Robin Mesnage; George Renney; Gilles-Eric Séralini; Malcolm Ward; Michael N. Antoniou

“ In the liver of rats administered with Roundup, a trend in the accumulation of most fatty acids, in particular acylcarnitines, as well as a statistically significant elevation of cholesterol levels was observed (Table 3) . Proteome profiles further confirmed a marked change in lipid detoxifying metabolic processes (Table 1, Fig. 5) . In the pathology of NASH, oxidative stress acts as a second hit [31] , leading to lipid peroxidation, mitochondrial damage, hepatocellular injury, and finally chronic inflammation and fibrosis. ”

“ Previously we have shown that rats administered for 2 years with 0.1 ppb (50 ng/L glyphosate equivalent dilution; 4 ng/kg body weight/day daily intake) of a Roundup GBH formulation showed signs of enhanced liver injury as indicated by anatomorphological, blood/urine biochemical changes and transcriptome profiling. Here we present a multiomic study combining metabolome and proteome liver analyses to obtain further insight into the Roundup-induced pathology. ”

“ In short term acute exposures, some adjuvants can be considered as responsible of Roundup toxicity [42] . Populations of farmers exposed to adjuvants, such as solvents or petroleum distillates, possess a higher risk of developing hypospadias [43] and present with more allergic and non-allergic wheeze conditions [44] . However, as adjuvant composition is proprietary and not fully disclosed, it is not possible to attribute the toxicity of the whole agricultural herbicide formulation to a given component. ”

“ Elevation in oxidative stress markers is detected in rat liver and kidney after subchronic exposure to GBH at the United States permitted glyphosate concentration of 700 μg/L in drinking water [13] . Hepatic histological changes and alterations of clinical biochemistry are detected in rats consuming 4.87 mg/kg body weight (bw) glyphosate every 2 days over 75 days [14] . In farm animals, elevated glyphosate urinary levels are correlated with alterations in blood serum parameters indicative of liver and kidney oxidative stress and depletion in nutrient trace element levels [15] . ”

“ Given the insight molecular profiling methods can potentially provide into processes and mechanisms of toxicity, we have previously conducted a transcriptomics investigation of the same female cohort of animals subjected to ultra-low dose Roundup exposure, and which showed signs of liver and kidney damage at a anatomorphological and blood/urine biochemical level of function [28] . Our previous results showed alterations in the liver transcriptome reflective of fibrosis, necrosis, phospholipidosis, mitochondrial membrane dysfunction and ischemia [29] . ”

“ Bioinformatics analysis For the proteome, only peptides with TMT reporter ion signal intensities for all ten samples were used for further bioinformatics analysis. Any duplicate peptides were removed before the data was SumScale normalised. The two normalised data files were then merged together to give one 10vs10 file comparison. Any peptides which did not have intensity values in all twenty TMT reporter ion channels were filtered out and median values were taken of the control and treated samples respectively. A total of 1906 peptides were quantified across all liver samples. ”

“ Control and Roundup-treated animals were respectively euthanized at 701+/− 62 and 635+/− 131 days. Anatomopathological analysis of organs from these animals revealed that the liver was the most affected organ [18] . Roundup-treated female rats showed 3 times more anatomical signs of pathology (15 in 8 rats) than the control group (6 in 4 rats) [18] . Blood samples were collected from the tail vein of each rat under short isoflurane anesthesia after 1, 2, 3, 6, 9, 12, 15, 18, 21 and 24 months of treatment. Serum biochemical analysis showed increased levels of serum triglycerides (Fig. 1) . ”

“ When importing raw SRM data files from the TSQ vantage into Skyline software, a unique peak picking algorithm was used to confidently and accurately assign the best transition peaks for each peptide. Peptides were only taken forward for analysis if all assigned peaks were within a similar retention time period across all 60 samples. From the assigned peaks Skyline software was then used to produce a ‘total ratio’ between the Light and Heavy transitions for all of the 60 raw data files (3 × 10 control sample injection and 3 × 10 treated sample injections) . ”

“ Although our results cannot provide insight into the mechanisms of the pathologies resulting from chronic very low–dose glyphosate exposure, they highlight the need for future GBH toxicity studies where organ molecular profiles are determined prior to appearance of the overt pathologies observed at late-stage termination as in this instance. Indeed, the use of aged animals may have enhanced variability. ”

“ Herbicides act mostly by inhibiting plant enzymes responsible of photosynthesis, carotenoid synthesis, or amino acid synthesis [19] . Besides its well known interaction with EPSPS, it has been suggested that glyphosate could impact mitochondrial function by inhibiting succinate dehydrogenase [20] , or on steroid biosynthesis by inhibiting aromatase enzyme activity [21] . Molecular profiling techniques can be used to identify specific signatures of chemical toxicity and thus provide greater insight into organ pathological status [22] [23] . ”

“ They are endogenous analogue of glutathione (GSH) considered as markers for GSH depletion under oxidative stress conditions. A depletion of GSH was detected but did not reach statistical significance (FC = −1.64, p = 0.09) . Gamma glutamyl dipeptides, indicators of the production of reduced glutathione, are increased in our study. Out of 10 gamma glutamyl dipeptides detected, 9 had their levels elevated (3 being significantly increased) . ”

“ However, many suffer from NAFLD but which do not have any high risk factors and thus other contributors to disease, such as exposure to physiologically active environmental pollutants via contaminated food, cannot be excluded. ”

“ Shehata, A. A.; Schrodl, W. & Rodloff, A. (2013) , Glyphosate suppresses the antagonistic effect of Enterococcus spp. on Clostridium botulinum, vol. 20, pp. 74–78, 23396248 KrugerM.ShehataA. A.SchrodlW.RodloffA., pmid:23396248 1 2 Feise, R. J. (2002) , Do multiple outcome measures require p-value adjustment?, vol. 2, p. 8, 12069695 FeiseR. J., pmid:12069695 ↑ Prospective Studies, C. (2007) , Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55 000 vascular deaths, vol. ”