert(s) and locations within the genome; suggesting that the transgenic lines are not only unstable but also non-uniform. Consequently, the results of the molecular characterisation could differ from sample to sample of the same transgenic line. In other words, the transgenic lines may well not pass the DUS (distinctness, uniformity and stability) test, which is required by European legislation.
The new EU Directive 2001/18/EC on deliberate release of GMOs also requires information documenting genetic stability (Annex IIIB) as a condition for market approval. Genetic stability can only be demonstrated by 'event specific' molecular data of the kind carried out in the two studies. In view of the finding that practically every transgenic insert has rearranged from that reported in the company's original dossier, it would indicate that the transgenic lines have failed the test of genetic stability, and are no longer the same lines that were risk assessed, and in some cases, placed on the market. This has important safety implications. Rearrangements and deletions are signs of structural instability, which enhances horizontal gene transfer and recombination, with all the attendant risks [4]. This is particularly relevant as the molecular analyses have so far revealed a strong tendency for transgenic inserts to land in mobile genetic elements, such as retrotransposons and repeat sequences. Four out of six transgenic inserts analysed for flanking sequences identified repeat or retrotransposon sequences.
For either or both those reasons, it would be illegal, under European legislation, to grant those transgenic lines commercial approval; and the lines that have been approved must surely now be withdrawn.
The detailed comparisons on the findings in the four transgenic lines from the two studies are presented below, followed by comments on the additional transgenic lines investigated separately in the two studies.
Transgenic lines analysed in both studies
Bt 176 maize (Syngenta)
The Bt176 maize dossier was first submitted in 1994 by Ciba Geigy (Novartis) and approved under the old EU Directive for growing, seed production, import, processing and food/feed purposes since 23 January 1997 [5]. It was modified for tolerance to the herbicide glufosinate, male sterility and insect resistance. Two constructs were used to transform maize (see below).
French study
Only the simpler construct was analysed. Company data showed single insert containing the cauliflower mosaic virus (CaMV) 35S promoter (hereafter referred to as P35S) driving the bar gene (glufosinate tolerance) terminated by the CaMV 35S terminator (hereafter referred to as T35S) followed by the ampicillin resistance (bla) gene plus a bacterial promoter, and the plasmid origin of replication, ori.
Analysis revealed several fragments, all containing P35S: one with P35S joined to T35S, a second with P35S joined to an unknown sequence, and a third with P35S joined to the bar gene, with the T35S deleted (that means P35S could drive the expression of downstream maize genes).
At least three insertion sites were found for this construct.
Brussels study
This study [5] describes the line as being obtained by microprojective bombardment into immature embryos of inbred corn line CG00526 (Zea mays L.) using two different transforming plasmids. The plasmid pCIB4431 contains two copies of a synthetic truncated crylA(b) gene, having approximately 65% homology at nucleotide level with the native gene of Bacillus thuringiensis subsp. kurstaki strain HD1. The first copy is under the regulation of the maize phosphenlopyruvate caboxylase (PEPC) promoter (PPEPC) and the T35S. The second copy is under the regulation of the maize calcium-dependent protein kinase (CDPK) promoter(PCDPK), resulting in pollen-specific expression, and terminated with T35S. In addition, both copies were combined with the intron #9 derived from the maize PEPC gene to enhance expression in maize. The plasmid pCIB3064 contains the bar gene derived from Streptomyces hygroscopicus under the regulation of P35S and T35S. Both plasmids also contain a copy of the bla gene for amipicillin resistance under the control of a bacterial promoter.
There are still uncertainties about the copy number of the insert in event Bt176. Published results from Koziel et al [6] indicated that there may be as many as five copies of the crylA(b) gene present.
Data from Centrum Landbouwkundig Onderzoek, Mell, Belgium (CLO) revealed that the cry coding sequence showed 94% similarity with Genbank accession no. AF537267 for synthetic construct of crylAc gene. In comparison, the cry transgene showed only 65% homology at nucleotide level with the native gene of Bacillus thuringiensis subsp. kurstaki strain HD1. This suggests the company may have misreported or misidentified the transgene present.
The company's dossier claimed one single copy of transgene insert (P35S-bar-T35S), and gave no information on 5' or 3' flanking sequences. For the second transgene insert (T35S-int#9-crylAb-PPEPC-PCPDK- cry1Ab-int#9-T35S), it claimed 2 to 5 copies were present, but no information on flanking sequences was provided.
Other sources report that first transgene insert is present in at least 4 truncated copies, and depending on the source, the number of truncated copies differs. This is an indication of non-uniformity of the transgenic line as well as genetic instability. The second transgene insert is present in at least 5 copies.
There are basic agreements between the two studies on the rampant rearrangements that have occurred. There is also evidence of non-uniformity from the Brussels study.
Mon 810 (Monsanto)
Mon 810, modified for resistance to lepidopteran insects (butterflies & moths), was submitted by Monsanto in 1995 and approved under the old Directive for growing, import, seed production and processing into animal feeding stuffs and industrial purposes since 22 April 1998 [7]. In December 1997 food and food ingredients derived from Mon 810 maize were notified under Article 5 of the Regulation (EC) 238/97 (for novel foods). Several hybrids of Mon810 are still pending approval for marketing:
* T25 x Mon810 submitted under the old directive in April 1999. The Scientific Committee gave a favourable opinion on 6 June 2000
* Mon810 x K603 submitted 15 Jan 2003 under the new Directive 2001/18/EC for import and use in feed and industrial processing.
* Mon863x Mon 810 submitted under the new Directive 7 Feb. 2003 for import and use of grain and grain products. On 29 August 2002, the application was submitted under Regulation (EC) 258/97.
* MaisGard/RR (Mon810and GA21) submitted under the new Directive 13 Feb 2003 for import and use in feed and industrial processing. On 16 March 2000, maize application was submitted under Regulation (EC) 258/97.
French study
Company data showed that the insert has a P35S driving a crylAb synthetic gene with terminator T-nos. Maize heat shock protein intron is located between P35S and crylAb. Analysis revealed however, that T-nos and part of the 3' (tail) end of the crylAb gene have been deleted. T-nos is detected elsewhere in the genome, indicating that it may have moved from its original position.
The 5' (head) end of the insertion site shows homology to the long terminal repeats (LTR) of the maize alpha Zein gene cluster, but no homology to the maize genome was detected at the 3' site, indicating that there had been scrambling of the maize genome at the insertion site. The strong P35S promoter could therefore be driving the transcription of an unknown gene downstream.
Brussels study
Mon 810 was produced by transforming maize genotype HiII with two plasmid vectors, pV-ZMBK07 and pV-ZMGT10. The plasmid pV-ZMVK07 contains the crylAb gene isolated from Bacillus thuringiensis ssp. kurstaki, placed under control of the enhanced CaMV 35S promoter (e35S) and the T-nos. An intron from the maize heat-shock protein (hsp70) is located between the e35S promoter and the crylAb gene. The second plasmid pV-ZMGT10 contains the CP4 EPSPS gene from Agrobacterium strain CP4 and the gox gene cloned from Achromobacter strain LBAA. Both plasmids contain the nptII gene under control of a bacterial promoter. Molecular analysis by Monsanto showed that the nptII gene and the backbone sequences of pV-ZMBK07 are not integrated and that none of the DNA sequences from vector pV-ZMGT10 are present.
According to the company dossier, Mon 810 contains a single copy of the e35S promoter, the hsp70 intron and the crylAb gene. The absence of the 3'T-nos sequence was confirmed by CLO.
CLO determined the 5' junction, upstream from the e35S, and found that the DNA shows 88% identity with the 22kDa alpha Zein gene of maize.
The rearrangement of the insert was confirmed in both studies. A potentially serious discrepancy is that the French study found the insert flanked by the LTR of the Zein gene cluster at its 5'end, and not by the Zein gene, as found in the Brussels study. A minor discrepancy is in the P35S reported in the French study as opposed to e35S in the Brussels study, and the detecting of T-nos elsewhere in the maize genome in the French study.
T25 maize (Bayer)
Liberty-link maize event T25, modified for tolerance to the herbicide glufosinate, was submitted by AgrEvo (Bayer CropScience) in 1995 and approved for marketing since 22 April 1998 [8]. Products derived from T25 have been notified under Article 5 of the Regulation (EC) 258/97 on 21 October 1999.
A hybrid of T25, still pending approval for marketing, T25 x Mon 810, was submitted 29 April under the old Directive, and the Scientific Committee gave a favourable opinion on the dossier 6 June 2000.
French study
Company data showed that the insert includes a truncated ampicillin resistance bla gene in the plasmid vector pUC18, a P35S driving a synthetic pat gene (glufosinate tolerance) terminated by T35S. On analysis, the insert was found to have undergone further rearrangement, so that a second, truncated and rearranged P35S has been joined to the 5' (left, or head) end of the insert, while additional pUC18 sequences were found at the 3' (right, or tail) end.
Edges flanking the insert show homologies (similarities) with Huck retrotransposons (a class of mobile genetic elements) in the maize genome.
Brussels study
T25 was obtained by protoplast transformation of the parental line He/89 using plasmid pUC/Ac containing the pat gene from S. viridochromogenes Tu494 and controlled by P35S and T35S. The plasmid includes the bla gene for ampicillin resistance.
The company dossier claimed there was a single insert, and this was confirmed by CLO's analysis. The pat gene is "surrounded" by sequences from the plasmid vector pUC18. According to the dossier, a 2187 bp pUC fragment is present upstream of P35S. This fragment ends up in the bla gene followed by a 353bp fragment of the P35S, probably resulting from a duplication/recombination event. CLO confirmed these data, except that a shorter, 298 bp P35S promoter fragment was found. According to both the applicant and CLO, a fragment from pUC plasmid was found at the 3' end downstream of 35S terminator; but differences in length were reported.
Aventis submitted data that describe the host flanking sequences of the T25 line. A 151p(5') and a 121 bp (3') fragment show homology (94% identity) to maize alcohol dehydrogenase adh1 gene. This differs from the findings of the French study, which detected flanking sequences homologous with Huck retrotransposons.
Apart from this discrepancy, the nature of the rearrangement in the insert was confirmed in both studies.
GTS 40-3-2 (Monsanto)
This line was modified for tolerance to the herbicide glyphosate (Roundup Ready variety). According to UK's Advisory Committee for Novel Foods and Processes (ACNFP) [9], the Committee considered Monsanto's RR soybeans line 40-3-2 under its "voluntary scheme" in 1994 and gave it clearance for food safety on 20 February 1995. The event has been approved for planting and/or consumption in a number of countries worldwide and products from it consumed for a number of years.
French study
The company's original data showed a single insert with P35S driving a composite gene containing the N-terminal chloroplast transit peptide (CPT4) joined to modified EPSPS gene with T-nos terminator. Analysis provided by the Ministry of Midclass and Agriculture, Belgium, published by Windels et al [10] revealed that a 254bp piece of DNA homologous to the EPSPS gene and 534bp of unknown DNA have been joined to the 3'end of the insert.
It was not possible to identify the insertion site, indicating that substantial genome scrambling or deletion had taken place at the insertion site.
Brussels study
This study merely referred out to the ACNFP website. It appears that Monsanto was allowed to submit new data in 2000, and again in 2002. The first confirming that a 254bp piece of the EPSPS gene has been joined to the 3' end of the insert, the second claiming that "large portions" (29bp + 420bp) of the 543bp of unknown DNA found by Windels et al [9] was identical to soybean genomic DNA from the company's own "proprietary database", that has undergone rearrangement.
While the French study emphasized the rearrangement of the insert, both the UK ACNFP and the Brussel report have accepted Monsanto's new data and not questioned why they should differ so substantially from those presented in the company's original dossier.
Transgenic lines analysed in one study only
GA 21 maize (Monsanto)
French study
The line was modified for tolerance to the herbicide glyphosate (Roundup Ready). Company data indicated that the insert contains multiple copies of the cassette with the rice actin gene promoter (P-ract) driving the composite gene containing the N-terminal chloroplast transit peptide (CPT4) joined to modified EPSPS gene and T-nos. There were three complete cassettes flanked by a cassette with P-ract partially deleted at the 5' end, and one cassette with 3' deletion of EPSPS plus a lone P-ract at the 3'end. Analysis found partial deletion of P-ract and deletion of T-nos in two different cassettes.
The insertion site at the 3'end is flanked by sequences of pol polyprotein gene belonging to a PREM2-retrotransposon.
On 15 September 2003, Monsanto informed the European Commission that it was withdrawing its application for GA21 Roundup Ready maize and GA21 x MON810 MaisGard/Roundup Ready maize, for "commercial reasons".
Bt 11 maize (Syngenta)
Brussels study
This was notified in 1996 and approved under the old Directive for import and processing since 22 April 1998 [11]. The notifications for cultivation submitted in 1996 and 1998 are still pending. On 30 November 2000, the EU Scientific Committee on Plants gave a favourable opinion for cultivation. Up till now, the Commission has not received an updated version of these two notifications according to the requirements of Directive 2001/18/EC. In February 1999, Novartis submitted a new application, which is still pending. On 13 March 2002, the SCP gave a favourable opinion.
Food and food ingredient products derived from Bt11 crossed with the Northup King Company inbred line #2044 maize were notified on 20 Jan. 1998.
The plasmid used for transformation contains a synthetic truncated crylAb, isolated from Bacillus thuringiensis spp. kurstaki HDI, and a synthetic pat gene, isolated from Streptomyces viridochromogenes Tu494. Both coding sequences were placed under the regulation of P35S and the T-nos terminator from Agrobacterium tumefaciens. In addition, the promoter sequences of the pat and cry1Ab gene were combined with respectively intron Int II and Int VI derived from maize alcohol dehydrogenase adh1S gene to enhance expression. The event Bt11 maize was obtained by protoplast transformation with plasmid pZ01502 after digestion with restriction enzyme Not1 to remove the bla gene encoding ampicillin resistance.
The whole sequence of the insert was determined by TEPRAL, Strasbourg, France. The insert consists of a single copy of the vector fragment carrying both the crylAb and pat gene. "It was found that rearrangements have taken place into the insert compared to the original insert and that several parts of the plasmid have been truncated or unexpected inserted, e.g., t35S sequences….The presence of t35S fragments into the insert was confirmed by INRA."
Sequence analysis done by CLO with PCR using P35S specific primer in combination with a 3'T-nos specific primer, proved that the DNA segment present in between the two expression cassettes of the Bt11 insert is similar to the pUC vector backbone sequence.
Zimmermann et al [12] showed that next to the 5' P35S border of the crylAb, a maize 180bp knob-specific repeat sequence is present. In addition, CLO analysed the sequence that is present between the P35S sequence and the maize plant and demonstrated that a 1099 bp segment is present, homologous to the pUC backbone sequence and contains part of the lacZ coding sequence.
The junction regions at the 3' T- nos terminator border were amplified by CLO using a specific anchor primer. A 244 bp junction was amplified that contains 149 bp plant DNA that on BLAST sequence analysis, showed similarity to the maize 180bp knob associated tandem repeat. Independently from CLO, the 3' T-nos border region was also amplified by Ronning et al [13], confirming this finding. The remaining part of the amplified 3'T-nos junction is homologous with the pUC backbone sequences.
These data provide evidence that the Bt11 insert is integrated in the Zea mays 180bp knob associated tandem repeat locus. At the P35S border, an extensive piece 1099 bp of pUC backbone DNA was observed between the plant DNA and the P35S promoter, while at the 3'nos border only a small stretch of pUC backbone DNA is present.
According to TEPRAL, it is not certain if only one copy of the insert is present.
Preliminary data of INRA showed that a set of primers designed on the edge fragment of Bt 176 amplified sequences from both Bt176 and Bt11. These data were obtained on six different Bt11 plant seeds received by Syngenta, suggest contamination of Bt11 by Bt176.
Bt 11 is therefore neither genetically stable nor uniform, and should on no account be approved.
Event Ms8xRf3 canola (Aventis, Bayer)
This 'event' is really a composite of two different transformations, and was first notified in 1996 (C/BE/96/01) from PGS (now Bayer Cropscience) under the old Directive 90/220/EEC for cultivation, import, seed production and processing into animal feed stuffs and industrial purposes [14]. On 24 Jan 2003, the European Commission received an updated version according to the requirements of the new Directive 2001/18/EC. Oil derived from Ms8xRf3 products has been notified under Article 5 of the Regulation (EC 258/97) on 21 October 1999.
It is not clear whether the company's data were provided in the original 1996 dossier or in the updated version submitted 24 January 2003.
Ms8 was produced by Agrobacterium mediated transformation using plasmid pTHW107. This plasmid contains the barnase gene derived from Bacillus amyloliquefaciens and the bar gene derived from Streptomyces hygroscopicus. Barnase under regulation of a tapetum specific promoter PTA29 isolated from Nicotiana tabacum and the T- nos terminator of Agrobacterium tumefaciens. The bar gene is regulated by the PSsuAra promoter isolated from Arabidopsis thaliano and by the 3' end of the T-DNA gene 7 of A. tumefaciens.
The transgenic fertility restorer line Rf3 was obtained using plasmid pTHW118 containing a barstar gene derived from Bacillus amyloliquefaciens under regulation of the PTA29 promoter and the T-nos together with the same bar cassette as described for pTHW107.
According to the company dossier, the Ms8 insertion contains a single T-DNA copy. At the left border (3'end of the T-DNA) a 357 bp host sequence was retrieved. At the right border junction (5' of the TDNA) an 864 bp host sequence was retrieved. PCR amplification from the parental line showed co-linearity with the sequences found on both sides of the T-DNA insert. Molecular analysis done by the CLO confirmed that the adjacent DNA is plant DNA. Search in the database showed that part of the 5' flanking regions has over 82% similarity with Arabidopsis sequences.
Determination of the pre-insertion site was done by the applicant using DNA isolated from wild type oilseed rape. Alignment of wildtype sequence with the Rf3 transgene locus revealed that a fragment of 51 bp is present at the wildtype locus but missing from the transgene locus. At the right border 5 nucleotides (filler-DNA) are inserted. Alignment of the wildtype sequence with the Ms8 transgenic locus revealed that 19bp are missing at the target site. At right border junction 3 nucleotides of unknown origin are inserted.
Both Rf3 line and Ms8 line transgene is integrated in a single genetic locus. But the Rf3 event resulted in the insertion of one-TDNA copy arranged in an inverted repeat structure with a second incomplete T-DNA copy. Event Ms8 contains an intact single T-DNA copy. During insertion, typical rearrangements have occurred at the pre-insertion site. In both lines, the dossier claimed, the inserts are flanked by plant DNA showing high similarity with Arabidopsis DNA.
CLO analysis confirms data in dossier (1996) for the right border (RB) of the Rf3 insert, but no data were available for the truncated left border (LB) and the plant DNA rearrangement. For Ms8, CLO confirms data in dossier.
I cannot ascertain from the report whether rearrangement had occurred in the original inserts in the two events Ms8 and Rf3, as it is unclear if the company's data were provided in the original 1996 dossier or in the updated version submitted 24 January 2003. The characteristic inversions, duplications and deletions, insertions and scrambling of host genome DNA at the sites of insertions are evident.
We have explained why this line is unacceptable in other respects [15] and should not be approved for commercial release. This is a 'terminator' crop, engineered for male sterility, ostensibly to prevent transgene escape, but in reality to protect patented herbicide tolerant trait. It also prevents farmers from saving seeds, compelling them to buy the fertile hybrid every year. In reality, the crop spreads both the male sterility 'suicide' gene barnase in its pollen – which is highly toxic to all cells, mammalian included - as well as the herbicide tolerance trait, with potentially large impacts on agricultural and natural biodiversity including the soil biota. The results of UK government-sponsored Farm Scale Evaluations, recently released, have documented negative impacts on biodiversity from growing this transgenic crop [16].
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Agrobacterium & Morgellons Disease, A GM Connection?
CDC launch investigation on Morgellons' Disease
The Centers for Disease Control (CDC) in the United States announced the launch of an investigation on 'Morgellons Disease' in January 2008 [1], after receiving thousands of complaints from people with this bewildering condition, which it describes as follows [2]: "Persons who suffer from this unexplained skin condition report a range of cutaneous (skin) symptoms including crawling, biting and stinging sensations; granules, threads, fibers, or black speck-like materials on or beneath the skin, and/or skin lesions (e.g., rashes or sores). In addition to skin manifestations, some sufferers also report fatigue, mental confusion, short term memory loss, joint pain, and changes in visions."
Morgellons Disease first became known in 2001, when Mary Leitao created a web site describing the illness in her young son, which she named after a 17th century medical study in France describing similar symptoms [3]. Until then, people with Morgellons Disease have been diagnosed as cases of "delusional parasitosis", in which the symptoms are deemed entirely imaginary, and lesions allegedly due to self-inflicted wounds.
Indeed, the debate over Morgellons Disease has continued in the pages of medical and scientific journals right up to the CDC's announcement [4-7]
Dr. Michele Pearson, principal investigator for the CDC said [1] that the primary goals of the study are "to learn more about who may be affected with this condition, the symptoms they experience and to look for clues about factors that might contribute to the condition," adding that the condition is "complex", and "may be due to multiple factors."
In response to questions from journalists at the CDC press conference, Pearson said:
" We are aware that many patients have suffered from this condition. And, I can tell you that here at CDC, we have really been seeing an increasing number of these reports over the past year or so."
CDC's investigation is to be carried out in conjunction with Kaiser Permanente's Northern California Division of Research and the US Armed Forces Institute of Pathology.
Dr. Joe Selby, Director of the Kaiser Permanente's Northern California Division of Research, said the study would proceed in three stages. In the first stage, they will identify all members who may have seen a Kaiser Permanente physician with symptoms suggestive of this condition at any point during the 18 months between July 1 2006 and December 31, 2007, and determine whether they meet eligibility criteria for the study. In stage two, all eligible members will be invited to complete a comprehensive web based or telephone survey conducted by the CDC that examines the duration and severity of a variety of symptoms. And in stage three, those with active symptoms will be invited to the division of research for an extensive clinical examination including collection of skin biopsies, blood and urine samples.
In a paper [6] published in 2006, researchers from the Morgellons Research Foundation [3] identified the states of California, Texas and Florida as having the highest number of cases of Morgellons disease in the United States. Primary clusters were noted in Los Angeles and San Francisco (California) and Houston, Dallas and Austin (Texas). California accounted for 26 percent of cases in the US, but all 50 US states and 15 other nations, including Canada, the UK, Australia, and the Netherlands, have reported cases of Morgellons disease. The two main occupational groups reporting symptoms are nurses and teachers, with nurses outnumbering teachers three to one. The risk factor common to both groups is suspected to be the possibility of transmitted infectious agents.
Skin lesions and fibres may not be readily apparent in all individuals with the disease, as family members of patients often report similar systemic disease symptoms without skin symptoms. Families in which all members are affected often have suspected simultaneous exposure to an inciting agent. Contact with soil or waste products appears to be associated with the disease. Cases have been reported in cats and dogs, as well as horses.
What finally prompted CDC to investigate the disease? The Morgellons Research Foundation [3] was set up in 2002 in honour of Mary Leitao, the Foundation's executive director. It publicises the plight of patients with similar conditions and operates a registry of afflicted families. The Foundation also funds scientific research. It has a Medical Advisory Board of seven with M.D. degree and two with nursing degrees. In addition, it has a Board of Nursing with five other nurses, and a Scientific Advisory Board of six scientists, all with Ph.D. degree; one of which is Vitaly Citovsky. It may have been Citovsky's discovery last year that finally persuaded the CDC to announce an investigation.
The Agrobacterium connection
Vitaly Citovsky is a professor of molecular and cell biology at Stony Brook University in New York (SUNY). He is a world authority on the genetic modification of cells by Agrobacterium, a soil bacterium causing crown gall disease in plants, that has been widely used in creating genetically modified (GM) plants since the 1980s because of its ability to transfer a piece of its genetic material, the T-DNA on its tumour-inducing (Ti) plasmid to the plant genome (see later for details).
Citovsky's team took scanning electron microscope pictures of the fibres in or extruding from the skin of patients suffering from Morgellons disease, confirming that they are unlike any ordinary natural or synthetic fibres (see Fig. 1, assembled from Citovsky's website [8]).
Figure 1. Scanning electron microscope images of fibres from skin biopsies of patients with Morgellons Disease - a, white fibre with calcite, scale bar 10 mm; b, green fibre with alumina 'rock' protruding, scale bar 20 mm; c, various ribbon-like, cylindrical and faceted fibres all coated with minerals, scale bar 10 mm; d, skin lesion with fibres stabbing through the epidermis, scale bar 300 mm
They also analysed patients for Agrobacterium DNA. Skin biopsy samples from Morgellons patients were subjected to high-stringency polymerase chain reaction (PCR) tests for genes encoded by the Agrobacterium chromosome and also for Agrobacterium virulence (vir) genes and T-DNA on its Ti plasmid. They found that "all Morgellons patients screened to date have tested positive for the presence of Agrobacterium, whereas this microorganism has not been detected in any of the samples derived from the control, healthy individuals." Their preliminary conclusion is that "Agrobacterium may be involved in the etiology and/or progression" of Morgellons Disease.
The unpublished findings have been posted on a website [8] since January 2007. They were further publicized in the "first ever" Morgellons conference in Austin Texas, attended by 100 in March 2008 [9]. A growing list of people are registered with Morgellons Disease, totalling 12 106 worldwide recorded by Morgellons Research Foundation [3], as of 12 April 2008.
San Francisco physician, Raphael Stricker, one of only a few doctors who believe the disease is real, said [9]. "There's almost always some history of exposure to dirt basically either from gardening or camping or something." He is one of the co-authors on the Agrobacterium research done in SUNY, which reported finding Agrobacterium DNA in all 5 Morgellons patients studied. Stricker suggests it is transmitted by ticks, like Lyme disease, and in a recent survey of 44 Morgellons patients in San Francisco, 43 of them also tested positive for the bacterium causing Lyme disease. Another factor consistent with Agrobacterium being a causative agent, if not the causative agent, is that when patients are treated with antibacterials for their Lyme disease, remission of Morgellons symptoms is seen in most of them [6].
Stricker also told his audience that Agrobacterium lives in the soil, and is known to cause infections in animals and human beings with compromised immune systems. It can cause skin lesions when injected into Swiss mice, a strain that is immune deficient, he said.
At this point, the findings on the Agrobacterium connection are still preliminary, as only seven patients have been studied. Nevertheless, the implications are far-reaching if this connection is confirmed, as existing evidence (reviewed below) suggests a link between Agrobacterium and genetic engineering in the creation of new disease agents, and it is paramount for the CDC investigation to include this aspect, if only to rule it out.
Agrobacterium and the genetic engineering connection
Agrobacterium not only infects human and other animal cells, it also transfers genes into them. It was SUNY professor Citovsky and his team that made the discovery some years ago [10]. Until then, the genetic engineering community had assumed that Agrobacterium did not infect animal cells, and certainly would not transfer genes into them.
Agrobacterium was found to transfer T-DNA into the chromosomes of human cells.
In stably transformed HeLa cells, the integration occurred at the right border of the T-DNA, exactly as would happen when it is being transferred into a plant cell genome, suggesting that Agrobacterium transforms human cells by a mechanism similar to that involved in transforming plants cells (see Box 1). Human cancer cells, neurons and kidney cells were all transformed with the Agrobacterium T-DNA. Commenting on this research in 2001, Joe Cummins had warned of hazards to laboratory and farm workers [11] (i-sis news11/12)
The Agrobacterium vector system for gene transfer
Since the discovery in the 1970s that Agrobacterium can transfer genes into plants causing crown gall disease, the soil bacterium has been developed into a vector for inserting desirable genes into the plant genome to create transgenic (GM) plants [12].
Agrobacterium transfers T-DNA – a small region of approximately 5 to 10 percent of a resident tumour-inducing (Ti) or root-inducing (Ri) plasmid – into numerous species of plants; and as later turns out, also to fungi, algae, and even animal and human cells [13, 14] (see main text).
Transfer requires three major elements [13]: T-DNA border direct repeat sequences of 25 base pairs that flank the T-DNA and delineate the region transferred into the host, the virulence (vir) genes located on the Ti/Ri plasmid, and various genes on the bacterial chromosome. Plant genes are also involved in the successful integration of T-DNA [15]. The T-DNA contains oncogenes (cancer genes or gene for forming tumours) and genes for synthesizing opines; none of which is essential for T-DNA transfer, so they can be deleted and replaced with genes of interest and selectable markers.
Furthermore, the vir genes and T-DNA region need not be on the same replicating plasmid. This gave rise to the binary vector systems in which T-DNA and the vir genes are located on separate replicating units. The T-DNA containing unit is the binary vector and contains also the origin(s) of replication that could function both in E. coli and Agrobacterium tumefaciens, and antibiotic resistance marker genes used to select for the presence of the binary vector in bacteria. The replicating unit containing the vir genes is the 'helper' plasmid. Strains of Agrobacterium harbouring the two separate units are considered 'disarmed' if they do not contain oncogenes that could be transferred to a plant.
The association of Morgellons Disease with dirt and soil where Agrobacterium lives, the widespread use of Agrobacterium in genetic engineering of plants, and the ability of Agrobacterium to infect human cells, all point towards a possible role of genetic engineering in the aetiology of Morgellans disease via Agrobacterium.
Extensive genetic manipulation of Agrobacterium does have the potential to transform it into an aggressive human pathogen. Genetic engineering is nothing if not enhanced and facilitated horizontal gene transfer and recombination, which is widely acknowledged to be the main route for creating new pathogens. Mae-Wan Ho was among an international panel of scientists have raised this very issue in 1998, calling for a public enquiry into the possible contributions of genetic engineering biotechnology to the aetiology of infectious diseases which has greatly increased since genetic engineering began in the 1970s [16].
The epidemiological data of Morgellons Disease are very incomplete, and the Morgellons Research Foundation's registry of more than 12 000 families afflicted worldwide is almost certainly only a fraction of the emerging epidemic. Still, it is significant that the majority of the cases are in the United States, the first country to release GM crops and remaining the top producer ever since.
There are other findings implicating Agrobacterium in transgenic plants released into the environment, particularly during the early years of field trials, when knowledge was poor and safety measures not as stringent as they may be today.
Agrobacterium persists in transgenic plants and is a vehicle for gene escape
By the late 1990s, the Agrobacterium vector system became very widely used, and many GM crops created were commercially released.
Scientists at the Kinsealy Research and Development Centre in Dublin, Ireland, and the Scottish Crop Research Institute in Dundee, Scotland, were concerned that the inserted genes in plants would spread to wild populations by cross-pollination or by horizontal gene transfer to unrelated species, which was by then well-documented in the scientific literature.
They considered it "imperative" to address the risk posed in using Agrobacterium as a tool in genetic engineering [17], given its ability to transfer genes to plants. The transformation procedure involves inoculating the cells or tissue explants with Agrobacterium and co-cultivation the plant cells and bacterium for a short period, followed by the elimination of the bacterium with antibiotics.
However, if all the bacteria were not eliminated, then "release of these plants may also result in release of the Agrobacterium [with the foreign genes]", which will serve as a vehicle for further gene escape, at least to other Agrobacterium strains naturally present in the soil.
Although various antibiotics have been used to eliminate Agrobacterium following transformation, the researchers stated that "very few authors actually test to ensure that the antibiotics succeed."
The difficulty is compounded because the bacterium can remain latent within the plant tissue. So putting transgenic plant material into culture medium without antibiotics and finding no Agrobacterium is no guarantee that the transgenic plant is free of the bacterium, as was often assumed.
In their study, they investigated the ability of antibiotics to eliminate Agrobacterium tumefaciens after transformation in three model systems: Brassica (mustard), Solanum (potato), and Rubus (raspberry). The antibiotics carbenicillin, cefataxime and ticaracillin were used respectively to eliminate the bacterium at four times the minimum bactericidal concentration, as recommended. They found that none of the antibiotic succeeded in eliminating Agrobacterium.
The contamination levels increased from 12 to 16 weeks to such an extent that transgenic Solanum cultures senesced and died. Contamination in shoot material decreased over 16 to 24 weeks possibly because only the apical node was used in further culture, but even that did not eliminate Agrobacterium from all the samples; 24 percent remained contaminated at 24 weeks.
The binary vector was also present under non-selective conditions up to 6 months after transformation, where approximately 50 percent of contaminated material still harboured bacterial cells with the binary vector at high levels of about 107 colony forming units per gram. The researchers pointed out: "Here is where the possibility of gene escape arises. The presence of the disarmed Agrobacterium in the tissue would not be a problem if the binary vector had been lost, but now its survival and spread are real possibilities." The binary vector contains the foreign genes as well as antibiotic resistance marker gene(s).
There is no limit to the foreign genes that can be inserted into the binary vector. A few years earlier, a research group in Israel had inserted a viroid that causes disease in citrus fruits into the disarmed Ti plasmid of Agrobacterium and used that to infect and transform several plant species including tomato (Lycopersicon esculentum) Gynura aurantiaca, avocado (Persea americana), and grapefruit (Citrus paradisi) grafted on Troyer citrange (Pancirus trifoliate x C. sinensis) [18]. Extracts prepared from tissues of the infected plants 38-90 days after inoculation were plated on selective media and found to contain large amounts of the engineered bacteria.
The researchers warned of "newly formed combinations of persistently transmitted viruses" coupled with "the opportunistic and systemically moving Agrobacterium vector infectious to a wide host range might eventually cause infection and damage to crop plants or natural vegetation" that are "not presently visited by the traditional vectors of the virus disease."
In other words, Agrobacterium persisting in transgenic plants released into the environment has the potential to spread new diseases, and to plants that normally would not be infected by the disease agents. At the time, the researchers did not know that Agrobacterium would also infect animals and humans, and could spread new diseases to them as well.
Have these warnings been heeded by other researchers? There is no evidence they have been taken on board. Agrobacterium has since been shown to transform at least 80 different non-plant species including yeasts and other fungi, algae, mammalian and human cells, also the gram positive bacterium Streptomyces lividans. In a recent review, the researchers stated [14]: "Future research has to show whether Agrobacterium-mediated transformation contributed to horizontal gene transfer between microorganisms in the rhizosphere."
But there is already evidence suggesting that Agrobacterium can indeed engage in horizontal gene transfer with a wide range of bacteria in the soil. (For more on horizontal gene transfer see [19] Horizontal Gene Transfer from GMOs Does Happen, SiS 38)
Agrobacterium gene transfer mechanisms similar to conjugation in bacteria
Ho first alerted regulators to the potential of Agrobacterium contaminating GM plants to facilitate the escape of transgenes in 2003 (see Living with the Fluid Genome [20] and The Case for A GM-Free Sustainable World [21] ISIS publications). By then, Gayle Ferguson and Jack Heinemann at the University of Canterbury, Christchurh, New Zealand, had already pointed out in a review that the process whereby Agrobacterium injects T-DNA into plant cells strongly resembles conjugation, the normal mating process between bacteria [22].
Conjugation, mediated by certain bacterial plasmids, depends on a sequence called the origin of transfer (oriT) on the DNA transferred. All other functions - called tra for trans-acting functions - can be supplied from unlinked sources. Thus, 'disabled' plasmids with no trans-acting functions, can nevertheless be transferred by helper plasmids, the same as the binary vector system of Agrobacterium (Box 1). The resemblance does not stop there.
The left and right borders of T-DNA are similar to oriT and can be replaced by it. Furthermore, the disarmed T-DNA binay vector, lacking oncogenes as well as virulence genes, can be helped by similar genes belonging to many other pathogenic bacteria. The trans-kingdom gene transfer apparatus of Agrobacterium and the conjugative systems of bacteria are both involved in transporting macromolecules, not just DNA but also protein.
Thus, transgenic plants with contaminating Agrobacterium [20] "have a ready route for horizontal gene escape, via Agrobacterium, helped by the ordinary conjugative mechanisms of many other bacteria that cause diseases, which are present in the environment." In the process, new and exotic disease agents could be created.
Investigations on the role of Agrobacterium in Morgellons Disease urgently needed
The investigation launched by the CDC needs to clarify the role of Agrobacterium in the aetiology of Morgellons Disease as a matter of urgency. This should include:
* Molecular characterization of Agrobacterium DNA sequences in Morgellans Disease patients
* Design of suitable probes for diagnostic purposes and for monitoring soil samples and other suspected sources of infection
* Introduction of stringent tests for Agrobacterium contamination for all transgenic plants already released or about to be released into the environment.
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DNA in GM Food & Feed
Is GM DNA different from natural DNA?
"DNA is DNA is DNA," said a proponent in a public debate in trying to convince the audience that there is no difference between genetically modified (GM) DNA and natural DNA, "DNA is taken up by cells because it is very nutritious!"
"GM can happen in nature," said another proponent. "Mother Nature got there first."
So, why worry about GM contamination? Why bother setting contamination thresholds for food and feed? Why award patents for the GM DNA on grounds that it is an innovation? Why don't biotech companies accept liabilities if there's nothing to worry about?
As for GM happening in nature, so does death, but that doesn't justify murder. Radioactive decay happens in nature too, but concentrated and speeded up, it becomes an atom bomb.
GMDNA and natural DNA are indistinguishable according to the most mundane chemistry, i.e., they have the same chemical formula or atomic composition. Apart from that, they are as different as night and day. Natural DNA is made in living organisms; GMDNA is made in the laboratory. Natural DNA has the signature of the species to which it belongs; GMDNA contains bits copied from the DNA of a wide variety of organisms, or simply synthesized in the laboratory. Natural DNA has billions of years of evolution behind it; GMDNA contains genetic material and combinations of genetic material that have never existed.
Furthermore, GMDNA is designed – albeit crudely - to cross species barriers and to jump into genomes. Design features include changes in the genetic code and special ends that enhance recombination, i.e., breaking into genomes and rejoining. GMDNA often contains antibiotic resistance marker genes needed in the process of making GM organisms, but serves no useful function in the GM organism.
The GM process clearly isn't what nature does (see "Puncturing the GM myths", SiS22). It bypasses reproduction, short circuits and greatly accelerates evolution. Natural evolution created new combinations of genetic material at a predominantly slow and steady pace over billions of years. There is a natural limit, not only to the rate but also to the scope of gene shuffling in evolution. That's because each species comes onto the evolutionary stage in its own space and time, and only those species that overlap in space and time could ever exchange genes at all in nature. With GM, however, there's no limit whatsoever: even DNA from organisms buried and extinct for hundreds of thousands of years could be dug up, copied and recombined with DNA from organisms that exist today.
GM greatly increases the scope and speed of horizontal gene transfer
Horizontal gene transfer happens when foreign genetic material jumps into genomes, creating new combinations (recombination) of genes, or new genomes. Horizontal gene transfer and recombination go hand in hand. In nature, that's how, once in a while, new viruses and bacteria that cause disease epidemics are generated, and how antibiotic and drug resistance spread to the disease agents, making infections much more difficult to treat.
Genetic modification is essentially horizontal gene transfer and recombination, speeded up enormously, and totally unlimited in the source of genetic material recombined to make the GMDNA that's inserted into the genomes plants, animals and livestock to create genetically modified organisms (GMOs).
By enhancing both the rate and scope of horizontal gene transfer and recombination, GM has also increased the chance of generating new disease-causing viruses and bacteria. (It is like increasing the odds of getting the right combination of numbers to win a lottery by betting on many different combinations at the same time.) That's not all. Studies on the GM process have shown that the foreign gene inserts invariably damages the genome, scrambling and rearranging DNA sequences, resulting in inappropriate gene expression that can trigger cancer.
The problem with the GM inserts is that they could transfer again into other genomes with all the attendant risks mentioned. There are reasons to believe GM inserts are more likely to undergo horizontal transfer and recombination than natural DNA, chief among which is that the GM inserts (and the GM varieties resulting from them) are structurally unstable, and often contain recombination hotspots (such as the borders of the inserts).
After years of denial, some European countries began to carry out 'event-specific' molecular analyses of the GM inserts in commercially approved GM varieties as required by the new European directives for deliberate release, novel foods and traceability and labelling. These analyses reveal that practically all the GM inserts have fragmented and rearranged since characterised by the company. This makes all the GM varieties already commercialised illegal under the new regime, and also invalidates any safety assessment that has been done on them (see "Transgenic lines proven unstable", SiS 20 and "Unstable transgenic lines illegal", SiS 21). As everyone knows, the properties of the GM variety, and hence its identity, depend absolutely on the precise form and position of the GM insert(s). There is no sense in which a GM variety is "substantially equivalent" to non-GM varieties.
GMDNA in food & feed
In view of the strict environmental safety assessment required for growing GM crops in Europe, biotech companies are bypassing that by applying to import GM produce for food and processing only. Is GM food safe? There are both scientific and anecdotal evidence indicating it may not be: many species of animals were adversely affected after being fed different species of GM plants with a variety of GM inserts (see "GM food safe?" series, SiS 21), suggesting that the common hazard may reside in the GM process itself, or the GMDNA.
How reliably can GMDNA be detected?
DNA can readily be isolated and quantified in bulk. But the method routinely used for detecting small or trace amounts of GMDNA is the polymerase chain reaction (PCR). This copies and amplifies a specific DNA sequence based on short 'primers strings' of DNA that match the two ends of the sequence to be amplified, and can therefore bind to the ends to 'prime' the replication of the sequence through typically 30 or more cycles, until it can be identified after staining with a fluorescent dye.
There are many technical difficulties associated with PCR amplification. Because of the small amount of the sample routinely used for analysis, it may not be representative of the sample, especially if the sample is inhomogeneous, such as the intestinal contents of a large animal. The primers may fail to hybridise to the correct sequence; the PCR itself may fail because inhibitors are present. Usually, the sequence amplified is a small fraction of the length of the entire GM insert, and will therefore not detect any other GM fragment present. If the target sequence itself is fragmented or rearranged, the PCR will also fail. For all those reasons, PCR will almost always underestimate the amount of GMDNA present, and a negative finding cannot be taken as evidence that GMDNA is absent.
A new review on monitoring GM food casts considerable doubt over the reliability of PCR methods. Mistakes can arise if the sample is not large enough to give a reliable measure, or if the batch of grain sampled is inhomogeneous, or the PCR reaction not sensitive enough, or the data presented to the regulatory authorities simply not good enough. Consequently, the level of contamination is almost invariably underestimated.
There is an urgent need to develop sensitive, standardized and validated quantitative PCR techniques to study the fate of GMDNA in food and feed. Regulatory authorities in Europe are already developing such techniques for determining GM contamination. One such technique has brought the limit of detection down to 10 copies of the transgene (the GM insert or a specific fragment of it).
In contrast, the limit of PCR detection in investigations on the fate of GMDNA in food and feed is extremely variable. In one study commissioned by the UK Food Standards Agency, the limit of detection varied over a thousand fold between samples, with some samples requiring more than 40 000 copies of the GM insert before a positive signal is registered. Such studies are highly misleading if taken at face value, given all the other limitations of the PCR technique.
Despite that, however, we already have answers to a number of key questions regarding the fate of DNA in food and feed.
1. Does DNA break down sufficiently during food processing?
The answer is no, not for most commercial processing. DNA was found to survive intact through grinding, milling or dry heating, and incompletely degraded in silage. High temperatures (above 95 deg. C) or steam under pressure were required to degrade the DNA completely.
"The results imply that stringent conditions are needed in the processing of GM plant tissues for feedstuffs to eliminate the possibility of transmission of transgenes." The researchers warned.
They pointed out for example, that the gene aad, conferring resistance to the antibiotics streptomycin and spectinomycin, is present in GM cottonseed approved for growth in US and elsewhere (Monsanto's Bollgard (insect-protected) and Roundup Ready (herbicide tolerant)). Streptomycin is mainly used as a second-line drug for tuberculosis. But it is in the treatment of gonorrhoea that spectinomycin is most important. It is the drug of choice for treating strains of Neisseria gonorrhoeae already resistant to penicillin and third generation cephalosporins, especially during pregnancy. The release of GM crops with the blaTEM gene for ampicillin resistance is also relevant here, because that's where resistance to cephalosporins has evolved.
Another study found large DNA fragments in raw soymilk of about 2 000bp (base pairs, unit of measurement for the length of DNA), which degraded somewhat after boiling, but large fragments were still present in tofu and highly processed soy protein. Heating in water under acid conditions was more effective in degrading DNA, but again, the breakdown was incomplete (fragments larger than 900bp remaining).
It is generally assumed, incorrectly, that DNA fragments less than 200bp pose no risk, because they are well below the size of genes. But that's a mistake, as these fragments may be promoters (signals needed by genes to become expressed), and sequences of less than 10bp can be binding sites for proteins that boost transcription. The CaMV 35S promoter, for example, is known to contain a recombination hotspot, and is implicated in the instability of GM inserts.
2. Does DNA break down sufficiently rapidly in the gastrointestinal tract?
Although free DNA breaks down rapidly in the mouth of sheep and humans, it was not sufficiently rapid to prevent gene-transfer to bacteria inhabiting the mouth. DNA in GM food and feed will survive far longer. The researchers conclude: "DNA released from feed material within the mouth has potential to transform naturally competent oral bacteria."
Several studies have now documented the survival of DNA in food throughout the gastrointestinal tract in pig and mice, in the rumen of sheep and in the rumen and duodenum of cattle. The studies were variable in quality, depending especially on the sensitivity of the PCR methodology used to amplify specific sequences for detection. Nevertheless they suggest that GMDNA can transfer to bacteria within the rumen and in the small intestine. In neither sheep nor cattle was feed DNA detected in the faeces, suggesting that DNA breakdown may be complete by then.
The only feeding trial in human volunteers was perhaps the most informative. After a single meal containing GM soya containing some 3x1012 copies of the soya genome, the complete 2 266 bp epsps transgene was recovered from the colostomy bag in six out of seven ileostomy subjects (who had their lower bowel surgically removed). The levels were highly variable among individuals as quantified by a small 180bp PCR product overlapping the end of cauliflower mosaic virus (CaMV) 35S promoter and the beginning of the gene: ranging from 1011 copies (3.7%) in one subject to only 105 copies in another. This is a strong indication that DNA in food is not sufficiently rapidly broken down in transit through the gastrointestinal tract, confirming the results of an earlier experiment by the same research group.
No GMDNA was found in the faeces of any of 12 healthy volunteers tested, suggesting that DNA has completely broken down, or all detectable fragments have passed into the bloodstream (see later) by the time food has passed through the body. This finding is in agreement with the results from ruminants.
In general, the studies report that GMDNA degrades to about the same extent and at about the same rate as natural plant DNA. However, no quantitative measurements have been made, and GMDNA was often compared with the much more abundant chloroplast DNA, which outnumbers the transgene by 10 000 to one.
3. Does GMDNA get taken up by bacteria and other micro-organisms?
The answer is yes. The evidence was reported in the human feeding trial mentioned. The transgene was not detected in the content of the colostomy bag from any subject before the GM meal. But after culturing the bacteria, low levels were detected in three subjects out of seven: calculated to be between 1 and 3 copies of the transgene per million bacteria.
According to the researchers, the three subjects already had the transgene transferred from GM soya before the feeding trial, probably by having eaten GM soya products unknowingly. No further transfer of GM DNA was detected from the single meal taken in the trial.
The researchers were unable to isolate the specific strain(s) of bacteria that had taken up the transgene, which was not surprising, as "molecular evidence indicates that 90% of microorganisms in the intestinal microflora remain uncultured. …they can only grow in mixed culture, a phenomenon seen with other microorganisms."
Actually, GMDNA can already transfer to bacteria during food processing and storage. A plasmid was able to transform Escherichia coli in all 12 foods tested under conditions commonly found in processing and storage, with frequencies depending on the food and on temperature. Surprisingly, E. coli became transformed at temperatures below 5 degrees C, i.e. under conditions of storage of perishable foods. In soy drink this condition resulted in frequencies higher than those at 37 degrees C.
4. Do cells lining the gastrointestinal tract take up DNA?
The answer is yes. Food material can reach lymphocytes (certain white blood cells) entering the intestinal wall directly, through Peyer's patches. And fragments of plant DNA were indeed detected in cows' peripheral blood lymphocytes.
It is notable that in the human feeding trial, a human colon carcinoma cell line CaCo2 was directly transformed at a high frequency of 1 in 3 000 cells by an antibiotic resistance marker gene in a plasmid. This shows how readily mammalian cells can take up foreign DNA, as we have pointed out some years ago (see also below).
5. Does DNA pass through the gastrointestinal tract into the blood stream?
The answer is yes, as mentioned above, fragments of plant DNA was detected in cow's peripheral blood lymphocytes. However, attempts to amplify plant DNA fragments from blood have failed, most likely on account of the presence of inhibitors of the PCR amplification.
6. Does DNA get taken up by tissue cells?
The answer is yes, and this has been known since the mid 1990s. GMDNA and viral DNA fed to mice ended up in cells of several tissues, and when fed to pregnant mice, the DNA was able to cross the placenta, and enter the cells of the foetus and the newborn. These results were confirmed in 2001, when soya DNA, too, was found taken into the tissue cells of a few animals.
In general, abundant chloroplast sequences have been detected in the tissues of pig and chicken but not single gene DNA nor GMDNA. But rare events are most likely to go undetected, on account of the limitations of the PCR technique.
Recently, "spontaneous transgenesis" – the process of spontaneous uptake of foreign DNA resulting in gene expression - has been rediscovered by a team of researchers looking for new possibilities in gene therapy. They documented the phenomenon in several human B lymphocyte cell lines as well as peripheral blood B lymphocytes. The transgene in a plasmid was readily taken up and was found in many cell compartments including the nucleus, where gene transcription took place. The plasmid was not integrated into the genome, but the researchers say that its eventual integration cannot be ruled out.
7. Is GM DNA more likely to insert into genomes?
This is perhaps the most important question. There are reasons to believe GMDNA is more likely to insert into genomes after it is taken up into cells, chief among which, its sequence similarities (homologies) to a wide variety of genomes, especially those of viruses and bacteria. Such homologies are known to enhance horizontal gene transfer to bacteria up to a billion fold.
More significantly, the integration of non-homologous genetic material can occur at high frequencies when flanked by homologous sequences. A recent report highlights the importance of this "homology-facilitated illegitimate recombination", which increases the integration of foreign (non-homologou) DNA at least 105 fold when it was flanked on one side by a piece of DNA homologous to the recipient genome.
No experiment has yet been done to assess whether GMDNA is more likely to transfer horizontally than natural DNA. However, in the human feeding trial, where three ileostomy volunteers tested positive for the soya transgene in the bacteria cultured from their colostomy bag, the soya lectin gene Le was not detected in the bacterial cultures from any of the subjects.
The researchers found it necessary to remark, "Although the plant lectin gene was not detected in the microbial population…it is premature to conclude that the epsps transgene is more likely than endogenous plant genes to transfer into the microbial population."
But until this possibility has been adequately addressed, it cannot be ruled out.
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Superbug with Anthrax Genes
The Bacillus species causing anthrax and food poisoning are closely related to each other and to a third, Bacillus thuringiensis, whose toxin genes are extensively exploited to create genetically modified Bt-crops. ISIS has warned of the potential for dangerous recombinants to emerge; such a recombinant has now been identified. Dr. Mae-Wan Ho and Prof. Joe Cummins caution against growing Bt crops, especially in the Third World.
The three Bacillus bacteria all live in the soil and are so closely related that they may as well be regarded as a single species. B. anthracis, causes anthrax, B. cereus is linked to food poisoning, and B. thuriengiensis is extensively exploited as biopesticides in genetically engineered Bt crops, now widely cultivated in the United States, and increasingly being promoted in Third World countries. The three bacteria readily mate with one another and exchange plasmids (circular pieces of DNA) carrying specific toxin and virulence genes. They share very similar viruses (phages) that can integrate into the bacterial genome as 'prophage', and can hence also move toxin and virulence genes among them, many of them reside in the bacterial chromosome. Cummins has warned that dangerous recombinants could arise, from gene exchange between the bacteria and between the Bt plant debris and bacteria in the soil.
Now, an international team of infectious disease researchers led by Claire M. Fraser of the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA, have identified a recombinant between B. anthracis and B. cereus. They were alerted last year when two hospital patients in Texas died of severe pneumonia that appeared to be caused by inhalation anthrax, but neither patient was infected with B. anthracis. Instead, DNA tests showed that both patients were infected by a strain of B. cereus that normally causes mild food poisoning, which has somehow acquired the lethal anthrax genes.
When the Texas cases came to light, the CDC scientists were sequencing a strain of B. cereus isolated from a man in Louisiana who, in 1994, showed up with severe anthrax-like symptoms. The Texas and Lousiana patients were all metal workers who seemed to have inhaled the bacteria.
Anthrax is an acute fatal disease among mammals and B. anthracis became widely known as a biological weapon soon after September 11, 2001. It has two plasmids: pXO1 carrying the lethal toxin complex (edema factor, lethal factor and protective antigen), and pXO2 carrying the glutamic acid polymer that inhibits white blood cells from engulfing and digesting the bacterium. Until a few years ago, B. anthracis was thought to be distinct from B. cereus, because they look different and causes different diseases.
The researchers sequenced the B. cereus genome using draft genome sequences obtained and assembled by the company Celera, and the resulting sequence annotated through The Institute for Genomic Research (TIGR) Bioinformatics pipeline, set up by Craig Venter, the maverick scientist who founded Celera to sequence the human genome, succeeded only too well, and was sacked from the company in January 2002, after he remarked on there being too few genes to support the simplistic idea that organisms are hardwired in their genes.
It turns out that the culprit strain of B. cereus G9241 had acquired a plasmid very similar to the pXO1 of B. anthracis. In addition, analysis of seven other metabolic genes showed that the strain is closely related to, albeit distinct from B. anthracis.
The sequence of B. cereus G9241 genome reveals a mosaic structure, which could be due to the presence of a great number of what appears to be known and novel mobile genetic elements that can insert sequences from other sources. It also has a 119 110bp circular plasmid with high similarity to B. anthracis pXO1. There is, further, a cryptic phage of 29 886bp that encodes phage-like proteins and a plasmid replicon (replicating unit) similar to B. anthracis plasmid pXO2. It also carries genes that, if functional, should provide the strain with resistance to b-lactam, chloramphenicol and macrolide antimicrobial agents.
When injected into mice, B. cereus G9241 proved to be 100% lethal, as was B. anthracis, but it killed the mice almost twice as fast. All the mice injected with an ordinary B. cereus strain survived the experiment.
As a result of these findings, the researchers concluded that, "it may not be appropriate to consider B. anthracis, as currently defined, as the only species capable of causing inhalation anthrax-like disease."
Another noteworthy feature is that at least two isolates of B. cereus (ATCC 14579) and M 1550) are extremely closely related to and cluster with B. thuringiensis. A number of delta endotoxins from B. thuringiensis strains are implicated in allergies and other illnesses, or known to be immunogenic. What sort of disease agent might emerge from B. cereus if it acquired endotoxin genes either from B. thuringiensis or from Bt crop debris in the soil? This question is especially pertinent in view of the substantial changes in the genetically modified Bt genes that are completely untested and hence unknown in toxicities.
Countries, especially those in the Third World, where farmers live next to their fields, should be particularly wary about growing Bt crops.
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The soil bacterium, Bacillus thuringiensis (Bt), has proven to be a rich source of toxins for killing insect pests. Most of the toxin genes now being used in genetically modified (GM) crops are produced in sporulating Bt, and belong to the Cry family: designated Cry1, Cry 2 etc. up to at least Cry 41. The Cry genes are further distinguished as Cry1A, Cry1B etc for substantial sequence variations, and labeled Cry1Aa, Cry1Ab etc for very small differences in sequence. The Cry gene toxins target specific insect cell receptor proteins and create pores that lead to osmotic lysis of the insect gut cells. Only a few Cry genes have found favour in GM crops. Along with the Cry genes, Cyt genes have been characterized that are distinct from Cry genes and act by breaking open the insect's blood cells.
In recent years, vegetative insecticidal proteins (VIP) have been found to have potent, broad-spectrum activity against insects. VIP genes are not homologous to Cry and Cyt genes, and bind to cell membrane proteins different from the other toxins.
Syngenta Corporation, producers of chemical and biological pesticides, has patented the VIP genes for use in transgenic crop plants and microbes. Syngenta's United States patent 6 429 360 covers the use of Bt-VIP genes and their synthesis and alteration to improve performance in crop plants. Syngenta's patent provided evidence that VIP3A toxin produced apoptotic type of cell death, including the production of membrane-bound apoptotic bodies and activation of endonuclease enzymes that cleave chromatin into discrete fragments.
Apoptosis (meaning petals falling from a flower) is a form of programmed cell death common to all cells with discrete nuclei. It is a part of normal development, but the VIP3A toxin uses programmed cell death to destroy the cells of the insect gut. In order to function fully in the plant cells, the Bt-VIP3A gene is modified in its coding sequence; a strong promoter added, as well as an intron to facilitate transfer of the pre-messenger RNA from nucleus to cytoplasm; and the usual transcription terminator and polyA addition sequences.
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The insect VIP3A receptor was identified and its characteristic "death" recognition sequence was characterized. Organisms whose cells have nuclei generally have receptors with death signals and the insect VIP3A receptor is a unique member of the class of sequences.
Syngenta has petitioned the United States Environmental protection Agency (EPA) for commercial release of event COT102 cotton containing a synthetic VIPA3 gene. Presumably, corn containing the VIP3A gene will be proposed for commercial release. The EPA report of the Syngenta petition for tolerance in or near food reported that the VIPA3 toxin was homologous to the VIP3A toxin in numerous Bt strains. However, the petition failed to mention the numerous change in DNA sequence including promoter, introns, terminator and polyA signal, which were reported in the Syngenta patent for VIP genes. Mammalian acute toxicity studies were done using the VIPA3 toxin produced in bacteria, not the toxin produced in modified corn or cotton. The VIPA3 toxin in cotton is assumed to be substantially equivalent to the toxin produced in bacteria but, as in the case of most other commercial Bt cry toxins, the toxin protein is allowed to diverge significantly from the bacterial toxin so long as the protein remains active against insect cells and is immunologically similar to the toxin produced in cotton. The toxin tested by Syngenta showed no overt acute toxicity and there was no indication that it was allergenic. Sequence analysis showed no overt similarity to known toxins. The practice of putting forward Bt toxins produced in bacteria as equivalent to the Bt toxins produced in crops was criticised earlier. The practice is unsound and should, at least, be made very clear in the government announcements on the safety testing of GM crops bearing genes for Bt toxins.
The EPA report notes: "Once in the insect gut, the VIPA3 protein binds to specific receptors (different from those by Cry 1A proteins) and forms ion specific pores." There was no discussion, in the EPA report of the apoptosis and binding to death sequences receptors mentioned in the Syngenta patent. Indeed, the claim that the VIP3A toxin had no obvious homology to mammalian toxins seems to have ignored the homology of all apoptosis receptor death sequences. The contrast between the Syngenta patent and the EPA report is perplexing because the patent document was well supported with experiments while the EPA report provided little scientific evidence for its claims.
In conclusion, the Bt toxins of the VIP gene family provide potent broad spectrum insect control. The toxins have been reported to act by binding to death sequences and triggering apoptosis in insect cells. At the very least, the potential impact of such toxins on the receptors and death sequences in mammalian cells should be fully evaluated before GM crops bearing the toxins enter the mammalian food chain.
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GM Microbes Invade North America
A number of GM microbes are being widely deployed since their first release six years ago.
Sinorhizobium meliloti is a bacterium added to soil or inoculated into seeds to enhance nodule formation and nitrogen fixation in the roots of legumes. It was released for commercial production in 1997.
The other commercial GM microbes are designated as bio-pesticides. These include GM Agrobacterium radiobacter k1026, used to prevent crown gall disease in fruit and vegetable plants, and Pseudomonas fluorescens modified with a number of different Cry delta-endotoxin genes from different subspecies of Bacillus thruingiensis (Bt). The modified P. fluorescens cultures are killed by heat pasteurization and provides a persistent biopesticide preparation that degrades much slower in sunlight than Bt.
Neither the people selling nor those using the preparations are necessarily aware that the microbes are genetically modified, however. Even organic farmers may be using them inadvertently.
The legume symbiont, Sinorhizobium meliloti, is tremendously important for fixing nitrogen from the air into plant roots and the soil. Legumes signal to the bacterium by exuding flavonoids from their roots, activating the expression of nodulation genes in the bacterium, resulting in the production of Nod factors that regulate the formation of nitrogen fixing root nodules [1]. The S. meliloti genome has been fully sequenced. It is unusual in containing three chromosomes (or a chromosome and two very large plasmids), all of them contributing to the symbiosis with the plant roots [2]. The genetically modified commercial strain (RMBPC-2) has genes added that regulate nitrogenase enzyme (for nitrogen fixation) along with genes that increase the organic acid delivered from the plant to the nodule bacterium. It also has the antibiotic resistance marker genes for streptomycin and spectinomycin [3]. The commercial release was permitted in spite of concerns about the impact of the GM microbe on the environment.
Evidence supporting the initial concerns has accumulated but that has not dampened the use of the GM microbe. For example, a recent review reports that GM S. meliloti strains persisted in the soil for six years, even in the absence of the legume hosts. Horizontal gene transfer to other soil bacteria and microevolution of plasmids was observed [4]. Other studies showed that a soil micro arthropod ingested GM S. meliloti, and a GM E. coli in the arthropod gut facilitated gene transfer to a range of bacteria [5].
There is little doubt that the antibiotic resistance markers for streptomycin and spectinomycin will be transferred to soil bacteria and to a range of animal pathogens. For example, the resistance genes for streptomycin could be observed to transfer from their insertion as transgenes in plant chloroplast to infecting bacterium Actinobacter sp. [6] when homologous gene sequences were present.
The antibiotics spectinomycin and streptomycin are used extensively in human and animal medicine. Spectinomycin is used to treat human gonorrhea [7] and bovine pneumonia [8]. Streptomycin is used to treat human tuberculosis [9] and Meniere's disease [10] and as a pesticide on fruits and vegetables [11]. Thus, the commercial release of GM Sinorhizobium meliloti has resulted in the establishment of the GM microbe in the soil in millions of acres of cropland, where it can spread antibiotic resistance genes for antibiotics that are extensively in use in medicine and agriculture.
Agrobacterium radiobacter k1026 [12] is a bio-pesticide derived from A. radiobacter k84, a natural bacterium used to control the crown gall disease of fruits and ornamental trees and shrubs. Crown gall disease is due to the bacterium Agrobacterium tumefaciens that causes tumors to form on the plant stems, and is the most common vector employed in plant genetic engineering.
GM Agrobacterium radiobacter releases a chemical warfare agent bacteriocin (agrocin) against A. tumefaciens. Bacteriocin is a novel nucleic acid derivative that prevents the crown gall tumors from forming in the infected plants. The GM A. radiobacter has an engineered deletion in the genes controlling plasmid transfer so that the 'male' bacterium cannot transfer its plasmid, but it can act as a 'female' to receive a plasmid transfer. However, recent research suggests that retrotransfer of genetic material can occur from 'female' recipient to 'male' donor bacterium [13].
Pseudomonas flourescens strains modified with Cry delta endotoxin genes from Bacillus thuringiensis are killed before being marketed [14]. The killed GM bacteria are more persistent than are the conventionall B. thuringiensis sprays. The main fallacy in the approval of these biopesticides is to suppose that bacteria cannot enjoy sex (conjugation) after death, they do.
Soil bacteria are also easily transformed with cell lysates (squashed dead cells) and function in their genetically modified form in soil microcosms [15]. P. fluorescens and A. tumefacians are both transformed in soil [16]. Soil Pseudomonas and Actinobacter can also take up genes from transgenic plants [17]. So, the combination of transgenic crops and GM biopesticides can create genetic combinations capable of devastating the soil microflora and microfauna.
In conclusion, GM microbes have begun to be ubiquitous invaders of the North America ecosystem. This massive invasion took place with little or no public awareness and input, and with very little monitoring of the impact of the invasion. The environmental risk assessments of the commercial microbes were rudimentary and frequently erroneous. We may have a bio-weapons equivalent of a time bomb on our hands.
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|Anonymous Coward
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Thank you so much! It is coming and very ominous... We are in some heap o trouble...
Cheerios are a product now to be avoided. It's consumption causes extreme gas and explosive, irritable bowel. My guess, the GM grain and the GM sugar beet processed for sugar. I will never consume cheerios again...
Every loaf of bread, every can of peas, I am incredibly worried about our food supply. Thank you so much again for posting this valuable information.
Organic crops are at risk, labeling is at present not required here, and no testing has been done at all on long-term effects here in the USA.
Good news however: Germany just banned GM crops! And California just passed legislation that legally protects its farmers from less than ethical Monsanto patent litigation.
Google Monsanto and codex alimentarius:
[link to www.google.com]
In a scary world, this is the scariest scenario of all! Monsanto wants to own the world's food supply and spray Roundup all over the entire planet.
Google roundup and illness:
[link to www.google.com]
Good Job OP!
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Yes the GMO sugar beet is new and is about to release a terrible problem for all products using the sugar. GMO wheat is no different, it has major issues.
People really need to OPEN up those eyes and minds to see what is actually going on with the GMO foods. The real science does not equal the Corporation science. This is a dangerous product and is not the same as nature supplied food.
Obviously this is going over the heads or people truly do not give a hoot. Lack of aliens in the writings I guess.
GM Grapevines & Toxic Wines
A number of genetically modified (GM) grapes have been created, though none is yet commercialised. There were 25 field test releases in USA between 1999 and 2005, and a small deluge of commercial releases is expected any time now.
The bulk of the test releases were of GM grapes resistant to diseases including powdery mildew, Botrytis , Agrobacterium , Clostridium , Xylella , nepovirus and closterovirus. There was one application for improved fruit quality, but the transgene was designated confidential business information.
The disease resistance genes included synthetic antimicrobial peptides encoded by synthetic genes. The majority of the applications for release permits were from Cornell, California and New York State Universities, the rest were from vintners or wine research companies [1].
In Europe, Italy conducted trials of grape modified with a gene regulating the plant hormone auxin. Germany tested grapes resisting fungal diseases , and France tested grapes resisting nepovirus [2].
Australia has field-tested grapes modified for fruit colour or quality [3], most of them carrying antibiotic resistance genes as selectable markers, which would very likely spread to other organisms during wine making. There has been a hiatus in the commercial approval of GM crops recently despite a very large number of field trials. Bureaucrats may regard the low frequency of commercial approvals to be a "log jam" and facilitate a flood of new approvals without warning. It is something that the concerned public should be prepared for.
GM grapes potential hazards not addressed
GM grapes carry all the potential hazards of other GMOs [4-6] ( Horizontal Gene Transfer - The Hidden Hazards of Genetic Engineering ; ISIS Report; GM Food Animals Coming , SiS 32), but because GM grape juice and GM wine come as a clear liquids, many people may assume it is safe to drink; not so.
DNA from a GM grape persisted for over a year after wine fermentation, contradicting claims that wine fermentation eliminates DNA [6]. GM DNA in wine carries all the risks of horizontal gene transfer and recombination: creating new viruses and bacteria that cause diseases, triggering cancer in the case of GM DNA with strong promoters jumping into the genome of human cells Other potential hazards from GM grapes are toxins and allergens from the transgene products, or from unexpected metabolic disturbances to the host plant.
Toxic synthetic antimicrobial peptides
Chardonnay grapes have been modified with genes for magainin and peptidyl-glycine-leucine carboxyamide, both synthetic antibiotic peptides originally from frog skin, and neomycin resistance and GUS were included as selectable markers. The GM grape was found to be more resistant to bacteria than to fungi [7, 8]. The toxicity of the GM grape to mammals has not yet been investigated.
Patent applications have been made for producing transgenic grapes with a synthetic version of a cecropin-like toxin, shiva 1. The transgenic grapes resisted bunch rot, powdery mildew and downy mildew [9, 10]. Shiva 1 is an experimental synthetic antibiotic peptide for treating mammals, and treatment of rabbits revealed a narrow range between effective and toxic doses [11]. Great caution is needed in evaluating GM grape with genes for synthetic toxins that could endanger domestic and wildlife, as well as human beings.
Grapevine fan leaf virus resistance was achieved by transforming grape with a coat protein gene from the fan leaf nepovirus [12,13]. The coat protein gene conferred resistance most likely through an RNAi suppression of virus replication [14] ( Subverting the Genetic Text , SiS 24).
Disease resistance with bacteria and viral genes
Grapevines are susceptible to infections with Agrobacterium causing crown gall (tumour) disease (the same Agrobacterium in a disarmed form that's commonly used in genetic modification of plants). The plant tumours are initiated when the bacterium injects plant cells with a DNA Ti (tumour-inducing) plasmid carrying genes for plant cell proliferation. A portion of the Ti plasmid is transferred into the plant cell genome, and depends on the forming a DNA single strand intermediate that is protected from degradation in the plant cell by a coating of vir gene protein produced by the bacterium. The protected single strand of DNA integrates into the plant chromosome and begins activating genes that initiate tumour formation. Agrobacterium -resistant grape vine was made by inserting a gene for a mutant form of the vir gene into the plant genome. The transgenic grape produces the mutant vir protein continuously, which attaches to the infecting part of the Ti plasmid, causing it to be inactivated and destroyed rather than being integrating into the chromosome [15].
Grape bunch rot ( Botrytis ) is caused by the fungus Botrytis cinerea , while the fatal Pierce's disease is caused by the bacterium Xylella . Both pathogens use enzymes that degrade the plant cell wall to invade the grape tissue. A gene for an inhibitor of the pathogen wall-digesting enzyme (polygalacturonase inhibiting protein) from pear fruit was used to transform grape vines. The transgenic DNA included the CaMV promoter, a TMV enhancer, the pear gene, and octopine synthase terminator, accompanied by the GUS gene and a neomycin resistance gene as selectable markers. The transgenic grape was reported to resist both the bacterial gene and the fungal gene [16].
Cyanide-producing grape
Grape has also been genetically modified to resist insects by making them produce hydrogen cyanide when attacked by insects. Cyanogenic plants are characterized by the liberation of HCN in the course of tissue injury, due to the hydrolysis of cyanogenic glucosides. Most of our knowledge of cyanogenicity comes from Sorghum bicolor , which contains large quantities of the cyanogenic glucoside, dhurrin. Prussic acid, a derivative of cyanide, is also a serious potential problem. Crop species most commonly associated with prussic acid poisoning are sorghum, Johnsongrass, and Sudangrass. Grain sorghum typically has more potential for toxic levels of prussic acid than forage sorghum or Sudangrass. Young, rapidly growing plants are the most likely to contain high levels of prussic acid. Cyanide is more concentrated in young leaves than in older leaves or stems. New sorghum growth following drought or frost is dangerously high in cyanide. Generally, any stress condition that retards normal plant growth may increase prussic acid content. Hydrogen cyanide is released when plant leaves are damaged by trampling, cutting, crushing, chewing, or wilting. Drought-stunted plants accumulate cyanide and can possess toxic levels at maturity. Prussic acid poisoning is most commonly associated with regrowth following a drought-ending rain, or the first fall frost. New growth from frosted or drought-stressed plants is palatable, but dangerously high in cyanide. After a killing frost, at least four days should pass before grazing to allow released hydrogen cyanide to dissipate.
A multigenic trait responsible for biosynthesis of the secondary metabolite, dhurrin cyanogenic glucoside was engineered in grapevine with three genes sequences from sorghum ( Sorghum bicolor ): two cytochrome P450s (CYP79A1 and CYP71E1) and a UDPG-glucosyltransferase (sbHMNGT). The grapevine was modified using a two-step process involving whole plant transformation followed by hairy root transformation. The two step process make sure that the whole plant could be transformed with the dhurrin pathway, while the secondary transformation of the hairy root culture allowed fuller study of the dhurrin produced in roots which had been challenged with the root pathogenic insect Phyloxera .
One dhurrin-positive line was tested and found to release cyanide upon maceration. Co-culture of a cyanogenic hairy root line or a non-cyanogenic line with the specialist rootsucking, gall-forming, aphid-like insect, grapevine phylloxera ( Daktulosphaira vitifoliae ) gave no evidence that the cyanogenic plant tissue was protected from insect infestation. Consistently high levels of dhurrin accumulation may be required for that to occur [17]. We are not sure at all about the ultimate purpose of the modified grape but will certainly avoid drinking juice and wine made from it, provided that it is labeled as such. If it is not labeled, we may all have no choice over cyanide poisoning.
For crying out loud
The modification of grapevines has gone beyond the humdrum of Bt and herbicide tolerance of most GM food crops. The new emphasis is on synthetic genes and proteins, and virus resistance using coat protein gene modifications. The introduction of a cyanogenic toxin into grapevine may be a sign that genetic engineers are growing ever more daring in the recognition that regulators are standing with them against the public. Clearly these new developments are crying out for GM labelling at the very least, and a clean sweep of the regulatory regimes would not come amiss.
[link to www.i-sis.org.uk]
|vet ikke
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"Let Them Eat Cake!"- The Nutricide of America
[link to www.godlikeproductions.com]
When the Lamb opened the third seal, I heard the third living creature say, "Come!" I looked, and there before me was a black horse! Its rider was holding a pair of scales in his hand.
Then I heard what sounded like a voice among the four living creatures, saying, "A quart of wheat for a day's wages, and three quarts of barley for a day's wages, and do not damage the oil and the wine!"
-Revelation 6:5, 6:6
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This should get pinned, glued, nailed and stapled!
I am not sure if I am scared to death or happy with this information.
Why the hell is nobody giving this thread some 5 star ratings?????
How much more truth do people need before they realize we have been had. This information is laid out so well. I feel like I am back in school reading text books.
This is reality, our food supply is done for if we do not figure out what to do with the Codex and these chemical companies.
Seriously this is terrifying information that needs to be read by every human on the earth. True Doom and takeover.
Great thread OP and keep going.
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How Bad Science and Big Business Put the World at Risk from Viral Pandemics
Xenotransplantation - the transplant of animal organs into human beings - is a multi-billion dollar business venture built on the anticipated sale of patented techniques and organs, as well as drugs to overcome organ-rejection (1). It has received strong criticism and opposition from scientists warning of the risks of new viruses crossing from animal organs to human subjects and from there to infect the population at large. But regulators are adopting a permissive attitude for clinical trials to go ahead. Scientific reports of virus crossing from pig to human cells (2) and of viral infections in humans subjects transplanted with baboon livers (3) are being ignored or dismissed, while inconclusive, widely faulted papers are taken as evidence that no viruses are found in xenotransplant patients (4). This audit exposes the shoddy science that puts the world at risk of viral pandemics for the sake of corporate profit, and concludes that xenotranplantation should not be allowed to continue in any form. Instead, effort should be devoted to developing safer, more sustainable and affordable alternatives that are already showing promise and will be more likely to benefit society as a whole in the industrialized west as well as in the Third World.
Key words: transgenic pigs, hyperacute rejection, xenozoonosis, endogenous retroviruses, xenotropism, viral infections, recombinant viruses, PERV, pig HEV, baboon endogenous virus, simian foamy virus.
A multi-billion dollar business venture
Xenotransplantation - the transplant of organs or tissues between species – has become a major issue within the past ten years. Biotech companies are developing genetically engineered 'humanized' pigs to meet the demand for spare body parts in the industrialized world. A multi-billion dollar market is anticipated from the sale of patented techniques and organs, as well as existing and new drugs to overcome organ-rejection (1). Still at the experimental phase, it has received strong criticism and opposition from mainstream scientists warning of the risks of new viruses crossing from animal donor organs to human subjects, and from there to infect the population at large. But these warnings have done little to dampen the enthusiasm for continued research well into clinical trials.
The world-leader in xenotransplant research is the UK biotech company Imutran based in Cambridge, now a subsidiary of Novartis. Novartis already own the rights to Cyclosporine A, the main anti-rejection drug given to transplant patients to suppress the immune system. Since acquiring Imutran, Novartis have pledged $1 billion for research in xenotransplantation, and thereby to dominate a projected $11billion a year market for organs and associated immune-suppressive drugs.
An estimated 10 000 pigs and nearly five hundred primates have been in the UK, with very little accomplished. Xenotransplantation is in crisis. At the bottom of the crisis lies some shoddy science that puts the world at risk of viral pandemics for the sake of profit. At least one company, PPL, which produced Dolly the cloned sheep, is reported to be winding up xenotransplantation research, on the possibility that pig virus could infect humans (5).
'Humanized' transgenic pigs as organ donors
Transgenic pigs, rather than our close relatives primates, were considered as organ donors because there are greater ethical objections to using primates, many of which are endangered protected species (6). As pigs are already farmed for food, it was thought that there would be less ethical concern, and that pigs could also be more easily controlled for viral infections and consistent quality. Nevertheless, large numbers of primates are exploited and made to suffer as experimental transplant recipients; and primate to human transplant clinical trials have been authorized in the United States.
The first hurdle in transplanting organs between distant species, as in the case of pig to human, is hyperacute rejection (HAR) of the donor organ by the host. This reaction is swift and severe, and depends on naturally occurring, pre-existing antibodies. Naturally occurring human anti-pig antibodies predominantly recognize the carbohydrate antigen galactose-alpha-(1,3)-galactose attached to cell-surface proteins. Both IgM and IgG (different classes of immunoglobulins or antibodies) in the human blood contain antibodies that bind this antigen; which may comprise up to 1% of the IgG. The enzyme for making the galactose-alpha-(1,3)-galactose exists in all mammals except humans, old world monkeys and the great apes. The binding of these antibodies to the antigens triggers a cascade of reactions – complement activation – that results in destruction of the donor organ and cells within minutes. Induced antibodies against the foreign graft, xenograft, are responsible for organ rejection in the longer term.
There are three possible ways to block HAR: by depleting the pre-existing antibodies, by reducing antigen expression in the donor cells, and by inhibiting complement activation. Of these, the last option appeared to be the only clinically viable strategy in combating HAR. One of the five candidate proteins that proved most promising is the decay accelerating factor (DAF), which blocks an early step in complement activation. Transgenic pigs containing hDAF were therefore produced.
Lack of documentation and molecular characterization of the transgenic pigs
The first and only report in the scientific literature on the experiment creating the transgenic pigs with hDAF was a note (7) less than one and a half pages long, published in Transplantation Proceedings in 1994. It contained no molecular genetic documentation of the construct such as genetic map to indicate whether unknown sequences are present, or the promoter-enhancer sequences used. It did not state how the 'minigene' construct was introduced, whether by itself, or spliced into a vector. Later papers (6, 8, 9, 10) up to year 2000, all referred back to the same experiment with no further elaboration.
About 2500 fertilized eggs were injected with the minigene. Of 85 surrogate mothers implanted with embryos, 49 delivered litters with 311 piglets, 49 of which were transgenic, ie, contained human DNA, with one to 30 copies of the gene. Only 33 expressed the gene for hDAF, however. The rate of success is thus no better than 1.3%.
There was considerable variability in expression of hDAF in the transgenic animals, not only between animals, but also between organs from the same animal (6). Liver expression was found in 90% of transgenic pigs, and expression in the heart was the least frequently detected (18/30). But high expression did not guarantee expression in endothelial cells (cells on the surfaces of the organ). No correlation was found between the number of copies of the gene integrated and hDAF expression. Animals with the highest gene copy number (13 copies) expressed very low levels of hDAF in all transplantable tissues. The two most promising lines incorporated between 6-8copies of the gene and expressed hDAF on parenchyma (inside) and endothelium (surfaces) of all the transplantable organs. In 75% of the organs, gene expression levels of hDAF was greater than in equivalent human tissues.
These results underscore the unpredictable, uncontrollable nature of the transgenic process and the low rate of success. There were no attempts to characterise the transgenic inserts, nor to create stable transgenic lines before transplant experiments were carried out; thus compromising not only the reproducibility of the experimental findings, but also the safety of the procedure, particularly with regard to the stability of the transgenic inserts (see box 1) and the potential for creating new viruses (see box 3). Plans were made (8, 9) to use whole yeast artificial chromosomes (YACs) containing large segments of the human genome to optimize gene expression, but it is not clear if such procedures have been carried out. Introducing YACs may mean that uncharacterized human genome DNA, including endogenous human viruses (see box 3), will be transferred into the transgenic pigs, which could increase the potential for generating new recombinant viruses (see box 3).
The 1997 review (6) admitted that production of an ideal hDAF expressing pig was not complete, and that all organs used for xenotransplantation were derived from heterozygous pigs, ie, pigs having hDAF gene(s) on one of a pair of chromosomes. The review did not state whether these were zero-generation transgenic pigs, or transgenic pigs from later generations bred from the original. The ideal pig, according to the authors, would express high levels of hDAF on organs and cells lining the organs, and would be bred to homozygosity, ie, having hDAF genes on both of a pair of chromosomes. That means the transgenic pigs would 'breed true'. Only one line was reported to fulfill these criteria in 1997. But the authors pointed out that "breeding to homozygosity might cause undesirable effects on the stability and health of the pig". This conceals a major technical problem with creating transgenic lines. Transgenic organisms, plants as well as animals, are genetically unstable and do not breed true (see box 1).
Box 1
Transgenesis is unpredictable and uncontrollable, and trangenes are unstable
The instability of transgenic plants is well-known and actively researched. Transgenic constructs typically integrate at random into the host genome, and in a scrambled configuration, consisting of repeats, rearrangements and deletions (11). There is no reason to expect transgenic animals to be different. Indeed, integration of transgenic construct was reported to be random in the transgenic pigs, and the expression of the transgene depended on the site of integration (8).
Transgene integration was examined by fluorescence in situ hybridization (FISH) (12), a technique that enables the inserts to be seen on chromosomes. Routine 'slot blot' analysis of total transgenic pig DNA was done first to identify pigs with hDAF DNA. According to the strength of the signal, one line, E14, was estimated to contain 40 copies of the hDAF transgene; while another, A74, contained 6 copies, and a third, C50, two copies. These were referred to as "heterozygous founders lines". Again, it is not stated how many generations were bred after transgenesis. And it is not clear why the "lines" are heterozygous and not homozygous.
In the cross E14 x A74, one from a litter of 13, and another from a litter of four were the only piglets that showed a signal similar to that of one parent. In the cross C50 x A74, three out of 9, and 4 out of 11, respectively showed signals that were similar to those of the parents. But when analyzed by FISH, only 4 piglets were actually found to have inherited any transgenes from their parents. Thus, the actual transmission of transgenes is 4/37 or 10.8%. This is much lower than the 75% predicted (assuming both parents were heterozygous), and is typical of the instability of transgenic inserts, which can become lost in subsequent generations. This raises the question as to whether the lost insert can be tranferred again, unintentionally, to unrelated species, a process referred to as horizontal gene transfer, with its own attendant hazards (13).
Intended xenotransplant recipient animals were not screened for viruses and postmortems of transplant recipients did not include examination for viral infections
In the first experiment, eight hearts from transgenic pigs were transplanted into non-immune suppressed cynomolgus monkeys (Macaca fascicularis) of unspecified origins, and without pre-screening for viral infections or endogenous viruses. The median survival was 5.1 days (97-126h) with no reported hyperacute rejection. Five of the ten controls that received control hearts survived a surprising mean of 86.4h, while the other five survived for only 2.6h, as typical of HAR.
In a second experiment, ten cynomolgus monkeys receiving heart xenotransplants from transgenic pigs were dosed with a regimen of immune suppressing drugs: 80-180mg/kg/day of cyclosporine and 10-20mg/kg of cyclophosphamide on alternate days. Methylprednisolone was also administered at 1mg/kg. This regimen produced median survival of 40 days (2 to 62 days). The five non-transgenic hearts were rejected hyperacutely (median 55 mins). Five animals from the transgenic heart group had to be euthanased (killed out of compassion, to relieve suffering) due to gastrointestinal toxicity, resulting in severe diarrhoea. All hearts were reported to be normal with no evidence of complement or immunoglobulin deposition.
In immune-suppressed animals, rejection was considered not the primary cause of graft failure. Only two out of ten were due to rejection, while drug toxicity resulted in 50% having to be euthanized. That accounted for seven of the ten xenotransplant recipients. So, what did the remaining three die of? The report did not specify. There was no indication that post-mortem examination for viral infections had been carried out.
An experiment involving a single transgenic pig heart transplanted to a baboon was described in a subsequent paper (10). The animal survived 39 days with an immunosupressive regimen of cyclophosphamide, cyclosporine A, mycophenolate mofetila and cortecosteroids. It was reported to be active and energetic until day 39, when it underwent sudden and rapid decline, leading to collapse almost immediately following the routine administration of drugs. The cause of death was recorded as "unclear". Postmortem examination was limited to ascertaining that organ rejection was not to blame. Again, no investigations for viral infections were reported.
Hazards of cross-species viruses arising from xenotransplantation
The problem of infectious viruses arising from xenotransplantation was first raised by Robin Weiss and his coworkers at the Institute of Cancer Research (2). They showed that a pig endogenous retrovirus (see Box 2) can infect cultured human cells. And once the virus has gone through a complete life-cycle in human cells, it is then able to infect a wide range of other human cells. Many copies of pig endogenous retroviruses (PERV) exist in the pig genome and it will be extremely difficult, if not impossible, to breed pigs free of PERV. Robin Weiss argued that accidents have already occurred (reported in ref. 1). Millions have become infected with the monkey SV40 virus through polio and adenovirus vaccines made in monkey kidney cells. Many viruses lying dormant in animals, in particular herpes viruses and retroviruses, can become activated and deadly in humans. Activation of animal viruses might be favoured under transplant conditions, which compromise many barriers to natural infection. Robin Weiss stressed that virus adaptation or recombination with other retroviruses in the new host cannot be dismissed.
Box 2
What are endogenous retroviruses and why are they dangerous?
A retrovirus is a RNA virus that is reverse-transcribed into complementary DNA (cDNA) and integrated into the host cell genome to replicate and complete its life-cycle. Endogenous retroviruses (ERVs) are elements in the genomes of all higher organisms including human beings, which are very similar to the genomes of retroviruses. They are flanked by long terminal repeats (LTRs) and carry genes coding for structural and coat proteins of the virus as well as the reverse transcriptase and integrase enzymes (required for reverse-transcription and integration of the viral genome into the host genome) (14).
There are two theories on how ERVs may have evolved. Howard Temin, Nobel laureate who co-discovered the enzyme reverse transcriptase, suggested that they have evolved from retro-transposons – mobile genetic elements with reverse transcriptase - which are part of the genomes of all higher organisms. Alternatively, ERVs may have evolved from exogenous viruses, foreign viruses that have become integrated into the genome. There is no reason to believe that these alternatives are mutually exclusive. Exogenous viruses, which may have arisen from retrotransposons, can indeed re-invade the genome of higher organisms to become endogenous retroviruses. In general, most endogenous retroviruses appear to have been acquired millions of years ago, but there is evidence that new retroviruses can be acquired. Under certain circumstances, endogenous retroviruses can also give rise to infectious retroviruses, although most ERVs are in a dormant, non-infectious state.
Some ERVs have retained their ability to code for virus that can infect the cells of other species, a phenomenon known as xenotropism, and this is of particular safety concern with regard to xenotransplantation. For example, xenotropic retroviruses in mice have been described that cannot replicate in mouse cells, but can propagate profusely in human cells in culture. Also, chick and pig ERVs rarely replicate in their own species but readily infect cultured cells of other species, including those of humans. Likewise, a cat ERV replicates in human cells, as does one from baboon, although neither replicates in its own host species.
Another important safety consideration is that the creation of transgenic pigs with human genes, such as hDAF, to suppress hyperacute rejection, actually increases the potential for creating infectious cross-species viruses. It suppresses the body's defense against bacteria and viral infections, and also provides more opportunities for the viruses to gain access to the host cells (see Box 3).
Box 3
Transgenic pigs increase the likelihood of generating cross-species viruses
Robin Weiss (15) points out that many animal viruses with lipid envelopes are sensitive to inactivation by the human complement cascade. The virus undergoes lysis (breaking open), triggered by the binding of anti-alpha-Gal antibodies to alpha-Gal on the viral envelope. Viruses grown in non-primate cells are sensitive to inactivation by fresh human serum, whereas the same viruses propagated in human cells are not because they have lost the alpha-Gal. Other enveloped viruses grown in animal cells are also sensitive to lysis by human complement, including arenavirus, paramyxovirus, alphavirus and the rhabdoviral pig pathogen, vesicular stomatitis virus. If alpha-Gal is on the host cell, then the viral envelope becomes sensitive to rapid lysis by human serum. In other words, virus inactivation occurs by precisely the same mechanism as hyperacute rejection of xenograft. So, modifications to make pig xenografts resistant to hyperacute rejection may also make any enveloped viruses of pigs similarly resistant to breakdown in the human host.
The key proteins are CD46 (membrane cofactor protein, MCP-1), CD55 (decay accelerating factor, DAF) and CD59 (prolectin). They all inhibit downstream steps in the complement cascade, and several transgenic pig herds have been developed expressing one or more of these human genes. All of these are present in the envelope of HIV, the AIDS virus, and protect the virus from lysis.
CD46 is the cell-surface receptor for measles virus and CD55 can serve as a binding receptor for Echo and Coxsackie B picornaviruses. Coxsackie B virus causes myocarditis and might endanger the pig heart in an immune suppressed recipient of a xenograft. Transgenic pigs may therefore also provide an opportunity for animal viruses to adapt to a human host range. Coxsackie B virus, for example, can be adapted to grow in mice, and in some human cell cultures, it increases its infectivity a million-fold by adopting the CD55 receptor. If pigs were to harbour picornaviruses that use the pig equivalent of CD55, such viruses may readily adapt to recognize human CD55 in transgenic pigs that express both pig and human equivalents. These viruses would then be pre-adapted to transmit to the xenograft recipient and to be transmitted from human-to-human. There is already concern that mice transgenic for human poliovirus receptor should not escape and become a non-human reservoir for a human pathogen.
Animal morbilliviruses (measles-related viruses such as canine distemper virus and rinderpest virus) might become pre-adapted for human transmission in CD46 transgenic pigs. Morbilliviruses are known to jump host species as in the recent epidemic in seals and dolphins. In Australia, a vet and a stable-hand died after an autopsy of a horse with a new type of morbillivirus which in turn was probably acquired from fruit bats.
Human tumour tissue transplanted into immunodeficient mice frequently becomes infected by endogenous xenotropic mouse retrovirus. Two or three distinct pig retroviruses can infect some human cells in culture.
Researchers are identifying many new pig viruses. One pig virus, closely related to human hepatitis virus E (16), was found in the majority of pigs, three months or older, in herds from mid-western United States. This raised concerns over the creation of cross-species pathogens in xenotransplantation.
Risks considered disproportionate to benefits by many scientists
Jonathan Allen, a virologist on FDA's advisory subcommittee, accused the FDA of the failure to adhere to the precautionary principle (see ref. 1). It may take decades for a xenozoonosis – infectious diseases arising from cross-species viruses - like the AIDs virus or Human T-cell Leukemia Virus to spread and become detected. The FDA's requirement that all future xenotransplant recipients be monitored for infectious diseases over their life time, and prohibiting them and their close contacts from donating blood, amount to shutting the barn door after the horse has bolted.
The American Society of Transplant Physicians also want tougher guidelines, and accuse the capital-hungry biotech companies of excessive hype, and creating unrealistic expectations among patients, fuelling pressure to proceed to clinical trials.
Fritz Bach, xenotransplant scientist from Harvard among others, called for a moratorium in 1998, as potential risk of xenotransplants would affect the general public who are being exposed without informed consent. He argued for a wide "informed" public debate on whether such trials should be allowed to proceed at all, as it is an ethical question.
According to the United Network for Organ Sharing, the number of transplants increased from 12 000 to 20 000 between 1988 and 1996; while the number on the waiting list soared from 16 000 to 50 000 and the number of deaths rose from about 1 000 to 3 000 (17). David Sachs of Harvard Medical School estimated that more than 400 000 could benefit from heart transplants when the official waiting list in 1996 was 3 698. Many on the waiting list are for repeat procedures to replace failed transplants. Was Sachs' estimate overblown? Did it reflect the over-enthusiasm on the part of the medical establishment for spare-organ trafficking rather than real demand or benefit? Chronic rejection is the major cause of the loss of allotransplants from unrelated human donors. So it can be predicted that xenotransplants will be much worse.
A new study published in the British Medical Journal suggests that even transplants from unrelated humans save lives only in patients on the verge of death (18). The study was carried out in Germany. Researchers looked back at 889 patients listed for a first heart transplant in 1997. The patients were categorised into groups with low, medium or high risk of dying and compared the mortality of those on the waiting list with those who had a transplant. It turns out that there were no differences in mortality for the low and medium risk groups. Only in the sickest patients was there an improvement in survival due to the transplant.
There is evidence for cross-species viruses in xenotransplant recipients
Evidence that baboon viruses have arisen in two human subjects transplanted with baboon livers emerged two years ago (3). DNA of two retroviruses, the simian foamy virus (SFV) and baboon endogenous virus (BaEV), were found in many tissues of the patients. The presence of baboon mitochondrial DNA (evidence of baboon cells) were also founded in the same tissues, suggesting that baboon leukocytes harboring latent or active viral infections had migrated from the xenografts to distant sites in the human transplant recipients. The authors stressed, "The persistence of SFV and BaEV in human recipients throughout the posttransplant period underscores the potential infectious risks associated with xenotransplantation."
These were the first baboon-to-human liver transplants. One was performed in June 1992 in an HIV-infected 35-yr old man, who survived 70 days, and the second, in Jan. 1993, in a 62-yr man who also received donor bone marrow intravenously and survived for 27 days. Both patients had hepatitis B virus-associated liver cirrhosis. Neither transplanted baboon liver functioned normally. In addition, both patients developed kidney failures and multiple post-transplant infectious complications. Both received an immunosuppressive regimen of FD-506 prednisone and cyclophosphamide. The two adult male baboon donors were screened against a panel of simian and human viruses and were negative for Simian T-cell Leukemia Virus, Simian Immunodeficiency Virus and simian retrovirus.
Antibodies against SFV were detected in samples from both donor baboon samples prior to transplantation, whereas the human patients were non-reactive during several time points after receiving the transplant. However, a faint positive result was recorded at day 22 in patient 2. The absence of antiviral antibody in the patients may be due to insufficient time in case of patient 2, and AIDs in patient 1. Furthermore, immunosuppressants may have suppressed the antiviral response.
Nevertheless, SFV DNA was detected by PCR (Polymerase Chain Reaction) probes in tissues from both patients. In patient 2, SFV DNA was detected in the liver graft on day 24 but not day 12. The liver sample from patient 1 on day 16 was negative for the viral DNA, but positive results in both lymph node and kidney were obtained on the day 70. DNA sequence analyses confirmed that the SFV in the transplant recipients were closely related to the baboon virus rather than those of other primates. The life cycle of SFV includes integration of viral DNA into the host genome.
Baboon mitochondrial DNA and BaEV were simultaneously detected in every sample in which SFV was present.
The authors stated, "These findings demonstrate the potential for both exogenous and endogenous viruses to reside in human recipients of animal organs for a significant period after transplantation. It is possible that these circulating xenogeneic cells could also act as conduits for new human infections….Since retroviruses commonly exist as persistent latent infections, with an incidence of disease that varies because of both host and viral factors, the possibility that baboon foamy viruses might cause disease in humans remains a consideration in discussing future animal sources for xenotransplantation. Theoretically, other yet to be characterized viruses carried by baboons might also be transmitted to human recipients." (p.824).
As mentioned earlier, none of the published papers up to year 2000 from the Imutran group gave any indication that post-mortem pathological investigations included tests for viral infections.
A brief note (less than one page) from Imutran-Novartis, published later in the same year, reported an experiment in which pig alveolar macrophages (PAM) from pig blood, infected with pig cytomegalovirus, PCMV, were cultivated for up to 15 passages together with human cell lines, and monitored for the presence of PCMV (19) at three time points: passages 5, 10 and 15. It reported "no evidence of PCMV infection of the human cells at passage 15, the farthest time point in this study, despite evidence that PAM and PCMV were present in the co-culture up to at least passage 10. On the basis of this evidence, PCMV is unlikely to be a significant zoonotic agent in clinical xenotransplantation of pig organs to human."
The experimental results were equivocal, to say the least. It is bad science to draw any conclusions on the basis of such limited, inconclusive data. Cell culture conditions are obviously different from the conditions in which a xenograft is transplanted into a living body. Furthermore, positive indications for PCMV were obtained at both passages 5 and 10.
Clinical trials to go ahead based on faulted study
White and Nicholson (20) reviewed xenotransplantation research at the end of 1999, and concluded that xenograft rejection cannot be prevented without significant immune suppression and toxic side-effects. They highlighted the risk of pig endogenous retrovirus transmission, but state that some of the important issues will never be solved "until carefully regulated clinical trials are allowed to begin." They take at face-value a report (4) published by Imutran/Novartis and other biotech companies claiming no retroviral cross-infection in patients exposed to pig tissues or receiving pig xenografts; and which has been criticized by many scientists.
The study tracked 160 patients in 9 countries exposed to living pig tissue over a 12-year period. One hundred and thirty one patients had their blood "filtered" and re-circulated through pig spleens, kidneys, livers, or devices made with pig liver cells; 15 received pig skin grafts for burns, and 14 received injections of pig pancreas cells for diabetes.
As pointed out by Peter Collignon of the Infectious Diseases Unit, Canberra Hospital, Australia (21), pig endogenous viral (PERV) genes were detected in 30 of the patients, and pig cells persisted in 23 xenotransplant recipients for up to 8.5 years. Although the authors found no active infection, the possibility of infection remains in the four patients with positive antibodies to PERV, and in another four patients with unexplained symptoms (skin rashes). In addition, lack of antibodies to PERV may not exclude the existence of infection, as for example, prion diseases (which include mad cow disease) cannot be detected by antibody or cellular immune responses. Immune suppressive drugs could also prevent the development of anti-viral antibodies (3). Collignon asked, "Who would have predicted that so many patients only transiently exposed to pig tissue would have persistent pig cells (and PERV) in their blood?" Even though the authors claim that there is no conclusive evidence of human infection by PERV (4), they admit that "PERV infection [cannot] be excluded."
Emanuel Goldman, Professor of Microbiology and Molecular Genetics at New Jersey Medical School in Newark noted that a majority of the samples tested were from patients whose blood had been flushed through pig organs/tissues, and recirculated into their bodies for very short periods - of the order of minutes to hours. Such data are hardly relevant to the kinds of conditions that would apply in whole organ xenotransplants. Data from the 14 subjects who received pig pancreatic islet cells could be taken more seriously. But, as with the burn victims, important information about these patients' exposure times to the xenografts and health and immunological status was missing (22).
Moreover, Goldman pointed out that the patients in the study were treated, and serum samples handled and stored in 9 separate countries, making quality control almost impossible. Looking for PERV RNA is always suspect with serum stored for several years. Plasma samples are frozen at -70C and thawed at very high temperatures. Many viruses are very unstable; it is unknown whether such extreme temperature changes might alter PERV and affect test results.
Another problem with the study is that the PCR probes are only good for two genes of one PERV, and will not detect other viruses, such as Hepatitis E virus or Cytomegalovirus, nor recombinant viruses, which are hybrids of pig and human viruses. Finally, none of the patients have been exposed to transgenic pig tissues. And it has already been pointed out that transgenic pig tissue may be more likely to give rise to new viruses (see Box 3).
To address the risks of infection, the US Food and Drug Administration (FDA) established an Advisory Panel on Xenotransplantation, and the British government set up the UK Xenotransplantation Interim Regulatory Authority (UKXIRA) in 1997.
The report (4) on the lack of evidence for PERV infection in xenotransplant recipients allows the Novartis/Centers for Disease Control teams to conclude that only cautious progress in closely monitored, prospective clinical trials will help to assess the safety and efficacy of xenotransplantation. Both the FDA and UKXIRA are taking this same attitude, and are ready to approve small-scale human trials of pig cell therapy. To proceed on this basis not only exhibits flagrant violation of the precautionary principle, it is to adopt the anti-precautionary approach (23), where failure to rule out viral infection (due to faults in data collection or handling) is taken as evidence that there is no risk of viral infection.
Robin Weiss (24) compares the present situation in xenotransplantation to the short-lived Asilomar moratorium on genetic engineering declared in mid 1970s. The parallel is closer than perhaps he thinks, as some of us have indeed questioned whether the exponential growth in genetic engineering biotechnology since the 1970s may have contributed to the recent resurgence of drug and antibiotic resistance diseases (25). In genetic engineering as in xenotransplantation, species barriers are undermined, and conditions are created which favour the generation of new viruses through horizontal gene transfer and recombination.
It turns out that PERVs not only infect human cells but produce products of the infection that inhibit human immune cell functions. Thus, PERV infection in transplant recipients could lead to an immunodeficiency disease (26). The suitability of baboons as models for human transplantation was previously questioned on grounds that pig cells do not release PERVS when they contact baboon cells or following pig to baboon cell transplants (27). However, a subsequent study showed that human, gorilla, and Papio hamadryas primary skin fibroblasts, as well as baboon B-cell lines, are permissive for PERV infection (28). There are probably no barriers to the transfer of viruses across species under conditions of co-culture of cells, or xenotransplantation of tissues and organs.
British scientists have now found that cancer-causing retroviruses can also spread relatively easily across species in the wild (29). Mouse leukaemia viruses, close relatives of the cancer retroviruses known to infect pigs, were found in a range of mammalian species, suggesting that pig retroviruses may also be capable of infecting other animals - including humans - with relative ease. This has prompted the Western health authorities to impose a moratorium on all xenotransplant surgery.
Professor George Griffen, a member of the UK Xenotransplantation Interim Regulatory Authority, admitted that viruses jumping species from xenotransplant organs is possible, but draws attention to the 'fact' that "hundreds of pre-moratorium xenotransplant recipients have yet to show reactions to retroviruses."
Governments disregard scientific evidence to put their citizens at risk from cross-species viral pandemics
In January 2000, the US FDA's Xenotransplant Subcommittee met in Gaithersburg, Maryland to review its proposed guidelines to "indefinitely defer" blood and plasma donations from xenotransplant recipients and their "close contacts" (29).
Phil Noguchi, Director of FDA's Division of Cellular and Gene Therapies, acknowledged that xenotransplantation is "fraught with danger." Yet he revealed that there are currently 12 FDA-approved xenotransplant clinical trials going on in the U.S. Most, if not all, are industry-sponsored, and involve the use of pig cells to treat diabetes and neurological diseases, and whole pig livers and cells to perfuse the blood of patients with acute liver failure.
In order to perform such trials, companies must submit an Investigational New Drug (IND) application. But Jay Siegel, Director of FDA's Office of Therapeutics Research and Review indicated that he would be shocked if there weren't activities being done that are not under IND that should be.
Genzyme, a Cambridge, Massachusetts-based biotech company, had been treating about 100 burn patients per year since 1987 with a xenotransplant product called Epicel, regulated as a 'device'. The company use 3T3 mouse cells to grow layers of human skin,which are then applied to the patient. The mouse cells are allegedly irradiated to prevent them, and any viruses, from proliferating; though when pressed, Genzyme's President admitted that the company was still assessing the efficacy of its irradiation method. And, it had not performed FDA-required tests to determine whether its mouse cells could infect human cells. Most shocking was the company's admission that it had not kept a registry of the patients it treated, nor followed up to see whether any of them might have developed signs of illness or infection. Genzyme said it would be "impractical" to try to find these patients. The FDA seemed to have no knowledge of this situation.
Andrew Dayton of the FDA's Division of Transfusion Transmitted Diseases, and architect of the guidelines, acknowledged that if a xenotransplant-related virus entered the blood supply by mistake, the results would be "disastrous" and the necessary withdrawal of contaminated blood products would cause serious blood shortages.
While some Subcommittee members seemed to downplay the threat of infection by pig viruses, virologist Jonathan Allan commented that, for FDA to recognize infectious disease risks in non-human primates, but not in pigs, is arbitrary. Prem Paul, a veterinary researcher at Iowa State University, warned that new pig viruses were continually being discovered; they had not been extensively studied; and the potential existed for them to mutate and infect humans.
British veterinary pathologist David Onions concurred. He warned that pig parvovirus can change hosts and escape inactivation treatments; and has already been found in Porcine Factor 8 used to treat hemophiliacs.
In May, 2000, a new US Public Health Service (PHS) Guideline on Infectious Disease Issues in Xenotransplantation was published (30). It involves a complex series of measures to store tissue samples for future study and to establish a national xenotransplant database – something that should have been done before clinical trials were approved. As it is, they will only serve to detect disease and virus after it is too late.
The PHS acknowledge that viruses from animals used in xenotranplantation could infect patients, their offspring, health workers and the general public. And even admits that, "all xenotransplantation products pose a risk of infection and disease to humans", "baboon endogenous retrovirus in human recipients of baboon [livers] has been documented", "new viruses capable of infecting humans have been identified in pigs", "all species pose infectious disease risks", and "[xenotransplant recipients] may represent a biohazard to healthy livestock".
According to the PHS guidelines, the sponsors are entrusted to design and monitor xenotransplant trials, tailor complex informed consent documents, educate workers, to effectively screen source animals for viruses, maintain proper documentation, and reliably report crucial information about patient and animal health to federal agencies. There is no mention of who will be held responsible if a novel virus is unleashed, and no emergency procedures to deal with an outbreak have been proposed, even though the PHS acknowledges that "airborne transmission of infectious agents" is possible.
PHS further suggests that some animals from xenotransplant facilities may be considered "safe for human food use or as feed ingredients", in flagrant disregard of the fact that the safety of transgenic food is yet to be established, and the international community has found it necessary to negotiate and agree a Biosafety Protocol regulating the safe use and transfer of genetic engineered products under the UN Convention on Biological Diversity.
If xenotransplantation is to go ahead, it will involve levels of animal suffering unacceptable to the majority of people. As it is extremely inefficient, it will also generate many abnormal failures and surplus animals which have to be disposed of safely. There is as yet no documented, true-breeding transgenic line established to-date.
PHS, in their current guidelines, state that Americans have neither endorsed nor rejected xenotransplantation. But documents obtained through the Freedom of Information Act reveal otherwise. In response to its 1996 draft guideline, PHS received over 160 comments: 115 against xenotransplantation, 29 in favor, and 19 neither for nor against, with 8 of these strongly opposing the use of nonhuman primates. Furthermore, the Food and Drug Administration received almost 6,000 postcards, and over 350 letters protesting its April 1999 guidelines on the use of non-human primates in xenotransplant trials.
The Campaign for Responsible Transplantation (CRT), an international coalition of physicians, scientists, and 90 public interest groups, have denounced the PHS Guidelines as irrational and in violation of the precautionary principle.
Conclusion: stop xenotransplantation for safer, more humane and effective alternatives
Our investigations have revealed how bad science has been involved in the xenotransplant project from the start:
* lack of proper documentation of the transgenic process and characterization of the transgenic pigs
* lack of quality control
* failure to obtain well-characterized stable transgenic lines before transplantation experiments were attempted
* failure to screen for viral infections in experimental xenograft recipients
* use of inconclusive studies to push for clinical trials in humans
* systematic disregard of existing scientific evidence of cross-species viruses arising from xenografts
It is nothing short of a scandal to allow xenotranplantation to go ahead in the light of existing scientific evidence, especially when there are safer, more humane and effective alternatives (17).
Much can be done to increase human organ donation in the short term, especially if an assurance can be made to the donor that the organ will be offered free of commercial interest to the recipient. The use of artificial organs and human cells and tissues will both avoid the risk of cross-species viral epidemics.
One of the most exciting recent development is the possibility of regenerating organs and tissues from the patients' own stem cells (31), cells which retain the ability to multiply and differentiate into a number of different cell types even in the adult. This would avoid immune rejection as well as viral epidemics. We reject the claim that human embryonic stem cells have to be used, which are obtained from human embryos created solely for the purpose. It has now been demonstrated that adult human liver cells can be derived from stem cells originating in the bone marrow (which normally produce blood cells) or circulating outside the liver. This raises the possibility that bone-marrow stem cells, either from a donor or from the patient could be used to generate liver cells for replacing damaged tissue, thus obviating the need for organ transplant altogether (32). Better yet, why not find out how to encourage adult stem cells to regenerate in situ? These alternatives are infinitely preferable to xenotransplantation in being safe, humane, sustainable and affordable; and hence more likely to benefit society as a whole in the industrialized west as well as in the Third World.
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Key words: Superviruses, HIV, SIV, interleukin, GM crops
Researchers have been creating one deadly virus after another in the laboratory, and the latest is 'SHIV', a hybrid between the human and monkey AIDS virus containing human interleukin genes that suppress immune response against viruses. At the same time, GM crops engineered with interleukin genes are being grown in open field trials.
In January this year, researchers in Canberra Australia created a GM mouse-pox virus that killed all its victims simply by inserting into it a gene coding for interleukin 4, a protein belonging to the cytokine family that regulates thymus (T ) helper-cells in the immune response. This deadly virus also killed half of the mice that have been immunized against the mouse-pox virus [1] (See "Genetic engineering superviruses" ISIS News 9/10, July 2001).
Unbeknownst to most of the world, researchers in Kyoto University, Japan, have created far worse. In order to investigate the role of cytokines in the progression of AIDS disease, they made 'SHIV' - a chimeric virus containing several genes from the human virus, HIV, in a basic frame of the monkey virus, SIV - which is capable of infecting both human and monkey cells. Into this SHIV, they insert various human cytokine genes in order to investigate how the virus replicate in cell cultures and in experimental macaque monkeys infected with the GM virus.
In the first experiment reported last year [2], the human gene for interleukin 6, IL-6, was inserted into a SHIV with a deletion in one of the genes from the HIV sequence in the chimeric virus. The deletion slowed the replication of the SHIV, but does not abolish it. IL-6 is known to be elevated in AIDS disease and to contribute to immunological abnormalities in HIV-infected patients. The resultant GM virus successfully replicated in a human thymus cell line as well as in monkey and human peripheral blood mononuclear cells, with high levels of expression of IL-6. Surprisingly, the inserted gene was stable for at least four passages in the human thymus cell line, and it was suggested that the IL-6 gene in the GM virus might make the virus grow faster.
In a second report just published [3], the researchers inserted the human gene for interleukin 5, IL-5, into two SHIVs with deletions in different HIV genes. Again, the GM virus replicated stably in human thymus-derived cell lines as well as monkey peripheral blood mononuclear cells, with very high expression of IL-5, especially in one of the GM viruses.
The researchers stated, "The replication of both SHIVs having IL-5 appeared to be faster than that of the parental viruses without the IL-5 gene. These results show that co-expression of IL-5 stimulates SHIV replication in vitro. Thus, it is expected that expression of IL-5 will also have an effect on viral replication and pathogenicity in vivo." (p.1051, italics ours)
By the admission of the researchers themselves, chimeric viruses such as the SHIV constructions with interleukins are potentially threatening to both humans and primates. Unintended release of these viruses through infection of human beings or escape of experimental monkeys injected with the virus should be matters of grave concern. These GM viruses can readily recombine with all kinds of viruses to give them at least one gene – the interleukin gene – that would make them more virulent.
Do the risks of producing SHIV chimeras with interleukins outweigh the potential benefits that may result from learning about the role of interleukin in facilitating virus multiplication? We do not think so. Such laboratory experiments should never have been approved on grounds of both safety and animal welfare.
In a parallel development, interleukins are being produced in GM crop plants. Field trials of a crop engineered with an interleukin gene had been carried out in the county where one of us reside. The approval has been given without considering the risks associated with pollution of surface and ground water by the protein following plant-wounding or breakage of rootlets. Birds and mammals readily consume sucking insects feeding on the test crop. Interleukin genes may spread by pollen to crop plants and weeds consumed by human beings, livestock as well as wild mammals. The interleukins consumed may lead to suppression of immune response and immune memory, thereby promoting the spread of viral diseases.
In addition, all kinds of viruses may pick up the interleukin gene from the GM crops, to become more lethal than nature's worst.
As field trials and production sites for GM crops producing pharmaceuticals are not made public, the first recognition of their presence near a community may be devastating viral diseases spreading through human, domestic and wild animal populations.
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Poison Pharm Crops Near You
Pharm crops are crops genetically modified to produce gene products that are pharmaceutically active. Such bio-pharmaceuticals are frequently active in minute quantities and expensive to produce in cell cultures or whole animals, and so the companies turn to crop plants.
The range of products currently being produced using mammalian genes introduced into crop plants includes vaccines, immune control proteins such as cytokines, growth hormones and enzymes [1, 2]. There have been a number of field trials of pharm crops in North America but it is difficult to determine the full extent of the trials because they are not regulated in the way that genetically modified food crops are.
In Canada, the field trials are regulated and monitored by the Canadian Food Inspection Agency (CFIA) and the regulation of the products as drugs is not considered until the crop is ready for commercial production. At such a time, the Therapeutic Products Directorate of Health Canada reviews the safety of the product to humans. Thus, the environmental and health impacts of the crops are completely ignored.
In a field test in Ontario near the city of London, the pharm crop is tobacco genetically modified with the gene for the human cytokine, interleukin 10, combined with the cauliflower mosaic virus promoter and the transcription terminator from Agrobacterium [3]. Interleukin 10 is known to be a powerful immunosuppressive. The modified tobacco plants had previously been selected to contain low alkaloid content and to be male sterile (to produce little or no pollen). The field trials were presumed to be safe and approved by CFIA because it was believed that no transgenic tobacco pollen would be produced to fertilize tobacco or weedy relatives.
The CFIA was constituted a few years ago from bureaucrats in Agriculture Canada, with a strong bias towards genetically modified food crops and no apparent expert knowledge of pharmaceuticals and their impact on humans. There was little or no effort to monitor release of interleukin 10 from the tobacco plants in the field, which may follow wounding of the plant parts, normal breakage of feeder roots, damage by sucking insects and other predators. Post harvest root breakdown will also release significant amounts of the immunosuppressant to surface and groundwater and pollute drinking water wells (both dug wells and drilled wells).
Those exposed to the juices of wounded transgenic tobacco as well as those exposed to surface and groundwater from the test plots might become compromised in their ability to resist viral infections.
The possibility that entire fields of plants containing trillions of human interleukin 10 genes may transfer that gene to human viruses should not be ignored. A gene homologous to human interleukin 10 in cytomegalovirus was found to be powerfully immunosuppressive [4]. In other words, a virus with interleukin 10 could be deadly, as it disarms our immune system during an infection.
Furthermore, the human interleukin 10 gene could be mobilized by recombination through contact with insect Baculovirus both in the plant and in the soil. Baculovirses are known to cause nonpathogenic infections of human cells [5] and in that way recombination could create further "superviruses" by contact between baculovirus and any of number of human viruses.
A super virus was accidentally created when another immunosuppressive cytokine, interleukin 4, was combined with mouse pox virus [6]. Viruses with interleukin 10 could become "doomsday" pathogens.
This dangerous field experiment in Canada was undertaken with little public knowledge and discussion. Neither of those responsible for regulating or for testing such dangerous biopharmaceuticals appears to have any regard for the hazards involved. Unfortunately, similar field trials may well have been conducted in the United States and in Europe with equal disregard for environmental and health impacts.
ISIS has been trying to draw attention to this regulatory loophole repeatedly since 1998, and to demand that such bio-pharmaceuticals should be produced in strictly contained facilities [7].
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We have repeatedly warned against using food crops to produce gene drugs and industrial chemicals since 1998 [1-3]. The inevitable contamination of our food supply has now come to light. But the more insidious pollution of our soil, water and air has yet to be assessed [3]. Poisons can seep through the plant roots and dissolve in ground water. Pollen carrying the offending drugs and chemicals could be inhaled. Wild and domestic animals of all kinds are likely to feed on the crops.
On November 11, the US government ordered the biotech company, ProdiGene, to destroy 500,000 bushels of soybeans contaminated with GM maize, engineered to produce a drug not approved for human consumption [4]. The US Department of Agriculture (USDA) refused to give details on the protein involved because it is deemed 'confidentual business information'.
It could be one of the following [5]: the HIV glycoprotein gp120, a blood-clotting agent (aprotinin), a digestive enzyme (trypsin), an industrial adhesive (a fungal enzyme, laccase), vaccines for hepatitis B, vaccine for a pig disease, transmissible gastroenteritis.
USDA records show that ProdiGene has received 85 test permits for experimental open-air trials of pharm crops and chemical crops in at least 96 locations.
The 'edible' AIDS vaccine with the HIV glycoprotein gp120 gene [6] has been condemned as dangerous by a number of AIDS virologists [7-9] because the gp120 gene and gene product can undermine our immune system and generate new viruses and bacteria that cause diseases.
A day later, the US government disclosed that ProdiGene did the same thing in Iowa back in September. The USDA ordered 155 acres of nearby corn to be incinerated for fear of contamination [10,11].
This is just the tip of the iceberg. The true extent of the contamination remains unknown owing to the secrecy surrounding more than 300 field trials of such crops across the country since 1991. Still others sites are in Canada [3]. The chemicals these plants produce include vaccines, growth hormones, clotting agents, industrial enzymes, human antibodies, contraceptives, immune suppressive cytokines and abortion-inducing drugs.
The majority of engineered biopharmaceuticals are being incorporated into maize. ProdiGene, the company at the centre of the current scandal has the greatest number of pharm crops and projects that 10 percent of the US maize will be devoted to biopharm products by 2010.
Far from supporting even weak containment strategies such as buffer zones, ProdiGene has told its shareholders it is hoping to "gain regulatory approval to lessen or abandon these requirements altogether".
Trials in other countries have also come to light. According to a recent report by Genetically Engineered Food Alert, a US-based coalition of environmental and consumer advocacy groups, Puerto Rico is one of four main centres in the US for these tests. The other three are the states of Nebraska, Wisconsin and Hawaii.
Another report by the same group reveals that these plants are by no means the only experimental GM crops grown in Puerto Rico. This Caribbean island has been host to 2,296 USDA-approved GM open-air field tests as of January 2001, making Puerto Rico host to more GM food experiments per square mile than any US state, except Hawaii.
Puerto Rico is not a state. Its residents are US citizens but have no voice or vote in the US Congress or in the UN.
Puerto Rico Farmers Association president Ramon Gonzalez revealed that he plants GM crops in his farm in the town of Salinas. He said that genetically modified crops in Puerto Rico are commercial and include a herbicide-resistant soya made by Monsanto (Roundup-ready) and a variety of corn that produces its own bio-pesticide, or Bt corn.
According to Gonzalez, the harvested GM crops planted there are sold as seed to be planted elsewhere. "Puerto Rico is the preferred place to make seed because our weather permits us to have up to four harvests a year."
Local regulatory agencies seem to be unaware of the issue. A spokeswoman for the Puerto Rico Environmental Quality Board said that as Puerto Rico has no laws or regulations for GM crops, it has no mandate to intervene or investigate.
USDA spokesman Jim Rogers is reported to have said, "Nobody's going to know all the possible risks", and "We mitigate these risks to what we feel is appropriate" [12].
On the contrary, we do know enough of the risks for such crops to be banned immediately. The USDA and other government regulators have been warned, and they should be held liable for all damages along with the companies involved.
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|Real Estate Mutual Funds Jivi
User ID: 784535
United States
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User ID: 781339
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SHIT LOAD OF TEXT!!Quoting: vet ikke
Anyone who reads it deserves a medal.
That said, of course genetically modified food is bad for you, there is a reason nature doesn't put these things in itself.
One who chooses selfish actions has their reward, one who chooses selfless acts, also have their reward.
|Dems Trapped by Their Russia Conspiracy, 1681
|42m 22s
|Let's talk about real conspiracies
|17h 20m
|Conspiracy : Can't Flip Channels to Avoid Commercials anymore ALL TV Stations in on it
|15h 16m
|Its officially boring - no doom no actual conspiracy discussion. Just trump dindus and incels
|5h 5m
|The conspiracy of low T
|14h 14m
|Related Topic: GM foods (Science)