https://doi.org/10.4081/ejh.2022.3428
Glyphosate, AMPA and glyphosate-based herbicide exposure leads to GFAP, PCNA and caspase-3 increased immunoreactive area on male offspring rat hypothalamus
Submitted: 21 April 2022
Accepted: 25 July 2022
Published: 13 October 2022
Accepted: 25 July 2022
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All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
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Glyphosate, aminomethylphosphonic acid (AMPA), and glyphosate-based herbicides altered the neuroendocrine axis, the content of brain neurotransmitters, and behavior in experimental animal models. Glyphosate alone, AMPA or Roundup® Active were administered to postpartum female rats, from P0 to P10, and their water consumption was measured daily. The immunoreactivity for glial fibrillary acidic protein (GFAP), proliferating cell nuclear antigen (PCNA) and caspase-3 was measured in the anterior, medial preoptic, periventricular, supraoptic and lateroanterior hypothalamic nuclei of P0-P10 male pups after exposure, via lactation, to these xenobiotics. Puppies exposed to glyphosate had a moderate level of GFAP with no overlapping astrocyte processes, but this overlapping was observed after Roundup® Active or AMPA exposure. After being exposed to Roundup® Active or AMPA, PCNA-positive cells with strong immunoreactivity were found in some hypothalamic nuclei. Cells containing caspase-3 were found in all hypothalamic nuclei studied, but the labeling was stronger after Roundup® Active or AMPA exposure. Xenobiotics significantly increased the immunoreactivity area for all of the markers studied in the majority of cases (p<0.05). AMPA or Roundup® Active treated animals had a greater area of PCNA immunoreactivity than control or glyphosate alone treated animals (p<0.05). The effects observed after xenobiotic exposure were not due to increased water intake. The increased immunoreactivity areas observed for the markers studied suggest that xenobiotics induced a neuro-inflammatory response, implying increased cell proliferation, glial activation, and induction of apoptotic pathways. The findings also show that glyphosate metabolites/adjuvants and/or surfactants present in glyphosate commercial formulations had a greater effect than glyphosate alone. In summary, glyphosate, AMPA, and glyphosate-based herbicides altered GFAP, caspase-3, and PCNA expression in the rat hypothalamus, altering the neuroendocrine axis.
Dechartres J, Pawluski JL, Gueguen, MM, Jablaoui A, Maguin E, Rhimi M, et al. Glyphosate and glyphosate-based herbicide exposure during the peripartum period affects maternal brain plasticity, maternal behaviour and microbiome. J Neuroendocrinol 2019;31:e12731.
Kwiatkowska M, Michałowicz J, Jarosiewicz P, Pingot D, Sicińska P, Huras B, et al. Evaluation of apoptotic potential of glyphosate metabolites and impurities in human peripheral blood mononuclear cells (in vitro study). Food Chem Toxico 2020;135:110888.
DOI: https://doi.org/10.1016/j.fct.2019.110888
Van Bruggen AHC, He MM, Shin K, Mai V, Jeong KC, Finckh MR, et al. Environmental and health effects of the herbicide glyphosate. Sci Total Environ 2018;616-617:255-68.
DOI: https://doi.org/10.1016/j.scitotenv.2017.10.309
Fuchs B, Saikkonen K, Helander M. Glyphosate-modulated biosynthesis driving plant defense and species interactions. Trends Plant Sci 2021;26:312-23.
DOI: https://doi.org/10.1016/j.tplants.2020.11.004
Erban T, Stehlik M, Sopko B, Markovic M, Seifrtova M, Halesova T, et al. The different behaviors of glyphosate and AMPA in compost-amended soil. Chemosphere 2018;207:78–83.
DOI: https://doi.org/10.1016/j.chemosphere.2018.05.004
Okada E, Allinson M, Barral MP, Clarke B, Allinson G. Glyphosate and aminomethylphosphonic acid (AMPA) are commonly found in urban streams and wetlands of Melbourne, Australia. Water Res 2020;168:115139.
DOI: https://doi.org/10.1016/j.watres.2019.115139
Soukup ST, Merz B, Bub A, Hoffmann I, Watzl B, Steinberg P, et al. Glyphosate and AMPA levels in human urine samples and their correlation with food consumption: results of the cross-sectional KarMeN study in Germany. Arch Toxicol 2020;94:1575-84.
DOI: https://doi.org/10.1007/s00204-020-02704-7
Agostini LP, Dettogni RS, dos Reis RS, Stur E, dos Santos EVW, Ventorim DP, et al. Effects of glyphosate exposure on human health: Insights from epidemiological and in vitro studies. Sci Total Environ 2020;705:135808.
DOI: https://doi.org/10.1016/j.scitotenv.2019.135808
Cattani D, Cesconetto PA, Tavares MK, Parisotto EB, De Oliveira PA, Rieg CEH, et al. Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress. Toxicology 2017;387:67-80.
DOI: https://doi.org/10.1016/j.tox.2017.06.001
Gallegos CE, Bartos M, Bras C, Gumilar F, Antonelli MC, Minetti A. Exposure to a glyphosate-based herbicide during pregnancy and lactation induces neurobehavioral alterations in rat offspring. Neurotoxicology 2016;53:20-8.
DOI: https://doi.org/10.1016/j.neuro.2015.11.015
Maddalon A, Galbiati V, Colosio C, Mandic-Racevic E. Glyphosate-based herbicides: Evidence of immune-endocrine alteration. Toxicology 2021;459:152851.
DOI: https://doi.org/10.1016/j.tox.2021.152851
de Souza JS, Laureano-Melo R, Herai RH, da Conceição RR, Oliveira KC, et al. Maternal glyphosate-based herbicide exposure alters antioxidant-related genes in the brain and serum metabolites of male rat offspring. Neurotoxicology 2019;74:121-31.
DOI: https://doi.org/10.1016/j.neuro.2019.06.004
Lanzarin GAB, Venâncio CAS, Monteiro SM, Félix LM. Behavioural toxicity of environmental relevant concentrations of a glyphosate commercial formulation - RoundUp® UltraMax - During zebrafish embryogenesis. Chemosphere 2020;253:126636.
DOI: https://doi.org/10.1016/j.chemosphere.2020.126636
Teleken JL, Gomes ECZ, Marmentini C, Moi MB, Ribeiro RA, Balbo SL, et al. Glyphosate-based herbicide exposure during pregnancy and lactation malprograms the male reproductive morphofunction in F1 offspring. J Dev Orig Health Dis 2020;11:146-53.
DOI: https://doi.org/10.1017/S2040174419000382
Riaño-Quintero C, Gómez-Ramírez E, Hurtado-Giraldo H. Glyphosate commercial formulation effects on preoptic area and hypothalamus of cardinal neon Paracheirodon axelrodi (Characiformes: Characidae). Neotrop Ichthyol 2019;17:1–8.
DOI: https://doi.org/10.1590/1982-0224-20190025
Spinaci M, Nerozzi C, Tamanini Cl, Bucci D, Galeati G. Glyphosate and its formulation Roundup impair pig oocyte maturation. Sci Rep 2020;10:1-10.
DOI: https://doi.org/10.1038/s41598-020-68813-6
Zanardi MV, Schimpf MG, Gastiazoro MP, Milesi MM, Muñoz-deToro M, Varayoud J, et al. Glyphosate-based herbicide induces hyperplastic ducts in the mammary gland of aging Wistar rats. Mol Cell Endocrinol 2020;501:110658.
DOI: https://doi.org/10.1016/j.mce.2019.110658
Serra L, Estienne A, Vasseur C, Froment P, Dupont J. Review: Mechanisms of glyphosate and glyphosate-based herbicides action in female and male fertility in humans and animal models. Cells 2021;10:3079 .
DOI: https://doi.org/10.3390/cells10113079
Romano RM, Oliveira JM, Oliveira VM, Oliveira IM, Torres YR, Bargi-Souza P, et al. Could glyphosate and glyphosate-based herbicides be associated with increased thyroid diseases worldwide? Front Endocrinol 2021;12:627167.
DOI: https://doi.org/10.3389/fendo.2021.627167
Poppola S, Sakpa C. Hormones of pituitary-gonadal axis and histology of pituitary glands following oral treatment of male Wistar rats with glyphosate. J Biomed Res Clin Pract 2018;1:164-9.
DOI: https://doi.org/10.46912/jbrcp.57
Fu H, Gao F, Wang X, Tan P, Qiu S, Shi B, et al. 2020. Effects of glyphosate-based herbicide-contaminated diets on 2 reproductive organ toxicity and hypothalamic-pituitary-ovarian axis 3 hormones in weaned piglets. Environ Pollut 2020;272:115596.
DOI: https://doi.org/10.1016/j.envpol.2020.115596
Martínez MA, Ares I, Rodríguez JL, Martínez M, Martínez MR, Anadón A. Neurotransmitter changes in rat brain regions following glyphosate exposure. Environ Res 2018;161:212-9.
DOI: https://doi.org/10.1016/j.envres.2017.10.051
Dell’Armelina PR, Ribeiro MO, Sandini TM, Adari ELR, Bernardi MM, Spinosa HS. Perinatal glyphosate-based herbicide impaired maternal behavior by reducing the striatal dopaminergic activity and delayed the offspring reflex development. Atas de Saúde Ambiental 2019;7:130-56.
de Araújo-Ramos AT, Passoni MT, Romano MA, Romano RM, Martino-Andrade AJ. Controversies on endocrine and reproductive effects of glyphosate and glyphosate-based herbicides: a mini-review. Front Endocrinol 2021;12:627210.
DOI: https://doi.org/10.3389/fendo.2021.627210
Baxter PS, Dando O, Emelianova K, He X, McKay S, Hardingham GE, et al. Microglial identity and inflammatory responses are controlled by the combined effects of neurons and astrocytes. Cell Rep 2021;34:108882.
DOI: https://doi.org/10.1016/j.celrep.2021.108882
Cabezas R, El-Bachá RS, González J, Barreto GE. Mitochondrial functions in astrocytes: neuroprotective implications from oxidative damage by rotenone. Neurosci Res 2012;74:80-90.
DOI: https://doi.org/10.1016/j.neures.2012.07.008
Giovannoni F, Quintana FJ. The role of astrocytes in cns inflammation. Trends Immunol 2020;41:805-19.
DOI: https://doi.org/10.1016/j.it.2020.07.007
Julien O, Wells JA. Caspases and their substrates. Cell Death Differ 2017;24:1380-9.
DOI: https://doi.org/10.1038/cdd.2017.44
Lossi L, Castagna C, Merighi A. Caspase-3 mediated cell death in the normal development of the mammalian cerebellum. Int J Mol Sci 2018;19:3999.
DOI: https://doi.org/10.3390/ijms19123999
Benachour N, Séralini GE. Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells. Chem Res Toxicol 2019;22:97-105.
DOI: https://doi.org/10.1021/tx800218n
Chaufan G, Coalova I, Ríos-De Molina M. Glyphosate commercial formulation causes cytotoxicity, oxidative effects, and apoptosis on human cells: Differences with its active ingredient. Int J Toxicol 2014;33:29-38.
DOI: https://doi.org/10.1177/1091581813517906
Clair É, Mesnage R, Travert C, Séralini GÉ. A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels. Toxicol In Vitro 2012;26:269-79.
DOI: https://doi.org/10.1016/j.tiv.2011.12.009
Dodel RC, Du Y, Bales KR, Ling Z, Carvey PM, Paul SM. Caspase-3-like proteases and 6-hydroxydopamine induced neuronal cell death. Mol Brain Res 1999;64:141-8.
DOI: https://doi.org/10.1016/S0169-328X(98)00318-0
Martínez MA, Rodríguez JL, Lopez-Torres B, Martínez M, Martínez-Larrañaga MR, Maximiliano JE, et al, Ares I. Use of human neuroblastoma SH-SY5Y cells to evaluate glyphosate-induced effects on oxidative stress, neuronal development and cell death signaling pathways. Environ Int 2020;135:105414.
DOI: https://doi.org/10.1016/j.envint.2019.105414
Mesnage R, Clair E, Gress S, Then C, Székács A, Séralini GE. Cytotoxicity on human cells of Cry1Ab and Cry1Ac Bt insecticidal toxins alone or with a glyphosate-based herbicide. J Appl Toxicol 2013;33:695-9.
DOI: https://doi.org/10.1002/jat.2712
McKenzie B, Fernandes JP, Doan MAL, Schmitt LM, Branton WG, Power C. Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis. J Neuroinflammation 2020;17:253.
DOI: https://doi.org/10.1186/s12974-020-01902-5
Glushakova OY, Glushakov AO, Borlongan CV, Valadka AB, Hayes RL, Glushakov AV. Role of caspase-3-mediated apoptosis in chronic caspase-3-cleaved tau accumulation and blood-brain barrier damage in the corpus callosum after traumatic brain injury in rats. J Neurotrauma 2018;35:157-73.
DOI: https://doi.org/10.1089/neu.2017.4999
Ino H, Chiba T. Expression of proliferating cell nuclear antigen (PCNA) in the adult and developing mouse nervous system. Mol Brain Res 2000;78:163-74.
DOI: https://doi.org/10.1016/S0169-328X(00)00092-9
Dechartres J, Pawluski JL, Gueguen MM, Jablaoui A, Maguin E, Rhimi M, et al. Glyphosate and glyphosate‐based herbicide exposure during the peripartum period affects maternal brain plasticity, maternal behaviour and microbiome. J Neuroendocrinol 2019;31:e12731.
DOI: https://doi.org/10.1111/jne.12731
Panza SB, Vargas R, BalboSL, Bonfleur ML, Granzotto DCT, Sant’Ana DMG, et al. Perinatal exposure to low doses of glyphosate-based herbicide combined with a high-fat diet in adulthood causes changes in the jejunums of mice. Life Sci 2021;275:119350.
DOI: https://doi.org/10.1016/j.lfs.2021.119350
Ait-Bali Y, Ba-M’hamed S, Gambarotta G, Sassoè-Pognetto M, Giustetto M, Bennis M. Pre- and postnatal exposure to glyphosate-based herbicide causes behavioral and cognitive impairments in adult mice: evidence of cortical ad hippocampal dysfunction. Arch Toxicol 2020;94:1703-23.
DOI: https://doi.org/10.1007/s00204-020-02677-7
Coveñas R, Duque E, Mangas A, Marcos P, Narváez J. Neuropeptides in the monkey (Macaca fascicularis) brainstem. In: Mangas A, Coveñas R, Geffard M, editors, Brain molecules: from vitamins to molecules for axonal guidance. Trivadrum: Transworld Research Network; 2007. pp. 1-26.
Duque-Díaz, E, Díaz-Cabiale Z, Narváez JA, Coveñas R. Mapping of enkephalins and adrenocorticotropic hormone in the squirrel monkey brainstem. Anat Sci Int 2017;92:275-92.
DOI: https://doi.org/10.1007/s12565-016-0333-2
Guntern R, Vallet PG, Bouras C, Constantinidis J. An improved immunohistostaining procedure for peptides in human brain. Experientia 1989;45:159-61.
DOI: https://doi.org/10.1007/BF01954858
Paxinos G, Watson C. The rat brain in stereotaxic coordinates. Elsevier: Amsterdam; 2009.
Mateos J, Pascau J. Image processing with ImageJ. Pack Publishing; 2015.
Li H, Hong T, Zhu Q, Wang S, Huang T, Li X, et al. Paraquat exposure delays late-stage Leydig cell differentiation in rats during puberty. Environ Pollut 2019;255:113316.
DOI: https://doi.org/10.1016/j.envpol.2019.113316
Turkmen R, Birdane YO, Demirel HH, Kabu M, Ince S. Protective effects of resveratrol on biomarkers of oxidative stress, biochemical and histopathological changes induced by sub-chronic oral glyphosate-based herbicide in rats. Toxicol Res 2019;8:238-45.
DOI: https://doi.org/10.1039/C8TX00287H
Gallegos CE, Bartos M, Gumilar F, Rasman-Vozari R, Minetti A, Baier CJ. Intranasal glyphosate-based herbicide administration alters the redox balance and the cholinergic system in the mouse brain. Neurotoxicology 2020;77:205-15.
DOI: https://doi.org/10.1016/j.neuro.2020.01.007
Hashim AR, Bashir DW, Yasin NAE, Galal MK, El-Gharbawy S. Ameliorative effect of N-acetylcysteine against glyphosate-induced hepatotoxicity in adult male albino rats: histopathological, biochemical, and molecular studies. Environ Sci Pollut 2021;28:42275-89.
DOI: https://doi.org/10.1007/s11356-021-13659-2
Stur E, Aristizabal-Pachon AF, Peronni KC, Agostini LP, Waigel S, Chariker J, et al. Glyphosate-based herbicides at low doses affect canonical pathways in estrogen positive and negative breast cancer cell lines. PLoS One 2019;14:e0219610.
DOI: https://doi.org/10.1371/journal.pone.0219610
George J, Shukla Y. Emptying of intracellular calcium pool and oxidative stress imbalance are associated with the glyphosate-induced proliferation in human skin keratinocytes HaCaT cells. ISRN Dermatol 2013;2013:825180.
DOI: https://doi.org/10.1155/2013/825180
González-Magaña A, Blanco FJ. Human PCNA structure, function and interactions. Biomolecules 2020;10:570.
DOI: https://doi.org/10.3390/biom10040570
McComb S, Chan PK, Guinot A, Hartmannsdottir H, Jenni S, Dobay MP, et al. Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7. Sci Adv 2019;5:eaau9433.
DOI: https://doi.org/10.1126/sciadv.aau9433
Gui YX, Fan XN, Wang HM, Wang G, Chen SD. Glyphosate induced cell death through apoptotic and autophagic mechanisms. Neurotoxicol Teratol 2012;34:344-9.
DOI: https://doi.org/10.1016/j.ntt.2012.03.005
Gómez E. Apoptosis and cell proliferation sites in the brain of P. brachypomus exposed to sublethal concentrations of Roundup Active. J Surg Clin Pract 2021;5:1-2.
Burguillos MA, Deierborg T, Kavanagh E, Persson A, Hajji N, Garcia-Quintanilla A, et al. Caspase signalling controls microglia activation and neurotoxicity. Nature 2011;472:319-24.
DOI: https://doi.org/10.1038/nature09788
Ministerio de Vivienda, Ciudad y Territorio de Colombia. Resolución 1435 (in Spanish). 2007. Available from: https://www.minvivienda.gov.co/normativa/resolucion-1435-2017
Mesnage R, Benbrook C, Antoniou MN. Insight into the confusion over surfactant co-formulants in glyphosate-based herbicides. Food Chem Toxicol 2019;128:137-45.
DOI: https://doi.org/10.1016/j.fct.2019.03.053
Szepanowski F, Szepanowski LP, Mausberg AK, Albrecht P, Kleinschnitz C, Kieseier BC, et al. Differential impact of pure glyphosate and glyphosate-based herbicide in a model of peripheral nervous system myelination. Acta Neuropathol 2018;136:979-82.
DOI: https://doi.org/10.1007/s00401-018-1938-4
Mesnage R, Antoniou MN. Ignoring adjuvant toxicity falsifies the safety profile of commercial pesticides. Front Public Health 2018;5:361.
DOI: https://doi.org/10.3389/fpubh.2017.00361
Ait Bali Y, Ba-Mhamed S, Bennis M. Behavioral and immunohistochemical study of the effects of subchronic and chronic exposure to glyphosate in mice. Front Behav Neurosci 2017;11:146.
DOI: https://doi.org/10.3389/fnbeh.2017.00146
Limberger C, Ferreira PCL, Fontella FU, Laydner AC, Oliveira J, Bortoluzzi-Salles G, et al. Glyphosate-based herbicide alters brain aminoacid metabolism withou taffecting blood-brain barrier integrity. Alzheimer’s Dement 2020;16:e043847.
DOI: https://doi.org/10.1002/alz.043847
Martinez A, Al-Ahmad AJ. 66. Effects of glyphosate and aminomethylphosphonic acid on an isogeneic model of the human blood-brain barrier. Toxicol Lett 2019;304:39-49.
DOI: https://doi.org/10.1016/j.toxlet.2018.12.013
Mitra A, Guèvremont G, Timofeeva E. Stress and sucrose intake modulate neuronal activity in the anterior hypothalamic area in rats. PLoS One 2016;11:e0156563.
DOI: https://doi.org/10.1371/journal.pone.0156563
Pu Y, Yang J, Chang L, Qu Y, Wang S, Zhang K, et al. Maternal glyphosate exposure causes autism-like behaviors in offspring through increased expression of soluble epoxide hydrolase. Proc Natl Acad Sci USA 2020;117:11753-9.
DOI: https://doi.org/10.1073/pnas.1922287117
Ethics Approval
This study was approved by the Research Commission of the Universidad de Santander (Bucaramanga, Colombia) under act n. 009-VIISupporting Agencies
Ministerio de Ciencia y Tecnología (MINCIENCIAS) from Colombia (129977758110)How to Cite
Duque-Díaz, E. ., Hurtado Giraldo, H., Rocha-Muñoz, L. P., & Coveñas, R. (2022). Glyphosate, AMPA and glyphosate-based herbicide exposure leads to GFAP, PCNA and caspase-3 increased immunoreactive area on male offspring rat hypothalamus . European Journal of Histochemistry, 66(4). https://doi.org/10.4081/ejh.2022.3428
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