Bon,
l'article est là.
Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize
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Abstract
The health effects of a Roundup-tolerant genetically modified maize (from 11% in the diet), cultivated with or without Roundup, and Roundup alone (from 0.1 ppb in water), were studied 2 years in rats. In females, all treated groups died 2–3 times more than controls, and more rapidly. This difference was visible in 3 male groups fed GMOs. All results were hormone and sex dependent, and the pathological profiles were comparable. Females developed large mammary tumors almost always more often than and before controls, the pituitary was the second most disabled organ; the sex hormonal balance was modified by GMO and Roundup treatments. In treated males, liver congestions and necrosis were 2.5–5.5 times higher. This pathology was confirmed by optic and transmission electron microscopy. Marked and severe kidney nephropathies were also generally 1.3–2.3 greater. Males presented 4 times more large palpable tumors than controls which occurred up to 600 days earlier. Biochemistry data confirmed very significant kidney chronic deficiencies; for all treatments and both sexes, 76% of the altered parameters were kidney related. These results can be explained by the non linear endocrine-disrupting effects of Roundup, but also by the overexpression of the transgene in the GMO and its metabolic consequences.
2. Materials and methods a écrit :2.2. Plants, diets and chemicals
The varieties of maize used in this study were the R-tolerant NK603 (Monsanto Corp., USA), and its nearest isogenic non-transgenic control. These two types of maize were grown under similar normal conditions, in the same location, spaced at a 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 glyphosate, EPA Reg. 524-537), and another field of NK603 was not treated with R. Corns 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 in 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 have 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 glyphosate, approval 2020448, Monsanto, Belgium). Herbicides levels were assessed by glyphosate measurements in the different dilutions by mass spectrometry.
2.3. 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 (45–65%) 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 moved. This 2 year life-long experiment was conducted in a GPL environment according to OECD guidelines. After 20 days of acclimatization, 100 male and 100 female animals were randomly assigned on a weight basis into 10 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 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 of R or 50 ng/L of glyphosate, the contaminating level of some regular tap waters), 0.09% of R (400 mg/kg, US MRL of glyphosate in some GM feed) and 0.5% of R (2.25 g/L, 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 that may arise, food and water consumption, and individual body weights.
2.6. Statistical analysis
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 (Eriksson et al., 2006b) 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 ( [Weljie et al., 2011] and [Wiklund et al., 2008]). 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 (Eriksson et al., 2006a). Prior to analysis, variables were mean-centered and unit variance scaled.
Fig. 1. Mortality of rats fed GMO treated or not with 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, 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 administrated in drinking water at 3 increasing doses, same symbols (environmental (A), MRL in agricultural GMOs (B) and half of minimal agricultural levels (C), see Section 2). 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 represented 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.
=> sous les histogrammes: 0=groupe contrôle. 11, 22, 33 sont les groupes avec 11%, 22% et 33% d'OGM dans leur nourriture. Idem lignes fines, moyennes et grasses sur les courbes. En plus de l'OGM, on leur a donné du round-up dans l'eau (groupes A,B,C, doses définies ci-dessus. En haut, OGM non traité, au milieu OGM traité, en bas Round-up sans OGM.
En grisé: rats morts. en noir, rats euthanasiés car trop grosses tumeurs => règles éthiques anti-souffrance.
Fig. 2. Largest non-regressive tumors in rats fed GMO treated or not by Roundup, and effects of Roundup alone. The symbols of curves and treatments are explained in the caption of Fig. 1. 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 largest tumors; white, small internal tumors; grey, metastases.
Fig. 5. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) for biochemical data (females fed 33% GMO versus controls). (A) OPLS-DA regression coefficients for predictive component, with jack-knifed confidence intervals at 99% confidence level, indicate discriminant parameters versus controls at month 15 (Abbreviations: 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). (B) In this case, 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. Profiles evidence kidney ion leakages and sex hormonal imbalance versus controls.
For biochemical measurements in rats, statistical analysis was performed on the results obtained from 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). OPLS-DA 2-class models were 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 (Fig. 5A), when their regression coefficients were significant at 99% confidence level. Thus, in treated females, kidney failures appeared at the biochemical level (82% of the total disrupted parameters). Ions (Na, Cl) or urea increased in urine. Accordingly, the same ions decreased in serum (Fig. 5B) as did the levels of P, K and Ca. Creatinine or clairance 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.
3. Résultats a écrit :The lifespan of the control group of animals corresponded to the mean rat lifespan, but as is frequently the case with most mammals including humans (WHO, 2012), males on average died before females, except for some female treatment groups. All treatments in both sexes enhanced large tumor incidence by 2–3-fold in comparison to our controls but also for the number of mammary tumors in comparison to the same Harlan Sprague Dawley strain (Brix et al., 2005), and overall around 3-fold in comparison to the largest study with 1329 Sprague Dawley female rats (Chandra et al., 1992). In our study the tumors also developed considerably faster than the 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, underlining the inadequacy of the standard 90 day feeding trials for evaluating GM crop and food toxicity (Séralini et al., 2011).
=> la majorité des tumeurs est observées après 18 mois. Donc les tests industriels standards (90 jours= 3 mois) sont clairement à revoir.
The similar pathological profiles provoked by the GM maize containing R residues may thus be explained at least by R residues themselves, knowing that the medium dose of the R treatment corresponds to acceptable levels of this pesticide residues in GMOs.
Interestingly, in the groups of animals fed with the NK603 without R application, similar effects with respect to enhanced tumor incidence and mortality rates were observed.
=> tumeurs chez les rats alimentés aux OGM traités ou non, y compris aux niveau "résidu acceptable".
Et dans la conclusion:
[...]We propose that agricultural edible GMOs and formulated pesticides must be evaluated very carefully by long term studies to measure their potential toxic effects.
=> Demande de tests de long terme, qui montrent ce que cachaient les tests standards à 90 jours.
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je ne déforme pas le propos, j'en tire les conséquences logiques;
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