The antioxidant and neuroprotective effects of melatonin on glyphosate-based herbicide-induced affective and cognitive impairments in rats

ABSTRACT

Several studies have found that exposure to glyphosate-based herbicides (GBH) can lead to memory impairment and affective disorders. Oxidative stress (OS) is one of the mechanisms that can explain the neurotoxicity of GBH. Consequently, molecules with an antioxidant effect may have neuroprotective potential against GBH-related brain damage. In this work, we aim to evaluate the neuroprotective effect of melatonin (MEL) against affective and cognitive disorders caused by GBH. For this experiment, male Wistar rats received, during the peripubertal period, a daily administration of GBH (75 mg/kg) or GBH  + MEL (4 mg/kg). At the end of the treatment, the open field, elevated plus maze, and forced swimming tests were used to explore anxiety levels and depression-like symptoms. While the Y-Maze and Morris Water Maze have been used to detect cognitive deficits, we also measured markers of OS, specifically catalase, nitric oxide, and lipid peroxidation, in the hippocampus and prefrontal cortex. Our results revealed that MEL reduces anxiety and depressive behavior, as well as the cognitive deficits caused by GBH. In addition, co-administration of MEL and GBH induced an improvement in the various markers of OS. The positive action of MEL against OS is therefore one of the mechanisms explaining its neuroprotective power against GBH.

KEYWORDS:

• Glyphosate-based herbicides
• Melatonin
• anxiety
• depression
• memory
• oxidative stress

Introduction

The widespread use of pesticides in agriculture is increasingly endangering the environment, as these substances are infiltrating water, food, and soil. Notably, glyphosate-based herbicides (GBH) emerged as the most commonly utilized organophosphate pesticide globally, and their usage has substantially increased in recent years [1]. On the other hand, the heightened usage of GBH results in persistent human and animal exposure to this pesticide through the consumption of food and drinking water [2]. Research indicates that GBH can permeate various body regions by traversing the intestinal epithelial barrier [3], leading to notable accumulations, particularly in cases of prolonged exposure to low doses of the pesticide [4].

Prolonged exposure to GBH presents a significant health threat to both humans and animals, as it has been associated with the onset of various adverse effects, including hepatic cancer and disruptions to the endocrine system [5–7]. Furthermore, concerns have been raised about the capacity of GBH to penetrate the blood-brain barrier (BBB), prompting apprehensions about their involvement in neurological disorders [8]. Clinical evidence links the escalating use of GBH to conditions such as anxiety, depression, Parkinson, autism, and memory impairments [9,10]. Simultaneously, animal experiments have substantiated the neurotoxic effects associated with this pesticide [11–13].

Even though the precise mechanism leading to affective and cognitive disorders induced by GBH is not fully comprehended, there is solid evidence that mood and memory disturbances can arise due to microbial degradation in the intestines [14], neuroinflammation [15], and/or imbalances in neurotransmitter systems triggered by this pesticide [8,11–13]. Certainly, oxidative stress (OS) appears to be a pivotal mechanism contributing to the neurotoxic effects of GBH. Additionally, existing literature emphasizes the involvement of OS, including heightened free radicals and imbalances in oxidant and antioxidant levels, in the pathophysiology of mood and memory disturbances [16–20]. Our previous work demonstrated that OS induced by GBH may play a role in behavioral alterations. In this sense, an ideal candidate for mitigating the adverse effects of GBH would be a molecule possessing antioxidant properties that can influence both physiological and behavioral functions.

Melatonin (MEL) is a neurohormone produced by the pineal gland in mammals [21]. Functioning as a multitasking molecule, it regulates various physiological processes, including circadian rhythms, energy metabolism, feeding behavior, immunity, sexual functions, and reproduction [22]. Moreover, this indoleamine can engage with other systems and/or neurotransmitters to modulate behaviors such as anxiety and depression, as evidenced in numerous studies [23,24]. Beyond its crucial role in ameliorating neurobehavioral deficits, MEL has the capability to neutralize free radicals, enhance the antioxidant defenses of cells, and reduce the activity of pro-oxidant enzymes [25,26].

To our knowledge, there have been no studies to date that have explored the effectiveness of MEL in mitigating GBH-induced neurobehavioral alterations in adolescent rats. Given this gap in research, the objective of the present study was to evaluate the impact of MEL administration on affective and cognitive disorders resulting from the subchronic administration of GBH in male Wistar rats, with a particular focus on the potential involvement of OS in the hippocampus (HPC) and prefrontal cortex (PFC).

Material and methods

Reagents

The herbicide applied in this study is BARBARIAN SUPER 360 (Barclay Chemicals Manufacturing Ltd.), Melatonin (N-acetyl-5-methoxytryptamine) was obtained from Sigma-Aldrich (St. Louis, France).

Animals and study design

Male WISTAR rats (1-month-old), supplied by the Ibn Tofail University breeding center, were maintained under controlled conditions of temperature (22 ± 2°C), with a 12-hour light/dark cycle, and had free access ad libitum to food and water. Rats received a daily injection for 4 weeks. During the peripubertal period (PND 30 to PND 58) [27], the rats were subdivided into three different groups of 6 animals: (1) Control group, rats receiving 0.9% NaCl, (2) GBH group, animals treated with GBH 75 mg/kg; (3) GBH + MEL group, animals injected with GBH (75 mg/kg) 1 hour before the administration of MEL (4 mg/kg). MEL was dissolved in a 1% ethanol solution and administered intraperitoneally at 4:00 pm (2 hours before the dark phase), while GBH (glyphosate concentration 360 g/l in the form of glyphosate isopropylamine salt) was diluted in a saline solution and injected subcutaneously, basis on the Glyphosate no-observed adverse effect level (NOAEL) for subchronic toxicity [28]. The volumes administered are adjusted twice a week according to the animals’ weight. All experimental procedures were in accordance with the University Ethics Committee guidelines. The timeline of the current experiment is presented in Figure 1.

Figure 1. Timeline of the glyphosate-based herbicide (GBH) and melatonin treatment study. The Open Field Test (OFT), elevated plus maze Test (EPM), forced swimming Test (FST), Y-maze test, and Morris water maze test (MWM). Nitric oxide (NO), lipid peroxidation (LPO), Catalase (CAT).

Neurobehavioral tests

Anxiety-like measurement

In this work, we used the open field test (OFT) and the elevated maze test (EPM) to determine the anxious behavior of the animals after different treatments.

Open field test (OFT): The box (100-L × 100-W × 40-H cm) is divided into two regions, central and peripheral. Each rat was monitored for 10 minutes to measure the time spent in the center area (TCA), the number of visits to the center (NRC), and the number of total squares (NTS). The exploration of the center area reflects the anxiety level, and the number of total squares indicates locomotion [29].

Elevated plus maze test (EPM): The apparatus consists of two opposing open arms (50 × 10 cm) and two closed arms (50 × 10 × 40 cm). Each animal was allowed to explore them for 5 minutes. A low level of exploration of the open arms indicates an increase in anxious behavior [30].

Depression-like measurement

We used the forced swimming test (FST) to evaluate the state of depressive illness [31]. The total duration of immobility (TIM) for 5 minutes was scored; an important level of immobility is a sign of depressive-like behavior.

Cognitive measurement

Y‑Maze test

This test is used to assess working memory in rodents. The Y maze consists of three identical arms A, B, and C (45 × 12 × 35 cm), for 8 minutes, the sequences of the three arm entries were recorded. The following formula is used to calculate the percentage of alternation: % alternation = [(number of alternations)/(total arm entries − 2)] × 100.

Morris water maze test

The Morris pool, first developed by R. Morris [32], is a fundamental tool in the study of memory in rodents. The animal is dropped into a circular pool filled with water made opaque by milk or white paint that contains an escape platform. The acquisition phase lasted 4 days. The time required for the animal to reach the platform placed in the north-west quadrant was recorded. The probe test was carried out after the last day of training. The platform was then removed from the pool, and the time spent in the north-west quadrant was measured during a single 60 second attempt.

Oxidative stress indices

After behavioral analysis, rats were euthanized by decapitation (without anesthesia). The HPC and PFC were collected on ice, homogenized in ice-cold 0.1 M phosphate-buffered saline (PBS) pH 7.4, and centrifuged at 1500 rpm for 10 minutes. The supernatant was stored at −80°C until use.

Determination of nitric oxide

Nitric oxide was tested using the Griess reagent [33]. The mixture of 100 μL of Griess reagent, 300 μL of the sample, and 2.6 mL of distilled water was incubated for 30 minutes at room temperature, and then the optical density was measured at 548 nm.

Lipid peroxidation assay

The reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA) is used to determine the concentration of thiobarbituric acid reactive substances (TBARS), which is one of the markers of lipid peroxidation (LPO). The reaction product is quantified at 532 nm [34].

Catalase activity

The decrease in H₂O₂ absorbance at 240 nm was recorded every 30 seconds for 2 minutes. CAT activity was expressed as IU/min/g of tissue [35].

Statistical analysis

All measurements were analyzed by an observer blind to the treatments. Figures were made using GraphPad Prism 6 software. Data were represented as mean ± standard error of the mean (SEM), and comparisons between groups were made using a one-way analysis of variance (ANOVA), followed by a post-hoc Tukey (Version 22 SPSS). A repeated measures ANOVA is used for the MWM; the comparison of groups is judged to be statistically significant if p < 0.05.

Results

Effect of GBH and MEL on the levels of anxiety-like measured in OFT

The results summarized in Figure 2(a,b) show that rats treated with GBH for four weeks showed significantly decreased TCA (−48%) and NRC (−36%), as compared to normal controls (p < 0.01 and p < 0.05, respectively). However, MEL administration significantly increased the TCA (+71%) and NRC (+73%) in comparison with GBH-treated rats (p < 0.05 and p < 0.01, respectively). Besides, the NTS was unaffected by any treatment (p > 0.05) (Figure 2(c)).

Figure 2. Effects of GBH (75 mg/kg) and MEL (4 mg/kg) administration on anxiety associated behaviors adolescent male rats. (a) Total amount time spent in the center (TCA); (b) number of returns into center area of the arena in the open-field behavior apparatus (NRC); and (c) number of total squares (NTS) in the open field test. The data are presented as mean ± S.E.M of 6 animals/group. *p <0.05, **p <0.01 compared with the control group; and ^$p <0.05, ^$$p <0.01 compared with the GBH group.

Effect of GBH and MEL on the levels of anxiety-like measured in EPM

Our results presented in Figure 3(a,b) show that the GBH group showed a significant decrease in EOA compared to the control group (p < 0.05). Moreover, MEL induces a significant increase in the time and number of entries into the open arms of the EPM compared to the group exposed to GBH alone (p < 0.05 and p < 0.01, respectively). In addition, no change was observed in the TEA parameter following different treatments (Figure 3(c)).

Figure 3. Effects of GBH (75 mg/kg) and MEL (4 mg/kg) administration on anxiety associated behaviors adolescent male rats. (a) Total amount of time spent in exposed arms (TOA); (b) number of entries in exposed arms (EOA); and (c) total number of arms entries (TEA) in the elevated plus maze. The data are presented as mean ± S.E.M of 6 animals/group. *p <0.05 compared with the control group; and ^$p <0.05, ^$$p <0.01 compared with the GBH group.

Effect of GBH and MEL on depressive-like performances measured by FST

According to the results of the FST (Figure 4), there was a significant increase in the immobility time of rats exposed to GBH compared to the control group (p < 0.01). In contrast, the group treated with MEL and GBH showed a significant time reduction in immobility compared with the GBH group (p < 0.05).

Figure 4. Effects of GBH (75 mg/kg) and MEL (4 mg/kg) administration on depression-related behavior of adolescent male rats subjected to the forced swimming test. immobility time expressed in seconds. The data are presented as mean ± S.E.M (n = 6). **p <0.01 compared with the control group; and ^$p <0.05 compared with the GBH group.

Effect of GBH and MEL on memory

Y-Maze test

Exposure to GBH induced a significant reduction in the percentage of alternations compared to the control group (p < 0.01), while the combination of MEL and GBH treatment induced an increase in the percentage of alternations compared to the GBH-exposed group (p < 0.05) (Figure 5).

Figure 5. Effects of GBH (75 mg/kg) and MEL (4 mg/kg) administration on spontaneous alternation percentage measured in Y-maze after 4 weeks of treatment in adolescent male rats. The data are presented as mean ± S.E.M of 6 animals/group. **p <0.01 compared with the control group; and ^$p <0.05 compared with the GBH group.

Morris Water Maze

Spatial learning

On the first day of the acquisition phase, we observed that the GBH group spent significantly more time finding the platform compared to the group treated with the MEL (p < 0.01). Whereas our results show no significant variation on the three other days (Figure 6(a)).

Figure 6. Effects of GBH (75 mg/kg) and MEL (4 mg/kg) administration on (a) latency to reach the hidden platform on each of the 4 days of the learning phase. (b) Percentage of time spent in the correct quadrant in the probe trial expressed as %. The data are presented as mean ± S.E.M of 6 animals/group. ^$p <0.05, ^$$p <0.01 compared with the GBH group.

Percentage of Time Spent in the correct quadrant

The results shown in Figure 6(b) indicate that MEL treatment induced a significant increase in the percentage of time spent in the correct quadrant compared to the group exposed to GBH (p < 0.05).

Effect of GBH and MEL on oxidative stress

Lipid peroxidation in the hippocampus and prefrontal cortex

According to the results in Figure 7(a,b) in the HPC, there was a significant increase in TBARS levels observed in the GBH group compared to the control group (p < 0.01). Similarly, MEL treatment induced no significant variation in TBARS levels compared to the GBH group, with a significant difference compared to the control group (p < 0.05). Concerning the PFC, the animals in the groups treated with GBH, significantly presented a higher level of TBARS compared to the control. Simultaneous treatment with MEL and GBH induced a significant reduction in TBARS in the PFC compared to the administration of GBH alone (p < 0.05).

Figure 7. Effects of GBH (75 mg/kg) and MEL (4 mg/kg) administration on TBARS levels (a,b), the nitric oxide (NO) levels (c,d) and catalase (CAT) activity (e,f) in the hippocampus (HPC) and prefrontal cortex (PFC) of adolescent male rats. The data are presented as mean ± S.E.M of 6 animals/group. *p <0.05, **p <0.01 compared with the control group; and ^$p <0.05, ^$$p <0.01 compared with the GBH group.

Nitric oxide concentrations in the hippocampus and the prefrontal cortex

The results presented in Figure 7(c,d) show the differences in NO concentration obtained in different structures. In the HPC, treatment with GBH induced an increase in NO levels compared to the control group (p < 0.01), whereas MEL in association with GBH caused a decrease in NO compared to rats treated with GBH (p < 0.05). Similarly, GBH caused a significant increase in NO in the PFC compared to control rats (p < 0.01). At the same time, rats treated with MEL showed significantly reduced levels compared with those exposed to GBH alone (p < 0.01).

Catalase activity in the hippocampus and the prefrontal cortex

As shown in Figure 7(e,f), the CAT activity was not affected by any of the treatments in the HPC. On the other hand, there is a statistically significant difference between the treated groups in PFC. Exposure to GBH induced a significant decrease in CAT activity compared with the control group (p < 0.01), while treatment with MEL and GBH significantly increased CAT activity compared to the rats receiving GBH alone (p < 0.01).

Discussion

The excessive use of glyphosate-based herbicides poses a major risk of human exposure. Our work explores for the first time the neuroprotective potential of MEL against the neurotoxic effects of GBH on affective and cognitive impairment. This protective mechanism could involve the inhibition of OS induced by GBH in neurons of the HPC and PFC.

Our results show that subchronic exposure to GBH during adolescence is associated with anxiety and depression behaviors, as measured in the OFT, EPM, and FST. In line with our findings, the implication of GBH in the induction of anxious and depressive behavior has been demonstrated in mice after subchronic (50 mg/kg) oral exposure [36]. However, our study demonstrates that the co-administration of MEL and GBH induces a significant anxiolytic and antidepressant effect. A significant improvement in the performance of the rats in the OFT and EPM tests was noted, and MEL also reduced immobility time in the FST compared to the GBH-treated rats.

On the other hand, our work affirms the involvement of GBH in cognitive impairment, manifested by a significant reduction in the performance of rats measured in the Y maze, which reflects the state of working memory, and in the MWM, which examines learning ability and spatial memory. Consistent with our findings, previous studies have shown that recurrent exposure to GBH negatively impacts recognition memory in mice. Specifically, in the Y maze test, there was a notable reduction in spontaneous alternation following prolonged administration at doses of 250 and 500 mg/kg [37]. Furthermore, glyphosate influenced the latency to reach the platform in the MWM test and the time spent in the original target quadrant on the trial day [38]. This work shows that MEL treatment improves cognitive deficits by increasing the percentage of alternation in the Y maze and by improving spatial memory, compared with the GBH group.

In this study, we demonstrated the ability of GBH to induce LPO in the PFC and HPC of rats via the generation of NO after subchronic administration of this herbicide. Furthermore, the oxidative damage induced by GBH was also supported in our study by the decrease in CAT enzyme activity in the PFC of rats. Several studies have shown that GBH is capable of crossing the BBB and inducing changes in various brain structures [8]. In line with our findings, it has been shown that chronic exposure to Roundup® increased TBARS levels and decreased GSH content in rat HPC [39]. In addition, administration of GBH at 375 mg/kg by oral gavage for 8 weeks induced a significant increase in the MDA levels in the brain [40]. Also, the effect of GBH has been associated with an increase in the release of NO, as detected in pure Schwann cell culture [41]. The study by [42] has shown that GBH can alter the activity of the CAT in the brains of adult intoxicated rats. Glyphosate, alone or in combination with other pesticides, has been shown to act at the mitochondrial level in substantia nigra, causing a loss of mitochondrial membrane potential and a reduction in concentrations of cardiolipin, a phospholipid that plays a major role in the electron transport chain and is extremely sensitive to OS due to its high fatty acid composition [43].

Our work presents OS as one of the possible neuroprotective mechanisms of MEL against the neurotoxic effects of GBH expressed at the affective and cognitive levels. Our results confirm that co-administration of MEL and GBH reduced NO and TBARS levels in the HPC and PFC. and contributed to improving CAT activity in the PFC. Due to its ability to cross the BBB [44], MEL can act at the brain level to counteract the damage caused by GBH. MEL is a major direct scavenger of free radicals, so it prevents the overproduction of free radicals, protecting various biological molecules from oxidative damage [45]. In addition, MEL activates nuclear factor erythroid 2-related factor 2 (Nrf2), which controls the expression of a group of genes involved in antioxidant defenses [46]. It was observed that MEL increased the nuclear translocation of Nrf2 in the brain of rats exposed to AlCl₃, leading to a reduction in MDA levels and increasing the activity of antioxidant enzymes such as CAT and SOD [47]. Additionally, MEL preserves the integrity of mitochondrial membranes, protecting them from the loss of cardiolipin and its peroxidation, thus supporting the normal physiological functions of mitochondria [44,47]. Recently, adult rats perinatally (GD5 to PND15) exposed to GBH showed a decrease in serum MEL levels and a significant increase in OS in the striatum [48]. GBH may therefore act on endogenous MEL, which may further exacerbate the neurotoxic effects of GBH.

Conclusion

In summary, our findings indicate that MEL may mitigate the emotional and cognitive deficits induced by GBH, potentially by suppressing OS in the HPC and PFC of adolescent rats.

Ethical approval

All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee (Local Institutional Research Committee).

Disclosure statement

The authors have no relevant financial or non-financial interests to disclose.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.