{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T18:59:20Z","timestamp":1785351560946,"version":"3.55.0"},"reference-count":53,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T00:00:00Z","timestamp":1760054400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004442","name":"National Science Centre","doi-asserted-by":"crossref","award":["2020\/04\/X\/NZ7\/00338"],"award-info":[{"award-number":["2020\/04\/X\/NZ7\/00338"]}],"id":[{"id":"10.13039\/501100004442","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Glyphosate, a widely used herbicide, is under scrutiny for its potential neurotoxic effects. This study investigated whether oral exposure to glyphosate, even at doses currently considered safe in Europe, alters the neurochemical profile of enteric nervous system (ENS) neurons in the porcine jejunum. Fifteen immature female pigs were allocated to three groups: control (C), low-dose (G50; 50 \u00b5g\/kg b.w.\/day), and higher-dose (G500; 500 \u00b5g\/kg b.w.\/day). Following 28 days of exposure, jejunal samples were subjected to double-labelling immunofluorescence staining for neuronal markers, including Hu C\/D and PACAP, CGRP, CART, nNOS, or VAChT. Results revealed dose-dependent neurochemical alterations across all enteric plexuses, with glyphosate increasing the number of neurons expressing PACAP, CGRP, CART, and nNOS, while reducing VAChT-positive neurons. The effect of glyphosate on enteric neurons appeared largely uniform across different plexus types, with more pronounced changes at the higher dose and only minor regional variation. Overall, the findings suggest that glyphosate exposure, even within regulatory limits, may alter the neurochemical profile of enteric neurons in a broadly uniform manner, potentially reflecting responses to oxidative stress or early neurotoxic effects, as reported in previous studies. This study challenges current safety thresholds and emphasises the need to reassess regulatory guidelines, particularly in the context of chronic exposure and potential risks to vulnerable populations.<\/jats:p>","DOI":"10.3390\/ijms26209840","type":"journal-article","created":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:50:16Z","timestamp":1760107816000},"page":"9840","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Neurochemical Remodelling of the Enteric Nervous System Neurons in the Porcine Jejunum Following Low-Dose Glyphosate Exposure"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7593-9552","authenticated-orcid":false,"given":"Katarzyna","family":"Palus","sequence":"first","affiliation":[{"name":"Department of Clinical Physiology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-718 Olsztyn, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aleksandra","family":"Karpiesiuk","sequence":"additional","affiliation":[{"name":"Department of Clinical Physiology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-718 Olsztyn, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6508-2965","authenticated-orcid":false,"given":"Barbara","family":"Jana","sequence":"additional","affiliation":[{"name":"Division of Reproductive Biology, Institute of Animal Reproduction and Food Research of the Polish, Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1038\/nrgastro.2012.32","article-title":"The enteric nervous system and neurogastroenterology","volume":"9","author":"Furness","year":"2012","journal-title":"Nat. Rev. Gastroenterol. Hepatol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"110673","DOI":"10.1016\/j.fct.2019.110673","article-title":"The impact of low and high doses of acrylamide on the intramural neurons of the porcine ileum","volume":"132","author":"Palus","year":"2019","journal-title":"Food Chem. Toxicol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1438","DOI":"10.1016\/S0016-5085(99)70295-7","article-title":"Plasticity in the enteric nervous system","volume":"117","author":"Giaroni","year":"1999","journal-title":"Gastroenterology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1007\/978-3-319-27592-5_16","article-title":"Integrated neural and endocrine control of gastrointestinal function","volume":"891","author":"Furness","year":"2016","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"956","DOI":"10.1172\/JCI76308","article-title":"Emerging roles of gut microbiota and the immune system in the development of the enteric nervous system","volume":"125","author":"Kabouridis","year":"2015","journal-title":"J. Clin. Investig."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e13580","DOI":"10.1111\/nmo.13580","article-title":"Neurochemical characterization of the enteric neurons within the porcine jejunum in physiological conditions and under the influence of bisphenol A (BPA)","volume":"31","author":"Gonkowski","year":"2019","journal-title":"Neurogastroenterol. Motil."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5328","DOI":"10.1007\/s12011-021-03092-4","article-title":"Impact of Heavy Metal Toxicity on the Gut Microbiota and Its Relationship with Metabolites and Future Probiotics Strategy: A Review","volume":"200","author":"Bist","year":"2022","journal-title":"Biol. Trace Elem. Res."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Palus, K., Chmielewska-Krzesi\u0144ska, M., Jana, B., and Ca\u0142ka, J. (2024). Glyphosate-induced changes in the expression of galanin and GALR1, GALR2 and GALR3 receptors in the porcine small intestine wall. Sci. Rep., 14.","DOI":"10.1038\/s41598-024-59581-8"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Ga\u0142\u0119cka, I., and Ca\u0142ka, J. (2024). Oral Exposure to Microplastics Affects the Neurochemical Plasticity of Reactive Neurons in the Porcine Jejunum. Nutrients, 16.","DOI":"10.3390\/nu16142268"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Palus, K., and Ca\u0142ka, J. (2019). Influence of Acrylamide Administration on the Neurochemical Characteristics of Enteric Nervous System (ENS) Neurons in the Porcine Duodenum. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21010015"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Karpiesiuk, A., and Palus, K. (2021). Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Physiological and Pathological Processes within the Gastrointestinal Tract: A Review. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22168682"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"903","DOI":"10.1053\/j.gastro.2003.11.046","article-title":"Nitric oxide in gastrointestinal health and disease","volume":"126","author":"Shah","year":"2004","journal-title":"Gastroenterology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1002\/(SICI)1096-9861(19970224)378:4<454::AID-CNE2>3.0.CO;2-1","article-title":"Vesicular acetylcholine transporter (VAChT) protein: A novel and unique marker for cholinergic neurons in the central and peripheral nervous systems","volume":"378","author":"Arvidsson","year":"1997","journal-title":"J. Comp. Neurol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Makowska, K., and Gonkowski, S. (2018). The Influence of Inflammation and Nerve Damage on the Neurochemical Characterization of Calcitonin Gene-Related Peptide-Like Immunoreactive (CGRP-LI) Neurons in the Enteric Nervous System of the Porcine Descending Colon. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19020548"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/j.aanat.2018.06.002","article-title":"Acrylamide-induced alterations in the cocaine- and amphetamine-regulated peptide transcript (CART)-like immunoreactivity within the enteric nervous system of the porcine small intestines","volume":"219","author":"Palus","year":"2018","journal-title":"Ann. Anat."},{"key":"ref_16","first-page":"255","article-title":"Environmental and health effects of the herbicide glyphosate","volume":"616\u2013617","author":"He","year":"2018","journal-title":"Sci. Total Environ."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.toxlet.2009.07.008","article-title":"Toxicokinetics of glyphosate and its metabolite aminomethyl phosphonic acid in rats","volume":"190","author":"Castellano","year":"2009","journal-title":"Toxicol. Lett."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Marino, M., Mele, E., Viggiano, A., Nori, S.L., Meccariello, R., and Santoro, A. (2021). Pleiotropic outcomes of glyphosate exposure: From organ damage to effects on inflammation, cancer, reproduction and development. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222212606"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Costas-Ferreira, C., Dur\u00e1n, R., and Faro, L.R.F. (2022). Toxic Effects of Glyphosate on the Nervous System: A Systematic Review. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23094605"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.tox.2014.03.001","article-title":"Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: Involvement of glutamate excitotoxicity","volume":"320","author":"Cattani","year":"2014","journal-title":"Toxicology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"741","DOI":"10.1136\/jech-2015-207005","article-title":"Differences in the carcinogenic evaluation of glyphosate between the International Agency for Research on Cancer (IARC) and the European Food Safety Authority (EFSA)","volume":"70","author":"Portier","year":"2016","journal-title":"J. Epidemiol. Community Health"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"108851","DOI":"10.1016\/j.envres.2019.108851","article-title":"Comparative cyto- and genotoxicity assessment of glyphosate and glyphosate-based herbicides in human peripheral white blood cells","volume":"179","author":"Nagy","year":"2019","journal-title":"Environ. Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1080\/1547691X.2020.1804492","article-title":"The impact and toxicity of glyphosate and glyphosate-based herbicides on health and immunity","volume":"17","author":"Peillex","year":"2020","journal-title":"J. Immunotoxicol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"7757","DOI":"10.1039\/D4FO00660G","article-title":"Effects of glyphosate exposure on intestinal microbiota, metabolism and microstructure: A systematic review","volume":"15","author":"Guerra","year":"2024","journal-title":"Food Funct."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.neuro.2019.08.006","article-title":"Gut microbiota and neurological effects of glyphosate","volume":"75","author":"Cruz","year":"2019","journal-title":"Neurotoxicology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"109846","DOI":"10.1016\/j.ecoenv.2019.109846","article-title":"Toxic effects of glyphosate on intestinal morphology, antioxidant capacity and barrier function in weaned piglets","volume":"187","author":"Qiu","year":"2020","journal-title":"Ecotoxicol. Environ. Saf."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e14507","DOI":"10.1111\/nmo.14507","article-title":"Glyphosate affects the neurochemical phenotype of the intramural neurons in the duodenum in the pig","volume":"35","author":"Palus","year":"2023","journal-title":"Neurogastroenterol. Motil."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1002\/bdd.2510160502","article-title":"Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals","volume":"16","author":"Kararli","year":"1995","journal-title":"Biopharm. Drug Dispos."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.ntt.2017.10.004","article-title":"Behavioral impairments following repeated intranasal glyphosate-based herbicide administration in mice","volume":"64","author":"Baier","year":"2017","journal-title":"Neurotoxicol. Teratol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Izumi, Y., O\u2019Dell, K.A., and Zorumski, C.F. (2023). The herbicide glyphosate inhibits hippocampal long-term potentiation and learning through activation of pro-inflammatory signaling. Sci. Rep., 13.","DOI":"10.1038\/s41598-023-44121-7"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"212","DOI":"10.1016\/j.envres.2017.10.051","article-title":"Neurotransmitter changes in rat brain regions following glyphosate exposure","volume":"161","author":"Ares","year":"2018","journal-title":"Environ. Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"110183","DOI":"10.1016\/j.neuropharm.2024.110183","article-title":"Glyphosate impairs both structure and function of GABAergic synapses in hippocampal neurons","volume":"262","author":"Chiantia","year":"2025","journal-title":"Neuropharmacology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1016\/j.etap.2009.07.009","article-title":"Antioxidant defense system in rats simultaneously intoxicated with agrochemicals","volume":"28","author":"Astiz","year":"2009","journal-title":"Environ. Toxicol. Pharmacol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"114129","DOI":"10.1016\/j.envpol.2020.114129","article-title":"Glyphosate exposure induces inflammatory responses in the small intestine and alters gut microbial composition in rats","volume":"261","author":"Tang","year":"2020","journal-title":"Environ. Pollut."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"117685","DOI":"10.1016\/j.envpol.2021.117685","article-title":"Negative impacts of microcystin-LR and glyphosate on zebrafish intestine: Linked with gut microbiota and microRNAs?","volume":"286","author":"Ding","year":"2021","journal-title":"Environ. Pollut."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"119350","DOI":"10.1016\/j.lfs.2021.119350","article-title":"Perinatal exposure to low doses of glyphosate-based herbicide combined with a high-fat diet in adulthood causes changes in the jejunums of mice","volume":"275","author":"Panza","year":"2021","journal-title":"Life Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e06027","DOI":"10.1016\/j.heliyon.2021.e06027","article-title":"Changes in microtubule stability in zebrafish (Danio rerio) embryos after glyphosate exposure","volume":"7","year":"2021","journal-title":"Heliyon"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1007\/s12264-019-00407-3","article-title":"Neuroprotective Effects of Brain-Gut Peptides: A Potential Therapy for Parkinson\u2019s Disease","volume":"35","author":"Dong","year":"2019","journal-title":"Neurosci. Bull."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1016\/j.bbrc.2006.06.117","article-title":"CART peptide promotes the survival of hippocampal neurons by upregulating brain-derived neurotrophic factor","volume":"347","author":"Wu","year":"2006","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1038\/nrn2214","article-title":"Nitric oxide in the central nervous system: Neuroprotection versus neurotoxicity","volume":"8","author":"Calabrese","year":"2007","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1157","DOI":"10.1189\/jlb.0310149","article-title":"The regulatory role of nitric oxide in proinflammatory cytokine expression during the induction and resolution of inflammation","volume":"88","author":"Kobayashi","year":"2010","journal-title":"J. Leukoc. Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1371","DOI":"10.1016\/0016-5085(93)90345-D","article-title":"Role of calcitonin gene-related peptide and nitric oxide in the gastroprotective effect of capsaicin in the rat","volume":"104","author":"Lambrecht","year":"1993","journal-title":"Gastroenterology"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Rytel, L., Gonkowski, I., Grzegorzewski, W., and Wojtkiewicz, J. (2021). Chemically-Induced Inflammation Changes the Number of Nitrergic Nervous Structures in the Muscular Layer of the Porcine Descending Colon. Animals, 11.","DOI":"10.3390\/ani11020394"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Bulc, M., Ca\u0142ka, J., and Palus, K. (2020). Effect of Streptozotocin-Inducted Diabetes on the Pathophysiology of Enteric Neurons in the Small Intestine Based on the Porcine Diabetes Model. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21062047"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1489","DOI":"10.1002\/jat.4651","article-title":"Evaluation of the potential role of glutamatergic, cholinergic, and nitrergic systems in the dopamine release induced by the pesticide glyphosate in rat striatum","volume":"44","author":"Faro","year":"2024","journal-title":"J. Appl. Toxicol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Makowska, K., and Gonkowski, S. (2023). Changes Caused by Bisphenols in the Chemical Coding of Neurons of the Enteric Nervous System of Mouse Stomach. Int. J. Environ. Res. Public Health, 20.","DOI":"10.3390\/ijerph20065125"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Kandpal, M., Indari, O., Baral, B., Jakhmola, S., Tiwari, D., Bhandari, V., Pandey, R.K., Bala, K., Sonawane, A., and Jha, H.C. (2022). Dysbiosis of Gut Microbiota from the Perspective of the Gut-Brain Axis: Role in the Provocation of Neurological Disorders. Metabolites, 12.","DOI":"10.3390\/metabo12111064"},{"key":"ref_48","first-page":"37","article-title":"Microbiota-gut-brain axis and its therapeutic applications in neurodegenerative diseases. Signal Transduct","volume":"9","author":"Loh","year":"2024","journal-title":"Target Ther."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Mesnage, R., Calatayud, M., Duysburgh, C., Marzorati, M., and Antoniou, M.N. (2022). Alterations in infant gut microbiome composition and metabolism after exposure to glyphosate and Roundup and\/or a spore-based formulation using the SHIME technology. Gut Microbiome, 3.","DOI":"10.1017\/gmb.2022.5"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Bombardi, C., Rambaldi, A.M., Galiazzo, G., Giancola, F., Gra\u00efc, J.M., Salamanca, G., Cozzi, B., and Chiocchetti, R. (2021). Nitrergic and Substance P Immunoreactive Neurons in the Enteric Nervous System of the Bottlenose Dolphin (Tursiops truncatus) Intestine. Animals, 11.","DOI":"10.3390\/ani11041057"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1016\/j.marpolbul.2014.01.021","article-title":"Glyphosate persistence in seawater","volume":"85","author":"Mercurio","year":"2014","journal-title":"Mar. Pollut. Bull."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"16383","DOI":"10.1007\/s11356-021-16957-x","article-title":"Assessing glyphosate in water, marine particulate matter, and sediments in the Lagoon of Venice","volume":"29","author":"Feltracco","year":"2022","journal-title":"Environ. Sci. Pollut. Res. Int."},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Palus, K., Bulc, M., and Ca\u0142ka, J. (2017). Changes in Somatostatin-Like Immunoreactivity in the Sympathetic Neurons Projecting to the Prepyloric Area of the Porcine Stomach Induced by Selected Pathological Conditions. Biomed. Res. Int., 2017.","DOI":"10.1155\/2017\/9037476"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/20\/9840\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T04:23:37Z","timestamp":1760243017000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/20\/9840"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,10]]},"references-count":53,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["ijms26209840"],"URL":"https:\/\/doi.org\/10.3390\/ijms26209840","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,10]]}}}