The present study showed that oral exposure of mice to glyphosate or the commercial formulation (Trop®) based on glyphosate caused a significant reduction in body weight without changing the organ weight/animal weight ratio. Although the weight of such organs had not been affected, there was a significant change in oxidative stress markers in the liver, lung and kidney, a factor that may be associated with changes in the p53 gene, as well as in the expression of Bax, Blc2 and p53 proteins in these tissues. The reduction of the p53 gene associated with the increased Bcl2 protein and oxidative stress may, in turn, be responsible for the genotoxic effect seen in the bone marrow after exposure of animals to glyphosate or the commercial formulation (Trop®).
Jasper et al (2012), when administering a dose of the herbicide Roundap® corresponding to 500 mg/kg of body weight, noticed a significant weight reduction of approximately 10% in relation to the initial weight over 15 days of treatment. Studies report that the oral ingestion of glyphosate with surfactant can induce severe gastrointestinal injuries, which can be responsible for the reduction in food intake, thus, the weight reduction seen in animals exposed to glyphosate (Dai et al. 2016) is likely to be due to the change in palatability (Daruich and Zirulnik and Gimenez 2001).
Food or water intake was not assessed in the present study, but only the weight of the animals, however, this may be an explanation for a significant reduction in the weight of the animals, which was dose dependent and higher in the group treated with glyphosate 200 mg/kg. Therefore, it can be inferred that weight loss is directly correlated to the active glyphosate principle rather than the surfactant found in the formulation.
Determining the weight of organs such as the liver, the kidney and the lung can provide indications of each organ function and the action of the substance tested on the organism. Bali, Ba-Mhamed and Bennis (2017) showed that the exposure of Swiss mice to low doses of glyphosate for 30 days cause changes in the mass of the brain, heart, lung, kidney, adipose tissue, or gall bladder. This effect was not seen in the present study about the liver, lung, and kidney weight (Table 2), however, a significant loss in body weight (Fig. 1) occurred. This might have happened due to the short time of herbicide exposure, that is, only 5 days.
Figure 2 shows an increase in oxidative stress caused by the exposure to both glyphosate and the commercial formulation (Trop®) based on glyphosate. Oxidative stress is more evident in the pulmonary and renal tissues, since there was an increase in lipid peroxidation associated with a reduction in catalase enzyme activity and in total antioxidant defenses. Similar results were seen in the liver of mice treated with a single dose of either glyphosate or Trop® Zancanaro et al. 2019; Zhang et al. 2021 and in fish exposed to the herbicide Roundup® Glusczak et al. (2006) for 96 hours, as well as after maternal exposure to glyphosate-Roundup® with an increase in the levels of lipid peroxidation (TBARS) in the liver tissue of the babies of herbicide-exposed mothers (Sinhorin et al. 2014).
The treatment with either glyphosate or Trop® for 5 days reinforces the hypothesis of antioxidant defense consumption, that is, when the production of reactive species surpasses the antioxidant defense capacity, the phenomenon of oxidative stress starts, resulting in morphological and functional disturbance of the injured cell, which can trigger molecular changes, such as the appearance of micronucleated cells, which shows a genotoxic effect (Fig. 5). These data may suggest that either glyphosate or commercial formulations based on glyphosate can induce cellular changes capable of promoting different types of cancer. In this sense, an epidemiological study by De Ross et al (2005) showed a high incidence of multiple myeloma in glyphosate-exposed individuals.
According to Zang et al (2020) and Córdova et al (2019) evaluations on genotoxicity and oxidative stress in aquatic planarians (Dugesia schubarti) exposed to glyphosate formulations, that is, Roundup® Original, Roundup® Transorb and Roundup® ready, showed that all formulations induced DNA damage and oxidative stress. The activity of superoxide dismutase (SOD) increased between 4 and 16 hours of treatment, whereas TBARS levels decreased between 4 and 16 hours. Regarding the three formulations tested, CAT activity remained decreased at all periods of exposure. These results showed that glyphosate–based herbicide formulations can cause damage to DNA, in addition to changing the oxidative balance in planaria, which indicates that this herbicide toxicity is not restricted to plants.
Studies have reinforced the hypothesis that increased expression of the p53 protein can normally prevent the transformation process, whereas the decrease in p53 expression predisposes the cell to change (Eliyahu et al. 1989; Baugh et al. 2018). In cells that have the mutated p53 gene and inactivation of the p53 protein, the cell cycle does not occur, which is necessary for DNA repair. These genetically unstable cells tend to accumulate additional chromosomal mutations and rearrangements, which leads to a rapid proliferation of cell clones with mutated DNA and neoplastic transformation (Cavalcanti Júnior and Klumb and Maia 2002). Therefore, chemical substances capable of modifying the expression of p53 may be responsible for the appearance of cell lesions that culminate in the emergence of tumors.
The results of the present study showed a correlation among a reduction in the p53 gene, a reduction in Bax expression, and an increase in Bcl2 with oxidative stress. Figures 2, 3 and 4 show that a greater reduction in the p53 gene, liver and lung occurred in all treatments, and in the kidney submitted to the treatments with Trop®, which implied in a significant reduced Bax and increased Bcl2 associated with more pronounced oxidative stress. This is likely to have been triggered due to changes in the expression of p53, which is considered a sensor of cellular stress. The induction of oxidative stress is, thus, usually accompanied by the activation of p53, which works as a pro-oxidant factor to promote toxicities mediated by oxidative stress, favoring the death of mutated cells (Velez et al. 2011; Liu et al. 2020). This was rather reported in renal cells treated with cisplatin, which showed an inhibition of p53 reduced oxidative stress and, thus, a decreased cisplatin-induced cytotoxicity (Yuan et al. 2015).
The present study showed a reduction of the p53 gene that did not significantly reduce oxidative stress, resulting in less induction of apoptosis and greater cell survival, an effect evidenced by the reduction in Bax expression and increase in Bcl2 expression in lung, liver and kidney. Table 3 shows an inverse correlation between Bax and Bcl2, since the ratio between the two proteins was higher than that of the control group, especially in the pulmonary and renal tissues whose antioxidant defenses are lower. It can be inferred that there was an overexpression of Bcl2 and a reduction in Bax expression, which might reduce the apoptotic index and favor the proliferation of modified cells. Studies show that the higher the Bcl2/Bax ratio, the greater the cell survival and the appearance of mutations such as those that occur in cases of chronic lymphocytic leukemia or in lung tumors (Brambilla et al. 1996; Vucicevic et al. 2016).
On the other hand, Zhang et al. (2021) found an increase in Bax associated with a reduction in Bcl2 in the ovaries of mice treated with glyphosate. These differences in the data found by Zhang et al. (2021) and the present study may be associated with the administered dose and the evaluated tissue. In the present study, was used a dose of 50 and 200 mg/kg, while Zhang et al. (2021) used a dose of 250 and 500 mg/kg in a slightly longer treatment time (7 days).
Ikumawoyi et al (2019) reported that substances capable of up-regulating the expression of pro-apoptotic proteins (p53 and Bax) and down-regulating the expression of anti-apoptotic proteins (Bcl2) reduce the appearance of mutations that may cause the emergence of micronuclei. However, substances that have an opposite effect, as shown in the present study, can also favor the appearance of micronuclei, and can be considered pre-carcinogenic substances.
Table 3 shows a significant increase in Bcl2/Bax ratio in the lung and kidney when submitting the animals to treatment with glyphosate or the commercial formulation based on glyphosate. This may have favored the emergence of the genotoxic effect visualized in the bone marrow with the presence of micronucleated cells.
In this sense, the results of the present study suggest that oral exposure to either glyphosate or the commercial formulation (Trop®) based on glyphosate can induce cell damage by inhibiting the p53 gene, which results in greater oxidative stress and, thus, the emergence of the genotoxic effect visualized by increased micronucleated cells in the bone marrow. Similar results were obtained with the exposure of glial cells to the monocrotophic organophosphate (Tripathi et al. 2017) and after the exposure of fish to the organophosphate phorate (Pacheco and Hackel 2002). The genotoxic effect shown in the present study may be associated with an increase of the Bcl2/Bax ratio, especially in the lung and kidney (Table 3). Brambilla et al (1996) and Vucicevic et al (2016) reported that the greater this ratio the greater the survival of mutated cells.
The present study showed a significant increase in the micronuclei of the bone marrow erythrocytes when exposing the animals to either glyphosate or Trop® at a dose of 200 mg/kg, which corroborates with the results by Zang et al. (2020), thus, indicating a genotoxic effect of this herbicide in mammals.
It is worth mentioning that Pacheco and Hackel (2002) proved the genotoxic activity by testing the micronucleus in peripheral blood cells of individuals exposed to agrochemicals, such as fungicides, insecticides, and herbicides. Another study by Cavalcante, Martinez and Sofia (2008) showed that glyphosate, one of the herbicides most used by farmers, produces genotoxic damage in erythrocytes and gills of the fish Prochilodus lineatus. Bortoli, Azevedo and Silva (2009) showed the genotoxic potential of pesticides used in soybean fields in view of the significant increase in cells with micronuclei in herbicide-exposed workers.
The present study showed the occurrence of a significantly higher micronucleus in groups of animals treated with higher doses, which indicates that the genotoxic action may be dose dependent, characterizing the presence of mutagenic activity in these doses, like those found in animals treated with cyclophosphamide, a drug known as being cytotoxic and genotoxic. 58 Compounds that induce the appearance of a genotoxic effect may be responsible for the emergence of tumors since this effect is related to changes in the tumor suppressor gene p53 (Dural et al. 2020).
In conclusion the results obtained in the present study were summarizes in Fig. 6. Exposure to either the glyphosate herbicide or commercial formulations based on glyphosate induce genotoxicity mediated by an increased oxidative stress caused by the inhibition of the p53 gene, reduction in expression of the pro-apoptotic protein Bax, and increased expression of the anti-apoptotic protein Bcl2, which might exert a pro-oxidant effect after exposure to the herbicide. This is a dose-dependent effect that can be evidenced in short exposures. However, further studies that evaluate the pro-oxidant involvement of p53, as well as Bax and Bcl2 proteins regarding the exposure to glyphosate and the mechanisms associated with it must be carried out in herbicide-exposed workers so that the genotoxic effects and their possible correlation with the increase in the number of the cases of cancer can be proven in humans.