QUERCETIN AMELIORATES OXIDATIVE STRESS, NEURAL DAMAGE OF BRAIN AND BEHAVIORAL IMPAIRMENT OF RAT WITH FLUORIDE EXPOSURE

QUERCETIN AMELIORATES OXIDATIVE STRESS, NEURAL DAMAGE OF BRAIN AND BEHAVIORAL IMPAIRMENT OF RAT WITH FLUORIDE EXPOSURE

Mesram Nageshwar, Kurmeti Sudhakar and Karnati Pratap Reddy ^*

Department of Zoology, Osmania University, Hyderabad - 500007, Telangana, India.

ABSTRACT: Excess fluoride causing fluorosis, can enter in to brain, disturb oxidative status and induce neural damage. Quercetin, antioxidant flavonoid stabilizes oxidative milieu of brain by quenching free radicals. The present study aimed to report ameliorative effect of quercetin on NaF induced oxidative stress, histological alterations in the brain and behavioural changes of rats. Rats were divided into four groups. The first group served as normal which received tap water. The second group was intoxicated with sodium fluoride (20 ppm/kg bw) through drinking water. The third group was given 20 ppm sodium fluoride in drinking water, along with treatment of quercetin (20 mg/kg bw) through oral gavage. The fourth group was treated with quercetin (20 mg/kg bw) through oral gavage. All animals were maintained for 60 days. Then, the antioxidant enzyme levels were measured, histopathological and behavioural studies were carried out. The results showed that the quercetin treatment significantly reversed the NaF induced increased lipid peroxidation (LPO) and decreased superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels in the brain homogenate. Quercetin also reversed the NaF induced Maze learning impairment, hot plate latency and histological alterations in cerebral cortex, hippocampal CA1 and CA2 regions in the brain. The study showed that the concomitant administration of quercetin protects rat brain from sodium fluoride induced oxidative stress, neural damage of brain and behavioural alterations.

Behaviour, Cerebral cortex, Hippocampus, Oxidative stress, Quercetin

INTRODUCTION: Flavonoids are polyphenol compounds present in plants and vegetables. Pharmacological studies show flavonoids play vital role in human health and have antioxidant properties including cardiovascular protection, cataract prevention and hepatoprotective as well as anticancer activities ¹. Quercetin is a flavonoid present in plants including vegetables, fruits, red wine, barcoli, apples and onions ^{2 - 3}.

Quercetin is mainly responsible for the antioxidant activity of edible plants, which may protect against oxidative stress diseases such as diabetes, cancer and neurodegenerative disorders ⁴. Nabavi et al., ⁵ reported that quercetin significantly attenuates oxidative damage induced by sodium fluoride by significantly restoring SOD and GSH levels and reducing LPO.

Several studies have reported quercetin is capable of crossing the blood brain barrier and reaching the central nervous system, essential for the treatment of neurodegenerative diseases ⁶. Quercetin not only acts as an antioxidant with ability of direct hydrogen-donating properties to quench reactive oxygen species but also may exert modulatory actions on the endogenous antioxidative defense system by interactions with intracellular signaling cascades ⁷. The anti-carcinogenic activity of quercetin inhibit cancer cell enhancement and modify the cellular signaling pathway in carcinogenesis and ameliorated the histological alteration in kidney and hepatic tissue ⁸. Jiang et al., ⁹ stated quercetin treatment reduced histopathological damage in spinal cord injury.

Fluorosis is a one of the most complicated neurodegenerative diseases and it is a major problem in several countries in the world. Chronic administration of sodium fluoride induced neurodegeneration in central nervous system ^{10 - 12}. Fluoride has capability to enter blood brain barrier and alter activity of free radical scavenging enzymes such as super oxide dismutase, lipid peroxidation and glutathione ¹³. Free radicals play major role to damage biological systems in singlet oxygen species. Neural system is highly vulnerable to fluoride and it is observed that the fluoride accumulates in brain before skeletal system. Fluorosis alters the neuronal and cerebrovascular integrity resulting in cognitive and behavioral deficits in both humans and animals ¹⁴. Several reports demonstrated neurodegeneration leads to cognitive impairment, motor coordination dysfunction, beta amyloid plaques formation and pathological abnormalities ^{15 - 16}. IQ scores of children living in endemic fluorosis area are less compared to children living in non-endemic area ^17-18. The severity of the adverse effects of sodium fluoride on the behaviour is directly correlated with the concentration of Fluoride ion in particular regions of the brain and in the plasma.

Furthermore, disturbances in brain development, longer latency of the pain reaction and conditioned reflex observed in rats at high concentration of Fluoride treatment ¹⁹. The histological reports in rat exposed to fluoride seemed thickening and loss of dendrites, swelling of mitochondria, dilation of the endoplasmic reticulum in neurons and impaired hippocampus synaptic interface structure ²⁰. Guan et al., ²¹ reported histopathological changes such as loss of purkinje cells, oligodendrocytes, demyelination in dendrites, mitochondria dysfunction, decreased Nissl granules in neurons of fluoride exposed animals. Recently we have reported that quercetin reversed monoamines epinephrine, nor-epinephrine and acetylcholine levels in developing rat brain exposed to pre and post natal rats with NaF ²². This study reports the neuroprotective effect of quercetin against sodium fluoride-induced oxidative stress, neuronal damage in the brain and behavioural changes in rat.

MATERIALS AND METHODS:

Chemicals: Quercetin, sodium fluoride, hematoxylin-eosin stain and congo red stain were purchased from sigma chemicals. All others chemicals were purchased from local firms for biochemical analysis.

Animals: The study was carried out on albino wistar rats (weighted about 250 - 300 g). Animals were housed in conventional condition at a temperature 24 ± 2 ºC with a relative humidity of 60 ± 5% and a 12-h / 12-h light / dark cycle and maintained as per ethical committee guidelines. Animals were allowed in animal house one week before starting the experiment. The rats were divided randomly to four experimental groups of 10 animals each. The experimental period was 60 days. Group-I Control: these rats received only tap water. Group-II sodium fluoride: these rats received 20 ppm sodium fluoride through drinking water. Group-III Sodium fluoride + Quercetin: these rats _{­­­­­­­­}received 20 ppm sodium fluoride through drinking water along with  quercetin (20 mg/kg bw) through orally with gavage. Group-IV Quercetin: these rats received quercetin (20 mg/kg bw) through orally with gavage. The brain was dissected and stored at -20 ºC for biochemical analysis, 10% formaldehyde used to store brain for histopathalogicals studies.

Methods:

Biochemical analysis:

Lipid peroxidation (LPO): Lipid peroxidation in brain tissue was measured by modified method of Garcia et al., ²³. 1 ml of 10% homogenate was added to 1 mL of 20% TCA and heated at 70 °C for 10 min, and cooled at room temperature and centrifuged at 3000 rpm for 10 min. 400 µL of supernatant was mixed with 200 µL of 0.5% TBA reagent in a test tube covered with a glass marble and heated in a boiling water bath for 10 min and the tubes cooled to room temperature. The absorbance of the pink coloured trimethine condensation product was measured at 533 nm using a spectrophotometer. The results were expressed as nano mole MDA/gm weight of tissue.

Superoxide Dismutase (SOD): Superoxide dismutase activity in brain tissue was estimated by modified protocol of Marklund and Marklund ²⁴. The assay system in a final volume of 1.0 ml consisting 600 µl of 83.3 mM Tris-HCl buffer, (pH 8.2), 100 µl of 0.5 mM DETPA, 50 µl of enzyme, 50 µl of Tris-EDTA, 50 µl of 0.01 N HCl, 100 µl of H₂O₂ was mixed well and the reaction was initiated by adding the 50 µl of 3.97 mM Pyrogallol. Increased absorbance was read at 420 nm using a spectrophotometer. The enzyme activity was expressed as Units/mg protein.

Catalase (CAT): Catalase activity was assayed following method of Aebi ²⁵. A 10% tissue homogenate was prepared in 0.1M phosphate buffer (pH 7.4). Reaction mixture containing 200 µl phosphate buffer (0.1M, pH 7.4) and 50 µl of tissue extract, 250 µl of 0.006M H₂O₂ was added and decreased in optical density was measured at 240 nm. One unit of activity is equal to the µ moles of degraded H₂O₂ /min/mg/ protein.

Glutathione (GSH): GSH was determined by the using method of Ellman ²⁶. 1.0 ml of supernatant was treated with 0.5 ml of Ellman reagent and 0.3 ml of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm. The enzyme activity was expressed as µg/mg protein.

Behavioural Studies:

Maze Learning Test: A maze is a puzzle in the form of a complex branching passage through which the starved animals try to find the food/goal. Maze test is the process of learning a route typically by rats through a maze in order to obtain reinforcement. This process is a popular experiment in behavioural laboratory and it is the main method of studying spatial learning. The rats were starved for 12 h before the maze learning experimentation. All experimental groups were trained two days before the experimentation, the animals were tested and the results were analyzed time in minutes ²⁷.

Hot Plate Test: Hot plate test was performed according to the method ²⁸. Rats were placed on the hot plate the temperature of floor consisting of a 52.5 ± 0.15 °C (Analgesiometer - Eddy's Hot Plat). The response latency to either a hind-paw lick or a jump was recorded. In the absence of a response, animals were removed from the hot plate at 60 sec (cut-off time) and 60 sec latency was assigned as the passive response.

Histopathology: Brain sections were fixed in freshly prepared 10% formalin, processed routinely, and embedded in paraffin. Paraffin sections 5μm thick were prepared and stained with hematoxylin and eosin ²⁹ and Congo red ³⁰ for histopathological examination. Sections were examined using a light microscope (Magnification 40X).

Statistical Analyses: The results are expressed as the mean ± standard error of the mean (SEM). Comparison of means were conducted using one way analysis of variance followed by least significant difference post hoc test to compare means between the different groups. Differences were considered as significant (P <0.05). Statistical analyses were performed using SPSS version 20 software.

RESULTS:

Oxidative Stress Markers:

Effect on LPO Content, SOD, CAT and GSH Levels: As shown in Graph 1, brain LPO (Graph 1a) content was significantly increased (P < 0.05) in terms of MDA in NaF treated animals as compared with control group. It is significantly reduced (P < 0.05) in the NaF + Quercetin and Quercetin treated groups. The NaF treated group showed a significantly reduced (P < 0.05) levels of SOD (Graph 1b), CAT (Graph 1c) and GSH (Graph 1d) as compared with the control group, whereas their levels were significantly increased (P < 0.05) in NaF + Quercetin and Quercetin treated groups.

Behavioural Studies:

Maze Learning Test and Hotplate Test: As shown in Graph 2, spatial memory (Graph 2a) was assessed in terms of transfer latency on elevated plus maze. In the maze learning experiment the goal achieving time significantly (P < 0.05) increased was indicated in NaF treated group compared to that in the control group. Whereas NaF + Quercetin and Quercetin treatments significantly (P < 0.05) reduced the goal achieving time compared to NaF intoxicated group. The paw withdrawal in hot plate test (Graph 2b) latency period significantly (P<0.05) decreased in NaF exposure rats compared to control rats, and NaF + Quercetin and Quercetin treated rats showed significantly (P<0.05) increased the paw withdrawal latency period compared to NaF treated rats.

GRAPH 1: EFFECT OF QUERCETIN TREATMENT ON LPO (A) CONTENT, SOD (B), CAT (C) ACTIVITY AND GSH (D)LEVELS IN RATS SUBJECTED TO FLUORIDE TREATMENT FOR 60 DAYS. ^*P < 0.05 AS COMPARED TO CONTROL GROUP AND ^#P < 0.05 AS COMPARED TO FLUORIDE TREATED GROUP. DATA EXPRESSED AS THE MEAN ± S.E.M (n = 6) AND RESULTS EXPRESSED IN NANO mole mda/gm WEIGHT OF TISSUE (LPO), UNITS/MG PROTEIN (SOD), µ moles /min/mg PROTEIN (CAT) AND µg/mg PROTEIN (GSH)

GRAPH 2: EFFECT OF QUERCETIN TREATMENT ON COGNITIVE IMPAIRMENT (MAZE LEARNING TEST) AND LATENCY PERIOD (HOTPLATE TEST) IN RATS SUBJECTED TO FLUORIDE TREATMENT FOR 60 DAYS. ^*P < 0.05 AS COMPARED TO CONTROL GROUP AND ^#P < 0.05 AS COMPARED TO FLUORIDE TREATED GROUP. DATA EXPRESSED AS THE MEAN ± S.E.M (n = 6) AND RESULTS SHOWN IN TIME IN MINUTE AND SECONDS

Histopathology: Hematoxylin-eosin stain of cerebral cortex and hippocampus (CA1, CA2) regions of rat brain treated with sodium fluoride and quercetin are shown in Fig. 1 and 2. The shape of the cell, size of the cell, number of neurons and nuclear membrane were markedly damaged in the cerebral cortex and CA1, CA2 hippocampus regions in NaF treated rat brain.  This damage was markedly reversed up to normal in the NaF + Quercetin and Quercetin treated groups.

FIG. 1: MORPHOLOGICAL CHANGES IN THE CEREBRAL CORTEX REGION IN BRAIN (40X) (HEMATOXYLIN-EOSIN STAINING). THE CEREBRAL CORTEX IN THE CONTROL, SODIUM FLUORIDE, NaF + QUERCETIN AND QUERCETIN GROUPS, RESPECTIVELY. BLACK COLORED ARROW MARK SHOWING THE NORMAL CELLS WITH REGULAR SHAPE AND SIZE IN CONTROL, NaF + QUERCETIN AND QUERCETIN GROUPS AND YELLOW COLORED ARROW MARK SHOWING THE CELLS WITH IRREGULAR SHAPE AND NUCLEAR MEMBRANE WAS INDISTINCT IN THE NaF TREATED GROUP

FIG. 2: MORPHOLOGICAL CHANGES IN THE HIPPOCAMPAL CA1, CA2 REGION IN BRAIN (40X) (HEMATOXYLINE-EOSIN STAINING). THE HIPPOCAMPUS IN THE CONTROL, SODIUM FLUORIDE, NaF + QUERCETIN AND QUERCETIN GROUPS, RESPECTIVELY. BLACK COLORED ARROW MARK SHOWING THE NORMAL CELLS WITH REGULAR SHAPE AND SIZE IN CONTROL, NaF + QUERCETIN AND QUERCETIN GROUPS AND YELLOW COLORED ARROW MARK SHOWING THE CELLS WITH IRREGULAR SHAPE AND NUCLEAR MEMBRANE WAS INDISTINCT IN THE NaF TREATED GROUP

The histological sections with congo red stain of cerebral cortex and hippocampal CA1, CA2 regions of rat brain treated with sodium fluoride and quercetin (Magnification 40X) are shown in Fig. 3 and 4. In congo red stain, amyloid plaque formation and irregular shape of neurons were observed in NaF intoxicated rat cerebral cortex and CA1, CA2 hippocampus regions of the brain. The NaF + Quercetin and Quercetin treated groups were detected with decreased amyloid plaque formation and neuron regular shapes.

FIG. 3: BRAIN HISTOPATHOLOGICAL STUDIES IN CEREBRAL CORTEX BY CONGO RED STAIN IN CONTROL, NaF, NaF + QUERCETIN, QUERCETIN ALONE TREATED GROUPS. (MAGNIFICATION 40X). BLACK COLORED ARROW MARK SHOWING THE NEURONS ARRANGED CLOSELY, CIRCULAR SHAPE AND NUCLEAR MEMBRANE WAS CLEAR IN CONTROL, NaF + QUERCETIN AND QUERCETIN GROUPS AND YELLOW COLORED ARROW MARK SHOWING THE AMYLOID PROTEIN PLAQUES FORMATION, CELLS WITH IRREGULAR SHAPE AND NUCLEAR MEMBRANE WAS INDISTINCT IN THE NaF TREATED GROUP

FIG. 4: BRAIN HISTOPATHOLOGICAL STUDIES IN HIPPOCAMPAL CA1 AND CA2 REGION BY CONGO RED STAIN IN CONTROL, NaF, NaF + QUERCETIN AND QUERCETIN ALONE TREATED GROUPS. (MAGNIFICATION 40X). BLACK COLORED ARROW MARK SHOWING THE NEURONS ARRANGED CLOSELY, CIRCULAR SHAPE AND NUCLEAR MEMBRANE WAS CLEAR IN THE HIPPOCAMPAL CA1 AND CA2 REGIONS OF CONTROL, NaF + QUERCETIN AND QUERCETIN GROUPS AND YELLOW COLORED ARROW MARK SHOWING THE AMYLOID PROTEIN PLAQUES FORMATION, CELLS WITH IRREGULAR SHAPE AND NUCLEAR MEMBRANE WAS INDISTINCT IN THE NaF TREATED GROUP

DISCUSSION: Sodium fluoride induced neurodegeneration is associated with the oxidative stress, histopathological and behavioural alterations in brain which is seen to be well ameliorated with the pretreatment with quercetin. This study provide evidences for the neuroprotective effect of the most abundant polyphenolic compound quercetin on sodium fluoride induced neurodegeneration in rat. It is observed that quercetin prevents sodium fluoride induced oxidative stress, behavioral and histopathological alteration in rat brain. Oxidative stress occurs when there is an imbalance between the reactive oxygen species production and the antioxidant defense systems and the production of ROS overcomes the antioxidant capacity of the cell. The central nervous system is vulnerable to oxidative stress. The brain utilizes great amounts of oxygen, consequently promoting the formation of oxygen free radicals and reactive oxygen species. Lipid peroxidation is considered as the main molecular mechanisms involved in the oxidative damage to cell structures and in the toxicity process that lead to cell death ³¹. Lipid peroxidation was the consequence of toxic metabolites that produced highly reactive species, disruption of the intracellular membranes and cellular damage and it leads to neurodegeneration ².

Some studies demonstrated significantly increased lipid peroxidation products MDA and 4-hydroxynonenal in the soft tissues intoxicated with sodium fluoride ³². Superoxide dismutase is a master eukaryotic regulator of oxygen radicals, with cancer, cell biology and brain pathology ³³. The antioxidant enzymes SOD and CAT play an important role in the enzymatic mechanisms. Superoxide dismutase converts the highly effective superoxide radicals into H₂O₂ (hydrogen peroxide). In the oxidative stress, catalase is responsible for the breakdown of hydrogen peroxide.

Therefore, the effects of the SOD and CAT are complementary to each other ³⁴. Previous studies demonstrated superoxide dismutase activity decrease in sodium fluoride intoxicated rats liver and erythrocytes ^{2, 5}. Glutathione is a tripeptide an essential antioxidant, which is responsible for detoxification of hydroxyl and superoxide free radicals in the brain. in our lab also reported that the lipid peroxidation lipid peroxidation, superoxide dismutase, catalase and glutathione levels were altered in sodium fluoride intoxicated rats ³⁵. In the oxidative stress process glutathione is oxidized to make disulphide link to form oxidized glutathione. Catalase and Glutathione levels were depleted in sodium fluoride intoxicated rat liver, brain and kidney ^{13, 32}.

In the present study, we observed elevation in lipid peroxidation levels in the sodium fluoride intoxicated rat brain which is followed by a significantly decline superoxide dismutase, catalase and glutathione levels. The levels of brain lipid peroxidation were found to be restored in the rat treated with NaF + Quercetin and superoxide dismutase, catalase and glutathione levels were also restored. Quercetin provide to be significantly better in restoring the altered activity of antioxidant enzymes, superoxide dismutase, catalase, glutathione and  lipid peroxidation towards their normal values in brain. This role of quercetin especially decreasing oxidative stress in brain by directly scavenging reactive oxygen species is further correlated with present results.

Another central effect that we highlighted was reversal of spatial learning ability of quercetin against sodium fluoride induced cognitive impairment. Our results have shown that sodium fluoride exposure caused an increase in goal reach time, as observed in the elevated puzzle maze task. The learning process is significantly restored in Quercetin treated groups. Normal learning ability was found in control and only quercetin treated rats. In fact, several studies have shown that fluoride exposure decreases cognitive process ³⁶. High fluoride water supply affects the children’s IQ as the fluoride affects structural damage to the central nervous system to such extent of functional impairment. Several studies indicated a lowering of IQ is associated with fluoride exposure ³⁷. The mean IQ level of students exposed to high F drinking water was significantly lower than that of the students exposed to a lower F level drinking water ³⁸. The present results indicated quercetin improves learning process of rats.

The hot plate evaluates central pain attenuation in a rodent using thermal stimulus known to be mediated through central processing pathways. The nociceptive pain in mice is triggered through the release of inflammatory mediators such sympatho-mimetic amines, prostaglandins, and several cytokines, including IL-1, TNF- and IL-8 derived, from peritoneal macrophages and mast cells ³⁹. The paw (plantar surface) withdrawal response on hot plate with constant temperature, thermal stimulus was measured as nociceptive response in quercetin and sodium fluoride treated rats. Present results were showed significantly decreased in thermal sensitivity response in sodium fluoride intoxicated rat where as the latency period reversed in quercetin treated groups and these results were similar to Gunn et al., ²⁸, Sudhakar et al., ^35.

In the present study, we observed marked morphological changes and β-amyloid plaques formation in the neurons of cerebral cortex and hippocampal CA1 and CA2 region of rat brain treated with Quercetin - NaF. The neurons had irregular shapes, shrunken and the nuclear membrane. The neurons were arranged closely, had a circular shape, nuclear membrane was clear in the control, NaF + Quercetin and Quercetin groups. The hippocampus and the cerebral cortex are the key structures of memory formation. Previous studies demonstrated that shrinkage in the pyramidal neurons, neuronal degeneration, and change in cell morphology have been reported in model of Alzheimer’s disease ^{40 - 41}.

Further investigation find β-amyloid plaques formation in the CA1 region ⁴². Morphological analysis showed that the structure of neurons and mitochondria changed predominantly in the hippocampal CA3 region and cortex of AD model rats ¹⁶. The fluoro-aluminium compounds produce neuronal injury in the CA1 and CA4 parts of the hippocampus of rat ²⁰. The intracellular space, neutrophil infiltration and neuronal necrosis was observed in the hippocampus region in aluminum fluoride treated animals ⁴³.

Guan et al., ²¹ reported pathological changes including decreased in the number of purkinje cells, demyelination, thickening and disappearance of dendrites in the brain of fluoride exposed rats. Quercetin administration reduces histopathological congestion, edema, neutrophil infiltration and structural disruption in spinal cord injury ⁹. Chan-Min et al., ⁷ studied the morphological alterations of liver including leukocyte infiltration and extensive hepatocellular necrosis ameliorated by quercetin treated animals. According to Kandaswamy et al., ⁴⁴ normal polygonal cells around the portal canal and sinusoid showed a layer of fenestrated endothelial cells. Kupffer cells are normal and few macrophages in the periportal zones and no apoptosis seen in liver treated with quercetin. Thus, these results indicate that quercetin could be an effective approach for attenuating the neurotoxicity via modulation of histological alteration induced by NaF.

CONCLUSION: NaF induced oxidative stress, behavioral and histological alterations in rat brain were significantly attenuated with quercetin treatment. Fluoride increases the production of free radicals which acts through oxidation of membrane lipids. Due to the destruction of membrane, neural cells get damage in terms of number, size and shape, thus leads to neurodegeneration which ultimately alter the behavior of rats. Quercetin prevents the excess generation of free radicals or eliminates the excess free radicals. Hence, it maintains normal neural cell membrane and oxidative status. Thus it can protect from fluoride induced neurodegeneration.

ACKNOWLEDGEMENT: Authors acknowledge the partial funding under UGC-BSR award (19-118/2014) and DSA-SAP-II and DST-FIST of Department of Zoology, OU.

CONFLICT OF INTEREST: We declare that we have no conflict of interest.

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    How to cite this article:

    Nageshwar M, Sudhakar K and Reddy KP: Quercetin ameliorates oxidative stress, neural damage of brain and behavioral impairment of rat with fluoride exposure. Int J Pharm Sci Res 2018; 9(8): 3247-56. doi: 10.13040/IJPSR.0975-8232.9(8).3247-56.

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