{"paper_id":"d1be145d-ee72-4aae-8ba7-0425ad7f699a","body_text":"Glyphosate (GLP),  N -(phosphonomethyl)glycine,\nis an active ingredient (ActI) of the most common herbicides used\nin contemporary agriculture, forestry, industrial weed control, lawn,\ngarden, and aquatic environments. By slow, reversible, and competitive\ninhibition of 5-enolpyruvynyl-shikimate-3-phosphate synthase (EPSPS,\nEC 2.5.1.19), responsible for the biosynthesis of aromatic amino acids\nin plants 1  and several strains of bacteria,\nyeast, algae, and fungi, GLP acts as one of the most effective and\nbroad-spectrum agrochemicals ever produced. 2 , 3  First\nsynthesized in 1950, then commercialized in the herbicide market in\n1974, GLP has become the most widely used, postemergent, nonselective\nweedkiller worldwide. 4\nAgricultural\nuse of genetically engineered (GE) GLP-tolerant crops,\ncommercial GLP-surfactant (GLP-SH), and GLP-based herbicides (GBHs),\noften used as Roundup or RangerPro commercial formulations, applied\nwithin the so-called “green burn-downs” has modernized\nthe harvest, weed, and herbicide management. In recent years, GBH\nusage has increasingly been withdrawn in EU, the U.S.A., and then\nworldwide. 5  Significant causes include\nenvironmental pollution of GBH, the development of herbicide-resistant\nweeds (superweeds) and microorganisms (superbugs), as well as overconsumption\nof GE organisms and GBH-contaminated products. 6 − 8  Concerningly,\nthe relevant epidemiological and environmental contamination risk\nand human toxicity are driven by the growing reports on the health\nissues among farmers and occupational workers of GBH factories. 9 , 10\nThe most poisonous xenobiotics present in GBHs are GLP metabolites,\ne.g., (aminomethyl)phosphonic acid (AMPA), dyes, antifoaming agents,\ninert ingredients, and adjuvants, e.g., ethoxylates and polyoxyethyleneamine\n(POEA), more toxic than GLP itself, 11 − 14  and ppb traces of heavy metals,\nincluding chromium, cobalt, lead, or nickel. 15  Notably, the toxicity of these coformulants is diverse and variant.\nAccording to the insightful review of pesticide variability, 16  ∼750 different GBH formulations on the\nmarket contain various combinations of the GLP active principle and\ncoformulants. Moreover, because of the legal regulations, the total\ncomposition of GBH is classified as confidential commercial information. 17  Hence, in commercial brochures and scientific\nreports, this heterogeneity of ingredients is usually simplified and\ndefective, which may cause confusion and potential risk, even though\nthe toxicological details of each ingredient included are well-described\nin the medical literature.\nThe current review article presents\nthe poisoning with GLP, GLP-SH,\nand GBHs impact on the endocrine, reproductive, and cardiopulmonary\nsystems, also mentioning its hepato- and nephrotoxic consequences.\nBesides, we introduce the potential carcinogenic effects of this poisoning.\nAilments of the gastrointestinal tract and nervous system, including\ngut dysbiosis and the dysregulation of microbiota-gut-brain-axis,\nare discussed in our review article concerning GLP and GBH toxicity, 18  whereas an extensive summary of novel technologies\napplied to GLP sensing is presented in our recent critical review. 19\n\nGLP and GBHs are remarkably poisonous to the gut microbiome (gut\nmicrobiota, GM) 20 , 21  and the neurological system 22 − 25  by affecting the microbiota-gut-brain (MGB) axis and GLP-mediated\ninhibition of acetylcholinesterase (AChE) and cholinergic neurotransmission. 22 , 26 − 29  GBHs have also been cautioned as endocrine system disruptors (ESDs). 30 − 34  However, over decades, this topic has become exceptionally controversial 35 , 36  ( Tables  1  and  2 ). Regardless of several studies demonstrating the\ndysregulating activity of GLP/GBH on the hypothalamic-pituitary-adrenal\n(HPA) axis, hypothalamic-pituitary-peripheric glands (HPP) axes, 23 , 37  steroidogenesis, 38 − 42  or reproductive system, 37 , 43 − 49  this ESD activity has been questioned by the EPA’s Endocrine\nDisruptor Screening Program (EDSP) and, subsequently, by the European\nFood Safety Authority (EFSA). In 2015, the EPA and EFSA eventually\ndeclined the direct interaction of GLP with estrogen, androgen, and\nthyroid (EAT) pathways involving modes of action. 50 − 52  Because of\nthe absence of the EPSP-metabolic pathway in vertebrates and the quick\nelimination of GLP, after its absorption and accumulation, from mammals,\nthe GLP half-life time (∼5 to 10 h) is relatively short. Therefore,\nGLP is expected to be slightly or minimally toxic. However, many distressing\nreports have recently pointed to the GLP and GBH pathogenesis, indicating\nboth direct and indirect influences on the intestinal tract and GM 25 , 53 , 54  and reproductive system, 43 , 55  as well as neurotoxicity and MGB-associated neurological disorders, 20 , 21  and carcinogenicity 43  ( Table  3 ).\nn. m.: not measured.\nn. m.: not measured.\nn. m.: not measured.\n\nThe endocrine system regulates metabolism, respiration,\nmood, mechanosensory perception and movement, growth, reproduction,\nsexual development by producing and secreting hormones 56 − 58  ( Figure  1 ). Endocrinology\nmentions two categories of endocrine system diseases, namely, (i)\nhormone imbalance, resulting from the failure of the endocrine feedback\nsystem and causing hyposecretion or hypersecretion, i.e., hormone\ndeficiency or hormone excess, respectively, and (ii) diseases resulting\nfrom infections, injuries, tumors, or genetic issues, which may lead\nto hormone imbalance. Major endocrine system diseases involve diabetes,\nhyper- or hypothyroidism, adrenal insufficiency, Cushing’s\ndisease, and sex hormone disorders, including hermaphroditism, hypogonadism,\nprecocious puberty, and multiple endocrine neoplasia. 56 − 59  Exposure to environmental toxicants and EDCs also dysregulates the\nhormonal balance homeostasis. 60 , 61  The EDCs, first reported\nin the 1990s, include pharmaceuticals, plastics (phthalates), pesticides,\ncosmetics, detergents, and phytoestrogens. Experimental pieces of\nevidence highlight the endocrine-disrupting activity of GLP and GBHs. 31 , 35 , 52  Nonetheless, according to the\nEPA’s EDSP and EFSA, there is no sufficient evidence to support\nthe endocrine-disrupting effects of GLP, as it exhibits no direct\ninteraction with the EAT pathways. 35 , 52  However, this\nissue has still been debated in the EU and Brazil. 62\nThe GBH impact on endocrine and reproductive systems. The scheme\npresents an overview of GLP and GBH toxicity in male and female reproductive\nsystems. By deviating the hormone production and activities, the herbicides\nimpact the development and functionality of Sertoli cells, Leydig\ncells, and sperm cells in seminiferous tubules of the testes and oocytes\nin ovaries. Besides, they cause, e.g., endometriosis and polycystic\nsyndrome, thus leading to infertility, embryotoxicity, and teratogenicity.\n(A–F) Hematoxylin and eosin (X 100) staining of ovarian sections\nof GLP-treated mice showing (A) the normal histological ovarian follicle\nstructure and (B, C) increased numbers of atretic follicles after\nmice treatment with (B) GLP and (C) Roundup. (D–F) Hematoxylin\nand eosin (X 200) staining showing (D) the normal ovarian interstitial\ncell structure and (E, F) the interstitial fibrosis after mice treatment\nwith (E) GLP and (F) Roundup. Black arrows indicate lesioned regions.\nAdapted with permission from ref ( 37 ). Copyright Elsevier 2018.\nTen key characteristics (KCs) of the EDCs have\nbeen classified\nto describe their mechanistic impacts on the endocrine system functionality. 63  (i) The EDCs interact or activate hormone receptors,\ne.g., androgen receptor (AR), estrogen receptors (ERα, ERβ),\nand progesterone receptor (ProgR). As revealed by preclinical studies,\nGLP plays an ambiguous role as a hormone mimic, 64 , 65  being either antiestrogenic in hormone- and dose-dependent manner\nactivity 41  or ER-antagonistic 66  in a ligand-independent and GLP-specific manner. 67  Contrariwise, (ii) as an EDC, GLP may antagonize\nhormone receptors. However, no clear evidence for the GLP-mediated\nantagonistic effect on hormone activity has so far been reported. 41 , 66  For instance, using liver cells (HepG2), genotoxic, antiestrogenic,\nand aromatase-disruptive activities of GLP were compared with these\nof Roundup Express, Bioforce (Extra 360), Grands Travaux 400, and\nGrands Travaux 450. Among these formulations, applied in subagricultural\ndilutions (0.5–5 ppm), the toxicity of pure GLP was the lowest\nor negligible, whereas the carcinogen, mutagen, and reprotoxic actions\nof these formulations depended on the GBH’s adjuvant content\nrather than on the GLP concentration. 41  These outcomes are, however, in contrast to other pesticides. 68  To continue, (iii) GLP regulates the gene expression\nof hormone receptors in a dose-dependent manner in hormone-dependent\ncancer cells. 66  However, another study\nexcluded this activity in Leydig cells. 42  Yet, studies on pre- and postnatal, 47  perinatal, 69  and prepubertal rats 70  provide no support for this conclusion. Moreover,\n(iv) GLP alters the signal transduction, cell cycle, and cellular\ngrowth in hormone-responsive cells without direct interaction with\nthe hormone receptor, 32 , 71  as revealed by a study on prepubertal\nrat-derived Sertoli cells. 12  Consecutively,\n(v) GLP and GBH induce epigenetic modifications in hormone-producing\nor hormone-responsive cells. Examples of the in vivo GLP hazardous\nepigenotoxicity include epimutations (epigenetic traits), DNA hypomethylation\nof oncogenes, 72  histone targeting and chromatin\nremodeling, 69  dose-dependent hypermethylation\nof the CpG islands of the ER gene promoters, 73  transgenerational (F1, F2, and F3) pathologies, 43  miRNA and circRNA dysregulation-associated metabolic and\nneurodevelopmental disorders (NDDs), 74 , 75  and organ\nmalformations and congenital anomalies. 76  Moreover, (vi) GLP and GBHs alter steroidogenesis and hormone synthesis\nby disrupting the expression of steroidogenic acute regulatory (StAR)\nprotein, 77 − 81  demonstrated in vivo. 40 − 42  Importantly, in a study on human\nplacental JEG cells, toxic and inhibitory activities of Roundup, dosed\nat the agricultural concentration, were superior to those of GLP. 38  Furthermore, (vii) GLP and GBHs interfere with\nhormonal balance throughout the body by altering hormone transport\nacross endocrine cell membranes or vesicle secretion. 31  Roundup indirectly influenced the plasma membrane-linked\nendocrine disruption in pregnant female rats, 82  male pubertal rats, 83 , 84  and perinatal mice 37  ( Figure  1 A– 1 F). However, contradictory\ndata have also been presented. 42 , 70 , 85 , 86  In addition, (ix) as EDC, the\nGLP and GBHs are expected to deviate from hormonal metabolism and\nclearance mechanisms, including first-pass metabolism in the liver\nand excretion in the kidney. Yet, no experimental data have confirmed\nthis mechanism. 31  Finally, (x) GLP and\nGBHs influence the fate of hormone-producing or hormone-responsive\ncells by direct or indirect changes in differentiation, proliferation,\napoptosis, DNA repair, hypoxia, mutagenesis, and migration of target\nof effector endocrine cells. 32 , 42 , 66 , 67 , 71 , 87 − 89\nThe hypothalamus coordinates the endocrine system.\nBy consolidating signals from upper cortical inputs, autonomic function\nand physical cues, and peripheral hormonal feedback, the hypothalamus\nprovides specific signal outputs to the pituitary gland that subsequently\nsupplies the endocrine system with hormones stimulating the peripheral\nglands. 90  As EDCs, GLP and GBHs dysregulate\nthe functionality of the hypothalamus-pituitary and its connections\nwith HPP glands axes, including adrenal (HPA), thyroid (HPTh), and\ngonadal axes, i.e., ovaries (HPO) and testes (HPT). 35  However, the EPA, EFSA, and the Organization of Economic\nCo-operation and Development (OECD) have recently questioned the endocrine-disrupting\nactivity of GLP and excluded GLP as an EDC. A comprehensive experimental\nreview conducted within EPA’s EDSP and the European Centre\nfor Ecotoxicology and Toxicology of Chemicals (ECETOC) critically\nverified an endocrine-modulating or adverse potential of GLP on steroidogenesis\nand the EAT pathways in humans, other mammals, and wildlife. 52 , 91  Contemporary contradictory reports highlight, though, the harmful\nGLP impact on steroidogenesis, gonadal, and thyrotropic axes, and\nthe reproductive system. 23 , 30 , 33 , 37 , 43 , 49 , 82\nFor\nexample, GBH induced dysregulation of the HPTh axis, causing osteoporosis,\nskeletal dysfunctionality, and hypothyroidism in Kalach 360 SL-fed\nrats female and offspring. Malfunctions in the osteocytes and thyroid\ncells’ activity altered the estrogen, calcium, phosphates,\nphosphatase alkaline, and vitamin D levels, as well as decreased triiodothyronine\nand thyroxine levels, associated with an increased plasma level of\nthyroid-stimulating hormone. These malfunctions led to subosteoporotic\nthinning and discontinuity of bone trabecular with a significant decrease\nin intertrabecular links. 92  The impact\nof excessive exposure to GLP or GBHs on the functionality of the HPTh\naxis was summarized elsewhere. 52  Examples\nof the impacts of these herbicides on the HPA, HPO, and HPT axes are\ndiscussed below.\nReproductive system diseases, also called generational pathologies,\ncomprise (i) genetic and congenital abnormalities, including epimutations\nand epigenetic fertility issues, (ii) functional or structural genital\ndisorders associated with disruption of the endocrine system and hormonal\ndisorders, (iii) disturbances of pregnancy and embryonal or fetal\ndevelopment, and parturition, (iv) infections, and (v) tumors. 93 , 94  The pesticides and herbicides belong to well-known toxins causing\nmenstrual cycle disturbances, infertility, subfertility, prolonged\ntime-to-pregnancy (TTP), spontaneous abortion, miscarriage, stillbirth,\nhemangioma birthmarks, congenital malformations in the offspring,\nas well as endocrine and hormonal issues, and musculoskeletal and\nneurobehavioral disorders (NBDs). 95 , 96\nEpigenetics is a\nbranch of genetics treating the inheritance of stable phenotype changes\nthat arise from affected gene activity or expression without alterations\nin the DNA sequence, manifested as epigenetic traits (epimutations)\nin a chromosome that result from environmental (extracellular) impacts\non the DNA methylation, chromatin remodeling, and transgenerational\nepigenetic inheritance, etc. 97  GLP and\nGBHs trigger epimutations. For instance, the ancestral environmental\nexposure of F0 female rats to GLP caused no or minor epigenotoxicity\nin the F0 and F1 generations but became severely toxic in the F2 and\nF3 offspring. The transgenerational pathology, including differential\nDNA methylation regions, caused prostate disease, obesity, kidney\ndisease, ovarian disease, and parturition abnormalities. 43  Organ malformations and GLP tissue residuals,\nputatively associated with congenital anomalies, were observed in\none-day-old piglets born by females exposed to GLP in the first 40\ndays of pregnancy. The organs most severely damaged in the piglets\nwere the lungs, liver, kidney, brain, gut wall, and heart, whereby\nthe highest GLP tissue concentration, quantified by enzyme-linked\nimmunosorbent assay (ELISA), was in the lungs and hearts, whereas\nthe lowest was in muscles. 76\nGLP\nand GBHs destroy the production and functionality of gonads ( Figure  1 A). Roundup attenuated\nprogressive motility and destroyed the mitochondrial integrity of\nhuman sperm, 44  whereas GLP alone decreased\nthe sperm’s motility and caused sperm DNA fragmentation. 45  Moreover, GLP negatively affected sperm mitochondrial\nrespiration efficiency and worsened the harmful effect of dihydroxytestosterone\non sperm mitochondria. 98  Agent-specific\nimpacts were evaluated, as well. In pigs, both herbicides caused dose-dependent\ndecreases in sperm motility, viability, mitochondrial activity, and\nacrosome integrity but no changes in the DNA structure were observed.\nHowever, the toxicity of Roundup was more profound than that of GLP\nalone. 11 , 15\nGLP and GBH affect signaling pathways\nin cells responsible for adrenal gland steroidogenesis. The adult\nmale rats’ exposure to Roundup triggered apoptosis, reduced\nsystemic levels of corticosterone and adrenocorticotropic hormone\nreceptors, and altered the level of StAR protein phosphorylation.\nThe serum concentration of testosterone was decreased, as well as\naromatase levels and luteinizing hormone (LH) and follicle-stimulating\nhormone (FSH) gonadotropins deviated in male rat offspring of the\nperinatally GLP-exposed females. 99  GLP\nand Roundup endocrine cytotoxicity were evaluated in male estuarine\ncrabs ( Neohelice granulate ). Both herbicides decreased\nsperm count in spermatophores from the vas deferens and inhibited\nthe secretion and/or transduction of the androgenic gland hormone,\nthus dysregulating spermatogenesis. 100  Neuroendocrine\nand immune toxicity of GLP was demonstrated in lizards ( Salvator\nmerianae ). Blood morphology of the GLP-treated lizards revealed\nan elevated level of plasma corticosterone, decreases in the total\nwhite blood cell count and natural antibodies titres, and an increase\nin the lobularity index, thus indicating immunosuppression and symptoms\nof chronic infection, although differential white blood cell count,\nheterophils/lymphocytes index, and complement system have not deviated. 101\nGBHs affect testes development,\nleading to changes in testosterone levels, seminiferous tubules, and\npuberty progression ( Figure  1 A). In prepubertal rats, Roundup decreased testosterone levels\nwithout affecting corticosterone or estradiol levels, and it altered\nseminiferous tubules and germinal epithelium in a dose-dependent manner. 84  Feeding prepubertal male rat offspring GLP-containing\nsoy milk had toxic effects. GLP reduced testosterone levels and Sertoli\ncell numbers and increased the percentage of degenerated Sertoli and\nLeydig cells. Additionally, it reduced spermatid numbers, increased\nepididymal tail mass, and decreased seminiferous tubule diameter. 102  Perinatal mouse exposure to GLP or Roundup\nat acceptable daily intake concentrations in drinking water had agent-specific\noutcomes. GLP, but not Roundup, deviated from testis morphology, decreased\ntestosterone serum levels, and reduced undifferentiated spermatogonia\nnumbers by 60% in the GLP group. It was associated with the downregulation\nof the  Sal4  gene and the up-regulation of the  Nano3  gene related to germ cell differentation, as well\nas the  Bax  and  Bcl2  genes, involved\nin apoptosis. 34  Moreover, maternal gestational\nexposure to Roundup altered masculinization of male offspring masculinization.\nAt 60 days old, males from Roundup-treated dams showed increased sexual\npartner preference scores, elevated serum testosterone and estradiol\nlevels, LH and FSH mRNA expression, LH and FSH gonadotropin protein\ncontent in the pituitary gland, deviated sperm production, and testicular\nmorphology alterations. They also experienced an early onset of puberty. 99  Similar outcomes were observed in a study on\nattenuating the effects of Roundup on male mouse offspring from females\nexposed to Roundup in drinking water from the fourth day of pregnancy\nto the end of the lactation period. In F1 males from the GBH group,\ntesticular descent was delayed, spermatozoa in the cauda epididymis\nwere reduced, seminiferous epithelium height was decreased, intratesticular\ntestosterone levels were increased, and the HPT axis was dysregulated. 33\nExposure of both prepubertal and postpubertal\nmale rats to GBHs promotes mammary gland development by increasing\ncollagen fiber organization and terminal end buds. Additionally, GBH-treated\nrats exhibited higher levels of mast cell infiltration, ERα\nexpression, and proliferation index than control rats. 70  Roundup induced Ca2+-dependent oxidative stress\nand activated multiple endoplasmic reticulum stress-response pathways,\nleading to Sertoli cell death and reduced spermatogenesis in prepubertal\nrat testes. Exposure to GLP alone produced similar effects. 12  A study comparing GLP, POEA, and GBHs (Roundup\nand Glyphogan) at concentrations ranging from environmental- to agricultural-use\nlevels in an immature Sertoli cell line (TM4) revealed that the GBH\nformulation caused mitochondrial dysfunction, disrupted cellular detoxification\nsystems, and led to lipid droplet accumulation and necrosis. Overexposure\nto POEA resulted in excessive lipid accumulation, suggesting that\ncell death followed immediate penetration and overload of the formulants\ninside the cells. 103  Contradictory results\nemerged from comparing the GBH formulation (Glyfonova) and an equivalent\namount of GLP on rat testes and androgen functionality. GLP had no\nsignificant impact on testes or testosterone synthesis, whereas Glyfonova\nonly slightly upregulated the steroidogenic genes Cyp11a1 and Cyp17a1,\nrelated to aromatase ( Figure  1 A). 104\nPerinatal exposure\nof rats to Roundup Transorb during a critical\nperiod of sexual differentiation led to HPT axis dysfunction, including\nincreased LH and FSH mRNA expression levels, elevated LH protein in\nthe pituitary gland, higher serum LH concentrations in adult male\noffspring, and subsequent pro-angiogenic effects. This dysregulation\nboosted blood testosterone levels, enhanced sperm production, and\nincreased the weight of reproductive organs. 99  In rats exposed to Roundup in utero and postnatally, there was documented\nevidence of an increase in anogenital distance. 30  Meanwhile, exposure of prepubertal rats to Roundup resulted\nin an antiandrogenic effect, lowering systemic testosterone levels\nand inhibiting male puberty entry. 84  Male\nmice exposed to Roundup during gestation and lactation experienced\ndelayed testis descent and decreased spermatozoa in the cauda epididymis. 33  Lastly, when adult rats were orally administered\ntechnical-grade Roundup, it disrupted the transcription of StAR mRNAs,\nleading to lipid droplet accumulation in the adrenal gland, increased\ngland weight, and reduced levels of corticosterone, adrenocorticosterone,\nand phosphorylated CREB. 79\nTreatment with GLP or\nGBH deviates from the functionality of the HP-ovaries axis, thus triggering\novarian failure and deteriorating the quality of oocytes ( Figure  1 ). In mice, pure\nGLP dysregulated metaphase II oocyte quality, disrupted the microtubule\norganizing center, formation of a spindle fiber, and chromosomal alignment,\nand chelated zinc cations, which decreased its intracellular content\nand caused reactive oxygen species (ROS)-mediated embryo damage. 105  A comparison of GLP and Roundup activities\nin pig oocytes revealed that Roundup impaired oocyte development and\nblastocyst rate deviated steroidogenesis in cumulus cells and increased\nintracellular levels of ROS, wherein the Roundup impact was higher\nthan that of an equivalent amount of pure GLP. 106  Disrupting activity of orally administered technical grade\nGLP and Roundup on ovaries was demonstrated in pregnant mice and their\nfetuses during the gestation period (first 19 days). The body, ovaries,\nliver weight, and mature follicles in treated mice decreased, whereas\natretic follicles and interstitial fibrosis increased. Both progesterone\nand estrogen levels were significantly changed, as well as the expression\nlevels of  GnRH  (gonadotropin-releasing hormone),  LHR ,  FSH ,  3β-HSD , and  Cyp19a1  genes at the hypothalamic-pituitary-ovarian\n(HPO) axis. The herbicide treatment induced oxidative stress, manifested\nby increased T-AOC, CAT, and glutathione peroxidase (GSH-Px) activity\nand high malondialdehyde (MDA) content in the serum and ovaries.\nFinally, prenatal exposure to GLP altered the sex ratio of the litter 37  ( Figure  1 A– 1 F).\nExtensive studies on rats\nhave demonstrated that excessive exposure to GLP or GBH destroyed\nthe uterus’s development and functionality, as well as morphological\nand physiological features 46 − 49  ( Figure  1 ). For example, in adult ovariectomized rats subcutaneously\ninjected with a GBH formulation, there were no changes in uterine\nweight or epithelial proliferation, but the GBH injection increased\nthe luminal epithelial cell height and downregulated the ERα\nmRNA and protein levels in luminal epithelial cells, whereas the ERα\nwas upregulated in the stroma. Moreover, the GBH injection upregulated\nERβ and ProgR expression levels. 107  In another study, the GBH exposure deviated the activity of ERα,\nProgR, homeobox protein Hox-A10 (HOXA10), and Wnt7a that regulate\nuterine organogenetic differentiation, causing luminal epithelial\nhyperplasia and increases in the stromal and myometrial thickness. 108  Ovarian follicular dynamics, associated with\nincreased proliferation of granulosa and theca cells, was altered,\nexpression of FSHR and GDF9 mRNA was downregulated, and proliferative\nactivity of the uteri cells was decreased in GBH-exposed prepubertal\nlambs. Noteworthy, none of these outcomes were in the lambs treated\nwith GLP or AMPA. 109\nEarly postnatal\nexposure to GBH induced lasting morphological changes in the female\nrat mammary gland, including a fibroblastic-like stroma, a higher\npercentage of hyperplastic ducts, and increased expression of steroid\nhormone receptors 110  ( Figure  1 ). In prepubertal rats, GBH\nincreased uterine sensitivity to estradiol, leading to endometrial\nhyperplasia characterized by increased luminal and glandular epithelial\nheight and stromal nuclei density. 46  In\nneonatal rats exposed to GBH, alterations in endometrial decidualization\nat implantation sites were associated with dysregulated expression\nlevels of estrogen and progesterone receptors (ER and ProgR), as well\nas endocrine pathway-regulating markers (HOXA10) and proliferation\nmarkers. 47 , 48  In another study involving rat females exposed\nto either pure GLP or GBH from gestational day 9 until weaning, herbicide\nexposure induced preimplantation losses in the F1 generation, increased\n17β-estradiol serum levels, and upregulated ERα expression.\nGLP specifically downregulated ProgR mRNA expression. Additionally,\nHOXA10 and Lif genes were downregulated in herbicide-treated rats. 49  In weaned pigs, GLP and Roundup administered\nthrough feed had insignificant effects on the vulvar size and the\nindex of reproductive organs. However, they altered the uterine and\novarian ultrastructure and disrupted the synthesis and secretion of\nLH, FSH, GnRH, and testosterone. Roundup also caused an imbalance\nin hydrogen peroxide and MDA levels in reproductive organs 23  ( Figure  1 ). In cows, GLP directly stimulated estradiol secretion from\ngranulosa cells, while both GLP and Roundup had varying effects on\noxytocin and progesterone secretion from luteal cells, leading to\ndeviations in the estrous cycle and uterine contractions that could\nresult in infertility. Additionally, both formulations decreased prostaglandin\nsecretion from endometrial cells but did not directly affect the basal\nand oxytocin-stimulated force of the motor functions of the myometrium. 111\nGLP\nand GBH directly impact embryonic and fetal development, as evidenced\nby various experimental findings 112  ( Figure  1 ). For instance,\nGLP led to carbonic anhydrase inhibition and ROS-triggered cellular\napoptosis in zebrafish embryos, resulting in multiorgan and body malformations. 113  Lizards treated with Roundup and Panzer Gold\nformulations at different stages of embryonic development (3–5\nand 33 days) exhibited embryonic and hematological alterations in\ntheir blood samples. 114  Embryotoxicity\nand teratogenicity in  Xenopus laevis , three GBH formulations (Roundup, Kilo Max, and Enviro Glyphosate)\nwere higher than those of GLP alone. These GBHs caused cardiac and\nabdominal edema and altered gut formation and axial malformations.\nIn  X. laevis  embryos, GLP and GBHs induced cephalic\nabnormalities, abnormal neural crest development, and anterior-posterior\naxis shortening, resulting in cranial cartilage deformities at the\ntadpole stages. Notably, the highest teratogenic indices indicated\nthat Roundup and Enviro Glyphosate caused the most severe harm. 115\nGBH exposure similarly affected chicken\nembryos, leading to the gradual loss of rhombomere domains, decreased\noptic vesicles, and the development of microcephaly, which was linked\nto increased endogenous retinoic acid activity. These effects underscore\nthe direct impact of GBHs on the early morphogenesis of the vertebrate\nnervous system. 116  In contrast, a 52 week\nstudy in an avian model revealed that cumulative GBH exposure affected\nthe overall composition of gut microbiota, suppressed the development\nof beneficial microflora, reduced hepatic catalase activity, and lowered\nmale testosterone levels. However, reproductive physiology, including\nmaturation, testis size, and egg production, remained intact. 117  Regarding teratogenicity, perinatal oral exposure\nto GLP led to excessive lipoperoxidation and an overload of antioxidant\nenzyme systems in maternal and fetal serum and livers at 21 days of\ngestation. 118  Transgenerational and multigenerational\ntoxicity of orally administered GBHs was also reported. A study involving\nrat dams (F0) and two offspring generations (F1 and F2) revealed more\npronounced effects in the F2 generation. While there were no changes\nin body weight or the onset of vaginal opening in the F1 offspring,\nthe F2 offspring showed delayed growth, lower fetal weight and length,\nhigher placental weight and placental index, and congenital morphological\nanomalies, despite a lower number of implantation sites. 119\nFurthermore, cesarean sections were performed\non rat dams exposed\nto oral administration of Roundup from day 6 to 15 of pregnancy, revealing\nvarious outcomes, including corpora lutea, implantation sites, resorptions,\nand living and dead fetuses. Fetal examination confirmed external\nand skeletal malformations, while analysis of the dams showed numerous\ninternal alterations and a high (50%) mortality rate among dams treated\nwith 1000 mg/kg Roundup. 120  Finally, the\noral treatment of rat dams with Roundup during pregnancy (21–23\ndays) and lactation (21 days) adversely affected male offspring. That\nincluded a reduction in sperm production and quality during adulthood,\na dose-dependent decrease in serum testosterone levels at puberty,\nand spermatid degeneration during both periods. Female offspring only\nexhibited a delay in vaginal canal opening. 83\nThe influence of herbicides, pesticides,\ninsecticides, fungicides, and fumigants on congenital disabilities\namong applicators’ children was also investigated ( Figure  1 ). A population study\ninvolving 695 families and 1532 children conducted between 1997 and\n1998 in the Red River Valley, Minnesota, revealed that the congenital\ndisability rate was 31.3 per 1000 births in the first year of life\nand 47.0 per 1000 births within the first 3 years or later. A higher\nnumber of these defects were associated with conceptions in the spring.\nNotably, adverse neurologic and developmental neurobehavioral disorders\n(NBDs) were more prevalent among children of users of the phosphine\nfumigant and GBH. 121  These findings align\nwith in vivo data indicating a harmful link between sustained exposure\nof dams to the GBH MGB axis and the impairment of hippocampal neuroplasticity,\nlearning, and memory, as well as the development of anxiety, autism-like\nbehavior, and depression-like behavior in the offspring later in life. 25 , 122 , 123  In neonate rats, gestational\nexposure to pure GLP led to dose-dependent NBDs in reflex development,\nmotor activity, and cognitive functions, indicated by inhibiting the\nWnt5a-CaMKII noncanonical signaling pathway. 124  Finally, a miRNA microarray-based investigation of the association\nbetween GLP and NDDs in postnatal rats revealed upregulation of 55\ngenes and downregulation of 19 genes involved in the etiology of NDDs\nin the prefrontal cortex, particularly participating in neurogenesis,\nneuron differentiation, and brain development. 74\nContradictory outcomes were reported by a meta-analysis\ninvestigating the association between human exposure to GMO GBH-treated\ncorps in South America and reproductive system diseases, including\ncongenital disabilities, abortions, preterm deliveries, childhood\ndiseases, or altered sex ratios, as well as congenital malformations\nand disabilities. Except for attention-deficit hyperactivity disorder\namong children of GLP appliers, no significant associations were observed,\nwhich excludes the direct risk of human embryo- or teratogenicity\nof GLP or GBH. 125\nIn contrast, the\nOntario Farm Family Health Study (OFFHS), published\nin 1997 by the Canadian Census of Agriculture, provided evidence of\nthe putative impacts of pesticides or herbicides, including GLP, on\nthe human reproductive system. In this retrospective study, pesticide-exposed\nfarm couples were surveyed about their farm activities, reproductive\nexperiences, and occupational health risks. Particular attention was\npaid to the relationship between male health, within 3 months before\nconception through the month of conception, and miscarriage, preterm\ndelivery, small-for-gestational-age births, and altered sex ratio.\nIdentification of 3984 eligible pregnancies among 1898 couples (64%\nresponse) ruled out the significant association between male exposure\nto classified pesticides (including GLP) and the probability of small-for-gestational-age\nbirths or altered sex ratio. However, the combined use of various\nchemicals (GLP, atrazine, organophosphates, 4-[2,4-dichlorophenoxy]\nbutyric acid, and insecticides) increased the risk of reproductivity\ncomplications and a continuation of the study focusing on miscarriage\nwas strongly suggested. 126\nIn the\nretrospective cohort evaluation, surveyed during 1991–1992,\nthe OFFHS examined the influence of exposure to any of 13 pesticides\non TTP. The 2012 planned pregnancies were analyzed in terms of the\nconditional fecundability ratio. In men’s exposure only to\npesticide-related activity, three pesticides were associated with\na 17–30% increase in fecundability. In contrast, six pesticides,\nincluding GLP, were associated with decreased fecundability in the\nwomen-only pesticide exposure case. 127  According\nto another OFFHS study in 2001, targeting 2110 women who provided\n3936 pregnancies, including 395 spontaneous abortions, preconception\npesticide exposure to GBH (3 months before and up to a month of conception)\nwas linked with a moderate risk of early abortion (<20 weeks) and\nan increased risk of late abortion. 128\nDirect association between exposure to GLP and TTP (measured in\nmonths) was assessed in 2592 fertile Colombian women from five regions\nexposed to different uses of GLP, applied by aerial spraying for illicit\ncrop eradication. Retrospective interviews with the women regarding\ntheir reproductive health, life, and work, revealed no significant\nGLP effect on the TTP measured as fecundability odds ratios. 129\nIn another birth-cohort study conducted\nin Central Indiana on 71\nCaucasian women with singleton pregnancies, maternal GLP exposure\nwas tested in terms of its pathological influence on exposure risk,\nfrequency, and pathways as well as increased fetal exposure risk,\nfetal growth indicators, and pregnancy length. Liquid chromatography\ncoupled with mass spectrometry (LC-MS) determination of urine and\nresidential drinking water obtained from the subjected women showed\nGLP levels above the limit of detection of 0.1 ng/mL (the linear dynamic\nconcentration range of 0.5–7.2 ng/mL) in 93% of the women,\nwith a mean urinary GLP level of 3.4 ng/mL. In drinking water samples,\nGLP was undetectable. Although there were no correlations with fetal\ngrowth indicators, including birth weight and head circumference,\nan elevated GLP urine concentration was significantly correlated with\nshortened gestational length. However, despite geographical limitations\nand lack of racial and/or ethnic diversity, the study reported direct\nproof of the perinatal GLP exposure-associated threat on shortened\npregnancy. 130\nMoreover, the impact\nof an abused GLP or GBH on human congenital\ndisabilities was assessed. In the late 1990s, the relation of the\nselected congenital malformations occurrence upon occupational paternal\nexposure to pesticides was assessed in a case-referent study conducted\nin 8 hospitals of Comunidad Valenciana, Spain, with 261 matched pairs.\nNo statistically significant associations between the father’s\nexposure to GBHs (including glufosinate) and the congenital disabilities\nin the first trimester of pregnancy were shown. 131\nIn contrast, a cross-sectional study conducted in\nRed River Valley,\nMinnesota, U.S.A., during 1997–1998, among 1532 children of\n695 pesticide applicator families, revealed unsettled data about the\nharmful impact of pesticides on the congenital disability rate. In\nthe first year of life, this rate counted 31.3 births per 1000, with\n83% of the total congenital disabilities reported by medical records.\nIn the first three years of life or later, the rate increased to 47\nper 1000. Neurologic and developmental NBDs refer to children of the\napplicators exposed to fumigant phosphine. NBDs were observed in\nchildren of the GLP group of the analyzed workmen. 121\nThe association between maternal residential proximity\n(1000 m)\nand gestational exposure (month of conception) to 59 different agricultural\npesticides and birth malformations was examined in infants with neural\ntube defects (NTDs), anencephaly, and spina bifida. In this two-control\nstudy, conducted in California in 1987–1991, the odds ratios\nwere computed using conventional single- and multiple-pesticide models\nand hierarchical multiple-pesticide logistic regression in infants\nwith NTDs. There was no association between GLP use and the NTD group\nin multiple-pesticide models. In contrast, an odds ratio was significant\nfor the proximity to GLP and the occurrence of NTDs in the single-pesticide\nmodel. Elevated risks of NTDs, anencephaly, and spina bifida subtypes\nwere also linked with carbamates, benzimidazole, and OPs. 132\nInfant cases of anencephaly (73), spina\nbifida (123), cleft lip\nwith or without cleft palate (277), or cleft palate only (117) were\nsubjects of another interview-based study that surveyed pregnant mothers\nwho were residentially exposed to agricultural pesticide applications\nin San Joaquin Valley, California, in the years 1997–2007.\nAs many as 35% of the interviewed mothers were threatened with the\nproxy activities of 52 chemical groups and 257 agricultural chemicals.\nHowever, there were no significant associations between maternal exposure\nduring early pregnancy to GLP or GBHs crop spraying and these infant\nmalformations. 133\nResearchers in\na study conducted in the same geographical region\nexamined 156 cases of infants and/or fetuses for pesticide-associated\ngastroschisis. A survey of 30 women exposed to GLP during pregnancy,\namong 22 pesticide groups and 36 specific pesticides, found no conclusive\ncause-and-effect link between agricultural exposure to GLP and gastroschisis. 134  Gestational exposure to GLP/GBHs was not associated\nwith a persistent cough, bronchitis, asthma, allergies, or hay fever\nin newborns, as observed in the analysis of 5853 pregnancies in the\nOFFHS study. 135 , 136  Eventually, in the Agricultural\nHealth Study (AHS) conducted in Iowa and North Carolina from 1993\nto 1997, researchers examined 2246 women pesticide applicators and\ntheir infants to investigate the association between maternal exposure\nto pesticide use and low birth weight. Only 3% of the infants had\nlow birth weight (less than 2500 g), and no significant birth weight\nloss was attributed to early pregnancy exposure to GLP or GBHs. 137\nGlobally, ∼18 million people die yearly from cardiovascular\ndiseases (CVDs). CVDs are a group of heart or blood vessel disorders.\nMajor causal factors of the CVDs relate to inappropriate diet, GM\nmalfunctions, stress, 138  epigenetics, 139  congenital heart defect, 140  environmental pollution, 141  substance\nabuse, 142  warfare agents intoxication, 143 − 146  and occupational agrochemical exposure. 147 − 150\nCardiotoxicity\nof GLP, GLP-SH, and GBHs was investigated in vivo and in humans ( Figure  2 ). In vivo studies\nconfirmed the aggravating contribution of these agents to CVDs associated\nwith developmental heart toxicity, GM dysregulation, arrhythmias,\natherogenicity, and ventricular or aortic malformations. Remarkably,\nexposure of zebrafish embryos to a GLP solution caused structural\nabnormalities in the atrium, ventricle, and body vasculature, irregular\nheart looping, situs inversion, and a decrease in the heartbeat rate.\nMoreover,\nin situ hybridization and Mef2/mef2ca immunohistochemistry, performed\nduring early cardiac patterning stages, confirmed the deviation of\ncardiomyocytic development. 151  In contrast,\na comparative study on the cardiotoxicity of GLP and Roundup, conducted\non guinea pig hearts and human cardiomyocytes, confirmed the proarrhythmogenic\nproperties of Roundup. At a relatively high concentration of 100 μM,\nRoundup significantly affected heart rate and reduced ventricular\ncontractility and cardiomyocytic viability. In molecular terms, Roundup’s\ndepressive impact on contractility was caused by concentration-dependent\nblocking of the CaV1.2 cardiac calcium channel. No such impacts of\n100 μM GLP were observed, excluding the cardiotoxic properties\nof GBH’s adjuvants. 152  Similarly,\nstudies on rat and rabbit adults and offspring excluded developmental\ncardiotoxicity and cardiovascular malformations related to the GLP\ntreatment applied during pregnancy. 153  However,\nstudies in vivo and in humans affirmed putative GLP- or GBH-related\nrisk of atherosclerosis and tachycardia. In rats, 75-day-long oral\nand inhalation exposure to GLP in three concentrations resulted in\na fatty streak, as demonstrated by histopathological examination.\nGLP exhibited a clear atherogenic potential. However, there was no\ndose- and exposure route-dependent alteration of the right and left\nventricle thicknesses or in the collagen density. 154  Oral administration of GLP and Roundup significantly increased\nthe urinary level of homocysteine, a risk factor for CVD, related\nto a deviated  Prevotella  sp. abundance in the gut. 53  Moreover, electrophysiological analysis of rats\nand rabbits treated with GLP and GBH showed electrical abnormalities,\npresumably resulting from a Roundup superfusion-induced reduction\nof intracellular calcium uptake. Beyond this excitability alteration,\nRoundup increased the incidence of arrhythmias in a dose-dependent\nmanner. Nonetheless, a control group treated with GLP alone showed\nnone of the above symptoms. This result suggests that, most likely,\nGBH surfactants and adjuvants, but not GLP itself, may cause life-threatening\nQT (ventricular repolarization) prolongation, atrioventricular conduction\nblocking, and arrhythmias. 155\nThe GBH impact\non the cardiovascular system. (A) Irregular electrocardiogram\n(ECG) on admission to the hospitalization of a 30-year-old woman who\nswallowed Roundup. The ECG, acquired approximately 4 h after syncope,\nshows sinus rhythm at 75 beats per minute with first-degree atrioventricular\n(AV) block (PR 260 ms), LBBB (QRS 200 ms), and significantly prolonged\nQT (670 ms). The patient recovered after 2 days of hospitalization.\nLBBB: left bundle branch block. Adapted with permission from ref ( 161 ). Copyright Elsevier 2019.\nClinical studies\non GLP/GBH intoxication primarily refer to evaluation of occupational\nrisk in farmlands and herbicide factories. Alongside the plasmatic\nand urine GLP level determination, the QT prolongation was suggested\nto be monitored as the most common symptom of GBH intoxication to\nascertain the risk of cardiovascular disease among farmers and GBH-factories\nworkers. 156  Prolonged PR intervals (called\nfirst-degree atrioventricular block) also belong to these symptoms.\nProlongation of the PR interval, denoting the time from the beginning\nof atrial depolarization to the onset of ventricular depolarization, 157  was analyzed in patients exposed to GLP herbicide\nformulations, including GLP ammonium salt herbicides and glyphosate\nisopropylamine (GLP-IPA) salt herbicides. As reported, of the two\ngroups, GLP-IPA poisoning caused more fatality because of a higher\nincidence of QT prolongation and a higher tendency for PR prolongation. 158  The QT interval was evaluated via a retrospective\ncohort study of 153 patients with acute GLP-SH ingestion as an early\npredictor factor for predicting mortality from GLP-SH intoxication.\nThe 19 fatal cases were reported. A comparison of the electrocardiograms\nrevealed that the nonsurvivors’ QT intervals were significantly\nlonger than survivors, followed by intraventricular conduction and\nfirst-degree atrioventricular block. 159  Likewise, a retrospective analysis of 232 GLP-SH-poisoned patients,\nincluding 29 deaths, showed significantly increased levels of lactate\nin nonsurvivors when compared to survivors. Additionally, this increase\nwas markedly associated with 30 days of mortality, altered levels\nof potassium, and a prolonged QT interval. These findings suggest\nthe usefulness of acidosis levels and QT interval measurements in\nthe early prognosis of GBH-linked CVDs. 160  Similarly, GLP cardiotoxicity mirrors acute sodium channel blocker\noverdose, leading to cardiogenic syncope, symptomized by diffuse electrophysiological\ndepolarization and repolarization conduction abnormalities, including\nprolonged QTc, intraventricular block, and AV conduction delay ( Figure  2 ). An electrocardiogram\nexamination of a 30-year-old woman exposed to high-concentration GLP\nrevealed that the exposure caused a syncopal episode in the left bundle\nbranch block that evolved into a type I Brugada pattern and life-threatening\narrhythmia 161  ( Figure  2 A).\nGBH hemotoxicity was encountered\nin the clinical cases of acute GBH poisoning. In 2013, Roundup, Pistol\nEV, Glyper, Grivolax, Verdis, or their mixtures, were used in 13 cases\nof suicide attempts, symptomized with oropharyngeal ulceration, nausea\nand vomiting, acidosis, respiratory issues, cardiac arrhythmia, hyperkalemia,\nimpaired renal function, hepatic toxicity, and altered unconsciousness.\nIn fatal cases, characterized by the 4146 mg/L GLP blood concentration\n(range of 690–7480 mg/L), cardiogenic shock, cardiorespiratory\narrest, hemodynamic disturbance, and intravascular disseminated coagulation\nwere dominated. 162  Moreover, accidental\nand deliberate oral ingestions of GLP-trimesium (Touchdown) caused\nthe deaths of a 6-year-old boy and a 34-year-old woman, respectively.\nThe post-mortem examination revealed cardiopulmonary alterations,\nincluding edema and erosion of the mucus membranes of the airways\nand gastrointestinal tract, pulmonary and cerebral edemas, and deformation\nof the right atrium and ventricle of the heart. 163  Moreover, refractory respiratory failure and cardiogenic\nshock were fatal in the suicidal case of a 57-year-old woman who died\nfrom swallowing a GLP-SH. 164  Furthermore,\na 65-year-old woman suffered from hyperkalemia, hypoxemia, and hypotension\nafter the accidental ingestion of GLP-surfactant. The intoxication\nsymptoms included increased creatinine levels, acute kidney injury,\nhemoconcentration, bicarbonate and lactate acidosis, and pneumonitis.\nThe patient was detoxified using continuous hemofiltration and direct\nhemoperfusion. 165  Moreover, persistent\nventricular tachycardia and metabolic acidosis developed in a 47-year-old\nGLP-SH-poisoned man who recovered after extracorporeal membrane oxygenation\nwas applied within 4 h of the cardiopulmonary collapse. 166  Similarly, a 52-year-old man, intoxicated with\nGLP-POEA, experienced circulatory shock and refractory hypotension.\nDespite the nonresponsiveness to vasopressors, the patient recovered\nafter a 5-h-long intravenous (I.V.) fat emulsion treatment, 167  one of the most efficient therapies verified\nin a clinical survey, which included 64 patients. 168\nLung diseases refer to pathologies of airways, air\nsacs, and vascular and neuromuscular elements of respiration, leading\nto airway obstruction, lung compliance, and the blockage of gas exchange.\nMajor causative factors of respiratory diseases include autoimmune\nrisks, allergens, infections, sepsis, cold, burns, smoking, air pollution,\nheavy metals, coal dust, asbestos, combat gases, and persistent exposition\nto agrochemicals. 169 − 182\nEnvironmental or occupational pesticide exposure’s\nmost common pulmonary symptoms include cough, wheezing, dyspnea, breathlessness,\nchest tightness, chills, fevers, and sweats. Regarding occupational\ndisorders, asthma, chronic bronchitis, chronic obstructive pulmonary\ndisease (COPD), and pneumonia are most frequent among agricultural\nworkers. 180  Herbicide exposition-related\nlung disease case studies mentioned asthma, COPD, acute fibrinous\nand organizing pneumonia, pulmonary fibrosis, and lung cancer, as\nreviewed elsewhere 181 , 182  ( Figure  3 ). Mechanisms of agrochemical-caused respiratory\npathophysiology associated with oxidative stress, inhibition of the\nparasympathetic system followed by airway hyperactivity, immunological\nalterations, including macrophage infiltration and eosinophil abscesses,\nand allergic response. 180 − 182  The risk of agricultural airborne\nexposure to GLP involves inhaling the GLP containing eroded sediments\nand dust. Granulometric extraction of loess soil uncovered that GLP\nand AMPA were highly concentrated in soil particles of micrometer\nsize, which positively correlated with clay, organic matter, and silt.\nThe median half-life of GLP in the soil is between 2 and 197 days.\nSince the GLP decay in the soil is slow because of low soil moisture\ncontent, the health risk of off-site GLP inhalation increases significantly,\nwhich enhances the GLP airborne toxicity. 183 , 184\nThe\nGBH impact on the pulmonary system. (A–L) Immunohistochemical\nstaining of T lymphocytes on mice lung tissue using CD-3 antibody,\na pan-T lymphocyte marker. Expression of T lymphocytes in lung sections\nexposed for 1, 5, and 10 days to (A–C) control, (D–F)\nLPS (lipopolysaccharide), (G–I) GLP, and (J–L) LPS and\nGLP combination. The most intense T lymphocyte expression around lung\nperivascular regions (rectangles) was detected after (K) 5- and (L)\n10-day exposure to the LPS and GLP combination. (H, I) No impact of\npure GLP. Magnification ×400, scale bar 50 μm. B –\nbronchus; PA – pulmonary artery. Adapted with permission from\nref ( 188 ). Copyright\nElsevier 2021.\nIncreased airborne concentration and slow dissemination\nin the\nsoil of GLP may affect ground cover vegetation, e.g., impair immunity\nand the population of insects. GLP insectotoxicity was demonstrated\nfor two evolutionary-distant species:  Galleria mellonella  (butterfly) and  Anopheles gambiae  (mosquito). Mechanisms\nunderlying this activity were linked with GLP-inhibited melanization,\ni.e., the production of melanin, a black pigment involved in UV protection,\nthermoregulation, reactive species scavenging, and antimicrobial immunity.\nThe GLP exposure indirectly attenuated insect immunity against  Cryptococcus neoformans , a major fungal pathogen causing\nmeningoencephalitis, and the  Plasmodium falciparum  parasite. Besides, GLP decreased the size of melanized nodules in\nthe butterfly hemolymph and perturbed the midgut microbiome of the\nmosquito. 185  The mechanistic association\nbetween life-threatening infection with  C. neoformans  and GLP-inhibited melanization was confirmed in mice. 186  GLP-induced pulmonary pathology and inflammation\nwere explored by measuring murine models’ cellular and humoral\nresponses and lung functionality that inhaled GLP-enriched air samples\ncollected on herbicide-sprayed farms. The GLP-rich air and GLP alone\ninhalation increased the level of eosinophil and neutrophils, mast\ncell degranulation, and TSLP and interleukin (IL) IL-33, IL-13, and\nIL-5 production. Both samples induced pulmonary (IL-13)-dependent\ninflammation and promoted Th2-type cytokine, providing evidence of\na risk of GLP-induced occupational respiratory disorders. 187  Moreover, proinflammatory outcomes of the GLP\ntreatment were evaluated in the presence of endotoxin (lipopolysaccharide,\nLPS), a potent inflammatory agent. Levels of neutrophils, myeloperoxidase,\ntumor necrosis factor-α (TNF-α), IL-6, ICAM-1, and TLR-2\nexpression in mice exposed to the LPS-GLP comintation were higher\nthan in mice exposed to either of these agents alone 188  ( Figure  3 A– 3 L). Respiratory toxicities of GLP,\nPOEA, a GLP-POEA mixture, and Roundup were compared in rats by using\nintratracheal administration. The POEA-containing preparations elicited\na more rapid and prolonged respiration effect than the preparation\nwith GLP alone. Noteworthy, within 1 h of treatment, all preparations\nappeared fatal. However, the mortality of the POEA preparations was\nhigher than that of the GLP group.\nAdditionally, oral administration\nof POEA-containing preparations\nresulted in diarrhea and blood-stained weeping from noses, whereas\nanimals of the GLP group expressed only diarrhea. Only 24-h treatment\nwith POEA ended with death. Oral or intratracheal exposure to POEA\nand GLP caused lung hemorrhages and lung epithelial cell damage. 189  Finally, respiratory disturbances caused by\nGLP-SH exposure were verified in humans. As reported in a clinical\ncase report, oral intoxication with GLP-SH caused a blood pressure\ndrop, metabolic and respiratory acidosis, respiratory distress, hypoxia,\nand altered consciousness. Further hospitalization uncovered sinus\ntachycardia, cardiomegaly free hilar congestion, and eventually acute\npulmonary edema and respiratory failure. 190\nPulmonary and respiratory\ndisorders are major symptoms of human death triggered by GBH poisoning.\nFor example, in a suicidal case of GLP-SH ingestion, typical chemical\npneumonitis and respiratory failure were associated with acute pancreatitis,\nwhich developed on the first day and lasted for 10 days. 191  Moreover, in a clinical investigation conducted\nduring 1992–1996, 36 out of 53 patients exhibited aspiration\npneumonitis-associated laryngeal and mucosal injuries, which were\nconsidered potentially life-threatening. 192  Finally, a case of GLP-SH-involved suicidal attempt of a 52-year-old\nwoman reported aspiration pneumonitis and intestinal ileus. After\nthe recovery, the woman suffered from a sudden upper-airway obstruction\noriginating with fibrinous laryngotracheobronchitis. 193\nLiver (hepatic) diseases\naccount for ∼2 million deaths per year globally. 194 , 195  These pathologies’ primary mechanisms refer to hepatic inflammation,\noxidative DNA damage resulting from infection, and obesity as well\nas alcohol, pharmaceutical, and drug abuse. Many controversies have\narisen about whether GBP toxicity relates to formulants (surfactants,\ne.g., POEA and heavy metals) or GLP itself 196 , 197  ( Figure  4 A and  4 B). In vitro studies on the destructive impact of\nGBH in hepatoblastoma (HepG2), adenocarcinoma (A594), and neuroblastoma\n(SH-SY5Y) cell lines revealed that the ethoxylated formulants and\ntheir mixtures with GLP-IPA salt significantly inhibited proliferation\nof these cells, whereas GLP, the ActI, alone was not cytotoxic at\nall. 198\nThe GBH impact on the liver and the kidneys.\n(A, B) Assessment\nof GBH hepatotoxicity in common carp treated with GLP (0, 5, and 50\nmg/L) for 45 days. (A) Activities of alanine transaminase (ALT) and\naspartate transaminase (AST) in plasma samples collected after 15,\n30, and 45 days of exposure. (B) Hematoxylin and eosin staining of\nliver sections to assess histopathological changes. Red, yellow, and\ngreen arrows indicate respective hepatocyte swelling, cytoplasmic\nvacuolation, and increased fatty changes. Adapted with permission\nfrom ref ( 196 ). Copyright\nElsevier 2021. (C–E) Assessment of GLP nephrotoxicity in human\nurine samples. The dot plots illustrate the urinary levels of (C)\nkidney injury molecule (KIM-1), a renal tubular injury biomarker,\n(D) neutrophil gelatinase-associated lipocalin (NGAL), an acute kidney\ninjury biomarker, and (E) albumin-to-creatine ratio (ACR), a biomarker\nof albuminuria, in participants enrolled in three different studies:\nHealthy Start, Starting Early, and Preventing Environmental Exposures\nin Pregnancy Study (PEEPS). Despite GLP detectability in urine (limit\nof detection of 0.1 ng/mL) of 11.1% of children at various ages, the\nmultivariable regression models excluded the significant associations\nof these GLP exposures with any kidney injury biomarkers. Adapted\nwith permission from ref ( 227 ). Copyright Elsevier 2020.\nMoreover, contradictory in vivo results were demonstrated.\nPure\nGLP was hepatoxic to wall lizards ( Podarcis siculus ), important predators of herbivorous insects. Oral administration\nof low doses of GLP to a lizard caused fibrotic formation and a loss\nof liver functions. These malfunctions were associated with oxidative\nstress, manifested by the dysregulation of Cu/Zn superoxide dismutase,\nGSH-Px, metallothionein, and tumor suppressor protein 53, and upregulation\nof ERα and vitellogenin, thus showing the xenoestrogenic activity\nof GLP. 199  Studies on rats confirmed GLP\nand GBH hepatotoxicity. 28-day feeding rats with GLP caused weight\nloss, triggered primary DNA damage in the liver cells and leukocytes,\nlowered thiobarbituric reactive substances in the liver and plasma,\nand dysregulated AChE and GSH-Px activity in the liver and plasma. 200  Two-year chronic exposure to ultralow (0.1\nppb) Roundup, administrated via drinking water, occurred as hepatoxic\nand nephrotoxic. Transcriptome microarray analysis confirmed the GBH-related\ndisruption of spliceosome and chromatin, lipotoxicity, phospholipidosis,\nand abnormal enlargement and necrosis of the liver and kidney cells\nassociated with anatomical symptoms, including fibrosis and ischemia. 201  Similarly treated rats had symptoms of steatohepatitis.\nThe\nproteome analysis confirmed the GBH-induced disturbance of organonitrogen\nmetabolism and fatty acid beta-oxidation, hallmarked by peroxisomal\nproliferation, steatosis (fatty liver disease), and necrosis. The\nmetabolome analysis confirmed lipotoxicity and oxidative stress related\nto the glutathione and ascorbate free radical scavenger system. Likewise,\nthe progression of steatohepatitis was associated with the GBH-induced\nalteration of biomarker levels of the nonalcoholic fatty liver disease\nbiomarkers. 202  Finally, the hepatotoxicity\nof GLP and Roundup has been confirmed in metagenomics and metabolomics\nprofiling-based studies of rats exposed to these herbicides. These\nexposures caused markedly increased levels of gastrointestinal, hepatic,\nand oxidative stress biomarkers associated with shikimate and 3-dehydroshikimate,\nreflective of the inhibition of EPSPS of the shikimate pathway. These\noutcomes suggest a severe herbicidal impact on rat gut microbiota,\nalthough it must be highlighted that Roundup’s toxicity was\nhigher when compared to GLP. 203  Particularly,\nregarding liver biochemistry, the multiomics approach confirmed a\nherbicidal-caused deviation of nicotinamide (vitamin B 3 ) metabolism that naturally prevents hepatic steatosis by increasing\nthe redox potential. 204  These results were\nreinforced in a comparative 12-month study on hemo- and hepatotoxicity\nof GLP and Roundup in rabbits, designed for the real-life risk simulation\n(RLRS) approach. Toxicities of GLP and Roundup were evaluated versus\na mixture of common endocrine disruptors and xenobiotics containing\nGLP, bisphenol, and triclosan as well as phthalates and paraben derivatives.\nAs a result, GLP displayed only redox perturbations in blood homeostasis,\nwhereas there were no effects of GLP on liver tissue. This relatively\nminor outcome was contradictory to the effects of Roundup and a mixture\nof endocrine disruptors that distorted blood redox equilibrium and\ncaused oxidative stress manifested by decreases in the activities\nof SOD and glutathione reductase and increases in the total antioxidant\ncapacity and activities of GSH and GSH-Px. Overall, these findings\nconfirmed the RLRS approach applied to the hemo- and hepatoxicity\nof Roundup and common xenobiotics, whereby the harm of pure GLP was\nthe lowest or negligible. 205\nAdditionally,\nthe subacute exposure of rats to Roundup caused adverse\ninflammatory effects. The Roundup treatment elevated levels of C-reactive\nprotein, cytokines IL-1β, IL-6, TNF-α, and prostaglandin-endoperoxide\nsynthase in the liver and adipose tissue. These results correlated\nwith histological analysis showing the formation of vacuoles, fibroid\ntissue, and glycogen depletion, thus suggesting the progression of\nfatty liver disease, multiorgan inflammation, and liver scarring. 206\nPreclinical studies uncovered GLP- and\nGBH-induced hepatotoxicity,\ncongestive hepatopathy, and liver fibrosis. These disorders were confirmed\nin patients with nonalcoholic steatohepatitis (NASH) and biopsy-determined\nnonalcoholic fatty liver disease (NAFLD), the most common chronic\nliver disease in developed countries nowadays 194 , 195  ( Figure  4 ). Particularly,\nhigh-performance liquid chromatography (HPLC) examination of urine\nprofiles revealed a significant increase in GLP excretion in NASH\npatients compared with non-NASH patients. These results and a dose-dependent\nGLP exposure-fibrosis stage correlation suggest that NASH patients\nare more susceptible to fibrosis progression and the development of\ncirrhosis and hepatocellular carcinoma. 207\nClinical cases of GLP-related hepato- and nephrotoxicity in\nhumans\nmainly refer to commercial GBHs. As documented, multiple symptoms\nof GBH intoxication include respiratory failure, GI dysfunction, neurological\ndisorders, 208  and disruption of the liver\nand kidneys. 209\nKidney (renal)\ndisease, also called nephropathy, results from inflammatory (nephritis)\nor noninflammatory (nephrosis) renal malfunction. Two significant\ntypes of kidney disease are distinguished, namely, chronic kidney\ndisease (CKD), lasting longer than three months, 210  and acute kidney injury, i.e., a sudden, severe impediment\nto renal function. 211  In 2017, the global\nburden of CKD reached 1.2 million deaths, making it the twelfth leading\ncause of death ( Figure  4 ). Major CKD causes include diabetes, hypertension, cardiovascular\ndisease, xanthine oxidase deficiency, retention of analgesics or nephrotoxins,\ndeposition of antibodies (glomerulonephritis), as well as lupus, sepsis,\npolycystic kidney disease, kidney stones, and infections of the urinary\ntract. 212 , 213  In vivo toxicogenomic studies confirmed\nthe nephrotoxic effects of the GLP and Roundup formulations. For example,\nhyaline cysts, mineralization pelvis, epithelial pelvis, kidney tubular\nfibrosis, and kidney tubular degeneration were observed in rats treated\nwith these herbicides. These malformations were associated with genotoxic\nalterations and DNA damage in the kidney. 214\nEpidemiological\nreports have hallmarked the pesticide- or herbicide-induced kidney\ndiseases in farmers worldwide, including in Sri Lanka and India, 215 − 218  and the U.S.A. 219 , 220  A critical summary of global\npesticide CKD epidemics, including GLP and GBH, was presented elsewhere. 221  Noteworthy, the environmental use of GLP and\nGBH-related CDK risk had arisen since 1994 when GLP became a potential\ncausal factor for CKD of unknown etiology in rice paddy farming areas\nin the dry zones of Sri Lanka (called Sri Lanka Agricultural Nephropathy).\nThe putative GLP-induced CKD outbreak was associated with the GLP\nspraying, particularly with the consumption of hard water and exceptional\nmetal chelating properties of GLP. As revealed by inductively coupled\nplasma mass spectrometry and ELISA analyses, samples of drinking water\nfrom serving wells and abandoned wells contained traces of GLP and\nmetals, including Ca, Mg, Ba, Sr, Fe, Ti, and V. 215 , 216  Besides, urine profiles of the farmer patients living in endemic\nareas revealed urinary Sb, As, Cd, Co, Pb, Mn, Ni, Ti, and V concentrations\nexceeding the reference ranges. Moreover, GLP and creatinine urinary\nlevels were elevated compared to the nonendemic controls. 217  Finally, LC-MS analyses of topsoil samples\nfrom agricultural fields, water samples from nearby shallow wells\nand lakes, and sediment samples from lakes confirmed the presence\nof GLP complexes with Fe and Al and strong retention of GLP in soil\nand groundwater. 218  Nephrotoxicity of GLP-metal\ncomplexes is well documented by studies in vivo and in humans. 222 − 224  In 2014, a clinical case report demonstrated airborne GLP-induced\nhepatorenal dysfunction in workers of GLP-producing factories. 225  However, contradictory results were obtained\nby comparing GLP and Roundup renal toxicity in male rats. By measuring\nlevels of kidney biomarkers, including serum urea and creatinine,\nplasma cystatin-C and neutrophil gelatinase-associated lipocalin (NGAL),\nand oxidative stress indices related to activities of several kidney\nmembrane-bound enzymes, we showed that Roundup-exposed rats accumulated\nmore xenobiotics (including GLP, the ActI) than the group exposed\nto GLP alone. This increased accumulation was associated with nephrotoxicity,\nhallmarked by deviated levels of the biomarkers, whereas GLP administrated\nalone displayed no effect on renal function. 226  Eventually, GLP potential nephrotoxicity was evaluated in children\nby measuring urinary levels of kidney injury biomarkers including\nalbuminuria, NGAL, and kidney injury molecule-1. Despite GLP detectability\nin the children’s urine, there was no evidence of GLP-induced\nrenal injury 227  ( Figure  4 C– 4 E).\nClinical cases of GBH intoxication involve severe GBH nephrotoxicity. 228  In the case of an intentionally intoxicated\n22-year-old man, the poisoning caused a cute hemolysis, acidosis,\nand compensatory respiratory alkalosis. Besides, the GLP-SH increased\nthe permeability of the erythrocyte membrane by disturbing the lipid\nbilayer and consequent hypotonic hemolysis, which resulted in multiorgan\ncollapse, including frequent ventricular tachycardia, acute renal\nfailure, rhabdomyolysis, coagulation dysfunction, and urinalysis.\nThe patient recovered after alkaline diuresis, emergency plasmapheresis,\nand blood component transfusion. 229\nConsiderable controversy regarding\ncancer risk associated with both the use and misuse of GBH has recently\narisen among scientists, authorities, and society. 230 , 231  Chronologically, in 2014, EFSA classified GLP as “unlikely\nto pose a carcinogenic hazard to humans”. A year later, the\nInternational Agency for Research on Cancer (IARC) stated that GLP\nis a “probable human carcinogen” (Group 2A), 232  whereas, in 2016, the EPA concluded that it\nis “not likely to be carcinogenic to humans”. 233  In 2017, the European Chemical Agency (EChA)\ndenied a support link between GLP and animal cancer. 234  However, the Joint Meeting of Pesticide Residues (JMPR)\nagreed on the possibility that GLP “is cancerogenic in mice\nat very high doses”. 235  In 2018,\nthe AHS cohort study declined any association of GLP with any solid\ntumors or lymphoid malignancies, including non-Hodgkin lymphoma (NHL)\nand its subtypes, with only some evidence of increased risk of acute\nmyeloid leukemia. 236  These discrepancies\noriginate from various sources of epidemiological data, lack of established\ncriteria for statistical analyses and meta-analyses, overconfidence\nin common cancer etiology in experimental animals and humans, as well\nas differences in toxicological impacts and human incidence exposures\nto pure GLP and GBH formulations. 231 , 236 − 238  Despite clear GLP-induced cancer cases in rodents, including hemangiosarcomas,\nhemangiomas, kidney and liver adenomas, malignant lymphomas, NHLs,\nskin keratoacanthomas, adrenal cortical carcinomas, and skin basal\ncell tumors, summarized in 2020, 231  some\nof these reports have already been questioned. 238  Although the recent epidemiological evidence has recalled\nthe carcinogenic potential of GLP in humans, 239  the GLP-induced oxidative damage, chromosomal alterations in human\nlymphocytes, and stimulation of cell proliferation, i.e., the major\ncausative factors of cancer, cannot be underestimated. 240 − 245  These results are consistent with comparative toxicogenomics conducted\non rats exposed to GLP and three Roundup formulations used in the\nEU (MON 52276), United Kingdom (Roundup ProBio, MON 76473), and the\nUnited States (Roundup PROMAX, MON 76207). Generally, these herbicides\ncaused formulation- and organ-specific genotoxicity in the liver and\nkidney. Significant carcinogenesis-associated alterations were observed\nin the epigenome (differences in CpG methylation and levels of miR-10,\nmiR-17, miR-22, and miR-30), DNA damage-associated TP53 protein activation,\ndeviated circadian rhythm regulation, oxidative stress, and unfolded\nprotein response. Importantly, Roundup formulations were far more\ngenotoxic than GLP. However, GLP, in particular, caused apurinic/apyrimidinic\nDNA damage in the liver. 214  In this context,\nworth mentioning is that the augmented risk of cutaneous melanoma\nassociated with occupational exposure to the sun, GBH, and fungicides\nwas alerted among human subjects in Italy and Brazil. 246\n\nGBH intoxication triggers two major pathological clinical effects.\nFirst, it affects the GM and impacts the MGB and HPA axes. This dysbiosis\nincreases GI motility, resulting in fecal and urinary incontinence,\nmiosis, diaphoresis, and diaphragmatic failure. Second, after entering\nthe nervous system, GLP, an OP, phosphorylates the serine hydroxyl\ngroups in AChE, thus inhibiting the hydrolysis of ACh, which causes\ndysregulation of cholinergic neurotransmission and overstimulation\nof muscarinic and nicotinic receptors in skeletal muscles. 254\nSo far, no antidote has been developed\nfor GLP-SH poisoning, and\nthe therapy is mainly symptomatic and supportive. 255 , 256  Pharmacological therapy supplies the use of competitive ACh antagonists\nor pseudoreversible inhibitors of cholinesterase. In the case of established\nOP exposure, therapy includes pretreatment. 254  Carbamate pyridostigmine (the only FDA-approved substance for this\npretreatment) is investigated most widely. However, the CNS remains\nunprotected because of its incapability to cross the blood–brain\nbarrier (BBB). 27 , 257  In typical therapy of OP poisoning,\nthree FDA-approved therapeutics are used, vis., atropine, pralidoxime\nor obidoxime, and diazepam, among which atropine is the most common. 257  Another group of therapeutics includes enzyme-based\nGLP inactivators. They enable GLP transformation and biodegradation\nin the bloodstream before the GLP crosses the BBB. The transformation\nrate constants span from 0.08 to 100 h –1  (butyrylcholinesterase)\nthrough 4–340 min –1  (paraoxonases) to 5–2100\ns –1  (phosphotriesterase or organophosphorus hydrolase). 254  Other therapeutics, including non-FDA-approved\noximes, alkaloids, anti-NDMA agents, MgSO 4  magnesium sulfate\nand NaHCO 3 , and antibody-enzyme-nanoparticle conjugates,\nhave been reviewed elsewhere. 254\nHPLC-MS and ELISA have been employed in conventional medical diagnostics\nof GLP or GBH poisoning. They are mainly used for the toxicological\ndetermination and degradation of GLP in the blood and urine. Emergency\ntreatment of acute GBH intoxication involves HDF- or DHP-assisted\ngastric lavage with a large amount of normal saline followed by active\ncharcoal administration. 165  Further detoxification\nincludes charcoal hemoperfusion and pulse therapies of cyclophosphamide\nand methylprednisolone, followed by dexamethasone application. In\ncases of hypoxemia, glucocorticoid and cyclophosphamide pulse therapies\nare applied. 256  Intensive care is required\nin severe GLP-SH intoxication, symptomized by dehydration, oliguria,\nparalytic ileus, hypovolemic shock, cardiogenic shock, pulmonary edema,\nhyperkalemia, and metabolic acidosis. 165 , 249  In the case\nof hyperkalemia, the urine and blood concentrations of potassium,\nGLP, and AMPA can be decreased by an enema with polystyrenesulfonate 251  or intragastric cathartic and charcoal administration. 252\nPharmacological treatment is applied\nto injuries to the digestive\nsystem, including constipation and overall digestive peristalsis. 253  However, colon resection and colostomy are\nthe only solutions for mild colonic distention and peritonitis. 209  In cases of hypotension, vasopressor-based\ntherapy can be supported by hemodialysis, 164 , 250  and I.V. lipid emulsion application. 167 , 168  For multiorgan\nfailure, including cardiopulmonary and acute kidney failure, extracorporeal\nmembrane oxygenation 166  or alkaline diuresis,\nemergency plasmapheresis, and blood component transfusion 229 , 258 , 259  are recommended.\n\nModern toxicology and epidemiology have been revolutionized by\nultrasensitive analytical tools of precision (personalized) theranostics\nand wearable or smartphone-assisted artificial intelligence-excelled\nsensors or drug delivery systems. 260  Regarding\nGLP poisoning and GLP above’s modes of actions, i.e., dysbiosis\nand the inhibition of AChE, these devices, electronically powered,\ndecision-making, and user-friendly, shall enable self-handled or point-of-care\nprofessional-assisted evaluation of the harm followed with rapid and\nprecise determining and removing the herbicide and coformulants. In\ntechnological terms, these devices will be constructed as biocompatible\nchemo- and (bacteria cell)-based electronic biohybrids and nanorobots,\ncapable of measuring the GBH analyte or GBH pathology-associated biomarkers\nin skin, when implanted or wearable as a chip, in body, when swallowed,\nor in body fluids liquid, when used as a portable, smart device. 19 , 261 − 263  Finally, it is expected that chemisorptive\nor adsorptive tissue-specific systems will be manufactured to detect\nand capture the GBH xenobiotics with molecular recognition-provided\nselectivity and sensitivity, which will be followed by harmless, on-site,\nand nature-mimicking microbial biodegradation of GLP. 264 , 265","source_license":"CC-BY-4.0","license_restricted":false}