Fluoride-Induced Oxidative Stress in Rat’s Brain and Its Amelioration by Buffalo (Bubalus bubalis) Pineal Proteins and Melatonin
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- Bharti, V.K. & Srivastava, R.S. Biol Trace Elem Res (2009) 130: 131. doi:10.1007/s12011-009-8320-2
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Fluoride (F) becomes toxic at higher doses and induces some adverse effects on various organs, including brain. The mechanisms underlying the neurotoxicity caused by excess fluoride still remain unknown. The aims of this study were to examine F-induced oxidative stress (OS) and role of melatonin (MEL) and buffalo pineal proteins (PP) against possible F-induced OS in brain of rats. The 24 rats were taken in present study and were divided into four groups: control, F, F + PP, and F + MEL. The F group was given 150 mg/L orally for 28 days. Combined 150 ppm F and 100 μg/kg BW (i.p.) PP and F (150 ppm) + MEL (10 mg/kg BW, i.p.) were also administered. The activities of enzymatic, viz., superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GR), and non-enzymatic, viz., reduced glutathione (GSH) concentration, and the levels of malondialdehyde (MDA) in the brain tissue were measured to assess the OS. Fluoride administration significantly increased brain MDA compared with control group, while GSH levels were decreased in fluoride-treated groups, accompanied by the markedly reduced SOD, GPx, GR, and SOD activity. Buffalo PP and MEL administration caused brain MDA to decrease but caused SOD, GPx, GR, GSH, and CAT activities to increase to significant levels in F-treated animals. Together, our data provide direct evidence that buffalo PP and MEL may protect fluoride-induced OS in brain of rats through mechanisms involving enhancement of enzymatic and non-enzymatic antioxidant defense system. Therefore, this study suggested that PP and MEL can be useful in control of neurotoxicity induced by fluoride.
KeywordsAntioxidantsBrainBuffalo pineal proteinsEnzymesFluorideMelatoninOxidative stressRat
Oxidative damage due to overproduction of reactive free radicals plays a key role in the pathogenesis of many diseases in animals, including neurodegenerative processes [1, 2]. Oxidative stress (OS) is implicated as one of the primary factors that contribute to the development of various neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, AIDS, dementia, etc. . In spite of numerous biological defense systems, increased free radicals generation has the potential to result in OS  and promotes cellular injury and tissue damage. This is not surprising, since the brain (neural cells) is especially susceptible to OS and subsequent damage to cells (including cell death). Antioxidant defense mechanisms in the brain are not sufficient to prevent age-related increase in oxidative damage and that exogenous intake of antioxidants might be beneficial for preserving brain function. Therefore, supplementations of antioxidants are needed to enhance these antioxidative enzymes level for preventing OS . A growing number of studies have been designed to test the antioxidant effect of some agents to prevent neurodegenerative disease caused by OS. Antioxidants that effectively enter the nervous system improve antioxidant defense system, act as antioxidants themselves, and are well absorbed from the GI tract would be ideal candidates for protection of the brain and nervous system from OS/damage . Pineal gland actions that have directly been identified include its ability to directly neutralize a number of toxic reactants and stimulate antioxidative enzymes [7, 8]. In recent years, evidence correlating OS and neurodegenerative diseases has sparked a keen interest in the antioxidative effects of the pineal-derived neurohormone, melatonin (MEL) and pineal protein (PP) . MEL, the chief secretary product of the pineal gland, is a direct free radical scavenger and indirect antioxidant [10, 11]. Various experimental studies have demonstrated the neuroprotective effects of MEL, based on its antioxidant activity [12, 13]. Most investigators have invoked MEL as the primary mediator of antioxidative properties of pineal glands [12, 14], since mammalian pineal body secretes biologically active proteins, peptides, enzymes, and they have many physiological roles of pineal gland . These proteinaceous materials were believed to contain active peptides, serotonin, MEL, and other pineal indoles. However, reports regarding the role of PP are scarce, but few studies revealed these proteins as potent regulators of physiological functions [16–18]. To find other active molecules as antioxidants, further research is required on pineal glands secretion other than MEL. Literature regarding the physiological role of PP and peptides in OS is completely lacking. However, these proteins may affect directly antioxidant defense system or indirectly via other pineal gland secretions. Therefore, to test this hypothesis, the present study was designed to investigate whether antioxidants levels (enzymatic or non-enzymatic) are changed in response to PP and MEL administration in fluoride-induced OS in rats. To the best of my knowledge, this study is the only reference on the effect of PP on antioxidant defense system in the brain of fluoride-induced OS.
Materials and Methods
This study was conducted at the Neurophysiology Laboratory of the Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar (U.P.), India. This is located at an altitude of 172 m above the mean sea level at a latitude of 28.20° North and longitude of 79.24° East. The climate is subtropical. All the procedures, conducted on the experimental animals, were duly approved by the Institutional Animal Ethics Committee and Committee for the Purpose of Control and Supervision of Experiments on Animals.
The chemical, glass, and plastic were procured as per requirement from different suppliers. All chemicals used in the study were of analytical grade from HiMedia, Loba Chemie (Mumbai, India), Sigma Chemical Co., and SRL Chemicals, India. MEL was procured from Sigma Chemical Co; however, buffalo (Bubalus bubalis) PP were supplied by the Neurophysiology Laboratory, Division of Physiology and Climatology, IVRI, Izatnagar.
Distribution of Experimental Rats to Different Treatments
Body weight (g)
Route of administration
141.66 ± 3.57b
Drinking water + normal saline
123.33 ± 1.05a
NaF + normal saline
150 ppm F
Oral with drinking water intra-peritoneal
F + PP
142.17 ± 5.66b
NaF + pineal proteins
150 ppm F, 100 μg/kg BW
Oral with drinking water intra-peritoneal
F + MEL
137.50 ± 4.79b
NaF + melatonin
150 ppm F, 10 mg/kg BW
Oral with drinking water intra-peritoneal
Rats were housed in polypropylene cages, and rice husk was used as the nesting material. Throughout the study, each cage was identified by a slip according to group and for recording study, schedule, animal number, details of treatments, etc. All the animals had free access to standard laboratory animal diet and clean water, arrangements for which were made in the cages. The animals were checked daily for the health and husbandry conditions.
Before the start of the experiment, appropriate dose of fluoride (F), MEL, and PP were optimized in pilot trial. Fluoride level in drinking water was calculated, and thereafter required, concentration of F was made by addition of sodium fluoride daily for 28 days (Table 1). Dose of PP (100 μg/kg BW, i.p) and MEL (10 mg/kg BW, i.p.) was calculated based on body weight, and thereafter, they were dissolved in suitable vehicle before administration (daily for 28 day). Solutions for administration in experimental animals were prepared daily to minimize possible instability of the chemicals in the mixture. However, feed intake and water consumption were recorded daily.
Daily observations were taken for the behavioral changes, clinical signs of toxicity, and mortality, if any, throughout the experimental period. The samples were collected at the end of experiment (28 day). The rats were euthanized using ether at the end of experiments, and brain was collected immediately and cleaned, rinsed in chilled saline, blotted, and weighed. Thereafter, 200 mg of sample was weighed and taken in 2 ml of ice-cold saline. Another 200 mg of sample was weighed separately and taken in 2 ml of 0.02 M ethylenediaminetetraacetic acid (EDTA) for GSH estimation. Organ homogenates were prepared using IKA homogenizer (Germany), under ice-cold condition. Thereafter, cell-free supernatant were collected and transferred to precooled microfuge tube in duplicate and stored at below −20°C. These supernatants were used for estimation of various parameters.
Cell-free supernatant of tissues homogenates were taken for the analysis of total proteins, lipid peroxidation (LPO), and different oxidative related parameters (enzymatic and non-enzymatic), viz., catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) activity, as well as non-enzymatic reduced glutathione (GSH) concentration. LPO and reduced GSH were measured on the day of tissue collection. Absorbances of all the blood and tissue biochemical estimations were read, using Double Beam UV-VIS Spectrophotometer (UV 5704 SS, ECIL, India).
Membrane peroxidative damage of brain tissues due to free radicals was determined in terms of malondialdehyde (MDA) production by the method of Rehman .
The concentration of GSH (μM/g wet tissues) in brain tissues were estimated by evaluating free-SH groups, using 5,5′-dithiobis(2-nitrobenzoic acid) method described by Sedlak and Lindsay .
Activities of CAT enzymes were estimated by spectrophotometric method as described by Bergmeyer  and were expressed as nanomolar H2O2 utilized per minute per milligram protein.
SOD activities in brain were estimated as per method described by Madesh and Balasubramanian . It involves generation of superoxide by Pyrogallol autoxidation and the inhibition of superoxide-dependent reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) to its formazan, measured at 570 nm. The reaction was terminated by the addition of dimethyl sulfoxide, which helps to solubilize the formazan formed. The color evolved is stable for many hours and is expressed as SOD units [one unit of SOD is the amount (μg) of protein required to inhibit the MTT reduction by 50%].
GPx activities were determined by the method of Paglia and Valentine . The reaction mixture contained phosphate buffer (pH 7.0) containing EDTA, nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), GSH, sodium azide, and GR. The reaction was initiated by the addition of H2O2 to the reaction mixture. The change in the absorbance was read at 540 nm at minute interval for 4 min. The enzyme activity has been expressed as nanomolar per minute per milligram proteins (U/mg protein), and one unit of enzyme activity is defined as 1 nM of substrate (NADPH) utilized/min/mg protein at 25°C.
The enzyme activities were assayed by the method of Goldberg and Spooner . The reaction mixture contained phosphate buffer (0.12 M, pH 7.2), EDTA (15 mM), and GSSG (65.3 mM). To these, cell-free supernatant was added and then incubated at room temperature. Thereafter, NADPH (9.6 mM) was added, and immediate decrease in OD per minute was recorded at 340 nm for 3 min. The enzyme activity has been expressed as nanomolar NADPH oxidized to NADP per minute per milligram protein by using the molar extinction coefficient of 6,200/M/cm at 340 nm.
Protein contents in brain homogenates were determined and calculated by the method of Lowry et al. .
Differences between groups were statistically analyzed by one-way analysis of variance (ANOVA), and the differences between the means of groups were separated by least significant difference test. All data were presented as mean ± standard error. Values of p < 0.05 were regarded as significant. A computer program (SPSS 10.01, SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Comparisons were also made with age-adjusted ANOVA models.
Effect of Different Treatments on Lipid Peroxidation (LPO), Catalase (CAT), and Superoxide Dismutase (SOD) in Brain of Female Rats
LPO (nM MDA/ml)
CAT (nM min−1 mg protein−1)
4.17 ± 0.150a
89.14 ± 5.19b
1.84 ± 0.130b
10.26 ± 0.130c
51.41 ± 4.95a
1.10 ± 0.085a
4.74 ± 0.160b
80.42 ± 6.72b
1.62 ± 0.110a
4.63 ± 0.180b
79.06 ± 6.63b
4.54 ± 0.460c
Effect of Different Treatments on Glutathione Peroxidase (GPx), Glutathione Reductase (GR), and Reduced Glutathione (GSH) in Brain of Female Rats
GR (nM min−1 mg protein−1)
GPx (nM min−1 mg protein−1)
GSH (μM/g tissue)
97.69 ± 4.56b
107.49 ± 7.79c
12.30 ± 0.59b
40.39 ± 4.13a
62.96 ± 2.93a
7.51 ± 0.48a
118.56 ± 4.40c
113.37 ± 10.24c
13.02 ± 0.33b
85.56 ± 3.09b
79.96 ± 5.74b
7.99 ± 0.42a
Similarly, in PP-treated rats, there was a significant (p < 0.05) increase in the activity of CAT (−9.78%), GPx (5.47%), SOD (−57.81%), and GR (21.36%) and levels of GSH (5.85%) in brain in F administered rats as compared to control rats (Tables 2 and 3). These data strongly support the protective action of PP against fluoride-induced OS in rats.
In the present study, we demonstrate that treatment of buffalo PP and MEL in vivo conferred neuroprotection against fluoride-mediated OS in rats. In vivo evidence from this current study indicates that these compounds likely attenuate fluoride-induced OS in brain via inhibition of free radical generation.
Moreover, many neurodegenerative diseases are characterized by OS-induced damage and consequently neuronal dysfunction and death. This impairment probably is due to the vulnerability of the brain cells to increased OS . We observed low levels of enzymatic as well as non-enzymatic antioxidant defense in brain of fluoride-treated animals. Fluoride exposure might decrease the defense capacity of brain to the OS and therefore elevated the LPO and inhibited activities of the CAT, GR, GPx, and GSH [27–30]. The brain contains high concentrations of polyunsaturated fatty acids having high oxygen demand, which makes them more prone to oxidative damage by free radicals . In present study, decrease of SOD activities in brain of fluoride-treated rats can be attributed to a direct action of fluoride on the enzymes (competitive inhibition), as well as free radicals rather due to increased free radicals alone .
The decrease in the levels of GSH in brain observed in our study may be due to increased utilization of GSH by GPx in detoxification of H2O2 generated by fluoride-induced OS . Fluoride might have elevated LPO and inhibited the activities of antioxidants enzymes. This altered antioxidant status and high LPO in F-treated rats may be due to increased free radical generation . Thus, the reactive oxygen detoxifying capacity was reduced, resulting in OS in brain. These events may be implicated in the impaired and poor coordination of brain on other organ functions and may lead to poor health of the animal under OS . These results indicate that brain OS may play an important role in brain function affected by exposure to F.
Previous studies have shown that these antioxidant systems are disrupted by fluoride . Similar findings were observed in different tissues by Shanthakumari et al.  in fluoride-treated rats. Chinoy and Patel  administered 10 mg of NaF/kg body weight during 30 days to female mice and found that cerebral levels of GSH decreased, as well as the activities of SOD, GPx, and CAT. Venkataraman et al.  also reported similar findings in rats. Shivarajashankara et al.  showed that rats receiving100 ppm fluoride (as NaF) in drinking water for 4 months have increased levels of MDA and decreased activity of erythrocyte SOD in rats.
The most plausible hypothesis for the observed significant decrease in antioxidants enzymes in fluoride-treated group is that fluoride affects the pineal’s ability to synthesize MEL in rats. Fluoride may affect the enzymatic conversion of tryptophan to MEL. Although MEL was the hormone investigated in this project, fluoride may also affect the synthesis of MEL precursors, (e.g., serotonin) or other pineal products (e.g., 5-methoxytryptamine) .
It is well known that the protection of cells from oxidative damage can be accomplished through non-enzymatic and enzymatic antioxidant systems . Fluoride-induced OS was inhibited by administration of exogenous MEL and PP. This protection was manifested by reduced levels of LPO, increased level of GSH (non-enzymatic), and increased activities of enzymatic, viz., GPx, CAT, GR, and SOD in brain of rats. These results suggest that MEL and PP may protect the fluoride-induced oxidative damage for its ability to prevent LPO and replenishing the body antioxidants levels. Endogenous enzymatic and non-enzymatic mechanisms are important defense mechanisms to prevent OS . When these defense mechanisms become incompetent to scavenge free radicals, tissue damage begins. There may be two possibilities for the induction of GPx activities: The increase in GPx activity is elicited by the antioxidative activity of PP and MEL, which protects the enzyme from inactivation by hydroxyl radicals, and an enzyme reduction is mediated by changes in GPx gene expression . Results of this study clearly show that PP are having antioxidant role as MEL and could prevent OS-induced neurodegenerative diseases.
Our results substantiate previous studies that have shown modifications in brain antioxidant status after MEL administration. Barlow-Walden et al.  reported high GPx activity in brain after MEL administration. Pablos et al.  observed enhanced GPx activities in chick brain exposed to MEL. GPx is a major antioxidative defense mechanism in the central nervous system . MEL crosses the blood–brain barrier, shows a decrease in its nocturnal peaks in blood with age that has been associated with the development of neurodegenerative disorders, and has been shown to be harmless at high concentration . Our results demonstrated that MEL significantly inhibits MDA elevations, but the extent of reduction in MDA level did not reach to the level in control group, indicating that MEL could provide a protection from OS. Antioxidant actions of MEL probably derive from its stimulatory effect on SOD, GPx, GR, and glucose-6-phosphate dehydrogenase and its inhibitory action on nitric oxide synthesis . MEL stabilizes the cell membranes, making them more resistant to oxidative attack . Therefore, MEL may have potential utility in the treatment of neurodegenerative disorders where OS is a participant [41, 44].
In present study, PP has shown better antioxidant properties (GSH, GPx, and GR) as compared to MEL. This dominant effect of PP over MEL under F-induced OS might be due to its effect on MEL synthesis and antioxidant properties. A decrease in free radical production is followed by a gradual normalization of LPO, GSH, and antioxidant enzymes . Interestingly, increases in GSH and GPx were highly correlated in the present study, suggesting that induction of GPx may be mediated through redox mechanisms similar to GSH. GSH is a central component in the antioxidant defenses of cells, acting directly to detoxify ROS and also as a substrate for several peroxidases . In several instances, protection against toxic heavy metals has been shown to be associated with GSH efflux from the cell . Increased sensitivity to chemical toxicity also occurs when cells are depleted of GSH . Thus, protection of the neuronal cells from F toxicity through a GSH-dependent mechanism could be a reasonable cause for the reduction of GSH level in brain under F-induced OS.
CAT is not very important to the brain and is not present in brain mitochondria, where much oxygen radicals are generated and also low levels of CAT in most brain regions . Recently, it was thought that the most important H2O2-removing enzymes in brain and other animal tissues are the GPx [50, 51]. It removes H2O2 by coupling its reduction to H2O2 with oxidation of GSH. In light of this view, PP has better antioxidant ability than MEL, as the former had more effect on GPx than MEL.
The pineal functions are modulated by some neuropeptides. The presence of immunoreactive nerve fibers of these proteins in the pineal gland has been shown in several mammalian species. These peptides of different chain length might be present in different concentrations in PP . These peptides influence the pineal serotonin, N-acetyltransferase activity and MEL synthesis . Therefore, this physiological basis of MEL synthesis might be influenced by PP administration and brings better effect as compared to MEL administration. This means, PP has two effects, one direct and second indirectly via influence on MEL synthesis. One could speculate that treating the rat with MEL and PP could progressively lower the tissue damage by OS to the extent seen with control rats.
Similar effect was seen on GR activity in F + PP and F + MEL-treated rats; however, buffalo PP had better antioxidant effect as expressed by brain GSH, GR, and GPx. Together, our data provide direct evidence that systemic administration of buffalo PP and MEL protects against fluoride-induced OS in brain of rats through mechanisms involving enhancement of enzymatic and non-enzymatic antioxidant defense system. Thus, present findings testify that PP and MEL enhance the antioxidant defense system, which can contribute to better health of human and animals.
We solemnly acknowledge the Indian Veterinary Research Institute, Izatnagar-243122, U.P. (India) for providing financial assistance in the form of Institute Senior Research Fellowship to the first author. I also acknowledge the tireless efforts of our lab and animal shed assistants.