This report describes the first long-term (2-year) rodent (rat) feeding studyinvestigating possible toxic effects arising from consumption of an R-tolerant GM maize(NK603) and a complete commercial formulation of R herbicide. The aims of thisinvestigation were essentially twofold. First, to evaluate whether the signs oftoxicity, especially with respect to liver and kidney functions, seen after 90 days'consumption of a diet containing NK603 R-tolerant GM maize [3, 7] escalated into serious ill health or dissipated over an extended period oftime. Second, to determine if low doses of full commercial R formulation at permittedlevels were still toxic, as indicated by our previous in vitro studies [8, 9]. The previous toxicity study with NK603 maize employed only this GM crop thathad been sprayed with R during cultivation [3]. However, in our study presented here, in addition to extending the treatmentperiod from 90 days to 2 years and in order to better ascertain the source of any illhealth observed, we included additional test feeding groups. These consisted of NK603maize grown without as well as with R application and R alone administered via drinkingwater. Furthermore, we used three levels of dosing in all cases rather than the twopreviously used [3], in order to highlight any dose response effects of a given treatment. It isalso important to note that our study is the first to conduct blood, urine, and organanalyses from animals treated with the complete agricultural formulation of R and notjust G alone, as measured by the manufacturer [35].

Our data show that the signs of liver and kidney toxicity seen at 90 days from theconsumption of NK603 GM maize [3, 7] do indeed escalate into severe disease over an extended period. Furthermore,similar negative health effects were observed in all treatment groups (NK603 GM maizewith or without R application and R alone).

What is also evident from our data is that ill effects were not proportional to the doseof either the NK603 GM maize ± R or R alone. This suggests that the observeddisease may result from endocrine disruptive effects, which are known to benon-monotonic. Similar degrees of pathological symptoms occurred from the lowest to thehighest doses, suggesting a threshold effect [36]. This corresponds to levels likely to arise from consumption or environmentalexposure, such as either 11% GM maize in food, or 50 ng/L G equivalent of R-formulation,a level which can be found in some contaminated drinking tap waters and which fallswithin authorized limits.

Death in male rats was mostly due to the development of severe hepatorenalinsufficiencies, confirming the first signs of toxicity observed in 90-day feedingtrials with NK603 GM maize [7]. In females, kidney ion leakage was evident at a biochemical level at month15, when severe nephropathies were observed in dead male animals at postmortem, at theanatomopathological level. Early signs of toxicity at month 3 in kidney and liver werealso observed for 19 edible GM crops containing pesticide residues [1]. It is known that only elderly male rats are sensitive to chronic progressivenephropathies [37]. Therefore, the disturbed kidney functional parameters may have been inducedby the reduced levels of phenolic acids in the GM maize feed used in our study, sincecaffeic and ferulic acids are beneficial to the kidney as they prevent oxidative stress [38, 39]. This possibility is consistent with our previous observation that plantextracts containing ferulic and caffeic acids were able to promote detoxification ofhuman embryonic kidney cells after culture in the presence of R [40]. It is thus possible that NK603 GM maize consumption, with its reduced levelsof these compounds, may have provoked the early aging of the kidney physiology,similarly to R exposure causing oxidative stress [41]. Disturbances in global patterns of gene expression leading to disease viaepigenetic effects cannot be excluded, since it has been demonstrated that numerouspesticides can cause changes in DNA methylation and histone modification, therebyaltering chromatin compaction and thus gene expression profiles [42].

Disturbances that we found to occur in the male liver are characteristic of chronictoxicity, confirmed by alterations in biochemical liver and kidney function parameters.The observation that liver function in female animals was less negatively affected maybe due to the known protection from oxidative stress conferred by estrogen [43]. Estrogen can induce expression of genes such as superoxide dismutase andglutathione peroxidase via the MAP kinase-NF-kB signaling pathway, thus providing anantioxidant effect [43]. Furthermore, liver enzymes have been clearly demonstrated as sex-specific intheir expression patterns, including in a 90-day rat feeding trial of NK603 GM maize [7]. However, in a long-term study, evidence of early liver aging was observed infemale mice fed with R-tolerant GM soy [12]. In the present investigation, deeper analysis at an ultrastructural levelrevealed evidence of impediments in transcription and other defects in cell nuclearstructure that were comparable in both sexes and dose-dependent in hepatocytes in alltreatments. This is consistent with the well-documented toxic effect of very lowdilutions of R on apoptosis, mitochondrial function, and cell membrane degradation,inducing necrosis of hepatocytes, and in other cell lines [8, 9, 44, 45].

The disruptions of at least the estrogen-related pathways and/or enhancement ofoxidative stress by all treatments need further confirmation. This can be addressedthrough the application of transcriptomic, proteomic, and metabolomic methods to analyzethe molecular profile of kidneys and livers, as well as the GM NK603 maize [46–48]. Other possible causes of observed pathogenic effects may be due to disturbedgene expression resulting from the transgene insertional, general mutagenic, ormetabolic effects [49, 50] as has been shown for MON810 GM maize [51, 52]. A consequent disruption of general metabolism in the GMO cannot be excluded,which could lead, for example, to the production of other potentially active compoundssuch as miRNAs [53] or leukotoxin diols [54].

The lifespan of the control group of animals corresponded to the mean for the strain ofrat used (Harlan Sprague-Dawley), but as is frequently the case with most mammals,including humans [55], males on average died before females, except for some female treatmentgroups. All treatments in both sexes enhanced large tumor incidence by two- to threefoldin comparison to our controls and also the number of mammary tumors in comparison to theHarlan Sprague-Dawley strain [56] and overall around threefold in comparison to the largest study with 1,329Sprague-Dawley female rats [57]. This indicates that the use of historical data to compare our tumor numbersis not relevant, first, since we studied the difference with concurrent controlschronologically (and not only at the end of the experiment, as is the case in historicaldata), and second, since the diets of historical reference animals may have beencontaminated with several non-monitored compounds including GMOs and pesticides atlevels used in our treatments. In our study, the tumors also developed considerablyfaster than in controls, even though the majority of tumors were observed after 18months. The first large detectable tumors occurred at 4 and 7 months into the study inmales and females, respectively, further underlining the inadequacy of the standard90-day feeding trials for evaluating GM crop and food toxicity [1]. Future studies employing larger cohorts of animals providing appropriatestatistical power are required to confirm or refute the clear trend in increased tumorincidence and mortality rates seen with some of the treatments tested in this study. Asalready stated, our study was not designed as a carcinogenicity study that would haverequired according to OECD the use of 50 rats per sex per group. However, we wish toemphasize that the need for more rats to provide sufficient statistical power may bebiased by the presence of contaminants in the diets used in gathering historical controldata, increasing artificially the background of tumors, which would inappropriately becalled in this case ‘spontaneous’ or due to the genetic strain. Forinstance, toxic, hormonal disrupting or carcinogenic levels of pesticides, PCBs,plasticizers, dioxins, or heavy metals may contaminate the diets or drinking water usedfor the establishment of ‘spontaneous’ tumors in historical data [58–62].

In females, induced euthanasia due to suffering and deaths corresponded mostly to thedevelopment of large mammary tumors. This was observed independently of the cancer gradebut according to impact on morbidity. These appeared to be related to the varioustreatments when compared to the control groups. These tumors are generally known to bemostly estrogen-dependent [63]. We observed a strikingly marked induction of mammary tumors in groupsadministered R alone, even at the very lowest dose (50 ng/L G equivalent dilution inadjuvants). At this concentration in vitro, G alone is known to induce humanbreast cancer cell growth via estrogen receptors [64]. In addition, R with adjuvants has been shown to disrupt aromatase, whichsynthesizes estrogen [19], and to interfere with estrogen and androgen receptors in cells [8]. Furthermore, R appears to be a sex endocrine disruptor in vivo inmales [10]. Sex steroid levels were also modified in treated rats in our study. Thesehormone-dependent phenomena are confirmed by enhanced pituitary dysfunction in treatedfemales. An estrogen-modified feedback mechanism may act at this level [65, 66]. The similar pathological profiles provoked by the GM maize + R diet may thusbe explained at least in part by R residues present in this feed. In this regard, it isnoteworthy that the medium dose of the R treatment tested (400 mg/Kg G equivalent)corresponds to acceptable residue levels of this pesticide in some edible GMOs.

Interestingly and perhaps surprisingly, in the groups of animals fed with the NK603 GMmaize without R application, similar effects with respect to enhanced tumor incidenceand mortality rates were observed. For instance, comparing the 11% GMO-treated femalegroup to the controls, the assumption that the tumors are equally distributed isrejected with a level of significance of 0.54% with the Westlake exceedance test [67]. The classical tests of Kolmogorov-Smirnov (one-sided) andWilcoxon-Mann-Whitney reach α values of significance, which are respectively of1.40% and 2.62%.

A possible explanation for this finding is the production of specific compound(s) in theGM feed that are either directly toxic and/or cause the inhibition of pathways, which inturn generates toxic effects. This is despite the fact that the variety of GM maize usedin this study was judged by industry and regulators as being substantially equivalent tothe corresponding non-GM closest isogenic line [3, 30]. As the total chemical composition of the GM maize has not been measured indetail, the use of substantial equivalence as a concept in risk assessment isinsufficient to highlight potential unknown toxins and therefore cannot replacelong-term animal feeding trials for GMOs.

A cause of the ill effects resulting from NK603 GM maize alone observed in this studycould be the fact that it is engineered to overexpress a modified version of theAgrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS-CP4) [3], which confers R tolerance. The modified EPSPS is not inhibited by G, incontrast to the wild-type enzyme in the crop. This enzyme is known to drive the firststep of aromatic amino acid biosynthesis in the plant shikimate pathway. In addition,estrogenic isoflavones and their glycosides are also products of this pathway [68]. A limited compositional analysis showed that these biochemical pathways werenot disturbed in the GM maize used in our study. However, our analysis did reveal thatthe levels of caffeic and ferulic acids in the GM diet, which are also secondarymetabolites of the plant shikimate pathway, but not always measured in regulatory tests,were significantly reduced. This may lower their protective effects againstcarcinogenesis and mammalian tumor formation [69, 70]. Moreover, these phenolic acids, and in particular ferulic acid, may modulateestrogen receptors or the estrogenic pathway in mammalian cells [71]. This does not exclude the possibility of the action of other unknownmetabolites. This explanation also corresponds to the fact that the observed effects ofNK603 GM maize and R were not additive but reached a threshold. This implies that boththe NK603 maize and R may cause hormonal disturbances in the same biochemical andphysiological pathways.