Urinary excretion of herbicide co-formulants after oral exposure to roundup MON 52276 in rats

The toxicity of surfactants, which are an integral component of glyphosate-formulated products is an underexplored and highly debated subject. Since biomonitoring human exposure to glyphosate co-formulants is considered as a public health priority, we developed and validated a high-resolution mass spectrometry method to measure the urinary excretion of surfactants present in Roundup MON 52276, the European Union (EU) representative formulation of glyphosate-based herbicides. Quantification was performed measuring the 5 most abundant compounds in the mixture. We validated the method and showed that it is highly accurate, precise and reproducible with a limit of detection of 0.0004 μg/mL. We used this method to estimate the oral absorption of MON 52276 surfactants in Sprague-Dawley rats exposed to three concentrations of MON 52276 via drinking water for 90 days. MON 52276 surfactants were readily detected in urine of rats administered with this commercial Roundup formulation starting from a low concentration corresponding to the EU glyphosate acceptable daily intake. Our results provide a first step towards the implementation of surfactant co-formulant biomonitoring in human populations

DUF3380 domain from a Salmonella phage endolysin shows potent N -acetylmuramidase activity

Bacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novel Salmonella bacteriophage endolysin, Gp110, which comprises an uncharacterized domain of unknown function (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 has N-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents

Glyphosate does not substitute for glycine in proteins of actively dividing mammalian cells

Glyphosate (N-phosphonomethyl glycine) and its commercial herbicide formulations have been shown to exert toxicity via various mechanisms. It has been asserted that glyphosate substitutes for glycine in polypeptide chains leading to protein misfolding and toxicity. However, as no direct evidence exists for glycine to glyphosate substitution in proteins, including in mammalian organisms, we tested this claim by conducting a proteomics analysis of MDA-MB-231 human breast cancer cells grown in the presence of 100 mg/L glyphosate for 6 days. Protein extracts from three treated and three untreated cell cultures were analysed as one TMT-6plex labelled sample, to highlight a specific pattern (+/+/+/−/−/−) of reporter intensities for peptides bearing true glyphosate treatment induced-post translational modifications as well as allowing an investigation of the total proteome

The surfactant co-formulant POEA in the glyphosate-based herbicide RangerPro but not glyphosate alone causes necrosis in Caco-2 and HepG2 human cell lines and ER stress in the ToxTracker assay

The toxicity of co-formulants present in glyphosate-based herbicides (GBHs) has been widely discussed leading to the European Union banning the polyoxyethylene tallow amine (POEA). We identified the most commonly used POEA, known as POE-15 tallow amine (POE-15), in the widely used US GBH RangerPro. Cytotoxicity assays using human intestinal epithelial Caco-2 and hepatocyte HepG2 cell lines showed that RangerPro and POE-15 are far more cytotoxic than glyphosate alone. RangerPro and POE-15 but not glyphosate caused cell necrosis in both cell lines, and that glyphosate and RangerPro but not POE-15 caused oxidative stress in HepG2 cells. We further tested these pesticide ingredients in the ToxTracker assay, a system used to evaluate a compound's carcinogenic potential, to assess their capability for inducing DNA damage, oxidative stress and an unfolded protein response (endoplasmic reticulum, ER stress). RangerPro and POE-15 but not glyphosate gave rise to ER stress. We conclude that the toxicity resulting from RangerPro exposure is thus multifactorial involving ER stress caused by POE-15 along with oxidative stress caused by glyphosate. Our observations reinforce the need to test both co-formulants and active ingredients of commercial pesticides to inform the enactment of more appropriate regulation and thus better public and environmental protection

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

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

(Poly)phenol-related gut metabotypes and human health: an update

Dietary (poly)phenols have received great interest due to their potential role in the prevention and management of non-communicable diseases. In recent years, a high inter-individual variability in the biological response to (poly)phenols has been demonstrated, which could be related to the high variability in (poly)phenol gut microbial metabolism existing within individuals. An interplay between (poly)phenols and the gut microbiota exists, with (poly)phenols being metabolised by the gut microbiota and their metabolites modulating gut microbiota diversity and composition. A number of (poly)phenol metabolising phenotypes or metabotypes have been proposed, however, potential metabotypes for most (poly)phenols have not been investigated, and the relationship between metabotypes and human health remains ambiguous. This review presents updated knowledge on the reciprocal interaction between (poly)phenols and the gut microbiome, associated gut metabotypes, and subsequent impact on human health

Multi-omics phenotyping of the gut-liver axis reveals metabolic perturbations from a low-dose pesticide mixture in rats.

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

Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme