Robin Mesnage, Mariam Ibragim, Daniele Mandrioli, Laura Falcioni, Eva Tibaldi, Fiorella Belpoggi, Inger Brandsma, Emma Bourne, Emanuel Savage, Charles A Mein, Michael N Antoniou; “Comparative Toxicogenomics of Glyphosate and Roundup Herbicides by Mammalian Stem Cell-Based Genotoxicity Assays and Molecular Profiling in Sprague-Dawley Rats”, Toxicological Sciences, 2021; DOI: 10.1093/toxsci/kfab143.
Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remain controversial. As GBHs are more cytotoxic than glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs, namely MON 52276 (European Union), MON 76473 (United Kingdom), and MON 76207 (United States) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (at 0.5, 50, and 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased miR-30, whereas miR-10 levels were increased. DNA methylation profiling of liver revealed 5727 and 4496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals, respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate. FULL TEXT
Benbrook, Charles, & Benbrook, Rachel (2021). “A minimum data set for tracking changes in pesticide use.” In R. Mesnage & J. Zaller (Eds.), Herbicides: Elsevier and RTI Press.
A frequently asked but deceptively simple question often arises about pesticide use on a given farm or crop: Is pesticide use going up, down, or staying about the same? Where substantial changes in pesticide use are occurring, it is also important to understand the factors driving change. These might include more or fewer hectares planted, a change in the crop mix, a higher or lower percentage of hectares treated, or higher or lower rates of application and/or number of applications. Or, it might arise from a shift to other pesticides applied at a higher or lower rate and/or lessened or greater reliance on nonpesticidal strategies and integrated pest management (IPM). Questions about whether pesticide use is changing and why arise for a variety of reasons. Rising use typically increases farmer costs and cuts into profit margins. It generally raises the risk of adverse environmental and/or public health outcomes. It can accelerate the emergence and spread of organisms resistant to applied pesticides. If the need to spray more continues year after year for long enough, farming systems become unsustainable. Lessened reliance on and use of pesticides, on the other hand, are typically brought about and can only be sustained by incrementally more effective prevention-based biointensive IPM systems (bioIPM).1–3 Fewer pesticide applications and fewer pounds/kilograms of active ingredient applied reduce the impacts on nontarget organisms and provide space for beneficial organisms and biodiversity to flourish. Such systems reduce the odds of significant crop loss in years when conditions undermine the efficacy of control measures, leading to spikes in pest populations and the risk of economically meaningful loss of crop yield and/or quality. FULL TEXT
Benbrook, Charles, Perry, Melissa J., Belpoggi, Fiorella, Landrigan, Philip J., Perro, Michelle, Mandrioli, Daniele, Antoniou, Michael N., Winchester, Paul, & Mesnage, Robin; “Commentary: Novel strategies and new tools to curtail the health effects of pesticides;” Environmental Health, 2021, 20(1); DOI: 10.1186/s12940-021-00773-4.
BACKGROUND: Flaws in the science supporting pesticide risk assessment and regulation stand in the way of progress in mitigating the human health impacts of pesticides. Critical problems include the scope of regulatory testing protocols, the near-total focus on pure active ingredients rather than formulated products, lack of publicly accessible information on co-formulants, excessive reliance on industry-supported studies coupled with reticence to incorporate published results in the risk assessment process, and failure to take advantage of new scientific opportunities and advances, e.g. biomonitoring and “omics” technologies.
RECOMMENDED ACTIONS: Problems in pesticide risk assessment are identified and linked to study design, data, and methodological shortcomings. Steps and strategies are presented that have potential to deepen scientific knowledge of pesticide toxicity, exposures, and risks.
We propose four solutions:
(1) End near-sole reliance in regulatory decision-making on industry-supported studies by supporting and relying more heavily on independent science, especially for core toxicology studies. The cost of conducting core toxicology studies at labs not affiliated with or funded directly by pesticide registrants should be covered via fees paid by manufacturers to public agencies.
(2) Regulators should place more weight on mechanistic data and low-dose studies within the range of contemporary exposures.
(3) Regulators, public health agencies, and funders should increase the share of exposure-assessment resources that produce direct measures of concentrations in bodily fluids and tissues. Human biomonitoring is vital in order to quickly identify rising exposures among vulnerable populations including applicators, pregnant women, and children.
(4) Scientific tools across disciplines can accelerate progress in risk assessments if integrated more effectively. New genetic and metabolomic markers of adverse health impacts and heritable epigenetic impacts are emerging and should be included more routinely in risk assessment to effectively prevent disease.
CONCLUSIONS: Preventing adverse public health outcomes triggered or made worse by exposure to pesticides will require changes in policy and risk assessment procedures, more science free of industry influence, and innovative strategies that blend traditional methods with new tools and mechanistic insights.
Mesnage R, Mazzacuva F, Caldwell A, Halket J, Antoniou MN. “Urinary excretion of herbicide co-formulants after oral exposure to roundup MON 52276 in rats.” Environmental Research. 2021 Jun;197:111103. DOI: 10.1016/j.envres.2021.111103.
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. FULL TEXT
Mesnage, R., Teixeira, M., Mandrioli, D., Falcioni, L., Ibragim, M., Ducarmon, Q. R., Zwittink, R. D., Amiel, C., Panoff, J. M., Bourne, E., Savage, E., Mein, C. A., Belpoggi, F., & Antoniou, M. N.; “Multi-omics phenotyping of the gut-liver axis reveals metabolic perturbations from a low-dose pesticide mixture in rats;” Communications Biology, 2021, 4(1), 471; DOI: 10.1038/s42003-021-01990-w.
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. FULL TEXT
Mesnage, R, Teixeira, M, Mandrioli, D., Falcioni, L., Ducarmon, QR, Zwittink, RD, Mazzacuva, F, Caldwell, A, Halket, J, Amiel, C., Panoff, J. , Belpoggi, F., & Antoniou, MN; “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;” Environmental Health Perspectives, 2021 (in press); DOI: 10.1289/EHP6990.
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 FULL TEXT.
Robinson, Claire, Portier, Christopher J., ČAvoŠKi, Aleksandra, Mesnage, Robin, Roger, Apolline, Clausing, Peter, Whaley, Paul, Muilerman, Hans, & Lyssimachou, Angeliki; “Achieving a High Level of Protection from Pesticides in Europe: Problems with the Current Risk Assessment Procedure and Solutions;” European Journal of Risk Regulation, 2020, 11(3), 450-480; DOI: 10.1017/err.2020.18.
The regulation of pesticides in the European Union (EU) relies on a network of hard law (legislation and implementing acts) and soft law (non-legally binding guidance documents and administrative and scientific practices). Both hard and soft laws govern how risk assessments are conducted, but a significant role is left to the latter. Europe’s pesticide regulation is one of the most stringent in the world. Its stated objectives are to ensure an independent, objective and transparent assessment of pesticides and achieve a high level of protection for health and environment. However, a growing body of evidence shows that pesticides that have passed through this process and are authorised for use may harm humans, animals and the environment. The authors of the current paper – experts in toxicology, law and policy – identified shortcomings in the authorisation process, focusing on the EU assessment of the pesticide active substance glyphosate. The shortcomings mostly consist of failures to implement the hard or soft laws. But in some instances the law itself is responsible, as some provisions can only fail to achieve its objectives. Ways to improve the system are proposed, requiring changes in hard and soft laws as well as in administrative and scientific practices. FULL TEXT
Mesnage, Robin, & Antoniou, Michael N.; “Computational modelling provides insight into the effects of glyphosate on the shikimate pathway in the human gut microbiome;” Current Research in Toxicology, 2020, 1, 25-33; DOI: 10.1016/j.crtox.2020.04.001.
The herbicide active ingredient glyphosate can affect the growth of microorganisms, which rely on the shikimate pathway for aromatic amino acid biosynthesis. However, it is uncertain whether glyphosate exposure could lead to perturbations in the population of human gut microbiota. We have addressed this knowledge gap by analysing publicly available datasets to provide new insights into possible effects of glyphosate on the human gut microbiome. Comparison of the abundance of the shikimate pathway in 734 paired metagenomes and metatranscriptomes indicated that most gut bacteria do not possess a complete shikimate pathway, and that this pathway is mostly transcriptionally inactive in the human gut microbiome. This suggests that gut bacteria are mostly aromatic amino acid auxotrophs and thus relatively resistant to a potential growth inhibition by glyphosate. As glyphosate blocking of the shikimate pathway is via inhibition of EPSPS, we classified E. coli EPSPS enzyme homologues as class I (sensitive to glyphosate) and class II (resistant to glyphosate). Among 44 subspecies reference genomes, accounting for 72% of the total assigned microbial abundance in 2144 human faecal metagenomes, 9 subspecies have class II EPSPS. The study of publicly available gut metagenomes also indicated that glyphosate might be degraded by some Proteobacteria in the human gut microbiome using the carbon–phosphorus lyase pathway. Overall, there is limited experimental evidence available for the effects of glyphosate on the human gut microbiome. Further investigations using more advanced molecular profiling techniques are needed to ascertain whether glyphosate and glyphosate-based herbicides can alter the function of the gut microbiome with consequent health implications. FULL TEXT
Mesnage, Robin, “Effects of dietary exposures to pesticide residues on the gut microbiome,” 2019, Presented 10/30/2019 at the London Microbiome Meeting 2019, Great Hall, King’s College London Strand Campus.
The gut microbiota: a major player in the toxicity of environmental pollutants?
Tsiaoussis, J., Antoniou, M. N., Koliarakis, I., Mesnage, R., Vardavas, C. I., Izotov, B. N., Psaroulaki, A., & Tsatsakis, A.; “Effects of single and combined toxic exposures on the gut microbiome: Current knowledge and future directions;” Toxicology Letters, 2019, 312, 72-97; DOI: 10.1016/j.toxlet.2019.04.014.
Human populations are chronically exposed to mixtures of toxic chemicals. Predicting the health effects of these mixtures require a large amount of information on the mode of action of their components. Xenobiotic metabolism by bacteria inhabiting the gastrointestinal tract has a major influence on human health. Our review aims to explore the literature for studies looking to characterize the different modes of action and outcomes of major chemical pollutants, and some components of cosmetics and food additives, on gut microbial communities in order to facilitate an estimation of their potential mixture effects. We identified good evidence that exposure to heavy metals, pesticides, nanoparticles, polycyclic aromatic hydrocarbons, dioxins, furans, polychlorinated biphenyls, and non-caloric artificial sweeteners affect the gut microbiome and which is associated with the development of metabolic, malignant, inflammatory, or immune diseases. Answering the question ‘Who is there?’ is not sufficient to define the mode of action of a toxicant in predictive modeling of mixture effects. Therefore, we recommend that new studies focus to simulate real-life exposure to diverse chemicals (toxicants, cosmetic/food additives), including as mixtures, and which combine metagenomics, metatranscriptomics and metabolomic analytical methods achieving in that way a comprehensive evaluation of effects on human health. FULL TEXT