Effect of sodium fluoride on neuroimmunological ...

http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, 2014; 24(1): 31–36 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2013.843224

RESEARCH ARTICLE

Y. P. Reddy1, S. K. Tiwari2, A. P. Shaik3, A. Alsaeed3, A. Sultana4, and P. K. Reddy1 1

Neurobiology Lab, Department of Zoology, University College of Sciences, Osmania University, Hyderabad, Andhra Pradesh, India, Cancer & Genetics Research Complex, Department of Urology, College of Medicine, University of Florida, Jacksonville, FL, USA, 3College of Applied Medical Sciences, King Saud University, Riyadh, Kingdom of Saudi Arabia, and 4Molecular Imaging Lab, Department of Radiological Sciences, King Saud University, Riyadh, Kingdom of Saudi Arabia

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Abstract

Keywords

Aims: This study was designed to evaluate the effect of sodium fluoride (NaF) in inducing neuroimmunological, oxidative and antioxidative damage. Methods: Twenty-four male Wistar rats broadly grouped into four groups containing six rats in each were fed with drinking water containing 20 ppm, 60 ppm, 100 ppm and 0.8 ppm (control) NaF. After 90 days, rats were sacrificed to assess the level of fluoride content and various neurotransmitters in brain. The levels of CD4, natural killer (NK) cells and IgG1 were assessed in blood and spleen. In addition, lipid peroxidation coupled with the levels of various antioxidative enzymes was also recorded. Results: Increase in the NaF concentration resulted in increased fluoride deposition in brain tissue. This increased fluoride content led to increased levels of certain neurotransmitters such as epinephrine, histamine, serotonin and glutamate and decreased levels of norepinephrine, acetylcholine and dopamine in a dose-dependent manner. NaF exposure led to the decrease in the levels of CD4, NK cells and IgG1 coupled with marked increase in lipid peroxidation and impairment of the antioxidative defense system. Conclusion: The result of the study emphasizes the toxic role of high NaF doses on the neurological and immunological functions.

Antioxidative defenses, neuroimmunological effect, oxidative stress, sodium fluoride

Introduction Fluoride has proven toxic effects when consumed at higher concentrations (ATSDR, 2003; Weinstein & Davidson, 2004; WHO, 2002). Although several reports have also demonstrated its important role in developmental process of the body (Chirumari & Pratap, 2007; Niu et al., 2008), there exist many studies on its toxic effects as well (Dhar & Bhatnagar, 2009; Zhang et al., 2006). Fluoride has been shown to cause cytotoxic effects and interferes with many enzyme systems by disrupting oxidative phosphorylation, glycolysis, coagulation and neurotransmission. Fluoride has been shown to inhibit Naþ/Kþ ATPase leading to hyperkalemia and acetylcholinesterase causing cholinergic signs (Bronstein et al., 2010). Besides causing fluorosis, epidemiological studies have suggested fluoride intake to increase the susceptibility of cancer (Connett, 2004, 2009), infertility (NRC, 2006), and premature aging (Dunipace et al., 1995). World Health Organization (WHO) guideline value and the permissible Address for correspondence: Prof. P. K. Reddy, Neurobiology Lab, Department of Zoology, University College of Sciences, Osmania University, Hyderabad 500 007, Andhra Pradesh, India. Tel: +91 40 27682218. E-mail: [email protected]

History Received 6 February 2013 Revised 7 September 2013 Accepted 7 September 2013 Published online 16 October 2013

limit of fluoride as per Bureau of Indian Standard (BIS) is 1.5 mg/L (average between 0.8 and 1.2 mg/L, i.e. not more than 0.7–1.0 ppm) and these levels are known to prevent tooth decay (WHO, 2002). Fluoride accumulation over a period of time has been shown to cause significant neurological damages and neurodegenerative disorders (Bhatnagar et al., 2006; Chirumari & Pratap, 2007). The problem of fluorosis in India is roughly estimated to affect 66.6 million people of which an estimated 6 million are children below the age of 14 years (Ayoob & Gupta, 2006). Sodium fluoride (NaF) (relative molecular mass 41.99; CAS No.7681-49-4) is colorless to white solid and moderately soluble in water. It is used to fluoridate water, as an industry standard for fluoridation and as an additive in toothpastes to prevent cavities. It has also been used in laundry, metallurgy and organic synthesis of many chemicals including fluorocarbons. Apart from this NaF is also used in medicine in some diagnostic procedures employing radiolabeled fluoride (NHMRC, 2007). The lethal dose for a 70 kg (154 lb) human is estimated at 5–10 g (Aigueperse et al., 2005). Although studies undertaken to date have shown a link between NaF and various metabolic and neurological diseases (Bhatnagar et al., 2006; Carton, 2006; Chinoy & Shah, 2004),

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Effect of sodium fluoride on neuroimmunological parameters, oxidative stress and antioxidative defenses

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the molecular mechanism remains highly elusive (Barbiera et al., 2010). Further, its precise effect on the mediators of the nervous system and the primary target cells of both the celland humoral-mediated immunity is still warranted. Therefore, this study was designed to assess the effect of NaF on the effectors of the neuronal and immune system in an experimental-induced fluorosis rodent model.


Materials and methods Male Wistar rats (n ¼ 24) aged four months and weighing 150–200 g were procured from National Centre for Laboratory Animal Supplies (NCLAS), Hyderabad, India. All the animals were maintained under standard laboratory conditions in a light–dark cycle (12:12 h) with the temperature between 12 C and 25 C. The rats were divided into four experimental groups (control, 20 ppm, 60 ppm and 100 ppm) with six animals in each group, respectively. Drinking water containing 20 ppm, 60 ppm and 100 ppm NaF (Sigma, St. Louis, MO) was administered to three groups for 90 days, whereas the rats in the control group were fed with tap water containing permissible limit of fluoride (0.8 ppm) for the same period. The animals had free access to the laboratory chow and water throughout the duration of the study and sacrificed on 90th day of the experiment by cervical dislocation. The study protocol was approved by Animal Ethics committee of Department of Zoology, Osmania University, Hyderabad, India (CPCSEA No: 383/01/a/ CPCSE). Tissue samples from brain were collected aseptically in sterile buffered saline (pH 7.0) and processed for spectrofluorimetric quantification of neurotransmitters (acetylcholine, dopamine, epinephrine, norepinephrine, serotonin, histamine and glutamate) for assessing the effect of NaF on neuronal parameters. Similarly, blood samples from the infraorbital sinus and spleen samples (after aspectic splenectomy) collected in EDTA-coated vacutainers (BD Biosciences, Gurgaon, India) was used for determining its effect on immunological parameters such as CD4 cells, IgG1 levels and Natural killer (NK) cells in paired blood and spleen samples. All the samples were transported to the laboratory within 30 min of collection. Neurological effects of NaF Estimation of fluoride levels in brain The fluoride content in the brain tissue was measured by Orion 720A þ meter with ion selective fluoride electrode model 9409 along with using a reference electrode model 9001 and using standard protocol described elsewhere (Madhusudhan & Piler, 2009). Total Ionic Strength adjusting buffer (TISAB) was used to quantify the fluoride in tissues and blood. Samples were digested with nitric acid and perchloric acid in 1:1 ratio for 12 h with a pH maintained at 4.5–5.0 with sodium hydroxide and sodium citrate. Estimation of neurotransmitters The levels of various neurotransmitters were assessed in the brain tissue of all the experimental animals as per the protocol described by Kari et al. (1978).

Toxicol Mech Methods, 2014; 24(1): 31–36

Immunological effects of NaF Analysis of CD4 cells, IgG1 & NK cells in rat spleen and blood Expression of cluster of differentiation 4 (CD4), IgG1 and Natural killer (NK) cells was carried out in experimental animals of all the four groups on Three-color flow cytometer FACS Calibur (Becton & Dickinson, San Diego, CA). Fluorescent antibodies pertaining to rat CD4-PE, IgG1FITC and NK cells (CD56-PE) were procured from Pharmingen (Becton & Dickinson, San Diego, CA) and processed as per the instruction manual. Briefly, 20 mL of the antibody was taken in 5 mL polystyrene round-bottomed tube (Cat. No. 352003, Becton & Dickinson, San Diego, CA), to which 50 mL of the anticoagulated rat blood and/or homogenized spleen was added and incubated in dark for 15–20 min. Following which the tubes were briefly vortexed for 3–5 s and lysed using 1X FACS lysing solution (BD FACS lysing solution, Cat. No. 349202, Becton & Dickinson, San Diego, CA). The reaction mixture was then further incubated in dark for 10–12 min and washed twice with 2 mL sheath solution (BD FACSFlow Cat. No. 342003, Becton & Dickinson, San Diego, CA) to subsequently remove the debris. Finally, the precipitate was resuspended in 450 mL of the sheath solution for flowcytometric acquisition. Acquisition and analysis of the processed samples was performed using CELLQuest software (Becton & Dickinson, San Diego, CA). Assessment of oxidative stress in brain, blood and spleen The levels of key cellular constituents responsible for oxidative stress in all the experimental animals was determined using specific protocol namely Bhuyan et al. (1981) for lipid peroxidation (LPO), Marklund & Marklund (1974) for superoxide dismutase (SOD), Aebi (1983) for catalase and Martinez et al. (1979) for glutathione peroxidase (GPx) activity, respectively. Statistical analysis The results of all experiments, that is, fluoride levels, neurotransmitter levels, antioxidant enzymes and immune parameters, are expressed as Mean SD. Statistical analysis were performed using the Pearson’s correlation test for oxidative stress and antioxidant data using StatistiXL (version 1.8) software (StatistiXL, Nedlands, Western Australia; http:// www.statistixl.com/downloads/downloading.aspx) and comparison of means (ANOVA) for neurochemical parameters using Graphpad Software (La Jolla, CA). A p value of 50.05 was considered as statistically significant.

Results Determination of fluoride content With an increase in the concentration of NaF (on 90th day), there was an exponential increase (p50.05) in the fluoride content in the brain tissue in the treatment group compared to the control group (Figure 1). While the control group had fluoride levels of 0.6 mg/gm wet tissue, the exposed groups showed levels of 1.56, 1.98 and 2.40 mg/gm wet tissue upon

Neuroimmunological effects of NaF in Wistar rats


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Figure 1. Analysis of the fluoride content in the brain tissue of control and experimental rats.

Figure 2. Levels of various neurotransmitters in control and experimental groups.

treatment with 20 ppm, 60 ppm and 100 ppm of NaF, respectively.

54.84%) and dopamine (20 ppm: 64.18%; 60 ppm: 46.64%; 100 ppm: 20.54%) in treated rats compared to controls.

Neurological parameters

Immunological parameters

Levels of neurotransmitters

Expression of CD4 cells, IgG1 & NK cells in rat spleen and blood

A significant difference in the levels of various neurotrasmittters between the treatment and control animals respectively (p50.05) was demonstrated (Figure 2). There was a dose-dependent increase in the levels of epinephrine, histamine, serotonin and glutamate and a corresponding decrease in the levels of norepinephrine, acetylcholine and dopamine with increase in the NaF concentration. When compared to control rats, on the 90th day, a significant (p50.05) increase in the epinephrine (20 ppm: 206.27%; 60 ppm: 230.74%; 100 ppm: 310.76%), histamine (20 ppm: 133.70%; 60 ppm: 147.53%; 100 ppm: 158.48%), serotonin (20 ppm: 124.26%; 60 ppm: 199.58%; 100 ppm: 389.39%) and glutamate levels (20 ppm: 139.24%; 60 ppm: 180.50%; 100 ppm: 192.35%) were found. However, there was a significant (p50.05) decrease on the 90th day in the levels of norepinephrine (20 ppm: 74.72%; 60 ppm: 50.84%; 100 ppm: 45.71%), acetylcholine (20 ppm: 84.30%; 60 ppm: 78.79%; 100 ppm:

Flow cytometric analysis of the expression of primary target cells of both cell- and humoral-mediated immunity, namely, CD4 cells, IgG1 and NK cells, respectively, showed drastic reduction in the levels of expression in the fluoride-treated rats than among the controls (Figure 3). There was a significant change (p50.05) in CD4 cells, a marker for cell-mediated immunity in treated animals versus control group in blood (20 ppm: 17.94%; 60 ppm: 39.37%; 100 ppm: 74.88%) and spleen (20 ppm: 14.85%; 60 ppm: 37.73%; 100 ppm: 69.09%). The humoral immunity mediator immunoglobulin (IgG1) levels showed a consistent decrease (p50.05) in both blood (20 ppm: 73.6%; 60 ppm: 75.22%; 100 ppm: 80.05%) and spleen (20 ppm: 32.88%; 60 ppm: 47.75%; 100 ppm 73.57%) compared to control rats. There was a decrease (p50.05) in NK cells in blood (20 ppm: 41.28%; 60 ppm: 59.12%; 100 ppm: 72.17%) and spleen (20 ppm:


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Figure 3. Effect of NaF on CD4, NK cells and IgG1.

Figure 4. Oxidative stress and levels of various antioxidant enzymes in control and treated rats.

52.56%; 60 ppm: 63.47%; 100 ppm: 75.19%. The decrease in CD4 cell count, IgG1 levels and NK cells occurred in a dosedependent manner, that is, maximum depletion occurring at higher dose of NaF. Oxidative stress and antioxidant enzymes With the increase in the concentration of NaF, a decreasing trend in the levels of various antioxidant enzymes (SOD, GPx, and Catalase) was seen in a dose-dependent manner

(Figure 4). In addition, a marked increase in the concentration of the MDA was also observed in a dose-dependent fashion indicating intensified lipid peroxidation. A positive correlation was seen in all of the oxidative stress and antioxidant parameters with statistically significant differences in some parameters (Figure 4). The LPO levels were found to be significantly (p50.05) increased in all the three tissues: blood (20 ppm: 105%; 60 ppm: 109%; 100 ppm: 112%), spleen (20 ppm: 101.66%; 60 ppm: 105.66%; 100 ppm: 120.98%) and brain (20 ppm: 48%; 60 ppm: 43%; 100 ppm: 64.87%)


DOI: 10.3109/15376516.2013.843224

compared to controls. Similarly, catalase levels significantly decreased (p50.05) in all tissues, that is, blood (20 ppm: 83%; 60 ppm: 60.66%; 100 ppm: 43.63%), spleen (20 ppm: 92.57%; 60 ppm: 87.42%; 100 ppm: 81.71%) and brain (20 ppm: 48%; 60 ppm: 43%; 100 ppm: 64%) The SOD levels were found to decrease significantly (p50.05) in all the three tissues: blood (20 ppm: 95.40%; 60 ppm: 87.62%; 100 ppm: 77.94%), spleen (20 ppm: 94.73%; 60 ppm: 84.98%; 100 ppm: 81.14%) and brain (20 ppm: 48%; 60 ppm: 43%; 100 ppm: 64.87%) compared to controls. Similarly, GPx levels were found to decrease significantly (p50.05) in all the three tissues: blood (20 ppm: 96.45%; 60 ppm: 90.95%; 100 ppm: 84.09%), spleen (20 ppm: 98.96%; 60 ppm: 90.60%; 100 ppm: 80.99%) and brain (20 ppm: 48%; 60 ppm: 43%; 100 ppm: 64.87%) compared to control.

Discussion Fluoride toxicity in humans has been an area of intense research in the past 2–3 decades (ATSDR, 2003; WHO, 2002; Isaacson, 2008). With the availability of ample data demonstrating its effect on the central nervous system (CNS) and immune system (Das et al., 2006; Gibson, 1992; Loftenius et al., 1999; Tang et al., 2008; Trivedi et al., 2007), it is increasingly important to understand the molecular events leading to disease pathology. The present study reports the neuroimmunomodulatory and oxidative effects of NaF in Wistar rats. An increase in the fluoride concentrations or exposure time lead to increased fluoride deposition in the brain corroborating the results of a previous study by Tsunoda et al. (2005) who reported a significant fluoride accumulation in cerebrum of mice at 25 ppm and a study by Burgstahler et al. (1996) who showed that fluoride accumulates in various parts of rat brain, especially in the hippocampus. Fluoride accumulation was also observed in brain of rats exposed to chronic high fluoride intake through drinking water (Mullenix et al., 1995). Fluoride was postulated to cause neurotoxicity in laboratory animals and a recent experimental study on rat showed that NaF in vitro caused neurotoxicity principally targeting hippocampal neurons (Zhang et al., 2008). This could be due to the ability of NaF to cause imbalance in the various neurotransmitters i.e. increased levels of glutamate, epinephrine, serotonin and histamine and decreased levels of acetylcholine, norepinephrine and dopamine in a dosedependent manner. Confirming the opinion expressed by Brugstahler (2002) on anomalous fluoride effect on central nervous system, decreased levels of dopamine in this study are probably a result of decreased metabolism. The levels of serotonin were higher suggesting an increase in the activity of serotonergic system. Besides these, reduced norepinephrine, and elevated levels of epinephrine were observed – the presence of norepinephrine in the brain allows the organism to become alert, and guards against the intensification of reflex reactions and other behavior. The elevated levels of epinephrine could be due to a blockage of the pathway that transforms epinephrine into norepinephrine or possibly due to suppression of the relevant metabolic enzymes, causing the brain levels of epinephrine to increase, and the levels of norepinephrine to decrease. Acetyl choline levels seen in this study

Neuroimmunological effects of NaF in Wistar rats

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might be a result of the damage to the brain tissue of the rats caused by fluoride poisoning which is responsible for poor learning ability. Besides these changes, histamine levels were elevated in all the experimental groups over control, this is an indication of allergic manifestations caused by fluoride. Further investigations are warranted to determine the mechanistic pathway by which fluoride alters the kinetics of these neurotransmitters. Although there have been a number of reports of the effect of NaF on the immunological parameters (Das et al., 2006; Gibson, 1992; Jain & Susheela, 1987; Loftenius et al., 1999; Trivedi et al., 2007), none of the studies hitherto have shown their effects on the markers of cell-mediated immunity [CD4, natural killer (NK) cells] with only a few reports on the IgG levels (Jain & Susheela, 1987). Therefore, our study for the first time reports the effect of different concentrations of NaF on CD4, NK cells in addition to IgG1 levels. The results obtained clearly demonstrated that with an increment in the fluoride concentration in the treatment group, there was decrease in the levels of primary cells of the immune response namely CD4 cells, IgG1, natural killer (NK) cells compared to the control group (Figure 2) indicating immunosuppressive activities of NaF as determined by flow cytometer. The results pertaining to IgG1 levels in our study are in concert with those reported previously in a study by Jain & Susheela (1987) which showed immunosuppressive activity of NaF on the IgG1 levels. However, none of the studies to date have reported the effect of NaF on CD4 and NK cells. Oxidative stress is reported to play a mainstream role in enhancing the neurotoxicity of the animal (Brambilla et al., 2008). Therefore, this study also estimated the level of various antioxidant enzymes in blood, brain and spleen of all the experimental animals. The results of the study emphasized that with the gradual increase in the fluoride concentration (20 ppm, 60 ppm and 100 ppm) among the treated rats, the catalase, superoxide dismutase and glutathione peroxidase levels decreased coupled with increased lipid peroxidation activity (Figure 4), the study also reports that significant correlation exists between fluoride and oxidative stress and antioxidant parameters. Our results are in agreement with previous studies by Patel & Chinoy (1998) who reported that fluoride impaired the functioning of enzymes such as SOD, Catalase thereby increasing the level of lipid peroxidation. Chinoy et al. (2005) reported significant decrease in the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in mice fed with varying concentrations of NaF in diet. Finally, the results of this study supports the notion that though fluoride at low concentrations act as a supplement to the body, its consumption at higher doses exert many deleterious effects, i.e. activate antioxidant enzymes, increase arachidonic acid release, distort cell morphology, induce apoptosis, inhibit protein synthesis and cell cycle progression as well as chromosomal aberrations (Thrane et al., 2001). This study for the first time demonstrated the effect of NaF on CD4 cells, NK cells and IgG1. In conclusion, this study showed that NaF exhibited neuroimmunological and oxidative stress potential. In addition, altered levels of neurotransmitters may cause some of the symptoms of fluoride-induced neurotoxicity.

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Fluorosis severely affects the quality of life ultimately causing several metabolic and degenerative disorders. Its alarming upsurge in the recent years is a matter of deep concern and warrants intense research. The information obtained in this study provides further impetus to understand in better way the molecular and biochemical basis of neurological and immune disorders.

Acknowledgements


The authors thank the technical staff of Dept. of Zoology, Osmania University, Hyderabad, India, and College of Applied Medical Sciences, King Saud University, Saudi Arabia for their kind patronage.

Declaration of interest The authors disclose that they do not have any commercial associations that might create a conflict of interest in connection with this manuscript. YPR would like to acknowledge the University Grants Commission for awarding Teacher Fellowship during 10th plan period. This work is partly supported with financial assistance from UGC in the form of DRS-SAP lll. The authors are also thankful to the Deanship of Scientific Research, King Saud University Riyadh for funding part of the work through the research Group project No RGP-VPP-259.

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