In this study, we observed tissue congestion, inflammation, an increase in the number of nucleoli-organizing regions (NORs) not associated with increased thickness of the epithelium in the tongue mucosa, and dysplasia of the oesophageal epithelium and small and large intestine in animals exposed to GBH. With the exception of an increase in the number of mast cells in the tongue and the number of micronuclei in polychromatic erythrocytes in the bone marrow, no parameters were influenced by the sex of the animals. The gastric mucosa did not suffer damage upon exposure to GBH.
The kidney and gastrointestinal tract have been identified as the target organs of glyphosate in ruminants. In the case of the digestive tract, mucosal irritation was detected by histopathological examination (EFSA 2015). Thus, in this study, we investigated the digestive tract and possible impact of inhalation exposure (more common in occupational exposure) and oral exposure to food contaminated by GBH (more common in paraoccupational exposure) on this organ system.
Burning, erosion, ulceration and haemorrhage of the oral mucosa have been reported in cases of incidental acute exposure to glyphosate (Mui 1993; Sribanditmongkol et al. 2012; Deo and Shetty 2012). Our study detected karyomegaly in cytological smears, which can be a reaction to tissue damage or even to a chronic inflammatory process. However, the group exposed to low concentration by inhalation had the highest number of cases with karyomegaly, highest intensity of congestion and lowest number of cases of inflammation. This result demonstrates direct damage caused by the effects of GBH on the epithelium; however, probably due to the low dosage of exposure, an inflammatory process was not detected. However, a greater number of cases with congestion and low inflammation were observed in animals exposed to high concentration of GBH (regardless of the route of exposure). Tissue congestion is the initial change in the inflammatory process in the tissue (Punchard 2004). High concentration of GBH can provoke a reaction in the tissue that stimulates more profound vascular alteration than that resulting from recruitment of inflammatory cells. A greater number of cases of inflammation were detected among animals exposed to low and medium oral concentrations, showing that the oral route and lower concentrations provide direct contact of GBH with the mucosa and can cause a local inflammatory response.
Mast cells reside in normal connective tissue and are associated with various pathological processes, such as allergies, tissue angiogenesis (formation of new vessels), and the inflammatory process (Parizi et al. 2010). We observed a greater number of mast cells in the oral mucosa in females and in groups exposed orally. These data show that GBH can stimulate an oral allergic reaction, especially after direct contact and in females. Studies evaluating other herbicides demonstrated that herbicides may be systemic allergens (Cushman and Street 1982; Yasunaga et al. 2015) and are allergenic to the skin, respiratory tract (Fukuyama et al, 2009) and oral cavity (Parizi et al. 2020).
NORs are markers of cell proliferation (Parizi et al. 2020). An increase in the number of NORs in the mucosa of the tongue observed in the groups exposed to GBH indicates toxicity to the oral epithelium that stimulated cell proliferation to repair possible cell destruction caused by GBH. However, in the group exposed to high oral concentration, the number of NORs was decreased compared to that in the control group. This decrease may be due to severe damage to the epithelium caused by a high oral concentration that prevents the stimulation of regenerative cell proliferation. We observed an increase in NORs in most groups exposed to GBH; however, this increase was insufficient to increase the thickness of the tongue epithelium. These data confirm that GBH causes a high level of cell destruction and that cell proliferation does not increase the thickness of the epithelium at this level of damage.
Case reports in humans described bleeding from the gastric mucosa after deliberate ingestion of glyphosate (Sribanditmongkol et al. 2012; Deo and Shetty 2012); however, another study showed ulceration of the oesophageal mucosa without gastric lesions in two cases of incidental ingestion of glyphosate (Chen et al. 2013). The authors speculated that the absence of gastric lesions in these cases may be due to the type of epithelium, presence of acid or mucus or other local factors and suggested that glyphosate may be considered a caustic agent of medium intensity (Chen et al. 2013). In our study, lesions of the gastric epithelium were not detected, possibly because even oral concentrations used in the present study were considerably lower, and GBH was inhaled or ingested along with feed rather than ingested alone in large quantities, as described in other studies. This consideration may also explain the absence of oesophageal lesions, such as erosions, ulcerations and inflammation, in the exposed animals.
An in vitro study with muscle fibres of the jejunum of rats detected a decrease in the motor activity even if glyphosate was applied at very low concentrations (Chlopecka et al. 2014). Dilation of the small intestine was observed after incidental ingestion of glyphosate in humans (Sribanditmongkol et al. 2012; Deo and Shetty 2012). In another study in weaned piglets, no change in intestinal morphology was detected after ingesting water containing glyphosate added in various concentrations (Qiu et al. 2020). A study in Sprague-Dawley rats demonstrated that long-term exposure to glyphosate in tap water was toxic to the intestinal microbiome, and dysbiosis was manifested only in treated females (Lozano et al. 2018). Dysbiosis of the intestinal microbiota is associated with a number of clinical conditions, such as inflammatory bowel disease or colorectal cancer (Cho and Blaser 2012). Induction of intestinal dysbiosis by glyphosate may explain the inflammation in the intestinal mucosa detected in exposed animals in the present study; however, we did not observe differences between sexes in this parameter reported in other studies. Greater intensity of inflammation and higher incidence of animals with lymphoid hyperplasia in the small intestine may be explained by higher concentrations of glyphosate detected in the small intestine after oral exposure (Chlopecka et al. 2014); a similar phenomenon may occur in inhalation exposure, since we did not detect differences between routes of exposure.
The carcinogenicity of glyphosate is a matter of controversy in the literature. IARC of World Health Organization (WHO) considers glyphosate to be "probably carcinogenic to human" (category 2A) (IARC 2015), and some studies in rodents have detected benign and malignant neoplasms in the thyroid, liver, kidney, pancreas, testis and pituitary gland (Greim et al. 2015); however, reviews of the toxicological profile of this herbicide in other studies suggested a lack of carcinogenic risk for glyphosate (Willians et al. 2016; Tarazona et al. 2017). EFSA considered that human studies were too limited to associate glyphosate with neoplasms (Portier et al. 2016). The grade of precancerous lesions in epithelial dysplasia is associated with a lower likelihood (mild dysplasia) or greater likelihood (moderate and severe dysplasia) of progression to carcinoma. Our study is the first to demonstrate that GBH has carcinogenic potential in the oesophagus and small and large intestine, especially at higher concentrations, and that the oral exposure route can lead to moderate dysplasia, which corresponds to a higher risk of progression to carcinoma.
The oesophageal epithelium is similar to the oral cavity epithelium, i.e., stratified squamous epithelium; however, dysplasia was detected only in the oesophagus. This phenomenon may be due to mechanical washing of the oral cavity by saliva, which reduces the time of exposure to glyphosate in the mouth.
Similar to carcinogenicity, the genotoxicity of glyphosate is also a matter of controversy according to the literature. Some studies have shown that glyphosate does not present a significant genotoxic risk under normal conditions of human or environmental exposure (Heydens 2008; Kier and Kirkland 2013). Other studies have shown that products containing glyphosate can be genotoxic in reptiles and mammals (Prasad et al. 2009; Lopez Gonzalez et al. 2013), increase DNA damage scores in the comet test in the erythrocytes and gill cells of fish (Moreno et al. 2014) and induce DNA strand breaks (also observed in the comet assay) in the gills and liver cells of eels exposed to glyphosate in water (Guilherme et al. 2011). Additionally, an increase in the incidence rate of chromosomal aberrations and induction of micronuclei depended on the concentration and time of exposure (1 to 3 days) to glyphosate in mammals (Prasad et al. 2009). This variability in the results may be due to differences in the purity of the active agent and the nature of the tested inert components, which can increase the toxicity of the herbicide (Prasad et al. 2009). Our study used the most common commercial worldwide brand and not the pure herbicide because it is not used in pure form to spray crops. Thus, comparison with the control groups in the present study detected an increase in the micronuclei in the groups exposed to GBH regardless of the route of exposure. This finding is in agreement with detection of dysplasia in the oesophagus and small and large intestine, indicating that GBH has genotoxic and carcinogenic potential at concentrations relevant to human exposures.
Gender can interact with exposure to xenobiotic agents and influence the toxicokinetics, toxicodynamics and results of the exposure. Sex-specific differences in response to xenobiotics can result from the differences in behaviour, exposure, anatomy, physiology, biochemistry and genetics, which can influence the responses to environmental chemicals and adverse reactions to drugs (Gochfeld 2017). Our study detected differences in some parameters between the sexes, including a higher incidence of cells with karyomegaly and mast cells in the oral cavity of females. These data show that females are more reactive and have a greater risk of oral allergic reaction upon exposure to GBH. Men and women were shown to differ in susceptibility to the development of immunological and allergy disorders and in the ability to fight infections. Women are more susceptible to the development of allergies and autoimmune diseases for multiple reasons, possibly associated with sex hormones and X chromosome factors (Laffont and Guéry 2019). These considerations may explain the higher cellular reactivity and greater number of mast cells in females exposed to GBH detected in the present study. We also observed that females had a higher number of micronuclei upon exposure to low oral GBH concentration, as micronuclei were not detected in males under these conditions; however, exposure to high concentrations by inhalation resulted in an increase in micronucleus formation in males. This result suggests that low concentrations are sufficient to cause DNA damage in females but not in males and that high concentrations can lead to cell destruction, explaining that the formation of micronuclei in females has not been detected. Other genotoxicity tests should be performed to provide a better explanation of various sex-dependent effects of various concentrations of GBH.
It is necessary to consider that pesticide formulations are mixtures of adjuvants and active ingredients (which in our case is glyphosate), and adjuvants may be as or more toxic than the active ingredient itself, or may potentiate the damage caused by the active ingredient (Mesnage et al. 2013). There is no description of the inert ingredients (adjuvants) added to the formulation in the package insert of the product that we use, so we cannot exclude the action of these products on the tissue damage observed in our study.
Further studies that evaluate the internal dose of glyphosate for each animal associated with the evaluation of the glyphosate concentration in the feed, as well as different concentrations of environmental exposure may provide a better understanding of the effects on the digestive tract, in addition to a better definition of the dose response.