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ISIS Report 07/03/08

USDA FONSI for Transgenic Poplars Absurd & Dangerous

Prof. Joe Cummins and Dr. Mae-Wan Ho expose cavalier risk assessment that ignores existing evidence and takes absence of evidence as evidence of absence

A fully referenced version of this report was submitted to USDA on behalf of ISIS.

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The Animal and Plant Health Inspection Service of the United States Department of Agriculture (APHIS/USDA) received a permit application (APHIS number 06–250–01r) from Oregon State University to conduct field tests using clones of transgenic poplars and hybrids. Permit application 06–250–01r includes trees with 95 transgenic constructs categorized only by their intended traits, with little or no other details: reproductive sterility genes, genes affecting stature or reduced light response, genes modifying tree physiology, and activation tagging mutants aimed at developing “experimental domesticates”.

An Environment Assessment (EA) [1] prepared by APHIS for this permit was open for public comment for 30 days; and on 4 February 2008 APHIS published their evaluation of public comments and awarded the open field experiments with transgenic poplars the verdict FONSI [2](finding of no significant impact). Their response to public comments were aimed largely at those submitted by the Institute of Science in Society (ISIS) [3] (Unregulated Release of GM Poplars and Hybrids, SiS 36), and showed that APHIS’ reviewers support field tests particularly in areas where evidence of safety is entirely absent or insufficient.  In such areas, the reviewers presumed that the transgenic trees are safe, and defy the public to produce evidence of hazard, thereby turning environmental assessment on its head.  As the transgenic trees are novel, the purpose of APHIS’ EA was to provide evidence that they are safe, and where evidence of hazard is absent, it cannot be taken to be evidence that hazard is absent. The review even resorted to the irrelevant remark that Science in Society, published by ISIS, was not a credible science source, as it was a journal of  “Marxist thought and analysis”, thereby also exposing the reviewers’ ignorance of both Marxism and Science in Society.  We challenge any rational reader to find anything Marxist in Science in Society. Furthermore, the articles in question are all reviews of the scientific literature published in professional journals, which have been ignored by APHIS.

We give some examples below.

Barnase A used in producing sterile poplars is a potent toxin 

Indeed, the toxicity of Barnase A was dismissed by just such a remark. The FONSI document stated [2]: “APHIS disputes the claim that the toxicity of barnase to mammals is well known. The articles the commenter cites are not from a credible science source but from a journal of Marxist thought and analysis. As stated above, no adverse effects to wildlife or humans are expected from expressing the barnase in the developing flowers. Because the hazard of the protein is extremely low and has been consumed by animals and humans with no adverse effects [no references cited here], APHIS reasonably concludes that there should not be a significant adverse effect on wildlife from the expression of barnase and barstar in this GE field release.”

APHIS is wrong. Barnase is a potent toxin. Two scientific studies listed in at least one of the Science in Society reports document that barnase is toxic for rat kidneys at 15 ppm (parts per million) [4] and to a range of human cells at 40 to 1000 ppb (parts per billion) [5]. It is also highly toxic for plant cells, which is why it is used for destroying cells producing flowers by putting it under the control of a promoter supposed to be specific for flowering tissues. Unfortunately, the applicants’ own research show that the promoter is not so specific for flowering tissue, and the expression of barnase in vegetative cells had drastic effects on plant growth and development, even when the barnase gene is accompanied by the gene for barstar, the specific inhibitor of baranse [5]. When the barnase gene is used to transform poplar cells without the barstar gene, no living transformants were obtained. Thus barnase is one of the most potent cytotoxins. APHIS maintains that barnase is present only in the flowers of the transgenic poplar, but they are fully aware that barnase is present throughout the plant: in the leaves, roots and stems of the transgenic poplars, and only to some extent neutralized by barstar. Animals feeding on the stem, leaves and roots could be affected. As the leaves, stems and roots fall to the forest floor and begin to decay, it is likely that the barnase-barstar complex will dissociate and affect animals and microorganisms in the soil.

APHIS maintained that the use of barnase is safe because the gene for barnase is already in some released food crops such as maize and oilseed rape, and there have been no reports of injury from those consuming the modified crops. However, APHIS is fully aware that the modified crops are not labelled in the market and there have been no scientific efforts to follow the impact of the modified food or feed crops on human or animal populations.

Diphtheria toxin A-chain also far from safe 

According to APHIS, “There should be very limited exposure of animals to the A-chain component of DTA. That is because the A-chain is primarily expressed in the cells of developing flowers. During flower development there would be very few cells expressing the protein and for only a short period of time. There could be a very low level of expression in vegetative tissues based on the promoter being used to drive the gene.”

Diphtheria toxin is produced normally by Corynebacterium diphtheriae when it is infected by the phage (bacterial virus) β. The gene that codes for the toxin is carried by the phage and is inserted into the bacterial chromosome along with the phage genome. The active toxin gene product is cleaved into two, the A and B chains. The A chain is the toxin that prevents protein synthesis in the cell, thereby killing it.  The B chain is necessary for the A chain to gain access into a cell. The gene for the A chain toxin is the one inserted into the poplar to make the trees sterile, and so it should be functioning only within the cell that expresses the toxin.

However, there are many non-toxin producing Corynebacterium species present in the human population and in the general environment that may be lacking the phage b, or else carrying a defective phage with a mutation in the A chain. These bacteria can be transformed into active toxin producing strains by acquiring a phage or recombining directly or via a phage with the A chain in the poplar [6]. The chromosomal attachment sites (for the phage that carries diphtheria toxin) of several Corynebacterium species are identical but for a short variable section [7], and this will facilitate horizontal gene transfer and recombination to generate toxin-producing strains.  Several Corynebacterium species of medical importance are found in the soil, while Corynebacterium diphtheria has been found in cattle [8]. It is clear that APHIS has ignored well documented aspects of diphtheria toxin biology in pronouncing diphtheria toxin A chain safe.

MADS-box genes of questionable safety:

APHIS disagreed that the introduction of an additional MADS-box gene into poplar trees should be presumed unsafe because they are homologues of animal homeotic genes and may be active in animals. APHIS claimed there is no scientific reason to believe that plant MADS proteins could have a significant impact on animals.

We shall quote from an important publication on plant MADS-box genes [9]: “ Floral homeotic genes that control the specification of meristem and organ identity in developing flowers have been isolated from both Arabidopsis thaliana and Antirrhinum majus. Most of these genes belong to a large family of regulatory genes and possess a characteristic DNA binding domain known as the MADS-box. Members of this gene family display primarily floral-specific expression and are homologous to transcription factors found in several animal and fungal species.”

Homology normally refers to similarity of DNA sequence, which means they are likely to have similar functions. APHIS’ claim is entirely unjustified.

RNA interference ubiquitous and hazardous

According to APHIS: “..one comment points out the use of RNA interference (RNAi) gene therapy and its potential for adverse health effects based on studies with mice. RNAi involves the use of a small interfering double stranded RNA of approximately 21-15 nucleotides that is complimentary to a known messenger RNA that is used to block its expression. In this case Populus RNAi genes have been used in an attempt to induce sterile flowers.”

APHIS’ response was: “The safety of nucleic acids is widely accepted. Both RNA and DNA are part of all food products that we consume. “ This is a misleading and ignorant statement of truly gigantic proportions. If all nucleic acids, regardless of sequences were equivalent, there would be no genetics and no genetic engineering. No wonder APHIS’ reviewers are dismissing scientific findings that RNAi frequently has pleiotropic (i.e. unintended) effects, including death [10-12]. Small RNA molecules are now known to be ubiquitous in regulatory circuits, their actions depending not only on sequence specificity to some extent, but also on minutely precise timing and location of expression, which is beyond the ken of the crude genetic engineering that can be done in the laboratory.  This, more than anything else, exposes the entire regulatory programme of APHIS to be a complete sham, as we have documented [13].

The gibberellin gene family and phytochrome receptor genes

APHIS commented  “Thus while there is no reason to believe that the light response and gibberellin genes will have any untoward effects, the experiment is conducted in a way that even if they did, the material will be confined to the test site.”

APHIS failed to refer to any published study on transgenic phytochromes or gibberellins. There does not seem to have been any effort to study the impact of the transgenic trees in truly contained conditions first. There have been numerous instances where modified crops or their pollen have escaped from open field trials. It is highly unlikely that serious untoward consequences of gibberellins and phytochrome receptor genes or other genes and sequences contained in the 95 constructs would be contained in an open field trial. But this is typical of the cavalier attitude of APHIS.

Activation tagging and unintended insertion mutagenesis

Activation tagging is insertional mutagenesis, using insertion vectors that contain a strong transcription enhancer to up-regulate a gene near the insertion site. The insertions appear randomly in the genome, resulting in a gain of function that represents a dominant mutation. APHIS noted the commenter (ISIS) does not believe that it is safe for field test releases because “it is likely to cause unintended insertional mutagenesis in a range of microorganisms and animals that interact with the transgenic plants.” APHIS disagrees with this comment: “The commenter did not provide any refereed citations that would substantiate their assumption. The inserted DNA used for the activation tagging is stably integrated into the Poplar genome. It is no more likely to cause unintended insertional mutagenesis in another organism than any other DNA within the Poplar. “

The activation tagged mutants of poplar to be released in the field test were modified  using Agrobacterium to transfer the genes for insertion and the T sequences. These mutant strains certainly bear the T DNA border sequences typical of Agrobacterium transformation, which are special breakpoints, i.e., recombination hotspots. It has been found that Agrobacterium T DNA can transform human cells including cancer cells as well as kidney and neuron cells by inserting into the cells’ genome [14]. Thus the genes transformed into poplars are certainly different from the native poplar genes in being flanked by the borders of the T DNA, which make them more prone to ‘break and run’, and worse yet, to re-insert into another site in the same genome or into the genome of another cell. This possibility has not been investigated by dedicated research. But, contrary to APHIS claim, our comments were accompanied by supporting references. In particular, we referred to a detailed review on the hazards of transgenic nucleic acids including the possibilities of insertion mutagenesis [15] (SLIPPING THROUGH THE REGULATORY NET: ‘Naked’ and ‘free’ nucleic acids).

Horizontal gene transfer cannot be denied

The issue of horizontal gene transfer and recombination has been raised in connection with the diphtheria A toxin and activation tagging above. Actually, this danger is generic to transgenic DNA, as first pointed out in some of ISIS’ earlier publications [15, 16] (Genetic Engineering Dream or Nightmare), and more generally acknowledged since. Several of the references cited by APHIS’ EA [1] to dismiss horizontal gene transfer actually provided the very evidence denied. In a separate report [17] (Horizontal Gene Transfer from GMOs Does Happen, SiS 38), we update the evidence on the horizontal transfer of transgenic DNA from GM plants, which not only confirms that it does happen, but also that it is greatly underestimated in bacteria, and that eukaryotic genomes may be even softer targets for integrating transgenic DNA.

Conclusion

APHIS reviewers do not seem to be at all well informed about the scientific literature and they have provided an uncritical review of the application for field-testing genetically modified poplar, disregarding all serious safety issues that we and others have raised. Opining that our publication Science in Society is of ‘Marxist thought and analysis’ and ignoring the fact that the articles were reviews of the scientific literature is simply adding insult on injury. This comment seems to be the off-hand response of some dim-witted bureaucrat, but could in reality serve as a threat to all who participate in APHIS’ public comment.  Such comments from a government bureaucracy though groundless, is not to be taken lightly, as it can trigger wide-ranging government actions in the name of counter-terrorism that may include travel restrictions and other invasive measures. APHIS should withdraw the FONSI ruling from the poplar field tests. And we must demand that APHIS retract the comment on Science in Society being of “Marxist thought and analysis.”

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