Robin Mesnage; Sarah Z. Agapito-Tenfen; Vinicius Vilperte; George Renney; Malcolm Ward; Gilles-Eric Séralini; Rubens O. Nodari; Michael N. Antoniou

“ Our results suggest that expression of the EPSPS-CP4 transgene alters the oxidative environment in cells, and the increased levels of antioxidant enzymes are likely to be a response to oxidative burst by reactive oxygen species (ROS) in order to maintain proper physiological function. Glutathione metabolism was significantly altered in the NK603 when Roundup was sprayed during cultivation. Glutathione is known to be an important antioxidant in most living organisms, preventing damage to important cellular components caused by several environmental pollutants, including agrochemicals [59] . ”

“ Abstract Glyphosate tolerant genetically modified (GM) maize NK603 was assessed as ‘substantially equivalent’ to its isogenic counterpart by a nutrient composition analysis in order to be granted market approval. We have applied contemporary in depth molecular profiling methods of NK603 maize kernels (sprayed or unsprayed with Roundup) and the isogenic corn to reassess its substantial equivalence status. Proteome profiles of the maize kernels revealed alterations in the levels of enzymes of glycolysis and TCA cycle pathways, which were reflective of an imbalance in energy metabolism. ”

“ Our molecular profiling results show that NK603 and its isogenic control are not substantially equivalent. The application of genetic engineering (GE) to modify edible crops is often advocated as one of the most important scientific advances to improve farming systems and feed the world in a more sustainable manner [1] . GE has been used to create crops adapted to abiotic stress, resistant to pathogens, with a longer shelf life, or with enhanced nutritional properties. ”

“ The set of parameters and analyses necessary to declare a GMO as substantially equivalent to its conventional counterpart is still vague and focuses on a restricted set of compositional variables, such as the amounts of protein, carbohydrate, vitamins and minerals. GMOs are then declared substantially equivalent when sufficient similarities appear for those selected variables [6] . Remarkably, while a majority of GMO crops have been modified to withstand and thus accumulate a herbicide without dying, analysis for residues for such pesticides are neglected in compositional assessment [7] . ”

“ The increasing literature reporting application of omics methods to assess proteome, metabolome and transcriptome profiles in GMO crops shows strong evidence of distinct grain proteomes in other GM maize events, such as MON810 Bt insecticide producing maize [11] [12] [46] . Although the majority of studies have focused on insect-resistant maize (e.g., MON810 event) and most likely because this was the first GM maize to enter the food and feed market, there has also been one previous metabolomics study investigating NK603. ”

“ Pathway enrichment analysis of differentially expressed proteins in NK603 and NK603+Roundup feed samples was mainly assigned to carbohydrate and energy metabolism (Table 1) . Most of the proteins, including enzymes, associated with these pathways were overexpressed in GM samples (Additional file 5) . An increased expression of some proteins involved in glycolysis (FDR adjusted p-value = 4.2e-7) , and in particular in the synthesis of pyruvate from D-glyceraldehyde 3-phosphate can be indicative of an increased demand for energy. ”

“ The lines connecting the different dots are proportional to the divergence between the different variables of the dataset. A relatively high correlation is depicted by the short edges. It shows similar trends in metabolome and proteome profiles, and also between the two cultivations, indicating that the most variant sources of biological information were similar. The projection of individual protein or metabolites on a 2-dimensional space (Fig. 2C) showed a mix pattern indicating that no particular subsets of variables are driving the clustering of groups. ”

“ Recent technologies used to ascertain the molecular compositional profile of a system, such as transcriptomics, proteomics, metabolomics, epigenomics and mirnomics, collectively referred to as “omics technologies”, are used extensively in basic and applied science [8] . ”

“ Figure 3 shows the statistical significance of differential protein/metabolite levels by volcano plots along with respective fold changes. While only one protein is newly produced as a result of the transgene insertion, a total of 117 proteins and 91 metabolites have been altered in maize by the genetic transformation process and insertion of the EPSPS-CP4 cassette (Isogenic vs NK603 panel, Fig. 3) . One protein (B4G0K5) and 31 metabolites had their expression significantly altered by the spraying of the Roundup pesticide (NK603 vs NK603+Roundup (R) panel, Fig. 3) . ”

“ The STITCH v5.0 beta web tool [71] was used to investigate metabolite-protein interactions on maize endogenous pathways. The list of disturbed proteins and metabolites, including the protein EPSPS, was uploaded and the metabolic networks was studied using STITCH v5.0 initial parameters.
Additional Information How to cite this article: Mesnage, R. et al. An integrated multi-omics analysis of the NK603 Roundup-tolerant GM maize reveals metabolism disturbances caused by the transformation process. Sci. ”

“ Tsujinaka, S. & Konishi, F. (2009) , Polyamine-rich food decreases age-associated pathology and mortality in aged mice, vol. 44, pp. 727–732, 19735716, pmid:19735716 ↑ Couto, N.; Wood, J. & Barber, J. (2016) , The role of glutathione reductase and related enzymes on cellular redox homoeostasis network, vol. 95, pp. 27–42, 26923386, pmid:26923386 ↑ Milligan, A. S.; Daly, A.; Parry, M. A. J.; Lazzeri, P. A. & Jepson, I. (2001) , The expression of a maize glutathione S-transferase gene in transgenic wheat confers herbicide tolerance, both in planta and in vitro, vol. ”