Introduction
The widespread use of pesticides in various domains increases the risk of environmental contamination by different xenobiotics which can be potentially noxious for non-target organisms including human beings. The use of organophosphate (OP) Glyphosate (Gly) has been globally expanded. Indeed, around two-thirds of the total volume of Glyphosate-based herbicides (GBH) used has been delivered to the environment during the last decade (Myers et al., 2016), in step with the increased adoption of genetically engineered crops such as corn, soy and canola. This GBH is the active ingredient present in Roundup® (Monsanto Company, St. Louis, MO, USA), currently the most heavily used herbicide worldwide (Powles et al., 1997).
Organophosphate (OP) exposure has been reported to be linked with mood disorders, such as depression and anxiety (Landrigan et al., 1999; London et al., 2005; Lee et al., 2007). Previous studies have demonstrated the potential risks of these pesticides, including their neurotoxic effects on developing organisms (Eaton et al., 2008). Furthermore, epidemiological studies have reported that long-term exposure to OPs elicits many neurological disturbances (Steenland et al., 1994; Roldan-Tapia et al., 2006). While classified under the herbicide class phosphonomethyl amino acids, Gly is often mischaracterized as an OP. This is likely due to the molecular structure being an organic molecule containing a phosphorus atom. However, clinical reports describing incidents of human ingestion of Gly do not reflect the classic symptoms for OP poisoning (salivation, lacrimation, urination, and defecation (Costa, 2008). Conversely, Seneff et al. (2015) highlighted the strong correlations between the increasing application of Gly in agriculture and the apparent surge of several neurological diseases at different ages. Abnormal EEG activity characterized by limb tremor (akinesia and rigidity) observed in Parkinsonian syndrome have been reported after professional exposure or accidental ingestion of a commercial mixture of Gly (Barbosa et al., 2001; Malhotra et al., 2010; Wang et al., 2011). In addition, data derived from structural MRI studies highlighted in a subject exposed to Gly changes in the T2 signal in substantia nigra, periaqueductal gray and globus pallidus, revealing likely lesions in these structures (Barbosa et al., 2001). Moreover, in human studies, Gly has been detected in the brain and cerebro-spinal fluid following commercial mixtures exposure, attesting that the active component can traverse the blood brain barrier (Menkes et al., 1991; Sato et al., 2011).
Research on animal models confirmed the neurotoxic effects of Gly. Indeed, Negga et al. (2012) revealed that exposure to GBH cause neuronal death, especially of GABAergic and dopaminergic neurons in the nematode Caenorhabditis elegans. It was also reported that Gly induces acetylcholinesterase activity alteration in the brain and the muscle of fishes exposed either to pure Gly or Roundup® (Modesto and Martinez, 2010; Menéndez-Helman et al., 2012; Sandrini et al., 2013; Samanta et al., 2014). An increasing body of literature revealed that Gly was able to provoke oxidative stress in specific rat brain regions such as substantia nigra, cerebral cortex and hippocampus (Astiz et al., 2009; Cattani et al., 2014).
In addition, the exposure to the GBH Roundup® during pregnancy and lactation provokes decrease in glutamate uptake and metabolism within glial cells, and increasing glutamate release in the synaptic cleft in the hippocampus of orally exposed rat’s offspring, leading to glutamate excitotoxicity (Gallegos et al., 2016).
Apart from the prenatal or postnatal period, the adolescence age is also a critical phase of brain development (Van Waes et al., 2011). Indeed, adolescence is a critical ontogenic period characterized by the maturation of brain processes that underlie higher cognitive functions, social and emotional behaviors (Spear, 2000). This period is often one of increased vulnerability and adjustment (Steinberg, 2005). In humans and rodents, adolescence is a time of extensive neural reorganization/pruning, in which long-term deleterious effects due to drug and environmental factor exposures are greater than at later times in life (Spear, 2000; Mendola et al., 2002).
The evident structural and neurochemical changes during adolescence are characterized by increases in functional connectivity between regions of the prefrontal cortex and several areas of the limbic system, especially the dopaminergic and serotoninergic systems, concomitant with changes in emotional and cognitive function regulation (Wahlstrom et al., 2010; Naneix et al., 2012; Spear, 2013). Moreover, during adolescence, brain areas continue to develop, such as the amygdala, hippocampus, and prefrontal cortex (Giedd and Rapoport, 2010). Thus, disturbing serotoninergic and dopaminergic systems in the aforementioned brain regions may result in permanent behavioral alterations associated with motor and emotional functions. In light of this, an important determinant that remains to be investigated is whether GBH exposure, following exposure to a Gly-containing herbicide, acts directly on the adolescent and adult brain resulting in subsequent sensorimotor and emotional function controlled by serotoninergic and dopaminergic systems.
Discussion
Because of the scarce information on the neurobehavioral effects of GBH in mammals, the present research was designed to gain insights concerning the effects of acute or repeated exposures to GBH on developing brain of juvenile and adult mice.
Our results showed that subchronic and chronic exposure to GBH in mice induced a significant decline in body weight gain for both treated groups. These results are in agreement with those obtained using comparable doses in adult mice (Jasper et al., 2012) and in rabbits (Yousef et al., 1995). According to Chahoud et al. (1999), weight loss is the main important indicator of toxicity, which may be associated with Roundup®’s capacity to provoke reactive oxygen species production. In addition, the decrease in the food consumption and the anorexic effect of Gly could lead to lower relative organs’ weight (ROW; Beuret et al., 2005). Thus, the assessment of ROW clearly revealed that GBH exposure induced a significant diminution of brain and liver weights after 6 weeks of treatment, while it affected brain, kidneys, liver and lung’s weight following 12 weeks of treatment. Several studies showed that Gly residues in the kidney, liver and lung were comparable to those found in the urine. This means that the Gly does not pass through the urine without affecting the organism (Krüger et al., 2014; Seneff et al., 2015). These results are consistent with previous studies in F344/N rats and B6C3F1 mice (World Health Organisation (WHO), United Nations Environment Programme, the International Labour Organisation, 1994). Similar results have been found in animals exposed to other pesticides such as deltamethrin, fenvalerate and diazinon (Kalender et al., 2006; Kilian et al., 2007). Indeed, in the same studies, the decrease of body weight gain recorded was due to malabsorption of nutrients induced by the gastro-intestinal tract impairment or inhibition of protein synthesis.
Acute exposure to GBH showed no change in locomotor activity. In contrast, subchronic and chronic treated animals showed a hypoactive profile. Our results are similar to those obtained by Hernández-Plata et al. (2015), who showed that repeated intraperitoneal injections of 100 or 150 mg/kg of Gly causes hypoactivity in adult male rats and those of Gallegos et al. (2016) in prenatally treated rats. Likewise, rats treated with methylparathion OP (Sun et al., 2006), diisopropylfluorophosphate pesticide (Bushnell et al., 1991) or dichlorvos-exposed (Binukumar et al., 2010) exhibited decreases in locomotor activity. Corroborating these findings, several OPs have been linked to movement and coordination deficits in humans following occupational exposures (Lotti and Moretto, 2005; Ehrich and Jortner, 2010). Further, many studies have reported that the locomotor activity reduction is positively correlated with the loss of dopaminergic neurons in the substantia nigra, as well as DA receptors (Bano et al., 2014; Gallo et al., 2015). Indeed, both the nigrostriatal and mesolimbic dopaminergic systems are implicated in the control of motor and motivated behaviors, and they have been shown to be susceptible to herbicides. For example, exposure to pesticides has frequently been defined as a major contributor to the risk of PD development (Hatcher et al., 2008).
During adolescence, Peak levels of extracellular dopamine are observed (Philpot et al., 2009) and that firing of dopamine cells and neurotransmitter turnover in target regions is higher in adolescents than adults (Tarazi et al., 1998; Moll et al., 2000; Placzek et al., 2009; McCutcheon et al., 2012). Moreover, increased studies reported the vulnerability of the nigrostriatal dopaminergic system following exposure to pesticides during critical periods of neurodevelopment, thus compromising the integrity and viability of this system (Cory-Slechta et al., 2005a,b). In light of this, the alterations of the dopamine system seen in our study coupled with locomotor hypoactivity are particularly interesting, when compared with previous work that demonstrated a reduction in the TH levels that paralleled motor activity deficiency after paraquat and maneb pesticide (Thiruchelvam et al., 2000). In agreement with our results, the exposure to Gly-containing pesticide leads to degeneration of GABA and dopamine neurons in Caenorhabditis elegans (Negga et al., 2012) and induces loss of cardiolipin content and mitochondrial transmembrane potential, especially in SNc, with a concomitant increase in fatty acid peroxidation (Astiz et al., 2009). According to Jackson et al. (2002) and Ricci et al. (2003), mitochondrial transmembrane potential loss reflects an early stage of apoptosis and release of many mitochondrial proteins into the cytosol during the first steps of programmed cell death. Additionally, Gui et al. (2012) reported a cell death in the dopaminergic cell line PC12 by Gly through apoptotic and autophagic mechanisms. Recently, Hernández-Plata et al. (2015) showed a reduction of the D1-DA receptor binding in the nucleus accumbens, accompanied by a significant decrease in ambulatory activity observed after Gly administration. Furthermore, following only 150 mg/kg of Gly administration, it was reported that striatal extracellular DA levels decreased by 50% in the microdialysis sample (Hernández-Plata et al., 2015).
On the other hand, the results revealed that subchronic and chronic exposure to the GBH induced an anxiogenic-like behavior. Similarly to our data, Sánchez-Amate et al. (2001) and Chen et al. (2011) found that repeated exposure of rats to another OP chlorpyrifos induces anxiogenic-like behavior. Conversely, Gallegos et al. (2016) observed that prenatal exposure to 100 or 200 mg/kg of GBH produced an anxiolytic-like effect in rats. These differential results may probably due to the different exposure periods. In fact, our study focused mainly on juvenile and adult age, whereas Gallegos et al. (2016) paid more attention to the prenatal exposure period.
Moreover, our results revealed that GBH exposed mice showed significant depression-like behavior. Our findings were in agreement with Chen et al. (2014) who showed that adolescent rats exposed to chlorpyrifos exhibited depression-like phenotype. Moreover, this result shows similarities to those of other OPs (Aldridge et al., 2004; Roegge et al., 2008). Indeed, previous researches that investigated the effects of methamidophos and diazinon at neonatal age and adulthood detected behavioral changes associated with depression (Lima et al., 2009) and changes in neurochemical markers of 5-HT function (Lima et al., 2011).
It is well established that several neuronal systems, in particular the serotoninergic system innervating cortical and limbic brain structures, are involved in the pathophysiology of anxiety and depression related behaviors (Azmitia and Segal, 1978; Vertes, 1991). Indeed, it has been shown that 5-HT system sends anatomical connections to the amygdala (Bobillier et al., 1976; Sadikot and Parent, 1990) and the ventral mPFC (Steinbusch, 1981; O’Hearn and Molliver, 1984) as pivotal structures contributing to emotional disturbances. Moreover, besides the changes in 5-HT transporters and their receptor activity, there is an increase in the release of 5-HT from the DRN in adolescence in comparison with adult age (de Jong et al., 2006), and the levels of 5-HT in several brain areas are also increased. Thus, The alteration of serotoninergic homeostasis during its development may result in a pattern of modified brain connections, leading to the loss of synapses and dendritic arborization, which normally occurs from puberty to adulthood (Barros et al., 2006; Zhang et al., 2013) and permanent behavioral alterations may be induced in adults (Whitaker-Azmitia, 2005; Ansorge et al., 2008).
Furthermore, the ventral mPFC is of special interest because it is strongly implicated in the expression of behavioral and autonomic responses to emotionally relevant stimuli, and imaging studies highlight abnormalities in the structure and function of this region in patients with mood disorder (Kennedy et al., 2001; Drevets et al., 2008). In agreement with these findings, our immunohistochemical data have shown a decrease of immunoreactive-neurons of the DRN and serotoninergic fibers in the BLA and in the ventral mPFC of mice following subchronic and chronic GBH exposure. Our results joined those obtained by Anadón et al. (2008) showing that Gly administered orally in rats decreases 5-HT and dopamine levels in the frontal cortex, midbrain and striatum. All these suggested that GBH induces depression and anxiety-like behavior observed in the treated groups.
On the other hand, the findings support a crucial role for perturbed amygdala 5-HT and enhanced glutamatergic mechanisms in the pathophysiology of mood disorders. Indeed, previous evidence supports the possibility that emotional disorders involve neuronal hyperexcitability in the amygdala (Tran et al., 2013). In support, Tran et al. (2013) showed that 5-HT depletion leads to the hyperexcitability of the central amygdala as the output of the nucleus, elucidating the anxiety-like behavior. Moreover, findings indicate that the depletion of 5-HT causes ventral mPFC neuron stimulation that excites principal BLA neurons elucidating anxiety phenotype (Russchen, 1982; McDonald, 1998). According to these findings, our results show that obvious serotoninergic circuitry disruptions observed in the DRN, the BLA and the ventral mPFC could underlie some structural and functional alterations potentiating the hyperexicitability of the amygdala and promote anxiety-like behavior.
While the mechanism underlying GBH-induced TH and 5-HT immunoreactivity reduction is not evaluated in the present study, it appears to be largely mediated by reactive oxygen species. As Gly can act as protonophore increasing mitochondrial membrane permeability to protons and Ca2+ (Olorunsogo, 1990), it can trigger the production of reactive oxygen species resulting in oxidative stress (de Liz Oliveira Cavalli et al., 2013). In human cell lines, both Gly and Roundup increased necrosis and apoptosis (Gasnier et al., 2009; Mesnage et al., 2013). Similarly, in rats, treatment with Gly and Roundup generated oxidative stress, induced lipid peroxidation (Astiz et al., 2009; El-Shenawy, 2009) and thus affected the oxidation-antioxidation homeostasis, promoting apoptosis and cellular death (Shimada et al., 1998; Yang and Sun, 1998), thereby inducing the anxiety and a depression-like phenotype and reducing motor development in the animals exposed to GBH.
In conclusion, we report for the first time the neurotoxic effect of subchronic and chronic subtoxic Roundup® exposure in mice. Overall the results reported in the present study indicate that, as previously described for other OPs, GBH exposure during the juvenile and adult period leads to an alteration in the activity level of animals and their affective performances paralleled by the dopaminergic and serotoninergic system impairment. These results support the epidemiological findings that pesticide-exposed populations are susceptible to neurobehavioral changes.