Manuela Malatesta

Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize.

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.

Ultrastructural analysis of testes from mice fed on genetically modified soybean.

We have considered the possible effects of a diet containing genetically modified (GM) soybean on mouse testis. This organ, in fact, is a well known bioindicator and it has already been utilized, for instance, to monitor pollution by heavy metals. In this preliminary study, we have focussed our attention on Sertoli cells, spermatogonia and spermatocytes by means of immunoelectron microscopy. Our results point out that the immunolabelling for Sm antigen, hnRNPs, SC35 and RNA Polymerase II is decreased in 2 and 5 month-old GM-fed mice, and is restored to normal at 8 months. In GM-fed mice of all ages considered, the number of perichromatin granules is higher and the nuclear pore density lower. Moreover, we found enlargements in the smooth endoplasmic reticulum in GM-fed mice Sertoli cells. A possible role played by traces of the herbicide to which the soybean is resistant is discussed.

Ultrastructural analysis of pancreatic acinar cells from mice fed on genetically modified soybean

No direct evidence that genetically modified (GM) food may represent a possible danger for health has been reported so far; however, the scientific literature in this field is quite poor. Therefore, we investigated the possible effects of a diet containing GM soybean on mouse exocrine pancreas by means of ultrastructural, morphometrical and immunocytochemical analyses. Our observations demonstrate that, although no structural modification occurs in pancreatic acinar cells of mice fed on GM soybean, quantitative changes of some cellular constituents take place in comparison to control animals. In particular, a diet containing significant amount of GM food seems to influence the zymogen synthesis and processing.

A long-term study on female mice fed on a genetically modified soybean: effects on liver ageing

Liver represents a suitable model for monitoring the effects of a diet, due to its key role in controlling the whole metabolism. Although no direct evidence has been reported so far that genetically modified (GM) food may affect health, previous studies on hepatocytes from young female mice fed on GM soybean demonstrated nuclear modifications involving transcription and splicing pathways. In this study, the effects of this diet were studied on liver of old female mice in order to elucidate possible interference with ageing. The morpho-functional characteristics of the liver of 24-month-old mice, fed from weaning on control or GM soybean, were investigated by combining a proteomic approach with ultrastructural, morphometrical and immunoelectron microscopical analyses. Several proteins belonging to hepatocyte metabolism, stress response, calcium signalling and mitochondria were differentially expressed in GM-fed mice, indicating a more marked expression of senescence markers in comparison to controls. Moreover, hepatocytes of GM-fed mice showed mitochondrial and nuclear modifications indicative of reduced metabolic rate. This study demonstrates that GM soybean intake can influence some liver features during ageing and, although the mechanisms remain unknown, underlines the importance to investigate the long-term consequences of GM-diets and the potential synergistic effects with ageing, xenobiotics and/or stress conditions.

Fine structural analyses of pancreatic acinar cell nuclei from mice fed on genetically modified soybean

We carried out ultrastructural morphometrical and immunocytochemical analyses on pancreatic acinar cell nuclei from mice fed on genetically modified (GM) soybean, in order to investigate possible structural and molecular modifications of nucleoplasmic and nucleolar constituents. We found a significant lowering of nucleoplasmic and nucleolar splicing factors as well as a perichromatin granule accumulation in GM-fed mice, suggestive of reduced post-transcriptional hnRNA processing and/or nuclear export. This is in accordance to already described zymogen synthesis and processing modifications in the same animals.

Reversibility of hepatocyte nuclear modifications in mice fed on genetically modified soybean

In the literature, the reports on the effects of a genetically modified (GM) diet are scanty and heterogeneous; in particular, no direct evidence has so far been reported that GM food may affect human or animal health. Hepatocytes represent a suitable model for monitoring the effects of a GM diet, the liver potentially being a primary target. In a previous study, we demonstrated that some modifications occur in hepatocyte nuclei of mice fed on GM soybean. In order to elucidate whether such modifications can be reversed, in the present study, 3 months old mice fed on GM soybean since their weaning were submitted to a diet containing wild type soybean, for one month. In parallel, to investigate the influence of GM soybean on adult individuals, mice fed on wild type soybean were changed to a GM diet, for the same time. Using immunoelectron microscopy, we demonstrated that a one-month diet reversion can influence some nuclear features in adult mice, restoring typical characteristics of controls in GM-fed animals, and inducing in control mice modifications similar to those observed in animals fed on GM soybean from weaning. This suggests that the modifications related to GM soybean are potentially reversible, but also that some modifications are inducible in adult organisms in a short time.

Answers to critics: Why there is a long term toxicity due to a Roundup- tolerant genetically modified maize and to a Roundup herbicide

Our recent work (Séralini et al., 2012) remains to date the most detailed study involving the life-long consumption of an agricultural genetically modified organism (GMO). This is true especially for NK603 maize for which only a 90-day test for commercial release was previously conducted using the same rat strain (Hammond et al., 2004). It is also the first long term detailed research on mammals exposed to a highly diluted pesticide in its total formulation with adjuvants. This may explain why 75% of our first criticisms arising within a week, among publishing authors, come from plant biologists, some developing patents on GMOs, even if it was a toxicological paper on mammals, and from Monsanto Company who owns both the NK603 GM maize and Roundup herbicide (R). Our study has limits like any one, and here we carefully answer to all criticisms from agencies, consultants and scientists, that were sent to the Editor or to ourselves. At this level, a full debate is biased if the toxicity tests on mammals of NK603 and R obtained by Monsanto Company remain confidential and thus unavailable in an electronic format for the whole scientific community to conduct independent scrutiny of the raw data. In our article, the conclusions of long-term NK603 and Roundup toxicities came from the statistically highly discriminant findings at the biochemical level in treated groups in comparison to controls, because these findings do correspond in an blinded analysis to the pathologies observed in organs, that were in turn linked to the deaths by anatomopathologists. GM NK603 and R cannot be regarded as safe to date.

Hepatoma tissue culture (HTC) cells as a model for investigating the effects of low concentrations of herbicide on cell structure and function

Previous studies on mice fed genetically modified (GM) soybean demonstrated modifications of the mitochondrial functions and of the transcription/splicing pathways in hepatocytes. The cause(s) of these alterations could not be conclusively established but, since the GM soybean used is tolerant to glyphosate and was treated with the glyphosate-containing herbicide Roundup , the possibility exists that the effects observed may be due to herbicide residues. In order to verify this hypothesis, we treated HTC cells with 1-10mM Roundup and analysed cellular features by flow cytometry, fluorescence and electron microscopy. Under these experimental conditions, the death rate and the general morphology of HTC cells were not affected, as well as most of the cytoplasmic organelles. However, in HTC-treated cells, lysosome density increased and mitochondrial membranes modified indicating a decline in the respiratory activity. Moreover, nuclei underwent morpho-functional modifications suggestive of a decreased transcriptional/splicing activity. Although we cannot exclude that other factors than the presence of the herbicide residues could be responsible for the cellular modifications described in GM-fed mice, the concordance of the effects induced by low concentrations of Roundup on HTC cells suggests that the presence of Roundup residues could be one of the factors interfering with multiple metabolic pathways.

Biochemical and Ultrastructural Features of Human Milk and Nipple Aspirate Fluids

Breast duct epithelium produces, secretes, and metabolises several biologically important compounds, which are found in breast secretions obtained in physiologic and pathologic conditions (milk and nipple aspirate fluids, respectively). In order to preliminarily evaluate the ultrastructural morphology of the cells found in Type II nipple aspirate fluids (NAF) and correlate it with the biochemical profile of the extracellular fluid present in these breast secretions and in human milk, we analyzed 72 NAFs from nonlactating premenopausal women affected by various breast diseases and 10 normal milk samples. Although several constitutive proteins were detected in all samples examined, the preliminary biochemical analyses and electrophoretic profiles revealed characteristic behaviours for several biologic constituents, suggesting a possible basic mechanism of production by breast epithelial cells during both physiologic and pathologic conditions. The ultrastructural analysis of milk cellular components give preliminary evidence of the apocrine secretion mechanism peculiar of breast gland, whereas Type II NAF cells appeared as biosynthetically active cells, showing a possible modified secretion mechanism. Our multidisciplinary approach seems to support the hypothesis that cellular and biochemical behaviour of Type II NAF may be an useful tool to identify aberrated breast epithelial cells in nonlactating women that might be prone to premalignant transformation. J. Clin. Lab. Anal. 14:330–335, 2000.

DADLE induces a reversible hibernation-like state in HeLa cells.

Abstract[d-Ala(2)-d-Leu(5)-Enkephalin] (DADLE) can induce hibernation when injected into ground squirrels in summer and is able to increase the survival time of explanted organs such as liver and lung. Since cell metabolism is a target of this peptide, we have treated HeLa cells with DADLE and investigated its possible effect on transcription and proliferation as well as the resumption of metabolic activity after treatment. The labelling for Pol I, Pol II and for splicing factors such as snRNPs and SC-35 decreased after treatment as did the nucleolar labelling for UBF. In treated cells, several spherical nuclear bodies were found to be labelled for hnRNPs. In parallel, the number of proliferating cells decreased after treatment with DADLE. After recovery, there was a gradual resumption of cell function: transcription and splicing factors had a distribution similar to that of controls; proliferation resumed; nuclear bodies, representing storage sites for RNPs, disappeared.