protective effect of resveratrol on fluoride induced alteration in protein and nucleic acid...
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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
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
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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
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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
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
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***
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
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***
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
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Table 3: Effect of resveratrol on fluoride-induced alteration of pronase, trypsin and cathepsin enzyme activities in cerebrum,
cerebellum, pons and medulla of rat brain
Groups of
animals
Pronase Activity
(µmoles of tyrosine/min/mg of protein)
Trypsin Activity
(µmoles of tyrosine/min/mg of protein)
Cathepsin Activity
(µmoles of tyrosine/min/mg of protein)
Cerebrum Cerebellum Pons Medulla Cerebrum Cerebellum Pons Medulla Cerebrum Cerebellum Pons Medulla
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***
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
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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)
Cerebrum Cerebellum Pons Medulla Cerebrum Cerebellum Pons Medulla Cerebrum Cerebellum Pons Medulla
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
F-treated 15.65±1.97
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***
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
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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)
Cerebrum Cerebellum Pons Medulla Cerebrum Cerebellum Pons Medulla Cerebrum Cerebellum Pons Medulla
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
F-treated 22.37±1.13
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***
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
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Table 6: Change in SOD, catalase, GPx, GST and GR activities following fluoride exposure with or without resveratrol in cerebrum,
cerebellum, pons and medulla of rat brain
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*
**
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
Page 37 of 38
Accep
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Man
uscr
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
Page 38 of 38
Accep
ted
Man
uscr
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)
Cerebrum Cerebellum Pons Medulla Cerebrum Cerebellum Pons Medulla Cerebrum Cerebellum Pons Medulla
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***
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