GM NK603 corn research generates debate in Europe and the US

Design of GMO corn 'equivalence' study flawed: US animal scientist

By Jane Byrne

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Related tags Genetically modified food Genetically modified organism

A new study on GM corn, involving French researcher Séralini, whose previous work has generated controversy, has spurred scientific debate on both sides of the Atlantic.

The study​, ​published in the Nature​ journal, Scientific Reports,​ alleges that GM NK603 corn and its corresponding non-GMO corn variety are not substantially equivalent.

The establishment of compositional ‘substantial equivalence’ is a key starting point requested by regulatory agencies for assessing the safety of a GMO crop and food.

Dr Michael Antoniou at King’s College London, who led the research in question, commented:

“Our study clearly shows that the GM transformation process results in profound compositional differences in NK603, demonstrating that this GMO corn is not substantially equivalent to its non-GMO counterpart. 

“The marked increase in putrescine and especially cadaverine is a concern since these substances are potentially toxic, being reported as enhancers of the effects of histamine, thus heightening allergic reactions, and both have been implicated in the formation of carcinogenic nitrosamines with nitrite in meat products.“

He added that their findings disprove industry and regulatory agency claims that NK603 is ‘substantially equivalent’ to its non-GMO counterpart and suggest that a more thorough evaluation of the safety of consuming products derived from this GMO corn on a long term basis should be undertaken.

Spotlight now on official regulation of GM crops?

Peter Melchett, policy director at the UK’s Soil Association, argued that this dramatic new research throws real doubts on the official regulation of GM crops worldwide:

Using the very latest scientific techniques (‘omics’ technologies -  transcriptomics, proteomics, metabolomics, epigenomics and mirnomics), which were not available when GM crops were first introduced, scientists have shown that GM crops can be significantly different from their non-GM equivalents. 

“The researchers also found that spraying with Roundup (which is routinely applied to many GM crops) also altered the molecular profile of the crop. This throws into disarray all GM safety testing, which is based on the assumption that GM crops are usually ‘substantially equivalent’ to non-GM versions, and therefore do not need any extensive safety testing.”

GMO istock
Researchers call for further safety testing of products derived from NK603 GM corn. Image credit: istock

Experimental design problems

FeedNavigator asked prominent geneticist, Alison Van Eenennaam, who is based in the Department of Animal Science at the University of California, to assess the study. She told us: “To perform such an evaluation requires a common agreement as to what substantial equivalence means, and what constitutes an appropriate comparator(s). Unfortunately, not such common understanding exists.”

Furthermore, she noted: “Unfortunately, there are a number of experimental design problems with the Mesnage et al. (2016) paper that complicate the interpretation of the results, and as concerning there appear to be confounders that further complicate the analyses.”

Those flaws in design, claimed Van Eenennaam, include the following:

  • Only a single replicate of each treatment (n=1) at a single location (over two years) is analyzed with no biological replication or randomization of locations to remove site variability.
  • The data from the two cultivations in different years were inexplicably merged prior to analysis which made it impossible to determine if results or trends were consistent or reproducible between years
  • No inclusion of non-GM reference-varieties (conventional commercial hybrids) representative of those that would be normally grown in the areas where the field trials are performed to put some figures and context to the natural biological variation in the composition of non-GM corn comparators
  • No discussion of correction for multiple comparisons (by chance one in every 20 comparisons would be expected to be significant at the p<0.05). If doing multiple comparisons, it is necessary to do a multiple-comparison correction
  • There appears to be evidence of different levels of fungal (Gibberella moniliformis ​Maize ear and stalk rot fungus) protein contamination between the three groups. [See Supplemental Dataset 5 where Tubulin alpha chain​ OS=Gibberella moniliformis (strain M3125 / FGSC 7600)] appears as the  protein that had the biggest fold change between control and GM lines. If there were differing levels of fungal infestation among the groups, this would also confound the data.

The University of California scientist also wrote an extensive reaction to the study in blog format, which can be accessed here​ ​- reaction to that piece was swift on Twitter.

Other experts have weighed in on the study, with comments posted on the UK’s Science Media Center​.

'The science is good as far as it goes'

Johnjoe McFadden, professor of molecular genetics at the University of Surrey, said:

“The science is good as far as it goes. But the analysis only emphasizes the inadequacy of the ‘substantial equivalence principle’. How equivalent does it need to be? If you perform this detailed level of analysis on any perturbation of any organism you will detect this level of change – organisms are extraordinary sensitive and, for example, similar changes are produced when treated with e.g. pesticide or herbicides or when attacked by pests.

“I would expect that practically any perturbation to an organism will generate a response that can be detected by these powerful techniques – that is after all what life does.

“So all it shows is that GM, like pesticides, herbicides, drought, predation or even growing in a different field will produce a response by the organism. If GM was banned on these grounds, then so would all herbicide pesticides and indeed anything that causes a change (which is everything).”

Dr Joe Perry, former chair of the European Food Safety Authority (EFSA) GMO Panel, said:

“In contrast with compositional analysis, which is done for every application, and reported by EFSA, and which involves proper replicated field trials, this study appears to have been done with single, unreplicated plots.

“Therefore it is not possible to say with any certainty whether the differences reported are due to differences between the treatments or differences between the two fields (or two plots within the fields) used.

“In other words the basic tenets of experimental design seem not to have been followed. For that reason I could not yet describe this as a thorough piece of science.

“Further details about the conduct of the experiment would be useful to confirm or otherwise this initial impression."

Source: Scientific Reports 6, Article number: 37855 (2016)

Published online ahead of print: doi:10.1038/srep37855

Title: An integrated multi-omics analysis of the NK603 Roundup-tolerant GM maize reveals metabolism disturbances caused by the transformation process

Authors: R Mesnage, SZ Agapito-Tenfen, V Vilperte, G Renney, M Ward, GE Séralini, R O Nodari and MN Antoniou

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Response from lead author of study to comments posted by Jane Byrne from Science Media Centre website

Posted by Dr Michael Antoniou,

I would like to respond to the comments posted from the Science Media Centre website.

Dr Joe Perry is incorrect in claiming that our study was done with single, unreplicated plots. In reality there were two cultivations of maize over two growing seasons, and the results were consistent over both, as presented (supplementary online Additional File 1).

The compositional analyses of GM crops performed by the GMO producer companies and submitted to regulators in support of market authorisation are extremely superficial, looking at major elements such as total proteins, carbohydrates and fats. They do not examine the types of proteins or metabolites that are present, yet these factors can determine whether a GM crop is substantially equivalent to the non-GM crop and safe to eat.

Claims that different growing conditions could account for the differences found between the GM and non-GM crops are incorrect. As clearly stated in the Materials and Methods section of our paper all these factors were carefully controlled for, thus minimizing the possibility of their being significant contributors to the changes found in the GM crop. A typical soil type analysis of the growing areas is shown in the supplementary online data (Additional File 1). The plots of land on which the different crops were grown were similar and consisted of an imperfectly drained field with a coarse loam surface texture and fine loam subsoil.

We also minimized the possibility that different growing seasons may be responsible for the differences. As mentioned in our article, “the fold changes observed in the comparisons of the NK603 maize sprayed with Roundup, the unsprayed NK603 maize and the isogenic control corn were highly correlated between the two cultivations performed during two different growing seasons” (see also supplementary online Additional File 4).

However, even though our experimental design takes into account the effect of the growing season, further experiments conducted under different environmental conditions would be needed to determine the full range of effects of the GM transformation process on this maize type.

Prof Johnjoe McFadden seems to imply that our analysis is too detailed, but the methods employed are both cutting-edge and widely used in both research and diagnosis. If he is implying that differences in pesticide or herbicide use could be responsible for the changes in the GM maize, he is incorrect, since these factors were taken into account. Our analysis did indeed reveal an effect resulting from the application of Roundup herbicide on the GM Roundup-tolerant maize, but this factor did not make such a large difference as the GM process itself (see Figure 2 in our paper).

In addition, we conducted a detailed analysis of pesticide residues in the GM and non-GM maize (Additional File 2) and found there were none above levels of detection. There were no differences in pesticide or fertilizer use between the GM and non-GM maize, except that Roundup herbicide was applied to one cultivation of the GM maize, in accordance with how this type of maize is designed to be grown. Therefore the differences observed in the GM maize cannot be attributed to differences in pesticide use or presence.

However, even if the toxicological relevance of the differences remains unclear, what is clear is that the use of these molecular profiling tools allows a better understanding of the composition of GM plants and thus could improve the risk assessment of the non-target effects of genetic modification. In fact, genetic engineers cannot control or predict the effects of genetic engineering on plants and currently these are not measured at the molecular level.

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Re: Response from lead author of study to Jane Byrne and A. van Eenennaam

Posted by Dr Michael Antoniou,

Regarding the Figure from our supplementary online data cited in my previous posting, the figure number is listed as "Additional file 4".

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Response from lead author of study to Jane Byrne and A. van Eenennaam

Posted by Dr Michael Antoniou,

Jane Byrne states that we “allege” that GM NK603 corn is not substantially equivalent to its non-GM counterpart. However, in science we do not “allege”. We allow the evidence to do the talking for us and simply present the data with our interpretation, which anyone in the field evaluating the same dataset in an objective manner would corroborate.

Alison Van Eenennaam makes several misleading statements about our study design. We would like to correct them as follows:

1. There were two independent cultivations of the GM and non-GM maize (n=2, not n=1 as stated).
2. The data from the two cultivations was not “inexplicably merged” as claimed. As shown in our supplementary data online (Figure xxx), the datasets from the two cultivations were independently analysed and the outcomes were found to be very similar. Thus they were ultimately merged into a single dataset.
3. We did not include non-isogenic non-GM “reference varieties” of maize since the objective of the study was to highlight the effect of the GM transformation process. With this objective in mind, the only scientifically valid comparator is the isogenic non-GM counterpart grown under the same conditions. Including non-isogenic non-GM reference varieties serves only to increase the range of variables in the investigation and as a result masks rather than highlights the effect on the maize of the GM transformation process. This would negate the objective of the study. This was clearly stated in the Discussion section of our paper.
4. As made clear in the Materials and Methods and Results sections of our paper, correction for multiple comparison was indeed undertaken, using the post-statistical analysis Benjamini-Hochberg adjustment.
5. We were aware that two proteins (out of more than 150 maize proteins that had their levels significantly altered by the GM transformation process) detected in our proteomics analysis, matched a protein in Gibberella moniliformis, a known maize pathogen.

Our finding of fungal proteins in our maize samples is not surprising since the crops were grown under open field, not sterile, conditions, which were intended to mimic real world farming scenarios of cultivated maize that will enter the human and animal feed supply. Thus it is expected there will be environmental interaction with the plants.
The claim that different levels of fungal infestation, as indicated by the presence of the G. moniliformis proteins, could confound the data is incorrect for two reasons:

(i) No other proteins related to pathogenic response have been observed in the entire dataset as would be expected, based on observations in other proteomic investigations looking at changes in plants in response to pathogen infection (Pechanova and Pechan, 2015).
(ii) In the case of a pathogen infection in control samples, these samples would be expected to show enzymes and metabolites related to an increase in oxidative stress or feedback enzymes to fight high oxidative cell environments. This is because the recognition of any invading pathogen results in a coordinated activation of plant defence mechanisms, including a strong and rapid oxidative response (Campo et al, 2004; Pechanova and Pechan, 2015). However, we observed the opposite – our results suggest that the GM samples, not the control samples, had increased oxidative stress.

We did consider the two pathogen proteins in our dataset. In fact, we raised concerns about any other mycotoxin contamination even before we performed the proteomics analysis. We performed a mycotoxin analysis but did not include this data in the manuscript because it was outside the scope of the work presented. This analysis revealed the presence of the fumonisin group of mycotoxins, but at only trace amounts (less than 200 µg/kg of fumonisin B1 and B2) in the corn samples. Levels did not differ between varieties. The fumonisin class of mycotoxin is produced by G. moniliformis, also known as Fusarium verticillioides or F. moniliforme. As only very low levels of the fumonisins were found to be present this in turn implies low levels of contamination with this fungal pathogen.

There is indeed evidence that the presence of this pathogen can change polyamine metabolism in plants. However, such changes occur at levels of contamination, as measured by fumonisin levels, at least 1,000 times higher than the levels we detected (Campos-Bermudez et al, 2013). Moreover, these studies show that the higher the levels of pathogen infection, the higher the levels of polyamine compounds. However, again, we observed the opposite. There were higher levels of mycotoxin in the control (non-GM) corn than in GM corn and yet the non-GM corn had lower levels of polyamines. Therefore the elevated levels of polyamines in the GM corn samples were not caused by the presence of the fungal pathogen.

Thus the suggestion that differing fungal infestation levels could confound the data is incorrect.

In summary, our results clearly show that the presence of fungal pathogen proteins suggesting infestation of the maize with G. moniliformis, which were found in our dataset of more than 150 proteins whose levels were significantly altered in the GM compared to the non-GM isogenic corn samples, did not contribute to the observed metabolic changes. Hence we are confident that our molecular profiling results obtained by application of state-of-the-art proteomics and metabolomics analyses of NK603 GM corn and its isogenic control clearly and unequivocally reveal that they are not substantially equivalent.

Pechanova O, Pechan T. Maize-Pathogen Interactions: An Ongoing Combat from a Proteomics Perspective. Int J Mol Sci. 2015 Nov 30; 16(12):28429-48.
Campo S, Carrascal M, Coca M, Abián J, San Segundo B. The defense response of germinating maize embryos against fungal infection: a proteomics approach. Proteomics. 2004 Feb;4(2):383-96.
Campos-Bermudez VA, Fauguel CM, Tronconi MA, Casati P, Presello DA, Andreo CS. Transcriptional and Metabolic Changes Associated to the Infection by Fusarium verticillioides in Maize Inbreds with Contrasting Ear Rot Resistance. PLoS ONE. 2013 8(4): e61580.

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