The Full Monty by GMO Pundit: The Full Monty on Animal Feeding Trials of GM foods.

GM Crop Animal Feeding Safety Testing and Crop Chemical Safety Profiling Papers.

Preface:
A readable summary of this post is given here.

A much more readable update on this issue (March 2010)) is given at Academics Review

Section 1—Top 20 Urban Myths about GM Grops

Late June 2007
This post has been transferred to GMO Pundit's main page here , and is being updated there.

What you'll find here:

Key review articles on testing of GM food and feed safety, and on animal feeding trials with GM food and feed.
Link to a summary of affiliations of scientists publishing on food safety, to enable their degree of independence to be scrutinised, plus links to original papers where available.
Collected list of published articles presenting GM animal feeding tests, and papers relating directly to experimental testing of GM food safety.
Base-line studies on variability and unpredictable outcomes from conventional breeding
List as in Exhibit 3 with summaries of articles added.

Exhibits 3 and 5 are large documents, but the point of presenting so many papers is to refute assertions that such studies are seldom published.

Important papers are bolded in the lists below.

Exhibit 1.

KEY REVIEW articles on GM food nutritional safety evaluation.

NAS Report- Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects (2004) by Food and Nutrition Board (FNB) Institute of Medicine (IOM) Board on Agriculture and Natural Resources (BANR) Board on Life Sciences (BLS))

REVIEW ARTICLE
Studies on feeds from genetically modified plants (GMP) – Contributions to nutritional and safety assessment
G. Flachowsky K. Aulrich, H. Bohme, I. Halle
Institute of Animal Nutrition, Federal Agricultural Research Centre (FAL),
Bundesallee 50, D-38116 Braunschweig, Germany
Institute of Organic Farming, Federal Agricultural Research Centre (FAL),
Trenthorst 32, D-23847 Westerau, Germany

Abstract
Since 1997, 18 studies with feeds from genetically modified plants (GMP) in the nutrition of dairy cows, growing bulls, growing and finishing pigs, laying hens, chicken for finishing as well as growing and laying quails were conducted at the Federal Agricultural Research Centre (FAL) in Braunschweig (Germany).
The majority of the experiments (16) were undertaken with GMP of the so-called first generation (plants with input traits and without substantial changes in composition) such as Bt-maize, Pat-maize, Pat-sugar beet, Gt-soybean, Gt-potatoes and Bt-potatoes. Two studies were carried out with GMP of the second generation (plants with output traits or with substantial changes in their chemical composition) such as an altered fatty acids profile in rapeseed or inulin potatoes. In all experiments, feeds from GMP were compared with their isogenic counterparts.
The iso- and transgenic feeds were analysed for their composition (proximates, fibre fraction, amino acids, fatty acid pattern, minerals) and undesirable substances (e.g., mycotoxins). Animal studies were carried out for nutritional and safety assessment such as digestibility, feed intake, health and performance of target animal species and quality of food of animal origin. Reproduction was studied in a 10-generation experiment with quails and a 4-generation experiment with laying hens.
Duration of experiments and number of animals were limited in some cases due to small amounts of GM-feed available for experimentation.

Attention was drawn to the fate of DNA during feed processing (silage making, oil extraction), the digestive tract of animals (slaughtering of animals 0, 4, 8, 12 and 24 h after feeding) and in animal body (samples from several organs and tissues).
In agreement with more than 100 animal studies available to date, results show no significant differences in the nutritional value of feeds from GMP of the first generation in comparison with non-GMP varieties. To date, no fragments of recombinant DNA have been found in any organ tissue sample from animals fed GMP. The lower content of mycotoxins in Bt-maize and side effects in GMP of the second generation are of safety concern.
The results indicate that routine feeding studies with target animal species add little to nutritional assessment of feed from GMP of the first generation, but they are of public interest and important safety assessment. These studies will play a more important role in nutritional and safety assessment of feeds from GMP with output traits. Proposals for such studies were made on the basis of previous experiments.

5. Conclusions
The results presented in Table 28 are in agreement with more than 100 international
experiments, recently reviewed by Flachowsky et al. (2005a). The following conclusions
can be drawn:
– Genetically modified plants without substantial changes in their composition (those of the first generation) do not significantly differ in their nutritional value from those of the isogenic variety.
– A transfer of recombinant DNA from feed into the animal body was not detected. To date, no residues of recombinant DNA have been found in any organ or tissue.
– Correspondingly, routine feeding studies with the target animal species generally add little to the nutritional and safety assessment (EFSA, 2004) but they are of public concern.
– As an approach for nutritional and safety assessment of feeds from GMP of the first generation a decision tree has been proposed (Flachowsky and Aulrich, 2001).
– Strategies for nutritional and safety assessment which are developed for those of the first generation cannot directly applied for GMP with substantial changes of the constituents
(second generation).
– Case by case studies with animals will gain much more importance. Proposals for nutritional assessment of feeds from GMP with output traits were submitted (Flachowsky and Bohme, 2005). Such feeding studies should be combined with safety studies in target animal species.

Animal Feed Science and Technology
133 (2007) 2–30

REVIEW ARTICLE.

Animal nutrition with feeds from genetically modiﬁed plants
GERHARD FLACHOWSKY, ANDREW CHESSON, & KAREN AULRICH

Abstract

Plant breeders have made and will continue to make important contributions toward meeting the need for more and better feed and food. The use of new techniques to modify the genetic makeup of plants to improve their properties has led to a new generation of crops, grains and their by-products for feed. The use of ingredients and products from genetically modiﬁed plants (GMP) in animal nutrition properly raises many questions and issues, such as the role of a nutritional assessment of the modiﬁed feed or feed additive as part of safety assessment, the possible inﬂuence of genetically modiﬁed (GM) products on animal health and product quality and the persistence of the recombinant DNA and of the ‘novel’ protein in the digestive tract and tissues of food-producing animals. During the last few years many studies have determined the nutrient value of GM feeds compared to their conventional counterparts and some have additionally followed the fate of DNA and novel protein. The results available to date are reassuring and reveal no signiﬁcant differences in the safety and nutritional value of feedstuffs containing material derived from the so-called 1st generation of genetically modiﬁed plants (those with unchanged gross composition) in comparison with non-GM varieties. In addition, no residues of recombinant DNA or novel proteins have been found in any organ or tissue samples obtained from animals fed with GMP.

These results indicate that for compositionally equivalent GMP routine-feeding studies with target species generally add little to nutritional and safety assessment. However, the strategies devised for the nutritional and safety assessment of the 1st generation products will be much more difﬁcult to apply to 2nd generation GMP in which signiﬁcant changes in constituents have been deliberately introduced (e.g., increased fatty acids or amino acids content or a reduced concentration of undesirable constituents). It is suggested that studies made with animals will play a much more important role in insuring the safety of these 2nd generation constructs.

Keywords: Animal nutrition, feed, genetically modiﬁed plants, nutritional assessment, DNA, protein degradation

REVIEW ARTICLE in Archives of Animal Nutrition February 2005; 59(1): 1 – 40
GERHARD FLACHOWSKY1, ANDREW CHESSON2, & KAREN AULRICH3
1Institute of Animal Nutrition, Federal Agricultural Research Centre (FAL), Braunschweig, Germany, 2College of Medical and Life Sciences, School of Biological Sciences, University of Aberdeen, Aberdeen, Scotland, UK, and 3Institute of Organic Farming, Federal Agricultural Research Centre (FAL), Trenthorst, Germany
(Received 11 October 2004; accepted 1 December 2004)

REVIEW ARTICLE
PROTEOMIC PROFILING AND UNINTENDED EFFECTS IN GENETICALLY MODIFIED CROPS
Sirpa O. Kärenlampi and Satu J. Lehesranta (ISB Report.)

It is generally accepted that traditional food is safe for the majority of consumers. For the introduction of a new variant or cultivar developed from a traditional crop plant, maximum limits have been set in some cases, e.g., for potato and oilseed rape, to the content of known toxins. The requirements are much more stringent if the crop is developed by using genetic engineering. Why is it so? In a majority of cases seen so far, a new gene, often derived from other plants or microbial species, has been introduced to a non-predetermined location in the plant genome. It is quite feasible to ask the question whether the new gene products are safe or not. Therefore, for all genetically modified crop plants, the safety of the newly introduced proteins needs to be demonstrated before the plants can be released into the market.

Another point of concern is the random integration of the new gene into the plant genome. Both the new gene itself and its site of integration may give rise to unintended adverse effects. For example, transgene integration might interrupt regulatory sequences or open reading frames leading to novel fusion proteins and, thereby, modify plant metabolism.1 These modifications could compromise the safety of the food crops by, for instance, leading to the production of new allergens or toxins. Having the gene and the integration site well characterised should provide a good basis for the safety assessment. However, it is a common practice today to perform a large number of analyses, so-called targeted analyses, to demonstrate that the characteristics of the novel crop are comparable with those of the conventional counterpart, in addition to the intended alterations. Targeted analyses include key macronutrients, micronutrients, antinutrients, and toxins. In certain cases, toxicity studies on experimental animals are advised. And yet, the question about the unintended effects does not seem to be covered in a way that would escape all criticism...continues at GMO Pundit (or alternatively at the original at title link just above)

See also
Catchpole, Gareth S., and others (2005 ) Hierarchical metabolomics demonstrates substantial compositional similarity between genetically modified and conventional potato crops.
PNAS October 4, 2005 vol. 102 no. 40 14458-14462
which is a pioneering study which deals with a different but related approach to food "profiling", of potatoes that assesses food safety. This study is further discussed in a GMO Pundit Post about a letter by Chris Preston to Agbioview.

The current concepts of genetics which can explain much of the detected natural variability in protein and metabolic fingerprints of plants suggested in the previous reports is discussed in Natural GMOs Part 26.

REVIEW ARTICLE.
The promise of metabolomics [detailed metabolite fingerprinting or profiling]

[Fingerprinting and profiling technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). Pundit note]

David A Bender
Department of Biochemistry Molecular Biology, University College London, Gower Street, London WC1E 6BT

"...Of more direct relevance to nutritional sciences, metabolomic techniques will permit identification of biomarkers of health (as opposed to disease), and will therefore permit more precise estimation of appropriate levels of nutrient intake—levels of
Within the European Union, since April 2004, all food and animal feed containing more than 0.9% of ingredients from genetically modified organisms must be so labelled, regardless of whether or not there is any GM material in the final product.14 This means products such as flour, oils and glucose syrups, which were previously considered to be substantially equivalent’ to material from traditional sources, since they contained no modified DNA, will have to be
labelled as GMif they are from a GM source. This will increase public concern about the safety of GM foods.

Christou and Twyman15 reviewed the potential of GM crops to alleviate food insecurity in less developed countries by providing higher yields, resistance to pests and diseases (so increasing the effective yield considerably), ability to grow under adverse conditions (so increasing the land available for crops) and improved nutritional quality. They concluded with the observation that genetic modification of crops is highly politically sensitive. Within the European Union environmental concerns have led to a trade war with the USA, and several southern African countries have refused American food aid that included genetically modified cereals or soy beans.

Metabolic profiling will permit more precise determination of whether a novel or GM product is indeed ‘substantially identical’, or whether there are (possibly significant) differences in hitherto disregarded trace compounds, and should provide valuable evidence to inform consumers about safety.16

Many potentially hazardous compounds occur in plant foods naturally, and sometimes improved varieties that result from traditional plant breeding methods may contain unexpectedly high amounts of toxins. Beier17 cites the case of a new variety of celery that was bred for resistance to pests and wilting (and so had a longer shelf life). Its pest resistance was due to higher than normal concentrations of psoralens, which causes a photosensitive dermatitis through contact with the skin, and its introduction was associated with a significant increase of cases of photodermatitis among grocery employees.Metabolic profiling would, presumably, have been able to detect this abnormal concentration of a toxic compound before the new variety was released into general cultivation..."

Journal of the Science of Food and Agriculture Volume 85, Issue 1 , Pages 7 - 9 Published Online: 3 Nov 2004

REFERENCES TO BENDER QUOTATION

14 Anonymous, Regulation no 1829/2003 on Genetically Modified Food and Feed. Off J EU L268:1–23 (2003).

15 Christou P and Twyman RM, The potential of genetically enhanced plants to address food insecurity. Nutr Res Rev 17:23–42 (2004).

16 Cellini F, Chesson A, Colquhoun I, Constable A, Davies HV, Engel KH, Gatehouse AM, Karenlampi S, Kok EJ, Leguay JJ, Lehesranta S, Noteborn HP, Pedersen J and Smith M, Unintended effects and their detection in genetically modified crops. Food Chem Toxicol 42:1089–1125 (2004).

The commercialisation of GM crops in Europe is practically non-existent at the present time. The European Commission has instigated changes to the regulatory process to address the concerns of consumers and member states and to pave the way for removing the current moratorium. With regard to the safety of GM crops and products, the current risk assessment process pays particular attention to potential adverse effects on human and animal health and the environment.

This document deals with the concept of unintended effects in GM crops and products, i.e. effects that go beyond that of the original modification and that might impact primarily on health. The document first deals with the potential for unintended effects caused by the processes of transgene insertion (DNA rearrangements) and makes comparisons with genetic recombination events and DNA rearrangements in traditional breeding.

The document then focuses on the potential value of evolving "profiling" or "omics" technologies as non-targeted, unbiased approaches, to detect unintended effects. These technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). The technologies are described, together with their current limitations. Importantly, the significance of unintended effects on consumer health are discussed and conclusions and recommendations presented on the various approaches outlined.

17 Beier RC, Natural pesticides and bioactive components in foods. Rev Environ Contam Toxicol 113:47–137 (1990).

WORLD HEALTH ORGANISATION
Modern food biotechnology, human health and development: An evidence-based study

FSANZ
GM Foods: Safety Assessments of Genetically Modified Foods
2005 – Source: Australian Government Agency, Food Standards Australia New Zealand

REVIEW ARTICLE
Safety assessment and feeding value for pigs, poultry and ruminant animals of pest protected (Bt) plants and herbicide tolerant (glyphosate, glufosinate) plants: interpretation of experimental results observed worldwide on GM plants

ABSTRACT

New varieties of plants resistant to pests and/or tolerant to specific herbicides such as maize, soybean, cotton, sugar beets, canola, have been recently developed by using genetic transformation (GT). These plants contain detectable specific active recombinant DNA (rDNA) and their derived protein. Since they have not been selected for a modification of their chemical composition, they can be considered as substantially equivalent to their parents or to commercial varieties for their content in nutrients and anti-nutritional factors. However, insect protected maize is less contaminated by mycotoxins than its parental counterpart conferring a higher degree of safety to animal feeds. The new feeds, grain and derivatives, and whole plants have been intensively tested in vivo up to 216 days for their safety and their nutritional equivalence for monogastric farm animals (pig, poultry) and ruminants (dairy cows, steers, lambs). The present article is based on the interpretation and the summary of the scientific results published in original reviewed journals either as full papers (33) or as abstracts (33) available through September 2003. For the duration of the experiments adapted to the species, feed intake, weight gain, milk yield and nutritional equivalence expressed as feed conversion and/or digestibility of nutrients have never been affected by feeding animals diets containing GT plants. In addition, in all the experimental animals, the body and carcass composition, the composition of milk and animal tissues, as well as the sensory properties of meat are not modified by the use of feeds derived from GT plants. Furthermore, the health of animals, their physiological characteristics and the survival rate are also not affected.
The presence of rDNA and derived proteins can be recognized and quantified in feeds in the case of glyphosate resistant soybean and canola and in the case of insect protected maize. However, rDNA has never been recovered either in milk, or in liver, spleen and muscles tissues of animals, or in rumen bacteria. On the basis of these data, it can be suggested that in vivo tests on high producing animals are necessary and sufficient to evaluate the safety and the nutritional value of new GT plants.
Keys words: GM plants, Safety, Feeding value, Farm animals

RIASSUNTO
SICUREZZA E VALORE NUTRITIVO DI ALIMENTI MODIFICATI GENETICAMENTE PER RESISTENZA AGLI INSETTI (BT) E TOLLERANZA AGLI ERBICIDI (GLIFOSATE, GLUFOSINATE) DESTINATI A SUINI, POLLI E RUMINANTI: INTERPRETAZIONE DEI RISULTATI SPERIMENTALI OTTENUTI A LIVELLO MONDIALE.

Aimé Aumaitre (2004) INRA. Saint Gilles, France
ITAL.J.ANIM.SCI. VOL. 3, 107-121, 2004 107

Corresponding author: Prof. Aimé Aumaitre. INRA. 35 590 Saint Gilles, France - Tel.+ 33 223 485041 - Fax: + 33
223 485080 - Email: aumaitre--AT--saint-gilles.rennes.inra.fr
Paper received September 25, 2003; accepted December 1, 2003

Mycotoxin content and safety of GM foods.

The benefit of Bt [GM] corn’s reduction of mycotoxin damage has been virtually ignored in policy debates anywhere in the world. As adoption of agricultural biotechnology continues to increase on a global scale, policy makers worldwide should consider the economic and health impacts of this secondary benefit of transgenic pest-protected crops. A superb recent paper by F Wu remedies deficiency in policy debate, and the following draws very heavily on F Wu's important scholarship. Reduction of mycoxin contamination is a clear parameter for food and feed safety, particularly in developing countries. Further details and extensive discussion in a GMO Pundit Post here.

Mycotoxin reduction in Bt corn: potential economic, health, and regulatory impacts

Abstract
Genetically modified (GM) Bt corn, through the pest protection that it confers, has lower levels of mycotoxins: toxic and carcinogenic chemicals produced as secondary metabolites of fungi that colonize crops. In some cases, the reduction of mycotoxins afforded by Bt corn is significant enough to have an economic impact, both in terms of domestic markets and international trade. In less developed countries where certain mycotoxins are significant contaminants of food, Bt corn adoption, by virtue of its mycotoxin reduction, may even improve human and animal health. This paper describes an integrated assessment model that analyzes the economic and health impacts of two mycotoxins in corn: fumonisin and aflatoxin. It was found that excessively strict standards of these two mycotoxins could result in global trade losses in the hundreds of millions $US annually, with the US, China, and Argentina suffering the greatest losses. The paper then discusses the evidence for Bt corn’s lower levels of contamination of fumonisin and aflatoxin, and estimates economic impacts in the United States. A total benefit of Bt corn’s reduction of fumonisin and aflatoxin in the US was estimated at $23 million annually. Finally, the paper examines the potential policy impacts of Bt corn’s mycotoxin reduction, on nations that are making a decision on whether to allow commercialization of this genetically modified crop.
Keywords Bt corn - economic impacts - health impacts - mycotoxin reduction - regulatory policy

Felicia Wu
Environmental, Occupational Health, Graduate School of Public Health, University of Pittsburgh, 130 DeSoto St.,
Pittsburgh, PA, 15261, USA
Transgenic Research (2006) 15:277–289 Springer 2006
DOI 10.1007/s11248-005-5237-1

Two earlier reviws by Wu reviews of fumonisin reduction in Bt-corn, see Felicia Wu et al., The Economic Impact of Bt Corn Resulting from Mycotoxin Reduction, 23 J. TOXICOLOGY, TOXIN REVS. 397 (2004) , and Felicia Wu, Mycotoxin Risk Assessment for the Purpose of Setting International Regulatory Standards, 38 ENVTL. SCI. & TECH. 4049 (2004).

Health and Food Safety: The Benefits of Bt-Corn .
Drew L. Kershen
FOOD & DRUG LAW JOURNAL, v. 61 # 2 (June 2006) pages 197-236) published 27th June.
(A hyperlink to a full pdf copy of the review will follow within a few days courtesy of Professor Kershen)

GMO Pundit Post on Movement of DNA between species and from the gut contents to (human) intestinal cells.

Exhibit 2.

GMO Pundit post on institutional affiliations of scientists who publish test results are listed in a GMO Pundit post hyperlinked here, which also has links to the original papers where available.

Exhibit 3.

Citation list of papers that test GM food safety in animal tests or directly and systematically measure safety parameters such as allergenicity or potential toxin fingerprinting:

Aeschbacher, K., L. Meile, R. Messikommer and C. Wenk. (2002) Influence of genetically modified maize on performance and product quality of chickens. Proc. Soc. Nutr. Physiol. 11:196.
Aeschbacher K, Messikommer R, Meile L, Wenk C (2005) Bt176 corn in poultry nutrition: Physiological characteristics and fate of recombinant plant DNA in chickens. Poultry Science 84:385-394
Ash, J., C. Novak, and S.E. Scheideler. (2003) The fate of genetically modified protein from Roundup Ready soybeans in laying hens. J. Appl. Poult. Res. 12:242:245.
Alexander TW, Sharma R, Deng MY, Whetsell AJ, Jennings JC, Wang YX, Okine E, Damgaard D, McAllister TA (2004) Use of quantitative real-time and conventional PCR to assess the stability of the cp4 epsps transgene from Roundup Ready (R) canola in the intestinal, ruminal, and fecal contents of sheep. Journal of Biotechnology 112:255-266
Ash J, Novak C, Scheideler SE (2003) The fate of genetically modified protein from Roundup Ready Soybeans in laying hens. Journal of Applied Poultry Research 12:242-245
Atkinson, H.J., Johnston, K.A., Robbins, M.,( 2004). Prima facie evidence that a phytocystatin for transgenic plant resistance to nematodes is not a toxic risk in the human diet. J. Nutr. 134, 431–434.
Aulrich K, Bohme H, Daenicke R, Halle I, Flachowsky G (2001) Genetically modified feeds in animal nutrition 1st communication: Bacillus thuringiensis (Bt) corn in poultry, pig and ruminant nutrition. Archives of Animal Nutrition-Archiv fur Tierernahrung 54:183-195
Bakan B, Melcion D, Richard-Molard D and Cahagnier B (2002) Fungal growth and Fusarium mycotoxin content in isogenic traditional maize and genetically modified maize grown in France and Spain. J Agric Food Chem 50(4): 728–731.
Baker, J M, Hawkins, N D, Ward, J L, Lovegrove, A, Napier,J A, Shewry, P R and Beale, M H.(2006) A metabolomic study of substantial equivalence of field-grown genetically modified wheat. Plant Biotechnology Journal Volume 4 Issue 4 Page 381 - July 2006 doi:10.1111/j.1467-7652.2006.00197.x
Barriere Y, Verite R, Brunschwig P, Surault F, Emile JC (2001) Feeding value of corn silage estimated with sheep and dairy cows is not altered by genetic incorporation of Bt176 resistance to Ostrinia nubilalis. Journal of Dairy Science 84:1863-1871
Baudo, M M, Lyons, Powers, S R, Pastori,G M, Edwards, K J, Holdsworth, M J, and Shewry, P R. (2006) Transgenesis has less impact on the transcriptome of wheat grain than conventional breeding Plant Biotechnology Journal Volume 4 Issue 4 Page 369 - July 2006 doi:10.1111/j.1467-7652.2006.00193.x
Benedict J, Fromme D, Cosper J, Correa C, Odvody G and Parker R (1998) Efficacy of Bt Corn Events MON810, Bt11 and E176 in Controlling Corn Earworm, Fall Armyworm, Sugarcane Borer and Aflatoxin. Texas A&M University System, College Station, TX .
Berberich, SA Ream, J.E., Jackson, T.L., Wood, R., Stipanovic, R., Harvey, P., Patzer, S., and Fuchs, R.L. (1996) The composition of insect-protected cottonseed is equivalent to that of conventional cottonseed. J. Agric. Food Chem. 44, 365–371.
Bohme H, Aulrich K, Daenicke R, Flachowsky G (2001) Genetically modified feeds in animal nutrition 2nd communication: Glufosinate tolerant sugar beets (roots and silage) and maize grains for ruminants and pigs. Archives of Animal Nutrition-Archiv fur Tierernahrung 54:197-207
Brake DG, Thaler R, Evenson DP (2004) Evaluation of Bt (Bacillus thuringiensis) corn on mouse testicular development by dual parameter flow cytometry. Journal of Agricultural and Food Chemistry 52:2097-2102.
Brake J, Vlachos D (1998) Evaluation of transgenic event 176 "Bt" corn in broiler chickens. Poultry Science 77:648-653.
Brake J, Faust MA, Stein J (2003) Evaluation of transgenic event Bt11 hybrid corn in broiler chickens. Poultry Science 82:551-559
Brake J, Faust M, Stein J (2005) Evaluation of transgenic hybrid corn (VIP3A) in broiler chickens. Poultry Science 84:503-512
Broll H, Zagon J, Butschke A, Leffke A, Spiegelberg A, Bohme H, Flachowsky G (2005) The fate of DNA of transgenic inulin synthesizing potatoes in pigs. Journal of Animal and Feed Sciences 14:337-340
Brown PB, Wilson KA, Jonker Y, Nickson TE. (2003). Glyphosate tolerant canola meal is equivalent to the parental line in diets fed to rainbow trout. J Agric Food Chem. 51:4268-72.
Catchpole, Gareth S., Manfred Beckmann, David P. Enot, Madhav Mondhe, Britta Zywicki, Janet Taylor, Nigel Hardy, Aileen Smith, Ross D. King, Douglas B. Kell, Oliver Fiehn and John Draper,(2005 ) ) Hierarchical metabolomics demonstrates substantial compositional similarity between genetically modified and conventional potato crops. Published online before print September 26, 2005, 10.1073/pnas.0503955102 PNAS October 4, 2005 vol. 102 no. 40 14458-14462. Discussed in a GMO Pundit Post about a letter by Chris Preston to Agbioview.
Chambers, P.A., Duggan, P.S., Heritage, J., Forbes, J.M. (2000). The fate of antibiotic resistance marker genes in transgenic plant feed material fed to chickens. J. Antimicrob. Chemother. 49, 161–164.
Chen ZL, Gu H, Li Y, Su Y, Wu P, Jiang Z, Ming X, Tian J, Pan N, Qu LJ. (2003) Safety assessment for genetically modified sweet pepper and tomato. Toxicology. 2003 Jun 30;188(2-3):297-307.
Chen X, Zhuo Q, Piao J, Yang X. (2004) [Immunotoxicologic assessment of transgenetic rice][Article in Chinese]. Wei Sheng Yan Jiu. 33:77-80.
Chesson A, Flachowsky G (2003) Transgenic plants in poultry nutrition. Worlds Poultry Science Journal 59:201-207
Chowdury, E.H., Kuribara, H., Hino, A., Sultana, P., Mikami, O., Shimada, N., Guruge, K.S., Saito, M.,Nakayima, Y. (2003). Detrection of corn intrinsic and DNA fragments and Cry1Ab protein in the gastrointestinal contents of pigs fed genetically modified corn Bt11. J. Anim. Sci. 81, 2546–2551.
Chowdhury EH, Mikami O, Murata H, Sultana P, Shimada N, Yoshioka M, Guruge KS, Yamamoto S, Miyazaki S, Yamanaka N, Nakajima Y (2004) Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11. Journal of Food Protection 67:365-370
Chowdhury EH, Shimada N, Murata H, Mikami O, Sultana P, Miyazaki S, Yoshioka M, Yamanaka N, Hirai N, Nakajima Y.(2003). Detection of Cry1Ab protein in gastrointestinal contents but not visceral organsof genetically modified Bt11-fed calves. Vet Hum Toxicol. 2003 Mar;45(2):72-5.
Chrenkova M, Sommer A, Ceresnakova Z, Nitrayova S, Prostredna M (2002) Nutritional evaluation of genetically modified maize corn performed on rats. Archives of Animal Nutrition-Archiv fur Tierernahrung 56:229-235
Cleveland, Thomas E, Patrick F Dowd, Anne E Desjardins, Deepak Bhatnagar, Peter J Cotty (2003). United States Department of Agriculture - Agricultural Research Service research on pre-harvest prevention of mycotoxins and mycotoxigenic fungi in US crops, Pest Management ScienceVolume 59, Issue 6-7 , Pages 629 - 642
Corpillo D, Gardini G, Vaira AM, Basso M, Aime S, Accotto GP, Fasano M (2004) Proteomics as a tool to improve investigation of substantial equivalence in genetically modified organisms: the case of a virus-resistant tomato. Proteomics. 2004 Jan;4(1):193-200.
Cromwell GL, Lindemann MD, Randolph JH, Parker GR, Coffey RD, Laurent KM, Armstrong CL, Mikel WB, Stanisiewski EP, Hartnell GF.( 2002). Soybean meal from roundup ready or conventional soybeans in diets for growing-finishing swine. J Anim Sci. 80:708-15.
Daenicke R, Aulrich K, Flachowsky G. (1999). GMO in animal feedstuffs: nutritional properties of Bt-maize... Mais: Fachzeitschrift uber Forschung, Produktionstechnik, Verwertung und Okonomik 135-137
de la Campa, Regina, David C. Hooker, J. David Miller, Arthur W. Schaafsma and Bruce G. Hammond (2005) Modeling effects of environment, insect damage, and Bt genotypes on fumonisin accumulation in maize in Argentina and the Philippines, Mycopathologia ISSN: 0301-486X (Paper) 1573-0832 (Online) DOI: 10.1007/s11046-005-2150-3
Volume 159, Number 4 June 2005 Pages: 539 - 552
Deaville, E.R. and B.C. Maddison. (2005). Detection of transgenic and endogenous plant DNA fragments, in the blood, tissues, and digesta of broilers. J. Agric. Food Chem. 53:10268-10275.
Donkin SS, Velez JC, Totten AK, Stanisiewski EP, Hartnell GF (2003). Effects of feeding silage and grain from glyphosate-tolerant or insect-protected corn hybrids on feed intake, ruminal digestion, and milk production in dairy cattle. Journal of Dairy Science 86:1780-1788
Dowd, Patrick F (2000). Indirect Reduction of Ear Molds and Associated Mycotoxins in Bacillus thuringiensis Corn Under Controlled and Open Field Conditions: Utility and Limitations, Journal of Economic Entomology Volume 93, Issue 6 (December 2000) pp. 1669–1679
Dowd PF (2001) Biotic and abiotic factors limiting efficacy of Bt corn in indirectly reducing mycotoxin levels in commercial fields. J Econ Ent 94(5): 1067–1074.
Dowd, Patrick F. (2004) Considering the Importance of Insect Resistance in Corn Ears in Relation to the Contribution of Insects to the Mycotoxin Problem (Mar. 2, 2004) (paper presented at the 40th Annual Illinois Corn Breeder’s School) (technical abstract available at http://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115-160750);
Dowd, P.F. (2004) Technical Abstract: Reliability of a ComputerProgram for Predicting Mycotoxin Levels in Midwestern U.S.A. Maize (May 21, 2004) (submitted to the Int’l Iupac Symposium on Mycotoxins and Phycotoxins), available at http://www.ars.usda.gov/research/publications/ublications.htm?SEQ_NO_115=159116.
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Described under
Scientists propose better profiling for GM crops- Stephen Daniells, NutraIngredients, June 11, 2007
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von Wettstein D, Warner J, Kannangara CG (2003) Supplements of transgenic malt or grain containing (1,3-1,4)-beta-glucanase increase the nutritive value of barley-based broiler diets to that of maize. British Poultry Science 44:438-449
Vazquez Padron, R.I., Moreno Fierros, L., Neri Bazan, L., De la Riva, G.A., Lopez Revilla, R., (1999). Intragastric and intraperitoneal administration of Cry1Ac protoxin from Bacillus thuringiensis induces systemic and mucosal antibody responses in mice. Life Sci. 64, 1897–1912.
Vazquez Padron, R.I., Gonzalez Cabrera, J., Garcia Tovar, C., Neri Bazan, L., Lopez Revilla, R., Hernandez, M., Morena Fierros, L., De la Riva, G.A. (2000). Cry1Ac protoxin from Bacillus thuringiensis sp. kurstaki HD73 binds to surface proteins in the mouse small intestine. Biochem. Biophys. Res. Commun. 271, 54–58.
Williams WP, Windham GL, Buckley PM and Daves CA (2002) Aflatoxin accumulation in conventional and transgenic corn hybrids infested with southwestern corn borer
(Lepidoptera: Crambidae). J Agric Urban Entomol 19(4): 227–236.
Yonemochi, C., H. Fujisaki, C. Harada, T. Kusama, and M. Hanazumi. 2002. Evaluation of transgenic event CBH 351 (Starlink) corn in broiler chicks. Animal Sci. J. 73:221-228.
Zdunczyk Z, Frejnagel S, Fornal J, Flis M, Palacios MC, Flis B, Zagorski-Ostoja W (2005) Biological response of rat fed diets with high tuber content of conventionally bred and transgenic potato resistant to necrotic strain of potato virus (PVYN) Part I. Chemical composition of tubers and nutritional value of diets. Food Control 16:761-766
Zdunczyk Z, Juskiewicz J, Fornal J, Mazur-Gonkowska B, Koncicki A, Flis B, Zimnoch-Guzowska E, Zagorski-Ostoja W (2005) Biological response of rat fed diets with high tuber content of conventionally bred and transgenic potato resistant to necrotic strain of potato virus (PVYN). Part II. Caecal metabolism, serum enzymes and indices of non-specific defence of rats. Food Control 16:767-772
Zhuo Q, Chen X, Piao J, Gu L (2004) [Study on food safety of genetically modified rice which expressed cowpea trypsin inhibitor by 90 day feeding test on rats] [Article in Chinese]. Wei Sheng Yan Jiu. 33:176-9.
Zhuo Q, Chen X, Piao J, Han C (2004) [Study on the teratogenicity effects of genetically modified rich which expressed cowpea trypsin inhibitor on rats] [Article in Chinese]. Wei Sheng Yan Jiu. 33:74-7.
Zhu Y, Li D, Wang F, Yin J, Jin H (2004) Nutritional assessment and fate of DNA of soybean meal from Roundup Ready or conventional soybeans using rats. Archives of Animal Nutrition-Archiv

Exhibit 4. Baseline studies on variability and unexpected outcomes from conventional breeding.

The genetics of plant metabolism

Joost J B Keurentjes, Jingyuan Fu, C H Ric de Vos, Arjen Lommen4, Robert D Hall, Raoul J Bino, Linus H W van der Plas, Ritsert C Jansen, Dick Vreugdenhil & Maarten Koornneef, Laboratory of Genetics, Wageningen University, Arboretumlaan 4, NL-6703 BD Wageningen, The Netherlands.

Variation for metabolite composition and content is often observed in plants. However, it is poorly understood to what extent this variation has a genetic basis. Here, we describe the genetic analysis of natural variation in the metabolite composition in Arabidopsis thaliana. Instead of focusing on specific metabolites, we have applied empirical untargeted metabolomics using liquid chromatography–time of flight mass spectrometry (LC-QTOF MS). This uncovered many qualitative and quantitative differences in metabolite accumulation between A. thaliana accessions. Only 13.4% of the mass peaks were detected in all 14 accessions analyzed. Quantitative trait locus (QTL) analysis of more than 2,000 mass peaks, detected in a recombinant inbred line (RIL) population derived from the two most divergent accessions, enabled the identification of QTLs for about 75% of the mass signals. More than one-third of the signals were not detected in either parent, indicating the large potential for modification of metabolic composition through classical breeding.

We observed considerable quantitative and qualitative variation in the mass profiles of the different accessions... On average, we detected 964 mass peaks per accession, with a minimum of 826 (Col) and a maximum of 1,337 (Cvi). We detected a total of 2,475 different mass peaks; 706 were unique to single accessions, and only 331 were present in all 14 accessions (Fig. 1a). We found an average of 50 unique mass peaks per accession, with a minimum of 14 (Bay-0) and a maximum of 235 (Cvi).

To uncover loci controlling the observed variation in metabolic profiles, we subsequently analyzed an RIL population derived from a cross between Landsberg erecta (Ler) and Cape Verde Islands (Cvi)13...We found it striking that 853 of a total of 2,129 mass peaks identified in the RIL population were not detected in either parent (Fig. 2a). Although the number of lines analyzed in the RIL population (160 lines measured in duplicate) exceeded that of the number of parental lines (five replicates of each parent measured in duplicate), making the chance of detecting mass peak intensities around the threshold level higher, the observed ratio did not differ much when the threshold was increased modestly (data not shown). This suggests that many metabolites not present in either parent are produced as a result of the recombination of the genomes of the two parents.

Nature Genetics
Published online: 4 June 2006; doi:10.1038/ng1815

Review
Plant breeding: importance of plant secondary metabolites for protection against pathogens and herbivores
M. Wink

Chemical protection plays a decisive role in the resistance of plants against pathogens and herbivores. The so-called secondary metabolites, which are a characteristic feature of plants, are especially important and can protect plants against a wide variety of microorganisms (viruses, bacteria, fungi) and herbivores (arthropods, vertebrates). As is the situation with all defense systems of plants and animals, a few specialized pathogens have evolved in plants and have overcome the chemical defense barrier. Furthermore, they are often attracted by a given plant toxin. During domestication of our crop and food plants secondary metabolites have sometimes been eliminated. Taking lupins as an example, it is illustrated that quinolizidine alkaloids are important as chemical defense compounds and that the alkaloid-free varieties (ldquosweet lupinsrdquo), which have been selected by plant breeders, are highly susceptible to a wide range of herbivores to which the alkaloid-rich wild types were resistant. The potential of secondary metabolites for plant breeding and agriculture is discussed.

Theoretical and Applied Genetics
DOI: 10.1007/BF00303957
Volume 75, Number 2, January 1988, p 225 - 233

Geographic and evolutionary diversification of glucosinolates among near relatives of Arabidopsis thaliana (Brassicaceae)

Glucosinolates are biologically active secondary metabolites that display both intra- and interspecific variation in the order Brassicales. Glucosinolate profiles have not been interpreted within a phylogenic framework and little is known regarding the processes that influence the evolution of glucosinolate diversity at a macroevolutionary scale. We have analyzed leaf glucosinolate profiles from members of the Brassicaceae that have diverged from Arabidopsis thaliana within the last 15 million years and interpreted our findings relative to the phylogeny of this group. We identified several interspecific polymorphisms in glucosinolate composition. A majority of these polymorphisms are lineage-specific secondary losses of glucosinolate characters, but a gain-of-character polymorphism was also detected. The genetic basis of most observed polymorphisms appears to be regulatory. In the case of A. lyrata, geographic distribution is also shown to contribute to glucosinolate metabolic diversity. Further, we observed evidence of gene-flow between sympatric species, parallel evolution, and the existence of genetic constraints on the evolution of glucosinolates within the Brassicaceae.

Windsor, A.J. et al. Phytochemistry 66, 1321–1333 (2005).

Global impact of mutation-derived varieties

B.S. Ahloowalia1, M. Maluszynski and K. Nichterlein
Euphytica,Volume 135, Number 2 January 2004 p 187 - 204

Abstract
During the past seventy years, world wide more than 2250 varieties have been released that have been derived either as direct mutants or from their progenies. Induction of mutations with radiation has been the most frequently used method for directly developed mutant varieties. The prime strategy in mutation-based breeding has been to upgrade the well-adapted plant varieties by altering one or two major traits, which limit their productivity or enhance their quality value. In this paper, the global impact of mutation-derived varieties on food production and quality enhancement is presented. In addition, the economic contribution of the selectedmutant varieties of rice, barley, cotton,groundnut, pulses, sunflower, rapeseed and Japanese pear is discussed. In several mutation-derived varieties, the changed traits have resulted in synergistic effect on increasing the yield and quality of the crop, improving agronomic inputs, crop rotation, and consumer acceptance. In contrast to the currently protected plantvarieties or germplasm and increasing restrictions on their use, the induced mutants have been freely available for plant breeding. Many mutants have made transnational impact on increasing yield and quality of several seed-propagated crops. Induced mutations will continue tohave an increasing role in creating cropvarieties with traits such as modified oil, protein and starch quality, enhanced uptake of specific metals, deeper rooting system, and resistance to drought, diseases andsalinity as a major component of theenvironmentally sustainable agriculture.Future research on induced mutations would also be important in the functional genomics of many food crops

Exhibit 5.
Selected GM Food safety testing papers citations together with paper summaries (a work in Progress):

Aeschbacher K, Messikommer R, Meile L, Wenk C (2005) Bt176 corn in poultry nutrition: Physiological characteristics and fate of recombinant plant DNA in chickens. Poultry Science 84:385-394
Abstract: A genetically modified Bt176 corn hybrid, which contains an insecticidal protein against the European corn borer, and its conventional, nonmodified counterpart were evaluated in 4 separate trials to verify substantial equivalence in feeding value and animal performance. Thirty-six individually kept laying hens and 3 replicates of 94 broiler chickens each, assigned to 12 cages, were fed 2 different hen and broiler diets containing either 60% conventional or 60% Bt176 corn. The nutrient compositions of the 2 corn hybrids and the 2 corn diets revealed no major differences. Furthermore, metabolism and performance data revealed no significant differences between the birds that received the conventional, nonmodified corn, and those that received the modified corn diets. The detection of the genetic modification, by PCR, in feed obtained from insect-resistant Bt corn, in tissues and products from animals fed Bt corn is described. In all evaluated chicken tissues of muscle, liver, and spleen, the corn-chloroplast ivr gene fragment was amplified. It can be deduced from these findings and from other studies that the transfer of DNA fragments into the body is a normal process that takes place constantly. Nevertheless, no recombinant plant DNA fragments such as recombinant bla or cry1A(b) fragments could be found. Bt-gene specific constructs from the Bt corn were not detected in any of the poultry samples, neither in organs, meat, nor eggs.

Alexander TW, Sharma R, Deng MY, Whetsell AJ, Jennings JC, Wang YX, Okine E, Damgaard D, McAllister TA (2004) Use of quantitative real-time and conventional PCR to assess the stability of the cp4 epsps transgene from Roundup Ready (R) canola in the intestinal, ruminal, and fecal contents of sheep. Journal of Biotechnology 112:255-266
Abstract: The stability of transgenic DNA encoding the synthetic cp4 epsps protein in a diet containing Roundup Ready (RR)® canola meal was determined in duodenal fluid (DF) batch cultures from sheep. A real-time TaqMan® PCR assay was designed to quantify the degradation of cp4 epsps DNA during incubation in DF at pH 5 or 7. The copy number of cp4 epsps DNA in the diet declined more rapidly (P <> 0.05) by corn line. In Study 2, there was no effect of corn line on overall ADFI (as-fed basis) or GY ratio. In addition, overall ADG of barrows fed the four corn lines did not differ (P > 0.05); however, overall ADG of gilts fed corn DK647 was greater (P <> 0.05) of corn line on carcass yield or fatness measurements in either study. Differences between barrows and gilts for growth and carcass traits were generally similar for both studies and in line with previous research. Overall, these results indicate that Roundup Ready corn (NK603) gives equivalent animal performance to conventional corn for growing pigs.

Ash J, Novak C, Scheideler SE (2003) The fate of genetically modified protein from Roundup Ready Soybeans in laying hens. Journal of Applied Poultry Research 12:242-245
Abstract: A study was conducted to determine the extent of genetically modified (GM) protein from Roundup Ready Soybeans in tissues and eggs of laying hens. Because a breakdown of the modified portion of protein was expected due to the digestive process of the hen, an immunoassay test was run. By using a double antibody sandwich format specific for the CP4 EPSPS protein, a qualitative test was performed to determine the presence of modified proteins in various samples. Raw soybeans, soybean meal, complete diet, whole egg, egg albumen, liver, and feces from laying hens were collected from two independent commercial egg producers. Roundup Ready soybeans, soybean meal, and complete diets ere determined to contain the GM proteins. Whole egg, egg albumen, liver, and feces were all negative for GM protein. In conclusion, the digestive process of the laying hen effectively broke down the GM protein from the soybean meal portion of the diet, hence no modified protein was found in the liver, egg; or feces in this brief field trial.

Aulrich K, Bohme H, Daenicke R, Halle I, Flachowsky G (2001) Genetically modified feeds in animal nutrition 1st communication: Bacillus thuringiensis (Bt) corn in poultry, pig and ruminant nutrition. Archives of Animal Nutrition-Archiv fur Tierernahrung 54:183-195
Abstract: Investigates the substantial equivalence of a transgenic Bacillus thuringiensis (Bt) corn and nontransgenic hybrid Cesar and a nutrition psychology for poultry, pigs and ruminants. Details of experiments conducted on corn, laying hens, broilers, pigs and ruminants; Overview on the cultivation of transgenic plants; Information on the concept of substantial equivalence.

Baker, J M, Hawkins, N D, Ward, J L, Lovegrove, A, Napier,J A, Shewry, P R and Beale, M H.(2006) A metabolomic study of substantial equivalence of field-grown genetically modified wheat. Plant Biotechnology Journal Volume 4 Issue 4 Page 381 - July 2006 doi:10.1111/j.1467-7652.2006.00197.x
The 'substantial equivalence' of three transgenic wheats expressing additional high-molecular-weight subunit genes and the corresponding parental lines (two lines plus a null transformant) was examined using metabolite profiling of samples grown in replicate field trials on two UK sites (Rothamsted, Hertfordshire and Long Ashton, near Bristol) for 3 years. Multivariate comparison of the proton nuclear magnetic resonance spectra of polar metabolites extracted with deuterated methanol–water showed a stronger influence of site and year than of genotype. Nevertheless, some separation between the transgenic and parental lines was observed, notably between the transgenic line B73-6-1 (which had the highest level of transgene expression) and its parental line L88-6. Comparison of the spectra showed that this separation resulted from increased levels of maltose and/or sucrose in this transgenic line, and that differences in free amino acids were also apparent. More detailed studies of the amino acid composition of material grown in 2000 were carried out using gas chromatography-mass spectrometry. The most noticeable difference was that the samples grown at Rothamsted consistently contained larger amounts of acidic amino acids (glutamic, aspartic) and their amides (glutamine, asparagine). In addition, the related lines, L88-6 and B73-6-1, both contained larger amounts of proline and γ-aminobutyric acid when grown at Long Ashton than at Rothamsted. The results clearly demonstrate that the environment affects the metabolome and that any differences between the control and transgenic lines are generally within the same range as the differences observed between the control lines grown on different sites and in different years.

Barriere Y, Verite R, Brunschwig P, Surault F, Emile JC (2001) Feeding value of corn silage estimated with sheep and dairy cows is not altered by genetic incorporation of Bt176 resistance to Ostrinia nubilalis. Journal of Dairy Science 84:1863-1871
Abstract: A genetically modified Bt176 corn hybrid (Rh208Bt)-providing control of European corn borer damage-and the conventional isogenic hybrid (Rh208)-harvested as whole plant silage-were evaluated in three separate feeding trials to verify that the in vivo feeding value was substantially equivalent among modified and conventional hybrids. In the first trial, after a week of preexperiment, two sets of six Texel sheep, housed in digestibility crates, were fed silage sources of Rh208 and Rh208Bt; hybrids, and silage of three additional control varieties of low, intermediate, and high feeding value (Rh289, Adonis, and Adonis bm3) for 1 wk. Feed offered to sheep was adjusted to maintenance requirements based on metabolic body weight. Agronomic and biochemical traits were similar among the Rh208 and Rh208Bt; hybrids. Organic matter digestibility (67.1 and 67.6%), crude fiber digestibility (52.9 and 54.2%), and neutral detergent fiber digestibility (50.2 and 49.0%) were not significantly different among Rh208 and Rh208Bt hybrids. In the second trial, two sets of 24 Holstein cows were fed silage from Rh208 and Rh208Bt corn hybrids for 13 wk, 9 wk after calving, and including 2 wk of preexperiment. Fat-corrected milk yield (31.3 and 31.4 kg/d), protein content (31.7 and 31.6 g/kg) and fat content (36.7 and 37.0 g/kg) in milk of dairy cows were unaffected by hybrid source. Body weight gains of cattle were not different. However, intake was significantly higher in cows fed Rh208Bt silage. In the third trial, five midlactation multiparous Holstein cows were successively fed the silage from Rh208 and Rh208Bt corn hybrids 2 or 3 wk. Data were considered only for the last week of each period. There were no significant effects on protein fractions, fatty acid composition, or coagulation properties of milk between Rh208 and Rh208Bt fed cattle. Cattle and sheep can perform equally well with a conventional or a genetically modified Bt176 corn silage

Baudo, M M, Lyons, Powers, S R, Pastori,G M, Edwards, K J, Holdsworth, M J, and Shewry, P R. (2006) Transgenesis has less impact on the transcriptome of wheat grain than conventional breeding Plant Biotechnology Journal Volume 4 Issue 4 Page 369 - July 2006 doi:10.1111/j.1467-7652.2006.00193.x
Detailed global gene expression profiles have been obtained for a series of transgenic and conventionally bred wheat lines expressing additional genes encoding HMW (high molecular weight) subunits of glutenin, a group of endosperm-specific seed storage proteins known to determine dough strength and therefore bread-making quality. Differences in endosperm and leaf transcriptome profiles between untransformed and derived transgenic lines were consistently extremely small, when analysing plants containing either transgenes only, or also marker genes. Differences observed in gene expression in the endosperm between conventionally bred material were much larger in comparison to differences between transgenic and untransformed lines exhibiting the same complements of gluten subunits. These results suggest that the presence of the transgenes did not significantly alter gene expression and that, at this level of investigation, transgenic plants could be considered substantially equivalent to untransformed parental lines.

Bohme H, Aulrich K, Daenicke R, Flachowsky G (2001) Genetically modified feeds in animal nutrition 2nd communication: Glufosinate tolerant sugar beets (roots and silage) and maize grains for ruminants and pigs. Archives of Animal Nutrition-Archiv fur Tierernahrung 54:197-207
Abstract: To analyse substantial equivalence of genetically modified sugar-beets and maize, in which the glufosinate-tolerant (Pat) gene is inserted, crude nutrients, the amino acid and the fatty acid profiles as well as the composition of the NDF-fraction of maize grains were determined and compared with those of the corresponding non-transgenic cultivars. Due to the genetic manipulation differences in crude nutrient contents including sugar and starch were not detected, The amino acid profile of maize grains was analysed to be the same, Fatty acid profile and composition of cell wall constituents did not show any influences as well. Digestibility of Pat-sugar-beets and maize grains for pigs did not demonstrate meaningful differences as compared to the corresponding non-transgenic cultivars. Digestibility of sugar-beet roots and sugar-beet top silage for ruminants proved to be also in the scope of natural variance. As the digestibility of the macro nutrients remained unaffected, the Pat-gene introduction into both crops did not show an influence on the energetic feeding value. For pigs the ME-content of Pat-sugar-beets was determined to be 14.1 MJ/kg DM versus 13.7 MJ of the non-transgenic cultivars. ME-content of Pat-maize grains was 16.0 MJ/kg DM versus 15.8 MJ for controls. For ruminants the feeding value of Pat-sugar-beets was found to be 8.5 MJ NEL/kg DM or 13.2 MJ ME/kg DM, regardless of whether the Pat-gene was inserted or not. The corresponding energy values of sugar-beet top silage ranged between 5.2 and 5.5 MJ NEL/kg DM or 8.6 and 9.1 MJ ME/kg DM, with differences considered in the biological range

Brake DG, Thaler R, Evenson DP (2004) Evaluation of Bt (Bacillus thuringiensis) corn on mouse testicular development by dual parameter flow cytometry. Journal of Agricultural and Food Chemistry 52:2097-2102.
Abstract: The health safety of Bt (Bacillus thuringiensis) corn (Zea mays L.) was studied using mouse testes as a sensitive biomonitor of potential toxic effects. Pregnant mice were fed a Bt corn or a nontransgenic (conventional) diet during gestation and lactation. After they were weaned, young male mice were maintained on the respective diets. At 8, 16, 26, 32, 63, and 87 days after birth, three male mice and an adult reference mouse were killed, the testes were surgically removed, and the percentage of germ cell populations was measured by flow cytometry. Multigenerational studies were conducted in the same manner. There were no apparent differences in percentages of testicular cell populations (haploid, diploid, and tetraploid) between the mice fed the Bt corn diet and those fed the conventional diet. Because of the high rate of cell proliferation and extensive differentiation that makes testicular germ cells highly susceptible to some toxic agents, it was concluded that the Bt corn diet had no measurable or observable effect on fetal, postnatal, pubertal, or adult testicular development. If data from this study were extrapolated to humans, Bt corn is not harmful to human reproductive development.
Brake J, Vlachos D (1998) Evaluation of transgenic event 176 "Bt" corn in broiler chickens. Poultry Science 77:648-653.
Abstract: A 38-d feeding study evaluated whether standard broiler diets prepared with transgenic Event 176-derived "Bt" corn (maize) grain had any adverse effects on male or female broiler chickens as compared to diets prepared with nontransgenic (isogenic) control corn grain. No statistically significant differences in survival or BW were observed bet ia een birds reared on mash or pelleted diets prepared with transgenic corn and similar diets prepared using control corn. Broilers raised on diets prepared from the transgenic corn exhibited significantly better feed conversion ratios and improved yield of the Pectoralis minor breast muscle. Although it is not clear whether this enhanced performance was attributable to the transgenic corn per se, or due to possible slight differences in overall composition of the formulated diets, it was clear that the transgenic corn had no deleterious effects in this study

Brake J, Faust MA, Stein J (2003) Evaluation of transgenic event Bt11 hybrid corn in broiler chickens. Poultry Science 82:551-559
Abstract: A feeding study evaluated whether standard broiler diets prepared with grain derived from Syngenta Seeds NK Brand Bacillus thuringiensis (Bt) Corn hybrids had any adverse effects on male or female broiler chickens. Four kinds of corn grain were used in this study: (1) grain from the Bt-expressing field corn hybrid N7070Bt, (2) grain from the N7070Bt hybrid that had been sprayed with Liberty brand herbicide (glufosinate) according to manufacturer's instructions (N7070Bt + Liberty), (3) grain from standard N7070 (non-Bt isoline of N7070Bt) grain, and (4) a lot of North Carolina grown grain from the 2000 growing season (NC2000). The amino acid balance for the four lots of corn was similar relative to their crude protein content; however, the NC2000 corn had higher protein content. Diets with the higher protein NC2000 season corn were amended with a combination of sand, ground cardboard (Solka Floc), and poultry fat so that the metabolizable energy and crude protein content of the diluted diets would be similar to that of the isoline and transgenic diets. Growth of broilers was excellent with males being significantly heavier than females (2,497 g vs. 2,103 g) at 42 d of age. BW of live birds at 42 d was within 26 g for the three treatment groups fed corn that was from the same genetic background, i.e., the two Bt transgenic groups (N7070Bt, N7070Bt + Liberty), and the non-Bt N7070 isoline corn group, while BW for the NC2000 group was significantly lower by 93 g. There was no overall corn source effect on feed conversion ratio (FCR) among the isoline and transgenic corn sources to 42 d of age, but FCR was poorer for broilers consuming the commercial NC2000 corn. There was no overall effect of corn source on survivability to 42 d. Carcass analysis at 48 d demonstrated no differences in percentage carcass yield due to corn source among males and females. The transgenic N7070Bt and N7070Bt + Liberty hybrid diets supported excellent broiler chicken growth with mortality and FCR that were similar to that supported by the N7070 isoline control and better than rates from the commercial NC2000 corn without significant differences among treatment groups in carcass yield. It was clear that the transgenic corn had no deleterious or unintended effects on production traits of broiler chickens in this study

Brake J, Faust M, Stein J (2005) Evaluation of transgenic hybrid corn (VIP3A) in broiler chickens. Poultry Science 84:503-512
Abstract: A 49-d feeding study evaluated whether standard broiler diets prepared with Syngenta Seeds VIP3A transgenic derived corn grain had any unanticipated adverse effects on male or female broiler chickens as compared with diets prepared with nontransgenic (isoline) control corn grain. Two commercial lots of grain grown in North Carolina during the 1999 (NC 1999) and 2000 (NC 2000) seasons were included for reference purposes. Broiler growth was excellent with males reaching 3,466 g and females reaching 2,882 g at 49 d of age. Final BW of the VlP3A, isoline, and NC 1999 corn groups were within 21.1 g, whereas the NC 2000 group was 42.4 g lower than the lowest of this group. There was no overall corn source effect on adjusted feed conversion ratio (FCR) or mortality to 49 d of age. Carcass analysis demonstrated no differences in percentage yield due to corn source among males and females other than percentage wings in females. Comprehensive clinical chemical analyses of blood taken from representative birds at 49 d of age showed no differences due to corn sources. The transgenic VIP3A hybrid diets numerically supported the most rapid broiler chicken growth, the second lowest mortality rate and best FCR, without practical differences in carcass yield. The few differences found in this study were not unique to a given corn source but instead appeared to be distributed equally across the diet groups evaluated in the study. Although it was not clear whether small differences in performance were attributable to the transgenic corn per se or were due to possible slight differences in overall composition of the formulated diets, it was clear that the transgenic corn had no deleterious effects on broiler performance and carcass yield in this study.

Broll H, Zagon J, Butschke A, Leffke A, Spiegelberg A, Bohme H, Flachowsky G (2005) The fate of DNA of transgenic inulin synthesizing potatoes in pigs. Journal of Animal and Feed Sciences 14:337-340
Abstract: Silage from a genetically modified potato expressing the 1-SST (sucurose:sucrose 1-fructosyltransferase) and the l-FFT (fructan:fructan 1-fructosyltransferase) was used in a feeding experiment with pigs. After a feeding period of 42 days samples from various organs and digesta were collected and investigated with four different real time PCR systems, in order to identify the fate of the foreign DNA. No plant specific DNA or DNA specific for the genome alteration in the transgenic potato were detected in any organ. In contrast, chloroplast specific DNA was detected in the digesta of duodenum, jejunum, colon and rectum. The single-copy metallo-carboxypeptidase inhibitor gene sequence was detected only in samples from the stomach content of pigs fed the isogenic potato and in those from duodenum and jejunum of animals fed the transgenic one. No evidence for the integration of the foreign DNA into the host genome was observed.

Catchpole, Gareth S., Manfred Beckmann, David P. Enot, Madhav Mondhe, Britta Zywicki, Janet Taylor, Nigel Hardy, Aileen Smith, Ross D. King, Douglas B. Kell, Oliver Fiehn and John Draper,(2005 ) Hierarchical metabolomics demonstrates substantial compositional similarity between genetically modified and conventional potato crops. Free online, Published online before print September 26, 2005, 10.1073/pnas.0503955102
PNAS October 4, 2005 vol. 102 no. 40 14458-14462

There is current debate whether genetically modified (GM) plants might contain unexpected, potentially undesirable changes in overall metabolite composition. However, appropriate analytical technology and acceptable metrics of compositional similarity require development. We describe a comprehensive comparison of total metabolites in field-grown GM and conventional potato tubers using a hierarchical approach initiating with rapid metabolome "fingerprinting" to guide more detailed profiling of metabolites where significant differences are suspected. Central to this strategy are data analysis procedures able to generate validated, reproducible metrics of comparison from complex metabolome data. We show that, apart from targeted changes, these GM potatoes in this study appear substantially equivalent to traditional cultivars.

Discussion in Catchpole et al.

The nature of food in terms of safety cannot be assessed in an absolute manner. As a first pass in any compositional comparison, we suggest that a rapid but sensitive comprehensive and comparably inexpensive first screen can be provided by mass spectrometric fingerprinting, which may be complemented by more detailed analyses using GC-TOF or LC-MS, depending on the level of similarity to other cultivars as determined by statistical analysis.

A major finding from the present study was the large variation in metabolite profile between the conventional cultivars. These significant differences were never sought as desired traits in traditional breeding programs, and overall composition has not given cause for public safety concerns in conventionally bred cultivars. In the context of substantial equivalence, we show that the metabolite composition of field-grown inulin-producing potatoes were within the natural metabolite range of classical cultivars and were, in fact, very similar to the progenitor line Désirée, with the exception of the introduced genes and, therefore, the predictable up-regulation of fructans and their expected derivatives. In the comparative assessment framework, such metabolic side products might eventually be subjected to more detailed investigations if deemed necessary with respect to toxicity, abundance, and chemical structure.

The cultivar-based compositional heterogeneity we describe emphasizes the importance of comparison with a range of equivalent cultivars and not solely the parental line. ...This result indicated that metabolic changes caused through conventional breeding techniques were, in these cases, at least of a comparable magnitude to those resulting as an unintended effect of genetic engineering techniques.

Chen ZL, Gu H, Li Y, Su Y, Wu P, Jiang Z, Ming X, Tian J, Pan N, Qu LJ.(2003) Safety assessment for genetically modified sweet pepper and tomato.
Toxicology. 2003 Jun 30;188(2-3):297-307.

The coat protein (CP) gene of cucumber mosaic virus (CMV) was cloned from a Chinese CMV isolate, the CaMV promoter and NOS terminator added and the gene construct was transformed into both sweet pepper and tomato plants to confer resistance to CMV. Safety assessments of these genetically modified (GM) plants were conducted. It was found that these two GM products showed no genotoxicity either in vitro or in vivo by the micronucleus test, sperm aberration test and Ames test. Animal feeding studies showed no significant differences in growth, body weight gain, food consumption, hematology, blood biochemical indices, organ weights and histopathology between rats or mice of either sex fed with either GM sweet pepper or tomato diets compared with those with non-GM diets. These results demonstrate that the CMV-resistant sweet pepper and tomato are comparable to the non-GM counterparts in terms of food safety.

Chesson A, Flachowsky G (2003) Transgenic plants in poultry nutrition. Worlds Poultry Science Journal 59:201-207
Abstract: Studies on genetically modified (GM) feedstuffs, for poultry (and other livestock species) have not added any substance to public concerns in Europe about their safety for human or bird health. The compositions of maize lines engineered for insect resistance (Bt-maize) or herbicide tolerance (glyphosate) and herbicide-tolerant soybean have all proved to be essentially indistinguishable from their conventional counterparts. Consequently, and not surprisingly, comparative feeding studies with broilers and layers in which conventional maize (50 to 78%) or soybeans (27%) were replaced in feeds by transgenic varieties, also have failed to show differences of any significance in production parameters. These data indicate that feeding studies with target livestock species contribute very little to the safety assessment of crops engineered for input traits that have little or no detectable effect on chemical composition. However, comparative growth studies made with broiler chicks, particularly sensitive to any change in nutrient supply or the presence of toxic elements in their feed, can be used to screen for any unintended adverse consequence of the recombinant event not detected by compositional analysis. This does, however, depend on whether the GM plant can be matched to a parental line or another suitable control and its suitability for inclusion in broiler diets. The discovery that DNA fragments from the digestive tract can be found in the tissues of animals evoked interest in the fate of ingested transgenes. Plant DNA derived from feed has been detected in the muscle, liver, spleen and kidneys of broilers and layers, although not in eggs. However, no fragments of transgenic DNA or its expressed protein have been found to date in poultry meat or eggs or in any other animal tissues examined

Chowdhury EH, Mikami O, Murata H, Sultana P, Shimada N, Yoshioka M, Guruge KS, Yamamoto S, Miyazaki S, Yamanaka N, Nakajima Y (2004) Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11. Journal of Food Protection 67:365-370
Abstract: Sixteen multiparous Holstein cows averaging 74 d in milk were used in a replicated 4 x 4 Latin square to compare the effects on animal performance of feeding whole plant silage and grain from a glyphosate-tolerant corn hybrid (event NK603), a nontransgenic control hybrid, and two commercial nontransgenic hybrids (DK647 and RX740). The grain and silage from the four corn hybrids were produced using the same procedures and under similar agronomic conditions at the University of Illinois. On a dry matter (DM) basis, diets contained 30% corn silage and 27.34% corn grain produced either from event NK603, a nontransgenic control, or commercial hybrids. Apart from the DM content of silages, the chemical composition of both grain and silage produced from the four corn hybrids were substantially equivalent. Feeding diets that contained event NK603 and DK647 hybrids tended to decrease DM intake (DMI) compared with the control nontransgenic and RX740. The intakes of crude protein (CP), acid and neutral detergent fiber, and nonfiber carbohydrates were not different for cows fed event NK603 and control diets. The RX740 diet resulted in the highest intakes of fiber and CP, whereas the DK647 diet resulted in the lowest intake of CP. These differences in nutrient intake arose from small variations in both the DMI and the chemical composition of feed ingredients and experimental diets. Production of milk and 3.5% fat-corrected milk; milk fat, CP, and true protein percentage and yield; milk urea N; milk total solids percentage and yield; and somatic cell count were not affected by treatments. These data indicate that the stable insertion of the gene that confers tolerance to glyphosate (event NK603) in the corn line used in this experiment does not affect its chemical composition and nutritional value for lactating dairy cows when compared with conventional corn.

Chowdhury EH, Shimada N, Murata H, Mikami O, Sultana P, Miyazaki S, Yoshioka M,
Yamanaka N, Hirai N, Nakajima Y.(2003). Detection of Cry1Ab protein in gastrointestinal contents but not visceral organs
of genetically modified Bt11-fed calves.Vet Hum Toxicol. 2003 Mar;45(2):72-5.

The fate of insecticidal Cry1Ab protein was examined in the gastrointestinal (GI) contents and visceral organs of calves fed insect-resistant genetically modified maize Bt11. Twelve cross-breed (Japanese black x Holstein) calves were fed either Bt11 or non-genetically modified isoline maize for 90 d. Peripheral blood, rumen juice and feces were collected fortnightly, and GI contents and visceral organs were collected at slaughter at the end of the experiment.
Samples were checked for Cry1Ab protein by immunological methods, and visceral organs were examined pathologically. Trace amounts of Cry1Ab protein were detected in the GI contents but not in the liver, spleen, kidney, muscle or
mesenteric lymph nodes. No lesions were observed pathologically. Cry1Ab protein in the feces was degraded quickly at atmospheric temperature. These results suggested that only a trace amount of Cry1Ab protein survived passage through
the GI tract but was not transferred to liver, spleen, kidney, lymph nodes or muscles.

Chrenkova M, Sommer A, Ceresnakova Z, Nitrayova S, Prostredna M (2002) Nutritional evaluation of genetically modified maize corn performed on rats. Archives of Animal Nutrition-Archiv fur Tierernahrung 56:229-235
Abstract: Presents a study that determined the composition and nutritional value of conventional and transgenic maize with an introduced gene of glyphosate resistance that was fed to rats. Background on genetically modified crops; Details on the chemical analysis performed; Implications of the results.

Corpillo D, Gardini G, Vaira AM, Basso M, Aime S, Accotto GP, Fasano M
Proteomics as a tool to improve investigation of substantial equivalence in
genetically modified organisms: the case of a virus-resistant tomato.
Proteomics. 2004 Jan;4(1):193-200.

At present, the so-called "substantial equivalence" is the only widely accepted criterion for deciding whether or not a transgenic food is, from an alimentary point of view, to be considered totally correspondent to the "traditional" one from which it derives. Although never exactly defined, it deals with a comparison between the chemical composition of the two foods. A more in-depth analysis can be performed by one of the most suitable methods that allows for
the simultaneous screening of many components without prior identification, the analysis of the proteome. As a model for testing this kind of approach, we compared protein expression of two types of tomato plants, having the same genetic background, except for a virus resistance trait introduced by genetic engineering. When proteins extracted from seedlings of the two types were analyzed by two-dimensional electrophoresis, no significant differences, either
qualitative or quantitative, were detected, indicating that in this case the expression of major proteins was unmodified by the genetic manipulation. Fifteen proteins were identified by peptide mass fingerprinting.

Daenicke R, Aulrich K, Flachowsky G (1999) GMO in animal feedstuffs: nutritional properties of Bt-maize... Mais: Fachzeitschrift uber Forschung, Produktionstechnik, Verwertung und Okonomik 135-137
Abstract: Die Verfütterung gentechnisch veränderter Pflanzen oder von Teilen dieser Pflanzen und die anschließende Nutzung der tierischen Produkte werfen mannigfaltige Fragen auf. Die Diskussion hierüber wird bislang ähnlich dem Lebensmittelbereich weitgehend auf emotionaler Ebene geführt.

Donkin SS, Velez JC, Totten AK, Stanisiewski EP, Hartnell GF (2003) Effects of feeding silage and grain from glyphosate-tolerant or insect-protected corn hybrids on feed intake, ruminal digestion, and milk production in dairy cattle. Journal of Dairy Science 86:1780-1788
Abstract: Lactating dairy cows were used to determine effects of feeding glyphosate-tolerant or insect-protected corn hybrids on feed intake, milk production, milk composition, and ruminal digestibility. Corn resistant to European corn borer (Ostrinia nubilalis) infestation (Bt-MON810), or its nontransgenic control (Bt-CON), were planted in alternating fields, during two successive years. One-half of each strip was harvested for whole plant corn silage and the remainder was allowed to mature and harvested as grain. Effects of feeding diets containing either Bt-MON810 or Bt-CON grain and silage were determined in two experiments (1 and 2) conducted during successive years. In experiment 3, glyphosate-tolerant Roundup Ready corn (RR-GA21) or its nontransgenic control (RR-CON) corn were grown in alternating fields during one cropping season. Diets contained 42 to 60% corn silage and 20 to 34% corn grain from Bt-MON810, RR-GA21, or the appropriate nontransgenic counterpart; treatments were applied using a switchback design. Cows were fed ad libitum and milked twice daily. There were no differences for nutrient composition between silage sources or between grain sources within an experiment. Data for experiments 1 and 2 indicated similar dry matter intake (DMI), 4% fat-corrected milk (FCM) production, and milk composition between Bt-MON810 and Bt-CON diets. There were no differences for DMI, 4% FCM production, and milk composition between RR-GA21 and RR-CON diets. There was no difference in ruminal degradability, determined separately for corn silage and corn grain, for RR-GA21 or Bt-MON810-hybrids compared with their respective controls. These data demonstrate equivalence of nutritional value and production efficiency for corn containing Bt-MON810 compared with its control and for RR-GA21 corn compared with its control

Einspanier R, Lutz B, Rief S, Berezina O, Zverlov V, Schwarz W, Mayer J (2004) Tracing residual recombinant feed molecules during digestion and rumen bacterial diversity in cattle fed transgene maize. European Food Research and Technology 218:269-273
Abstract: The aim of this study was to trace selected nucleic acid and protein components of isogene versus Bt transgene maize within the bovine gastrointestinal tract (GIT). After feeding 22 cattle for 4 weeks with Bt176 maize, different plant genes and the recombinant protein CryIAb were quantified during digestion. Furthermore, a first initial characterization of rumen bacteria was approached, using 16rDNA gene sequencing comparing isogene- against transgene-fed animals. Ingesta samples of different GIT sections (rumen, abomasum, jejunum, colon) were analysed for chloroplast, maize invertase, zein and Bt toxin (CryIAb) gene fragments using quantitative real-time PCR. First, the initial gene dose of these maize genes was detected in maize silage. During digestion, a significant reduction of high-to-medium abundant plant gene fragments was shown depending on the dwell-time and the initial gene copy number. Immunoreactive CryIAb protein was quantified by ELISA in intestinal samples indicating a significant loss of that protein. Remarkable amounts of Bt toxin were found in all contents of the GIT and the protein was still present in faeces. For the first time, the influence of CryIAb transgene maize on rumen bacterial microflora was investigated compared to isogene material through analysis of 497 individual bacterial 16S rDNA sequences. In principle, specific bacterial leader-species could be identified in all bovine rumen extracts, but no significant influence of Bt176 maize feed was found on the composition of the microbial population. This investigation provides supplementing data to further evaluate the fate of novel recombinant material originating from transgene feed or food within the mammalian GIT

Ewen SWB, Pusztai A (1999) Effect of diets containing genetically modified potatoes expressing Galanthus nivalis lectin on rat small intestine. Lancet 354:1353-1354
Abstract: Diets containing genetically modified (GM) potatoes expressing the lectin Galanthus nivalis agglutinin (GNA) had variable effects on different parts of the rat gastrointestinal tract. Some effects, such as the proliferation of the gastric mucosa, were mainly due to the expression of the GNA transgene. However, other parts of the construct or the genetic transformation (or both) could also have contributed to the overall biological effects of the GNA-GM potatoes, particularly on the small intestine and caecum.

Fearing PL, Brown D, Vlachos D, Meghji M, Privalle L (1997) Quantitative analysis of CryIA(b) expression in Bt maize plants, tissues, and silage and stability of expression over successive generations. Molecular Breeding 3:169-176
Abstract: The range and stability of expression of the transgenic CryIA(b) protein was examined in Ciba Seeds Bt maize plants derived from Event 176. Specifically, CryIA(b) levels were determined for: (1) various plant tissues and developmental stages in three maize lines from 1993 field tests; (2) pollen and leaves from plants representing four backcross generations of two genotypes; (3) leaves of 6 precommercial hybrids; and (4) silage from one Bt maize hybrid. Significant levels were found only in pollen and leaves. Genetic background did not greatly impact the level seen in either tissue. CryIA(b) expression in maize plants derived from transformation Event 176 was stable over at least four successive generations. On a per acre basis, the highest amount of CryIA(b) protein (estimated to be 2-4 g CryIA(b) protein/acre) was found to occur at anthesis, consistent with the stage at which maximum plant vegetative biomass is reached. CryIA(b) was not detected in silage prepared from CryIA(b)-expressing plants. The maize-expressed CryIA(6) protein was found to have the expected size and to be immunoreactive with antibodies prepared against crystals from Bacillus thuringiensis subsp. kurstaki

Glencross B, Curnow J, Hawkins W, Kissil GWM, Peterson D (2003) Evaluation of the feed value of a transgenic strain of the narrow-leaf lupin (Lupinus angustifolius) in the diet of the marine fish, Pagrus auratus. Aquaculture Nutrition 9:197-206
Abstract: This study assessed the nutritional and biological value of a noncommercial, transgenic line of the Australian sweet lupin, Lupinus angustifolius, (cv. Warrah), produced to increase the methionine content of the seed. An initial experiment demonstrated that differences in the methionine content of the transgenic and nontransgenic control lupins had no apparent influence on the growth of juvenile red seabream fed practical diets. Re-evaluation of the nutritional characteristics of the lupin meals with subsequent digestibility studies allowed the determination of the digestible value of the protein and energy content of each of the varieties. The digestible protein content of either variety was similar, however significant differences in the digestible energy value of each variety existed (56.3% cf. 64.0%). This re-evaluation of the nutritional value of the genetically manipulated (GM) and non-GM lupin varieties enabled the reformulation of diets on a digestible protein and energy basis. A second growth trial was undertaken using sub-satietal pair-feeding regimes, with the experiment also involving protein-restrictive diets to allow expression of the differences in the methionine content of the transgenic and nontransgenic lupin meals. A significant benefit of the enhanced methionine level in the transgenic lupin was observed. It is argued that in the high-protein fish diets used, the importance of amino acid composition is relatively limited. Economic modelling of the potential value of the increased methionine in the transgenic lupin suggests that this will have limited benefit for aquaculture industries, and that greater value would be attributable to higher protein and energy levels in ingredients

Grant RJ, Fanning KC, Kleinschmit D, Stanisiewski EP, Hartnell GF (2003) Influence of glyphosate-tolerant (event nk603) and corn rootworm protected (event MON863) corn silage and grain on feed consumption and milk production in Holstein cattle. Journal of Dairy Science 86:1707-1715
Abstract: Two studies were conducted to evaluate the effect of a glyphosate-tolerant (event nk603) and a corn rootworm protected (event MON863) corn hybrid on feed intake and milk production compared with the nontransgenic hybrid and two reference hybrids. In Experiment 1, 16 multiparous Holstein cows were assigned to one of four treatments in replicated 4 x 4 Latin squares with 28-d periods. Diets contained 40% (dry matter [DM] basis) of either 1) glyphosate-tolerant corn silage (GT), 2) nontransgenic control corn silage, or 3) two nontransgenic reference hybrids which are commercially available. Each diet also contained 23% corn grain from the same hybrid that supplied the silage. At ensiling, rapid drying conditions prevailed and the GT hybrid was the last to be harvested which resulted in greater DM content at similar physiological maturity. The 4% fat-corrected milk (FCM) yield and DMI were reduced for cows fed the GT corn diet due to the higher DM content of the GT silage (37.1 vs. 33.2 kg/d and 4.05 vs. 3.61% of BW, respectively). There was no effect of the GT diet on milk composition or efficiency of 4% FCM production that averaged 1.43 kg/kg of DM intake for all diets. In Experiment 2, 16 multiparous Holstein cows were assigned to one of four treatments in replicated 4 x 4 Latin squares with 21-d periods. Diets contained 26.7% (DM basis) corn grain from either 1) corn rootworm protected (event MON863) corn hybrid, 2) nontransgenic control corn hybrid, or 3) the same two nongenetically enhanced reference hybrids used in Experiment 1. The 4% FCM yield (34.8 kg/d) and DM intake (4.06% of BW) were unaffected by diet. Efficiency of FCM production (average 1.32 kg/kg of DMI) was not affected by diet. In summary, these two studies indicated that insertion of a gene for glyphosate tolerance or corn rootworm protection into a corn hybrid did not affect its nutritional value (as measured by efficiency of milk production) for lactating dairy cows compared with conventional corn hybrids.

Gregersen, Per L., Henrik Brinch-Pedersen and Preben B. Holm, (2005). A Microarray-Based Comparative Analysis of Gene Expression Profiles During Grain Development in Transgenic and Wild Type Wheat. Transgenic Research Volume 14, Number 6 December 2005. Pages: 887 - 905, DOI: 10.1007/s11248-005-1526-y

Department of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg, Slagelse, Denmark

Abstract Global, comparative gene expression analysis is potentially a very powerful tool in the safety assessment of transgenic plants since it allows for the detection of differences in gene expression patterns between a transgenic line and the mother variety. In the present study, we compared the gene expression profile in developing seeds of wild type wheat and wheat transformed for endosperm-specific expression of an Aspergillus fumigatus phytase. High-level expression of the phytase gene was ensured by codon modification towards the prevalent codon usage of wheat genes and by using the wheat 1DX5HMW glutenin promoter for driving transgene expression. A 9K wheat unigene cDNA microarray was produced from cDNA libraries prepared mainly from developing wheat seed. The arrays were hybridised to flourescently labelled cDNA prepared from developing seeds of the transgenic wheat line and the mother variety, Bobwhite, at three developmental stages. Comparisons and statistical analyses of the gene expression profiles of the transgenic line vs. that of the mother line revealed only slight differences at the three developmental stages. In the few cases where differential expression was indicated by the statistical analysis it was primarily genes that were strongly expressed over a shorter interval of seed development such as genes encoding storage proteins. Accordingly, we interpret these differences in gene expression levels to result from minor asynchrony in seed development between the transgenic line and the mother line. In support of this, real time PCR validation of results from selected genes at the late developmental stage could not confirm differential expression of these genes. We conclude that the expression of the codon-modified A.␣fumigatus phytase gene in the wheat seed had no significant effects on the overall gene expression patterns in the developing seed.

Key words cDNA microarrays - gene expression - phytase - safety assessment - transgenic wheat

Gulden RH, Lerat S, Hart MM, Powell JR, Trevors JT, Pauls KP, Klironomos JN, Swanton CJ (2005) Quantitation of transgenic plant DNA in leachate water: Real-time polymerase chain reaction analysis. Journal of Agricultural and Food Chemistry 53:5858-5865
Abstract: Roundup Ready (RR) genetically modified (GM) corn and soybean comprise a large portion of the annual planted acreage of GM crops. Plant growth and subsequent plant decomposition introduce the recombinant DNA (rDNA) into the soil environment, where its fate has not been completely researched. Little is known of the temporal and spatial distribution of plant-derived rDNA in the soil environment and in situ transport of plant DNA by leachate water has not been studied before. The objectives of this study were to determine whether sufficient quantities of plant rDNA were released by roots during growth and early decomposition to be detected in water collected after percolating through a soil profile and to determine the influence of temperature on DNA persistence in the leachate water. Individual plants of RR corn and RR soybean were grown in modified cylinders in a growth room, and the cylinders were flushed with rain water weekly. Immediately after collection, the leachate was subjected to DNA purification followed by rDNA quantification using real-time Polymerase Chain Reaction (PCR) analysis. To test the effects of temperature on plant DNA persistence in leachate water, water samples were spiked with known quantities of RR soybean or RR corn genomic DNA and DNA persistence was examined at 5, 15, and 25 C. Differences in the amounts and temporal distributions of root-derived rDNA were observed between corn and soybean plants. The results suggest that rainfall events may distribute plant DNA throughout the soil and into leachate water. Half-lives of plant DNA in leachate water ranged from 1.2 to 26.7 h, and persistence was greater at colder temperatures (5 and 15 C).

Hamilton KA, Pyla PD, Breeze M, Olson T, Li MH, Robinson E, Gallagher SP, Sorbet R, Chen Y (2004) Bollgard II cotton: Compositional analysis and feeding studies of cottonseed from insect-protected cotton (Gossypium hirsutum L.) producing the Cry1Ac and Cry2Ab2 proteins. Journal of Agricultural and Food Chemistry 52:6969-6976
Abstract: Bollgard II cotton event 15985 producing the Cry1Ac and Cry2Ab2 proteins has been developed by genetic modification to broaden the spectrum of insects to which the plant is tolerant and to provide an insect resistance management tool to impede the onset of resistance. The purpose of this study was to evaluate the composition and nutrition of Bollgard II cotton, relative to the use for food and animal feed, compared to that of conventional cotton varieties. Compositional analyses were conducted to measure proximate, fiber, amino acid, fatty acid, gossypol, and mineral contents of cottonseed from a total of 14 U.S. field sites over two years. Compositional analysis results showed that the cottonseed and cottonseed oil from Bollgard II cotton were comparable in their composition to those of the conventional control cotton line and other commercial varieties. The composition data are supported by nutritional safety studies conducted with dairy cows, catfish, and quail. Results from these studies showed that Bollgard II performed similarly to the conventional control cotton varieties. These data demonstrate that Bollgard II cotton is compositionally and nutritionally equivalent to conventional cotton varieties. These data support the conclusion that Bollgard II cotton is as safe and nutritious as conventional cotton for food and feed use.

Hemre GI, Sanden M, Bakke-Mckellep AM, Sagstad A, Krogdahl A (2005) Growth, feed utilization and health of Atlantic salmon Salmo salar L. fed genetically modified compared to non-modified commercial hybrid soybeans. Aquaculture Nutrition 11:157-167
Abstract: The present paper represents a part of a major scientific effort aiming to reveal possible effects, nutritional or health related, of genetically modified (GM) feed ingredients for Atlantic salmon. For 3 months groups of post-smolt Atlantic salmon were treated with diets holding 130 g kg(1) of the protein as soybean, one which contained 800 g kg(-1) GM type RR (Roundup Ready((TM))), and compared with a standard counterpart (commercial hybrid, not isogenic line) analysed to be non-GM (nGM), and again compared with a standard fishmeal diet without soybean protein. All diets were composed to be within the category 'compositional equivalent' and held exactly the same proximate compositions, starch and sugar levels, above requirements for methionine and lysine, equal fatty acid profiles, vitamin, mineral and pigment contents. There was, however, a slight difference in levels of anti-nutrients between the three diets. The various dietary treatments resulted in more than tripling of fish weight in all groups. In addition no significant differences in feed utilization, whole body, liver and muscle proximate compositions, and no significant differences in muscle fatty acid profiles were measured. The diet without soybean of either type resulted in greater retention of lipid, but equal retention of protein (protein productive value). The relative sizes of liver, kidney, head-kidney and brain were the same in all dietary groups, while the relative size of the spleen showed significant differences between fish fed the genetically modified soy diet compared with fish fed the nGM soybeans. Fish fed the soy diets of either type also showed a somewhat reduced mean erythrocyte cell volume. All other haematological values were equal between diet groups. The detoxification system, measured as glutathione peroxidase (GPx) and lysozyme activities, showed equal values for all groups when measured in plasma and liver or head-kidney. The distal part of the intestine showed reduced sizes as an effect of soybean additions, without any differences between the GM and nGM type. Our results showed high growth, no mortality, haematological values within normal ranges, and efficient and equal responses in the detoxification system. This was a first indication that up to 130 g kg(-1) RR-soybean protein can safely be used in diets for Atlantic salmon. However, there is still a need to elucidate higher inclusion levels of GM feed ingredients, and why spleen index was reduced, and if this was a long- or short-term effect.

Hyun Y, Bressner GE, Ellis M, Lewis AJ, Fischer R, Stanisiewski EP, Hartnell GF (2004) Performance of growing-finishing pigs fed diets containing Roundup Ready corn (event nk603), a nontransgenic genetically similar corn, or conventional corn lines. Journal of Animal Science 82:571-580
Abstract: Two studies were conducted at two locations to evaluate growth performance and carcass characteristics of growing-finishing pigs fed diets containing either glyphosate-tolerant Roundup Ready (event nk603) corn, a nontransgenic genetically similar control corn (RX670), or two conventional sources of nontransgenic corn (RX740 and DK647). A randomized complete block design (three and four blocks in Studies 1 and 2, respectively) with a 2 x 4 factorial arrangement of treatments (two genders and four corn lines) was used. Study 1 used 72 barrows and 72 gilts (housed in single-gender groups of six; six pens per dietary treatment) with initial and final BW of approximately 22 and 116 kg, respectively. Study 2 used 80 barrows and 80 gilts (housed in single-gender groups of five; eight pens per dietary treatment) with initial and final BW of approximately 30 and 120 kg, respectively. Pigs were housed in a modified open-front building in Study 1 and in an environmentally controlled finishing building in Study 2. The test corns were included at a fixed proportion of the diet in both studies. Animals had ad libitum access to feed and water. Pigs were slaughtered using standard procedures and carcass measurements were taken. In Study 1, overall ADG, ADFI (as-fed basis), and gain:feed (G:F) were not affected (P > 0.05) by corn line. In Study 2, there was no effect of corn line on overall ADFI (as-fed basis) or GY ratio. In addition, overall ADG of barrows fed the four corn lines did not differ (P > 0.05); however, overall ADG of gilts fed corn DK647 was greater (P <> 0.05) of corn line on carcass yield or fatness measurements in either study. Differences between barrows and gilts for growth and carcass traits were generally similar for both studies and in line with previous research. Overall, these results indicate that Roundup Ready corn (NK603) gives equivalent animal performance to conventional corn for growing pigs.

Ipharraguerre IR, Younker RS, Clark JH, Stanisiewski EP, Hartnell GF (2003) Performance of lactating dairy cows fed corn as whole plant silage and grain produced from a glyphosate-tolerant hybrid (event NK603). Journal of Dairy Science 86:1734-1741
Abstract: Sixteen multiparous Holstein cows averaging 74 d in milk were used in a replicated 4 x 4 Latin square to compare the effects on animal performance of feeding whole plant silage and grain from a glyphosate-tolerant corn hybrid (event NK603), a nontransgenic control hybrid, and two commercial nontransgenic hybrids (DK647 and RX740). The grain and silage from the four corn hybrids were produced using the same procedures and under similar agronomic conditions at the University of Illinois. On a dry matter (DM) basis, diets contained 30% corn silage and 27.34% corn grain produced either from event NK603, a nontransgenic control, or commercial hybrids. Apart from the DM content of silages, the chemical composition of both grain and silage produced from the four corn hybrids were substantially equivalent. Feeding diets that contained event NK603 and DK647 hybrids tended to decrease DM intake (DMI) compared with the control nontransgenic and RX740. The intakes of crude protein (CP), acid and neutral detergent fiber, and nonfiber carbohydrates were not different for cows fed event NK603 and control diets. The RX740 diet resulted in the highest intakes of fiber and CP, whereas the DK647 diet resulted in the lowest intake of CP. These differences in nutrient intake arose from small variations in both the DMI and the chemical composition of feed ingredients and experimental diets. Production of milk and 3.5% fat-corrected milk; milk fat, CP, and true protein percentage and yield; milk urea N; milk total solids percentage and yield; and somatic cell count were not affected by treatments. These data indicate that the stable insertion of the gene that confers tolerance to glyphosate (event NK603) in the corn line used in this experiment does not affect its chemical composition and nutritional value for lactating dairy cows when compared with conventional corn.

Jennings JC, Kolwyck DC, Kays SB, Whetsell AJ, Surber JB, Cromwell GL, Lirette RP, Glenn KC (2003) Determining whether transgenic and endogenous plant DNA and transgenic protein are detectable in muscle from swine fed Roundup Ready soybean meal. Journal of Animal Science 81:1447-1455
Abstract: Questions regarding the digestive fate of DNA and protein from transgenic, feed have been raised in regard to human consumption and commercial trade of animal products (e.g., meat, milk, and eggs) from farm animals fed transgenic crops. Using highly sensitive, well-characterized analytical methods, pork loin samples were analyzed for the presence. of fragments of transgenic and endogenous. plant DNA and transgenic protein from, animals fed meal prepared from conventional or glyphosate-tolerant Roundup Ready (RR) soybeans. Pigs were fed diets containing 24, 19, and 14%, RR or conventional soybean meal during grower, early-finisher, and late-finisher phases of growth, respectively, and longissimus muscle samples were collected (12 per treatment) after slaughter. Total DNA was extracted from the samples and analyzed by PCR, followed by Southern blot hybridization for the presence of a 272-bp fragment of the cp4 epsps coding region (encoding the synthetic enzyme 5-enolpyruvylshikimate-3-phosphate synthase derived from Agrobacterium sp. strain CP4) and a 198-bp fragment of the endogenous soybean gene le1 (encoding soy lectin). Using 1 mug of input DNA per reaction, none of the extracted samples was positive for cp4 epsps or le1 at the limit of detection (LOD) for these PCR/Southern blot assays. The LOD for these assays was shown to be approximately one diploid genome equivalent of RR soybean DNA, even in the presence of 10 mug of pork genomic DNA. A 185-bp fragment of the porcine preprolactin (prl) gene, used as a positive control, was. amplified from all samples showing that the DNA preparations were amenable to PCR amplification. Using a competitive immunoassay with an LOD of approximately 94 ng of CP4 EPSPS protein/g of pork muscle, neither the CP4 EPSPS protein nor the immunoreactive peptide fragments were detected in loin muscle homogenates from pigs fed RR soybean meal. Taken together, these results show that neither small fragments of transgenic DNA nor immunoreactive fragments of transgenic protein are detectable in loin muscle samples from pigs fed a diet containing RR soybean meal.

Jennings JC, Whetsell AJ, Nicholas NR, Sweeney BM, Klaften MB, Kays SB, Hartnell GF, Lirette RP, Glenn KC (2003) Determining whether transgenic or endogenous plant DNA is detectable in dairy milk or beef organs. Bulletin of the International Dairy Federation 383:41-46
Abstract: The fate of transgenic DNA in products derived from farm animals fed genetically modified feed was assessed. Sensitive methods were developed to analyze milk for the presence of transgenic and plant DNA from cows fed a diet containing conventional or transgenic cottonseed or maize. Genomic DNA was extracted from milk and analyzed by PCR followed by Southern blot for fragments of the cry1Ac transgene and an endogenous cotton gene, acp1, from cows fed a diet containing whole cottonseed. Additionally, milk, liver, kidney, and spleen were assessed for fragments of the cry1Ab transgene and an endogenous maize gene, sh2, from animals fed a diet containing maize grain. No sample was positive for transgenic or plant DNA fragments at the limits of detection for the assays following detailed data evaluation criteria. Results for sh2 analyses of milk were, however, indeterminate. A fragment of a bovine gene, prl, was amplified from each DNA extract to show that all preparations were amenable to PCR. These results indicate that DNA, whether derived from conventional or transgenic feed, is not present at detectable levels in bovine milk or organs.

Jung HG, Sheaffer CC (2004) Influence of Bt transgenes on cell wall lignification and digestibility of maize stover for silage. Crop Science 44:1781-1789
Abstract: There have been inconsistent reports that maize (Zea mays L.) hybrids with the Bacillus thuringiensis (Bt) cry1 Ab transgene contain more lignin than non-Bt hybrids of similar genetic background. Our objective was to evaluate the impact of the cry1 Ab transgene on lignin concentration (using three different assays), yield, and forage quality traits of maize. Replicated trials were conducted at four locations in Minnesota with 12 commercial hybrids (three MON810 and three Bt11 cry1 Ab transgene event hybrids, and respective near-isogenic controls). Whole plants and the fourth elongated, above-ground internodes were harvested at silage maturity. Samples were analyzed for crude protein, starch, neutral detergent fiber (NDF), acid detergent fiber, 24- and 96-h in vitro ruminal NDF digestibility, and lignin (acid detergent, Klason, and acetyl bromide). European corn borers (Ostrinia nubilalis Hubner) were not controlled and damage was limited to the non-Bt hybrids, averaging 1.5 internodes plant(-1) with tunnels. Environment and environment x hybrid interactions affected all measures of maize performance and quality, but comparisons of non-Bt/Bt hybrid pairs, for both whole plants and internodes, found no consistent differences in yield, nutrient content, in vitro ruminal NDF digestibility, or lignin concentration. Differences in lignin concentration were infrequent, small in magnitude, and inconsistent between a few non-Bt/Bt hybrid pairs at individual locations. Two non-Bt/Bt hybrid pairs did not differ in lignin concentration at any location. Contrary to some earlier reports, presence of the cry1 Ab transgene did not alter lignin concentration or other forage quality traits of maize stover in commercial maize hybrids.

Kosieradzka I, Sawosz E, Pastuszewska B, Szwacka M, Malepszy S, Bielecki W, Czuminska K (2001) The effect of feeding diets with genetically modified cucumbers on the growth and health status of rats. Journal of Animal and Feed Sciences 10:7-12
Abstract: The composition of the fruits of non-transgenic or transgenic (GM) cucumbers with genes coding the synthesis of a sweet protein, thaumatin, were compared and effects of feeding the fruits in balanced diets to rats were determined. The transgenic cucumbers contained more protein (20.3 vs 17.9% DM) and less fibre (9.4 vs 11.4% DM) and also had lower Na, K, Ca and Mg contents and higher levels of Fe and Cu in ash than normal cucumbers. Feeding male rats of initial body weight 150 g for 5 weeks on isoprotein diets containing 0 or 15% lyophilized transgenic or non-transgenic cucumbers did not affect weight gain, apparent health status, or relative organ weights of animals. Protein digestibility was slightly but significantly lower (89.2 vs 90.0%), that of crude fibre was higher (28.2 vs 15.0%) in diets containing transgenic than non-transgenic cucumbers, while digestibility of fat and N-free extractives did not differ

Kosieradzka I, Sawosz E, Skomial J, Szopa J (2005) Transgenic potato tubers with overexpression of 14-3-3 protein in growing rat diets. 1. Selected hormone activities and liver function status. Journal of Animal and Feed Sciences 14:545-548
Abstract: In a 4-week experiment, growing rats were fed diets containing 30% transgenic potatoes with overexpression of PI4-3-3 protein, non-transgenic dehydrated potato tubers of the same variety (Desiree), or a control diet. The activity of dehydroepiandrosterone (DHEA) as well as increases in the concentrations of NO3- ions, Fe and Zn in the liver of rats fed diets containing transgenic potatoes were observed. The reaction might be related to oxidative stress linked to the altered chemical composition of potato tubers resulting from transgenesis, changed concentrations of some minerals or biologically active substances.

Le Gall G, Colquhoun IJ, Davis AL, Collins GJ, Verhoeyen ME.(2003). Metabolite profiling of tomato (Lycopersicon esculentum) using 1H NMR spectroscopy as a tool to detect potential unintended effects following a genetic modification. J Agric Food Chem. 2003 Apr 23;51(9):2447-56. Erratum in: J Agric Food Chem. 2004 May 19;52(10):3210.

The maize transcription factors LC and C1 were simultaneously overexpressed in tomato with the aim of producing lines with increased amounts of flavonols. The metabolite composition of these genetically modified tomatoes has been compared with that of azygous (nonmodified) controls grown side-by-side under the same conditions. It has been possible to observe metabolic changes in both types at different stages of maturity. (1)H NMR spectra showed that the levels of glutamic acid, fructose, and some nucleosides and nucleotides gradually increase from the immature to the ripe stage, whereas some amino acids such as valine and gamma-aminobutyric acid were present in higher amounts in unripe tomatoes. Apart from the significantly increased content of six main flavonoid glycosides (mainly kaempferol-3-O-rutinoside, with additional increases in kaempferol-3,7-di-O-glucoside (1), kaempferol-3-O-rutinoside-7-O-glucoside (2), kaempferol-3-O-glucoside, a dihydrokaempferol-O-hexoside (3), and naringenin-7-O-glucoside), the levels of at least 15 other metabolites were found to be different between the two types of red tomato. Among them were citric acid, sucrose, phenylalanine, and trigonelline. However, although statistically significant, these changes in mean values were relatively minor (less than 3-fold) and within the natural variation that would be observed in a field-grown crop. Nevertheless, this study clearly showed that NMR combined with chemometrics and univariate statistics can successfully trace even small differences in metabolite levels between plants and therefore represents a powerful tool to detect potential unintended effects in genetically modified crops.

Le Gall, Gwénaëlle, M. Susan DuPont, Fred A. Mellon, Adrienne L. Davis, Geoff J. Collins, Martine E. Verhoeyen, and Ian J. Colquhoun* Characterization and Content of Flavonoid Glycosides in Genetically Modified Tomato (Lycopersicon esculentum) Fruits J. Agric. Food Chem., 51 (9), 2438 -2446, 2003. 10.1021/jf025995e S0021-8561(02)05995-2
Abstract:
There is a growing interest in producing food plants with increased amounts of flavonoids because of their potential health benefits. Tomatoes contain small amounts of flavonoids, most of which are located in the peel of the fruit. It has been shown that flavonoid accumulation in tomato flesh, and hence an overall increase in flavonoid levels in tomato fruit, can be achieved by means of simultaneous overexpression of the maize transcription factors LC and C1. Fruit from progeny of two modified lines (2027 and 2059) was selected for a detailed analysis and individual identification of flavonoids, at different stages of maturity. Nine major flavonoids were detected in the flesh of transgenic ripe tomatoes. LC/NMR, LC/MS, and LC/MS/MS enabled us to identify these as kaempferol-3,7-di-O-glucoside (1), kaempferol-3-O-rutinoside-7-O-glucoside (2), two dihydrokaempferol-O-hexosides (3 and 4), rutin (5), kaempferol-3-O-rutinoside (6), kaempferol-3-O-glucoside (7), naringenin-7-O-glucoside (8) and naringenin chalcone (9), which were quantified by HPLC/DAD. All but 5, 6, and 9 were detected in tomato for the first time. The total flavonoid glycoside content of ripe transgenic tomatoes of line 2059 was about 10-fold higher than that of the controls, and kaempferol glycosides accounted for 60% of this. Kaempferol glycosides comprised around 5% of the flavonoid glycoside content of ripe control tomatoes (the rest was rutin and naringenin chalcone). The rutin concentration in both transgenic and control fruits was similar.

Keywords: Metabolomics; tomato; ripening; transgenic; GMO; flavonoids; kaempferol glycosides; naringenin-7-O-glucoside; dihydrokaempferol glycosides; transcription factors; LC; C1; Lycopersicon esculentum

Lehesranta,Satu J. Howard V. Davies, Louise V.T. Shepherd, Naoise Nunan, Jim W. McNicol, Seppo Auriola, Kaisa M. Koistinen, Soile Suomalainen, Harri I. Kokko and Sirpa O. Kärenlampi
Comparison of Tuber Proteomes of Potato Varieties, Landraces, and Genetically Modified Lines,
2005 First published online June 10, 2005; 10.1104/pp.105.060152
Plant Physiology, July 2005, Vol. 138, pp. 1690-1699

Crop improvement by genetic modification remains controversial, one of the major issues being the potential for unintended effects. Comparative safety assessment includes targeted analysis of key nutrients and antinutritional factors, but broader scale-profiling or "omics" methods could increase the chances of detecting unintended effects. Comparative assessment should consider the extent of natural variation and not simply compare genetically modified (GM) lines and parental controls. In this study, potato (Solanum tuberosum) proteome diversity has been assessed using a range of diverse non-GM germplasm. In addition, a selection of GM potato lines was compared to assess the potential for unintended differences in protein profiles. Clear qualitative and quantitative differences were found in the protein patterns of the varieties and landraces examined, with 1,077 of 1,111 protein spots analyzed showing statistically significant differences. The diploid species Solanum phureja could be clearly differentiated from tetraploid (Solanum tuberosum) genotypes. Many of the proteins apparently contributing to genotype differentiation are involved in disease and defense responses, the glycolytic pathway, and sugar metabolism or protein targeting/storage. Only nine proteins out of 730 showed significant differences between GM lines and their controls. There was much less variation between GM lines and their non-GM controls compared with that found between different varieties and landraces. A number of proteins were identified by mass spectrometry and added to a potato tuber two-dimensional protein map.

Discussion from Lehesranta et al.

The aim of this work is to gain insight into potato proteome diversity using a large selection of potato varieties and to assess the potential for 2-DE to detect significant changes in the proteome of transgenic potato.

It is clear that genotypic variation is extensive, with most of the proteins detected showing significant quantitative and qualitative differences between one or more varieties and landraces... Furthermore, the levels of many proteins, such as defense-related proteins, may be affected by environmental conditions in the field experiment, contributing to variation in protein expression.

Compared with the natural variation observed in the non-GM samples, the effects of transformation on the proteome were considerably less pronounced. Indeed, statistical analysis showed no clear differences between the protein patterns of the GM lines and their controls. No new proteins unique to individual GM lines were observed. Therefore, on the basis of this analysis, there was no evidence for any major changes in protein pattern in the GM lines tested. This in itself is interesting, as some of the lines, e.g. Mal1 and Sam35S, produced extremely stunted plants with low tuber yield (Kumar et al., 1996Go; Taylor et al., 1998Go, 2000Go). However, nine proteins showing significant differences in expression were detected, and seven could be tentatively identified. It would require observations over several years and climatic conditions to confirm that these are truly unintended effects. Furthermore, such changes need not reflect a risk, particularly where the proteins are known to exist in other varieties or where the levels fall within the limits of natural variation in non-GM material. Corpillo et al. (2004)Go used a similar approach to assess GM and non-GM tomato lines and also did not find any evidence of differences in protein expression caused by genetic modification.

Lutz B, Wiedemann S, Einspanier R, Mayer J, Albrecht C (2005) Degradation of Cry1Ab protein from genetically modified maize in the bovine gastrointestinal tract. Journal of Agricultural and Food Chemistry 53:1453-1456
Abstract: Immunoblotting assays using commercial antibodies were established to investigate the unexpected persistence of the immunoactive Cry1Ab protein in the bovine gastrointestinal tract (GIT) previously suggested by enzyme-linked immunosorbent assay (ELISA). Samples of two different feeding experiments in cattle were analyzed with both ELISA and immunoblotting methods. Whereas results obtained by ELISA suggested that the concentration of the Cry1Ab protein increased during the GIT passage, the immunoblotting assays revealed a significant degradation of the protein in the bovine GIT. Samples showing a positive signal in the ELISA consisted of fragmented Cry1Ab protein of approximately 17 and 34 kDa size. Two independent sets of gastrointestinal samples revealed the apparent discrepancy between the results obtained by ELISA and immunoblotting, suggesting that the antibody used in the ELISA reacts with fragmented yet immunoactive epitopes of the Cry1Ab protein. It was concluded that Cry1Ab protein is degraded during digestion in cattle. To avoid misinterpretation, samples tested positive for Cry1Ab protein by ELISA should be reassessed by another technique.

Malatesta M, Caporaloni C, Rossi L, Battistelli S, Rocchi MBL, Tonucci F, Gazzanelli G (2002) Ultrastructural analysis of pancreatic acinar cells from mice fed on genetically modified soybean. Journal of Anatomy 201:409-415
Abstract: No direct evidence that genetically modified (GM) food may represent a possible danger for health has been reported so far; however, the scientific literature in this field is quite poor. Therefore, we investigated the possible effects of a diet containing GM soybean on mouse exocrine pancreas by means of ultrastructural, morphometrical and immunocytochemical analyses. Our observations demonstrate that, although no structural modification occurs in pancreatic acinar cells of mice fed on GM soybean, quantitative changes of some cellular constituents take place in comparison to control animals. In particular, a diet containing significant amount of GM food seems to influence the zymogen synthesis and processing.

Malatesta M, Caporaloni C, Gavaudan S, Rocchi MBL, Serafini S, Tiberi C, Gazzanelli G (2002) Ultrastructural morphometrical and immunocytochemical analyses of hepatocyte nuclei from mice fed on genetically modified soybean. Cell Structure and Function 27:173-180
Abstract: No direct evidence that genetically modified (GM) food may represent a possible danger for health has been reported so far; however, the scientific literature in this field is still quite poor. Therefore, we carried out an ultrastructural morphometrical and immunocytochemical study on hepatocytes from mice fed on GM soybean, in order to investigate eventual modifications of nuclear components of these cells involved in multiple metabolic pathways related to food processing. Our observations demonstrate significant modifications of some nuclear features in GM-fed mice. In particular, GM fed-mice show irregularly shaped nuclei, which generally represents an index of high metabolic rate, and a higher number of nuclear pores, suggestive of intense molecular trafficking. Moreover, the roundish nucleoli of control animals change in more irregular nucleoli with numerous small fibrillar centres and abundant dense fibrillar component in GM-fed mice, modifications typical of increased metabolic rate. Accordingly, nucleoplasmic (snRNPs and SC-35) and nucleolar (fibrillarin) splicing factors are more abundant in hepatocyte nuclei of GM-fed than in control mice. In conclusion, our data suggest that GM soybean intake can influence hepatocyte nuclear features in young and adult mice; however, the mechanisms responsible for such alterations remain unknown.

Malatesta M, Biggiogera M, Manuali E, Rocchi MBL, Baldelli B, Gazzanelli G (2003) Fine structural analyses of pancreatic acinar cell nuclei from mice fed on genetically modified soybean. European Journal of Histochemistry 47:385-388
Abstract: We carried out ultrastructural morphometrical and immunocytochemical analyses on pancreatic acinar cell nuclei from mice fed on genetically modified (GM) soybean, in order to investigate possible structural and molecular modifications of nucleoplasmic and nucleolar constituents. We found a significant lowering of nucleoplasmic and nucleolar splicing factors as well as a perichromatin granule accumulation in GM-fed mice, suggestive of reduced post-transcriptional hnRNA processing and/or nuclear export. This is in accordance to already described zymogen synthesis and processing modifications in the same animals.

Nemeth A, Wurz A, Artim L, Charlton S, Dana G, Glenn K, Hunst P, Jennings J, Shilito R, Song P (2004) Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA. Journal of Agricultural and Food Chemistry 52:6129-6135
Abstract: An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.

Phipps RH, Humphries DJ (2002) Detection of transgenic DNA in milk from cows receiving herbicide tolerant (CP4EPSPS) soyabean meal. Livestock Production Science 74:269-273
Abstract: Ten Holstein /Friesian cows with an average liveweight of 630 kg and producing 25.3 kg milk/day received a total mixed ration (TMR) in which the forage component formed 55% of total DM and contained non-genetically modified (GM) grass and maize silage in a 1:3 DM ratio. In study weeks 1-3 the TMR DM also contained non-GM supplernents of 18.5% cracked wheat and 26.1% rapeseed meal. In weeks 4-12 soyabean meal, genetically modified (CP4EPSPS) to be herbicide tolerant, replaced rapeseed meal at 26.1% of the total diet in weeks 4-5 and 13.9% of the total diet in weeks 6-12. Weekly milk samples were taken from all cows. Samples were spiked with DNA extracted from the soyabean meal to establish the limit of detection (LOD) of transgenic DNA using a polymerase chain reaction (PCR) analyses designed to detect transgenic DNA fragments smaller than 200 bp. Subsequent PCR analyses carried out in duplicate on TMR and milk samples collected at weeks 3, 4 and 122 of the study used three DNA primer sets to establish the presence or absence of transgenic DNA, The LOD of transgenic DNS in milk was 7.5 mug/l. Feed and milk samples analysed at week 3 were negative. The TMR feed samples at weeks 4 and 12 were positive for CP4EPSPS soyabean meal DNA, but all milk samples were negative. The results showed that transgenic DNA could not be detected in milk from cows receiving upto 26.1% of their diet DM as herbicide (glyphosate)-tolerant soyabean meal.

Phipps RH, Deaville ER, Maddison BC (2003) Detection of transgenic and endogenous plant DNA in rumen fluid, duodenal digesta, milk, blood, and feces of lactating dairy cows. Journal of Dairy Science 86:4070-4078
Abstract: The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.

Phipps RH, Tingey AP, Abeyasekera S (2005) Effect of corn silage from an herbicide-tolerant genetically modified variety on milk production and absence of transgenic DNA in milk. Journal of Dairy Science 88:2870-2878
Abstract: Data from 60 multiparous Holstein cows were used in a 12-wk continuous design feeding trial. Cows were allocated to 1 of 4 experimental treatments (T1 to T4). In T1 and T2, the total mixed ration (TMR) contained either corn silage from the genetically modified (GM) variety Chardon Liberty Link, which is tolerant to the herbicide glufosinate ammonium, or its near isogenic nonGM counterpart, whereas the TMR used in T3 and T4 contained corn silage from the commercially available nonGM varieties Fabius and Antares, respectively. The objectives of the study were to determine if the inserted gene produced a marked effect on chemical composition, nutritive value, feed intake, and milk production, and to determine if transgenic DNA and the protein expressed by the inserted gene could be detected in bovine milk. The nutritive value, fermentation characteristics, mineral content, and amino acid composition of all 4 silages were similar. There were no significant treatment effects on milk yield, milk composition, and yield of milk constituents, and the dry matter (DM) intake of the GM variety was not significantly different from the 2 commercial varieties. However, although the DM intake noted for the nonGM near-isogenic variety was similar to the commercial varieties, it was significantly lower when compared with the GM variety. Polymerase chain reaction analyses of milk samples collected at wk 1, 6, and 12 of the study showed that none of the 90 milk samples tested positive, above a detection limit of 2.5 ng of total genomic DNA/mL of milk, for either tDNA (event T25) or the single-copy endogenous Zea mays gene, alcohol dehydrogenase. Using ELISA assays, the protein expressed by the T25 gene was not detected in milk.

Ruebelt MC, Lipp M, Reynolds TL, Schmuke JJ, Astwood JD, DellaPenna D, Engel KH, Jany KD. Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 3. Assessing unintended effects. J Agric Food Chem. 2006 Mar 22;54(6):2169-77.

The current procedures to assess the safety of food and feed derived from modern biotechnology include the investigation of possible unintended effects. To improve the probability of detecting unintended effects, profiling techniques such as proteomics are currently tested as complementary analytical tools to the existing safety assessment. An optimized two-dimensional gel electrophoresis (2DE) method was used as a proteomics approach to investigate insertional and pleiotropic effects on the proteome due to genetic engineering. Twelve transgenic Arabidopsis thaliana lines were analyzed by 2DE, and their seed proteomes were compared to that of their parental line as well as to 12 Arabidopsis ecotype lines. The genetic modification of the Arabidopsis lines, using three different genes and three different promoters, did not cause unintended changes to the analyzed seed proteome. Differences in spot quantity between transgenic and nontransgenic lines fell in the range of values found in the 12 Arabidopsis ecotype lines or were related to the introduced gene.

Ruebelt MC, Lipp M, Reynolds TL, Astwood JD, Engel KH, Jany KD. Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 2. Assessing natural variability. J Agric Food Chem. 2006 Mar 22;54(6):2162-8.

Proteomics is currently tested as a complementary tool for the safety assessment of genetically modified (GM) crops. Understanding the natural variability of the proteome is crucial for the interpretation of biological differences between transgenic and nontransgenic parental lines. The natural variation of seed protein profiles among a set of 12 Arabidopsis thaliana ecotypes was determined by utilizing two-dimensional electrophoresis (2DE). The total number of different resolved protein spots found among the 12 ecotypes was 931 with a range of 573 (Mt-0) to 653 (Condara) in any one ecotype. Although the ecotypes were grown side-by-side in an environmentally controlled growth chamber, almost half of the resolved spots varied with respect to their presence/absence, and 95% of the spots present in all ecotypes varied in spot quantity (2-53-fold). In the evaluation of unintended effects of genetic modification, it is concluded that the experimental design must account for existing natural variability, which, in the case of the expressed proteome, can be substantial.

Ruebelt MC, Leimgruber NK, Lipp M, Reynolds TL, Nemeth MA, Astwood JD, Engel KH, Jany KD. Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation. J Agric Food Chem. 2006 Mar 22;54(6):2154-61.

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations.
Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity ofa protein.

Reuter T, Aulrich K, Berk A (2002) Investigations on genetically modified maize (Bt-maize) in pig nutrition: Fattening performance and slaughtering results. Archives of Animal Nutrition-Archiv fur Tierernahrung 56:319-326
Abstract: A grower finisher performance trial with forty-eight pigs was designed to compare the growth performance of pigs fed diets containing either genetically modified (GM) Bt-maize (NX6262) or its parental maize (Prelude) line. During the experiment, the pigs were fed with a grower and a finisher diet both containing 70% maize investigated in a previously study which showed that they contained similar ME values and digestibility of crude nutrients. The pigs with an initial live weight of 23.9 +/- 3.0 kg were allotted to single boxes. During a 91 days growing period the pigs of both groups recorded equal performance in daily weight gain (DeltaW) 815 +/- 93 vs. 804 +/- 64g/d depending on equal amounts of feed intake 1.95 +/- 0.15 vs. 1.94 +/- 0.15 kg/d (parental vs. transgenic). The results confirm equal performance among growing-finishing pigs fed parental or genetically modified maize containing diets. For slaughtering the pigs were divided into 4 groups with a different duration of the finishing period. After slaughtering the carcass characteristic were registered.

Reuter T, Aulrich K, Berk A, Flachowsky G (2002) Investigations on genetically modified maize (Bt-maize) in pig nutrition: Chemical composition and nutritional evaluation. Archives of Animal Nutrition-Archiv fur Tierernahrung 56:23-31
Abstract: The objective of the present study was to determine the composition and the nutritional value of parental and transgenic maize seeds fed to pigs. The parental maize line was genetically modified to incorporate a gene from Bacillus thuringiensis (Bt) expressing a toxin against the European corn borer (Ostrinia nubilalis). Both (parental and transgenic) maize lines were analyzed for crude nutrients, starch, sugar, non-starch polysaccharides (NSP), amino acids, fatty acids, as well as for selected minerals. Furthermore, four complete diets were mixed and were analyzed for the same nutrients and some selected ingredients. The diets contained 70% maize to attain a high effect level. To evaluate the feeding value of one variety of genetically modified maize (transgenic) compared to the feeding value of the unmodified maize (parental) line, a balance study with twelve pigs was designed. Three collecting periods were used for each maize line each with six animals. The collected faeces were analyzed for crude nutrients. All measured parameters were virtually the same (e.g. crude protein 11.59% vs. 11.06% in DM), especially the digestibility of crude protein (85.8 +/- 2.3% vs. 86.1 +/- 1.8%), the amount of nitrogen-free-extract (92.8 +/- 0.6% vs. 93.2 +/- 0.6%) and the metabolizable energy (15.7 +/- 0.2% vs, 15.8 +/- 0.2% MJ/kg DM) for both maize lines. Compared to the parental line, the chemical composition and digestibility of crude nutrients and energy content were not significantly affected by the genetic modification of maize. Therefore, from the view of a nutritional assessment, the genetically modified maize can be regarded as substantially equivalent to the parental maize line.

Reuter T, Aulrich K (2003) Investigations on genetically modified maize (Bt-maize) in pig nutrition: fate of feed-ingested foreign DNA in pig bodies. European Food Research and Technology 216:185-192
Abstract: The passage and fate of ingested DNA in 48 pigs fed with diets containing (n = 12) parental or (n = 36) transgenic (Bt) maize were examined. Pigs were fattened from an initial live weight of 24 kg to approximately 108 kg. Animals fed transgenic maize were slaughtered in groups (n = 6) 4, 8, 12, 24, 48 and 72 h after feeding the last maize-containing diet. Those slaughtered at up to 12 h received no further feed, while those held for longer prior to slaughter received a diet in which maize was replaced by barley and wheat. Control animals were slaughtered at 4 and 8 h. DNA extracted from tissues and gut contents was examined by PCR for the presence of plant DNA and for any transgenic material. Recombinant DNA was detectable in the intestinal contents up to 48 h after the last feeding of a diet containing the transgenic maize. PCR amplification of plant gene spacers produced fragments of different sizes, dependent on feed source. The feed source of rectum samples depended on individual passage rate in the groups and their restriction analysis showed grain species-specific patterns. Recombinant or maize-specific DNA was not detectable in tissue samples of pigs. In contrast, plant DNA fragments were detectable in the investigated pig tissues.

Ridley WP, Sidhu RS, Pyla PD, Nemeth MA, Breeze ML, Astwood JD (2002) Comparison of the nutritional profile of glyphosate-tolerant corn event NK603 with that of conventional corn (Zea mays L.). Journal of Agricultural and Food Chemistry 50:7235-7243
Abstract: The composition of glyphosate-tolerant (Roundup Ready) corn event NK603 was compared with that of conventional corn grown in the United States in 1998 and in the European Union in 1999 to assess compositional equivalence. Grain and forage samples were collected from both replicated and nonreplicated field trials, and compositional analyses were performed to measure proximates, fiber, amino acids, fatty acids, vitamin E, nine minerals, phytic acid, trypsin inhibitor, and secondary metabolites in grain as well as proximates and fiber in forage. Statistical analysis of the data,was conducted to assess statistical significance at the p < p=".07)"> 0.05) for final live weights and feed conversion. Broilers fed diets containing MON863 corn had adjusted feed conversion similar to the nontransgenic control and the population of control and commercial diets. On a weight basis, there were no differences among diets for chill, fat pad, and thigh, drum, and wing weights. Differences (P <> 0.05) in the percentage of moisture, protein, and fat in breast meat or thigh meat across treatment diets. In experiment 2, there were no significant differences among diets for all broiler performance and carcass parameters evaluated. Broilers overall performed consistently and had similar carcass yields and meat compositions when fed diets containing MON863 corn or MON810 x MON863 corn as compared with their respective nontransgenic control and commercial diets, supporting a conclusion of similar feeding values among diets.

Séralini, G-E, Cellier, D and de Vendomois, JS (2007) New Analysis of a Rat Feeding Study with a Genetically Modified Maize Reveals Signs of Hepatorenal Toxicity Archives of Environmental Contamination and Toxicology 10.1007/s00244-006-0149-5

Abstract Health risk assessment of genetically modified organisms (GMOs) cultivated for food
or feed is under debate throughout the world, and very little data have been published on mid- or long-term toxicological studies with mammals. One of these studies performed under the responsibility of Monsanto Company with a transgenic corn MON863 has been subjected to questions from regulatory reviewers in Europe, where it was finally approved in 2005. This
necessitated a new assessment of kidney pathological findings, and the results remained
controversial. An Appeal Court action in Germany (Münster) allowed public access in June 2005 to all the crude data from this 90-day rat-feeding study. We independently re-analyzed these data. Appropriate statistics were added, such as a multivariate analysis of the growth curves, and for biochemical parameters comparisons between GMO-treated rats and the controls fed with an equivalent normal diet, and separately with six reference diets with different compositions. We observed that after the consumption of MON863, rats showed slight but dose-related significant variations in growth for both sexes, resulting in 3.3% decrease in weight for males and 3.7% increase for females. Chemistry measurements reveal signs of hepatorenal toxicity, marked also by differential sensitivities in males and females. Triglycerides increased by 24–40% in females (either at week 14, dose 11% or at week 5, dose 33%, respectively); urine phosphorus and sodium excretions diminished in males by 31–35% (week 14, dose 33%) for the most important results significantly linked to the treatment in comparison to seven diets tested. Longer experiments are essential in order to indicate the real nature and extent of the possible pathology; with the present data it cannot be concluded that GM corn MON863 is a safe product. [Reanalysis of existing studies , funded by Greenpeace] Further comment here

Taylor ML, Hartnell GF, Riordan SG, Nemeth MA, Karunanandaa K, George B, Astwood JD (2003) Comparison of broiler performance when fed diets containing grain from YieldGard (MON810), YieldGard x Roundup Ready (GA21), nontransgenic control, or commercial corn. Poultry Science 82:823-830
Abstract: This 42-day experiment was undertaken to compare the nutritional value of insect-protected corn event MON810 (YieldGard) and YieldGard x herbicidetolerant corn event GA21 (Roundup Ready) to their nontransgenic controls as well as four different commercial reference corns, when fed to growing Cobb x Cobb broilers. A randomized complete block design was used, an each treatment was replicated with five pens of males and five pens of females with 10 broilers per pen. Broilers were fed approximately 55% wt/wt corn during the first 20 d and approximately 60% wt/wt corn thereafter. The corn component of diets fed to broilers was supplied entirely with grain from the eight hybrids included in the experiment. Final live weights averaged 2.09 kg/bird fed YieldGard corn and 2.15 kg/bird fed YieldGard x Roundup Ready corn and were not different (P > 0.05) from final weights for birds fed control or commercial corn. Feed conversion was not affected (P > 0.05) by YieldGard (1.72) or YieldGard x Roundup Ready (1.77) corn feeding when compared with the feeding of other corn diets. Chill weights, fat pad, thigh weights, and wing weights were not affected by diets (P > 0.05). Differences (P <> 0.05) across all treatments in both experiments. In experiment 1, broilers fed diets containing Roundup Ready corn had similar feed conversion adjusted for mortalities to those fed the non-transgenic control and one of the commercial corn diets. Chill weights and thigh, drum, and wing weights were not affected by diets. Differences (P <> 0.05). Furthermore, there were no differences in the PCV and the analysed serum parameters between the control and experimental group. The results of maize DNA digestibility showed that the new variety takes the normal physiological passage along broiler GIT similar to the conventional line. In addition, Bt 176 maize DNA appears to be partially degraded in different parts of GIT comparable to the DNA of the control maize line. Results of the metabolic fate of maize DNA in broiler blood, muscles and organs indicated that only short DNA fragments (199 bp) derived from the plant chloroplast gene could be detected in the blood, skeletal muscles, liver, spleen and kidney, which disappeared after prolongation the fasting time. In heart muscle, bursa of Fabricius and thymus, no plant chloroplast DNA was found. Bt gene specific constructs from Bt 176 maize were not detected in any investigated blood or tissue samples.

Twardowski T, Potkanski A, Pruszynski SAK (2003) A note on silage from genetically modified maize tested for biological activity. Polish Journal of Environmental Studies 12:759-764
Abstract: Forage from genetically modified (GM) maize in two consecutive years (1999 and 2000) was ensiled in bins of 120 l volume in two combinations: with formic acid (85%) and without. In the samples of ensilage material GM maize, basic parameters have been determined such as pH and dry matter. The determination of biological activity of the components of translational apparatus in model translation systems showed the complete inactivation of biological activities. In addition, degradation of nucleic acids in examined silages was discovered.

Van Deynze, A., Bradford, K. J., and Van Eenennaam, A. Crop biotechnology: Feeds for livestock. Div.Agriculture and Natural Resources Publication 8145, Univ.of California-Davis Pub. 8145, 1-6. 2004.
Abstract: Most crops developed through biotechnology that are on the market today provide farmers with increased convenience and product quality while requiring fewer chemical inputs. According to the USDA Economic Research Service (http://www.ers.usda.gov/data/biotechcrops), herbicide- and insect-resistant biotech varieties accounted for about 85 percent of U.S. soybean acreage and 45 percent of corn acreage in 2003. Livestock eat the meal from approximately 70 percent of the soybeans and consume 80 percent of the corn grain and silage grown in the United States (Etherton et al. 2003), making the livestock industry a major user of biotech crops. Plant breeders are concentrating on enhancing grains or protein sources to produce feedstuffs that will improve feed utilization, performance, product quality, and health of livestock while reducing production costs and environmental impacts. It is likely that biotech crops of the future will play an important role in this arena. This publication discusses potential applications and safety issues associated with such products.

von Wettstein D, Warner J, Kannangara CG (2003) Supplements of transgenic malt or grain containing (1,3-1,4)-beta-glucanase increase the nutritive value of barley-based broiler diets to that of maize. British Poultry Science 44:438-449
Abstract: 1. A diet with addition to normal barley of malt from transgenic barley expressing a protein engineered, thermotolerant Bacillus (1,3- 1,4)-beta-glucanase during germination has previously been demonstrated to provide a broiler chicken weight gain comparable to maize diets. It also reduced dramatically the number of birds with adhering sticky droppings, but did not entirely eliminate sticky droppings. One of the objectives of the broiler chicken trials reported here was to determine if higher concentrations of transgenic malt could alleviate the sticky droppings. 2. Another aim was to investigate the feasibility of using mature transgenic grain containing the thermotolerant (1,3- 1,4)-beta-glucanase as feed addition and to compare diets containing transgenic grain to a diet with the recommended amount of a commercial beta-glucanase-based product. 3. Inclusion of 75 or 151 g/kg transgenic malt containing 4.7 or 98 mg/kg thermotolerant (1,3- 1,4)-beta-glucanase with 545 or 469 g/kg non-transgenic barley instead of maize yielded a weight gain in Cornish Cross broiler chickens indistinguishable from presently used maize diets. The gene encoding the enzyme is expressed in the aleurone with a barley alpha-amylase gene promoter and the enzyme is synthesised with a signal peptide for secretion into the endosperm of the malting grain. 4. Equal weight gain was achieved, when the feed included 39 g/kg transgenic barley grain [containing 66 mg/kg thermotolerant (1,3- 1,4)-beta-glucanase] and 581 g/kg non-transgenic barley instead of maize. In this case, the gene encoding the enzyme has been expressed with the D-hordein gene (Hor3-1) promoter during grain maturation. The enzyme is synthesised as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeted into the storage vacuoles. Deposition of the enzyme in the prolamin storage protein bodies of the endosperm protects it from degradation during the programmed cell death of the endosperm in the final stages of grain maturation and provides extraordinary heat stability. The large amount of highly active (1,3- 1,4)-beta-glucanase in the mature grain allowed the reduction of the transgenic grain ingredient to 0.2 g/kg diet, thus making the ingredient comparable to that of the trace minerals added to standard diets. 5. A direct comparison using transgenic grain supplement at the level of 1 g/kg of feed with the standard recommended addition of the commercial enzyme preparation Avizyme 1100(R) at 1 g/kg yielded equal weight gain, feed consumption and feed efficiency in birds fed a barley-based diet. 6. The production of sticky droppings characteristic of broilers fed on barley diets was avoided with all 9 experimental diets and reduced to the level observed with a standard maize diet by supplementation with transgenic barley. 7. The excellent growth and normal survival of the 400 broilers tested on barley diets supplemented with transgenic grain or malt showed the grain and malt not to be toxic. 8. The barley feed with added transgenic grain or malt containing thermotolerant (1,3- 1,4)-beta-glucanase provides an environmentally friendly alternative to enzyme additives, as it uses photosynthetic energy for production of the enzyme in the grain and thus avoids use of non-renewable energy for fermentation. The deposition of the enzyme in the protein bodies of the grain in the field makes coating procedures for stabilisation of enzyme activity superfluous. 9. Barley feed with the small amount of transgenic grain as additive to normal barley provides an alternative for broiler feed in areas where grain maize cannot be grown for climatic reasons or because of unsuitable soil and thus has to be imported

Zdunczyk Z, Frejnagel S, Fornal J, Flis M, Palacios MC, Flis B, Zagorski-Ostoja W (2005) Biological response of rat fed diets with high tuber content of conventionally bred and transgenic potato resistant to necrotic strain of potato virus (PVYN) Part I. Chemical composition of tubers and nutritional value of diets. Food Control 16:761-766
Abstract: In experiments on rats, nutritional properties of a diet with high content (40%) of potato tubers obtained from conventional or genetically-modified potato were determined. The potato cultivar Irga was transformed with viral genome sequences in order to improve its resistance to a necrotic strain of potato virus Y (PVYN). Four lines of genetically-modified potato were compared with non-transgenic somaclone from cv. Irga and three conventional cultivars Irga, Maryna and Ania. Potato tubers were autoclaved, dried and introduced to the diets which contained similar amount of protein (levelled through small addition of casein), fat, minerals and vitamins.

Genetic modification of potato had no effect on the chemical composition (e.g. crude protein, starch, dietary fibre content and amino acid composition of protein) and nutritional properties of tubers (diet intake, animal growth, protein utilisation). Higher differences between chemical composition (especially in crude protein and starch content) and biological response of rats were determined in the case of diets containing tubers from conventional potato cultivars Ania, Maryna and Irga. Obtained results indicate that transgenic potatoes with genetically improved resistance to PVYN are substantial and nutritional equivalence to the non-transgenic cultivar.

Zdunczyk Z, Juskiewicz J, Fornal J, Mazur-Gonkowska B, Koncicki A, Flis B, Zimnoch-Guzowska E, Zagorski-Ostoja W (2005) Biological response of rat fed diets with high tuber content of conventionally bred and transgenic potato resistant to necrotic strain of potato virus (PVYN). Part II. Caecal metabolism, serum enzymes and indices of non-specific defence of rats. Food Control 16:767-772
Abstract: The potential effect of genetic modification on nutritional properties of potatoes was determined in a rat experiment. The potato cultivar Irga was transformed with viral genome sequences in order to improve its resistance to a necrotic strain of potato virus Y (PVYN). Four clones of genetically-modified potato were compared with the conventional variety Irga and non-transgenic somaclone from cv. Irga. Autoclaved and dried potato tubers were introduced in a high amount (40%) to rat diets. The genetic status of the potato had no effect on the mass of the caecum and caecal digesta. The parameters tested were pH, dry matter content and bacterial enzyme activity, comprising a- and ß-glucosidase, a- and ß-galactosidase, and ß-glucuronidase. The results indicate that improvement of potato resistance to PVYN by genetic transformation had no negative effect on the ecosystem of the caecum of the rats, activity of serum enzymes (aspartate and alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase and creatine kinase) and non-specific defence mechanisms (lysozyme and ceruloplasmine level, number of bacteria taken up per cell and the percentage of the phagocytic cells in the serum).

Zhu Y, Li D, Wang F, Yin J, Jin H (2004) Nutritional assessment and fate of DNA of soybean meal from Roundup Ready or conventional soybeans using rats. Archives of Animal Nutrition-Archiv fur Tierernahrung 58:295-310
Abstract: This study was conducted to compare the safety of soybean meal prepared from genetically modified (GM) glyphosate-tolerant (Roundup Ready; RR) soybeans and conventional soybeans. Eighty Sprague-Dawley rats (40 males and 40 females) were randomly allotted to one of four groups according to sex and body weight for a 13-week feeding experiment. The rats were fed corn-based diets containing 60% conventional soybean meal, a mixture of 30% conventional and 30% RR soybean meal, 60% or 90% RR soybean meal. All diets were adjusted to an identical nutrient level except the 90% RR diet. The two soybean meals were similar in chemical analysis and amino acid composition.

Based on lists originally compiled by Wayne Parrot, October, 2005, updated and extended by GMO Pundit.

2 comments:

Anonymous said...: Very interesting research David, with such a volume of edivence here I find it disheartening that our political system rewards those who have no regard for science. How can Minister Kim Chance (WA Agriculture) then justify funds to anti-GM activists like Judy Carman to test the safety of GMs when the work has already been done over and over and by those who are independent.; 10:48 PM
GMO Pundit said...: My position on this is,
There is no intrinsic harm in extra testing provided that it is done with transparancy, lack of bias (preferably double-blind) by well qualified expert scientists, with rigorous oversight, and is not done in a way that causes unnecessary economic damage. In the WA case, I judge few, if any, of these conditions are satisfied or demonstrated though.; 9:46 AM

The Full Monty by GMO Pundit

Friday, May 12, 2006

The Full Monty on Animal Feeding Trials of GM foods.

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