https://orcid.org/0000-0003-1732-4741
Abstract
We describe a comprehensive repository describing a collection of data from a range of studies investigating the molecular mechanisms of toxicity of glyphosate, the glyphosate-based herbicide commercial formulation Roundup, and a mixture of glyphosate and 5 other most frequently used pesticides (azoxystrobin, boscalid, chlorpyrifos, imidacloprid and thiabendazole) present as residues in food products in Europe. The data were obtained by analysing tissues from rats exposed to the pesticides for 90 days via drinking water. The administration of the mixture of six pesticides was chosen to mimic a possible human exposure scenario. We compared conventional methods used in regulatory toxicity studies to evaluate the safety of pesticide exposure (gross pathology, serum biochemistry) to new molecular profiling methods encompassing the analysis of the caecum and blood metabolome, liver transcriptome, liver DNA methylation, liver small RNA profiles, and caecum metagenome of the exposed animals. Altogether, these investigations provided in-depth molecular profiling in laboratory animals exposed to pesticides revealing metabolic perturbations that would remain undetected by standard regulatory biochemical measures. Our results highlight how multi-omics phenotyping can be used to improve the predictability of health risk assessment from exposure to toxic chemicals to better protect public health.
Introduction
Herbicides containing glyphosate as an active principle are the most widely used pesticides in the world (Benbrook Citation2016). An increasing number of toxicological studies report adverse effects from glyphosate exposure at doses considered to be safe in regulatory tests (Mesnage and Zaller Citation2021a). Besides glyphosate, five other most frequently detected pesticide residues in human food products marketed in Europe are azoxystrobin, boscalid, chlorpyrifos, imidacloprid and thiabenzadole (EFSA Citation2019).
No data are currently available in the literature concerning the possible synergistic effects of these pesticides, administered as a mixture, in particular at doses which are considered to be safe by regulatory agencies. There has been only one chronic toxicity study has been published where rats were exposed to drinking water containing Roundup Grand Travaux Plus at a glyphosate concentration permitted for human consumption, and which showed marked alterations of the transcriptomic profile of liver and kidneys (Mesnage et al. Citation2015), and changes in proteome and metabolome profiles in blood, liver and kidneys reflective of non-alcoholic fatty liver disease and its progression to non-alcoholic steatohepatitis (Mesnage et al. Citation2017).
The present project aimed to investigate the molecular mechanisms by which glyphosate, a representative European Union (EU) Roundup formulation and a mixture of glyphosate and 5 other most frequent detected pesticides present food products in Europe (azoxystrobin, boscalid, chlorpyrifos, imidacloprid and thiabendazole) could be toxic in rats exposed for 90 days via drinking water. The administration of the mixture of six pesticides was chosen to mimic a possible human exposure scenario with animals receiving the EU acceptable daily intake for each substance. We compared conventional methods used in regulatory toxicity studies to evaluate the safety of pesticide exposure (gross pathology, serum biochemistry) to new molecular profiling methods encompassing the analysis of the caecum and blood metabolome, liver transcriptome, liver DNA methylation, liver small RNA profiles, and caecum metagenome of the exposed animals.
Materials and methods
The experiment was approved by the ad hoc commission of the Italian Ministry of Health (authorization N. 447/2018-PR). The experiment was conducted as previously described (Mesnage and Zaller Citation2021). Female Sprague–Dawley rats were exposed for 90 days to glyphosate or Roundup Bioflow MON 52276 (at 0.5, 50, and 175 mg/kg bw/day glyphosate), or to the pesticide mixture in tap drinking water and compared to an unexposed control group. Body weight, water and food consumption of experimental animals were measured before the start of the treatment and then weekly for 90 days. The details for each of the omics analyses can be found in the publications describing the effects of the pesticide mixture (Mesnage et al. Citation2021b), or glyphosate and MON 52276 on either the gut microbiota and serum metabolome (Mesnage et al. Citation2021c) or the liver molecular profiling (Mesnage et al. Citation2022). Below is a brief description of the methods employed.
For RNA-seq transcriptomics, the mRNA libraries were prepared using the NEBNext® Poly(A) mRNA Magnetic Isolation Module in combination with the NEBNext® Ultra™ II Directional RNA Library Prep Kit and indexed with NEBNext® Multiplex Oligos for Illumina® (96 Index Primers) (New England Biolabs, Ipswich, Massachusetts, USA). We performed the small RNA profiling using the QIAseq miRNA library kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. For Reduced Representation Bisulfite Sequencing, the Premium Reduced Representation Bisulfite Sequencing (RRBS) Kit (Diagenode) was used as per the manufacturer’s instructions. All libraries were of good quality and reads were generated using the Illumina NextSeq®500 v2.5 High-output 150 cycle kit. Metabolomics analysis was conducted on four independent instrument platforms: two different separate reverse phase ultrahigh performance liquid chromatography-tandem mass spectroscopy analysis (RP/UPLC-MS/MS) with positive ion mode electrospray ionization (ESI), a RP/UPLC-MS/MS with negative ion mode ESI, as well as by hydrophilic-interaction chromatography/UPLC-MS/MS with negative ion mode ESI. Shotgun metagenomics analysis of the gut microbiome was performed using the NEBNext® Ultra II FS DNA module and the NEBNext® Ultra II Ligation module before the libraries were sequenced on the NovaSeq6000 platform according to manufacturer’s protocols.
Supplemental Material
Download MS Excel (36.9 KB)Contributions statement
M.N.A. and R.M. conceived the study and drafted the manuscript.
Disclosure statement
RM has served as a consultant on glyphosate risk assessment issues as part of litigation in the US over glyphosate health effects.
Data availability statement
The results constitute a unique set of multiple omics datasets (transcriptomics, small RNA profiling, liver DNA methylation, metabolomics, the shotgun metagenomics) for which all the raw data was made available in public repositories (Table ). Body weight, water and food consumption of experimental animals, as well as liver and kidney histopathology, serum biochemistry was provided as a supplementary file.
Table 1. Summary of the datasets available for the different treatment groups.
Additional information
Funding
References
- Benbrook CM. 2016. Trends in glyphosate herbicide use in the United States and globally. Environ Sci Eur. 28(1):3.
- EFSA. 2019. The 2017 European Union report on pesticide residues in food. EFSA J. 17:e05743.
- Mesnage R, Arno M, Costanzo M, Malatesta M, Séralini GE, Antoniou MN. 2015. Transcriptome profile analysis reflects rat liver and kidney damage following chronic ultra-low dose Roundup exposure. Environ Health. 14:70.
- Mesnage R, Mandrioli D, Falcioni L, Belpoggi F, Brandsma I, Bourne B, Savage E, Mein CA, Antoniou MN. 2022. Comparative toxicogenomics of glyphosate and Roundup herbicides by mammalian stem cell-based genotoxicity assays and molecular profiling in Sprague-Dawley rats. Toxicol Sci. 186:83–101.
- Mesnage R, Renney G, Seralini GE, Ward M, Antoniou MN. 2017. Multiomics reveal non-alcoholic fatty liver disease in rats following chronic exposure to an ultra-low dose of Roundup herbicide. Sci Rep. 7:39328.
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- Mesnage R, Zaller JG, eds. 2021. Herbicides: chemistry, efficacy, toxicology, and environmental impacts. Emerging issues in analytical chemistry. Cambridge, MA: Elsevier. https://www.elsevier.com/books/herbicides/mesnage/978-0-12-823674-1