In this report we present the first multi-omics analysis of GM NK603 maize compared to a near isogenic non-GM counterpart. Based on analysis conducted by the developer Monsanto Company, NK603 maize was scored as ‘substantially equivalent’ to its isogenic control, which was a major contributor to this product being granted market approval for animal and human consumption in the European Union, United States, Brazil and several other nations. Although NK603 had comparable nutritional and compositional profiles when originally accessed by the developer company upon registration of their product, our analysis at a detailed, in-depth molecular profiling level shows that NK603 grains, with or without Roundup spraying during cultivation, are not equivalent to isogenic non-transgenic control samples (Fig. 2).

The concept of substantial equivalence has long being used in safety testing of GMO crops, but the term and the concept has no clear definition35. In 1993 the Organization for Economic Co-operation and Development (OECD) stated that the “concept of substantial equivalence embodies the idea that existing organisms used as food, or as a source of food, can be used as the basis for comparison when assessing the safety of human consumption of a food or food component that has been modified or is new”36. The vagueness of this term generates conflict among stakeholders to determine which compositional differences are sufficient to declare a GMO as non-substantially equivalent. However, the Codex Alimentarius Commission37 makes it clear that a safety assessment of a new food based on the concept of substantial equivalence “does not imply absolute safety of the new product; rather, it focuses on assessing the safety of any identified differences so that the safety of the new product can be considered relative to its conventional counterpart.” Thus, the concept of substantial equivalence should not be used as a proof of safety. However, it could be used as a first tier in risk assessment to detect any unintended effects of the GM transformation process. Unintended effects can be understood as the effects that go beyond the primary expected effects of the genetic modification, and represent statistically significant differences in the GMO compared with an appropriate control38. Unintended effects during transgenesis include rearrangements, insertion, or deletions during the genetic transformation or during the tissue culture stages of GMO development39,40. A comprehensive characterization of the GM plant at the molecular level could facilitate identification of unintended effects in GMO crops and could be used as a complementary analytical tool to existing safety assessment procedures41,42,43,44.

In general, our study design further highlights the importance of restricting comparison to the GMO crop and non-GMO isogenic comparator and cultivation of the two at the same location and season when the objective is to evaluate the effect of the GM transformation process. This is obligatory in order to reduce effects on plant metabolism arising from differing environmental conditions, which can make it difficult to attribute differences that are observed to the procedure of transgenesis. However, even though our experimental design takes into account the effect of the growing season, further experiments made under different environmental conditions would be needed to determine the full range of effects of the GM transformation process on NK603 phenotype. Indeed, virtually all traits are influenced by genotype–environment interactions. Neither genetic differences nor environmental variations alone can account for the production of a particular phenotypic variation. For example, a study of the expression of the transgene encoding a Bt toxin in the MON810 GM maize under different environmental conditions, has shown that the phenotype resulting from the GM transformation process is influenced by stressful environmental conditions45.

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 maize11,12,46. 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. Metabolite profiling of NK603 maize kernels were analyzed and approximately 3% of the metabolites detected showed statistically significant differences compared to the respective isogenic lines47. Two metabolites (γ-tocopherol and myo-inositol) were less abundant in NK60347. Interestingly, γ-tocotrienol and myo-inositol levels were also found to be significantly reduced in our study, and thus attributable to the genetic transformation. This suggests that some metabolic alterations are consistently reported despite a strong background triggered by environmental influence. In a study of two common MON810/non-GM variety pairs subjected to two farming practices (conventional and low-nitrogen fertilization), it was found that up to 37.4% of the variation was dependent upon the variety, 31.9% were the result of the fertilization treatment, and 9.7% was attributable to the GM character48.

Alterations can also be found in other plant tissues. For example, analysis of leaves of Brazilian varieties of MON810 Bt maize revealed a total of 32 differentially expressed proteins between GM and non-GM samples that were identified and assigned to carbohydrate and energy metabolism, genetic information processing and stress response9.

Our study revealed significant metabolome profile differences between NK603 that was either sprayed or not with Roundup during cultivation (Fig. 2). This was surprising since the single application of this herbicide was prior to development of the maize cobs. In addition, we did not detect glyphosate or AMPA residues in the test maize kernel samples (Additional File 1). This indicates that metabolic differences provoked by an early application of Roundup persisted throughout the life of the maize even in the absence of herbicide residues. At present we can only speculate as to the mechanisms that may explain these effects but they may have their basis in epigenetic programming of gene expression patterns with consequent longer term effects. The spraying of Roundup could have acted as a signal causing an alteration in gene expression patterns in the growing maize. A recent study that demonstrated marked epigenetic (DNA methylation) changes in A thaliana in response to treatment with carbendazim supports this possibility49. In addition, it has been demonstrated that epigenetic (DNA methylation and post-translational histone modification) patterns acquired in one cultivation can be transgenerationally inherited in an A thaliana model system50. However, further research would be needed to determine if epigenetic alterations provoked by pesticide exposure can hamper plant phenotypes across generations.

The maize kernels analysed in this study were previously used to feed laboratory animals that formed part of a chronic (2 year) study looking at potential toxic effects arising from the consumption of this NK603 Roundup-tolerant GM maize. A dry feed was formulated to contain 11%, 22%, or 33% of NK603 maize, cultivated either with or without Roundup application, or 33% of the near isogenic variety. Sprague Dawley rats fed for two years on these diets presented blood/urine biochemical changes indicative of an increased incidence of liver and kidney structure and functional pathology in the NK603-containing diet groups compared to non-GM controls51. Standard biochemical compositional analysis revealed no particular differences between the different maize types tested51. Metabolic disturbances observed in our study may help to understand the negative health effects suggested after the chronic consumption of this GM maize. Alterations in concentrations of metabolites in grains might be directly related to pathogenic effects due to some active compounds that are known to be toxic52. For instance, a soybean glycoprotein allergen (Gly m Bd 28 K fragment) was also found overexpressed in a proteomic study of Roundup Ready GM soybean seeds (MSOY 7575 RR event)13. In our study, cadaverin levels were significantly increased (Log2FC 4.81 for NK603 and 5.31 for NK603+Roundup). Cadaverin plays important roles in lysine biosynthesis53 and also glutathione metabolism54. Other similar biogenic amines, such as N-acetyl-cadaverine, N-acetylputrescine and putrescine were also found to be present at higher levels in NK603 in our investigation. Different polyamines have been reported to have different effects, which depend on various factors such as age, tissue or disease status55. In certain contexts some of these polyamines have been found to be protective whereas in other situations they can be a cause of toxicity. On the one hand, toxicological effects such as nausea, headaches, rashes and changes in blood pressure are provoked by the consumption of foods with high concentrations of polyamines56. Putrescine and cadaverine have been reported as potentiators of the effects of histamine, and both have been implicated in the formation of carcinogenic nitrosamines with nitrite in meat products57. On the other hand, certain polyamines can also have beneficial anti-inflammatory effects and have been found to be beneficial during aging in some rodent model systems58. Noticeably, these polyamines were not measured in the first compositional analysis of NK603 maize performed for regulatory purposes32. Overall, whether the increased levels of cadaverine and putrescine found in the NK603 maize samples can account for the signs of potential negative health effects upon its consumption by rats, as implied by the blood/urine biochemical analysis33, needs to be further analyzed in experiments using more quantitative methods.

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 agrochemicals59. Plant glutathione S-transferases (GSTs) are also widely known for their role in herbicide detoxification60. Enzymes involved in combating reactive oxygen species, ascorbate peroxidase, glutathione reductase, and catalase are expressed at a higher level in transgenic soybean seeds14. Levels of ROS and other free radicals in GM food and feed would have to be monitored and quantified by further experiments in order to conclude on their potential impact on the agronomic performances of the plant. Additionally, it is known that polyamines are typically elevated in plants under abiotic stress conditions61. Typically, when cellular polyamine content increases, the levels of hydrogen peroxide also increases, activating antioxidant systems. Unintended effects of the inserted EPSPS-CP4 transgene was linked to energy metabolism disturbances in other studies13,14,15. It can be hypothesized that the plant is searching for a new equilibrium to maintain heterologous EPSPS-CP4 metabolism within levels that can be tolerated by the plant.

Glyphosate, the active ingredient of Roundup herbicide, inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is the sixth enzyme of the shikimate pathway, and plays an essential role in the biosynthesis of aromatic amino acids and other aromatic compounds in plants62. The EPSPS has a binding site for phosphenolpyruvate (PEP) and it could be hypothesized that an overexpression of a heterologous EPSPS could provoke a metabolic imbalance by altering the metabolism of PEP. Alterations in intermediate metabolism are corroborated in our experiment by the fact that the network formed by altered proteins/metabolites is centred on some TCA cycle intermediates (Fig. 5) such as α-ketoglutarate. In fact, it is also known that EPSPS inhibition by glyphosate impairs carbon metabolism, in particular by inducing alternative respiration and aerobic fermentation63. In this latest study, the metabolic switch was explained by an accumulation of pyruvate. Thus, if EPSPS inhibition is able to alter intermediate metabolism, a comparable change in the opposite direction could be expected as a result of EPSPS overexpression.

This study is the first and most detailed multi-omics characterization of a widely commercialized GMO crop and its isogenic counterpart. In conclusion, our integrative statistical and bioinformatics analysis allowed us to suggest a mechanistic link between the proteome and metabolome alterations observed and the insertion of a particular transgene. The transformation process and the resulting expression of a transgenic protein cause a general disturbance in the GM plant and it is clear that NK603 maize is markedly different from its non-GM isogenic line at the proteome and metabolome levels. In addition, our data correlates with previous studies, which observed higher amounts of ROS that act as free-radicals promoting oxidative stress in those transgenic plant materials. We also confirm a metabolic imbalance in energy and carbohydrate metabolism. Although a clear mechanistic link between alterations in the GM feed and the possible health effects following long-term consumption of this product remains to be established, the evidence we present clearly shows that NK603 and non-GM isogenic maize are not substantially equivalent and the nutritional quality of GM feed might be hampered by metabolic imbalances related to plant energy and stress metabolism.