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[SANET-MG] DAS-59122-7 maize modified for corn rotworm protection



17 March 2007
Prof. Joe Cummins
Professor Emeritus of Genetics University of Western Ontario London, Canada and The institute of Science in Society , London, UK
738 Wilkins Street
London, Ontario N6C4Z9 Canada
E-email : jcummins@uwo.ca
DAS-59122-7 Maize Genetically Modified for Root Worm Protection and Herbicide Tolerance Dow Chemical company and it’s associated company Pioneer Hybrid Seed Company have developed maize variety Herculex RW for rootworm protection. The variety is based on the gene transformation event DAS-59122-7 which express unique binary proteins Cry34Ab1 and Cry35Ab1 derived from the soil bacterium Bacillus thuringiensis (Bt) Herculex. DAS-59122-7 (59122 for short) also expresses the PAT protein conferring resistance to the herbicide glufosinate ammonia These transgenes are inserted at a single locus in a maize chromosome along with control genes including promoters and terminators (1). An Application for the Determination of Nonregulated status for BT Cry 34/35Ab1 Insect resistant , Glufosinate Tolerant Corn: Corn Line 59122 was submitted by Dow Agrisciences/Pioneer Hybrid International for approval by the United States Department of Agriculture Animal and Plant Health Inspection Service (USDA/APHIS during December 2003 (2). That report noted that event 59122 had incorporated a synthetic approximation of the Cry 34Ab1 gene driven by a maize ubiquitin promoter and intron and terminated using a terminator sequence from potato protease inhibitor II. A synthetic approximation of the Cry 35Ab1 gene was driven by a wheat peroxidise promoter and terminated with the terminator from potato protease inhibitor II A synthetic approximation of the PAT gene for resistance to glufosinate was driven by the 35S CaMV promoter and terminated with 35S CaMV terminator. According to the report the genes were stably inserted at a single locus and there was no evidence of instability in 59122. The proteins synthesized from the synthetic genes were compared with proteins synthesized in bacteria and seemed identical (however later studies showed that the proteins were significantly different). The main human safety and environmental tests were done on protein derived from bacteria not the protein products of the synthetic approximations of the bacterial genes used to transform maize. USDA/APHIS carried out an environment assessment of maize event 59122. That assessment included potential impact on non target organisms including threatened and endangered species. The event was deemed to have inflicted no significant impact on the environment (3). However, the bulk of those tests were done with proteins isolated from bacteria ( a less costly procedure than isolating the proteins from transgenic maize) and those proteins were similar but identical to the proteins produced in 59122. A fuller comparison of the insecticidal proteins produced in 59122 with those in the bacteria producing the proteins used in safety testing showed that 4 amino acids in the C-terminal protein domain were different in Cry35Ab1 protein produced in recombinant maize from those produced in the Cry35Ab1 produced in bacteria and used in safety tests. These differences were considered negligible (4). In general, the C terminal domain of Bt toxins is involved in structural stability, ion channel gating , binding to membrane vesicles and determining insecticidal specificity (5). A study of Cry34/Cry35 insecticidal proteins from diverse Bt strains Cry 35Ab1 contains a segment similar to the to a beta-trefoil domains that may be a binding motif (6). Beta trefoil domain binds galactose , some examples of trefoil domain include the toxins from Clostridium botulinum, abrin and ricin (7). It seems very daring to ignore amino acid sequences in a domain known to be active in important functions. Even though most of the earlier regulatory approval had been based on safety tests with proteins obtained from bacteria rather than from 59122 forage and grain samples from maize 59122 were analysed and compared with near-isogenic non-GM maize lines to determine whether or not 59211 was substantially equivalent to un-modified maize. The maize lines were compared for fibre and minerals, for amino acids, fatty acids , vitamins, secondary metabolites and anti-nutrients. One parameter, carbohydrate, was significantly lower in 59122 treated with glufosinate and along with that finding 13 of 65 determinations including forage fibre, grain amino acids and vitamins were outside the levels for conventional maize. In spite of these findings 59122 was deemed substantially equivalent to unmodified maize (8). A sub chronic feeding study of rats fed 59122 grain compared with grain from a near isogenic line for 90 days was followed by study of weight , behaviour and organ necropsy. For the most part the differences between 59122 and unmodified maze fed animals were not significantly different. However, Levels of mean corpuscular haemoglobin, and haemoglobin concentration, red cell width, reticulocyte count and platelet count showed significant differences but these blood values were not deemed significant (9). Chickens were fed grain either from 59122 or from a near isogenic maize. Carcass and organ weights were measured after 42 days of feeding (there was no extensive necropsy of the organs). For the most part carcass and organ sizes were not significantly different in 50122 than they were in unmodified maize but the livers of female chickens were significantly enlarged in the transgenic maize fed animals . The statistically significant difference in liver weight was not considered biologically significant by the company researchers(1O). The novel approach used by company researchers , passing over significant differences caused by the consumption of 59122 with a few words of comfort should be a matter of concern to regulators. The significant differences between 59122 and near isogenic maize lines in the light of the relatively crude parameters that were measured cries out for fuller study, The use of proteins produced in bacteria from natural genes to test the safety of proteins rather than the proteins produced by synthetic approximations of natural genes such as those used in 59122 also cries out for fuller study. Presently, a much fuller safety evaluation of genetically modified maize , such as 59122 can be achieved using the micro array technology which is available , for example, maize(11). The use of such advanced technology might improve on the equivocal results claimed to be evidence that 59122 is safe for consumption in food and feed.
References
1. The Dow Chemical Company Product Safety Assessment Herculex RW Rootworm Protection 2006 http://www2.dupont.com/Biotechnology/en_US/products/herculex_rw/safety2.html 2. Hunst.P and Rood,T . Application for the Determination of Nonregulated Status for Bt Cry34/35 Ab1 insect resistant Glufosinate-Tolerant Corn: Corn Line 59122 2004 http://www.aphis.usda.gov/brs/aphisdocs/03_35301p.pdf 3. Smith,C. Environment Assessment for Dow/Pioneer Rootworm Resistant Corn 2005 http://www.aphis.usda.gov/brs/aphisdocs2/03_35301p_com.pdf 4. Gao Y, Schafer BW,Collins RA,Herman RA, Xu X,Gilbert JR,Ni W,Langer V and Tagliani LA. Characterization of Cry34Ab1 and Cry35Ab1 insecticidal crystal proteins expressed in transgenic corn plants and Pseudomonas fluorescens. J Agric Food Chem. 2004 Dec 29;52(26):8057-65 5. Saraswathy,N and Kumar,P. Protein engineering of delta endotoxins of Bacillus thuringiensis Electronic Journal of Biotechnology Microbial Biotechnology 2004,2,1-12 DOI: 10.2225/vol7-issue2-fulltext-3
http://www.ejbiotechnology.info/content/vol7/issue2/full/3/index.html
6. Schnepf HE,Lee S,Dojillo J,Burmeister P,Fencil K,Morera L,Nygaard L,Narva KE and Wolt JD. Characterization of Cry34/Cry35 binary insecticidal proteins from diverse Bacillus thuringiensis strain collections. Appl Environ Microbiol. 2005 Apr;71(4):1765-74 7. Mancheno JM, Tateno H, Goldstein IJ, Martinez-Ripoll M and Hermoso JA. Structural analysis of the Laetiporus sulphureus hemolytic pore-forming lectin in complex with sugars. J Biol Chem. 2005 Apr 29;280(17):17251-9 8. Herman RA,Storer NP,Phillips AM,Prochaska LM and Windels P. Compositional assessment of event DAS-59122-7 maize using substantial equivalence. Regul Toxicol Pharmacol. 2007 Feb;47(1):37-47. 9. Malley LA,Everds NE,Reynolds J,Mann PC,Lamb I,Rood T,Schmidt J, Layton RJ, Prochaska LM,Hinds M,Locke M, Chui CF,Claussen F,Mattsson JL and Delaney B. Subchronic feeding study of DAS-59122-7 maize grain in Sprague-Dawley rats. Food Chem Toxicol. 2007 Jan 25;In press doi:10.1016/j.fct.2007.01.013 10. McNaughtona,J,Robertsa,M,Riceb,D,Smith,B,Hindsb,M.Schmidtb,J.Lockec,M,Bryant,A,Rood.T, Laytonb,R,Lamb,I and Delaneyb,B. Feeding performancenext term in previous termbroiler chickens fednext term diets containing previous termDAS-59122-7next term maize grain compared to diets containing non-transgenic maize grain Animal Feed Science and Technology 2007,132, 227-239 11. Shi,C,Thümmler,F,Melchinger,A,Wenzel,G and Lübberstedt.T. Comparison of transcript profiles between near-isogenic maize lines in association with SCMV resistance based on unigene-microarrays Plant Science, 2006, 170,159-169

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