Dow Agroscience petitions for deregulating new GM maize tolerant to 2,4-D and Quizalofop
by Prof. Joe Cummins ISIS Report 09/02/12
The United States Department of Agriculture (USDA) Animal and Plant Inspection Service (APHIS) has announced that Dow AgroScience Company is seeking deregulation of a new genetically engineered corn (DAS -4027809), tolerant to broadleaf phenoxy auxin herbicides such as 2,4-D and grass herbicides such as quizalofop, and is soliciting public comments by February 27, 2012, to be submitted at: http://www.regulations.gov/#!searchResults;rpp=10;po=0;s=APHIS-2010-0103
The herbicide 2,4-D was discovered in the early 1940s . For many years 2,4-D and its relatives in the phenoxy auxin group were the primary chemical used to control broad leaf weeds. In recent years 2,4-D has become the third most widely used herbicide in the world behind glyphosate and atrazine. The development of 2,4-D resistant crops will greatly increase the use of the herbicide and greatly amplify the environmental pollution associated with this old herbicide. Introduction of genetically modified (GM) crops tolerant to it is a step backwards for health and the environment.
According to USDA Economic Research Service, 73 % of the area planted to maize in the United States is GM herbicide tolerant (HT) varieties . It has become increasingly clear that the current plantings of HT maize are plagued by weeds that have grown as tolerant to the herbicides as the HT maize.
As a solution, Dow Agrosciences are putting forward a GM maize variety DAS-40278-9 that is tolerant to the old herbicide 2,4-D and its relatives of the phenoxy auxin group, as well as the herbicide quizalofop, along with its chemical relatives of the aryloxyphenoxypropionate acetyl coenzyme A carboxylase (ACCase) inhibitor group; and has petitioned USDA/APHIS for non-regulated status . APHIS has duly prepared a draft environment assessment .
What is event DAS-40278-9 ?
DAS-40278-9 corn plants have been genetically modified to express the aryloxyalkanoate dioxygenase (AAD-1) protein. The AAD-1 protein is an enzyme with an alpha ketoglutarate-dependent dioxygenase activity which results in metabolic inactivation of the herbicides of the aryloxyalkanoate family. The aad-1 gene coding for the AAD-1 protein was derived from Sphingobium herbicidovorans, a gram-negative soil bacterium. The aad-1 gene was introduced into DAS-40278-9 corn  using Whiskers-mediated transformation (see Box).
The aad-1 gene sequence was adapted for expression in corn, with many changes in synonymous codons (coding for the same amino acids) and inserted into a plant expression cassette to make a plasmid pDAS1740. The final transformation fragment was a 6 236 base pair DNA which contained the matrix attachment region (MAR) from Nicotiana tobacum , maize ZmUbi1 promoter, synthetic plant optimized aad-1 gene, and 3’ untranslated region from maize peroxidase gene ma. The linear DNA fragment, without the remainder of the plasmid, was used to transform embryogenic cell suspensions of Hi-II corn using silicon carbide whisker fibres for direct DNA insertion. Selection of transformation events was based on tolerance to R-haloxyfop herbicide and regeneration of aad-1 maize plants. The plants were backcrossed and selfed to create elite inbred lines and hybrids containing the aad-1 gene. The DAS-40278-9 event was selected as the lead commercial candidate .
Whiskers transformation gene delivery technique
Whiskers transformation is a recent advance in plant genetic engineering. Silicon carbide whiskers are microfibres 10–80- mm long and 0.6-m m wide. The vigorous agitation of plant cells in the presence of whiskers results in the formation of micron-sized holes in the cell wall, thereby allowing the entry of macromolecules. Successful uptake and integration of DNA into maize cells following whiskers treatment has been studied . Such whiskers must be handled with care in the laboratory because inhalation of such fibres creates severe fibrotic lesions in the lung .
Safety of the AAD-1 protein in question
It is claimed that the data presented in the petition (DAS 2010, pages 76-101) indicate no material difference in compositional and nutritional quality of DAS-40278-9 corn compared to other commercially available corn apart from the presence of the AAD-1 protein. A few variables did show statistically significant differences between DAS-40278-9 corn and control corn grown at the same locations. However, these differences were deemed to be relatively small and none of the values were outside the range of natural variability of conventional corn reported by the International Life Sciences Institute Crop Composition Database . But it is not acceptable practice to dismiss significant differences between an isogenic non-GM maize and the modified strain grown in the same experimental location on the basis that maize grown under different conditions showed variation as large as the observed differences.
As indicated in the Dow petition  and in a fuller peer-reviewed study, acute and 28-day repeated dose toxicology studies in mice with aryloxyalkanoate dioxygenase (AAD-1) protein expressed in 2,4-D tolerant DAS-40278-9 maize, presented a negligible risk to humans . However, the AAD-1 studied was not produced in maize but instead in the gram negative soil bacterium Sphingobium herbicidovorans. It was cheaper to produce a protein by bacterial fermentation than it was to isolate that protein from maize. But the gene producing the protein was different from the gene used to transform maize. As indicated in the Dow petition the gene for AAD-1 protein was extensively altered by making synonymous changes in the DNA code to allow production of the protein in maize. Such altered gene changes are typical of all of the bacterial transgenes in commercial GM crops, as is the practice of using the proteins from the bacteria instead of from the GM crop to test for safety.
Biotechnology had long assumed that any genetic mutation that does not alter a protein sequence should have no impact on human health. But recent research has shown that such synonymous DNA changes can trigger disease in a number of ways. Silent mutations are now recognized to arise from transgenes synonymously modified and such 'silent' mutations seem to be caused by changes in the conformation of proteins brought about during translation of the novel transgenes . These silent mutations are ignored and go undetected in the GM crops, which are not labelled in food and feed. The AAD- protein tested to ascertain the safety of the GM maize is from a bacterium, and is definitely not the same as the AAD-1 protein in DAS-40278-9 Maize. Thus the safety of the GM protein remains untested
Horizontal gene flow assessment
According to the Dow petition , “There is no known mechanism for, or definitive demonstration of, DNA transfer from plants to microbes. Even if such a transfer were to take place, transfer of the aad-1 gene from line DAS-40278-9 would not present a human health or plant pest risk, based on the safety data presented in this petition. The gene encoding the AAD-1 protein is from a naturally occurring soil bacterium, Sphingobium herbicidovorans, and is already present in nature. Transfer recipients would, therefore, not pose a greater plant pest risk than the environmentally prevalent wild type microbes from which the genes originate.”
Contrary to the bland assurance from Dow, genetically modified genes can indeed jump species from plant to bacteria in the soil, and in the air via wounds according to new research. Horizontal gene transfer does happen, and at high frequencies; it is the greatest, most underestimated hazard from GMOs released into the environment (see  Scientists Discover New Route for GM-gene 'Escape', SiS 50). Dow is revealing an appalling degree of ignorance, wilful or otherwise.
Impact of increased use of phenoxy auxin and the aryloxyphenoxypropionate acetyl coenzyme A carboxylase inhibitor herbicides
Neither the Dow petition nor the Aphis environment assessment  deals adequately with the consequences of the increased use of the herbicides such as 2,4-D and quizalofop on human and environmental health. 2,4-D and its relatives have been studied extensively over more than seventy years that the herbicide has been in use, while quizalofop and its relatives have been used for about a decade and most of the studies of the environmental impact are done by corporate producers of the herbicides.
2,4-D toxicity well documented
A study of farm homes in Iowa in 2006 showed that 95% of the homes were polluted with detectable levels of 2,4-D ; 2,4-D was detected in 100 % of the surface drinking-water supplies of the Northern Great Plains of Canada . There is substantive evidence that 2,4 –D and its contaminant dioxins are implicated in soft tissue sarcoma and non-Hodgkin lymphoma. After Sweden banned the herbicide in the early 1970s the incidence of two cancers declined . Birth malformations and other adverse perinatal outcomes were observed in four US wheat-producing states. Infant death from congenital anomalies significantly increased in high-wheat counties for males but not for females. These results are especially of concern because of widespread use of chlorophenoxy herbicides . A significant increase in the use of 2,4-D is likely to increase the incidence of some cancers and birth defects.
Recent studies showed that 2,4-D was teratogenic to a South American toad, resulting in reduced body size, delayed development, microcephaly and abnormal cellular proliferation . At low concentration, 2,4-D stimulated transcription of the c-Myc cancer gene and induced apoptosis (cell suicide) in Syrian hamster embryo cells, suggesting that 2,4-D should be considered a hazard to humans . It has also been shown to affect the expression of many genes in human liver (hepatoma) cells, including those involved in DNA repair. Human hepatoma HepG2 cells were incubated with 2,4-D or nitrate alone for 24 h. Total RNA from treated and control cells were isolated, reverse transcribed and labelled, and hybridized to a human cDNA microarray. The hybridized microarray chips were scanned, quantified and analyzed to identify genes affected by 2,4-D or nitrate exposure. Low-level exposure altered the expression of many genes. The affected genes were those involved in stress response, cell cycle control, immunological and DNA repair . A 2005 report prepared by the Sierra Club of Canada made reference to over 75 peer-reviewed scientific articles on the toxicity of 2,4-D documenting genotoxicity, cancer, teratogenicity, neurotoxic, immunosuppressive, cytotoxic and hepatoxic impacts in humans and animals . In view of the numerous toxic effects of the herbicide, introducing GM maize resistant to 2,4-D is simply out of the question.
Because quizalofop is a relatively newly introduced herbicide, but the relatively few available publications (see below) already indicate that the chemical is potentially toxic. Witness evidence of harm also exists. A farmer exposed to quizalofop-p-ethyl presented with obstructive cholestasis. A complete workup disclosed no other cause of liver pathology, but liver biopsy established drug-induced hepatotoxicity . Evidence on developmental and reproductive toxicity of quizalofop has been reported by the Reproductive and Cancer Hazard Assessment Section Office of Environmental Health Hazard Assessment California Environmental Protection Agency. Seven studies investigating the potential for quizalofop-ethyl to cause reproductive or developmental toxicity were reviewed. Two studies, one done in rats and the other in rabbits, investigated developmental toxicity, while five chronic or subchronic feeding studies provided information of its effects on reproductive organs. There was a statistically significant decrease in the number of foetuses alive at the time of sacrifice of the dams on day 21 of gestation in the high dose group.
Effects of quizalofop-ethyl on female and male reproductive organs were assessed in two studies in dogs, two studies in rats and one study in mice. One study on dogs found testicular atrophy in two males. The two studies on rats did not show any clear evidence of effects on female reproductive organs; but one of them demonstrated a high incidence of testicular atrophy at the end of the 13 week treatment period. The only study in mice reviewed showed significantly increased ovarian weight in females at all dose levels tested, as well as bilateral testicular atrophy in males after exposure for 78 weeks. In addition to effects on testes and ovaries, quizalofop-ethyl has been repeatedly shown to affect the liver. There are also some indications of effects on kidney, adrenals, thyroid, thymus and blood .
DAS-40278-9 Maize is a step backward in terms of environmental pollution. The raison d'etre of the product is that it provides an alternative to GM crops which have grown useless as a consequence of weeds resistant to herbicides for which they have been made tolerant. However, replacing them with GM tolerance to a herbicide that has been around for over seventy years is surely a retrograde step, as during that time, a number of weeds resistant to it such as wild carrot and water hemp has already evolved; not to mention its documented toxicity. The plan to stack 2,4-D resistance with glyphosate simply multiplies the calamities in terms of herbicide toxicities and weed resistances.
It is a sure sign that the herbicide treadmill has run its course, and the only way ahead is organic, integrated pest-management and agro-ecological farming  (Food Futures Now: *Organic *Sustainable *Fossil Fuel Free , ISIS publication).
• USDA Economic Research Service ERS/USDA Data - Adoption of Genetically Engineered Crops in the U.S. 2012 http://www.ers.usda.gov/Data/BiotechCrops/
• Tagliani L. Dow AgroSciences. Petition for Determination of Nonregulated Status for Herbicide Tolerant DAS-40278-9 Corn. 2011 http://www.aphis.usda.gov/brs/aphisdocs/09_23301p.pdf
• Eck C. Dow AgroSciences Petition (09-233-01p) for Determination of Nonregulated Status of Herbicide-Tolerant DAS-40278-9 Corn, Zea mays, Event DAS-40278-9 Draft Environmental Assessment. 2011 http://www.aphis.usda.gov/brs/aphisdocs/09_23301p_dea.pdf
• Petolino J, Arnold N. Whiskers-Mediated Maize Transformation in M. Paul Scott (ed.), Methods in Molecular Biology: Transgenic Maize, vol. 526, Chapter 5. © Humana Press, a part of Springer Science + Business Media, USA 2009 DOI: 10.1007/978-1-59745-494-0_5
• Akiyama I, Ogami A, Oyabu T, Yamato H, Morimoto Y, Tanaka I. Pulmonary effects and biopersistence of deposited silicon carbide whisker after 1-year inhalation in rats. Inhalation Toxicology 2007, 19,141-7.
• Stagg NJ, Thomas J, Herman RA, Juberg DR. Acute and 28-day repeated dose toxicology studies in mice with aryloxyalkanoate dioxygenase (AAD-1) protein expressed in 2,4-D tolerant DAS-40278-9 maize. Regulatory Toxicology and Pharmacology 2011 Nov 13. [Epub ahead of print]
• Katsnelson A. Breaking the silence. Nature Medicine 2011, 17, 536-8. doi: 10.1038/nm1211-1536
• HoMW. Scientists Discover New Route for GM-gene “Escape”. Science in Society 50, 14-16, 2011
• Ward MH, Lubin J, Giglierano J, Colt JS, Wolter C, Bekiroglu N, Camann D, Hartge P, Nuckols JR. Proximity to crops and residential exposure to agricultural herbicides in Iowa. Environmental Health Perspectives 2006, 114, 893-7.
• Donald DB, Cessna AJ, Sverko E, Glozier NE. Pesticides in surface drinking-water supplies of the northern Great Plains. Environmental Health Perspectives 2007,115, 1183-91
• Hardell L. Pesticides, soft-tissue sarcoma and non-Hodgkin lymphoma--historical aspects on the precautionary principle in cancer prevention. Acta Oncology 2008, 47, 347-54.
• Schreinemachers DM. Birth malformations and other adverse perinatal outcomes in four U.S. Wheat-producing states. Environmental Health Perspectives 2003, 111, 1259-64.
• Aronzon CM, Sandoval MT, Herkovits J, Pérez-Coll CS. Stage-dependent toxicity of 2,4-dichlorophenoxyacetic on the embryonic development of a South American toad, Rhinella arenarum. Environmental Toxicology 2011, 26, 373-81. doi: 10.1002/tox.20564.
• Maire MA, Rast C, Landkocz Y, Vasseur P. 2,4-Dichlorophenoxyacetic acid: effects on Syrian hamster embryo (SHE) cell transformation, c-Myc expression, DNA damage and apoptosis. Mutation Research 2007, 631, 124-36.
• Bharadwaj L, Dhami K, Schneberger D, Stevens M, Renaud C, Ali A. Altered gene expression in human hepatoma HepG2 cells exposed to low-level 2,4-dichlorophenoxyacetic acid and potassium nitrate. Toxicology In Vitro 2005, 19, 603-19. • Sierra Club of Canada Overview of the toxic effects of 2,4-D 2005 http://www.sierraclub.ca/national/programs/health-environment/pesticides/2-4-D-overview.pdf
• Elefsiniotis IS, Liatsos GD, Stamelakis D, Moulakakis A. Case report: mixed cholestatic/hepatocellular liver injury induced by the herbicide quizalofop-p-ethyl. Environmental Health Perspectives 2007, 115, 479-
• Donald, J. Evidence on developmental and reproductive toxicity of quizalofop-ethyl California Environmental Protection Agency 1999 http://oehha.ca.gov/prop65/pdf/HIDQuiz.pdf
• Ho MW, Burcher S, Lim LC et al. Food Future Now *Organic*Sustainable *Fossil Fuel Free, ISIS/TWN, 2008. http://www.i-sis.org.uk/foodFutures.php