Comparative toxicogenomics of glyphosate and Roundup herbicides by mammalian stem cell-based genotoxicity assays and molecular profiling in Sprague-Dawley rats

Abstract

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.

Single-cell transcriptome analysis reveals liver injury induced by glyphosate in mice

“…A similar effect of GLY in causing oxidative stress has been reported in the SH-SY5Y cell line [41] and rat liver [17]. Mesnage et al observed activated oxidative stress (reflected by Srxn1 and Blvrb) in mammalian stem cell-based ToxTracker system by glyphosatebased herbicides but not glyphosate itself [23], In our study, the expression level of Srxn1 and Blvrb (Fig. 3G and Additional file 2: Table S9) was improved after GLY exposure.…”

“…In addition, upregulation of genes involved in ribonucleoprotein complex assembly and ribonucleoprotein complex subunit organization in both Hep1 and Hep2 suggests potential DNA damage of GLY (Fig. 3E), which has been reported previously [23]. Overall, GLY may cause oxidative stress, abnormal lipid metabolism, inflammatory response, and DNA damage, suggesting GLY-induced dysregulation of hepatocytes.…”

“…Jia et al reported expression level changes of genes involved in ion transport, lipid metabolism, and peroxisome proliferator-activated receptor signaling pathway in livers of tilapia by transcriptomic analysis [67]. Another study from Mesnage et al utilized multiple omics including transcriptomics showing that GLY-based herbicide MON 52276 increased hepatic steatosis and necrosis and both MON 52276 and glyphosate induced oxidative damage to DNA in rat livers [23]. Although providing valuable information in reflecting the toxic effects of GLY on the liver, these studies explored the transcriptomic changes as a whole without differentiating specific cell types.…”

“…GLY induced proliferation of T47D cells, a human hormone-dependent breast cancer cell line [20]. Besides, the adjuvants used to enhance water solubility and plant absorption of GLY are often detected in the environment and induce cell toxicity [21][22][23]. It was not quite clear how GLY degrades in mammals, but its metabolites such as aminomethylphosphonic acid (AMPA) were detected after GLY exposure in animals [24][25][26] and humans [27].…”