Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remains controversial. As GBHs are more cytotoxic that glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs namely MON 52276 (EU), MON 76473 (UK) and MON 76207 (USA) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (both at 0.5, 50, 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased mir-30 while miR-10 levels were increased. DNA methylation profiling of liver revealed 5,727 and 4,496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate.

Health effects of pesticides are not always accurately detected using the current battery of regulatory toxicity tests. We compared standard histopathology and serum biochemistry measures and multi-omics analyses in a subchronic toxicity test of a mixture of six pesticides frequently detected in foodstuffs (azoxystrobin, boscalid, chlorpyrifos, glyphosate, imidacloprid and thiabendazole) in Sprague-Dawley rats. Analysis of water and feed consumption, body weight, histopathology and serum biochemistry showed little effect. Contrastingly, serum and caecum metabolomics revealed that nicotinamide and tryptophan metabolism were affected, which suggested activation of an oxidative stress response. This was not reflected by gut microbial community composition changes evaluated by shotgun metagenomics. Transcriptomics of the liver showed that 257 genes had their expression changed. Gene functions affected included the regulation of response to steroid hormones and the activation of stress response pathways. Genome-wide DNA methylation analysis of the same liver samples showed that 4,255 CpG sites were differentially methylated. Overall, we demonstrated that in-depth molecular profiling in laboratory animals exposed to low concentrations of pesticides allows the detection of metabolic perturbations that would remain undetected by standard regulatory biochemical measures and which could thus improve the predictability of health risks from exposure to chemical pollutants.

Background. Health effects from exposure to glyphosate-based herbicides is an intense matter of debate. Toxicity including genotoxicity of glyphosate alone has been repeatedly tested over the last 40 years. Contrastingly, few studies have conducted comparative investigations between glyphosate and its commercial herbicide formulations, such as Roundup. We thus performed the first in-depth comparative toxicogenomic evaluation of glyphosate and a typical European Union Roundup formulation by determining alterations in transcriptome and epigenome profiles. Methods. Glyphosate and the European Union reference commercial formulation Roundup MON 52276 (both at 0.5, 50, 175 mg/kg bw/day glyphosate equivalent concentration) were administered to rats in a subchronic 90-day toxicity study. Standard clinical biochemistry and kidney and liver histopathology was performed. In addition, transcriptomics and DNA methylation profiling of liver and selective gene expression analysis of kidneys was conducted. Furthermore, a panel of six mouse embryonic reporter stem cell lines validated to identify carcinogenic outcomes (DNA damage, oxidative stress, and protein misfolding) were used to provide insight into the mechanisms underlying the toxicity of glyphosate and 3 Roundup formulations. Results. Histopathology and serum biochemistry analysis showed that MON 52276 but not glyphosate treatment was associated with a statistically significant increase in hepatic steatosis and necrosis. Similar lesions were also present in the liver of glyphosate-treated groups but not in the control group. MON 52276 altered the expression of 96 genes in liver, with the most affected biological functions being TP53 activation by DNA damage and oxidative stress as well as the regulation of circadian rhythms. The most affected genes in liver also had their expression similarly altered in kidneys. DNA methylation profiling of liver revealed 5,727 and 4,496 differentially methylated CpG sites between the control group and the group of rats exposed to glyphosate and MON 52276, respectively. Direct DNA damage measurement by apurinic/apyrimidinic lesion formation in liver was increased with glyphosate exposure. Mechanistic evaluations showed that two Roundup herbicides but not glyphosate activated oxidative stress and misfolded protein responses. Conclusions. Taken together, the results of our study show that Roundup herbicides are more toxic than glyphosate, activating mechanisms involved in cellular carcinogenesis and causing gene expression changes reflecting DNA damage. This further highlights the power of high-throughput omics methods to detect metabolic changes, which would be missed by relying solely on conventional biochemical and histopathological measurements. Our study paves the way for future investigations by reporting a panel of gene expression changes and DNA methylation sites, which can serve as biomarkers and potential predictors of negative health outcomes resulting from exposure to glyphosate-based herbicides.