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

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 ðBWÞ 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, c-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.

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

“…Our preceding analysis of the same animals investigated in this study indicated that glyphosate and to a greater degree Roundup MON 52276 gave rise to oxidative stress (Mesnage et al, 2021). The primary aim of this follow-up investigation was to determine if this oxidative stress resulted in toxic effects in the liver.…”

“…Treatment of animals was as previously described (Mesnage et al 2021). In brief, groups of 12 female Sprague-Dawley rats of 8 weeks of age were administered for 90 days with glyphosate and MON 52276 as Roundup BioFlow (Italy) at the same glyphosate equivalent dose via drinking water of 0.5 mg, 50 mg and 175 mg/kg body weight per day (mg/kg bw/day), which respectively represent the EU acceptable daily intake (ADI), the EU no-observed adverse effect level (NOAEL) and the US NOAEL (European Food Safety 2015).…”

“…With the need to compare the toxicity of pesticide active principles with their commercial formulations, we performed a subchronic study for 90-days in rats administered with glyphosate (0.5, 50, 175 mg/kg bw/day), or its representative EU commercial herbicide formulation Roundup MON 52276 at the same glyphosate equivalent doses. In a first report of results from this toxicity study, we described how glyphosate and MON 52776 altered the function of the rat gut microbiome and brought about changes in the serum metabolome suggestive of oxidative stress damage (Mesnage et al 2021). Alterations of the gut microbiome glyphosate and MON 52776 were also reported in male and female rats of the same strain treated from pregnancy (Mao et al 2018).…”

Could Glyphosate and Glyphosate-Based Herbicides Be Associated With Increased Thyroid Diseases Worldwide?

“…This is corroborated by studies where gut microbiota dysbiosis was correlated to Hashimoto's thyroiditis (102)(103)(104), Graves' disease (104), thyroid cancer and thyroid nodules (105). In parallel in the field of toxicology, recent studies indicate that GBH is able to affect the intestinal microbiome of mice (106), rats (107)(108)(109)(110)(111)(112), honeybees (113,114), Japanese quails (115) and Daphnia magna (116). A possible explanation for this toxicity is that some classes of bacteria require the shikimate pathway (5enolpyruvylshikimate-3-phosphate synthase, EPSPS) for their metabolism.…”

“…The glyphosate mechanism of action is through inhibition of the growth of weeds by interfering with the production of essential aromatic amino acids by inhibiting EPSPS, which is responsible for chorismate biosynthesis, an intermediate in the biosynthesis of phenylalanine, tyrosine and tryptophan (7). Then, from the moment glyphosate inhibits it, these bacteria are unable to maintain normal levels in the gut microbiome (111,(113)(114)(115). Thus, new studies evaluating the involvement of the gut-thyroid axis are relevant in advancing the knowledge of glyphosate toxicity on thyroid function.…”