Exposure to paraquat associated with periodontal disease causes motor damage and neurochemical changes in rats

Abstract

Exposure to paraquat is possibly involved with the development of several conditions, including neurodegenerative diseases, such as Parkinson’s disease (PD). This condition is mainly characterized by the loss of dopaminergic neurons in the nigrostriatal pathway and the development of classical motor symptoms. Etiology includes exposure to environmental factors, such as the paraquat exposure, and inflammatory diseases may exacerbate paraquat neurotoxicity. The aim of the study was to investigate whether the exposure to paraquat associated with the presence of periodontal disease is able to induce motor and biochemical changes in rats similar to that observed in PD. Adult male Wistar rats were sent to ligature. After 48 h, they were sent to daily treatment paraquat (1 mg/kg/day; 2 mL/kg; intragastric) or vehicle for 4 weeks. Twenty-four hours after the last administration, the open field test was performed. The rats were euthanized and the left hemimandibles and striatum were dissected for the analysis of dopaminergic and inflammatory markers. Only the combination of periodontal disease model plus paraquat exposure induced motor impairments. Remarkably, the paraquat exposure increased the ligature-induced alveolar bone loss in hemimandibles. Moreover, only the combination of periodontal disease and paraquat exposure induced the loss of dopaminergic neurons and astrocyte activation in the striatum.

Keywords

Paraquat Parkinson’s disease periodontal disease neuroinflammation agriculture chemicals

Introduction

Parkinson’s disease (PD) is considered the second more common neurodegenerative disease in the world with estimated 1% of prevalence in individuals over 60 years old.^1,2 Clinically, PD is characterized by motor and nonmotor symptoms mainly caused by the loss of dopaminergic neurons in the substantia nigra pars compacta and the consequent reduction of striatal dopamine levels.³

PD etiology is multifactorial, being associated with the combination of genetic and environmental factors. Genetic factors involving mutation of specific genes are associated with the early onset of the disease.^4,5 Environmental factors include the exposure to metals, solvents, pesticides, herbicides, among other substances.^6,7 In addition, other putative factors are traumatic brain injury⁸ and the presence of inflammatory diseases, such as oral health-related inflammatory diseases.^9,10

A meta-analysis showed that occupational exposure to pesticides causes a 60% increase in the risk of developing PD.¹¹ Considering such pesticides, paraquat stands out because it induces oxidative stress^12,13 due to the oxidation of glutathione and thioredoxin, which compose the main mechanism of its neurotoxicity.^14,15

Importantly, dopaminergic neurons are particularly sensitive to paraquat neurotoxicity that involves glial cell activation.^16,17 Moreover, oral paraquat administration in rodents induces motor impairments and dopaminergic dysfunction similar to those observed in PD.^18

–21 However, the commonly used paraquat doses in animal models are too high and they do not represent the dosage rural residents and workers are exposed to.²²

In this context, it is suggested that there are triggering factors like systemic inflammatory diseases that may enhance the paraquat neurotoxicity.¹⁶ Periodontal disease is considered the second most prevalent oral disease, reaching rates of 47.2% in adults and up to 70.1% in elderly.²³ In a study carried out in Baghdad, it was shown that periodontal disease is 2.48 times more frequent in rural areas.²⁴ This condition is characterized by a biofilm formation in the tooth and gingival interface affecting supporting tissues, causing bone tissue destruction, inflammatory process, and oxidative stress.^25

–28

Thus, periodontal disease may cause systemic effects, and some systemic diseases may predispose the individual to periodontal disease.²⁹ In this context, the hypothesis of a possible association between the exposure to a chronic low dose of paraquat and periodontal disease emerges as a predisposing factor for the PD development. Therefore, the present study aimed to investigate whether the exposure to paraquat associated with the presence of periodontal disease is able to induce motor and biochemical changes in rats similar to that observed in PD.

Materials and methods

Animals

Three-month-old male Wistar rats with initial weight 290–320 g were used. The animals were kept in a temperature-controlled environment at 22 ± 3°C in 12-h light–12-h dark cycle and free access to water and food. The Animal Ethics Committee of the State University of Ponta Grossa (PR) (#040/2016) approved all steps of the experimental protocol.

Experimental protocol

Forty animals were initially divided into two groups: periodontal disease and sham. Induction of periodontal disease through ligature insertion occurred on the first day of the protocol. For this procedure, the animals were intraperitoneally anesthetized with ketamine (75 mg/kg) and xylazine (15 mg/kg). After the anesthetic procedure, the animal was placed in the Doku apparatus and the cotton ligature was bilaterally inserted in the mandibular first molars. For the sham group, the same procedures were performed except for the cotton ligature insertion.

After 48 h of periodontal disease induction, the animals were divided into four groups (sham, paraquat, ligature, and ligature plus paraquat (n = 10 per group)) and the treatment with paraquat treatment (Gramoxone®) (1 mg/kg/day; 2 mL/kg; intragastric (gavage)) or vehicle (water) for 4 weeks was applied. Rats were daily weighed during this period. This dose was chosen in the light of occupational exposure and in view of the conclusion of the World Health Organization³⁰ and a previous study that used a chronic exposure to low doses of paraquat in rat.³¹

Twenty-four hours after the last paraquat or vehicle administration, the animals were tested in the open field arena. Subsequently, the animals were euthanized and the left hemimandibles and striatum were dissected for the measurement of bone loss and analysis of dopaminergic and glial cell markers by Western blot, respectively.

Open field test

The locomotor activity of rats was addressed in the open field test, as described by Hall.³² The animals were positioned in the center of a wooden arena (100 × 100 cm²) with walls 30 cm high and observed for 5 min. The floor of the box is divided into 25 longitudinal and transverse squares (20 × 20 cm²). The number of crossings and rearings was registered.

Bone loss measurement

Bone loss was measured by morphometric analysis, as described by Souza et al.³³ After euthanization, the left hemimandibles were removed and, subsequently, the pieces were prepared and stained with 1% blue methylene to mark the cementoenamel junction. Then, they were observed with stereoscopic magnifying glass and digitalized by camera. Digital images were analyzed by ImageJ 1.8.0 software using linear measurements (mm) in the center of each root (mandibular molars) following its long axis from the cementoenamel junction to the bone crest.

Western blot

The analysis was performed to investigate the loss of dopaminergic neurons and the activation of astrocytes and microglia cells. Striatal samples were mechanically homogenized in 50 mM Tris, 1 mM ethylenediaminetetraacetic acid, 100 mM sodium fluoride, 0.1 mM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail (Sigma-Aldrich, St Louis, USA). Lysates were centrifuged to eliminate cell debris (3000 r/min for 10 min at 4°C). The supernatant protein content was determined by the method of Lowry et al.,³⁴ using bovine serum albumin (BSA) as a standard. The samples were diluted to a final concentration of 4 μg/mL in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer. Samples (80 µg of protein) and prestained molecular weight standard (GE Healthcare Life Sciences, Marlborough, Massachusetts, USA) were separated on 12% resolving with 5% SDS-PAGE. After electrotransfer to PVDF membrane, the blockade with 3% BSA solution was performed. The membranes were incubated at 4°C overnight with the following primary antibodies: anti-tyrosine hydroxylase (TH), anti-glial fibrillary acidic protein (GFAP), and anti-ionized calcium-binding adaptor molecule 1 (IBA-1). Then, the membrane was washed and incubated with their respective peroxidase-conjugated secondary antibodies (Upstate Cell Signaling, SP, Brazil) for 1 h at room temperature. Anti-β-actin (1: 30,000, Upstate Cell Signaling) was stained as a protein loading control. Chemiluminescent detection (Merck KGaA, Darmstadt, Germany) was performed in a photodocumenter (ChemiDoc MP, Bio-Rad, La Jolla, California, USA). Relative optical density of Western blot bands was quantified using the ImageJ software. Each value is derived from the ratio of arbitrary units obtained by the protein band and the respective β-actin band. The results were expressed as arbitrary units.

Statistical analysis

Shapiro–Wilk test was performed to verify data normality. Statistical analysis was performed using two-way analysis of variance (ANOVA), and when an interaction was found, Tukey’s post hoc test was carried out. Data are expressed as mean ± standard error of the mean (SEM). Probability values p < 0.05 were considered statistically significant. All analyses were performed using the GraphPad Prism® 7.04 computer program (GraphPad, San Diego, California, USA).

Results

Open field

The animals were evaluated for motor behavior after the periodontal disease induction and the paraquat treatment. Two-way ANOVA revealed significant differences in the number of crossings. Tukey’s post hoc analysis demonstrated a decrease in the number of crossing by the ligature plus paraquat group when compared to those from the sham (p < 0.0001), ligature (p ≤ 0.0001), and paraquat (p = 0.0002) groups, evidencing an impaired horizontal locomotion (Figure 1(a)). The other groups did not differ significantly from the sham group.

Figure 1.

Effects of periodontal disease and paraquat exposure on the motor performance of rats in the open field. (a) Number of crossings and (b) number of rearings. Data are expressed as mean and ± SEM for 10 animals per group. *p < 0.05, **p < 0.01, and ****p < 0.0001 when compared to sham vehicle group. ^§§§§ p < 0.0001 when compared to sham paraquat group. ^### p < 0.001 and ^#### p < 0.0001 when compared to ligature vehicle group. SEM: standard error of the mean.

On the other hand, the analysis of the number of rearings indicated that the paraquat exposure per se promoted a reduction in this parameter (p = 0.0392) while the ligature induction caused an increase of the same parameter (p = 0.0105) when compared to the sham group. Importantly, it was evidenced that the animals submitted to the periodontal disease model plus paraquat exposure (ligature plus paraquat group) had a reduction of the number of rearings, corroborating the motor impairment shown in the number of crossings (p < 0.0001) (Figure 1(b)).

Bone loss measurement

The animals were evaluated for bone loss after the periodontal disease induction and paraquat treatment (Figure 2). Two-way ANOVA revealed significant differences, and Tukey’s post hoc analysis demonstrated that the periodontal disease induction was effective since the ligature group exhibited a significant increase in the alveolar bone loss when compared to the sham group (p < 0.0001). Also, it was found that the paraquat exposure caused spontaneous alveolar bone loss pointed out by the difference in the sham and paraquat groups (p = 0.0002). In addition, it should be highlighted that the association between the ligature-induced periodontal disease and the paraquat exposure exacerbated the alveolar bone loss (sham × ligature plus paraquat (p < 0.0001), ligature × ligature plus paraquat (p < 0.0001), and paraquat × ligature plus paraquat (p < 0.0001)).

Figure 2.

Effects of periodontal disease and paraquat exposure on alveolar bone loss in rats. Data are expressed as mean and ± SEM for 10 animals per group. ***p < 0.001 and ****p < 0.0001 when compared to sham vehicle group. ^§§§§ p < 0.0001 when compared to the sham paraquat group. ^# p < 0.05 and ^#### p < 0.0001 when compared to the ligature vehicle group. SEM: standard error of the mean.

Western blot analysis

To investigate the putative dopaminergic damage and the activation of astrocytes and microglia, Western blot analysis was carried out. Tukey’s post hoc test revealed a significant reduction of striatal TH levels in the ligature plus paraquat group when compared to the sham (p = 0.0002) and paraquat (p = 0.0001) groups. Of note, paraquat exposure alone was not able to induce dopaminergic damage in the striatum of rats, pointing to an important role of ligature association in this process (Figure 3(b)).

Figure 3.

Effects of periodontal disease and paraquat exposure on dopaminergic and glial activation markers in the striatum of rats. (a) Representative Western blot bands. (b) TH, (c) GFAP, and (d) IBA-1 levels. Data are expressed as mean and ± SEM for six animals per group. *p < 0.05 and ***p < 0.001 when compared to sham vehicle group. ^§§§ p < 0.001 when compared to sham paraquat group. ^# p < 0.05 when compared to vehicle ligature group. TH: tyrosine hydroxylase; GFAP: glial fibrillary acidic protein; IBA-1: onized calcium-binding adaptor molecule 1; SEM: standard error of the mean.

The astrogliosis processes were evaluated by the measurement of striatal GFAP levels. As shown in Figure 3(c), the association of the paraquat exposure and ligature was able to cause an increase in the striatal GFAP immunocontent when compared to the sham (p = 0.0283) and ligature (p = 0.0120) groups. In this way, our data demonstrate that the exposure to paraquat alone is not enough to induce striatal astrogliosis and that the ligature is an important factor for the inflammatory process development. On the other hand, striatal microgliosis was not affected by any treatments, since that there was no statistically significant difference in the IBA-1 immunocontent among any groups (Figure 3(d)).

Discussion

The present study investigated the effects of ligature-induced periodontal disease, the paraquat exposure, and the association of these factors on the motor function, alveolar bone loss, and striatal neurochemical changes. Regarding alveolar bone loss, it was demonstrated that the methodology employed was effective in inducing periodontal disease. In addition, it was evidenced that the paraquat exposure per se caused spontaneous alveolar bone loss and that this exposure was able to enhance ligature-induced bone loss.

The present results indicated that the isolated exposure to paraquat, as well as the association of exposure with ligature, was able to cause motor impairment, as shown by statistically significant changes in the parameters evaluated in the open field test. The results show alterations in the number of quadrant crossings and in the number of rearings, while the exposure to paraquat per se caused a reduction only in the number of rearings. Although the open field test is widely used to evaluate motor function in PD’s animal models^31,35,36 and epidemiologic studies link paraquat exposure to PD development.^6,37,38 It is important to highlight that this study aimed to investigate only the relation between the exposure to a chronic low dose of paraquat and the periodontal disease on the motor function in the open field. Thus, the characterization in this protocol as a novel animal model to study of motor features in PD needs more investigation.

By the analysis in the literature, it was shown that the administration of 10 and 20 mg/kg of paraquat orally was capable of causing motor modification highlighted in the open field.³⁹ Other studies have been shown similar results, demonstrating that the administration of 10 mg/kg by intraperitoneal injection was capable of causing motor modifications highlighted when tested in open field and also, dopaminergic neurons decrease microglia activation and antioxidant enzymes deficiency.^40,41

It is emphasized that it was not found studies in the literature that analyze the administration of paraquat in low doses (1 mg/kg).

A study that evaluated paraquat exposure in a Drosophila model found that there was a loss of motor function, but without degeneration of dopaminergic neurons. However, when associated with chronic exposure to another pesticide, degeneration was observed.⁴² This finding is in agreement with our study that only the association with the ligature was able to show dopaminergic degeneration.

Regarding Western blotting, it was evidenced that neither the paraquat exposure nor the ligature induction induced per se significant changes in striatal dopaminergic and glial cell parameters. This finding is in agreement with a study by Widdowson, where paraquat alone at a dose of 5 mg/kg was not able to cause changes in the open field, neuronal death and changes in the nigrostriatal pathway.⁴³ However, the association of these factors was able to induce the decrease of TH-positive neurons and astrocyte activation, as indicated by the increase in GFAP.

Previous studies have shown that paraquat crosses the blood–brain barrier via the dopamine transporter and this mechanism happens from the induction of oxidative stress that occurs from the oxidation of mitochondrial chain complex I, which results in inhibition of the ubiquitin–proteasome system.^44
–46 Paraquat also induces several cycles of reduction and oxidation that gradually promote mitochondrial dysfunction and apoptosis of dopaminergic cells.^47
–49 It has been shown that the activity of complex I is reduced in rat brain tissue after 2 h of paraquat administration.^2,50,51

In addition, it has been shown that paraquat may induce nitric oxide activity, which causes peroxynitrite anion formation, resulting in DNA damage, apoptosis, and neuronal death. Besides, this exposure can cause changes in energy metabolism, promoting cycling, oxidative stress, and progression of cell death.^2,50,52 Evidence points to the involvement of environmental exposure to pesticides, including paraquat, in PD development.^37,53,54 However, currently available studies have frequently used administration routes (intraperitoneal and intracerebral) and doses that do not represent the exposure of the worker in a rural area.^55

–61

Rojo et al. demonstrated that paraquat administered at different intranasal doses did not cause motor damage in rats.⁶² Regarding the oral route, the administration of paraquat at a dose of 5 mg/kg/day for 4 weeks does not cause behavioral and biochemical changes.⁴³

These data partially reinforce those of our study, where no behavioral and neurochemical changes were observed in animals daily exposed to paraquat (1 mg/kg/day for 4 weeks) except for the number of rearings. Conversely, an increased dose (10 mg/kg) and time of exposure induced behavioral changes, motor deficits, degeneration of dopaminergic neurons, and astrocytic activation.^19,20 Astrocyte activation was also highlighted in the presence of 2–100 µm in neurons cultures.⁶³ However, it should be highlighted that paraquat concentration in water is between 50 mg/mL, so the dose used in previous studies is considered high to mimic occupational exposure to paraquat.²²

Although there is evidence that periodontal disease may be related to some systemic diseases, such as type 2 diabetes, respiratory tract infections, gestational complications, and Alzheimer’s disease,^64

–68 there are no studies investigating its possible association with PD. In this sense, our work is the first one to demonstrate an association between repeated exposure to paraquat and periodontal disease in the PD etiology. In fact, our data showed that animals subjected to the periodontal disease model and daily exposure to paraquat developed motor impairment in the open field test, reduction of striatal protein TH levels, indicating a dopaminergic degeneration in this region, and an increase in GFAP immunocontent suggesting astrocyte activation.

Interestingly, paraquat’s oxidative stress induction mechanism has features in common with those induced by the periodontal disease.²⁵ The association of periodontal disease with neurodegenerative processes is based on the hypothesis that the lipopolysaccharide generated by oral pathogens alters the blood–brain barrier permeability. This leads to the passage of macrophages, proinflammatory mediators, and pro-oxidants to the central nervous system, culminating in the degenerative process.^25,69,70

In this context, our data suggest that the exposure to paraquat may potentiate periodontal disease and vice versa. In fact, the alveolar bone loss measurement was higher in animals subjected to the periodontal disease model and daily exposed to paraquat than in animals subjected to only one condition. In addition, astrogliosis, represented by increased GFAP, is commonly associated with neurodegeneration processes.^70,71 Of note, this has been evidenced after the administration of the periodontal disease pathogen.⁷² However, the present study is opposed to this, since only the association of paraquat exposure and the presence of periodontal disease caused astrogliosis.

Conclusion

This study demonstrated that the exposure to paraquat potentiated periodontal disease causing increased spontaneous alveolar bone loss. In addition, the association of daily exposure to paraquat with periodontal disease caused motor impairment, degeneration of dopaminergic neurons, and astrogliosis in the striatal region. Thus, our data reinforce those showing that the exposure to a low dose of paraquat is not able to cause motor changes in rodents and this association provides knowledge about the prevalence of PD and periodontal disease in the rural areas.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasília, Brazil.

ORCID iDs

LC Fernandes

AG Santos

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