The scientific risks of GMO release.

1. The scientific risks of GMO release. Dr. William H.L Stafford, Advanced Research Center for Applied Microbiology, Department of Biotechnology, University of the Western Cape.

2. GMOs and breeding • We have been modifying our food sources for thousands of years, selecting for favourable characteristics. These breeding techniques rely on fertlization by cross-pollination of the same species • Biotechnology uses the tools of genetic engineering to modify a plant with any chosen characteristic. Species barriers can be crossed- we can take a gene from one organism and place it in another totally unrelated organism and create transgenics or GMOs

3. GMOs © Strong promoter © Marker gene © Desired gene © Terminator Marker gene for antibiotic resistance (ampicillin, kanamycin) Viral Promoter eg. cauliflower mosaic virus (35SCamV)

4. GMOs (food crops) have been released -an experiment • Estimated 80 million hectares of GMOs planted to date. Mainly cotton, maize, canola, soya. • GMO crops that have Herbicide resistant genes or genes for insecticidal toxins account for more than 93% of the types of GM crops grown worldwide..

5. Scientific Risk Assessments Absence of Risk = Absence of Science.. • Absence of evidence is not evidence of absence! • ”No harms reported” is not the same as ”no harms exist”!

6. Environmental interactions Outcrossing and horizontal gene transfer Consumption of GMO plant: humans, birds, insects, amphibians, microbes Soil microbiota Stability and persistence of transgene product (eg. Insecticide, herbicide)

7. ENVIRONMENTAL RISKS Random integration into the genome • Despite its importance for safety assessment, applications submitted to the USDA requesting permission to commercialise a transgenic line provide neither the sequence of the genomic DNA flanking the inserted transgene nor a comparison with the original genome. • Truncations, rearrangements, tandem repeats at one or more sites (perhaps reflecting the instability of the gene constructs) have been reported. (Collonier, et al. 2003)

8. Sequence expected Sequence observed T25 maize - LibertylinkTM (Bayer)Tolerance to herbicide glufosinateConstruct content : truncated bla gene (bla*), pUC cloning vector (pUC), synthetic pat gene (pat), CaMV 35S promotor and terminator (P35S, T35S). pUC18 pat T35S bla* 35SCaMV pUC18 35SCaMV T35S pUC18 pat bla* Maize DNA (Presence of cloning vector + the 5 first bp of bla on the 3’ end ) bla* 35SCaMV • Part of pUC cloning vector containing truncated B-lactamase gene (bla) • DNA rearrangement: presence of a second truncated and rearranged P35S on the 5’ end. • Insertion site: the 5’ and 3’ ends of the insert show homologies with Huck retrotransposons.

9. Rearrangements, deletions and multiple insertions…. • Mon 810 maizeYieldGard (Monsanto) • Modified for resistance to lepidopteran insects (butterflies & moths). Company data showed insert has a P35S driving a CrylAb synthetic gene with terminator T-nos. Analysis revealed however, that T-nos and part of the 3’ (tail) end of the CrylAb gene have been deleted. T-nos has been detected elsewhere in the genome. The 5’ (head) end of the insertion site shows homology to the long terminal repeats (LTR) of the maize alpha Zein gene cluster. • GTS 40-3-2 Roundup ready soybean (Monsanto) • Modified for tolerance to herbicide glyphosate. Company data showed 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 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. • Bt 176 maize (Syngenta) • Modified for tolerance to herbicide glufosinate, male sterility and insect resistance. Company data showed insert contains P35S driving the bar gene (glufosinate tolerance) terminated by T35S, followed by the ampicillin resistance (bla) gene plus bacterial promoter, and plasmid origin of replication, ori. Analysis revealed several fragments, all containing CaMV 35S promoter, 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. There were at least three insertion sites. Collonier C, Berthier G, Boyer F, Duplan M-N, Fernandez S, Kebdani N, Kobilinsky A, Romanuk M, Bertheau Y. Characterization of commercial GMO inserts: a source of useful material to study genome fluidity. Poster courtesy of Pr. Gilles-Eric Seralini, Président du Conseil Scientifique du CRII-GEN, www.crii-gen.org

10. How do they occur ? • When transgenic DNA is introduced into the plant cell a wound-response produces DNA repair enzymes that use DNA fragments for DNA repair, resulting in its rearrangement of the plant DNA. • Twelve representative transgenic rice lines were analyzed, and found to have several rearrangements demonstrating transgenicinstability. The 35SCamV promoter was identified as recombination hotspot(Kohli et al.,1999).

11. Gene transfer and escape • Genes can spread from transgenic plants by ordinary cross-pollination to nontransgenic plants of the same or similar species, and also by horizontal gene transfer to unrelated species. • With selection, such elements have increased penetrance into the environment and cannot easily be contained or controlled once they have entered the wider environment!

12. Hybridisation, Outcrossing and escape: Gene transfer and escape happens • Herbicide resistant transgenes from GMO plants were 20 times more likely to escape and spread than the same gene obtained by mutagenesis. (Bergelson, J. et al. 1998.) • Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico (Quist D and Chapela IH. 2001). Several cobs tested positive for the CaMV 35S promoter and sequence analysis of insertion site by inverse PCR indicated diverse sequences. Follow-up studies by two Mexican government laboratories found evidence of the CaMV 35S promoter in 12% of plants sample from Oaxaca and the adjacent state of Puebla (Mann 2002). • Transgenic DNA containing the CaMV 35S promoter is unstable and can randomly insert into the genome.

13. Contamination Pollen flow may occur over large distances for some crops (km for maize, canola; only m for potato). Transport, storage, and processing of seeds and crops are also routes for contamination. • StarLink cry9 corn was approved for animal feed but not human consumption. It was discovered in a wide variety of processed foods. Despite a massive recall of food products and extraordinary efforts to cry9 transgenes still persisted at detectable levels in US corn supplies 3 years later (USDA 2003b).

14. Widespread contamination of seedlots • Seed purity has long been an important issue for agronomists and plant breeders. Test on non-GM canola seedlots were tested, the majority of tested seedlots contained at least trace amounts of genetically engineered herbicide-tolerance traits. In fact, 97% (32 of 33) of the seedlots tested by Friesen et al. (2003), and 59% (41 of 70) of the seedlot stested by Downey and Beckie (2002) had foreign transgenes present at detectable levels (above 0.01%). This level of contamination in pedigreed seed is disturbing since even stringent segregation systems were not sufficient to deliver pure non-GM canola seed to farmers in western Canada.

15. Human error and contamination • BT11 maize (Cry1Ab insecticidal toxin under the 35SCaMV promoter and pat gene for resistance to the herbicide glufosinate, Basta) was approved for commercial release and grown in the USA (2001) and later in South Africa (2003). • December 2004, the Syngenta informed the US government that it had just learned that the Bt11 corn had been mislabeled and 165 000 tons of Bt10 seed were grown in the US and the resultant maize was sold as in the US and abroad.

16. Although Bt10 is “functionally equivalent” to Bt11 there are differences: • Bt11 is approved for commercial release and human consumption in, Bt10 is not. • The transgene is inserted at a different position in the plant DNA. • Bt10 produces only about 1/7th the amount of the insecticidal protein as Bt11. • More than one Cry1Ab toxin protein produced in Bt11. • The most substantial difference between Bt10 and Bt11 is that Bt10 contains the bla gene for ampicillin resistance.

17. Effects on biodiversity • The most obvious effects of cross-pollination already identified are in creating herbicide-tolerant, or insecticidal weeds and superweeds and the loss of locally adapted (‘indigenous’) crop varieties. Studies with oilseed rape (Brassica napus) have shown that the Bt gene can be passed on to a wild, weedier relative (Brassica rapa) (Halfhill, M.D., et al. 2002.).

18. Horizontal Gene Transfer (HGT) • Horizontal transfer from the transgenic plants may spread the novel genes and gene-constructs to unrelated species- bactreia, fungi and viruses in the soil, worms, insects reptiles, birds, small mammals and human beings • Horizontal gene transfer has been rare the billions of years of our evolution, because of natural species barriers preventing genetic exchange and because there are mechanisms which inactivate or break down foreign DNA. • Horizontal gene transfer of genes from one species to another may be a major factor in evolutionary change (Syvanen, M. 1986)

19. HGT to soil microbes matters • Microorganisms dominate soil-borne communities, and largely determine ecosystem functions, such as nutrient cycling and decomposition. Their direct and indirect interactions with plants create strong feedback mechanisms, influencing primary production and vegetation dynamics. They are important in both plant pathogenicity and protection. • The majority (>99%) of the microbial world is uncultured. Thousands of bacterial species per gram of soil whose functions are unknown! • Bacteria can take up DNA by several ways- transformation (DNA uptake), transduction (virus) or conjugation (Hfr mating) HGT to bacteria can spread antibiotic resistance marker genes to pathogenic microbes and disrupt ecosystem function.

20. Factors that increase HGT to bacteria • Factors affecting the likelihood of uptake of DNA into soil-borne microbes include: the stability of the inserted genetic material, the presence of similar (homologous) sequences in resident microorganisms the length of time that GM material or DNA remains intact and the proximity of potential recipient species. • Agents that induce a stress response (J. Beaber et al., 2003)“SOS response promotes horizontal dissemination of antibiotic resistance genes,” • Agricultural practices (Continual selection eg. Herbicide. Ploughing, etc).

21. HGT to soil bacteria • Horizontal gene transfer (HGT) occurs at very low frequency (indetectable using some methods) • Experiments with the the nptII gene in transgenic potato plants coding for kanamycin resistance, detected HGT to Pseudomonas stutzeri and Acinetobacter BD413 at a frequency of 3x10-5 -1x10-4 despite the presence of a more than 106 fold excess of plant DNA. This dropped to 10-16–10-17 when no sequence similarity was present (de Vries J., Wackernagel W., 1998). HGT to bacteria is most efficient where sequence similarity is present. • The bla and npt genes used as antibiotic resistance markers in some GM crops show sequence similarity to bacterial genes. • The pat and epsps genes (T25 maize - LibertylinkTM (Bayer) and GA21 maize (Monsanto) confer resistance to the glufosinate and glyphosphate herbicides (Basta and Roundup) also show significant sequence similarity to bacterial genes.

22. HGT and virus recombination • New and successful variants of viruses do arise naturally by recombination with a frequency that varies depending on the virus family (e.g. Chenault and Melcher 1994; Revers et al 1996; Padidam et al 1999). Success of a given variant depends upon the conditions (selective pressures). • The CamV is a recombination hotspot and therefore subject to increased HGT. CamV is from pararetroviruses, family that includes Hepatitis. • Reactivation of dormant viruses and the generation of new viruses?

23. Effects of HGT • More important than the frequency of HGT is what happens to the resulting transgenic microorganisms. Without positive selection for the new trait, it will soon be lost and have no further impact on the system. However, even a low frequency event can have an important impact if selection is strong. • The novelty of a trait can also influence its potential impact, as completely novel genes might give rise to new genetic variants that are not possible within the normal genetic pool of the system.

24. Summary of ENVIRONMENTAL RISKS : • Generation of new bacterial pathogens and the spread of drug and antibiotic resistance marker genes among pathogens Reactivation and the generation of new viruses • Increased resistance to herbicides, leading to super-weed characteristics • Position effects with unwanted changes in gene expression and occurrence of cancers caused by random inserton (insertional mutagenesis). • Reduced biodiversity due to GMO selection, outcrossing and contamination • Unpredictable effects on genetic evolution and ecosystem function

25. High levels of expression of transgene • The CaMv is a strong promoter providing a high level of gene expression. • All living organisms that interact with the transgenic plant (bacteria-birds and human beings) are exposed to high levels of the expressed transgene that are new to their physiology. Adverse immunological or allergic responses can be expected. The GMO insecticidal toxins such as Bt have been shown to affect beneficial non-target organisms including lacewings, ladybirds and earthworms (Birch, A.N.E., et al. 1997, and Marvier, M. 2001.) Bt can persist in certain soil types for up to 234 days. • There is evidence of insect pests becoming resistant after only a few years after the transgenic crops were first released since Bt-toxin genes are expressed continuously at high levels throughout the growing season.

26. DNA persistence in the environment • DNA is not completely broken down in the gut. Genes can spread from ingested transgenic plant material to bacteria in the gut. Antibiotic resistant marker genes from genetically engineered bacteria can be transferred to indigenous gut bacteria (Netherwood T, et al. Technical report on the Food Standards Agency project G010008) • DNA can persist in the soil for years. Transgenes from GMO plants may be able to spread to soil bacteria, spreading antibiotic resistance marker genes among the pathogens. Practically every medical organization that has looked at GM crop safety has expressed concern, including the American Medical Association, World Health Organization, UK Royal Society, United Nations Food and Agriculture Organization, Pasteur Institute, European Food Safety Authority, and Codex Alimentarius • The EU has decided to prohibit GMOs with antibiotic resistance genes after the 31st December 2004 (directive 2001/18EC and Revising Directive 90/220/CEE

27. HEALTH RISKS: Expected and unexpected toxicity • Transgenic potatoes expressing GNA insecticide (“Snowdrop”, Galanthus nivalis, lectin) fed to rats resulted in increase in intestinal mucosal thickness and T-lymphocyte infiltration. (Ewen S. W. B, and Pusztai, A. 1999) • Monsanto's transgenic soya, has a 26.7% increase in a trypsin-inhibitor and has been shown to inhibit the growth rate of male rats. This raises the possibility that transgenic soya is responsible for the reported recent increase in soya allergy. • Human gene therapy experiments for severe combined immunodeficiency (SCID) caused by a single non-functional gene (adenosine deaminase) were halted by the FDA after a second treated child died of cancer. Molecular analysis showed that the T cells were a single clone derived from one original cell that has multiplied out of control. The retroviral vector used – mouse Moloney leukaemia virus – had inserted into a gene on chromosome 11 causing truncation gene trucation and oncogenesis.

28. Requirements for GMO release • Efficient and specific gene targeting to cells • Stable, single insertion of gene at defined site with no other DNA (viral promoters or antibiotic resistance genes) • Normal levels of expression of desired gene • Proven safety (consumption and the evironment). X The GMO crops on the market fulfill none of these criteria

29. Better methods available? • Site specific recombination using Zn finger protein linked to integrase-target gene. • Control under endogenous promoters. • Plastid engineering (Daniell et al., 2002) • Other marker genes, such as green fluorescent protein, or mannose (Joersbo et al., 1998) • Removing the antibiotic resistance genes before the plants are released for commercial use (Lamtham and Day, 2000; Zuo et al., 2001), so that these genes can be used during development and then eliminated from the final product. • Use of introns to prevent expression in bacteria while allowing plant expression (Libiakova et al., 2001).

30. A lack of labelling and monitoring The transgenic genes of GMO crops are covered by patent. Since these GMO crops can be considered novel inventions however the food-crop is considered “functionally equivalent” (mainly un broad nutritional grounds) and no labelling is required in many countries including as South Africa. If there are problems…… there is no labelling, no monitoring and ten there will be difficulties in tracking and establishing liability. Can we recall the releaseof a GMO from the environment? Which is more cost effective- proper risk assessment, monitoring &labelling or loss of markets (EU) and clean-up cost from contamination?

31. Future of GMOs • It is important to distinguish between contained use of transgenic organisms and their release to the environment. • It is vital that GMO crops are proven safe for through proper independent, long-term feeding trials and environmental impact assessments • It is essential to monitor GMOs since they have been released and we need to observe the effects in this experiment

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