Protective effect of resveratrol on fluoride induced alteration in protein and nucleic acid...

Accepted Manuscript

Title: Protective effect of resveratrol on fluoride inducedalteration in protein and nucleic acid metabolism, DNAdamage and biogenic amines in rat brain

Author: Sudipta Pal Chaitali Sarkar

PII: S1382-6689(14)00163-XDOI: http://dx.doi.org/doi:10.1016/j.etap.2014.07.009Reference: ENVTOX 2045

To appear in: Environmental Toxicology and Pharmacology

Received date: 18-4-2014Revised date: 14-7-2014Accepted date: 15-7-2014

Please cite this article as: Protective effect of resveratrol on fluoride inducedalteration in protein and nucleic acid metabolism, DNA damage and biogenicamines in rat brain, Environmental Toxicology and Pharmacology (2014),http://dx.doi.org/10.1016/j.etap.2014.07.009

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http://dx.doi.org/doi:10.1016/j.etap.2014.07.009

http://dx.doi.org/10.1016/j.etap.2014.07.009

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Graphical Abstract

F-

Resveratrol

Scavenges O2

- , H2O2, NO, OH

- Generates

Fluoride Treated

F + Vit. CF +Resveratrol

Control

Control F treated

F + Vitamin CF + Resveratrol

Cellular level

Rat Brain

Rat Brain Cellular level

Comet assay

Histological changes

Antioxidant enzymes (SOD, Catalase, GSH, GPx,

GST, GR) LPO, NO, Free amino acid N, Free •OH, Protein carbonyl content

Proteolytic enzyme activity, acidic, basic, neutral, total protein

Transaminase and RNase enzyme activities

DNA and RNA

Neurotransmitter level

Reduced Form

Graphical Abstract (for review)

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Highlights

Fluoride, the 13th most abundant element on the earth's crust, is a chemical ion of

the element fluorine.

Fluoride, a neurotoxic element impairs the brain function via oxidative stress.

Fluoride can alter the metabolic status of mammalian brain.

Resveratrol, a flavonoid that found abundantly in the skin of grapes, berries, and

peanuts.

Resveratrol may provide strong ameliorative effect on fluoride induced brain

toxicity.

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Protective effect of resveratrol on fluoride induced alteration in protein

and nucleic acid metabolism, DNA damage and biogenic amines in rat

brain

Sudipta Pal* and Chaitali Sarkar

Nutritional Biochemistry Laboratory

Department of Human Physiology

Tripura University (A Central University)

Suryamaninagar, West Tripura 799022

India

Key words: Fluoride, nucleic acids, proteolytic activity, biogenic amines, oxidative stress,

resveratrol

* Corresponding author

Sudipta Pal

Assistant Professor

Department of Human Physiology

Tripura University (A Central University)

Suryamaninagar

West Tripura 799022, India

E-mail:sudiptapal @tripurauniv.in

http://ees.elsevier.com/etap/viewRCResults.aspx?pdf=1&docID=4513&rev=1&fileID=139799&msid=DDDB2BAB-0B54-4472-8219-D05F99E47EA3

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ABSTRACT

Fluoride, a well-established environmental carcinogen, has been found to cause

various neurodegenerative diseases in human. Sub-acute exposure to fluoride at a dose of 20

mg/kg b.w./day for 30 days caused significant alteration in pro-oxidant/anti-oxidant status of

brain tissue as reflected by perturbation of reduced glutathione content, increased lipid

peroxidation, protein carbonylation, nitric oxide and free hydroxyl radical production and

decreased activities of antioxidant enzymes. Decreased proteolytic and transaminase

enzymes’ activities, protein and nucleic acid contents and associated DNA damage were

observed in the brain of fluoride intoxicated rats. The neurotransmitters dopamine (DA),

norepinephrine (NE) and serotonin level was also significantly altered after fluoride

exposure. Protective effect of resveratrol on fluoride-induced metabolic and oxidative

dysfunctions was evaluated. Resveratrol was found to inhibit changes in metabolic activities

restoring antioxidant status, biogenic amine level and structural organization of the brain. Our

findings indicated that resveratrol imparted antioxidative role in ameliorating fluoride-

induced metabolic and oxidative stress in different regions of the brain.

1. INTRODUCTION

Safe drinking water is the basic requirement of every human being. Human activities

continuously alter water quality by the addition of substances and wastes to the landscapes.

Although, groundwater is considered safe but it is also contaminated with soluble organic and

inorganic materials. Common inorganic contaminants are fluoride (F), nitrates and nitrites of

metals and various heavy metals like arsenic, lead, cadmium, mercury, etc. Fluoride is an

important industrial chemical which is mainly used in aluminium industries, in the

manufacture of fluoridated dental preparations and the fluoridation of drinking water (Lu et

al., 2000). Fluoride exists in drinking water in an ionic form and hence, rapidly passes

through the intestinal mucosa. Once absorbed, fluoride binds with Ca2+

ions, which may lead

to hypocalcaemia (Miki and Motoyama, 1989). Fluoride under certain conditions can affect

virtually every phase of human metabolism. Fluoride is toxic when consumed in excess but

of benefit when consumed within permissible limit (Guan et al., 2000). The fluoride

concentration in drinking water up to 1ppm is safe for human body but above this limit is

considered deleterious to health. Excess fluoride exposure leads to a condition known as

fluorosis which is of three types, viz., skeletal fluorosis (affecting bones), dental fluorosis

(affecting teeth), and non-skeletal fluorosis affecting soft tissues such as muscles, liver,

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kidney, lungs, blood, cells, reproductive cells, and gastrointestinal mucosa, nervous system

(brain and spinal cord) etc. (Wang et al., 2004a).

Chronic fluorosis may induce oxidative stress, leading to the generation of free

radicals. Excessive ROS production leads to macromolecule oxidation, resulting in free

radical attack of membrane phospholipids with resulting membrane damage via induction of

lipid peroxidation. Previous studies revealed that oxygen free radicals caused a decrease in

biological activities of some antioxidant enzymes like super oxide dismutase (SOD), catalase

and glutathione peroxidase (GPX) (Shanthakumari et al., 2004). However, the manner in

which the whole body effects are produced is still unclear. The adverse toxic effects of

fluoride are due to (a) enzyme inhibition, (b) collagen breakdown(c) gastric damage and (d)

disruption of immune system (Ahmed et al., 2000).

Brain is highly susceptible to oxidative stress because of presence of more unsaturated

fatty acids, high oxygen utilization, high iron content, and decreased activities of detoxifying

enzymes (Bharath et al., 2002). Oxidative damage of nuclear and mitochondrial DNA in

human brain is supposedly involved in mild cognitive impairments that establish relevance of

integrity of brain DNA to brain function (Scott and Pandita, 2006). Fluoride is known to be

neurotoxic and thus impairs brain functions (Chouhan and Flora, 2008) by oxidative stress

mediated damage of brain tissues (Navabi et al., 2012a) or by alteration in neurotransmitter

level (Pereira et al., 2011). Fluoride influences metabolic status of mammalian brain as

evidenced by significant dose-dependent reduction of acidic, basic, neutral, and total protein

contents in the cerebral hemisphere, cerebellum and medulla oblongata regions of mice brain

after oral administration of NaF (Trivedi et al., 2007). Antioxidant treatment consistently

protects cells from lipid peroxidation caused by fluoride exposure (Hassan et al., 2009),

suggesting that oxidative/nitrosative damage is the major mode of action of fluoride.

Resveratrol (3,5,4’-trihydroxy-trans-stilbene) is a plant derived polyphenolic

compound belonging to a class of stilbenes, found abundantly in the skin of grapes, berries,

and peanuts (Zhang et al., 2013). Over the years, this molecule has received considerable

attention for its anti-inflammatory, anti-tumor (Mates et al., 2008) and antioxidant properties

(El-Agamy, 2010), as well as its ability to increase lifespan in lower organisms and improve

general health in mammals (Baur et al., 2006). In addition, oral administration of resveratrol

efficiently reduced oxidative stress and maintained mitochondrial function (Xu et al., 2012).

The beneficial properties of resveratrol have been well defined. However, to our knowledge,

no study has ever been conducted in vivo with trans-resveratrol in brain tissue exposed to

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fluoride. The present study aimed to provide a better understanding of the action of

resveratrol in modulating fluoride-induced oxidative and metabolic dysfunctions in four

discrete regions of rat brain (cerebrum, cerebellum, pons and medulla), if any and provides a

justification for further clinical study of resveratrol to be used as a protective agent against

fluoride-induced brain toxicity.

2. MATERIALS AND METHODS

2.1. Materials

Sodium fluoride (NaF), bovine serum albumin (BSA), 5,5′-Dithio-bis-2-nitrobenzoic acid

(DTNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), reduced glutathione

(GSH), trichloroacetic acid(TCA), ethylene diamine tetraacetic acid (EDTA), 1-chloro 2,4-

dinitrobenzene (CDNB), resveratrol, 2-thiobarbituric acid (TBA) and other chemicals used in the

study are of analytical grade and were purchased from the Sigma Aldrich, MERCK and SRL.

Fig:1 3,5,4'-trihydroxy-trans-stilbene (Resveratrol)

2.2. Experimental Animals

Healthy adult male albino rats of Wistar strain, weighing 140 to 180 g were obtained from

Authorised Animal Supplier of CPCSEA and acclimatized under laboratory conditions for two weeks

before starting the experiment. They were provided with standard protein diet (18% casein diet) and

supplied with drinking water ad-libitum. Animals were kept in animal house by maintaining standard

conditions of temperature (220C to 25

0C) and humidity (50%) with alternating 12 hours light/dark

cycle. Animal ethical committee of Tripura University approved the protocols of the experiments.

Animals received humane care as per CPCSEA guidelines.

2.3. Experimental Design

For the present study, 30 male albino rats of Wistar strain were divided into four groups of

equal average body weight and kept in well ventilated cages. They were labelled namely Group I,

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Group II, Group III and Group IV. The dose of fluoride has been selected from the report of Ghosh et

al., (2002). The treatment schedule for the present study is given below:-

Group I - Control (received 0.9% NaCl only)

Group II- Fluoride-treated (NaF at a dose of 20 mg/kg b. w./day orally for 30 days)

Group III – Resveratrol supplemented (NaF at 20 mg/kg b. w./day orally for 30 days + resveratrol at

20 mg/kg b.w./day intraperitoneally for last 14 days of fluoride treatment)

Group IV – Positive control (NaF at 20 mg/kg b. w./day orally for 30 days+ vitamin C at 20 mg/kg

b.w./day orally for last 14 days of fluoride treatment). Vitamin C serves as a common antioxidant and

so it is used as positive control in the present study.

2.4. Sample collection

After animal treatment was over, rats were sacrificed by cervical dislocation following ether

anesthesia according to the guidelines proposed by the Institutional Animal Ethical Committee for the

purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment

and Forests, Government of India. Brains from the experimental animals were quickly excised,

washed in ice-cold saline, blotted dry and kept at -200C until analysis.

2.5. Preparation of tissue homogenate

A 5% tissue homogenate of different regions of brain (cerebrum, cerebellum, pons and

medulla) of rat was prepared using all glass homogenizer in different homogenizing buffer as per

protocols for performing biochemical experiments and kept at -20oC until biochemical analysis was

performed.

2.6. Body weight and organo-somatic index

Changes in organ weight in relation to body weight were represented by organo-

somatic index (OSI) (Chirumari and Reddy, 2007). OSI of brain was calculated using the

following formula.

2.7. Biochemical assays

2.7.1. Nucleic acid contents

RNA and DNA contents of brain tissue homogenate were estimated by the method of

Stroev and Makarova (1989), except that DNA was extracted with 0.8 M PCA at 70ºC. RNA

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and DNA were measured in the respective extracts by UV-absorption at 270 and 290 nm

respectively on Dynamica double beam UV-VIS spectrophotometer (model Halo DB-20).

Values were expressed as mg per 100 mg of tissue.

2.7.2. Acidic, basic, neutral and total protein contents

Acidic, basic, neutral and total proteins were extracted separately by the method of

Shashi et al. (1992) and Trivedi et al. (2006). Protein content was determined

spectrophotometrically by the method of Lowry et al. (1951) using bovine serum albumin as

standard.

2.7.3. Free amino acid nitrogen content

Free amino acid nitrogen content of brain tissue was determined by the method of

Rosen (1957) using leucine standard curve. Readings were taken in a spectrophotometer at

570 nm wavelength.

2.7.4. Pronase activity

The pronase activity in rat brain (cerebrum, cerebellum, pons and medulla) was

estimated by the method of Barman (1974). The enzyme activity was measured in a

spectrophotometer at 280 nm and was expressed in terms of μg of tyrosine per minute per

100 mg tissue protein.

2.7.5. Trypsin activity

Tissue trypsin activity was measured by the method of Green and Work (1953). The

readings were taken in a spectrophotometer at 280 nm. Trypsin activity was calculated from

the tyrosine standard curve and the activity was expressed as µmoles of tyrosine per min per

mg of protein.

2.7.6. Cathepsin activity

Cathepsin activity of rat brain was measured by the method of Pokrovsky et al.

(1989). The readings were taken in a UV-VIS spectrophotometer at 280 nm wavelength. The

enzyme activity was expressed in terms of tyrosine per minute per mg protein.

2.7.7. Assay of ribonucleolytic (RNase) activity

Tissue RNase activity was measured by the method of Jossefsson and Lagerstedt

(1962). The 5% tissue homogenate was incubated with RNA substrate for 30 minutes and

then added with protein precipitating reagent (PPR). The supernatant was collected after

centrifugation and the readings were taken in a spectrophotometer at 260 nm. RNase activity

was expressed in terms of µg of RNase/100 mg tissue.

2.7.8. Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) activities

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ALT and AST activities in rat brain tissue were determined using a standard kit (Coral

clinical systems, Goa, India) following the method of Reitman and Frankel (1957). Both the

enzyme activities were expressed as units per mg of tissue protein.

2.7.9. Reduced glutathione (GSH) content

GSH content was measured in different parts of rat brain by the method of Ellman

(1959). The absorbance of the final reaction mixture was read at 412 nm. Tissue glutathione

content was calculated from the standard curve of known GSH concentration.

2.7.10. Lipid peroxidation level

Lipid peroxidation level was measured by method of Buege and Aust (1978). The

optical density was read at 533 nm. The molar extinction co-efficient, 1.56 x 105 cm2/mmol of

malondialdehyde was used to calculate the malondialdehyde production.

2.7.11. Nitric oxide (NO) level

Nitric oxide level in rat brain was measured by the method of Raso et al. (1999). The

optical density was measured spectrophotometrically at 550 nm.

2.7.12. Tissue free hydroxyl radical (.OH) production

Free hydroxyl radical formation was assayed according to the method of Babbs and

Steiner (1990). Fast blue BB hemi (zinc chloride) salt was used for production of yellow

coloured product by reaction with methane sulfinic acid (MSA) which was measured at 425

nm spectrophotometrically.

2.7.13. Protein carbonyl content

Protein carbonyl content was determined according to the method of Levine et al.

(1994). The absorbance of the supernatant was recorded at 370 nm. The results were

expressed as nmol of 2,4- Dinitrophenylhydrazine incorporated/mg protein based on the

molar extinction co-efficient of 22,000/M/cm for alkaline aliphatic hydrazones.

2.7.14. Super oxide dismutase (SOD) activity

SOD activity was estimated according to the method of Martin et al. (1987). The

assay was based on SOD-mediated increase in the rate of auto-oxidation of hematoxylin in

aqueous alkaline solution, which yielded a chromophore with maximum absorbance at 560

nm. The enzyme activity was expressed as units per minute per mg of protein.

2.7.15. Catalase activity

Catalase activity in the brain was assayed following the procedure of Aebi (1984).

The enzyme activity was expressed as μM of H2O2 utilized per minute per mg of protein.

2.7.16. Glutathione peroxidase (GPx) activity

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Glutathione peroxidase activity was measured by the method used by Maiti and

Chatterjee (2000). The absorbance was recorded at 340 nm. The enzyme activity was

expressed as nmoles of NADPH oxidized per min per mg protein.

2.7.17. Glutathione S-transferase (GST) activity

GST activity was determined following the procedure of Warholm et al. (1985). The

increase in absorbance was noted at 340nm. The activity of GST was expressed as nmoles of

GSH-CDNB (1-chloro-2,4-dinitrobenzene) conjugate formed per min per mg of protein.

2.7.18. Glutathione reductase (GR) activity

The GR activity was determined in the tissue according to the method of Carlberg and

Mannervik (1985). The enzyme activity was measured at 340 nm and was expressed as

nmoles of NADPH oxidized per minute per mg of protein.

2.7.19. Comet Assay

To detect primary DNA damage, the alkaline comet assay was performed on the

whole brain samples according to Singh et al. (1988). The method involved electrophoretic

separation of nucleotides followed by staining with ethidium bromide. The stained nucleoids

were immediately observed at 40X objective under a Leica fluorescence microscope fitted

with a 515–560 nm excitation filter and a 590 nm barrier filter.

Comet Capture and Analysis

An automated image analysis software (Comet assay IV cell scoring software) was

used to analyse images, compute the integrated intensity profile for each cell, estimate the

comet cell components and evaluate the range of derived parameters. To quantify DNA

damage, the following comet parameters were evaluated: tail length, tail intensity (percentage

DNA) and tail moment. Tail length (i.e. the length of DNA migration) is related directly to

the DNA fragment size and is presented in micrometers. It was calculated from the centre of

the cell. Tail intensity is defined as the percentage of fluorescence migrated in the comet tail.

Tail moment was calculated as tail length ×% DNA in tail/100.

2.7.20. Brain biogenic amine level

The frozen brain tissues were weighed and homogenized in acidified butanol.

Dopamine (DA), norepinephrine (NE) and serotonin were estimated according to the

procedure of Schlumpf et al. (1974). Fluorescence was read by excitation at 395 nm and

emission at 485 nm for NE, and 330 nm and 375 nm for DA respectively. To determine 5-

HT, the butanol layer was extracted with 0.1N HCl in the presence of n-heptane and the acid

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layer was mixed with o-phthaldehyde. After boiling and cooling, fluorescence was read by

excitation at 360 nm and emission at 470 nm.

2.5.21. Protein assay

Protein contents in tissue homogenates and in the supernatant were determined by the

method of Lowry et al. (1951).

2.5.22. Histopathological Examination

Brain tissues fixed in formalin (10%) were dehydrated through a graded ethanol series

and embedded in paraffin wax (MP, 68ºC). Brain sections at 9 µm were stained with

Delafield’s haematoxylin and eosin stain and analysed using compound microscope.

2.5.23. Statistical analysis

Data were expressed as means ± SD. Data were analyzed using one-way analysis of

variance using statistica software (version 9) followed by multiple comparison t test to

compare the difference between means of two different groups. Differences between all

possible pair-wise comparisons were tested and p value <0.05 was considered significant

(Das, 1981).

3. RESULTS

3.1. Effect of sodium fluoride on body weight and organo-somatic index

The OSI of whole brain (Figure 2) indicates that fluoride treatment at the present dose

and duration decreased the whole brain weight in relation to body weight by 19.32%

(p<0.01). Resveratrol supplementation in fluoride-treated rats restored the OSI near to the

control value. This effect was almost similar to the effect of vitamin C supplementation.

3.2. Effect of fluoride on DNA, RNA and free amino acid nitrogen contents in rat brain

The changes in tissue DNA and RNA contents as well as free amino acid nitrogen

concentration in different regions of rat brain (cerebrum, cerebellum, pons and medulla) have

been represented in Table (1). Results indicated that DNA content decreases by 29.47%

(p<0.001), 28.67% (p<0.001), 36.83% (p<0.001) and 34.88% (p<0.001) in cerebrum,

cerebellum, pons and medulla respectively following exposure to fluoride. Resveratrol

supplementation in fluoride-intoxicated rats restored the reduced DNA content of proposed

areas of brain by 44.16% (p<0.001), 40.2% (p<0.001), 49.52% (p<0.001) and 38.02%

(p<0.001) respectively. Similarly, vitamin C exhibited partial protective effect against

fluoride-induced alteration of DNA content in the mentioned areas of brain. The restoration

was found to be 34.76 % (p<0.001), 37.65% (p<0.001), 52.48% (p<0.001) and 46.37%

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(p<0.001) in cerebrum, cerebellum, pons and medulla, respectively. Resveratrol exhibited

better beneficial effect than vitamin C in cerebral tissue to protect against DNA damage,

whereas in cerebellum and pons the effects are almost similar as vitamin C. On the other side

vitamin C restored the DNA content in medulla more efficiently than resveratrol. RNA

content of fluoride-challenged animals was also decreased significantly in cerebrum,

cerebellum, pons and medulla. The decrease was found to be 20.3% (p<0.001), 28.4%

(p<0.001), 33.7% (p<0.001) and 34.1% (p<0.001) respectively. Administration of resveratrol

restored the reduced RNA content in the above mentioned areas of rat brain by 20.82%

(p<0.001), 8.53% (p<0.05), 34.36% (p<0.001) and 28.96% (p<0.05), respectively. Vitamin C

also exhibited partial ameliorative effect in restoration of RNA content in those regions of rat

brain. The present study further revealed that free amino acid nitrogen content increases

appreciably in the studied brain regions of fluoride-exposed rats. Resveratrol has the ability to

restore free amino acid nitrogen content to the respective control level. The restoration was

more effective in cerebellum (44.12%) and pons (21.1%) in comparison to cerebral cortex

(16.37%) and medulla (30.41%). Similarly, vitamin C also exhibited partial protective effect

against fluoride-induced alteration of free amino acid N2 level. The restoration was found to

be 7.53%, 47.91%, 21.47% and 21.94% in cerebrum, cerebellum, pons and medulla,

respectively.

3.3. Effect of fluoride on acidic, basic, neutral and total protein contents

The acidic, basic, neutral, and total protein contents were significantly reduced in the

cerebral hemisphere, cerebellum and medulla oblongata regions of rat’s brain (Table 2) after

fluoride exposure. The decrease in acidic protein was found to be 77.93% (p<0.001), 71.33%

(p<0.001), 71.61% (p<0.001) and 72.56% (p<0.001), respectively in cerebrum, cerebellum,

pons and medulla regions of brain whereas the basic protein level was diminished by 67.57%

(p<0.001), 72.75% (p<0.001), 80.42% (p<0.001) and 84% (p<0.001), respectively in those

specific brain regions. The percentage reduction of neutral protein content in the above

mentioned regions of the brain was found to be 58.76% (p<0.01), 71.87% (p<0.001), 66.77%

(p<0.001) and 72.29% (p<0.001) as compared with the control group. Finally, the total

protein content was decreased by 74.24% (p<0.001), 71.87% (p<0.001), 71.48% (p<0.001)

and 74.35% (p<0.001) as compared with their respective control groups. Administration of

resveratrol along with fluoride ameliorated the above mentioned changes in different protein

contents in all experimental regions of the brain of rats (Table 2). Vitamin C also showed

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appreciable beneficial effects in restoration of depleted protein contents in different parts of

the brain.

3.4. Effect of fluoride on brain proteolytic enzymes

Fluoride exposure decreased the cathepsin activity by 17.09% (p<0.01), 33.51%

(p<0.001), 41.08% (p<0.001) and 55.93% (p<0.001) in cerebrum, cerebellum, pons and

medulla, respectively (Table 3). Resveratrol supplementation restored the enzyme activity in

a better efficacy in cerebellum (62.21% restoration) and medulla (59.27% restoration) than in

cerebrum (22.8%) and pons (37.9%) in fluoride-exposed rats. Vitamin C also restored the

enzyme activity by 31.43% (p<0.001), 65.13% (p<0.001), 35.13% (p<0.001) and 86.95%

(p<0.001) in cerebrum, cerebellum, pons and medulla, respectively.

Table (3) represented that the trypsin activity decreased significantly in different regions

of brain tissue by fluoride. Resveratrol counteracted fluoride-induced alteration of trypsin

activity by 84.89% (p<0.001), 63.04% (p<0.001), 94.42% (p<0.001) and 115.48% (p<0.001)

in cerebrum, cerebellum, pons and medulla respectively. Vitamin C supplementation also

exhibited appreciable protective effects against fluoride-induced decreased trypsin activity by

restoring 97.12%, 60.59% in cerebrum and cerebellum and completely in pons and medulla.

The pronase activity was also decreased significantly in all studied regions of rat brain

(Table 3). The decrease was found to be 25.21% (P<0.01), 21.04% (p<0.05), 50.15%

(p<0.001) and 59.18% (p<0.001) in cerebrum, cerebellum, pons and medulla, respectively.

Administration of resveratrol appreciably restored the decreased activity of pronase in all of

the above mentioned regions of rat brain. Vitamin C alone also exhibited almost similar

counteractive effects against fluoride-induced alteration of brain pronase activities.

3.5. Effect of fluoride on brain transaminase and RNase activities

Effect of fluoride on brain ALT and AST activity (Table 4) revealed that GPT

activity in the experimental group increases by 17.51% (p<0.001), 39.72% (p<0.001), 18.64%

(p<0.001) and 29.84% (p<0.001) in cerebrum, cerebellum, pons and medulla, respectively.

Resveratrol supplementation restored the increased ALT activity of the proposed areas of

brain by 18.1% (p<0.001), 21.55% (p<0.001), 22.92% (p<0.001) and 24.43% (p<0.001).

Similarly, vitamin C exhibited partial protective effect against fluoride-induced alteration in

ALT activity in the observed areas of the brain. Tissue AST activities also increased in

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fluoride-exposed animals by 40.34% (p<0.001), 31.87% (p<0.001), 26.67% (p<0.001) and

28.69% (p<0.001) in those above mentioned regions of brain. Resveratrol supplementation

restored the increased AST activity in the proposed areas of brain by 36.84% (p<0.001),

49.18% (p<0.001), 38.04% (p<0.001) and 32.32% (p<0.001), respectively. Vitamin C also

exhibited almost similar protective effects as resveratrol against fluoride-induced alteration of

the increased AST activity in the proposed areas of rat’s brain.

The tissue RNase activity increased significantly in all studied regions of rat brain

after fluoride exposure (Table 4). The increase was found to be 112.64% (P<0.001),

115.96% (p<0.001), 107.78% (p<0.001) and 109.17% (p<0.001) over the control in

cerebrum, cerebellum, pons and medulla, respectively. Administration of resveratrol

appreciably restored increased RNase activity in all of the above mentioned regions of rat

brain. Vitamin C alone also exhibited similar counteractive effect as resveratrol against

fluoride-induced alteration of brain RNase activities.

3.6. Effect of fluoride on brain variables suggestive of oxidative stress, glutathione

metabolism and tissue free hydroxyl radical (.OH) production

Changes in tissue GSH content and LPO level have been represented in Figure (3)

and (4). The results showed that GSH level decreases by 47.64% (p<0.001), 49.27%

(p<0.001), 46.51% (P<0.001) and 46.51 (p<0.001), respectively in cerebrum, cerebellum,

pons and medulla of fluoride exposed animals. Resveratrol supplementation almost

completely restored the depleted glutathione level in the observed areas of rat’s brain.

Vitamin C alone also exhibited appreciable beneficial effects in restoration of decreased

glutathione content in all of those mentioned regions of the brain.

Fluoride exposure also caused significant increase in malondialdehyde production in

all studied regions of rat brain. The increase was found to be 31.66% (p<0.001), 32.85%

(p<0.001), 31.72% (p<0.001) and 29.85% (p<0.001), respectively as compared to their

respective control value. A remarkable protection against overproduction of lipid peroxides

in all the above mentioned regions of brain was found in resveratrol supplemented group.

Enhanced lipid peroxidation in fluoride-intoxicated animals was also checked by vitamin C

supplementation.

Fluoride also significantly increased free •OH radical production (Table 5) in brain

tissue, which was appreciably counteracted by resveratrol supplementation. The restoration

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was found to be 45.89%, 50.53%, 37.73% and 32.97% of their control values of respective

brain regions. Supplementation of vitamin C appreciably prevented increased production of

free •OH radical in the above mentioned regions of brain.

Exposure to fluoride caused a significant elevation in NO level (Table 5) in cerebrum,

cerebellum, pons and medulla of rat brain. The increase was found to be 48.11% (p<0.001),

53.72% (p<0.001), 49.42% (p<0.001) and 28.7% (p<0.001), respectively. Resveratrol

supplementation in fluoride exposed animals caused partial restoration of NO level in the

areas of rat brain. Vitamin C supplementation restored the elevated NO level much better

than resveratrol in cerebrum and cerebellum, whereas this vitamin exhibited similar

counteractive effect as resveratrol in other two regions of brain.

Table (5) represented the result of the extent of protein carbonyl content on brain

tissue (cerebrum, cerebellum, pons and medulla) homogenate of the control and experimental

animals. Fluoride exposure significantly increased the extent of protein carbonyl content by

53.19%, 32.97%, 34.58%, and 37.48% in all the four brain regions respectively. Vitamin C

supplementation restored the elevated protein carbonyl content much better than resveratrol

in cerebrum, cerebellum, pons, and medulla regions of the brain.

Changes in the activities of antioxidant enzymes have been demonstrated in Table

(6). The SOD and catalase activities decreased significantly in cerebrum, cerebellum, pons

and medulla regions of brain of fluoride-exposed animals. Resveratrol alone was found to

have appreciable beneficial effect on fluoride-induced alteration of these two potential

antioxidant enzymes to their respective control values. Vitamin C supplemented group also

exhibited almost complete restoration of the decreased SOD and catalase activities after

fluoride exposure.

GPx activity was also inhibited by 40.43% (p<0.001), 32.12% (p<0.001), 45.37%

(p<0.001) and 46.5% (p<0.001), respectively in cerebrum, cerebellum, pons and medulla

regions of rat brain after fluoride treatment (Table 6). Resveratrol restored the decreased GPx

activity in those studied regions by 41.42%, 29.38%, 36.89% and 38.45% of their respective

control values. Fluoride exposure also decreased the activities of GST and GR markedly in

the studied regions of rat brain. Resveratrol supplementation also exhibited appreciable

counteractive effect in restoration of GST and GR activities in cerebrum, cerebellum, pons

and medulla of rat brain (Table 6).

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3.7. Effect of fluoride on DNA damage

The comet assay provided a simple and effective method for evaluating DNA damage

in animal cells. The quantitative and statistical data were generated using Comet assay IV cell

scoring software. Figure (5) showed the typical ethidium bromide stained comets captured at

various experimental conditions. Table (7) showed qualitative and quantitative analysis of

ethidium bromide stained comets on the basis of DNA damage. We assessed the fluoride

toxicity in brain tissue of the exposed animals using the comet assay as a highly effective tool

for the biomonitoring of DNA integrity. In fluoride exposed groups, the tail length and tail

intensity (Table 7) in brain tissue was significantly higher than in controls (p<0.01; p<0.01).

The head DNA content of negative control was 100% and no tail content was observed.

3.8. Effect of fluoride on brain biogenic amines

Brain biochemical variables namely norepinephrine (NE), dopamine (DA), and

serotonin (5-HT) in cerebrum, cerebellum, pons and medulla of rat brain decreased

significantly during fluoride exposure (Table 8). Monoamine neurotransmitter serotonin also

showed a drastic and significant (p<0.001) decrease in its level after fluoride exposure in

cerebrum and cerebellum. Beneficial effect of resveratrol supplementation was also noted

against fluoride-induced changes in biogenic amine level.

3.9. Effect of fluoride on histological structure of the brain

Histological analysis of whole brain after fluoride exposure is depicted in Figure (6).

Control animals showed normal cellular compositions in all parts with intact neuron and

cytoplasm (Fig. 6A). The ultrastructure showed normal oligodendrocytes and few astrocytes.

Fluoride exposure (Fig 6B) showed chromatolysis of nuclear material, shrinkage of some

Purkinje neurons and mild necrosis which is indicated by hyperchromasia and disintegrated

cytoplasm. Cytoplasm showed edema indicated by vacuoles at many instances. Many

neurons were shrunken, pyknotic and darkly stained with small nuclei and there was a

decrease in their overall cell number.

4. DISCUSSION

The present study aimed to evaluate the salubrious effects of resveratrol, a natural

antioxidant as a neuroprotective agent against fluoride-induced alteration in metabolism of

brain protein, nucleic acid content, proteolytic and transaminase activities, biogenic amine

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levels in addition to DNA damage, oxidative stress and histopathological changes of discrete

brain regions. Remarkable decrease in whole brain weight in relation to body weight as

indicated by decrease in organo-somatic index was noticed after sub-acute exposure to

fluoride. This may be due to retardation of animal growth symptomized by decreased appetite

and primary malnutrition caused by fluoride. Excess intake of fluoride might cause

maldigestion and malabsorption of nutrients by the GI tract, inducing malnutrition of brain

tissue, thus retarding their proper growth (Pushpalatha et al., 2005). Increased breakdown of

brain tissue proteins or depressed protein synthetic machinery had been suggested to be

involved in fluoride-induced alteration of organo-somatic index (Sarkar et al., 2014).

Significant reduction in DNA and RNA level in different brain regions by fluoride has

been demonstrated in the present study which is in conformity with our earlier observations

(Sarkar et al., 2014). Decreased synthesis of nucleic acids and improper attachment of mRNA

to the ribosome were supposed to be involved in fluoride-induced alteration of nucleic acid

and protein metabolism (Verma et al., 2007). Earlier studies revealed that fluoride can alter

DNA/RNA, DNA/protein and RNA/protein ratios which were indicative of disturbances in

translational as well as transcriptional processes, mitotic cell division and chromosomal

aberrations (Memon and Chinoy, 2000). DNA damage by fluoride involves oxidation, base

alteration and strand breaks (Temple et al., 2005), resulting in cellular dysfunction. This is

supported by the present observation where the fluoride-challenged cell appears as a comet

instead of its normal round shape. The increased comet length and percentage of DNA in

comet head, tail and tail moment also reflect significant damaging effects of fluoride on

cellular DNA. Fluoride can cause DNA damage in various cell types (He and Chen, 2006),

possibly ascribed to the involvement of oxidative stress (Wang et al., 2004b). Enhanced ROS

production due to mitochondrial dysfunction (Maassen et al., 2004) induced by fluoride is

supposed to be involved in neurodegeneration (Yamanaka et al., 1990). DNA damage by

fluoride involves two possible mechanisms i) direct fluoride attacks the free amide group of

DNA and ii) indirect fluoride-induced free radical attack to the hydrogen bonds of DNA

forming various DNA adducts (Flora et al., 2009).

RNA contents of different regions of brain decreased in fluoride-exposed rats. The

decreased RNA content in brain tissue by fluoride was also reported earlier (Sarkar et al.,

2014). This may be attributed to either increased ribonucleolytic activity or decreased RNA

synthesis in response to this toxicant. Ribonuclease (RNase) catalyzes the degradation of

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RNA into smaller components and thus influences the cellular content of RNA (Ghosh et al.,

1992). In the present study RNase activity of different regions of brain appears to show a

relation with the relative changes of the regional RNA contents. Reduced RNA content after

fluoride exposure may also be due to decreased protein synthesis in brain tissues. Suppressed

protein synthesis in rabbit brain by fluoride was supposed to be involved in decreased RNA

content in those tissues (Shashi et al., 1994). Thus it may be suggested that accumulation of

fluoride may affect the regulation of RNA metabolism in distinct brain regions.

Alteration in protein synthesis by fluoride has been indicated by decreased contents of

acidic, basic, neutral and total protein in cerebrum, cerebelleum, pons and medulla as

observed in our present study. It is ascribed to either increased breakdown of proteins or

decreased synthesis of it. Breakdown of proteins is considered as one of the important

metabolic stress caused by fluoride (Sarkar et al., 2014). Protein breakdown in fluorosis may

be attributed to inhibition of oxidative decarboxylation of branched chain amino acids (Chang

and Globder, 1978). Another suggestive mechanism in favour of fluoride-induced decreased

brain protein contents may be decreased ability of brain tissues to synthesize amino acids as a

result of suppressed activity of certain metabolic enzymes like glutamine synthetase and

methionine activating enzymes (Zhavoronkov, 1977). Despite elevated level of free amino

acid nitrogen in brain tissues as found in the present study, it is not properly utilized to

synthesize new proteins due to toxic insult and amino acids are rapidly mobilized by the

catalytic action of transaminases from the studied regions of rat brain to other areas, causing

less availability of substrates for synthesis of desired proteins in those specific brain regions.

As a consequence, activities of proteolytic enzymes like pronase, cathepsin and trypsin were

suppressed after fluoride exposure. Changes in the proteolytic enzyme activity showed

consistency with the changes in regional protein level indicating altered metabolic efficacy of

brain tissue due to fluoride toxicity.

Earlier studies have indicated the role of oxygen derived free radicals in fluoride

toxicity (Wessam and Abdel-Wahab, 2013). Intracellular antioxidant enzymes are considered

as the first line of cellular defence that prevents biological macromolecules (like DNA,

proteins etc.) from free radical-mediated damage (Ghosh et al., 2008), whereas glutathione

antioxidant system plays a crucial role as the second line of cellular defence. On the basis of

information largely derived from histological, chemical and molecular studies, it is apparent

that fluoride has the ability to interfere with the functions of the brain and other tissues by

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direct or indirect means. The indirect effect is mediated via alteration of cellular antioxidant

defence system leading to oxidative stress. Perturbation of GSH was one of the important

toxic effects of fluoride (Ghosh et al., 2008) which motivated excessive production of ROS at

the mitochondrial level, leading to damage of cellular components. Fluoride-induced

decreased GSH level (Figure 3) was found in all observed brain regions of rats,

corroborating our earlier observation (Sarkar et al., 2014). Depletion of endogenous

antioxidants may lead to overproduction of peroxidized lipid molecules in brain tissue as

indicated by enhanced level of malondialdehyde in studied brain regions (Figure 4).

Overproduction of lipid peroxides due to fluoride exposure causes destabilization in cellular

lipid substances, inducing oxidative damages especially of membrane structures. Increased

LPO level after fluoride exposure was also reported in earlier occasion (Hassan and Abdel-

Aziz, 2010). Brain tissues, rich in polyunsaturated fatty acids were more vulnerable to

oxidative stress than others (Yur et al., 2003).

Fluoride-induced free radical generation has been evidenced by overproduction of

free hydroxyl radicals and nitric oxide in observed brain regions (Table 5). Free hydroxyl

radicals, nitrogen species and lipid peroxidation products can trigger the excitotoxic process

causing imbalance in cellular functions. Inhibitory effect of fluoride on SOD and catalase

activities was supposed to be involved in overaccumulation of those potential toxic free

radicals in brain tissue. Earlier report established that sodium fluoride significantly increased

nitric oxide synthase activity (Xu et al., 2001) that would increase the intracellular NO

concentration. Interestingly, this is of particular importance because NO after combining with

superoxide radicals form potentially toxic peroxynitrite radical, which plays a major role in

neurodegenerative diseases, primarily by damaging mitochondrial energy production,

inhibiting glutamate re-uptake and stimulating lipid peroxidation (Bolanos et al., 1997).

Superoxide radical, the substrate for peroxynitrite formation was enhanced in response to

decreased antioxidant enzyme activity caused by fluoride (Vani et al., 2000). Decreased

antioxidant enzyme (like SOD, catalase, GST, GPx and GR) activities were found in fluoride-

exposed rat brain which is in conformity with our earlier studies (Sarkar et al., 2014).

In the present study, fluoride intoxication imposes protein carbonylation (Table 5) in

the brain tissue of experimental animals due to high susceptibility towards oxidative damage.

The modification of native amino acids side chains in proteins to carbonyl (aldehyde and

ketone) derivatives enhanced due to fluoride exposure. Excessive ROS production may lead

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to protein carbonylation, macro-molecular oxidation, resulting in free radical attack to

membrane proteins and phospholipids causing membrane damage via induction of lipid

peroxidation, mitochondrial membrane depolarization. Additionally, fluoride at the present

dose and duration also altered the level of brain biogenic amines like NE, DA and 5HT,

which confirm earlier observations (Chirumari and Reddy 2007). Perturbation of these

biogenic amines upon fluoride intoxication indicated significant alteration of physiological

activities of brain tissue (Flora et al., 2009). This may be attributed to less availability of

precursor amino acids in the brain cell to synthesize those neurotransmitters, as fluoride

induced mobilization of free amino acids from studied regions to other parts and resulted in

enhanced transamination reaction. The decreased biogenic amine level may either be due to

decreased activity of enzymes involved in their synthesis like DOPA decarboxylase,

dopamine β-hydroxylase and tyrosine hydroxylase or to the enhanced release of catechol-O-

methyl transferase caused by increased neuronal activity (Kaur et al., 2009). It has been

reported that the decrease in serotonin level may occur due to conversion of serotonin to

melatonin to combat against fluoride-induced oxidative stress (Grad and Rozencwaig, 1993).

Biochemical changes are associated with alteration in ultrastructure of brain upon

fluoride exposure. Fig (6B) showed that fluoride alters the normal ultrastructure of the brain

tissue as indicated by pyknotic or absent nerve cell nuclei and stretched dendrites that were not

found in control group (Fig 6A). These histopathological changes are in accordance with the

results of Shivarajashankara et al., (2002). It may be inferred that during NaF intoxication, F-

crossed the blood brain barrier and damaged the nerve cells.

The present study demonstrated that resveratrol prevented fluoride-induced increase

in MDA level in different brain regions and concomitantly restored GSH content in them,

albeit to a different degree. These effects reflected the ability of resveratrol to enhance the

scavenging and inactivation of H2O2 and hydroxyl radicals. Additionally, resveratrol activates

endogenous enzymatic antioxidants like superoxide dismutase, catalase, glutathione S-

transferase, glutathione reductase and glutathione peroxidase, thus increasing intracellular

enzymatic antioxidant level in brain tissue. Resveratrol has been found to be an effective

scavenger of hydroxyl, superoxide and metal-induced free radicals, as well as showing

antioxidant abilities in cells producing reactive oxygen species (Leonard et al., 2003).

Resveratrol not only scavenges free radicals but also increased the intracellular expression of

other naturally occurring enzymatic antioxidants (Farris et al., 2013). Specifically, resveratrol

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up-regulated expression of nuclear factor-E2-related factor-2 (Nrf2), a transcription factor,

which regulates several genes responsible for detoxification of reactive oxygen species

(Farris et al., 2013) for example the gene which regulates production of glutathione

synthetase, the rate limiting enzyme of glutathione synthesis. As a result, depleted GSH after

fluoride intoxication was replenished by resveratrol supplementation. The antioxidant effect

of this natural polyphenol may involve stabilization of membrane lipids and protein

molecules as indicated by restoration of lipid peroxidation, free hydroxyl radical level and

protein carbonylation efficacy of brain tissue towards normalcy after resveratrol

supplementation that maintains the functional dynamics of the plasma membrane.

The present study further revealed that resveratrol supplementation appreciably

counteracted fluoride-induced alteration in brain protein and nucleic acid metabolism. Free

radicals were considered as one of the major causative factors for DNA and RNA damage

and associated protein depletion. Amelioration of free oxygen radicals by resveratrol is

assumed to restore cellular DNA, RNA and protein levels. Accordingly, the protection

afforded by resveratrol against fluoride-induced metabolic alteration was likely attributable to

its antioxidant effect. Resveratrol, in this study has been linked to beneficial neuroprotective

effect, assuming that trans-resveratrol does accumulate in brain tissues and exhibits beneficial

effect against metabolic alterations. The protective effect of resveratrol also involved

promotion of proteolytic and transaminase enzyme activities in fluoride-intoxicated brain

tissue. By restoring their substrate level resveratrol imparted counteraction of those enzyme

activities towards control. Moreover, resveratrol counteracted fluoride-induced increased

RNase activity, thus restoring RNA level in all studied brain regions. Such protective effect

of resveratrol reflected its involvement in normalizing brain metabolic activities.

In addition, resveratrol counteracted NO overload in fluoride-intoxicated brain tissue.

Resveratrol by counteracting NO production may protect brain from oxidative injury through

a nitric oxide dependent mechanism. Notably, resveratrol stabilized the reduced level of

biogenic amines in different regions of fluoride-intoxicated rat brain. These effects of

resveratrol further supported its neuroprotective action against fluoride toxicity.

Neuroprotective effects of resveratrol against oxidative stress-induced neurodegenerative and

metabolic disorders were reported in earlier occasion (Quincozes-Santos et al., 2014).

Additionally, this polyphenol appreciably restored the ultrastructural details of rat brain

which was adversely affected by fluoride. Stabilization of ultrastructure of brain tissue

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towards normalcy by resveratrol indicates its appreciable beneficial effect against fluoride-

induced neurodeterioration.

5. CONCLUSION

This study confirmed the antioxidant and neuroprotective effects of resveratrol, a

natural flavonoid against fluoride-induced neurotoxicity by restoring the oxidant/antioxidant

homeostasis, protein and nucleic acid metabolism and biogenic amine levels in brain tissue of

rats. Additionally, the altered brain tissue architecture induced by fluoride was appreciably

rejuvenated by resveratrol supplementation. Thus, this flavonoid compound may be used as a

prospective protective agent against fluoride-induced brain toxicity. Our findings validated

further studies focused in understanding the detail mechanism of resveratrol in preventing

neurodeterioration caused by sub-acute fluoride exposure.

6. ACKNOWLEDGMENTS

This study has been supported by the grants from Department of Biotechnology

(DBT), Govt. of India. We also express our sincere thanks to the Co-ordinator, State Biotech

Hub, Tripura University (A Central University) for providing infrastructure facility needed

for the present work. We are also thankful to Prof. Durgadas Ghosh, Department of Zoology,

Tripura University for his kind help.

7. CONFLICT OF INTEREST

All of the authors declare that they have no conflict of interest.

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Fig 1: 3,5,4'-trihydroxy-trans-stilbene (Resveratrol)

Fig 2: Effect of resveratrol on fluoride-induced changes in organo-somatic index

Fig 3: Change in GSH content after fluoride exposure with or without resveratrol

supplementation

Values are Means±S.D. pa compared with control group and p

b compared with fluoride

treated group, *** indicates p<0.001, ** indicates p<0.01, * indicates p<0.05, # indicates

insignificant difference

***pa

***pb

***pa

***p

a

***pa

***pb

***pb

***pb

***pb

***pb

***p

b

***p

b

**pa

*p

b

*p

b

Figure

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Fig 4: Change in LPO level after fluoride exposure with or without resveratrol

supplementation


b compared with fluoride

treated group, *** indicates p<0.001, ** indicates p<0.01, * indicates p<0.05, # indicates

insignificant difference

Fluoride Treated

F + Vit. CF +Resveratrol

Control

Fig 5: Comet assay micrograph represents undamaged and damaged brain cell of control

sample, fluoride treated, fluoride and resveratrol supplemented and fluoride and vitamin C

supplemented group of experimental animals. Cells were photographed under the

fluorescence microscope using a 40x objective equipped with a 515-560 nm excitation filter

and a 590 nm barrier filter

***pa

***pa

***pb

***pb

***pb

***pb

***pb

***pb

***pb

***pb

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Fig 6: Histological changes in the rat brain after fluoride treatment with or without

resveratrol supplementation (arrows indicate degenerated neurons in fluoride treated group,

neurones were shrunken and darkly stained with small nuclei, A) vacuolation and B)

chromatin clumping (darkly stained). Regeneration found in “F+Resveratrol” and

“F+Vitamin C” supplemented groups, arrows indicate regaining proper shape of the neurones

towards normalcy)

Control F treated

F + Vitamin CF + Resveratrol

A

B

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Table 1: Change in DNA, RNA contents and free amino acid nitrogen following fluoride exposure with or without resveratrol in

cerebrum, cerebellum, pons and medulla of rat brain

Groups of

animals

DNA

(mg/100 mg of tissue)

RNA

(mg/100 mg of tissue)

Free amino acid N content

(µg of leucine/mg of tissue protein)

Cerebrum Cerebellum Pons Medulla Cerebrum Cerebellum Pons Medulla Cerebrum Cerebellum Pons Medulla

Control 0.072±0.006 0.183±0.012 0.075±0.003 0.08±0.002 0.123±0.007 0.174±0.009 0.086±0.003 0.085±0.001 200.95±3.16 234.84±1.41 247.27±3.42 315.37±3.42

F-treated 0.05±0.005

pa***

0.131±0.006

pa***

0.047±0.002

pa***

0.052±0.002

pa***

0.098±0.008

pa***

0.157±0.004

pa***

0.057±0.003

pa***

0.056±0.002

pa***

349.38±2.71

pa*** 377.4±1.7

pa***

351.83±1.94

pa***

474.45±3.89

pa***

F+ resveratrol

supplemented

0.073±0.002

pb***

0.183±0.004

pb***

0.071±0.005

pb***

0.072±0.003

pb***

0.119±0.003

pb***

0.170±0.007

pb*

0.077±0.004

pb***

0.073±0.002

pb*

300.22±2.61

pb***

276.33±2.91

pb***

242±2.71

pb***

387±1.24

pb***

F+Vit-C 0.068±0.004

pb***

0.18±0.01

pb***

0.072±0.002

pb***

0.076±0.003

pb***

0.12±0.003

pb***

0.17±0.005

pb**

0.076±0.002

pb***

0.07±0.003

pb***

279.91±1.9

pb***

255.16±2.59

pb***

289.66±1.23

pb***

389.08±1.49

pb***


b compared with fluoride treated group, *** indicates p<0.001, **indicates p<0.01,

* indicates p<0.05, # indicates insignificant difference

Table

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Table 2: Change in acidic, basic, neutral and total protein contents following fluoride exposure with or without resveratrol in cerebrum,

cerebellum, pons and medulla of rat brain

Groups

of

animals

Acidic protein

(mg%)

Basic protein

(mg%) Neutral protein

(mg%)

Total protein

(mg%)

Cerebru

m

Cerebellum Pons Medulla Cerebru

m

Cerebellu

m

Pons Medull

a

Cerebru

m

Cerebellu

m

Pons Medulla Cerebru

m

Cerebellu

m

Pons Medulla

Control 11.28±0.86 11.52±0.61 9.41±0.28 9.28±0.08 4.35±0.75 3.86±0.29 3.30±0.12 2.85±0.1 1.67±0.62 1.19±0.23 1.59±0.3 1.47±0.12 17.17±0.92 16.58±0.68 13.68±0.1 13.54±0.2

6

F-

treated

2.49±0.59

pa***

3.30±0.48

pa***

2.67±0.33

pa***

2.55±0.14

pa***

1.41±0.54

pa***

1.05±0.26

pa***

0.65±0.25

pa***

0.46±0.2

pa***

0.69±0.45

pa**

0.33±0.11

pa***

0.53±0.11

pa***

0.41±0.11

pa***

4.42±0.65

pa***

4.66±0.64

pa***

3.9±0.26

pa***

3.47±0.14

pa***

F+

resverat

rol

4.33±0.64

pb***

3.84±0.53

pb**

3.4±0.39

pb***

2.98±0.23

pb***

1.77±0.79

pb#

1.60±0.36

pb**

1.03±0.20

pb***

0.82±0.3

7 pb***

0.81±0.36

pb#

0.40±0.12

pb#

0.75±0.13

pb*

0.57±0.16

pb**

6.63±0.86

pb***

5.73±0.71

pb***

5.12±0.30

pb***

4.38±0.13

pb***

F+Vit-

C

6.86±0.89

pb***

9.34±0.43

pb***

5.93±0.32

pb***

5.60±0.14

pb***

3.08±0.84

pb***

2.22±0.47

pb***

1.72±0.17

pb***

1.3±0.24

pb***

0.98±0.4

pb#

0.72±0.13

pb***

0.89±0.17

pb**

0.70±0.15

pb***

10.65±1.08

pb***

12.29±0.64

pb***

8.40±0.42

pb***

7.71±0.14

pb***




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Table 3: Effect of resveratrol on fluoride-induced alteration of pronase, trypsin and cathepsin enzyme activities in cerebrum,


Groups of

animals

Pronase Activity

(µmoles of tyrosine/min/mg of protein)

Trypsin Activity


Cathepsin Activity



Control 0.023±0.001 0.024±0.003 0.014±0.001 0.020±0.001 0.029±0.003 0.030±0.001 0.024±0.001 0.023±0.001 0.013±0.001 0.013±0.005 0.013±0.001 0.011±0.001

F-treated 0.019±0.002

pa**

0.021±0.001

pa**

0.009±0.001

pa***

0.01±0.001

pa***

0.018±0.002

pa***

0.015±0.002

pa***

0.011±0.001

pa***

0.009±0.001

pa***

0.011±0.003

pa***

0.007±0.001

pa***

0.006±0.001

pa***

0.005±0.001

pa***

F+ resveratrol

supplemented

0.024±0.003

pb***

0.025±0.004

pb**

0.013±0.002

pb***

0.021±0.001

pb***

0.031±0.003

pb***

0.025±0.002

pb***

0.021±0.001

pb***

0.021±0.002

pb***

0.014±0.004

pb***

0.012±0.002

pb***

0.009±0.002

pb***

0.009±0.001

pb***

F+Vit-C 0.024±0.003

pb***

0.027±0.006

pb**

0.015±0.002

pb***

0.020±0.001

pb***

0.033±0.004

pb***

0.026±0.003

pb***

0.022±0.001

pb***

0.019±0.001

pb***

0.015±0.002

pb***

0.013±0.002

pb***

0.009±0.001

pb***

0.009±0.002

pb***




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Table 4: Effect of resveratrol on fluoride-induced alteration of RNase and transaminase enzyme activities in cerebrum, cerebellum, pons

and medulla of rat brain

Groups of

animals

RNase (µg of RNase/100 mg tissue) GPT (U/mg of tissue) GOT (U/mg of tissue)


Control 7.36±1.54 7.52±1.24 6.78±1.08 6.65±1.42 37.67±0.92 28.33±2.34 48±1.79 44.67±1.03 25.83±1.29 25.83±1.29 41.25±1.37 36.25±2.09


pa***

16.24±1.10

pa***

14.02±1.29

pa***

13.91±1.26

pa***

45.67±1.51

pa***

47±1.09

pa***

59±2.09

pa***

63.67±1.51

pa***

43.33±3.03

pa***

37.92±1.88

pa***

56.25±2.62

pa***

50.83±2.58

pa***

F+ resveratrol

supplemented 9.38±1.44

pb***

9.46±1.16

pb***

8.46±1.19

pb***

8.26±1.31

pb***

38.67±1.37

pb***

38.67±2.48

pb***

48±0.89

pb***

51.17±1.79

pb***

31.67±1.37

pb***

25.42±1.58

pb***

40.75±1.58

pb***

38.42±0.77

pb***

F+Vit-C 7.94±1.10

pb***

8.42±1.32

pb***

8.12±1.15

pb***

7.95±0.95

pb***

38.67±1.03

pb***

38.33±1.97

pb***

50.33±1.97

pb***

52±2.19

pb***

30.42±2.92

pb***

25.83±1.29

pb***

42.92±1.88

pb***

38.33±1.29

pb***




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Table 5: Effect of resveratrol on fluoride-induced changes in free •OH radical formation, protein carbonyl content and NO level in

cerebrum, cerebellum, pons and medulla

Groups of

animals

Free hydroxyl radical

(µmoles/g of tissue )

Protein carbonyl content

(nmoles/mg of protein)

NO level

(µmoles/mg of protein)


Control 12.49±0.56 14.64±0.52 9.75±1.31 8.49±0.87 3.56±0.52 3.12±0.74 2.84±0.68 2.72±0.71 26.65±1.09 20.66±1.68

29.53±1.16 45.21±1.29


pa***

25.11±0.90

pa***

18.62±1.2

pa***

18.14±1.18

pa***

7.54±0.82

pa***

7.18±0.46

pa***

6.50±1.09

pa***

6.42±1.12

pa***

51.37±2.17

pa***

44.65±1.41

pa***

58.40±2.08

pa***

63.40±2.81

pa***

F+

resveratrol-

supplemented

15.33±1.64,

pb***

16.68±1.53

pb***

13.52±1.48

pb**

13.64±1.39

pb***

5.26±0.62

pb***

5.28±0.52

pb***

4.29±0.41

pb***

4.52±0.52

pb***

29.32±1.54

pb***

22.39±1.52

pb***

47.52±2.12

pb***

44.4±1.47

pb***

F+Vit-C 15.74±0.86

pb***

16.07±1.05

pb***

13.6±0.74

pb**

13.48±1.27

pb***

5.71±0.75

pb***

5.63±0.62

pb***

4.32±0.64

pb***

4.64±0.42

pb***

30.21±1.13

pb***

21.74±1.28

pb***

49.73±1.63

pb***

53.33±1.72

pb***




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Table 6: Change in SOD, catalase, GPx, GST and GR activities following fluoride exposure with or without resveratrol in cerebrum,


Control NaF treated NaF treated + resveratrol NaF treated + Vit.C

Cer

ebru

m

Cer

ebel

lum

Po

ns

Med

ull

a

Cer

ebru

m

Cer

ebel

lum

Po

ns

Med

ull

a

Cer

ebru

m

Cer

ebel

lum

Po

ns

Med

ull

a

Cer

ebru

m

Cer

ebel

lum

Po

ns

Med

ull

a

SO

D

(U/m

g o

f

pro

tein

)

3.2

8±

0.1

9

3.2

0±

0.0

8

2.8

7±

0.0

7

2.7

6±

0.0

6

2.6

8±

0.0

9

pa *

**

2.5

9±

0.0

6

pa *

**

1.9

0±

0.0

9

pa *

**

1.7

8±

0.0

6

pa *

*

3.1

8±

0.1

9

pb*

**

3.1

0±

0.1

5

pb*

**

2.4

7±

0.1

1

pb*

**

2.1

1±

0.1

4

Pb#

3.1

5±

0.0

5

pb*

**

3.0

8±

0.0

7

pb*

**

2.4

8±

0.0

7

pb*

**

2.5

2±

0.0

5

pb*

Ca

tala

se

(µm

ole

s o

f

H2O

2

hy

dro

lyse

d

/min

/mg

of

pro

tein

)

12

1.4

9±

0.8

5

12

0.3

6±

0.6

8

11

2.2

4±

0.6

4

11

1.5

8±

0.9

1

75

.44

±0

.61

pa *

**

73

.37

±1

.07

pa *

**

63

.69

±0

.8

pa *

**

62

.08

±0

.95

pa *

**

11

2.0

6±

1.6

0

pb*

**

11

2.6

4±

1.5

2

pb*

**

10

3.0

8±

0.8

9

pb*

**

10

2.5

7±

1.0

5

pb*

**

11

4.8

9±

0.8

8

pb*

**

11

3.2

5±

0.8

7

pb*

**

10

5.3

±1

.15

pb*

**

10

4.9

2±

1.0

8

pb*

**

GP

x

(µm

ole

s o

f

NA

DP

H

ox

idiz

ed

/m

in/m

g

pro

tein

)

11

0.5

6±

0.7

8

10

5.4

6±

0.8

6

97

.85

±1

.42

96

.29

±0

.51

65

.86

±0

.84

pa *

**

71

.59

±0

.95

pa *

**

53

.46

±1

.03

pa *

**

51

.52

±0

.88

pa *

**

93

.14

±1

.75

pb*

**

92

.62

±1

.84

pb*

**

73

.18

±1

.17

pb*

**

71

.32

±1

.55

pb*

**

95

.41

±0

.81

pb*

**

93

.61

±0

.9

pb*

**

74

.96

±1

.12

pb*

**

72

.68

±0

.85

pb*

**

GS

T

(nm

ole

s

GS

H-

CD

NB

con

jug

ate

form

ed/m

i

n/m

g

pro

tein

)

45

.53

±0

.71

42

.12

±0

.43

38

.76

±0

.73

37

.1±

0.6

8

34

.91

±0

.79

pa *

**

34

.51

±1

.17

pa *

**

31

.15

±0

.89

pa *

**

30

.82

±0

.54

pa *

**

42

.13

±0

.90

pb*

**

39

.12

±0

.68

pb*

**

35

.12

±0

.89

pb*

**

35

.29

±1

.16

pb*

**

43

.07

±0

.44

pb*

**

39

.9±

0.7

7

pb*

**

36

.74

±0

.66

pb*

**

36

.19

±0

.5

pb*

**

GR

(nm

ole

s o

f

NA

DP

H

ox

idiz

ed

/m

in/m

g o

f

pro

tein

)

97

.35

±1

.18

94

.19

±1

.33

82

.34

±0

.67

80

.88

±1

.21

41

.11

±1

.35

pa *

**

39

.14

±1

.06

pa *

**

48

.46

±0

.71

pa *

**

51

.48

±0

.51

pa *

**

77

.89

±1

.87

pb*

**

76

.43

±3

.22

pb*

**

70

.50

±1

.41

pb*

**

68

.58

±2

.38

pb*

**

81

.52

±1

.26

pb*

**

80

.04

±1

.27

pb*

**

71

.36

±0

.58

pb*

**

70

.16

±0

.69

pb*

**




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ipt

Table 7: Result of alkaline comet assay parameters (tail length, tail intensity and tail movement) in brain cells of rat after fluoride

exposure with or without resveratrol with the help of CASP (Comet Assay Software Package)

Control NaF treated NaF treated + resveratrol NaF treated + Vit.C

Tail length (µm) 10.26±1.54 22.41±2.06 16.42±2.74 15.22±3.02

Tail intensity (µm) 1.04±0.42 1.99±0.26 1.52±0.48 1.43±0.34

Tail moment 0.13±0.004 0.25±0.005 0.19±0.004 0.18±0.006

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ipt

Table 8: Change in norepinephrine, dopamine, and serotonin contents of fluoride-exposed rat brain with or without resveratrol

supplementation in cerebrum, cerebellum, pons and medulla

Groups of

animals

Norepinephrine

(ng/g of tissue )

Dopamine

(ng/g of tissue)

Serotonin

(ng/g of tissue)


Control 412.39±2.66 648.12±2.93 532.54±3.12 540.4±23.24 890.32±2.03 951.48±2.58 934.64±2.44 926.14±4.21 1846.08±3.9 1586.08±5.2 1524.56±2.82 1516.25±3.42

F-treated 358.42±1.24

pa***

410.28±1.18

pa***

424.65±2.46

pa***

420±2.36

pa***

661.97±1.83

pa***

628.79±1.05

pa***

658.16±1.69

pa***

642.36±3.78

pa***

1364.08±3.95

pa***

1108.26±4.96

pa***

1097.82±2.38

pa***

1082.42±4.56

pa***

F+

resveratrol-

supplemented

398.12±1.56

pb***

510.24±3.24

pb***

424.36±2.58

pb***

512.38±2.94

pb***

794.24±3.62

pb***

884.26±2.63

pb***

862.18±2.41

pb***

872.42±2.63

pb***

1784.29±2.45

pb***

1442.38±2.85

pb***

1427.91±4.36

pb***

1426.72±2.94

pb***

F+Vit-C 402.34±2.78

pb***

522.62±4.18

pb***

432.45±1.72

pb***

516.42±3.36

pb***

806.44±2.96

pb***

896.32±3.52

pb***

880.42±3.84

pb***

891.33±2.82

pb***

1792.36±3.34

pb***

1469.46±3.39

pb***

1448.52±3.74

pb***

1451.61±3.36

pb***




Protective effect of resveratrol on fluoride induced alteration in protein and nucleic acid...

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Transcript of Protective effect of resveratrol on fluoride induced alteration in protein and nucleic acid...