Glyphosate: Hepatotoxicity, Nephrotoxicity, Hemotoxicity, Carcinogenicity, and Clinical Cases of Endocrine, Reproductive, Cardiovascular, and Pulmonary System Intoxication.

The endocrine system regulates metabolism, respiration, mood, mechanosensory perception and movement, growth, reproduction, sexual development by producing and secreting hormones 56 − 58 ( Figure 1 ). Endocrinology mentions two categories of endocrine system diseases, namely, (i) hormone imbalance, resulting from the failure of the endocrine feedback system and causing hyposecretion or hypersecretion, i.e., hormone deficiency or hormone excess, respectively, and (ii) diseases resulting from infections, injuries, tumors, or genetic issues, which may lead to hormone imbalance. Major endocrine system diseases involve diabetes, hyper- or hypothyroidism, adrenal insufficiency, Cushing’s disease, and sex hormone disorders, including hermaphroditism, hypogonadism, precocious puberty, and multiple endocrine neoplasia. 56 − 59 Exposure to environmental toxicants and EDCs also dysregulates the hormonal balance homeostasis. 60 , 61 The EDCs, first reported in the 1990s, include pharmaceuticals, plastics (phthalates), pesticides, cosmetics, detergents, and phytoestrogens. Experimental pieces of evidence highlight the endocrine-disrupting activity of GLP and GBHs. 31 , 35 , 52 Nonetheless, according to the EPA’s EDSP and EFSA, there is no sufficient evidence to support the endocrine-disrupting effects of GLP, as it exhibits no direct interaction with the EAT pathways. 35 , 52 However, this issue has still been debated in the EU and Brazil. 62 The GBH impact on endocrine and reproductive systems. The scheme presents an overview of GLP and GBH toxicity in male and female reproductive systems. By deviating the hormone production and activities, the herbicides impact the development and functionality of Sertoli cells, Leydig cells, and sperm cells in seminiferous tubules of the testes and oocytes in ovaries. Besides, they cause, e.g., endometriosis and polycystic syndrome, thus leading to infertility, embryotoxicity, and teratogenicity. (A–F) Hematoxylin and eosin (X 100) staining of ovarian sections of GLP-treated mice showing (A) the normal histological ovarian follicle structure and (B, C) increased numbers of atretic follicles after mice treatment with (B) GLP and (C) Roundup. (D–F) Hematoxylin and eosin (X 200) staining showing (D) the normal ovarian interstitial cell structure and (E, F) the interstitial fibrosis after mice treatment with (E) GLP and (F) Roundup. Black arrows indicate lesioned regions. Adapted with permission from ref ( 37 ). Copyright Elsevier 2018. Ten key characteristics (KCs) of the EDCs have been classified to describe their mechanistic impacts on the endocrine system functionality. 63 (i) The EDCs interact or activate hormone receptors, e.g., androgen receptor (AR), estrogen receptors (ERα, ERβ), and progesterone receptor (ProgR). As revealed by preclinical studies, GLP plays an ambiguous role as a hormone mimic, 64 , 65 being either antiestrogenic in hormone- and dose-dependent manner activity 41 or ER-antagonistic 66 in a ligand-independent and GLP-specific manner. 67 Contrariwise, (ii) as an EDC, GLP may antagonize hormone receptors. However, no clear evidence for the GLP-mediated antagonistic effect on hormone activity has so far been reported. 41 , 66 For instance, using liver cells (HepG2), genotoxic, antiestrogenic, and aromatase-disruptive activities of GLP were compared with these of Roundup Express, Bioforce (Extra 360), Grands Travaux 400, and Grands Travaux 450. Among these formulations, applied in subagricultural dilutions (0.5–5 ppm), the toxicity of pure GLP was the lowest or negligible, whereas the carcinogen, mutagen, and reprotoxic actions of these formulations depended on the GBH’s adjuvant content rather than on the GLP concentration. 41 These outcomes are, however, in contrast to other pesticides. 68 To continue, (iii) GLP regulates the gene expression of hormone receptors in a dose-dependent manner in hormone-dependent cancer cells. 66 However, another study excluded 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, (iv) GLP alters the signal transduction, cell cycle, and cellular growth in hormone-responsive cells without direct interaction with the hormone receptor, 32 , 71 as revealed by a study on prepubertal rat-derived Sertoli cells. 12 Consecutively, (v) GLP and GBH induce epigenetic modifications in hormone-producing or hormone-responsive cells. Examples of the in vivo GLP hazardous epigenotoxicity include epimutations (epigenetic traits), DNA hypomethylation of oncogenes, 72 histone targeting and chromatin remodeling, 69 dose-dependent hypermethylation of the CpG islands of the ER gene promoters, 73 transgenerational (F1, F2, and F3) pathologies, 43 miRNA and circRNA dysregulation-associated metabolic and neurodevelopmental disorders (NDDs), 74 , 75 and organ malformations and congenital anomalies. 76 Moreover, (vi) GLP and GBHs alter steroidogenesis and hormone synthesis by disrupting the expression of steroidogenic acute regulatory (StAR) protein, 77 − 81 demonstrated in vivo. 40 − 42 Importantly, in a study on human placental JEG cells, toxic and inhibitory activities of Roundup, dosed at the agricultural concentration, were superior to those of GLP. 38 Furthermore, (vii) GLP and GBHs interfere with hormonal balance throughout the body by altering hormone transport across endocrine cell membranes or vesicle secretion. 31 Roundup indirectly influenced the plasma membrane-linked endocrine disruption in pregnant female rats, 82 male pubertal rats, 83 , 84 and perinatal mice 37 ( Figure 1 A– 1 F). However, contradictory data have also been presented. 42 , 70 , 85 , 86 In addition, (ix) as EDC, the GLP and GBHs are expected to deviate from hormonal metabolism and clearance mechanisms, including first-pass metabolism in the liver and excretion in the kidney. Yet, no experimental data have confirmed this mechanism. 31 Finally, (x) GLP and GBHs influence the fate of hormone-producing or hormone-responsive cells by direct or indirect changes in differentiation, proliferation, apoptosis, DNA repair, hypoxia, mutagenesis, and migration of target of effector endocrine cells. 32 , 42 , 66 , 67 , 71 , 87 − 89 The hypothalamus coordinates the endocrine system. By consolidating signals from upper cortical inputs, autonomic function and physical cues, and peripheral hormonal feedback, the hypothalamus provides specific signal outputs to the pituitary gland that subsequently supplies the endocrine system with hormones stimulating the peripheral glands. 90 As EDCs, GLP and GBHs dysregulate the functionality of the hypothalamus-pituitary and its connections with HPP glands axes, including adrenal (HPA), thyroid (HPTh), and gonadal axes, i.e., ovaries (HPO) and testes (HPT). 35 However, the EPA, EFSA, and the Organization of Economic Co-operation and Development (OECD) have recently questioned the endocrine-disrupting activity of GLP and excluded GLP as an EDC. A comprehensive experimental review conducted within EPA’s EDSP and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) critically verified an endocrine-modulating or adverse potential of GLP on steroidogenesis and the EAT pathways in humans, other mammals, and wildlife. 52 , 91 Contemporary contradictory reports highlight, though, the harmful GLP impact on steroidogenesis, gonadal, and thyrotropic axes, and the reproductive system. 23 , 30 , 33 , 37 , 43 , 49 , 82 For example, GBH induced dysregulation of the HPTh axis, causing osteoporosis, skeletal dysfunctionality, and hypothyroidism in Kalach 360 SL-fed rats female and offspring. Malfunctions in the osteocytes and thyroid cells’ activity altered the estrogen, calcium, phosphates, phosphatase alkaline, and vitamin D levels, as well as decreased triiodothyronine and thyroxine levels, associated with an increased plasma level of thyroid-stimulating hormone. These malfunctions led to subosteoporotic thinning and discontinuity of bone trabecular with a significant decrease in intertrabecular links. 92 The impact of excessive exposure to GLP or GBHs on the functionality of the HPTh axis was summarized elsewhere. 52 Examples of the impacts of these herbicides on the HPA, HPO, and HPT axes are discussed below. Reproductive system diseases, also called generational pathologies, comprise (i) genetic and congenital abnormalities, including epimutations and epigenetic fertility issues, (ii) functional or structural genital disorders associated with disruption of the endocrine system and hormonal disorders, (iii) disturbances of pregnancy and embryonal or fetal development, and parturition, (iv) infections, and (v) tumors. 93 , 94 The pesticides and herbicides belong to well-known toxins causing menstrual cycle disturbances, infertility, subfertility, prolonged time-to-pregnancy (TTP), spontaneous abortion, miscarriage, stillbirth, hemangioma birthmarks, congenital malformations in the offspring, as well as endocrine and hormonal issues, and musculoskeletal and neurobehavioral disorders (NBDs). 95 , 96 Epigenetics is a branch of genetics treating the inheritance of stable phenotype changes that arise from affected gene activity or expression without alterations in the DNA sequence, manifested as epigenetic traits (epimutations) in a chromosome that result from environmental (extracellular) impacts on the DNA methylation, chromatin remodeling, and transgenerational epigenetic inheritance, etc. 97 GLP and GBHs trigger epimutations. For instance, the ancestral environmental exposure of F0 female rats to GLP caused no or minor epigenotoxicity in the F0 and F1 generations but became severely toxic in the F2 and F3 offspring. The transgenerational pathology, including differential DNA methylation regions, caused prostate disease, obesity, kidney disease, ovarian disease, and parturition abnormalities. 43 Organ malformations and GLP tissue residuals, putatively associated with congenital anomalies, were observed in one-day-old piglets born by females exposed to GLP in the first 40 days of pregnancy. The organs most severely damaged in the piglets were the lungs, liver, kidney, brain, gut wall, and heart, whereby the highest GLP tissue concentration, quantified by enzyme-linked immunosorbent assay (ELISA), was in the lungs and hearts, whereas the lowest was in muscles. 76 GLP and GBHs destroy the production and functionality of gonads ( Figure 1 A). Roundup attenuated progressive motility and destroyed the mitochondrial integrity of human sperm, 44 whereas GLP alone decreased the sperm’s motility and caused sperm DNA fragmentation. 45 Moreover, GLP negatively affected sperm mitochondrial respiration efficiency and worsened the harmful effect of dihydroxytestosterone on sperm mitochondria. 98 Agent-specific impacts were evaluated, as well. In pigs, both herbicides caused dose-dependent decreases in sperm motility, viability, mitochondrial activity, and acrosome integrity but no changes in the DNA structure were observed. However, the toxicity of Roundup was more profound than that of GLP alone. 11 , 15 GLP and GBH affect signaling pathways in cells responsible for adrenal gland steroidogenesis. The adult male rats’ exposure to Roundup triggered apoptosis, reduced systemic levels of corticosterone and adrenocorticotropic hormone receptors, and altered the level of StAR protein phosphorylation. The serum concentration of testosterone was decreased, as well as aromatase levels and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) gonadotropins deviated in male rat offspring of the perinatally GLP-exposed females. 99 GLP and Roundup endocrine cytotoxicity were evaluated in male estuarine crabs ( Neohelice granulate ). Both herbicides decreased sperm count in spermatophores from the vas deferens and inhibited the secretion and/or transduction of the androgenic gland hormone, thus dysregulating spermatogenesis. 100 Neuroendocrine and immune toxicity of GLP was demonstrated in lizards ( Salvator merianae ). Blood morphology of the GLP-treated lizards revealed an elevated level of plasma corticosterone, decreases in the total white blood cell count and natural antibodies titres, and an increase in the lobularity index, thus indicating immunosuppression and symptoms of chronic infection, although differential white blood cell count, heterophils/lymphocytes index, and complement system have not deviated. 101 GBHs affect testes development, leading to changes in testosterone levels, seminiferous tubules, and puberty progression ( Figure 1 A). In prepubertal rats, Roundup decreased testosterone levels without affecting corticosterone or estradiol levels, and it altered seminiferous tubules and germinal epithelium in a dose-dependent manner. 84 Feeding prepubertal male rat offspring GLP-containing soy milk had toxic effects. GLP reduced testosterone levels and Sertoli cell numbers and increased the percentage of degenerated Sertoli and Leydig cells. Additionally, it reduced spermatid numbers, increased epididymal tail mass, and decreased seminiferous tubule diameter. 102 Perinatal mouse exposure to GLP or Roundup at acceptable daily intake concentrations in drinking water had agent-specific outcomes. GLP, but not Roundup, deviated from testis morphology, decreased testosterone serum levels, and reduced undifferentiated spermatogonia numbers by 60% in the GLP group. It was associated with the downregulation of the Sal4 gene and the up-regulation of the Nano3 gene related to germ cell differentation, as well as the Bax and Bcl2 genes, involved in apoptosis. 34 Moreover, maternal gestational exposure to Roundup altered masculinization of male offspring masculinization. At 60 days old, males from Roundup-treated dams showed increased sexual partner preference scores, elevated serum testosterone and estradiol levels, LH and FSH mRNA expression, LH and FSH gonadotropin protein content in the pituitary gland, deviated sperm production, and testicular morphology alterations. They also experienced an early onset of puberty. 99 Similar outcomes were observed in a study on attenuating the effects of Roundup on male mouse offspring from females exposed to Roundup in drinking water from the fourth day of pregnancy to the end of the lactation period. In F1 males from the GBH group, testicular descent was delayed, spermatozoa in the cauda epididymis were reduced, seminiferous epithelium height was decreased, intratesticular testosterone levels were increased, and the HPT axis was dysregulated. 33 Exposure of both prepubertal and postpubertal male rats to GBHs promotes mammary gland development by increasing collagen fiber organization and terminal end buds. Additionally, GBH-treated rats exhibited higher levels of mast cell infiltration, ERα expression, and proliferation index than control rats. 70 Roundup induced Ca2+-dependent oxidative stress and activated multiple endoplasmic reticulum stress-response pathways, leading to Sertoli cell death and reduced spermatogenesis in prepubertal rat testes. Exposure to GLP alone produced similar effects. 12 A study comparing GLP, POEA, and GBHs (Roundup and Glyphogan) at concentrations ranging from environmental- to agricultural-use levels in an immature Sertoli cell line (TM4) revealed that the GBH formulation caused mitochondrial dysfunction, disrupted cellular detoxification systems, and led to lipid droplet accumulation and necrosis. Overexposure to POEA resulted in excessive lipid accumulation, suggesting that cell death followed immediate penetration and overload of the formulants inside the cells. 103 Contradictory results emerged from comparing the GBH formulation (Glyfonova) and an equivalent amount of GLP on rat testes and androgen functionality. GLP had no significant impact on testes or testosterone synthesis, whereas Glyfonova only slightly upregulated the steroidogenic genes Cyp11a1 and Cyp17a1, related to aromatase ( Figure 1 A). 104 Perinatal exposure of rats to Roundup Transorb during a critical period of sexual differentiation led to HPT axis dysfunction, including increased LH and FSH mRNA expression levels, elevated LH protein in the pituitary gland, higher serum LH concentrations in adult male offspring, and subsequent pro-angiogenic effects. This dysregulation boosted blood testosterone levels, enhanced sperm production, and increased the weight of reproductive organs. 99 In rats exposed to Roundup in utero and postnatally, there was documented evidence of an increase in anogenital distance. 30 Meanwhile, exposure of prepubertal rats to Roundup resulted in an antiandrogenic effect, lowering systemic testosterone levels and inhibiting male puberty entry. 84 Male mice exposed to Roundup during gestation and lactation experienced delayed testis descent and decreased spermatozoa in the cauda epididymis. 33 Lastly, when adult rats were orally administered technical-grade Roundup, it disrupted the transcription of StAR mRNAs, leading to lipid droplet accumulation in the adrenal gland, increased gland weight, and reduced levels of corticosterone, adrenocorticosterone, and phosphorylated CREB. 79 Treatment with GLP or GBH deviates from the functionality of the HP-ovaries axis, thus triggering ovarian failure and deteriorating the quality of oocytes ( Figure 1 ). In mice, pure GLP dysregulated metaphase II oocyte quality, disrupted the microtubule organizing center, formation of a spindle fiber, and chromosomal alignment, and chelated zinc cations, which decreased its intracellular content and caused reactive oxygen species (ROS)-mediated embryo damage. 105 A comparison of GLP and Roundup activities in pig oocytes revealed that Roundup impaired oocyte development and blastocyst rate deviated steroidogenesis in cumulus cells and increased intracellular levels of ROS, wherein the Roundup impact was higher than that of an equivalent amount of pure GLP. 106 Disrupting activity of orally administered technical grade GLP and Roundup on ovaries was demonstrated in pregnant mice and their fetuses during the gestation period (first 19 days). The body, ovaries, liver weight, and mature follicles in treated mice decreased, whereas atretic follicles and interstitial fibrosis increased. Both progesterone and estrogen levels were significantly changed, as well as the expression levels of GnRH (gonadotropin-releasing hormone), LHR , FSH , 3β-HSD , and Cyp19a1 genes at the hypothalamic-pituitary-ovarian (HPO) axis. The herbicide treatment induced oxidative stress, manifested by increased T-AOC, CAT, and glutathione peroxidase (GSH-Px) activity and high malondialdehyde (MDA) content in the serum and ovaries. Finally, prenatal exposure to GLP altered the sex ratio of the litter 37 ( Figure 1 A– 1 F). Extensive studies on rats have demonstrated that excessive exposure to GLP or GBH destroyed the uterus’s development and functionality, as well as morphological and physiological features 46 − 49 ( Figure 1 ). For example, in adult ovariectomized rats subcutaneously injected with a GBH formulation, there were no changes in uterine weight or epithelial proliferation, but the GBH injection increased the luminal epithelial cell height and downregulated the ERα mRNA and protein levels in luminal epithelial cells, whereas the ERα was upregulated in the stroma. Moreover, the GBH injection upregulated ERβ and ProgR expression levels. 107 In another study, the GBH exposure deviated the activity of ERα, ProgR, homeobox protein Hox-A10 (HOXA10), and Wnt7a that regulate uterine organogenetic differentiation, causing luminal epithelial hyperplasia and increases in the stromal and myometrial thickness. 108 Ovarian follicular dynamics, associated with increased proliferation of granulosa and theca cells, was altered, expression of FSHR and GDF9 mRNA was downregulated, and proliferative activity of the uteri cells was decreased in GBH-exposed prepubertal lambs. Noteworthy, none of these outcomes were in the lambs treated with GLP or AMPA. 109 Early postnatal exposure to GBH induced lasting morphological changes in the female rat mammary gland, including a fibroblastic-like stroma, a higher percentage of hyperplastic ducts, and increased expression of steroid hormone receptors 110 ( Figure 1 ). In prepubertal rats, GBH increased uterine sensitivity to estradiol, leading to endometrial hyperplasia characterized by increased luminal and glandular epithelial height and stromal nuclei density. 46 In neonatal rats exposed to GBH, alterations in endometrial decidualization at implantation sites were associated with dysregulated expression levels of estrogen and progesterone receptors (ER and ProgR), as well as endocrine pathway-regulating markers (HOXA10) and proliferation markers. 47 , 48 In another study involving rat females exposed to either pure GLP or GBH from gestational day 9 until weaning, herbicide exposure induced preimplantation losses in the F1 generation, increased 17β-estradiol serum levels, and upregulated ERα expression. GLP specifically downregulated ProgR mRNA expression. Additionally, HOXA10 and Lif genes were downregulated in herbicide-treated rats. 49 In weaned pigs, GLP and Roundup administered through feed had insignificant effects on the vulvar size and the index of reproductive organs. However, they altered the uterine and ovarian ultrastructure and disrupted the synthesis and secretion of LH, FSH, GnRH, and testosterone. Roundup also caused an imbalance in hydrogen peroxide and MDA levels in reproductive organs 23 ( Figure 1 ). In cows, GLP directly stimulated estradiol secretion from granulosa cells, while both GLP and Roundup had varying effects on oxytocin and progesterone secretion from luteal cells, leading to deviations in the estrous cycle and uterine contractions that could result in infertility. Additionally, both formulations decreased prostaglandin secretion from endometrial cells but did not directly affect the basal and oxytocin-stimulated force of the motor functions of the myometrium. 111 GLP and GBH directly impact embryonic and fetal development, as evidenced by various experimental findings 112 ( Figure 1 ). For instance, GLP led to carbonic anhydrase inhibition and ROS-triggered cellular apoptosis in zebrafish embryos, resulting in multiorgan and body malformations. 113 Lizards treated with Roundup and Panzer Gold formulations at different stages of embryonic development (3–5 and 33 days) exhibited embryonic and hematological alterations in their blood samples. 114 Embryotoxicity and teratogenicity in Xenopus laevis , three GBH formulations (Roundup, Kilo Max, and Enviro Glyphosate) were higher than those of GLP alone. These GBHs caused cardiac and abdominal edema and altered gut formation and axial malformations. In X. laevis embryos, GLP and GBHs induced cephalic abnormalities, abnormal neural crest development, and anterior-posterior axis shortening, resulting in cranial cartilage deformities at the tadpole stages. Notably, the highest teratogenic indices indicated that Roundup and Enviro Glyphosate caused the most severe harm. 115 GBH exposure similarly affected chicken embryos, leading to the gradual loss of rhombomere domains, decreased optic vesicles, and the development of microcephaly, which was linked to increased endogenous retinoic acid activity. These effects underscore the direct impact of GBHs on the early morphogenesis of the vertebrate nervous system. 116 In contrast, a 52 week study in an avian model revealed that cumulative GBH exposure affected the overall composition of gut microbiota, suppressed the development of beneficial microflora, reduced hepatic catalase activity, and lowered male testosterone levels. However, reproductive physiology, including maturation, testis size, and egg production, remained intact. 117 Regarding teratogenicity, perinatal oral exposure to GLP led to excessive lipoperoxidation and an overload of antioxidant enzyme systems in maternal and fetal serum and livers at 21 days of gestation. 118 Transgenerational and multigenerational toxicity of orally administered GBHs was also reported. A study involving rat dams (F0) and two offspring generations (F1 and F2) revealed more pronounced effects in the F2 generation. While there were no changes in body weight or the onset of vaginal opening in the F1 offspring, the F2 offspring showed delayed growth, lower fetal weight and length, higher placental weight and placental index, and congenital morphological anomalies, despite a lower number of implantation sites. 119 Furthermore, cesarean sections were performed on rat dams exposed to oral administration of Roundup from day 6 to 15 of pregnancy, revealing various outcomes, including corpora lutea, implantation sites, resorptions, and living and dead fetuses. Fetal examination confirmed external and skeletal malformations, while analysis of the dams showed numerous internal alterations and a high (50%) mortality rate among dams treated with 1000 mg/kg Roundup. 120 Finally, the oral treatment of rat dams with Roundup during pregnancy (21–23 days) and lactation (21 days) adversely affected male offspring. That included a reduction in sperm production and quality during adulthood, a dose-dependent decrease in serum testosterone levels at puberty, and spermatid degeneration during both periods. Female offspring only exhibited a delay in vaginal canal opening. 83 The influence of herbicides, pesticides, insecticides, fungicides, and fumigants on congenital disabilities among applicators’ children was also investigated ( Figure 1 ). A population study involving 695 families and 1532 children conducted between 1997 and 1998 in the Red River Valley, Minnesota, revealed that the congenital disability rate was 31.3 per 1000 births in the first year of life and 47.0 per 1000 births within the first 3 years or later. A higher number of these defects were associated with conceptions in the spring. Notably, adverse neurologic and developmental neurobehavioral disorders (NBDs) were more prevalent among children of users of the phosphine fumigant and GBH. 121 These findings align with in vivo data indicating a harmful link between sustained exposure of dams to the GBH MGB axis and the impairment of hippocampal neuroplasticity, learning, and memory, as well as the development of anxiety, autism-like behavior, and depression-like behavior in the offspring later in life. 25 , 122 , 123 In neonate rats, gestational exposure to pure GLP led to dose-dependent NBDs in reflex development, motor activity, and cognitive functions, indicated by inhibiting the Wnt5a-CaMKII noncanonical signaling pathway. 124 Finally, a miRNA microarray-based investigation of the association between GLP and NDDs in postnatal rats revealed upregulation of 55 genes and downregulation of 19 genes involved in the etiology of NDDs in the prefrontal cortex, particularly participating in neurogenesis, neuron differentiation, and brain development. 74 Contradictory outcomes were reported by a meta-analysis investigating the association between human exposure to GMO GBH-treated corps in South America and reproductive system diseases, including congenital disabilities, abortions, preterm deliveries, childhood diseases, or altered sex ratios, as well as congenital malformations and disabilities. Except for attention-deficit hyperactivity disorder among children of GLP appliers, no significant associations were observed, which excludes the direct risk of human embryo- or teratogenicity of GLP or GBH. 125 In contrast, the Ontario Farm Family Health Study (OFFHS), published in 1997 by the Canadian Census of Agriculture, provided evidence of the putative impacts of pesticides or herbicides, including GLP, on the human reproductive system. In this retrospective study, pesticide-exposed farm couples were surveyed about their farm activities, reproductive experiences, and occupational health risks. Particular attention was paid to the relationship between male health, within 3 months before conception through the month of conception, and miscarriage, preterm delivery, small-for-gestational-age births, and altered sex ratio. Identification of 3984 eligible pregnancies among 1898 couples (64% response) ruled out the significant association between male exposure to classified pesticides (including GLP) and the probability of small-for-gestational-age births or altered sex ratio. However, the combined use of various chemicals (GLP, atrazine, organophosphates, 4-[2,4-dichlorophenoxy] butyric acid, and insecticides) increased the risk of reproductivity complications and a continuation of the study focusing on miscarriage was strongly suggested. 126 In the retrospective cohort evaluation, surveyed during 1991–1992, the OFFHS examined the influence of exposure to any of 13 pesticides on TTP. The 2012 planned pregnancies were analyzed in terms of the conditional fecundability ratio. In men’s exposure only to pesticide-related activity, three pesticides were associated with a 17–30% increase in fecundability. In contrast, six pesticides, including GLP, were associated with decreased fecundability in the women-only pesticide exposure case. 127 According to another OFFHS study in 2001, targeting 2110 women who provided 3936 pregnancies, including 395 spontaneous abortions, preconception pesticide exposure to GBH (3 months before and up to a month of conception) was linked with a moderate risk of early abortion (<20 weeks) and an increased risk of late abortion. 128 Direct association between exposure to GLP and TTP (measured in months) was assessed in 2592 fertile Colombian women from five regions exposed to different uses of GLP, applied by aerial spraying for illicit crop eradication. Retrospective interviews with the women regarding their reproductive health, life, and work, revealed no significant GLP effect on the TTP measured as fecundability odds ratios. 129 In another birth-cohort study conducted in Central Indiana on 71 Caucasian women with singleton pregnancies, maternal GLP exposure was tested in terms of its pathological influence on exposure risk, frequency, and pathways as well as increased fetal exposure risk, fetal growth indicators, and pregnancy length. Liquid chromatography coupled with mass spectrometry (LC-MS) determination of urine and residential drinking water obtained from the subjected women showed GLP levels above the limit of detection of 0.1 ng/mL (the linear dynamic concentration range of 0.5–7.2 ng/mL) in 93% of the women, with a mean urinary GLP level of 3.4 ng/mL. In drinking water samples, GLP was undetectable. Although there were no correlations with fetal growth indicators, including birth weight and head circumference, an elevated GLP urine concentration was significantly correlated with shortened gestational length. However, despite geographical limitations and lack of racial and/or ethnic diversity, the study reported direct proof of the perinatal GLP exposure-associated threat on shortened pregnancy. 130 Moreover, the impact of an abused GLP or GBH on human congenital disabilities was assessed. In the late 1990s, the relation of the selected congenital malformations occurrence upon occupational paternal exposure to pesticides was assessed in a case-referent study conducted in 8 hospitals of Comunidad Valenciana, Spain, with 261 matched pairs. No statistically significant associations between the father’s exposure to GBHs (including glufosinate) and the congenital disabilities in the first trimester of pregnancy were shown. 131 In contrast, a cross-sectional study conducted in Red River Valley, Minnesota, U.S.A., during 1997–1998, among 1532 children of 695 pesticide applicator families, revealed unsettled data about the harmful impact of pesticides on the congenital disability rate. In the first year of life, this rate counted 31.3 births per 1000, with 83% of the total congenital disabilities reported by medical records. In the first three years of life or later, the rate increased to 47 per 1000. Neurologic and developmental NBDs refer to children of the applicators exposed to fumigant phosphine. NBDs were observed in children of the GLP group of the analyzed workmen. 121 The association between maternal residential proximity (1000 m) and gestational exposure (month of conception) to 59 different agricultural pesticides and birth malformations was examined in infants with neural tube defects (NTDs), anencephaly, and spina bifida. In this two-control study, conducted in California in 1987–1991, the odds ratios were computed using conventional single- and multiple-pesticide models and hierarchical multiple-pesticide logistic regression in infants with NTDs. There was no association between GLP use and the NTD group in multiple-pesticide models. In contrast, an odds ratio was significant for the proximity to GLP and the occurrence of NTDs in the single-pesticide model. Elevated risks of NTDs, anencephaly, and spina bifida subtypes were also linked with carbamates, benzimidazole, and OPs. 132 Infant cases of anencephaly (73), spina bifida (123), cleft lip with or without cleft palate (277), or cleft palate only (117) were subjects of another interview-based study that surveyed pregnant mothers who were residentially exposed to agricultural pesticide applications in San Joaquin Valley, California, in the years 1997–2007. As many as 35% of the interviewed mothers were threatened with the proxy activities of 52 chemical groups and 257 agricultural chemicals. However, there were no significant associations between maternal exposure during early pregnancy to GLP or GBHs crop spraying and these infant malformations. 133 Researchers in a study conducted in the same geographical region examined 156 cases of infants and/or fetuses for pesticide-associated gastroschisis. A survey of 30 women exposed to GLP during pregnancy, among 22 pesticide groups and 36 specific pesticides, found no conclusive cause-and-effect link between agricultural exposure to GLP and gastroschisis. 134 Gestational exposure to GLP/GBHs was not associated with a persistent cough, bronchitis, asthma, allergies, or hay fever in newborns, as observed in the analysis of 5853 pregnancies in the OFFHS study. 135 , 136 Eventually, in the Agricultural Health Study (AHS) conducted in Iowa and North Carolina from 1993 to 1997, researchers examined 2246 women pesticide applicators and their infants to investigate the association between maternal exposure to pesticide use and low birth weight. Only 3% of the infants had low birth weight (less than 2500 g), and no significant birth weight loss was attributed to early pregnancy exposure to GLP or GBHs. 137 Globally, ∼18 million people die yearly from cardiovascular diseases (CVDs). CVDs are a group of heart or blood vessel disorders. Major causal factors of the CVDs relate to inappropriate diet, GM malfunctions, stress, 138 epigenetics, 139 congenital heart defect, 140 environmental pollution, 141 substance abuse, 142 warfare agents intoxication, 143 − 146 and occupational agrochemical exposure. 147 − 150 Cardiotoxicity of GLP, GLP-SH, and GBHs was investigated in vivo and in humans ( Figure 2 ). In vivo studies confirmed the aggravating contribution of these agents to CVDs associated with developmental heart toxicity, GM dysregulation, arrhythmias, atherogenicity, and ventricular or aortic malformations. Remarkably, exposure of zebrafish embryos to a GLP solution caused structural abnormalities in the atrium, ventricle, and body vasculature, irregular heart looping, situs inversion, and a decrease in the heartbeat rate. Moreover, in situ hybridization and Mef2/mef2ca immunohistochemistry, performed during early cardiac patterning stages, confirmed the deviation of cardiomyocytic development. 151 In contrast, a comparative study on the cardiotoxicity of GLP and Roundup, conducted on guinea pig hearts and human cardiomyocytes, confirmed the proarrhythmogenic properties of Roundup. At a relatively high concentration of 100 μM, Roundup significantly affected heart rate and reduced ventricular contractility and cardiomyocytic viability. In molecular terms, Roundup’s depressive impact on contractility was caused by concentration-dependent blocking of the CaV1.2 cardiac calcium channel. No such impacts of 100 μM GLP were observed, excluding the cardiotoxic properties of GBH’s adjuvants. 152 Similarly, studies on rat and rabbit adults and offspring excluded developmental cardiotoxicity and cardiovascular malformations related to the GLP treatment applied during pregnancy. 153 However, studies in vivo and in humans affirmed putative GLP- or GBH-related risk of atherosclerosis and tachycardia. In rats, 75-day-long oral and inhalation exposure to GLP in three concentrations resulted in a fatty streak, as demonstrated by histopathological examination. GLP exhibited a clear atherogenic potential. However, there was no dose- and exposure route-dependent alteration of the right and left ventricle thicknesses or in the collagen density. 154 Oral administration of GLP and Roundup significantly increased the urinary level of homocysteine, a risk factor for CVD, related to a deviated Prevotella sp. abundance in the gut. 53 Moreover, electrophysiological analysis of rats and rabbits treated with GLP and GBH showed electrical abnormalities, presumably resulting from a Roundup superfusion-induced reduction of intracellular calcium uptake. Beyond this excitability alteration, Roundup increased the incidence of arrhythmias in a dose-dependent manner. Nonetheless, a control group treated with GLP alone showed none of the above symptoms. This result suggests that, most likely, GBH surfactants and adjuvants, but not GLP itself, may cause life-threatening QT (ventricular repolarization) prolongation, atrioventricular conduction blocking, and arrhythmias. 155 The GBH impact on the cardiovascular system. (A) Irregular electrocardiogram (ECG) on admission to the hospitalization of a 30-year-old woman who swallowed Roundup. The ECG, acquired approximately 4 h after syncope, shows sinus rhythm at 75 beats per minute with first-degree atrioventricular (AV) block (PR 260 ms), LBBB (QRS 200 ms), and significantly prolonged QT (670 ms). The patient recovered after 2 days of hospitalization. LBBB: left bundle branch block. Adapted with permission from ref ( 161 ). Copyright Elsevier 2019. Clinical studies on GLP/GBH intoxication primarily refer to evaluation of occupational risk in farmlands and herbicide factories. Alongside the plasmatic and urine GLP level determination, the QT prolongation was suggested to be monitored as the most common symptom of GBH intoxication to ascertain the risk of cardiovascular disease among farmers and GBH-factories workers. 156 Prolonged PR intervals (called first-degree atrioventricular block) also belong to these symptoms. Prolongation of the PR interval, denoting the time from the beginning of atrial depolarization to the onset of ventricular depolarization, 157 was analyzed in patients exposed to GLP herbicide formulations, including GLP ammonium salt herbicides and glyphosate isopropylamine (GLP-IPA) salt herbicides. As reported, of the two groups, GLP-IPA poisoning caused more fatality because of a higher incidence of QT prolongation and a higher tendency for PR prolongation. 158 The QT interval was evaluated via a retrospective cohort study of 153 patients with acute GLP-SH ingestion as an early predictor factor for predicting mortality from GLP-SH intoxication. The 19 fatal cases were reported. A comparison of the electrocardiograms revealed that the nonsurvivors’ QT intervals were significantly longer than survivors, followed by intraventricular conduction and first-degree atrioventricular block. 159 Likewise, a retrospective analysis of 232 GLP-SH-poisoned patients, including 29 deaths, showed significantly increased levels of lactate in nonsurvivors when compared to survivors. Additionally, this increase was markedly associated with 30 days of mortality, altered levels of potassium, and a prolonged QT interval. These findings suggest the usefulness of acidosis levels and QT interval measurements in the early prognosis of GBH-linked CVDs. 160 Similarly, GLP cardiotoxicity mirrors acute sodium channel blocker overdose, leading to cardiogenic syncope, symptomized by diffuse electrophysiological depolarization and repolarization conduction abnormalities, including prolonged QTc, intraventricular block, and AV conduction delay ( Figure 2 ). An electrocardiogram examination of a 30-year-old woman exposed to high-concentration GLP revealed that the exposure caused a syncopal episode in the left bundle branch block that evolved into a type I Brugada pattern and life-threatening arrhythmia 161 ( Figure 2 A). GBH hemotoxicity was encountered in the clinical cases of acute GBH poisoning. In 2013, Roundup, Pistol EV, Glyper, Grivolax, Verdis, or their mixtures, were used in 13 cases of suicide attempts, symptomized with oropharyngeal ulceration, nausea and vomiting, acidosis, respiratory issues, cardiac arrhythmia, hyperkalemia, impaired renal function, hepatic toxicity, and altered unconsciousness. In fatal cases, characterized by the 4146 mg/L GLP blood concentration (range of 690–7480 mg/L), cardiogenic shock, cardiorespiratory arrest, hemodynamic disturbance, and intravascular disseminated coagulation were dominated. 162 Moreover, accidental and deliberate oral ingestions of GLP-trimesium (Touchdown) caused the deaths of a 6-year-old boy and a 34-year-old woman, respectively. The post-mortem examination revealed cardiopulmonary alterations, including edema and erosion of the mucus membranes of the airways and gastrointestinal tract, pulmonary and cerebral edemas, and deformation of the right atrium and ventricle of the heart. 163 Moreover, refractory respiratory failure and cardiogenic shock were fatal in the suicidal case of a 57-year-old woman who died from swallowing a GLP-SH. 164 Furthermore, a 65-year-old woman suffered from hyperkalemia, hypoxemia, and hypotension after the accidental ingestion of GLP-surfactant. The intoxication symptoms included increased creatinine levels, acute kidney injury, hemoconcentration, bicarbonate and lactate acidosis, and pneumonitis. The patient was detoxified using continuous hemofiltration and direct hemoperfusion. 165 Moreover, persistent ventricular tachycardia and metabolic acidosis developed in a 47-year-old GLP-SH-poisoned man who recovered after extracorporeal membrane oxygenation was applied within 4 h of the cardiopulmonary collapse. 166 Similarly, a 52-year-old man, intoxicated with GLP-POEA, experienced circulatory shock and refractory hypotension. Despite the nonresponsiveness to vasopressors, the patient recovered after a 5-h-long intravenous (I.V.) fat emulsion treatment, 167 one of the most efficient therapies verified in a clinical survey, which included 64 patients. 168 Lung diseases refer to pathologies of airways, air sacs, and vascular and neuromuscular elements of respiration, leading to airway obstruction, lung compliance, and the blockage of gas exchange. Major causative factors of respiratory diseases include autoimmune risks, allergens, infections, sepsis, cold, burns, smoking, air pollution, heavy metals, coal dust, asbestos, combat gases, and persistent exposition to agrochemicals. 169 − 182 Environmental or occupational pesticide exposure’s most common pulmonary symptoms include cough, wheezing, dyspnea, breathlessness, chest tightness, chills, fevers, and sweats. Regarding occupational disorders, asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD), and pneumonia are most frequent among agricultural workers. 180 Herbicide exposition-related lung disease case studies mentioned asthma, COPD, acute fibrinous and organizing pneumonia, pulmonary fibrosis, and lung cancer, as reviewed elsewhere 181 , 182 ( Figure 3 ). Mechanisms of agrochemical-caused respiratory pathophysiology associated with oxidative stress, inhibition of the parasympathetic system followed by airway hyperactivity, immunological alterations, including macrophage infiltration and eosinophil abscesses, and allergic response. 180 − 182 The risk of agricultural airborne exposure to GLP involves inhaling the GLP containing eroded sediments and dust. Granulometric extraction of loess soil uncovered that GLP and AMPA were highly concentrated in soil particles of micrometer size, which positively correlated with clay, organic matter, and silt. The median half-life of GLP in the soil is between 2 and 197 days. Since the GLP decay in the soil is slow because of low soil moisture content, the health risk of off-site GLP inhalation increases significantly, which enhances the GLP airborne toxicity. 183 , 184 The GBH impact on the pulmonary system. (A–L) Immunohistochemical staining of T lymphocytes on mice lung tissue using CD-3 antibody, a pan-T lymphocyte marker. Expression of T lymphocytes in lung sections exposed for 1, 5, and 10 days to (A–C) control, (D–F) LPS (lipopolysaccharide), (G–I) GLP, and (J–L) LPS and GLP combination. The most intense T lymphocyte expression around lung perivascular regions (rectangles) was detected after (K) 5- and (L) 10-day exposure to the LPS and GLP combination. (H, I) No impact of pure GLP. Magnification ×400, scale bar 50 μm. B – bronchus; PA – pulmonary artery. Adapted with permission from ref ( 188 ). Copyright Elsevier 2021. Increased airborne concentration and slow dissemination in the soil of GLP may affect ground cover vegetation, e.g., impair immunity and the population of insects. GLP insectotoxicity was demonstrated for two evolutionary-distant species: Galleria mellonella (butterfly) and Anopheles gambiae (mosquito). Mechanisms underlying this activity were linked with GLP-inhibited melanization, i.e., the production of melanin, a black pigment involved in UV protection, thermoregulation, reactive species scavenging, and antimicrobial immunity. The GLP exposure indirectly attenuated insect immunity against Cryptococcus neoformans , a major fungal pathogen causing meningoencephalitis, and the Plasmodium falciparum parasite. Besides, GLP decreased the size of melanized nodules in the butterfly hemolymph and perturbed the midgut microbiome of the mosquito. 185 The mechanistic association between life-threatening infection with C. neoformans and GLP-inhibited melanization was confirmed in mice. 186 GLP-induced pulmonary pathology and inflammation were explored by measuring murine models’ cellular and humoral responses and lung functionality that inhaled GLP-enriched air samples collected on herbicide-sprayed farms. The GLP-rich air and GLP alone inhalation increased the level of eosinophil and neutrophils, mast cell degranulation, and TSLP and interleukin (IL) IL-33, IL-13, and IL-5 production. Both samples induced pulmonary (IL-13)-dependent inflammation and promoted Th2-type cytokine, providing evidence of a risk of GLP-induced occupational respiratory disorders. 187 Moreover, proinflammatory outcomes of the GLP treatment were evaluated in the presence of endotoxin (lipopolysaccharide, LPS), a potent inflammatory agent. Levels of neutrophils, myeloperoxidase, tumor necrosis factor-α (TNF-α), IL-6, ICAM-1, and TLR-2 expression in mice exposed to the LPS-GLP comintation were higher than in mice exposed to either of these agents alone 188 ( Figure 3 A– 3 L). Respiratory toxicities of GLP, POEA, a GLP-POEA mixture, and Roundup were compared in rats by using intratracheal administration. The POEA-containing preparations elicited a more rapid and prolonged respiration effect than the preparation with GLP alone. Noteworthy, within 1 h of treatment, all preparations appeared fatal. However, the mortality of the POEA preparations was higher than that of the GLP group. Additionally, oral administration of POEA-containing preparations resulted in diarrhea and blood-stained weeping from noses, whereas animals of the GLP group expressed only diarrhea. Only 24-h treatment with POEA ended with death. Oral or intratracheal exposure to POEA and GLP caused lung hemorrhages and lung epithelial cell damage. 189 Finally, respiratory disturbances caused by GLP-SH exposure were verified in humans. As reported in a clinical case report, oral intoxication with GLP-SH caused a blood pressure drop, metabolic and respiratory acidosis, respiratory distress, hypoxia, and altered consciousness. Further hospitalization uncovered sinus tachycardia, cardiomegaly free hilar congestion, and eventually acute pulmonary edema and respiratory failure. 190 Pulmonary and respiratory disorders are major symptoms of human death triggered by GBH poisoning. For example, in a suicidal case of GLP-SH ingestion, typical chemical pneumonitis and respiratory failure were associated with acute pancreatitis, which developed on the first day and lasted for 10 days. 191 Moreover, in a clinical investigation conducted during 1992–1996, 36 out of 53 patients exhibited aspiration pneumonitis-associated laryngeal and mucosal injuries, which were considered potentially life-threatening. 192 Finally, a case of GLP-SH-involved suicidal attempt of a 52-year-old woman reported aspiration pneumonitis and intestinal ileus. After the recovery, the woman suffered from a sudden upper-airway obstruction originating with fibrinous laryngotracheobronchitis. 193 Liver (hepatic) diseases account for ∼2 million deaths per year globally. 194 , 195 These pathologies’ primary mechanisms refer to hepatic inflammation, oxidative DNA damage resulting from infection, and obesity as well as alcohol, pharmaceutical, and drug abuse. Many controversies have arisen about whether GBP toxicity relates to formulants (surfactants, e.g., POEA and heavy metals) or GLP itself 196 , 197 ( Figure 4 A and 4 B). In vitro studies on the destructive impact of GBH in hepatoblastoma (HepG2), adenocarcinoma (A594), and neuroblastoma (SH-SY5Y) cell lines revealed that the ethoxylated formulants and their mixtures with GLP-IPA salt significantly inhibited proliferation of these cells, whereas GLP, the ActI, alone was not cytotoxic at all. 198 The GBH impact on the liver and the kidneys. (A, B) Assessment of GBH hepatotoxicity in common carp treated with GLP (0, 5, and 50 mg/L) for 45 days. (A) Activities of alanine transaminase (ALT) and aspartate transaminase (AST) in plasma samples collected after 15, 30, and 45 days of exposure. (B) Hematoxylin and eosin staining of liver sections to assess histopathological changes. Red, yellow, and green arrows indicate respective hepatocyte swelling, cytoplasmic vacuolation, and increased fatty changes. Adapted with permission from ref ( 196 ). Copyright Elsevier 2021. (C–E) Assessment of GLP nephrotoxicity in human urine samples. The dot plots illustrate the urinary levels of (C) kidney injury molecule (KIM-1), a renal tubular injury biomarker, (D) neutrophil gelatinase-associated lipocalin (NGAL), an acute kidney injury biomarker, and (E) albumin-to-creatine ratio (ACR), a biomarker of albuminuria, in participants enrolled in three different studies: Healthy Start, Starting Early, and Preventing Environmental Exposures in Pregnancy Study (PEEPS). Despite GLP detectability in urine (limit of detection of 0.1 ng/mL) of 11.1% of children at various ages, the multivariable regression models excluded the significant associations of these GLP exposures with any kidney injury biomarkers. Adapted with permission from ref ( 227 ). Copyright Elsevier 2020. Moreover, contradictory in vivo results were demonstrated. Pure GLP was hepatoxic to wall lizards ( Podarcis siculus ), important predators of herbivorous insects. Oral administration of low doses of GLP to a lizard caused fibrotic formation and a loss of liver functions. These malfunctions were associated with oxidative stress, manifested by the dysregulation of Cu/Zn superoxide dismutase, GSH-Px, metallothionein, and tumor suppressor protein 53, and upregulation of ERα and vitellogenin, thus showing the xenoestrogenic activity of GLP. 199 Studies on rats confirmed GLP and GBH hepatotoxicity. 28-day feeding rats with GLP caused weight loss, triggered primary DNA damage in the liver cells and leukocytes, lowered thiobarbituric reactive substances in the liver and plasma, and dysregulated AChE and GSH-Px activity in the liver and plasma. 200 Two-year chronic exposure to ultralow (0.1 ppb) Roundup, administrated via drinking water, occurred as hepatoxic and nephrotoxic. Transcriptome microarray analysis confirmed the GBH-related disruption of spliceosome and chromatin, lipotoxicity, phospholipidosis, and abnormal enlargement and necrosis of the liver and kidney cells associated with anatomical symptoms, including fibrosis and ischemia. 201 Similarly treated rats had symptoms of steatohepatitis. The proteome analysis confirmed the GBH-induced disturbance of organonitrogen metabolism and fatty acid beta-oxidation, hallmarked by peroxisomal proliferation, steatosis (fatty liver disease), and necrosis. The metabolome analysis confirmed lipotoxicity and oxidative stress related to the glutathione and ascorbate free radical scavenger system. Likewise, the progression of steatohepatitis was associated with the GBH-induced alteration of biomarker levels of the nonalcoholic fatty liver disease biomarkers. 202 Finally, the hepatotoxicity of GLP and Roundup has been confirmed in metagenomics and metabolomics profiling-based studies of rats exposed to these herbicides. These exposures caused markedly increased levels of gastrointestinal, hepatic, and oxidative stress biomarkers associated with shikimate and 3-dehydroshikimate, reflective of the inhibition of EPSPS of the shikimate pathway. These outcomes suggest a severe herbicidal impact on rat gut microbiota, although it must be highlighted that Roundup’s toxicity was higher when compared to GLP. 203 Particularly, regarding liver biochemistry, the multiomics approach confirmed a herbicidal-caused deviation of nicotinamide (vitamin B 3 ) metabolism that naturally prevents hepatic steatosis by increasing the redox potential. 204 These results were reinforced in a comparative 12-month study on hemo- and hepatotoxicity of GLP and Roundup in rabbits, designed for the real-life risk simulation (RLRS) approach. Toxicities of GLP and Roundup were evaluated versus a mixture of common endocrine disruptors and xenobiotics containing GLP, bisphenol, and triclosan as well as phthalates and paraben derivatives. As a result, GLP displayed only redox perturbations in blood homeostasis, whereas there were no effects of GLP on liver tissue. This relatively minor outcome was contradictory to the effects of Roundup and a mixture of endocrine disruptors that distorted blood redox equilibrium and caused oxidative stress manifested by decreases in the activities of SOD and glutathione reductase and increases in the total antioxidant capacity and activities of GSH and GSH-Px. Overall, these findings confirmed the RLRS approach applied to the hemo- and hepatoxicity of Roundup and common xenobiotics, whereby the harm of pure GLP was the lowest or negligible. 205 Additionally, the subacute exposure of rats to Roundup caused adverse inflammatory effects. The Roundup treatment elevated levels of C-reactive protein, cytokines IL-1β, IL-6, TNF-α, and prostaglandin-endoperoxide synthase in the liver and adipose tissue. These results correlated with histological analysis showing the formation of vacuoles, fibroid tissue, and glycogen depletion, thus suggesting the progression of fatty liver disease, multiorgan inflammation, and liver scarring. 206 Preclinical studies uncovered GLP- and GBH-induced hepatotoxicity, congestive hepatopathy, and liver fibrosis. These disorders were confirmed in patients with nonalcoholic steatohepatitis (NASH) and biopsy-determined nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disease in developed countries nowadays 194 , 195 ( Figure 4 ). Particularly, high-performance liquid chromatography (HPLC) examination of urine profiles revealed a significant increase in GLP excretion in NASH patients compared with non-NASH patients. These results and a dose-dependent GLP exposure-fibrosis stage correlation suggest that NASH patients are more susceptible to fibrosis progression and the development of cirrhosis and hepatocellular carcinoma. 207 Clinical cases of GLP-related hepato- and nephrotoxicity in humans mainly refer to commercial GBHs. As documented, multiple symptoms of GBH intoxication include respiratory failure, GI dysfunction, neurological disorders, 208 and disruption of the liver and kidneys. 209 Kidney (renal) disease, also called nephropathy, results from inflammatory (nephritis) or noninflammatory (nephrosis) renal malfunction. Two significant types of kidney disease are distinguished, namely, chronic kidney disease (CKD), lasting longer than three months, 210 and acute kidney injury, i.e., a sudden, severe impediment to renal function. 211 In 2017, the global burden of CKD reached 1.2 million deaths, making it the twelfth leading cause of death ( Figure 4 ). Major CKD causes include diabetes, hypertension, cardiovascular disease, xanthine oxidase deficiency, retention of analgesics or nephrotoxins, deposition of antibodies (glomerulonephritis), as well as lupus, sepsis, polycystic kidney disease, kidney stones, and infections of the urinary tract. 212 , 213 In vivo toxicogenomic studies confirmed the nephrotoxic effects of the GLP and Roundup formulations. For example, hyaline cysts, mineralization pelvis, epithelial pelvis, kidney tubular fibrosis, and kidney tubular degeneration were observed in rats treated with these herbicides. These malformations were associated with genotoxic alterations and DNA damage in the kidney. 214 Epidemiological reports have hallmarked the pesticide- or herbicide-induced kidney diseases in farmers worldwide, including in Sri Lanka and India, 215 − 218 and the U.S.A. 219 , 220 A critical summary of global pesticide CKD epidemics, including GLP and GBH, was presented elsewhere. 221 Noteworthy, the environmental use of GLP and GBH-related CDK risk had arisen since 1994 when GLP became a potential causal factor for CKD of unknown etiology in rice paddy farming areas in the dry zones of Sri Lanka (called Sri Lanka Agricultural Nephropathy). The putative GLP-induced CKD outbreak was associated with the GLP spraying, particularly with the consumption of hard water and exceptional metal chelating properties of GLP. As revealed by inductively coupled plasma mass spectrometry and ELISA analyses, samples of drinking water from serving wells and abandoned wells contained traces of GLP and metals, including Ca, Mg, Ba, Sr, Fe, Ti, and V. 215 , 216 Besides, urine profiles of the farmer patients living in endemic areas revealed urinary Sb, As, Cd, Co, Pb, Mn, Ni, Ti, and V concentrations exceeding the reference ranges. Moreover, GLP and creatinine urinary levels were elevated compared to the nonendemic controls. 217 Finally, LC-MS analyses of topsoil samples from agricultural fields, water samples from nearby shallow wells and lakes, and sediment samples from lakes confirmed the presence of GLP complexes with Fe and Al and strong retention of GLP in soil and groundwater. 218 Nephrotoxicity of GLP-metal complexes is well documented by studies in vivo and in humans. 222 − 224 In 2014, a clinical case report demonstrated airborne GLP-induced hepatorenal dysfunction in workers of GLP-producing factories. 225 However, contradictory results were obtained by comparing GLP and Roundup renal toxicity in male rats. By measuring levels of kidney biomarkers, including serum urea and creatinine, plasma cystatin-C and neutrophil gelatinase-associated lipocalin (NGAL), and oxidative stress indices related to activities of several kidney membrane-bound enzymes, we showed that Roundup-exposed rats accumulated more xenobiotics (including GLP, the ActI) than the group exposed to GLP alone. This increased accumulation was associated with nephrotoxicity, hallmarked by deviated levels of the biomarkers, whereas GLP administrated alone displayed no effect on renal function. 226 Eventually, GLP potential nephrotoxicity was evaluated in children by measuring urinary levels of kidney injury biomarkers including albuminuria, NGAL, and kidney injury molecule-1. Despite GLP detectability in the children’s urine, there was no evidence of GLP-induced renal injury 227 ( Figure 4 C– 4 E). Clinical cases of GBH intoxication involve severe GBH nephrotoxicity. 228 In the case of an intentionally intoxicated 22-year-old man, the poisoning caused a cute hemolysis, acidosis, and compensatory respiratory alkalosis. Besides, the GLP-SH increased the permeability of the erythrocyte membrane by disturbing the lipid bilayer and consequent hypotonic hemolysis, which resulted in multiorgan collapse, including frequent ventricular tachycardia, acute renal failure, rhabdomyolysis, coagulation dysfunction, and urinalysis. The patient recovered after alkaline diuresis, emergency plasmapheresis, and blood component transfusion. 229 Considerable controversy regarding cancer risk associated with both the use and misuse of GBH has recently arisen among scientists, authorities, and society. 230 , 231 Chronologically, in 2014, EFSA classified GLP as “unlikely to pose a carcinogenic hazard to humans”. A year later, the International Agency for Research on Cancer (IARC) stated that GLP is a “probable human carcinogen” (Group 2A), 232 whereas, in 2016, the EPA concluded that it is “not likely to be carcinogenic to humans”. 233 In 2017, the European Chemical Agency (EChA) denied a support link between GLP and animal cancer. 234 However, the Joint Meeting of Pesticide Residues (JMPR) agreed on the possibility that GLP “is cancerogenic in mice at very high doses”. 235 In 2018, the AHS cohort study declined any association of GLP with any solid tumors or lymphoid malignancies, including non-Hodgkin lymphoma (NHL) and its subtypes, with only some evidence of increased risk of acute myeloid leukemia. 236 These discrepancies originate from various sources of epidemiological data, lack of established criteria for statistical analyses and meta-analyses, overconfidence in common cancer etiology in experimental animals and humans, as well as differences in toxicological impacts and human incidence exposures to pure GLP and GBH formulations. 231 , 236 − 238 Despite clear GLP-induced cancer cases in rodents, including hemangiosarcomas, hemangiomas, kidney and liver adenomas, malignant lymphomas, NHLs, skin keratoacanthomas, adrenal cortical carcinomas, and skin basal cell tumors, summarized in 2020, 231 some of these reports have already been questioned. 238 Although the recent epidemiological evidence has recalled the carcinogenic potential of GLP in humans, 239 the GLP-induced oxidative damage, chromosomal alterations in human lymphocytes, and stimulation of cell proliferation, i.e., the major causative factors of cancer, cannot be underestimated. 240 − 245 These results are consistent with comparative toxicogenomics conducted on rats exposed to GLP and three Roundup formulations used in the EU (MON 52276), United Kingdom (Roundup ProBio, MON 76473), and the United States (Roundup PROMAX, MON 76207). Generally, these herbicides caused formulation- and organ-specific genotoxicity in the liver and kidney. Significant carcinogenesis-associated alterations were observed in the epigenome (differences in CpG methylation and levels of miR-10, miR-17, miR-22, and miR-30), DNA damage-associated TP53 protein activation, deviated circadian rhythm regulation, oxidative stress, and unfolded protein response. Importantly, Roundup formulations were far more genotoxic than GLP. However, GLP, in particular, caused apurinic/apyrimidinic DNA damage in the liver. 214 In this context, worth mentioning is that the augmented risk of cutaneous melanoma associated with occupational exposure to the sun, GBH, and fungicides was alerted among human subjects in Italy and Brazil. 246

Modern toxicology and epidemiology have been revolutionized by ultrasensitive analytical tools of precision (personalized) theranostics and wearable or smartphone-assisted artificial intelligence-excelled sensors or drug delivery systems. 260 Regarding GLP poisoning and GLP above’s modes of actions, i.e., dysbiosis and the inhibition of AChE, these devices, electronically powered, decision-making, and user-friendly, shall enable self-handled or point-of-care professional-assisted evaluation of the harm followed with rapid and precise determining and removing the herbicide and coformulants. In technological terms, these devices will be constructed as biocompatible chemo- and (bacteria cell)-based electronic biohybrids and nanorobots, capable of measuring the GBH analyte or GBH pathology-associated biomarkers in skin, when implanted or wearable as a chip, in body, when swallowed, or in body fluids liquid, when used as a portable, smart device. 19 , 261 − 263 Finally, it is expected that chemisorptive or adsorptive tissue-specific systems will be manufactured to detect and capture the GBH xenobiotics with molecular recognition-provided selectivity and sensitivity, which will be followed by harmless, on-site, and nature-mimicking microbial biodegradation of GLP. 264 , 265

Glyphosate (GLP), N -(phosphonomethyl)glycine, is an active ingredient (ActI) of the most common herbicides used in contemporary agriculture, forestry, industrial weed control, lawn, garden, and aquatic environments. By slow, reversible, and competitive inhibition of 5-enolpyruvynyl-shikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19), responsible for the biosynthesis of aromatic amino acids in plants 1 and several strains of bacteria, yeast, algae, and fungi, GLP acts as one of the most effective and broad-spectrum agrochemicals ever produced. 2 , 3 First synthesized in 1950, then commercialized in the herbicide market in 1974, GLP has become the most widely used, postemergent, nonselective weedkiller worldwide. 4 Agricultural use of genetically engineered (GE) GLP-tolerant crops, commercial GLP-surfactant (GLP-SH), and GLP-based herbicides (GBHs), often used as Roundup or RangerPro commercial formulations, applied within the so-called “green burn-downs” has modernized the harvest, weed, and herbicide management. In recent years, GBH usage has increasingly been withdrawn in EU, the U.S.A., and then worldwide. 5 Significant causes include environmental pollution of GBH, the development of herbicide-resistant weeds (superweeds) and microorganisms (superbugs), as well as overconsumption of GE organisms and GBH-contaminated products. 6 − 8 Concerningly, the relevant epidemiological and environmental contamination risk and human toxicity are driven by the growing reports on the health issues among farmers and occupational workers of GBH factories. 9 , 10 The most poisonous xenobiotics present in GBHs are GLP metabolites, e.g., (aminomethyl)phosphonic acid (AMPA), dyes, antifoaming agents, inert ingredients, and adjuvants, e.g., ethoxylates and polyoxyethyleneamine (POEA), more toxic than GLP itself, 11 − 14 and ppb traces of heavy metals, including chromium, cobalt, lead, or nickel. 15 Notably, the toxicity of these coformulants is diverse and variant. According to the insightful review of pesticide variability, 16 ∼750 different GBH formulations on the market contain various combinations of the GLP active principle and coformulants. Moreover, because of the legal regulations, the total composition of GBH is classified as confidential commercial information. 17 Hence, in commercial brochures and scientific reports, this heterogeneity of ingredients is usually simplified and defective, which may cause confusion and potential risk, even though the toxicological details of each ingredient included are well-described in the medical literature. The current review article presents the poisoning with GLP, GLP-SH, and GBHs impact on the endocrine, reproductive, and cardiopulmonary systems, also mentioning its hepato- and nephrotoxic consequences. Besides, we introduce the potential carcinogenic effects of this poisoning. Ailments of the gastrointestinal tract and nervous system, including gut dysbiosis and the dysregulation of microbiota-gut-brain-axis, are discussed in our review article concerning GLP and GBH toxicity, 18 whereas an extensive summary of novel technologies applied to GLP sensing is presented in our recent critical review. 19

GBH intoxication triggers two major pathological clinical effects. First, it affects the GM and impacts the MGB and HPA axes. This dysbiosis increases GI motility, resulting in fecal and urinary incontinence, miosis, diaphoresis, and diaphragmatic failure. Second, after entering the nervous system, GLP, an OP, phosphorylates the serine hydroxyl groups in AChE, thus inhibiting the hydrolysis of ACh, which causes dysregulation of cholinergic neurotransmission and overstimulation of muscarinic and nicotinic receptors in skeletal muscles. 254 So far, no antidote has been developed for GLP-SH poisoning, and the therapy is mainly symptomatic and supportive. 255 , 256 Pharmacological therapy supplies the use of competitive ACh antagonists or pseudoreversible inhibitors of cholinesterase. In the case of established OP exposure, therapy includes pretreatment. 254 Carbamate pyridostigmine (the only FDA-approved substance for this pretreatment) is investigated most widely. However, the CNS remains unprotected because of its incapability to cross the blood–brain barrier (BBB). 27 , 257 In typical therapy of OP poisoning, three FDA-approved therapeutics are used, vis., atropine, pralidoxime or obidoxime, and diazepam, among which atropine is the most common. 257 Another group of therapeutics includes enzyme-based GLP inactivators. They enable GLP transformation and biodegradation in the bloodstream before the GLP crosses the BBB. The transformation rate constants span from 0.08 to 100 h –1 (butyrylcholinesterase) through 4–340 min –1 (paraoxonases) to 5–2100 s –1 (phosphotriesterase or organophosphorus hydrolase). 254 Other therapeutics, including non-FDA-approved oximes, alkaloids, anti-NDMA agents, MgSO 4 magnesium sulfate and NaHCO 3 , and antibody-enzyme-nanoparticle conjugates, have been reviewed elsewhere. 254 HPLC-MS and ELISA have been employed in conventional medical diagnostics of GLP or GBH poisoning. They are mainly used for the toxicological determination and degradation of GLP in the blood and urine. Emergency treatment of acute GBH intoxication involves HDF- or DHP-assisted gastric lavage with a large amount of normal saline followed by active charcoal administration. 165 Further detoxification includes charcoal hemoperfusion and pulse therapies of cyclophosphamide and methylprednisolone, followed by dexamethasone application. In cases of hypoxemia, glucocorticoid and cyclophosphamide pulse therapies are applied. 256 Intensive care is required in severe GLP-SH intoxication, symptomized by dehydration, oliguria, paralytic ileus, hypovolemic shock, cardiogenic shock, pulmonary edema, hyperkalemia, and metabolic acidosis. 165 , 249 In the case of hyperkalemia, the urine and blood concentrations of potassium, GLP, and AMPA can be decreased by an enema with polystyrenesulfonate 251 or intragastric cathartic and charcoal administration. 252 Pharmacological treatment is applied to injuries to the digestive system, including constipation and overall digestive peristalsis. 253 However, colon resection and colostomy are the only solutions for mild colonic distention and peritonitis. 209 In cases of hypotension, vasopressor-based therapy can be supported by hemodialysis, 164 , 250 and I.V. lipid emulsion application. 167 , 168 For multiorgan failure, including cardiopulmonary and acute kidney failure, extracorporeal membrane oxygenation 166 or alkaline diuresis, emergency plasmapheresis, and blood component transfusion 229 , 258 , 259 are recommended.

GLP and GBHs are remarkably poisonous to the gut microbiome (gut microbiota, GM) 20 , 21 and the neurological system 22 − 25 by affecting the microbiota-gut-brain (MGB) axis and GLP-mediated inhibition 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 dysregulating activity of GLP/GBH on the hypothalamic-pituitary-adrenal (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 Disruptor Screening Program (EDSP) and, subsequently, by the European Food Safety Authority (EFSA). In 2015, the EPA and EFSA eventually declined the direct interaction of GLP with estrogen, androgen, and thyroid (EAT) pathways involving modes of action. 50 − 52 Because of the absence of the EPSP-metabolic pathway in vertebrates and the quick elimination of GLP, after its absorption and accumulation, from mammals, the GLP half-life time (∼5 to 10 h) is relatively short. Therefore, GLP is expected to be slightly or minimally toxic. However, many distressing reports have recently pointed to the GLP and GBH pathogenesis, indicating both 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 ). n. m.: not measured. n. m.: not measured. n. m.: not measured.