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Abstract

Background

The health effects of a Roundup-tolerant NK603 genetically modified (GM) maize (from 11% in the diet), cultivated with or without Roundup application and Roundup alone (from 0.1 ppb of the full pesticide containing glyphosate and adjuvants) in drinking water, were evaluated for 2 years in rats. This study constitutes a follow-up investigation of a 90-day feeding study conducted by Monsanto in order to obtain commercial release of this GMO, employing the same rat strain and analyzing biochemical parameters on the same number of animals per group as our investigation. Our research represents the first chronic study on these substances, in which all observations including tumors are reported chronologically. Thus, it was not designed as a carcinogenicity study. We report the major findings with 34 organs observed and 56 parameters analyzed at 11 time points for most organs.

Results

Biochemical analyses confirmed very significant chronic kidney deficiencies, for all treatments and both sexes; 76% of the altered parameters were kidney-related. In treated males, liver congestions and necrosis were 2.5 to 5.5 times higher. Marked and severe nephropathies were also generally 1.3 to 2.3 times greater. In females, all treatment groups showed a two- to threefold increase in mortality, and deaths were earlier. This difference was also evident in three male groups fed with GM maize. All results were hormone- and sex-dependent, and the pathological profiles were comparable. Females developed large mammary tumors more frequently and before controls; the pituitary was the second most disabled organ; the sex hormonal balance was modified by consumption of GM maize and Roundup treatments. Males presented up to four times more large palpable tumors starting 600 days earlier than in the control group, in which only one tumor was noted. These results may be explained by not only the non-linear endocrine-disrupting effects of Roundup but also by the overexpression of the EPSPS transgene or other mutational effects in the GM maize and their metabolic consequences.

Conclusion

Our findings imply that long-term (2 year) feeding trials need to be conducted to thoroughly evaluate the safety of GM foods and pesticides in their full commercial formulations.

Keywords:

Genetically modified; GMO; Roundup; NK603; Rat; Glyphosate-based herbicides; Endocrine disruption

Research

Empirical natural and social sciences produce knowledge (in German: Wissenschaften schaffen Wissen) which should describe and explain past and present phenomena and estimate their future development. To this end quantitative methods are used. Progress in science needs controversial debates aiming at the best methods as basis for objective, reliable and valid results approximating what could be the truth. Such methodological competition is the energy needed for scientific progress. In this sense, ESEU aims to enable rational discussions dealing with the article from G.-E. Séralini et al. (Food Chem. Toxicol. 2012, 50:4221–4231) by re-publishing it. By doing so, any kind of appraisal of the paper’s content should not be connoted. The only aim is to enable scientific transparency and, based on this, a discussion which does not hide but aims to focus methodological controversies. -Winfried Schröder, Editor of the Thematic Series “Implications for GMO-cultivation and monitoring” in Environmental Sciences Europe.

Background

There is an ongoing international debate as to the necessary length of mammalian toxicity studies, including metabolic analyses, in relation to the consumption of genetically modified (GM) plants [1]. Currently, no regulatory authority requires mandatory chronic animal feeding studies to be performed for edible genetically modified organisms (GMOs), or even short-term studies with blood analyses for the full commercial formulations of pesticides as sold and used, but only for the declared active principle alone. However, several 90-day rat feeding trials have been conducted by the agricultural biotechnology industry. These investigations mostly concern GM soy and maize that are engineered either to be herbicide-tolerant (to Roundup (R) in 80% of cases), or to produce a modified Bt toxin insecticide, or both. As a result, these GM crops contain new pesticide residues for which new maximum residue levels (MRL) have been established in some countries.

Though the petitioners conclude in general that no major physiological changes is attributable to the consumption of the GMO in subchronic toxicity studies [25], significant disturbances have been found and may be interpreted differently [6,7]. A detailed analysis of the data in the subchronic toxicity studies [25] has revealed statistically significant alterations in kidney and liver function that may constitute signs of the early onset of chronic toxicity. This may be explained at least in part by pesticide residues in the GM feed [6,7]. Indeed, it has been demonstrated that R concentrations in the range of 103 times below the MRL can induce endocrine disturbances in human cells [8] and toxic effects thereafter [9]. This may explain toxic effects seen in experiments in rats in vivo[10] as well as in farm animals [11]. After several months of consumption of an R-tolerant soy, the liver and pancreas of mice were affected, as highlighted by disturbances in sub-nuclear structure [1214]. Furthermore, this toxic effect was reproduced by the application of R herbicide directly to hepatocytes in culture [15].

More recently, long-term and multi-generational animal feeding trials have been performed, with some possibly providing evidence of safety, while others conclude on the necessity of further investigation because of metabolic modifications [16]. However, in contrast with the study we report here, none of these previous investigations have included a detailed follow-up of the animals, including multiple (up to 11) blood and urine sampling over 2 years, and none has investigated either the GM NK603 R-tolerant maize or Roundup.

Furthermore, evaluation of long-term toxicity of herbicides is generally performed on mammalian physiology employing only their active principle, rather than the complete formulations as used in agriculture. This was the case for glyphosate (G) [17], the declared active chemical constituent of R. It is important to note that G is only able to efficiently penetrate target plant organisms with the help of adjuvants present in the various commercially used R formulations [18]. Even if G has shown to interact directly with the active site of aromatase at high levels [19], at low contaminating levels, adjuvants may be better candidates than G to explain the toxicity or endocrine disruptive side effects of R on human cells [8,20] and also in vivo for acute toxicity [21]. In this regard, it is noteworthy that the far greater toxicity of full agricultural formulations compared to declared supposed active principles alone has recently been demonstrated also for six other major pesticides tested in vitro[22]. When G residues are found in tap water, food, or feed, they arise from the total herbicide formulation although little data is available as to the levels of the R adjuvants in either the environment or food chain. Indeed, adjuvants are rarely monitored in the environment, but some widely used adjuvants (surfactants) such as nonylphenol ethoxylates, another ethoxylated surfactant like POEA present in R, are widely found in rivers in England and are linked with disruption of wildlife sexual reproduction [23]. Adjuvants are found in groundwater [24]. The half-life of POEA (21 to 42 days) is even longer than for G (7 to 14 days) in aquatic environments [25]. As a result, the necessity of studying the potential toxic effects of total chemical mixtures rather than single components has been strongly emphasized [2628]. On this basis, the regular measurement of only G or other supposed active ingredients of pesticides in the environment constitute at best markers of full formulation residues. Thus, in the study of health effects, exposure to the diluted whole formulation may be more representative of environmental pollution than exposure to G alone.

With a view to address this lack of information, we performed a 2-year detailed rat feeding study. Our study was designed as a chronic toxicity study and as a direct follow-up to a previous investigation on the same NK603 GM maize conducted by the developer company, Monsanto [3]. A detailed critical analysis of the raw data of this subchronic 90-day rat feeding study revealed statistically significant differences in multiple organ function parameters, especially pertaining to the liver and kidneys, between the GM and non-GM maize-fed group [3,7]. However, Monsanto’s authors dismissed the findings as not ‘biologically meaningful’ [3], as was also the case with another GM corn [29]. The European Food Safety Authority (EFSA) accepted Monsanto’s interpretation on NK603 maize [30], like in all other cases.

Our study is the first and to date the only attempt to follow up Monsanto’s investigation and to determine whether the differences found in the NK603 GM maize-fed rats, especially with respect to liver and kidney function, were not biologically meaningful, as claimed, or whether they developed into serious diseases over an extended period of time.

The Monsanto authors adapted Guideline 408 of the Organization for Economic Co-operation and Development (OECD) for their experimental design [3]. Our study design was based on that of the Monsanto investigation in order to make the two experiments comparable, but we extended the period of observation from Monsanto’s 90 days to 2 years. We also used three doses of GMOs (instead of Monsanto’s two) and Roundup to determine treatment dose response, including any possible non-linear as well as linear effects. This allowed us to follow in detail the potential health effects and their possible origins due to the direct or indirect consequences of the genetic modification itself in the NK603 GM maize, or due to the R herbicide formulation used on the GM maize (and not G alone), or both. Because of recent reviews on GM foods indicating no specific risk of cancer [2,16], but indicating signs of hepatorenal dysfunction within 3 months [1,7], we had no reason to adopt a carcinogenesis protocol using 50 rats per group. However, we prolonged to 2 years the biochemical and hematological measurements and measurements of disease status, as allowed, for example, in OECD protocols 453 (combined chronic toxicity and carcinogenicity) and 452 (chronic toxicity). Both OECD 452 and 453 specify 20 rats per sex per group but require only 50% (ten per sex per group, the same number that we used in total) to be analyzed for biochemical and hematological parameters. Thus, these protocols yield data from the same number of rats as our experiment. This remains the highest number of rats regularly measured in a standard GM diet study, as well as for a full formulated pesticide at very low environmentally relevant levels.

We used the Sprague-Dawley strain of rat, as recommended for chronic toxicology tests by the National Toxicology Program in the USA [31], and as used by Monsanto in its 90-day study [3]. This choice is also consistent with the recommendation of the OECD that for a chronic toxicity test, rats of the same strain should be used as in studies on the same substance but of shorter duration [32]. We then also tested for the first time three doses (rather than the two usually employed in 90-day protocols) of the R-tolerant NK603 GM maize alone, the GM maize treated with R, and R alone at very low environmentally relevant doses, starting below the range of levels permitted by regulatory authorities in drinking water and in GM feed.

Overall, our study is the first in-depth life-long toxicology study on the full commercial Roundup formulation and NK603 GM maize, with observations on 34 organs and measurement of 56 parameters analyzed at 11 time points for most organs, and utilizing 3 doses. We report here the major toxicological findings on multiple organ systems. As there was no evidence in the literature on GM food safety evaluation to indicate anything to the contrary, this initial investigation was designed as a full chronic toxicity and not a carcinogenicity study. Thus, we monitored in details chronologically all behavioral and anatomical abnormalities including tumors. A full carcinogenicity study, which usually focuses only on observing incidence and type of cancers (not always all tumors), would be a rational follow-up investigation to a chronic toxicity study in which there is a serious suspicion of carcinogenicity. Such indications had not been previously reported for GM foods.

Our findings show that the differences in multiple organ functional parameters seen from the consumption of NK603 GM maize for 90 days [3,7] escalated over 2 years into severe organ damage in all types of test diets. This included the lowest dose of R administered (0.1 ppb, 50 ng/L G equivalent) of R formulation administered, which is well below permitted MRLs in both the USA (0.7 mg/L) [33] and European Union (100 ng/L) [34]. Surprisingly, there was also a clear trend in increased tumor incidence, especially mammary tumors in female animals, in a number of the treatment groups. Our data highlight the inadequacy of 90-day feeding studies and the need to conduct long-term (2 years) investigations to evaluate the life-long impact of GM food consumption and exposure to complete pesticide formulations.

Results

Biochemical analyses of the maize feed

Standard biochemical compositional analysis revealed no particular differences between the different maize types and diets, the GM and non-GM maize being classified as substantially equivalent, except for transgene DNA quantification. For example, there was no difference in total isoflavones. In addition, we also assayed for other specific compounds, which are not always requested for establishing substantial equivalence. This analysis revealed a consistent and statistically significant (p < 0.01) decrease in certain phenolic acids in treatment diets, namely ferulic and caffeic acids. Ferulic acid was decreased in both GM maize and GM maize + R diets by 16% to 30% in comparison to the control diet (889 ± 107, 735 ± 89, respectively, vs. control 1,057 ± 127 mg/kg) and caffeic acid in the same groups by 21% to 53% (17.5 ± 2.1, 10.3 ± 1.3 vs. control 22.1 ± 2.6 mg/kg).

Anatomopathological observations and liver parameters

All rats were carefully monitored during the experiment for behavior, appearance, palpable tumors, and infections. At least ten organs per animal were weighed and up to 34 analyzed postmortem, at the macroscopic and/or microscopic levels (Table 1). Due to the large quantity of data collected, it cannot all be shown in one report, but we present here the most important findings. There was no rejection by the animals of the diet with or without GM maize, nor any major difference in body weight (data not shown).

Table 1. Protocol used and comparison to existing assessment and to non-mandatory regulatory tests

The most affected organs in males were the liver, hepatodigestive tract, and kidneys (Table 2; Figure 1A,B,C,D,E,F,G,H,I). Liver abnormalities such as hepatic congestions and macroscopic and microscopic necrotic foci were 2.5 to 5.5 times more frequent in all treatments than in control groups, where only two rats out of ten were affected with one abnormality each. For instance, there were 5 abnormalities in total in the GMO 11% group (2.5 times higher than controls) and 11 in the GMO 22% group (5.5 times greater). In addition, by the end of the experiment, Gamma GT hepatic activity was increased, particularly in the GMO + R groups (up to 5.4 times higher), this probably being reflective of liver dysfunction. Furthermore, cytochrome P450 activity generally increased in the presence of R (either in drinking water or in the GM maize-containing diet) according to the dose and up to 5.7 times greater at the highest dose.

Table 2. Summary of the most frequent anatomical pathologies observed

thumbnailFigure 1. Anatomopathological observations in rats fed GMO treated or not by Roundup and effects of Roundup alone. Macroscopic (A to D) and microscopic (A’ and C’) photographs show male left kidneys and livers (E to I) and female pituitaries (J to M), in accordance to Table 2. The number of each animal and its treatment is specified. Macroscopic pale spots (I) and microscopic necrotic foci in liver (G clear-cell focus, H basophilic focus with atypia), and marked or severe chronic progressive nephropathies, are illustrated. In females, pituitary adenomas (K to M) are shown and compared to control (J, rat number and C for control). Apostrophes after letters indicate organs from the same rat.

Transmission electron microscopic observations of liver samples confirmed changes for all treated groups in relation to glycogen dispersion or appearance in lakes, increase of residual bodies and enlargement of cristae in mitochondria (Figure 2, panels 2 to 4). The GM maize-fed groups either with or without R application showed a higher heterochromatin content and decreased nucleolar dense fibrillar components, implying a reduced level of mRNA and rRNA transcription. In the GMO + R group (at the highest dose), the smooth endoplasmic reticulum was drastically increased and nucleoli decreased in size, becoming more compact. In the R alone treatment groups, similar trends were observed, with a partial resumption of nucleolar activity at the highest dose.

thumbnailFigure 2. Ultrastructure of hepatocytes in male rats from groups presenting the greatest degree of liver pathology. (1) Typical control rat hepatocyte (bar 2 μm except in 4). (2) Effects with Roundup at the lowest dose. Glycogen (G) is dispersed in the cytoplasm. L, lipid droplet; N, nucleus; R, rough endoplasmic reticulum. (3) Details of treatment effects with 22% dietary GMO (bar 1 μm). a, cluster of residual bodies (asterisks); b, mitochondria show many enlarged cristae (arrows). (4) Hepatocytes of animal fed GM maize (GMO) at 22% of total diet. Large lakes of glycogen occur in the cytoplasm. M, mitochondria.

Degenerating kidneys with turgid inflammatory areas demonstrated the increased incidence of marked and severe chronic progressive nephropathies, which were up to two fold higher in the 33% GM maize or lowest dose R treatment groups (Table 2; Figure 1, first line).

Biochemical analyses of blood and urine samples

Biochemical measurements of blood and urine were focused on samples taken at the 15th month time point, as this was the last sampling time when most animals were still alive (in treated groups 90% males, 94% females, and 100% controls). Statistical analysis of results employed OPLS-DA 2-class models built between each treated group per sex and controls. Only models with an explained variance R2(Y) ≥ 80%, and a cross-validated predictive ability Q2(Y) ≥ 60%, were used for selection of the discriminant variables (Figure 3), when their regression coefficients were significant at a 99% confidence level. Thus, in treated females, kidney failures appeared at the biochemical level (82% of the total disrupted parameters). Levels of Na and Cl or urea increased in urine with a concomitant decrease of the same ions in serum, as did the levels of P, K, and Ca. Creatinine and creatinine clearance decreased in urine for all treatment groups in comparison to female controls (Table 3). In GM maize-treated males (with or without R), 87% of discriminant variables were kidney-related, but the disrupted profiles were less obvious because of advanced chronic nephropathies and deaths. In summary, for all treatments and both sexes, 76% of the discriminant variables versus controls were kidney-related.

thumbnailFigure 3. Orthogonal partial least squares-discriminant analysis (OPLS-DA) for biochemical data (females fed 33% GMO versus controls). (A) First, detailed examples of significant discriminant variables distribution between females fed 33% GMO (bold line) and controls (dotted line). On X axis, animals; on Y axis, serum or urine biochemical values for Na, Cl, estradiol, testosterone. (B) Wider view of OPLS-DA regression coefficients for predictive component, with jack-knifed confidence intervals at 99% confidence level, indicating discriminant parameters versus controls at month 15. U, urinary; UEx, excreted in urine during 24 h; APPT, activated partial thromboplastin time; MCV, mean corpuscular volume; PT, prothrombine time; RBC, red blood cells; ALT, alanine aminotransferase; MCHC, mean corpuscular hemoglobin concentration; A/G, albumin/globulin ratio; WBC, white blood cells; AST, aspartate aminotransferase. Profiles evidence kidney ion leakages and sex hormonal imbalance versus controls.

Table 3. Percentage variation of parameters indicating kidney failures of female animals

Furthermore, in females (Table 3), the androgen/estrogen balance in serum was modified by GM maize and R treatments (at least 95% confidence level, Figure 3). For male animals at the highest R treatment dose, levels of estrogens were more than doubled.

Tumor incidence

Tumors are reported in line with the requirements of OECD chronic toxicity protocols 452 and 453, which require all ‘lesions’ (which by definition include tumors) to be reported. These findings are summarized in Figure 4. The results are presented in the form of real-time cumulative curves (each step corresponds to an additional tumor in the group). Only the growing largest palpable growths (above a diameter of 17.5 mm in females and 20 mm in males) are presented (for example, see Figure 5A,B,C). These were found to be in 95% of cases non-regressive tumors (Figure 5D,E,F,G,H,I,J) and were not infectious nodules. These arose from time to time; then, most often disappeared and were not different from controls after bacterial analyses. The real tumors were recorded independently of their grade, but dependent on their morbidity, since non-cancerous tumors can be more lethal than those of cancerous nature, due to internal hemorrhaging or compression and obstruction of function of vital organs, or toxins or hormone secretions. These tumors progressively increased in size and number, but not proportionally to the treatment dose, over the course of the experiment (Figure 4). As in the case of rates of mortality (Figure 6), this suggests that a threshold in effect was reached at the lower doses. Tumor numbers were rarely equal but almost always more than in controls for all treated groups, often with a two- to threefold increase for both sexes. Tumors began to reach a large size on average 94 days before controls in treated females and up to 600 days earlier in two male groups fed with GM maize (11 and 22% with or without R).

thumbnailFigure 4. Largest non-regressive tumors in rats fed GMO treated or not by Roundup and effects of Roundup alone. Rats were fed with NK603 GM maize (with or without application of Roundup) at three different doses (11%, 22%, and 33% in their diet; thin, medium, and bold lines, respectively) compared to the substantially equivalent closest isogenic non-GM maize (control, dotted line). Roundup was administered in drinking water at three increasing doses, same symbols, environmental (A), MRL in some agricultural GMOs (B), and half of minimal agricultural levels (C), see ‘Methods’). The largest tumors were palpable during the experiment and numbered from 20 mm in diameter for males and 17.5 mm for females. Above this size, 95% of growths were non-regressive tumors. Summary of all tumors are shown in the bar histograms: black, non-regressive large tumors; white, small internal tumors; grey, metastases.

thumbnailFigure 5. Examples of female mammary tumors observed. Mammary tumors are evidenced (A, D, H, representative adenocarcinoma, from the same rat in a GMO group) and in Roundup and GMO + Roundup groups, two representative rats (B, C, E, F, I, J fibroadenomas) are compared to controls. A normal representative rat in controls is not shown, only a minority of them having tumors up to 700 days, in contrast with the majority affected in all treated groups. (G) The histological control.

thumbnailFigure 6. Mortality of rats fed GMO treated or not with Roundup and effects of Roundup alone. The symbols of curves and treatments are explained in the caption of Figure 4. Lifespan during the experiment for the control group is represented by the vertical bar ± SEM (grey area). In bar histograms, the causes of mortality before the grey area are detailed in comparison to the controls (0). In black are the necessary euthanasia because of suffering in accordance with ethical rules (tumors over 25% body weight, more than 25% weight loss, hemorrhagic bleeding, etc.); and in hatched areas, spontaneous mortality.

In female animals, the largest tumors were in total five times more frequent than in males after 2 years, with 93% of these being mammary tumors. Adenomas, fibroadenomas, and carcinomas were deleterious to health due to their very large size (Figure 5A,B,C) rather than the grade of the tumor itself. Large tumor size caused impediments to either breathing or digestion and nutrition because of their thoracic or abdominal location and also resulted in hemorrhaging (Figure 5A,B,C). In addition, one metastatic ovarian cystadenocarcinoma and two skin tumors were identified. Metastases were observed in only two cases; one in a group fed with 11% GM maize and another in the highest dose of R treatment group.

Up to 14 months, no animals in the control groups showed any signs of palpable tumors, whilst 10% to 30% of treated females per group developed tumors, with the exception of one group (33% GMO + R). By the beginning of the 24th month, 50% to 80% of female animals had developed tumors in all treatment groups, with up to three tumors per animal, whereas only 30% of controls were affected. A summary of all mammary tumors at the end of the experiment, independent of size, is presented in Table 2. The same trend was observed in the groups receiving R in their drinking water (Figure 4, R treatment panels). The R treatment groups showed the greatest rates of tumor incidence, with 80% of animals affected (with up to three tumors for one female), in each group. Using a non-parametric multiple comparison analysis, mammary tumor incidence was significantly increased at the lowest dose of R compared to controls (p < 0.05, Kruskal-Wallis test with post hoc Dunn’s test). All females except one (with metastatic ovarian carcinoma) presented in addition mammary hypertrophies and in some cases hyperplasia with atypia (Table 2).

The second most affected organ in females was the pituitary gland, in general around two times more than in controls for most treatments (Table 2; Figure 1J,K,L,M). Again, at this level of examination, adenomas and/or hyperplasias and hypertrophies were noticed. For all R treatment groups, 70% to 80% of animals presented 1.4 to 2.4 times more abnormalities in this organ than controls.

The large palpable tumors in males (in kidney and mostly skin) were by the end of the experimental period on average twice as frequent as in controls, in which only one skin fibroma appeared during the 23rd month. At the end of the experiment, internal non-palpable tumors were added, and their sums were lower in males than in females. They were not significantly different from controls, although slightly increased in females (Figure 4, histogram insets).

Mortality

The rates of mortality in the various control and treatment groups are shown as raw data in Figure 6. Control male animals survived on average 624 ± 21 days, whilst females lived for 701 ± 20 days during the experiment, plus in each case, a 5-week starting age at reception of animals and a 3-week housing stabilization period. After mean survival time had elapsed, any deaths that occurred were considered to be largely due to aging. Before this period, 30% control males (three in total) and 20% females (only two) died spontaneously, while up to 50% males and 70% females died in some groups on diets containing the GM maize (Figure 6, panels GMO, GMO + R). However, the rate of mortality was not proportional to the treatment dose, reaching a threshold at the lowest (11%) or intermediate (22%) amounts of GM maize in the equilibrated diet, with or without the R application on the crop. It is noteworthy that the first two male rats that died in both GM maize-treated groups had to be euthanized due to Wilms’ kidney tumors that had grown by this time to over 25% of body weight. This was approximately a year before the first control animal died. The first female death occurred in the 22% GM maize feeding group and resulted from a mammary fibroadenoma 246 days before the first control female death. The maximum difference in males was five times more deaths occurring by the 17th month in the group consuming 11% GM maize and in females six times greater mortality by the 21st month on the 22% GM maize diet with and without R. In the female cohorts, there were two to three times more deaths in all treated groups compared with controls by the end of the experiment and deaths occurred earlier in general. Females were more sensitive to the presence of R in drinking water than males, as evidenced by a shorter lifespan (Figure 6, panels R). The general causes of death represented in histogram format within each of the panels in Figure 6, are linked mostly to mammary tumors in females and to problems in other organ systems in males.

Additional file 1. Biochemistry M15

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Additional file 2. List of blood and urine paramaters with sampling and unit detail

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Additional file 3. Mortality and tumors raw data

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Additional file 4. Rats identification

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Additional file 5. Raw data legends

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Discussion

This report describes the first long-term (2-year) rodent (rat) feeding study investigating possible toxic effects arising from consumption of an R-tolerant GM maize (NK603) and a complete commercial formulation of R herbicide. The aims of this investigation were essentially twofold. First, to evaluate whether the signs of toxicity, 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 of time. Second, to determine if low doses of full commercial R formulation at permitted levels 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 that had been sprayed with R during cultivation [3]. However, in our study presented here, in addition to extending the treatment period from 90 days to 2 years and in order to better ascertain the source of any ill health observed, we included additional test feeding groups. These consisted of NK603 maize grown without as well as with R application and R alone administered via drinking water. Furthermore, we used three levels of dosing in all cases rather than the two previously used [3], in order to highlight any dose response effects of a given treatment. It is also important to note that our study is the first to conduct blood, urine, and organ analyses from animals treated with the complete agricultural formulation of R and not just G alone, as measured by the manufacturer [35].

Our data show that the signs of liver and kidney toxicity seen at 90 days from the consumption 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 maize with or without R application and R alone).

What is also evident from our data is that ill effects were not proportional to the dose of either the NK603 GM maize ± R or R alone. This suggests that the observed disease may result from endocrine disruptive effects, which are known to be non-monotonic. Similar degrees of pathological symptoms occurred from the lowest to the highest doses, suggesting a threshold effect [36]. This corresponds to levels likely to arise from consumption or environmental exposure, 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 falls within authorized limits.

Death in male rats was mostly due to the development of severe hepatorenal insufficiencies, confirming the first signs of toxicity observed in 90-day feeding trials with NK603 GM maize [7]. In females, kidney ion leakage was evident at a biochemical level at month 15, when severe nephropathies were observed in dead male animals at postmortem, at the anatomopathological level. Early signs of toxicity at month 3 in kidney and liver were also observed for 19 edible GM crops containing pesticide residues [1]. It is known that only elderly male rats are sensitive to chronic progressive nephropathies [37]. Therefore, the disturbed kidney functional parameters may have been induced by the reduced levels of phenolic acids in the GM maize feed used in our study, since caffeic and ferulic acids are beneficial to the kidney as they prevent oxidative stress [38,39]. This possibility is consistent with our previous observation that plant extracts containing ferulic and caffeic acids were able to promote detoxification of human embryonic kidney cells after culture in the presence of R [40]. It is thus possible that NK603 GM maize consumption, with its reduced levels of 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 via epigenetic effects cannot be excluded, since it has been demonstrated that numerous pesticides can cause changes in DNA methylation and histone modification, thereby altering chromatin compaction and thus gene expression profiles [42].

Disturbances that we found to occur in the male liver are characteristic of chronic toxicity, confirmed by alterations in biochemical liver and kidney function parameters. The observation that liver function in female animals was less negatively affected may be due to the known protection from oxidative stress conferred by estrogen [43]. Estrogen can induce expression of genes such as superoxide dismutase and glutathione peroxidase via the MAP kinase-NF-kB signaling pathway, thus providing an antioxidant effect [43]. Furthermore, liver enzymes have been clearly demonstrated as sex-specific in their 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 in female mice fed with R-tolerant GM soy [12]. In the present investigation, deeper analysis at an ultrastructural level revealed evidence of impediments in transcription and other defects in cell nuclear structure that were comparable in both sexes and dose-dependent in hepatocytes in all treatments. This is consistent with the well-documented toxic effect of very low dilutions 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 of oxidative stress by all treatments need further confirmation. This can be addressed through the application of transcriptomic, proteomic, and metabolomic methods to analyze the molecular profile of kidneys and livers, as well as the GM NK603 maize [4648]. Other possible causes of observed pathogenic effects may be due to disturbed gene expression resulting from the transgene insertional, general mutagenic, or metabolic 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 compounds such as miRNAs [53] or leukotoxin diols [54].

The lifespan of the control group of animals corresponded to the mean for the strain of rat 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 treatment groups. All treatments in both sexes enhanced large tumor incidence by two- to threefold in comparison to our controls and also the number of mammary tumors in comparison to the Harlan Sprague-Dawley strain [56] and overall around threefold in comparison to the largest study with 1,329 Sprague-Dawley female rats [57]. This indicates that the use of historical data to compare our tumor numbers is not relevant, first, since we studied the difference with concurrent controls chronologically (and not only at the end of the experiment, as is the case in historical data), and second, since the diets of historical reference animals may have been contaminated with several non-monitored compounds including GMOs and pesticides at levels used in our treatments. In our study, the tumors also developed considerably faster than in controls, even though the majority of tumors were observed after 18 months. The first large detectable tumors occurred at 4 and 7 months into the study in males and females, respectively, further underlining the inadequacy of the standard 90-day feeding trials for evaluating GM crop and food toxicity [1]. Future studies employing larger cohorts of animals providing appropriate statistical power are required to confirm or refute the clear trend in increased tumor incidence and mortality rates seen with some of the treatments tested in this study. As already stated, our study was not designed as a carcinogenicity study that would have required according to OECD the use of 50 rats per sex per group. However, we wish to emphasize that the need for more rats to provide sufficient statistical power may be biased by the presence of contaminants in the diets used in gathering historical control data, increasing artificially the background of tumors, which would inappropriately be called in this case ‘spontaneous’ or due to the genetic strain. For instance, toxic, hormonal disrupting or carcinogenic levels of pesticides, PCBs, plasticizers, dioxins, or heavy metals may contaminate the diets or drinking water used for the establishment of ‘spontaneous’ tumors in historical data [5862].

In females, induced euthanasia due to suffering and deaths corresponded mostly to the development of large mammary tumors. This was observed independently of the cancer grade but according to impact on morbidity. These appeared to be related to the various treatments when compared to the control groups. These tumors are generally known to be mostly estrogen-dependent [63]. We observed a strikingly marked induction of mammary tumors in groups administered R alone, even at the very lowest dose (50 ng/L G equivalent dilution in adjuvants). At this concentration in vitro, G alone is known to induce human breast cancer cell growth via estrogen receptors [64]. In addition, R with adjuvants has been shown to disrupt aromatase, which synthesizes estrogen [19], and to interfere with estrogen and androgen receptors in cells [8]. Furthermore, R appears to be a sex endocrine disruptor in vivo in males [10]. Sex steroid levels were also modified in treated rats in our study. These hormone-dependent phenomena are confirmed by enhanced pituitary dysfunction in treated females. An estrogen-modified feedback mechanism may act at this level [65,66]. The similar pathological profiles provoked by the GM maize + R diet may thus be explained at least in part by R residues present in this feed. In this regard, it is noteworthy 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 GM maize without R application, similar effects with respect to enhanced tumor incidence and mortality rates were observed. For instance, comparing the 11% GMO-treated female group to the controls, the assumption that the tumors are equally distributed is rejected with a level of significance of 0.54% with the Westlake exceedance test [67]. The classical tests of Kolmogorov-Smirnov (one-sided) and Wilcoxon-Mann-Whitney reach α values of significance, which are respectively of 1.40% and 2.62%.

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

A cause of the ill effects resulting from NK603 GM maize alone observed in this study could be the fact that it is engineered to overexpress a modified version of the Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS-CP4) [3], which confers R tolerance. The modified EPSPS is not inhibited by G, in contrast to the wild-type enzyme in the crop. This enzyme is known to drive the first step 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 were not disturbed in the GM maize used in our study. However, our analysis did reveal that the levels of caffeic and ferulic acids in the GM diet, which are also secondary metabolites of the plant shikimate pathway, but not always measured in regulatory tests, were significantly reduced. This may lower their protective effects against carcinogenesis and mammalian tumor formation [69,70]. Moreover, these phenolic acids, and in particular ferulic acid, may modulate estrogen receptors or the estrogenic pathway in mammalian cells [71]. This does not exclude the possibility of the action of other unknown metabolites. This explanation also corresponds to the fact that the observed effects of NK603 GM maize and R were not additive but reached a threshold. This implies that both the NK603 maize and R may cause hormonal disturbances in the same biochemical and physiological pathways.

Conclusions

In conclusion, the consumption of NK603 GM maize with or without R application or R alone gave similar pathologies in male and female rats fed over a 2-year period. It was previously known that G consumption in water above authorized limits may provoke hepatic and kidney failure [33]. The results of the study presented here clearly indicate that lower levels of complete agricultural G herbicide formulations, at concentrations well below officially set safety limits, can induce severe hormone-dependent mammary, hepatic, and kidney disturbances. Similarly, disruption of biosynthetic pathways that may result from overexpression of the EPSPS transgene in the GM NK603 maize can give rise to comparable pathologies that may be linked to abnormal or unbalanced phenolic acid metabolites or related compounds. Other mutagenic and metabolic effects of the edible GMO cannot be excluded. This will be the subject of future studies, including analyses of transgene, G and other R residue presence in rat tissues. Reproductive and multigenerational studies will also provide novel insight into these problems. This study represents the first detailed documentation of long-term deleterious effects arising from consumption of a GMO, specifically a R-tolerant maize, and of R, the most widely used herbicide worldwide.

Taken together, the significant biochemical disturbances and physiological failures documented in this work reveal the pathological effects of these GMO and R treatments in both sexes, with different amplitudes. They also show that the conclusion of the Monsanto authors [3] that the initial indications of organ toxicity found in their 90-day experiment were not ‘biologically meaningful’ is not justifiable.

We propose that agricultural edible GMOs and complete pesticide formulations must be evaluated thoroughly in long-term studies to measure their potential toxic effects.

Methods

Ethics

The experimental protocol was conducted in an animal care unit authorized by the French Ministries of Agriculture and Research (Agreement Number A35-288-1). Animal experiments were performed according to ethical guidelines of animal experimentations (CEE 86/609 regulation), including the necessary observations of all tumors, in line with the requirements for a long-term toxicological study [32], up to a size where euthanasia on ethical grounds was necessary.

Concerning the cultivation of the maize used in this study, no specific permits were required. This is because the maize was grown (MON-00603-6 commonly named NK603) in Canada, where it is authorized for unconfined release into the environment and for use as a livestock feed by the Canadian Food Inspection Agency (Decision Document 2002-35). We confirm that the cultivation did not involve endangered or protected species. The GM maize was authorized for import and consumption into the European Union (CE 258/97 regulation).

Plants, diets, and chemicals

The varieties of maize used in this study were the DKC 2678 R-tolerant NK603 (Monsanto Corp., USA), and its nearest isogenic non-transgenic control DKC 2675. These two types of maize were grown under similar normal conditions, in the same location, spaced at sufficient distance to avoid cross-contamination. The genetic nature, as well as the purity of the GM seeds and harvested material, was confirmed by qPCR analysis of DNA samples. One field of NK603 was treated with R at 3 L ha−1 (WeatherMAX, 540 g/L of G, EPA Reg. 524-537), and another field of NK603 was not treated with R. Corn cobs were harvested when the moisture content was less than 30% and were dried at a temperature below 30°C. From these three cultivations of maize, laboratory rat chow was made based on the standard diet A04 (Safe, France). The dry rat feed was made to contain 11%, 22%, or 33% of GM maize, cultivated either with or without R, or 33% of the non-transgenic control line. The concentrations of the transgene were confirmed in the three doses of each diet by qPCR. All feed formulations consisted of balanced diets, chemically measured as substantially equivalent except for the transgene, with no contaminating pesticides over standard limits. All secondary metabolites cannot be known and measured in the composition. However, we measured isoflavones and phenolic acids including ferulic acid by standard HPLC-UV. All reagents used were of analytical grade. The herbicide diluted in the drinking water was the commercial formulation of R (GT Plus, 450 g/L of G, approval 2020448, Monsanto, Belgium). Herbicide levels were assessed by G measurements in the different dilutions by mass spectrometry.

Animals and treatments

Virgin albino Sprague-Dawley rats at 5 weeks of age were obtained from Harlan (Gannat, France). All animals were kept in polycarbonate cages (820 cm2, Genestil, France) with two animals of the same sex per cage. The litter (Toplit classic, Safe, France) was replaced twice weekly. The animals were maintained at 22 ± 3°C under controlled humidity (45% to 65%) and air purity with a 12 h-light/dark cycle, with free access to food and water. The location of each cage within the experimental room was regularly changed. This 2-year life-long experiment was conducted in a Good Laboratory Practice (GLP) accredited laboratory according to OECD guidelines. After 20 days of acclimatization, 100 male and 100 female animals were randomly assigned on a weight basis into ten equivalent groups. For each sex, one control group had access to plain water and standard diet from the closest isogenic non-transgenic maize control; six groups were fed with 11%, 22%, and 33% of GM NK603 maize either treated or not treated with R. The final three groups were fed with the control diet and had access to water supplemented with respectively 1.1 × 10−8% of R (0.1 ppb or 50 ng/L of G, the contaminating level of some regular tap waters), 0.09% of R (400 mg/kg G, US MRL of 400 ppm G in some GM feed), and 0.5% of R (2.25 g/L G, half of the minimal agricultural working dilution). This was changed weekly. Twice-weekly monitoring allowed careful observation and palpation of animals, recording of clinical signs, measurement of any tumors, food and water consumption, and individual body weights.

Anatomopathology

Animals were sacrificed during the course of the study only if necessary because of suffering according to ethical rules (such as 25% body weight loss, tumors over 25% body weight, hemorrhagic bleeding, or prostration) and at the end of the study by exsanguination under isoflurane anesthesia. In each case, detailed observations and anatomopathology was performed and the following organs were collected: brain, colon, heart, kidneys, liver, lungs, ovaries, spleen, testes, adrenals, epididymis, prostate, thymus, uterus, aorta, bladder, bone, duodenum, esophagus, eyes, ileum, jejunum, lymph nodes, lymphoreticular system, mammary glands, pancreas, parathyroid glands, Peyer’s patches, pituitary, salivary glands, sciatic nerve, skin, spinal cord, stomach, thyroid, and trachea. The first 14 organs (at least ten per animal depending on the sex, Table 1) were weighted, plus any tumors that arose. The first nine were divided into two parts and one half was immediately frozen in liquid nitrogen/carbonic ice. The remaining parts including other organs were rinsed in PBS and stored in 4% formalin before anatomopathological study. These samples were used for further paraffin-embedding, slides, and HES histological staining. For transmission electron microscopy, the kidneys, livers, and tumors were cut into 1 mm3 fragments. Samples were fixed in pre-chilled 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 M PBS pH 7.4 at 4°C for 3 h and processed as previously described [13].

Biochemical analyses

Blood samples were collected from the tail vein of each rat under short isoflurane anesthesia before treatment and after 1, 2, 3, 6, 9, 12, 15, 18, 21, and 24 months: 11 measurements were obtained for each animal alive at 2 years. It was first demonstrated that anesthesia did not impact animal health. Two aliquots of plasma and serum were prepared and stored at −80°C. Then, 31 parameters were assessed (Table 1) according to standard methods including hematology and coagulation parameters, albumin, globulin, total protein concentration, creatinine, urea, calcium, sodium, potassium, chloride, inorganic phosphorus, triglycerides, glucose, total cholesterol, alanine aminotransferase, aspartate aminotransferase, gamma glutamyl-transferase (GT), estradiol, and testosterone. In addition, at months 12 and 24, the C-reactive protein was assayed. Urine samples were collected similarly 11 times, over 24 h in individual metabolic cages, and 16 parameters were quantified including creatinine, phosphorus, potassium, chloride, sodium, calcium, pH, and clearance. Liver samples taken at the end made it possible to perform assays of CYP1A1, 1A2, 3A4, 2C9 activities in S9 fractions, with glutathione S-transferase and gamma-GT.

Statistical analysis

In this study, multivariate analyses were more appropriate than pairwise comparisons between groups because the parameters were very numerous, with samples of ten individuals. Kaplan-Meyer comparisons, for instance, were not used because these are better adapted to epidemiological studies. Differences in the numbers of mammary tumors were studied by a non-parametric multiple comparisons Kruskal-Wallis test, followed by a post hoc Dunn’s test with the GraphPad Prism 5 software.

Biochemical data were treated by multivariate analysis with the SIMCA-P (V12) software (UMETRICS AB Umea, Sweden). The use of chemometrics tools, for example, principal component analysis (PCA), partial least squares to latent structures (PLS), and orthogonal PLS (OPLS), are robust methods for modeling, analyzing, and interpreting complex chemical and biological data. OPLS is a recent modification of the PLS method. PLS is a regression method used in order to find the relationship between two data tables referred to as X and Y. PLS regression [72] analysis consists in calculating by means of successive iterations, linear combinations of the measured X-variables (predictor variables). These linear combinations of X-variables give PLS components (score vectors t). A PLS component can be thought of as a new variable – a latent variable – reflecting the information in the original X-variables that is of relevance for modeling and predicting the response Y-variable by means of the maximization of the square of covariance (Max cov2(X,Y)). The number of components is determined by cross validation. SIMCA software uses the nonlinear iterative partial least squares algorithm (NIPALS) for the PLS regression. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used in this study [73,74].

The purpose of discriminant analysis is to find a model that separates groups of observations on the basis of their X variables. The X matrix consists of the biochemical data. The Y matrix contains dummy variables which describe the group membership of each observation. Binary variables are used in order to encode a group identity. Discriminant analysis finds a discriminant plan in which the projected observations are well separated according to each group. The objective of OPLS is to divide the systematic variation in the X-block into two model parts, one linearly related to Y (in the case of a discriminant analysis, the group membership), and the other one unrelated (orthogonal) to Y. Components related to Y are called predictive, and those unrelated to Y are called orthogonal. This partitioning of the X data results in improved model transparency and interpretability [75]. Prior to analysis, variables were mean-centered and unit variance scaled.

Competing interests

The author(s) declare that they have no competing interests, and that, in contrast with regulatory assessments for GMOs and pesticides, they are independent from companies developing these products.

Authors’ contributions

GES directed and with JSV designed and coordinated the study. EC, RM, SG, and ND analyzed the data, compiled the literature, and participated in the drafting of the manuscript and final version. MM performed transmission electron microscopy. DH performed OPLS-DA statistical analysis. All authors read and approved the final manuscript.

Acknowledgements

We thank Michael Antoniou for English assistance, editing, and constructive comments on the manuscript. We gratefully acknowledge the Association CERES, for research on food quality, representing more than 50 companies and private donations, the Foundation ‘Charles Leopold Mayer pour le Progrès de l′Homme’, the French Ministry of Research, and CRIIGEN for their major support.

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French prosecutor: 9th suspect in Paris terror attacks being sought

France seeks EU security aid, launches new airstrikes on ISIS
France made an unprecedented demand Tuesday that its European Union allies support its military action against the Islamic State group after the attacks in Paris.
Tuesday, November 17, 2015 03:33PM

Authorities are seeking a ninth suspect in Friday’s attacks in Paris, a French prosecutor’s spokeswoman told ABC News. The spokeswoman did not reveal any details about the suspect’s identity.

France took unprecedented action Tuesday when it invoked the Mutual-Defense article of the EU Treaty. Now all 28 member nations must help France in its war against ISIS.

French Defense Minister Jean-Yves Le Drian said all 27 of France’s EU partners responded positively, and they could help “either by taking part in France’s operations in Syria or Iraq, or by easing the load or providing support for France in other operations.”

“Every country said: I am going to assist, I am going to help,” Drian said.

VIDEO: France carries out raids, names more potential attackers

A major action with heavily armed police is underway Monday, Nov. 16, 2015 in the Brussels neighborhood of Molenbeek amid a manhunt for a suspect of the Paris attacks.

Arriving for talks in Brussels with his EU counterparts, Greek Defense Minister Panagiotis Kammenos told reporters that the Paris attacks were a game-changer for the bloc.

“This is Sept. 11 for Europe,” he said.

French President Francois Hollande has vowed to forge a united coalition capable of defeating the jihadists at home and abroad. NATO allies were sharing intelligence and working closely with France, NATO chief Jens Stoltenberg said.

Noting that victims of the Paris attacks came from at least 19 nations, Hollande says the international community, led by the U.S. and Russia, must overcome their deep-seated divisions over Syria to destroy IS on its home turf.

 

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Islamic State threatens attack on Washington, other countries

isis fighter

An ISIS operative holds
a shoulder-fired anti-aircr
aft missile on the background of a black ISIS flag
(Picture sent by ISIS via Twitter)

slamic State warned in a new video on Monday that countries taking part in air strikes against Syria would suffer the same fate as France, and threatened to attack in Washington.

The video, which appeared on a website used by Islamic State to post its messages, begins with news footage of the aftermath of Friday’s Paris shootings in which at least 129 people were killed.

The message to countries involved in what it called the “crusader campaign” was delivered by a man dressed in fatigues and a turban, and identified in subtitles as Al Ghareeb the Algerian.

“We say to the states that take part in the crusader campaign that, by God, you will have a day, God willing, like France’s and by God, as we struck France in the center of its abode in Paris, then we swear that we will strike America at its center in Washington,” the man said.

It was not immediately possible to verify the authenticity of the video, which purports to be the work of Islamic State fighters in the Iraqi province of Salahuddine, north of Baghdad.

The U.S. Department of Homeland Security would not comment on the video but said it has not received information indicating a potential attack.

 

 

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ABC News

Devastation in France: What We Know About The Paris Attacks

Christian Hartmann/Reuters
WATCH At Least 1 American Killed in Paris Terror Attacks

A wave of devastating attacks on civilians across Paris Friday night has left at least 129 dead, including an American, and many more injured.

As France initiates a state of emergency and begins several days of mourning, here is a breakdown of what we know so far:

Where

Six separate attacks were carried out across Paris, beginning after 10 p.m. local time Friday night.

One of the targets was the Stade de France soccer stadium, where Germany was playing France in a game that French President Francois Hollande was attending. Hollande was evacuated from the stadium.

PHOTO: A French supporter reacts after invading the pitch of the Stade de France stadium at the end of a soccer match between France and Germany in Saint Denis, outside Paris, Nov. 13, 2015.

Christophe Ena/AP Photo
A French supporter reacts after invading the pitch of the Stade de France stadium at the end of a soccer match between France and Germany in Saint Denis, outside Paris, Nov. 13, 2015.more +

At least 80 were killed inside the Bataclan concert hall, where American rock band Eagles of Death Metal was playing. A witness in the theater told ABC News she heard gunmen shout “Syria!”

The four other attacks were at other locations in the 10th and 11th districts of Paris, where gunmen targeted bars and restaurants. Police said some attackers first sprayed cafes outside the venue with machine gunfire before continuing the assault inside the concert hall.

PHOTO: A woman is evacuated from the Bataclan theater after a shooting in Paris, Nov. 13, 2015.

Thibault Camus/AP Photo
A woman is evacuated from the Bataclan theater after a shooting in Paris, Nov. 13, 2015.

 

 

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ABC News

American College Student Killed in Paris Attacks

John Walton PA via AP

A 23-year-old American was killed in Friday night’s devastating six-part attack in Paris, according to California State University, Long Beach.

Nohemi Gonzalez, a CSU Long Beach student, was in Paris attending Strate College of Design for a semester abroad program, the school said.

Gonzalez was one of 17 CSU Long Beach students studying abroad at Strate, according to a school official. The 16 other students are safe, according to the school.

 

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Paris massacre: World mourns 120+ killed in series of terrorist attacks Live updates

French police patrol near the closed Galeries Lafayette department store in the shopping district the day after a series of deadly attacks in Paris, France, November 14, 2015 © Yves Herman
The world is mourning with France, the victims of Friday’s terror assaults in Paris that claimed the lives of at least 129 people. Security has been tightened up globally as Islamic State, who claimed responsibility for the massacre, threatened more violence.
  • 14 November 2015

    21:46 GMT

    A second person who was reportedly involved in the Paris massacre is “very likely” to have entered European Union via Greece, sources in the Greek government told Reuters. Earlier Greece’s deputy minister in charge of police said a Syrian man whose passport was found at one of the suicide bombing sites made it to the EU through Greece in October.

  • 21:33 GMT

    A spokesman for the Accor hotel chain, which operates the Pullman Hotel, also confirmed that the earlier incident was a false bomb alert.

  • 21:24 GMT

    A spokesman for the French Interior Ministry has told Reuters that the incident at the hotel in the center of Paris was a false alarm.

  • 21:22 GMT

    Heavily armed police have been deployed at the Pullman Hotel in Paris’ 15th district, according to witnesses, who said the officers were searching through rooms. An evacuation has also been reported to have taken place near the Eiffel Tower.

  • 21:08 GMT

    Police in Paris have evacuated an area around the Eiffel tower, Reuters reports, citing witnesses.

     

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Perfect storm: ‘Paris massacre may enflame public opinion on migrant crisis, NATO interventions’

People react and keep warm using thermal blankets near the Bataclan concert hall following fatal attacks in Paris, France, November 14, 2015. © Christian Hartmann
French President Hollande has declared a state of emergency across the country as Paris comes to grip with a series of terrorist attacks that led to the death of 127 people in the capital, which was placed under curfew for the first time since World War II.

RT interviewed several analysts for their opinions on what these deadly coordinated attacks in the French capital mean not only for the freedom of movement across Europe, but for the ongoing Syrian military operation, which has brought together various state actors – including the US and Russia – in an increasingly shrinking and volatile military theater.

 

Alain Corvez, Former Advisor to French Interior Ministry, provided insight into the question as to how it is possible that Islamic State has such a long reach – right into the very heart of Europe.

 

Corvez believes these attacks are “linked to the geopolitical system, and what we are doing in Iraq and in Syria is directly linked to attacks.”

 

I think all of the specialists and experts on terrorism knew that such an attack was possible,” he continued. “This is a world war against terrorists that we have to wage. And for the first time, President Hollande said we have to fight terrorism on a larger scale.”

‘France has been intervening in the Middle East since 1917’

Washington-based defense analyst Ivan Eland, asked whether this was the right time for nations to put aside their differences and join together in the fight against terrorism, he explained that it is important to consider the problem long-term it is important to ask “what are the root causes of this terrorism – we call them “blanket terrorism” – but there’s many different groups in the world with many different causes and most of them tend to be local causes, which become international causes when Western countries, or other countries, intervene where they shouldn’t.”

 

“France has been intervening in the Middle East since after World War I, when Britain and France divided it; it was the colonial master in Syria and now it’s taking a fairly assertive policy by bombing in Syria.

 

It’s no secret – this is not random – that France is the target.”

 

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‘Terrorists chose Paris as iconic target like twin towers’ – ex-CIA officer

 The Paris attacks are reminiscent to the terrorist gun spree in Mumbai in 2008, when separate groups of gunmen went on a shooting spree at different locations across the city. This tactic is a brutally efficient form of terrorism, Rice said.
The series of apparent Islamic State attacks in Paris can be compared to the 2001 destruction of the WTC towers in the US, says Jack Rice, a former CIA officer. The French capital is an iconic European city, and terrorists target icons.

READ MORE: Paris attacks: 150+ killed and many injured in a series of terrorist acts (LIVE UPDATES)

France has suffered one of the worst tragedies in its modern history with more than 150 people reported killed in seven separate gun and bomb attacks throughout the capital. The terrorist group Islamic State (IS, formerly ISIS/ISIL) claimed responsibility for the attacks.

“Part of the strength that a terrorist group has is to take down an icon,” Rice told RT. “If we go back to 9/11 in the United States, they took down the World Trade Center and parts of New York. And we’ve seen efforts around the world to do similar things.

 

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No need to get shirty! Air France executive is forced to climb a fence after staff attack him and rip off his shirt when he announces 2,900 job losses

  • Air France executives attacked after staff stormed company headquarters
  • Company plans to cut 2,900 jobs and 14 aircraft from its long-haul fleet
  • HR vice president and long-haul flights deputy had their shirts torn off

Air France managers have been forced to flee the company’s headquarters after being attacked by a baying mob of workers that tore their clothes off.

Hundreds of angry staff stormed the Air France building at the Charles de Gaulle International Airport in Roissy, near Paris, after the company announced plans to cut 2,900 jobs on Monday.

Two senior executives, Xavier Broseta, Vice President for Human Resources, and Pierre Plissonnier, deputy of Air France long-haul flights, both had their shirts ripped off their backs as they were evacuated through the crowds.

 

Under attack: A shirtless Xavier Broseta, Executive Vice President for Human Resources at Air France, is evacuated by security after employees interrupted a meeting with representatives staff at the Air France headquarters building at the Charles de Gaulle International Airport in Roissy, near Paris

Under attack: A shirtless Xavier Broseta, Executive Vice President for Human Resources at Air France, is evacuated by security after employees interrupted a meeting with representatives staff at the Air France headquarters building at the Charles de Gaulle International Airport in Roissy, near Paris

Shortly before the attack, Mr Broseta and Air France Chief Executive Frederic Gagey had outlined a drastic cost cutting plan, which would see 2,900 jobs cut by 2017.

The cuts include 1,700 ground staff, 900 cabin crew and 300 pilots, as part of efforts to lower costs, two union sources said.

Air France also confirmed in the meeting that it plans to shed 14 aircraft from its long-haul fleet, reducing the business by ten per cent, and that it wants to cancel its order for Boeing 787 Dreamliner aircraft.

This outraged staff, who are already at loggerheads with the company, and hundreds stormed the building, interrupting the meeting.

Mr Broseta and Mr Plissonnier were aided by security as they tried to escape the baying mob
 Published time: 25 Sep, 2015 12:20
© Seven LE DUC
The French animal rights group, Cause Animal Nord, has come under fire after a video emerged of the activists taking away a puppy from a crying homeless man in central Paris.

The video shows the man fighting for his dog, but eventually losing out as three members of the group, including the organization’s president, seized the puppy and ran away. The homeless man was left in tears.

The cruel act has been condemned by the media and internet users. A number took to social networks to express their disgust, while others left highly critical messages on the organization’s Facebook page.

View image on Twitter

3 French animal rights activists steal puppy from homeless man, and put it up for adoption under the name “Vegan”:h…

SARS: French scientists lose 2,300 samples of potentially deadly virus

Institut Pasteur realised tubes were missing during a routine inventory check. Pictured posed.
Institut Pasteur realized tubes were missing during a routine inventory check. Pictured posed.

 

The renowned Institut Pasteur in France has admitted that it has misplaced 2,349 vials containing samples of the potentially deadly SARS virus and, despite enlisting help from France’s drug and health safety agency, have been unable to find them.

 

The investigation by the ANSM at the unnamed laboratory failed to locate the samples, which have been missing since January. It has now filed a case to the prosecutor of Paris to investigate the disappearance.

The Institut Pasteur has been quick to reassure the public that the vials do not pose any risk, according to The Local.

“The tubes concerned have no infectious potential,” a statement said. “Independent experts referred by health authorities have qualified the risk as ‘nil’ in regards to available evidence and literature on the survival of the SAS virus.”

 

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Paris laboratory loses deadly SARS virus samples

©

Text by FRANCE 24

Latest update : 2014-04-16

The Pasteur Institute in Paris has asked the authorities to investigate the disappearance of more than 2,000 vials containing fragments of the SARS virus, while insisting that missing samples represent no danger to the public.

The institute said it discovered the loss of 29 boxes containing 2,349 tiny vials during a routine inventory check.

Professor Christian Bréchot, the head of the Pasteur Institute, said “human error” was the most likely explanation, but that they “did not want to rule anything out.”
“From the start, we’ve known that the samples are harmless,’’ he said.
Severe acute respiratory syndrome (SARS) is an airborne virus.  In a 2003 outbreak, it  spread to 30 countries infecting  8,273 people and causing a reported 775 deaths, the majority in Hong Kong, for a mortality rate of almost 10 per cent.
An outbreak of a SARS-like illness in 2013 caused a reported  40 cases worldwide, two of them in France, where a 65-year old man died from the illness.

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VOA

Syrian President Bashar al-Assad has no bigger ally than Iran.  So his government says it expects Iran will be invited to planned peace talks in Geneva “just like any other state.”

The international mediator to the conflict, Lakhdar Brahimi, agrees.

“Iran is a very important country in the region and they have to be present in a conference like this,” he said.

But while the United Nations is responsible for sending the invitations, Brahimi says he is working in consensus with the main organizing partners: Russia and the United States.

But the U.S. says Iran must first agree to the establishment of a transitional Syrian government by “mutual consent” which presumes that President Assad’s opponents would never agree to his joining an interim government and would thus end his rule.

  • Smoke rises from what activists said were explosive barrels thrown from helicopters on Daria outside Damascus, Jan. 12, 2014.

 

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The GIs who raped France: We know about the mass rape of German women by Stalin’s soldiers. Now a new book reveals American troops committed thousands of rapes on French women they were ‘liberating’

 

By Guy Walters

 

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Targets: Some American GIs saw French women as spoils of war according to an explosive new book

Targets: Some American GIs saw French women as spoils of war according to an explosive new book

 

The handsome American soldier was Elisabeth’s tenth client that evening. Working her trade on the top floor of a dingy apartment block in Paris, she felt that she had seen them all.

For the past four years, the men had been Germans, and now, since the city had been liberated in August, 1944, they were Americans. It made little difference.

Elisabeth held out three fingers of her hand to indicate the price of her body — three hundred francs.

‘Too much,’ said the soldier.

Elisabeth sighed. She had seen that before as well. Wearily, she kept the three fingers held up, almost as an insult.

There was no negotiation — three hundred was little enough as it was.

‘Two hundred,’ the soldier insisted.

‘Non,’ said Elisabeth. ‘Three hundred or nothing’.

The soldier approached her, hate in his eyes. Elisabeth glowered back, starting to feel scared.

‘In that case,’ said the soldier, ‘it will be nothing.’

The soldier then placed his huge hands around Elisabeth’s neck and started to squeeze. She struggled as hard as she could, lashing out, but it was in vain.

After a minute or so she slumped down, her lifeless body falling on to the stained sheets. The soldier then calmly removed his trousers and had sex with her. For nothing.

Afterwards, he went through Elisabeth’s belongings and stole her cash and jewellery. He then went round the block, found another prostitute and took her to dinner and the movies.

For the GI, it had been a swell evening. Paris was just as they said it was.

 

 

Even by the standards of war, this was a particularly grim episode. But while such barbaric murders were extremely rare, a new book reveals that the violation by American soldiers of the women whom they had been sent to Europe to free and assist was far more common than has first been thought.

It is, of course, a horrific fact of war that soldiers rape the women of the lands they conquer.

Many troops — but certainly not all — see female flesh as a justified spoil, something they deserve after fighting with the husbands, fathers and sons of the women they abuse.

Rape is also a way by which one nation signifies that it now has dominance over another.

Grateful: French women are seen waving American flags in Paris in August 1944 following the city's liberation, but the book alleges that thousands of French women were raped by GIs after freeing the city from the Nazis

Grateful: French women are seen waving American flags in Paris in August 1944 following the city’s liberation, but the book alleges that thousands of French women were raped by GIs after freeing the city from the Nazis

Welcome: A French woman embraces an American soldier as troops parade through Paris in August 1944

Welcome: A French woman embraces an American soldier as troops parade through Paris in August 1944

We can have your women, rape says, and there is nothing you can do because we are in charge.

Many thousands of German women and girls, for example, were raped by Russian troops in the battle for Berlin at the end of World War II.

Until now, we in the former Allied Western nations tend to regard rape as something carried out by countries other than ourselves.

Through films such as Saving Private Ryan and The Longest Day, we are conditioned to think of the Allied troops as being above such behaviour.

However, an explosive new book published by an American academic sensationally debunks that myth.

My book seeks to debunk an old myth about the GI, thought of as a manly creature that always behaved well — the GIs were having sex anywhere and everywhere.’

Professor Mary Louise Roberts

 

In What Soldiers Do, Professor Mary Louise Roberts of the University of Wisconsin argues that American GIs committed rape thousands of times during the War. And, more surprisingly still, many of their victims were French.

As Professor Roberts says: ‘My book seeks to debunk an old myth about the GI, thought of as a manly creature that always behaved well — the GIs were having sex anywhere and everywhere.’

In total, it is estimated that some 14,000 women were raped by American GIs in Western Europe from 1942 to 1945. In France, 152 American soldiers were tried for rape, of whom 29 were hanged.

But the statistics do not reveal the full story. There were undoubtedly thousands of rapes in France, many of which went unreported by the victims who were keen to avoid the dreadfully unfair stigma that rape carried with it during those days.

But why did the Americans rape their allies? For the average GI, France was as much an ‘erotic adventure’ as a military expedition, and the war was, in part, ‘sold’ to conscripted soldiers as an opportunity to meet attractive French women.

Many of the soldiers’ fathers had been in France during World War I, and had come back with lurid tales of the supposed looseness of French women.

All smiles: The population of Valognes welcoming US soldiers in 1944

All smiles: The population of Valognes welcoming US soldiers in 1944

Hotspot: The Channel port of Le Harve, pictured after a bombing raid during the war, was particularly badly affected by crime

Hotspot: The Channel port of Le Harve, pictured after a bombing raid during the war, was particularly badly affected by crime

Their sons, now off to fight in the same land, regarded France as essentially a giant brothel, with thousands of nubile French girls eager to be taken by manly GIs.

As Professor Roberts rightly observes, the average GI ‘had no emotional attachment to the French people or the cause of their freedom’.

Magazines aimed at the troops such as Stars And Stripes showed pictures of cheering women during liberation parades, accompanied by headlines such as ‘Here’s What We’re Fighting For’.

The magazine even published ‘useful’ French phrases, such as the translations for ‘I am not married’ and ‘You have charming eyes’.

It was almost as if the magazine was telling the GIs: come and get it, boys.

And that’s exactly what they did. Throughout the summer of 1944, from the moment they had pushed back the Germans during the D-Day landings in June, the Americans unleashed throughout northern France, in the words of Professor Roberts, a ‘tsunami of  male lust’.

‘Normandy women launched a wave of rape accusations against American soldiers,’ Roberts writes, ‘threatening to destroy the erotic fantasy at the heart of the operation. The spectre of rape transformed the GI from rescuer-warrior to violent intruder’.

Destroyed: To make matters worse the American troops were the same ones who had destroyed many French towns and cities in bombing campaigns that many thought was a display of US machoism

Destroyed: To make matters worse the American troops were the same ones who had destroyed many French towns and cities in bombing campaigns that many thought was a display of US machoism

Earth Watch Report  –  Epidemic  Hazards

10.05.2013 Epidemic Hazard France Nord-Pas-de-Calais, [Valenciennes, Douai and Tourcoing ] Damage level Details

Epidemic Hazard in France on Wednesday, 08 May, 2013 at 09:54 (09:54 AM) UTC.

Description
French health authorities say they have confirmed the country’s first case of a new respiratory virus related to SARS, in a traveler returning from the United Arab Emirates. The Health Ministry said in a statement Wednesday that the person is hospitalized in isolation and is under medical surveillance. It says Paris’ Pasteur Institute analyzed the virus and confirmed that it is a novel coronavirus, the first such case in France. The ministry gave no other details. Health Minister Marisol Touraine plans a news conference later Wednesday to elaborate. The new coronavirus, first identified last year, can cause acute pneumonia and kidney failure.
Biohazard name: nCOV (SARS-like virus)
Biohazard level: 4/4 Hazardous
Biohazard desc.: Viruses and bacteria that cause severe to fatal disease in humans, and for which vaccines or other treatments are not available, such as Bolivian and Argentine hemorrhagic fevers, H5N1(bird flu), Dengue hemorrhagic fever, Marburg virus, Ebola virus, hantaviruses, Lassa fever, Crimean-Congo hemorrhagic fever, and other hemorrhagic or unidentified diseases. When dealing with biological hazards at this level the use of a Hazmat suit and a self-contained oxygen supply is mandatory. The entrance and exit of a Level Four biolab will contain multiple showers, a vacuum room, an ultraviolet light room, autonomous detection system, and other safety precautions designed to destroy all traces of the biohazard. Multiple airlocks are employed and are electronically secured to prevent both doors opening at the same time. All air and water service going to and coming from a Biosafety Level 4 (P4) lab will undergo similar decontamination procedures to eliminate the possibility of an accidental release.
Symptoms:
Status: confirmed

Epidemic Hazard in France on Wednesday, 08 May, 2013 at 09:54 (09:54 AM) UTC.

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Updated: Wednesday, 08 May, 2013 at 13:08 UTC
Description
A 65-year-old Frenchman is hospitalized after contracting France’s first case of a deadly new respiratory virus related to SARS, and French health authorities said Wednesday they are trying to find anyone who might have been in contact with him to prevent it from spreading. It’s unclear how or where the man was infected with the novel coronavirus, which has killed 18 people in four countries and raised new public health concerns since being identified last year in the Middle East. It can cause acute pneumonia and kidney failure. The Frenchman fell ill after returning from a trip to the United Arab Emirates, the Health Ministry said. He has been under isolation and medical surveillance at a hospital in Douai in northern France since April 23, and is receiving respiratory assistance and blood transfusions, said Jean-Yves Grall, the government health director. Paris’ Pasteur Institute analyzed the man’s virus and confirmed that it is a novel coronavirus, the ministry announced Wednesday. France’s health minister, Marisol Touraine, said “this is an isolated case” in France but said authorities are “fully mobilized” to prevent it from spreading. Authorities are trying to reach anyone who was in contact with the patient before he was hospitalized, and a national hotline was established Wednesday for the public to call about the virus. WHO has advised countries to test any people with unexplained pneumonia.

Epidemic Hazard in France on Wednesday, 08 May, 2013 at 09:54 (09:54 AM) UTC.

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Updated: Thursday, 09 May, 2013 at 02:50 UTC
Description
The latest novel coronavirus case (NcoV), a Sars-like virus, has been linked to the UAE. According to international newswires on Wednesday, France confirmed its first case of a Sars-like virus in a traveller returning from the UAE. The report stated that the Paris-based Pasteur Institute, a leading biomedical research organisation, analysed the virus and confirmed that it was a novel coronavirus. According to the French Health Ministry statement, the person is in isolation in hospital and is under medical surveillance in an undisclosed location.

Epidemic Hazard in France on Wednesday, 08 May, 2013 at 09:54 (09:54 AM) UTC.

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Updated: Friday, 10 May, 2013 at 02:45 UTC
Description
Two people who had contact with a Frenchman who is seriously ill with the new SARS-like coronavirus have fallen sick and been admitted to hospital, health officials in northern France said on Thursday (9 May 2013). One is a patient who shared a ward with the 65-year-old man infected with the virus when he was in a hospital in the town of Valenciennes, northern France, at the end of April (2013), and the other is a doctor who treated him there. The 65-year-old carrier, who fell ill on his return from a trip to Dubai, has since been transferred to an isolated intensive care wing in a hospital in Douai, near the northern city of Lille, where he is in a critical condition. The ARS local health authority said the 2 other men were in individual rooms in separate hospitals, one in Lille and the other in the nearby town of Tourcoing and that tests had been carried out on both of them.”They show symptoms which require a special infectious diseases consultation,” the ARS said in a statement. “The results of the tests carried out on these 2 people will be known soon and will be made public.” As France reported the 65-year-old as its 1st case of the coronavirus on Wednesday (8 May 2013), the World Health Organization said it would send experts to visit a Saudi hospital from which the virus has spread, killing 7 people so far. The French case brought the total number of known infections worldwide to 31, of which 18 resulted in death. Coronavirus is from the same viral family that triggered the outbreak of severe acute respiratory syndrome (SARS) that swept the world from Asia in late 2003, killing 775 people. Despite there being no evidence so far of sustained human-to-human transmission, health experts’ concerns are growing over clusters of new cases.

Epidemic Hazard in France on Wednesday, 08 May, 2013 at 09:54 (09:54 AM) UTC.

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Updated: Friday, 10 May, 2013 at 12:07 UTC
Description
A Frenchman diagnosed with a new, deadly SARS-like virus known as coronavirus that has killed 18 people may have infected two other people, authorities in northern France said Thursday. French health authorities said Thursday they feared the country’s first case of a new SARS-like virus that has killed 18 people, mostly in Saudi Arabia, may have infected two other people. The 65-year-old man who came back to France from a holiday in Dubai was diagnosed with the deadly novel coronavirus, and is in intensive care in a hospital in the northern city of Douai, the health ministry said Wednesday. “This is the first and only confirmed case in France to date,” it added. But on Thursday the health authorities in the Nord/Pas-de-Calais region said two more people were undergoing tests after showing symptoms of the virus. They were a man who had shared a room in hospital with the patient, and a doctor who treated him.The unnamed patient, who was in Dubai from April 9 to 17, has been placed in isolation and is being given respiratory assistance and blood transfusions. The virus, known as nCoV-EMC, is a cousin of Severe Acute Respiratory Syndrome (SARS), which triggered a scare 10 years ago when it erupted in east Asia, leaping to humans from animal hosts and killing some 800 people. It was first detected in September 2012 and since then more than 30 cases have been reported in different countries, with 18 deaths. But the French health ministry says it does not appear to be very contagious. While it has been deadliest in Saudi Arabia, where 11 people have been killed by the virus, other cases have been reported in Jordan, Germany, Britain and now France. The World Health Organisation on Thursday reported two new confirmed cases in Saudi Arabia.