. 2021 Jan;129(1):17005.

doi: 10.1289/EHP6990. Epub 2021 Jan 27.

Use of Shotgun Metagenomics and Metabolomics to Evaluate the Impact of Glyphosate or Roundup MON 52276 on the Gut Microbiota and Serum Metabolome of Sprague-Dawley Rats

Affiliations

¹ Gene Expression and Therapy Group, Department of Medical and Molecular Genetics, King's College London, Faculty of Life Sciences & Medicine, Guy's Hospital, London, UK.
² Unité de Recherche Aliments Bioprocédés Toxicologie Environnements, University of Caen Normandy, Caen, France.
³ Ramazzini Institute, Bologna, Italy.
⁴ Center for Microbiome Analyses and Therapeutics, Leiden University Medical Center, Leiden, Netherlands.
⁵ Mass Spectrometry Facility, King's College London, London, UK.

PMID: 33502259
PMCID: PMC7839352
DOI: 10.1289/EHP6990

Use of Shotgun Metagenomics and Metabolomics to Evaluate the Impact of Glyphosate or Roundup MON 52276 on the Gut Microbiota and Serum Metabolome of Sprague-Dawley Rats

Robin Mesnage et al. Environ Health Perspect. 2021 Jan.

. 2021 Jan;129(1):17005.

doi: 10.1289/EHP6990. Epub 2021 Jan 27.

Affiliations

¹ Gene Expression and Therapy Group, Department of Medical and Molecular Genetics, King's College London, Faculty of Life Sciences & Medicine, Guy's Hospital, London, UK.
² Unité de Recherche Aliments Bioprocédés Toxicologie Environnements, University of Caen Normandy, Caen, France.
³ Ramazzini Institute, Bologna, Italy.
⁴ Center for Microbiome Analyses and Therapeutics, Leiden University Medical Center, Leiden, Netherlands.
⁵ Mass Spectrometry Facility, King's College London, London, UK.

PMID: 33502259
PMCID: PMC7839352
DOI: 10.1289/EHP6990

Abstract

Background: There is intense debate on whether glyphosate can inhibit the shikimate pathway of gastrointestinal microorganisms, with potential health implications.

Objectives: We tested whether glyphosate or its representative EU herbicide formulation Roundup MON 52276 affects the rat gut microbiome.

Methods: We combined cecal microbiome shotgun metagenomics with serum and cecum metabolomics to assess the effects of glyphosate [0.5, 50, $175 mg / kg body weight (BW) per day$ ] or MON 52276 at the same glyphosate-equivalent doses, in a 90-d toxicity test in rats.

Results: Glyphosate and MON 52276 treatment resulted in ceca accumulation of shikimic acid and 3-dehydroshikimic acid, suggesting inhibition of 5-enolpyruvylshikimate-3-phosphate synthase of the shikimate pathway in the gut microbiome. Cysteinylglycine, $γ -glutamylglutamine$ , and valylglycine levels were elevated in the cecal microbiome following glyphosate and MON 52276 treatments. Altered cecum metabolites were not differentially expressed in serum, suggesting that the glyphosate and MON 52276 impact on gut microbial metabolism had limited consequences on physiological biochemistry. Serum metabolites differentially expressed with glyphosate treatment were associated with nicotinamide, branched-chain amino acid, methionine, cysteine, and taurine metabolism, indicative of a response to oxidative stress. MON 52276 had similar, but more pronounced, effects than glyphosate on the serum metabolome. Shotgun metagenomics of the cecum showed that treatment with glyphosate and MON 52276 resulted in higher levels of Eggerthella spp., Shinella zoogleoides, Acinetobacter johnsonii, and Akkermansia muciniphila. Shinella zoogleoides was higher only with MON 52276 exposure. In vitro culture assays with Lacticaseibacillus rhamnosus strains showed that Roundup GT plus inhibited growth at concentrations at which MON 52276 and glyphosate had no effect.

Discussion: Our study highlights the power of multi-omics approaches to investigate the toxic effects of pesticides. Multi-omics revealed that glyphosate and MON 52276 inhibited the shikimate pathway in the rat gut microbiome. Our findings could be used to develop biomarkers for epidemiological studies aimed at evaluating the effects of glyphosate herbicides on humans. https://doi.org/10.1289/EHP6990.

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Figures

Figure 1 is a flow chart showing the seven steps of the Shikimate Pathway. The Pentose Phosphate Pathway leads to D-erythrose 4-phosphate and Phosphoenolpyruvate. Step 1: D-erythrose 4-phosphate and Phosphoenolpyruvate lead to 3-deoxy-arabino-heptulosonate 7-phosphate. Step 2: 3-deoxy-arabino-heptulosonate 7-phosphate leads to 3-dehydroquinate. Step 3: 3-dehydroquinate leads to shikimate. Step 4: Shikimate leads to shikimate 3-phosphate. Step 5: Shikimate 3-phosphate with 5-enolpyruvylshikimate-3-phosphate synthase (which is blocked by glyphosate) leads to 5-enolpyruvoyl-shikimate 3-phosphate. Step 6: 5-enolpyruvoyl-shikimate 3-phosphate leads to chorismate. Step 7: Chorismate leads to tyrosine biosynthesis, phenylalanine biosynthesis, tryptophan biosynthesis, menaquinone biosynthesis, ubiquinone biosynthesis, pyocyanine biosynthesis, and biosynthesis of terpenoids and polyketides. — **Figure 1.**
The shikimate pathway and its associated biosynthesis pathways. Information about the biosynthetic pathway associated with the shikimate pathways metabolites were retrieved from the KEGG Orthology database. Note: EPSPS, enolpyruvylshikimate-3-phosphate synthase; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 3 is a set of fourteen error bar graphs plotting shikimate, ranging from negative 1.5 to 0.5 in increments of 0.5; solanidine, ranging from negative 0.4 to 0.0 in increments of 0.2; 3-dehydroshikimate, ranging from negative 1.0 to 0.5 in increments of 0.5; 2-isopropylmalate, ranging from negative 0.50 to 0.50 in increments of 0.25; glyphosate, ranging from negative 1 to 2 in increments of 0.1; linolenoylcarnitine, ranging from negative 0.5 to 0.0 in increments of 0.5; carotene diol, ranging from negative 0.4 to 0.2 in increments of 0.2; glutarate, ranging from negative 1.00 to 0.25 in increments of 0.25; pimelate, ranging from negative 0.50 to 0.25 in increments of 0.25; cysteinylglycine, ranging from negative 0.5 to 0.5 in increments of 0.5; prolyglycine, ranging from negative 0.50 to 0.50 in increments of 0.25; valylglycine, ranging from negative 0.8 to 0.4 in increments of 0.4; N-accetylputrescine, ranging from negative 0.4 to 0.2 in increments of 0.2; and OH-trimethyllysine, ranging from negative 0.2 to 0.2 (y-axis) across a (control), b [Glyphosate (0.5 milligrams per kilogram body weight per day)], c [Glyphosate (50 milligrams per kilogram body weight per day)], d [Glyphosate (175 milligrams per kilogram body weight per day)], e [MON 52276 (0.5 milligrams per kilogram body weight per day)], f [MON 52276 (50 milligrams per kilogram body weight per day)], and g [MON 52276 (175 milligrams per kilogram body weight per day)] (x-axis) for adjusted lowercase p equals 7.1E-32; 2.1E-14; 2.1E-14; 4.0E-02; 9.3E-22; 8.5E-08; 1.6E-7; 0.04; 0.04; 2.1E-06; 0.003; 0.03; 0.03; and 0.04, respectively. — **Figure 3.**
Differences in the level of cecum metabolites after exposure to glyphosate or Roundup MON 52276. Female Sprague-Dawley rats were administered via drinking water to 0.5, 50, and $175 mg / kg BW per day$ glyphosate and MON 52276 at the same glyphosate-equivalent dose for 90 d. A total of 14 metabolites had adjusted $p < 0.05$ ( $a d j - p$ ) according to an ANOVA adjusted for multiple comparisons with the FDR procedure. Log-transformed abundance values are shown as box plots with the median, two hinges (the 25th and 75th percentiles), and two whiskers extending to the furthest observation $\leq 1.5$ times the interquartile range, along with individual values for each metabolite (solid circles). $n = 10$ per group. Note: ANOVA, analysis of variance; BW, body weight; FDR, false discovery rate.

Figure 4 is a set of thirty-three error bar graphs plotting glyphosate, ranging from negative 0.5 to 0.5 in increments of 0.5; 1-methylnicotinamide, ranging from negative 0.50 to 0.25 in increments of 0.25; 3-methylglutaconate, ranging from negative 0.2 to 0.4 in increments of 0.2; N-accetylisoleucine, ranging from negative 0.2 to 0.2 in increments of 0.2; 2,3-dihydroxy-5-methylthio-4-pentenoate, ranging from negative 0.1 to 0.1 in increments of 0.1, glutarate, ranging from negative 0.4 to 0.2 in increments of 0.2; 1-methylnicotinamide, ranging from negative 0.50 to 0.25 in increments of 0.25; ectoine, ranging from negative 0.50 to 0.25 in increments of 0.25; citrate, ranging from negative 0.10 to 0.15 in increments of 0.05; xanthurenate, ranging from negative 0.5 to 0.5 in increments of 0.5; leucine, ranging from negative 0.10 to 0.05 in increments of 0.05; taurine, ranging from negative 0.10 to 0.05 in increments of 0.05; glycerate, ranging from negative 0.3 to 0.2 in increments of 0.1; isoleucine, ranging from negative 0.10 to 0.05 in increments of 0.05; methionine sulfoxide, ranging from negative 0.2 to 0.2 in increments of 0.2; N-accetylmethionine sulfoxide, ranging from negative 0.5 to 0.5 in increments of 0.5; N-accetylhomocitrulline, ranging from negative 0.2 to 0.2 in increments of 0.1; deoxycholate, ranging from negative 0.5 to 1.0 in increments of 0.5; 3-acetylphenol sulfate, ranging from negative 0.50 to 0.50 in increments of 0.25; 4-hydroxycoumarin, ranging from negative 0.5 to 0.5 increments of 0.5; alpha-ketoglutarae, ranging from negative 0.2 to 0.1 in increments of 0.1; 3-hydroxybutyrate, ranging from negative 0.3 to 0.6 in increments of 0.3; 4-hydroxycinnamate, ranging from negative 0.4 to 0.4 in increments of 0.4; phosphate, ranging from negative 0.2 to 0.4 increments of 0.2; N-acetyl-1-methylhistidine, ranging from negative 0.2 to 0.4 in increments of 0.2; 1-methylguanidine, ranging from negative 0.5 to 1.0 increments of 0.5; glutamate, ranging from negative 0.2 to 0.1 in increments of 0.1; guanidinoacetate, ranging from negative 0.4 to 0.2 in increments of 0.2; alpha-hydroxyisocaproate, ranging from negative 0.2 to 0.4 in increments of 0.2; ribitol, ranging from negative 0.2 to 0.2 increments of 0.1; ribonate, ranging from negative 0.2 to 0.2 in increments of 0.1; kynurenine, ranging from negative 0.3 to 0.2 in increments of 0.1, and azelate, ranging from negative 0.3 to 0.6 in increments of 0.3 (y-axis) across a (control), b [Glyphosate (0.5 milligrams per kilogram body weight per day)], c [Glyphosate (50 milligrams per kilogram body weight per day)], d [Glyphosate (175 milligrams per kilogram body weight per day)], e [MON 52276 (0.5 milligrams per kilogram body weight per day)], f [MON 52276 (50 milligrams per kilogram body weight per day)], and g [MON 52276 (175 milligrams per kilogram body weight per day)] (x-axis) for adjusted lowercase p equals 6.5E-26, 0.004; 0.002; 0.002; 0.002; 0.002; 0.002; 0.005; 0.005; 0.01; 0.01; 0.01; 0.01; 0.02; 0.02; 0.02; 0.02; 0.02; 0.02; 0.02; 0.03; 0.03; 0.03; 0.03; 0.04; 0.04; 0.04; 0.04; 0.04; 0.05; 0.05; 0.05; and 0.05, respectively. — **Figure 4.**
Differences in the level of serum metabolites after exposure to glyphosate or Roundup MON 52276. A total of 33 metabolites had adjusted $p < 0.05$ ( $a d j - p$ ) according to an ANOVA adjusted for multiple comparisons with the FDR procedure. Log-transformed abundance values are shown as box plots with the median, two hinges (the 25th and 75th percentiles) and two whiskers extending to the furthest observation $\leq 1.5$ times the interquartile range, along with individual values for each metabolite (solid circles). $n = 10$ per group. Note: ANOVA, analysis of variance; BW, body weight; DMTPA, 2,3-dihydroxy-5-methylthio-4-pentenoate; FDR, false discovery rate.

Figure 5A is an error bar graph plotting alpha diversity (Shannon Index), ranging from 5.2 to 6.0 in increments of 0.2 (y-axis) across a (control), b [Glyphosate (0.5 milligrams per kilogram body weight per day)], c [Glyphosate (50 milligrams per kilogram body weight per day)], d [MON 52276 (0.5 milligrams per kilogram body weight per day)], and e [MON 52276 (50 milligrams per kilogram body weight per day)] (x-axis). Figure 5B nonmetric multidimensional scaling 2, negative 0.1 to 0.1 in increments of 0.1 (y-axis) across nonmetric multidimensional scaling 1, ranging from negative 0.1 to 0.2 in increments of 0.1 (x-axis) for a (control), b [Glyphosate (0.5 milligrams per kilogram body weight per day)], c [Glyphosate (50 milligrams per kilogram body weight per day)], d [MON 52276 (0.5 milligrams per kilogram body weight per day)], and e [MON 52276 (50 milligrams per kilogram body weight per day). — **Figure 5.**
(A) Alpha diversity and (B) beta diversity in the cecum microbiome of rats following 90 d of treatment with glyphosate or Roundup MON 52276. Alpha diversity was calculated from species count data using the diversity function of the R package Vegan. Beta diversity was estimated by calculating pair-wise dissimilarities between samples as the Bray-Curtis distance and plotted as a nonmetric multidimensional scaling (NMDS) plot. The statistical significance of this clustering was tested with a 10,000-times permutational multivariate ANOVA test. $n = 12$ per group. Note: ANOVA, analysis of variance; BW, body weight.

Figure 6A is a set of five stacked area graphs titled control, Glyphosate (0.5 milligrams per kilogram body weight per day), Glyphosate (50 milligrams per kilogram body weight per day), MON 52276 (0.5 milligrams per kilogram body weight per day), and MON 52276 (50 milligrams per kilogram body weight per day) plotting Abundance (percentage), 0 to 100 in increments of 25 (y-axis) for Firmicutes, Bacteriodetes, Proteoacteria, Actinobacteria, Spirochetes, Euryarchaeota, Fusobacteria, Cyanobacteria, and others. Figure 6B is a set of five stacked area graphs titled control, Glyphosate (0.5 milligrams per kilogram body weight per day), Glyphosate (50 milligrams per kilogram body weight per day), MON 52276 (0.5 milligrams per kilogram body weight per day), and MON 52276 (50 milligrams per kilogram body weight per day), plotting Abundance (percentage), 0 to 30 in increments 10 (y-axis) for Bacteroides capillosus, Ruminococcaceae bacterium D16, Eubacterium rectale, Clostridium phytofermentans, Clostridium proteoclasticum, Clostridium saccharolyticum, Prevotella ruminicola, and Ruminococcus albus, respectively. Figures 6C to 6F are error bar graphs plotting Shinella zoogleoides (percentage), ranging from 0.000 to 0.075 in increments of 0.025; Eggerthellaceae H G M 04355, ranging from 0.0 to 2.0 in increments of 0.5; Acinetobacter johnsonii (percentage), ranging from 0.002 to 0.006 in increments of 0.002; and Akkermansia muciniphila (percentage), ranging from 0.06 to 0.12 in increments of 0.02 (y-axis) across a (control), b [Glyphosate (0.5 milligrams per kilogram body weight per day)], c [Glyphosate (50 milligrams per kilogram body weight per day)], d [MON 52276 (0.5 milligrams per kilogram body weight per day)], and e [MON 52276 (50 milligrams per kilogram body weight per day)], respectively. Figure 6G is a set of eight error bar graphs plotting metC cyc-S-conjugate lyase (counts per million), ranging from 30 to 90 in increments of 30; K00870 protein kinase (counts per million), ranging from 0 to 150 in increments of 50; acetoacetyl-CoA reduction (counts per million), ranging from 0 to 60 in increments of 20; rihA ribonucleoside hydrolase (counts per million), ranging from 0 to 60 in increments of 20, pseC transaminase (counts per million), ranging from 0 to 125 in increments of 25; M G L L acylglycerol lipase (counts per million), ranging from 50 to 150 in increments of 50; rifL oxidoreductase (counts per million), 0 to 120 in increments of 30; and buk; butyrate kinase (counts per million), ranging from 40 to 160 in increments of 40 (y-axis) across a (control), b [Glyphosate (0.5 milligrams per kilogram body weight per day)], c [Glyphosate (50 milligrams per kilogram body weight per day)], d [MON 52276 (0.5 milligrams per kilogram body weight per day)], and e [MON 52276 (50 milligrams per kilogram body weight per day)], respectively. — **Figure 6.**
Shotgun metagenomics of rat cecal microbiome composition. Female Sprague-Dawley rats were administered via drinking water with 0.5, 50, and $175 mg / kg BW per day$ glyphosate and Roundup MON 52276 at the same glyphosate-equivalent dose for 90 d. Cecum content was isolated at the time of sacrifice at the end of the treatment period and processed for metagenomics analysis. The abundance of (A) the most abundant phyla or of (B) the eight species found at an average abundance of >1% is presented. Box plots show the relative abundance for the species (C) *Shinella zoogleoides*, (D) *Acinetobacter johnsonii*, (E) *Eggerthella* isolate HGM04355, and (F) *Akkermansia muciniphila*. (G) Additional box plots display the functional potential assessed by evaluating abundance of KEGG Orthology annotations. Log-transformed abundance values are shown as box plots with the median, two hinges (the 25th and 75th percentiles), and two whiskers extending to the furthest observation $\leq 1.5$ times the interquartile range, along with individual values for each metabolite (solid circles). $n = 12$ per group. Note: BW, body weight; cpm, counts per million; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figures 7A to 7D are line graphs plotting optical density begin subscript 600 nanometers end subscript, ranging from 0.0 to 2.5 in increments of 0.5 (y-axis) across glyphosate (grams per liter), ranging from 10 begin superscript negative 4 end superscript to 10 begin superscript 2 end superscript in increments of 10 begin superscript 1 end superscript (x-axis), respectively. — **Figure 7.**
*In vitro* bacterial growth of four different strains of *Lacticaseibacillus rhamnosus* exposed for 48 h to glyphosate alone or to commercial herbicide formulations [Roundup MON 52276, Roundup ${GT}^{+}$ ]. *L. rhamnosus* strains were (A) LB2, (B) LB3, (C) LB5, and (D) LB7. Values are shown as $mean \pm SD$ . $n = 3 - 4$ for each tested concentration. Statistical significance was evaluated using a Kruskal-Wallis test with Dunn’s multiple comparison post-test (ns, not significant; * $p < 0.05$ and ** $p < 0.01$ ). Note: OD, optical density; SD, standard deviation.

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References

1. Abraham W, inventor; Monsanto Technology LLC, assignee. Glyphosate formulations and their use for the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase. US patent 7,771,736. 10 August 2010.
1. Adam A, Marzuki A, Abdul Rahman H, Abdul Aziz M. 1997. The oral and intratracheal toxicities of ROUNDUP and its components to rats. Vet Hum Toxicol 39(3):147–151, PMID: 9167243. - PubMed
1. Anadón A, Martínez-Larrañaga MR, Martínez MA, Castellano VJ, Martínez M, Martin MT, et al. 2009. Toxicokinetics of glyphosate and its metabolite aminomethyl phosphonic acid in rats. Toxicol Lett 190(1):91–95, PMID: 19607892, 10.1016/j.toxlet.2009.07.008. - DOI - PubMed
1. Aristilde L, Reed ML, Wilkes RA, Youngster T, Kukurugya MA, Katz V, et al. 2017. Glyphosate-induced specific and widespread perturbations in the metabolome of soil Pseudomonas species. Front Environ Sci 5:34, 10.3389/fenvs.2017.00034. - DOI
1. Barrett KA, McBride MB. 2005. Oxidative degradation of glyphosate and aminomethylphosphonate by manganese oxide. Environ Sci Technol 39(23):9223–9228, PMID: 16382946, 10.1021/es051342d. - DOI - PubMed

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Use of Shotgun Metagenomics and Metabolomics to Evaluate the Impact of Glyphosate or Roundup MON 52276 on the Gut Microbiota and Serum Metabolome of Sprague-Dawley Rats

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Use of Shotgun Metagenomics and Metabolomics to Evaluate the Impact of Glyphosate or Roundup MON 52276 on the Gut Microbiota and Serum Metabolome of Sprague-Dawley Rats

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