Republished study: long-term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize.

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.

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.

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.

Effects of Roundup(®) and glyphosate on three food microorganisms: Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus.

Use of many pesticide products poses the problem of their effects on environment and health. Amongst them, the effects of glyphosate with its adjuvants and its by-products are regularly discussed. The aim of the present study was to shed light on the real impact on biodiversity and ecosystems of Roundup(®), a major herbicide used worldwide, and the glyphosate it contains, by the study of their effects on growth and viability of microbial models, namely, on three food microorganisms (Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus) widely used as starters in traditional and industrial dairy technologies. The presented results evidence that Roundup(®) has an inhibitory effect on microbial growth and a microbicide effect at lower concentrations than those recommended in agriculture. Interestingly, glyphosate at these levels has no significant effect on the three studied microorganisms. Our work is consistent with previous studies which demonstrated that the toxic effect of glyphosate was amplified by its formulation adjuvants on different human cells and other eukaryotic models. Moreover, these results should be considered in the understanding of the loss of microbiodiversity and microbial concentration observed in raw milk for many years.

A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels.

The major herbicide used worldwide, Roundup, is a glyphosate-based pesticide with adjuvants. Glyphosate, its active ingredient in plants and its main metabolite (AMPA) are among the first contaminants of surface waters. Roundup is being used increasingly in particular on genetically modified plants grown for food and feed that contain its residues. Here we tested glyphosate and its formulation on mature rat fresh testicular cells from 1 to 10000ppm, thus from the range in some human urine and in environment to agricultural levels. We show that from 1 to 48h of Roundup exposure Leydig cells are damaged. Within 24-48h this formulation is also toxic on the other cells, mainly by necrosis, by contrast to glyphosate alone which is essentially toxic on Sertoli cells. Later, it also induces apoptosis at higher doses in germ cells and in Sertoli/germ cells co-cultures. At lower non toxic concentrations of Roundup and glyphosate (1ppm), the main endocrine disruption is a testosterone decrease by 35%. The pesticide has thus an endocrine impact at very low environmental doses, but only a high contamination appears to provoke an acute rat testicular toxicity. This does not anticipate the chronic toxicity which is insufficiently tested, and only with glyphosate in regulatory tests.

Defined plant extracts can protect human cells against combined xenobiotic effects.

BACKGROUND: Pollutants representative of common environmental contaminants induce intracellular toxicity in human cells, which is generally amplified in combinations. We wanted to test the common pathways of intoxication and detoxification in human embryonic and liver cell lines. We used various pollutants such as Roundup residues, Bisphenol-A and Atrazine, and five precise medicinal plant extracts called Circ1, Dig1, Dig2, Sp1, and Uro1 in order to understand whether specific molecular actions took place or not. METHODS: Kidney and liver are major detoxification organs. We have studied embryonic kidney and hepatic human cell lines E293 and HepG2. The intoxication was induced on the one hand by a formulation of one of the most common herbicides worldwide, Roundup 450 GT+ (glyphosate and specific adjuvants), and on the other hand by a mixture of Bisphenol-A and Atrazine, all found in surface waters, feed and food. The prevention and curative effects of plant extracts were also measured on mitochondrial succinate dehydrogenase activity, on the entry of radiolabelled glyphosate (in Roundup) in cells, and on cytochromes P450 1A2 and 3A4 as well as glutathione-S-transferase. RESULTS: Clear toxicities of pollutants were observed on both cell lines at very low sub-agricultural dilutions. The prevention of such phenomena took place within 48 h with the plant extracts tested, with success rates ranging between 25-34% for the E293 intoxicated by Roundup, and surprisingly up to 71% for the HepG2. By contrast, after intoxication, no plant extract was capable of restoring E293 viability within 48 h, however, two medicinal plant combinations did restore the Bisphenol-A/Atrazine intoxicated HepG2 up to 24-28%. The analysis of underlying mechanisms revealed that plant extracts were not capable of preventing radiolabelled glyphosate from entering cells; however Dig2 did restore the CYP1A2 activity disrupted by Roundup, and had only a mild preventive effect on the CYP3A4, and no effect on the glutathione S-transferase. CONCLUSIONS: Environmental pollutants have intracellular effects that can be prevented, or cured in part, by precise medicinal plant extracts in two human cell lines. This appears to be mediated at least in part by the cytochromes P450 modulation.

Debate on GMOs health risks after statistical findings in regulatory tests.

We summarize the major points of international debate on health risk studies for the main commercialized edible GMOs. These GMOs are soy, maize and oilseed rape designed to contain new pesticide residues since they have been modified to be herbicide-tolerant (mostly to Roundup) or to produce mutated Bt toxins. The debated alimentary chronic risks may come from unpredictable insertional mutagenesis effects, metabolic effects, or from the new pesticide residues. The most detailed regulatory tests on the GMOs are three-month long feeding trials of laboratory rats, which are biochemically assessed. The tests are not compulsory, and are not independently conducted. The test data and the corresponding results are kept in secret by the companies. Our previous analyses of regulatory raw data at these levels, taking the representative examples of three GM maize NK 603, MON 810, and MON 863 led us to conclude that hepatorenal toxicities were possible, and that longer testing was necessary. Our study was criticized by the company developing the GMOs in question and the regulatory bodies, mainly on the divergent biological interpretations of statistically significant biochemical and physiological effects. We present the scientific reasons for the crucially different biological interpretations and also highlight the shortcomings in the experimental protocols designed by the company. The debate implies an enormous responsibility towards public health and is essential due to nonexistent traceability or epidemiological studies in the GMO-producing countries.

Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines.

BACKGROUND: Worldwide used pesticides containing different adjuvants like Roundup formulations, which are glyphosate-based herbicides, can provoke some in vivo toxicity and in human cells. These pesticides are commonly found in the environment, surface waters and as food residues of Roundup tolerant genetically modified plants. In order to know their effects on cells from liver, a major detoxification organ, we have studied their mechanism of action and possible protection by precise medicinal plant extracts called Dig1. METHODS: The cytotoxicity pathways of four formulations of glyphosate-based herbicides were studied using human hepatic cell lines HepG2 and Hep3B, known models to study xenobiotic effects. We monitored mitochondrial succinate dehydrogenase activity and caspases 3/7 for cell mortality and protection by Dig1, as well as cytochromes P450 1A1, 1A2, 3A4 and 2C9 and glutathione-S-transferase to approach the mechanism of actions. RESULTS: All the four Roundup formulations provoke liver cell death, with adjuvants having stronger effects than glyphosate alone. Hep3B are 3-5 times more sensitive over 48 h. Caspases 3/7 are greatly activated in HepG2 by Roundup at non-cytotoxic levels, and some apoptosis induction by Roundup is possible together with necrosis. CYP3A4 is specifically enhanced by Roundup at doses 400 times less than used in agriculture (2%). CYP1A2 is increased to a lesser extent together with glutathione-S-transferase (GST) down-regulation. Dig 1, non cytotoxic and not inducing caspases by itself, is able to prevent Roundup-induced cell death in a time-dependant manner with an important efficiency of up to 89%, within 48 h. In addition, we evidenced that it prevents Caspases 3/7 activation and CYP3A4 enhancement, and not GST reduction, but in turn it slightly inhibited CYP2C9 when added before Roundup. CONCLUSION: Roundup is able to provoke intracellular disruption in hepatic cell lines at different levels, but a mixture of medicinal plant extracts Dig1 can protect to some extent human cell lines against this pollutants. All this system constitutes a tool for studying liver intoxication and detoxification.

Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines.

Glyphosate-based herbicides are the most widely used across the world; they are commercialized in different formulations. Their residues are frequent pollutants in the environment. In addition, these herbicides are spread on most eaten transgenic plants, modified to tolerate high levels of these compounds in their cells. Up to 400 ppm of their residues are accepted in some feed. We exposed human liver HepG2 cells, a well-known model to study xenobiotic toxicity, to four different formulations and to glyphosate, which is usually tested alone in chronic in vivo regulatory studies. We measured cytotoxicity with three assays (Alamar Blue, MTT, ToxiLight), plus genotoxicity (comet assay), anti-estrogenic (on ERalpha, ERbeta) and anti-androgenic effects (on AR) using gene reporter tests. We also checked androgen to estrogen conversion by aromatase activity and mRNA. All parameters were disrupted at sub-agricultural doses with all formulations within 24h. These effects were more dependent on the formulation than on the glyphosate concentration. First, we observed a human cell endocrine disruption from 0.5 ppm on the androgen receptor in MDA-MB453-kb2 cells for the most active formulation (R400), then from 2 ppm the transcriptional activities on both estrogen receptors were also inhibited on HepG2. Aromatase transcription and activity were disrupted from 10 ppm. Cytotoxic effects started at 10 ppm with Alamar Blue assay (the most sensitive), and DNA damages at 5 ppm. A real cell impact of glyphosate-based herbicides residues in food, feed or in the environment has thus to be considered, and their classifications as carcinogens/mutagens/reprotoxics is discussed.