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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 70  |  Page : 320-326  

Ginkgo biloba ameliorates fluoride toxicity in rats by altering histopathology, serum enzymes of heme metabolism and oxidative stress without affecting brain mGluR5 gene


1 Department of Anatomy, Fathima Institute of Medical Sciences, Kadapa, Andhra Pradesh; Department of Research and Development, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, India
2 Department of Research and Development, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, India
3 Department of Anatomy, Viswabharathi Medical College and General Hospital, Kurnool, Andhra Pradesh, India

Date of Submission18-Dec-2019
Date of Decision05-Feb-2020
Date of Acceptance17-Mar-2020
Date of Web Publication28-Aug-2020

Correspondence Address:
Senthilkumar Sivanesan
Department of Research and Development, Saveetha Institute of Medical and Technical Sciences, Chennai - 602 105, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_534_19

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   Abstract 


Background: To evaluate the therapeutic potential of Ginkgo biloba extract (GBE) in experimental model of fluorosis. Objectives: To study the protective effect of GBE in fluoride toxicity by assessment of oxidative stress, serum biochemical parameters, acetylcholinesterase (AChE) activity, histopathology and brain mGluR5 gene expression. Materials and Methods: Fifteen adult male Wistar rats were randomly assigned to 5 groups (n = 3 rats in each group). Group 1 (control) received water, Groups 2–5 were treated with 100 ppm of sodium fluoride for 30 days, while the Groups 3, 4 and 5 were GBE treated with 50 mg/kg, 100 mg/kg and 200 mg/kg body weight for 15 days, after sodium fluoride treatment for 30 days. Results: Elevated serum delta aminolevulinic acid dehydratase and delta aminolevulinic acid synthatase levels in fluoride intoxicated rats were ameliorated by various doses of GBE treatment. Elevated serum glutathione and decreased oxidized gluatathione levels observed in fluoride intoxicated rats were also ameliorated by GBE treatment but effectively at 100 mg/kg dose. Reduced AChE activity of hippocampus in fluoride-induced toxicity was reverted by 50 mg/kg of GBE whereas other doses (100 and 200 mg/kg) caused significant inhibition of AChE activity in comparison with fluoride group. Fluoride group rats showed significant reduction of mGluR5 gene expression levels whereas in all GBE treatment groups those changes Were not significantly reverted. GBE treatment to fluoride intoxicated rats almost reverted the degenerative changes in liver and kidney caused by fluorosis. Conclusion: The present study concluded beneficial effects of GBE in experimental model of fluorosis.

Keywords: Acetylcholineesterase activity, Fluoride, Ginkgo biloba extract, glutathione, heme metabolism enzymes,reduced glutathione


How to cite this article:
Raju S, Sivanesan S, Gudemalla K. Ginkgo biloba ameliorates fluoride toxicity in rats by altering histopathology, serum enzymes of heme metabolism and oxidative stress without affecting brain mGluR5 gene. Phcog Mag 2020;16:320-6

How to cite this URL:
Raju S, Sivanesan S, Gudemalla K. Ginkgo biloba ameliorates fluoride toxicity in rats by altering histopathology, serum enzymes of heme metabolism and oxidative stress without affecting brain mGluR5 gene. Phcog Mag [serial online] 2020 [cited 2020 Sep 26];16:320-6. Available from: http://www.phcog.com/text.asp?2020/16/70/320/293781



SUMMARY

  • To summarize the findings, the current investigation provides evidence on the beneficial effects of Ginkgo biloba extract in experimental model of fluorosis through assessment of serum biochemical, hippocampal neurotransmitter activity, and histopathological studies.




Abbreviations used: AChE: Acetylcholinesterase; AD: Alzheimer's disease; ALA: Aminolevulinic acid; DALD: Delta aminolevulinic acid dehydratase; DALS: Delta aminolevulinic acid synthetase; GBE: Ginkgo biloba extract; GSH: Glutathione; GSSG: Oxidized glutathione; mGluR5: Metabotropic glutamate receptor5; MDA: Malondialdehyde; NAF: Sodium fluoride; ROS: Reactive oxygen species; RT-PCR: Reverse transcription polymerase chain reaction.


   Introduction Top


Prolonged intake of fluoride leads to fluorosis. Fluoride consumption in optimal amounts through drinking water can protect against the development of dental caries.[1] According to World Health Organization, 1.5 part per million of fluoride in drinking water is safe. The principal source of fluoride to the human body is drinking water.[2] Dental fluorosis causes mottling of enamel of the teeth, whereas skeletal fluorosis is characterized by swollen, deformed joints and enlarged bones. Chronic fluorosis can lead to neurodegenerative related problems such as Alzheimer's dementia with learning and memory impairments.[3],[4],[5] Chronic fluoride toxicity decreased the number of nicotinic acetylcholine receptors in rat brain[6] and also affected the expression of M1 and M3 muscarinic acetylcholine receptors.[4]

Reactive oxygen species (ROS) plays a significant role in the pathogenesis of chronic fluoride toxicity that increases oxidative stress in tissues.[7] Delta aminolevulinic acid dehydratase (DALD) is an enzyme that plays important role in hematopoiesis.[8] DALD, a rate limiting enzyme in heme biosynthesis has been found altered in people living in fluoride affected areas implicating a link between heme metabolism and fluorosis.[9] A recent study finding connected a link between iron overload and fluoride-induced hepatic oxidative stress.[10]

Fluoride alters the serum enzyme antioxidant biochemical markers like glutathione (GSH), oxidized glutathione (GSSG) levels. GSH is well known to protect the cellular system against harmful effects of lipid peroxidation.[11],[12] Elevated GSH levels, increased lipid peroxidation and altered antioxidant system were found in rats that received 100 ppm fluoride in drinking water for 4 months.[13] The hepatic content of GSH is increased with aging when rats were treated with fluoride.[14] Acetylcholinesterase (AChE) activity is essential to maintain normal brain physiological functions and necessary in the processing of learning and memory. Fluoride crosses the blood-brain barrier and accumulates in rat hippocampus that causes inhibition of the activity of cholinesterase. Fluoride penetrates the blood brain barrier, interacts with AChE located on cell membranes and hamper with their physiological functions and thus induce neurotoxicity.[15] mGluR5 is the subtype receptor of group I metabotropic glutamate receptor and plays a vital role in modulation of synaptic plasticity. The function of mGluRs in synaptic plasticity and synaptic transmission are responsible for the source of learning and memory, characterized in the hippocampus.[16] Evidence support reduced mGluR5 gene and protein expression in the hippocampus and cortex of fluoride intoxicated rats.[17] However, no significant changes in hippocampal mGluR5 gene expression levels were seen in mouse pups following maternal exposure of fluoride during gestation and lactation period which is contrasting.[18] Fluoride toxicity associated pathological alterations in the glomeruli and the proximal and distal collecting tubules of nephrons were reported earlier.[19]

Toxic effects of fluoride can be ameliorated by supplementation of phytochemical agents with potent antioxidant activity.[10],[20],[21]Ginkgobiloba belongs to ginkgoacece family and its leaf extract is composed of flavone glycosides, 24% (quercetin, kaempferol, isorhamnetin) and terpene lactones, 6% (ginkgolides and bilobalide).[22] Treatment with GBE reduced intracellular ROS accumulation, apoptosis and mitochondrial dysfunction and increased the cell viability.[23] In our previous published reports, we have shown the therapeutic efficacy of GBE in rat model of fluorosis by evaluating various serum biochemical markers, hematological parameters and neurobehavioral tests assessment.[24],[25] In the present work, we studied the histopathology, serum enzymes of heme metabolism and oxidative stress and brain mGluR5 gene expression levels.


   Materials and Methods Top


Chemicals

Sodium fluoride was purchased from Madras Fluorine Private Limited, India (Batch. No. 038P011, 99% pure). G.biloba leaves extract powder (Kshipra Biotech Private Limited, India Batch. No. KBPL/GBE/140101) and enzyme standards (Sigma Aldrich, USA) were procured as indicated. All other reagents and chemicals used in this study were of high pure analytical grade.

Animals

Fifteen adult male Wistar rats, weighing (120–160 g) were procured from Biogen Laboratory (CPCSEA Reg. No. 971/Po/RcBibt/S.2006), Bangalore, India and maintained in Centre for Laboratory and Animal Research (CLAR), Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai, India. Animals were housed in polypropylene cages and supplied with pellet feed and filtered water as adlibitum and maintained at room temperature of 22°C–24°C, 40%–60% humidity and natural light and dark cycle. The animal experiment was approved by the Institutional Animal Ethics Committee (Reference No: SU/CLAR/RD/019/2016) and the work involving rats were strictly followed as per guidelines of the CPCSEA, Government of India.

Experimental design

Fifteen male Wistar rats were randomly divided into 5 groups and each group had 3 rats. Group 1 control rats received water adlibitum , Group II (Fluoride) rats received water containing 100 ppm of fluoride for a period of 30 days adlibitum , Group III (fluoride + 50 mg GBE) rats received 100 ppm of fluoride water for 30 days adlibitum followed by GBE (50 mg/kg b. w) for 15 days, Group IV (fluoride + 100 mg GBE) rats received 100 ppm of fluoride water for 30 days adlibitum followed by GBE(100 mg/kg b. w) for 15 days and Group V (fluoride + 200 mg GBE) rats received 100 ppm of fluoride water for 30 days adlibitum followed by GBE (200 mg/kg b. w) for 15 days. Estimation of serum enzymes, reverse transcription polymerase chain reaction (RT-PCR) analysis of brain mGluR5 gene expression, AChE activity in hippocapmpus, and histopathology of liver and kidney were performed at the end of 45 days of experimental period.

Dose selection

Two hundred and twenty one milligram of sodium fluoride was dissolved in one liter of water to accomplish 100 ppm of fluoride.[26] Fluoride was administered to animals through drinking water (adlibitum ) in water feeding bottles. GBE was administered at the doses of 50 or 100 or 200 mg/kg orally using oral gavage needle fixed to syringe. The method of preparation of the G.biloba was water extract method according to the manufacturer instructions and the extract contained total flavone glycosides >24% and total terpene lactones >6%.

Serum enzyme assay

DALD: DALD activity and index of reactivation, was measured in serum by spectrophotometry, according to Sassa.[27]

Determination of delta-aminolevulinic acid synthetase (DALS): DALS activity was determined using the method of Marver et al .[28]

Determination of GSSG and reduced GSH: GSSG and GSH were measured in serum by spectrophotometry and results are expressed as nmol/mL of serum.

Determination of AChE activity in rat hippocampus: The activity of AChE in the hippocampus region was determined by the method described by Ellman et al.[29] as modified by Srikumar et al .[30] The activity is expressed as nmol Ach hydrolysed/min/mg protein.

Determination of gene expression by real-time PCR: Total RNA was isolated from the brain hippocampus regions of control and experimental rats using Trizol reagent (Thermo Fischer Scientifc, India). RNA concentration was determined by measuring absorbance at 260 nm using Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., USA). cDNA was prepared from 0.5 μg total RNA by reverse transcription using Roche PCR Kit. TaqMan based detection was used RT-PCR analysis. The primers used for amplification of mGLUR5 and β-actin were as follows:

  • mGluR5


  • forward, 5'-CACTCTTGCCCAACATCAC-3'

    reverse, 5'-CACAGCGTACCAAACCTTC-3'.

  • β-actin


forward, 5'-AGCCATGTACGTAGCCATCC-3'

reverse, 5'-ACCCTCATAGATGGGCACAG-3'.

Histopathology of liver and kidney

To assess histoarchitectural changes, small sections of liver and kidney from each of the experimental animals were taken and subjected to H and E staining. The pathological changes were viewed under the Microscope (Olympus CX23).

Statistical analysis

The data was analyzed by one-way analysis of variance followed by Student Newman Keuls's multiple comparison test (Sigma Plot 13 Software Inc., USA). P value (P < 0.05) was considered as statistically significant.


   Results Top


Results of serum enzyme parameters

Effect of Ginkgo biloba extract on delta aminolevulinic acid dehydratase in fluoride-induced toxicity

The fluoride group showed an elevated levels of DALD compared to control. It was found to be statistically significant (P < 0.001). GBE 50 mg group showed reduced levels of DLAD compared to fluoride group and it was not statistically significant. GBE 100 mg/kg group showed reduced levels of DALD compared to fluoride group and it was not statistically significant. GBE 200 mg/kg group showed further reduced levels of DALD compared to fluoride and it was statistically significant. Treatment with GBE 200 mg/kg showed improvement in DALD levels in fluoride-induced toxicity. 200 mg/kg GBE provided effective protection in fluoride toxicity in the present study [Figure 1]: Top panel].
Figure 1: The effect of GBE on serum delta aminolevulinic acid dehydratase and delta-aminolevulinic acid synthetase in fluoride intoxicated rats. GBE-50, GBE-100 and GBE-200 are the doses (mg/kg, p. o). The values are mean ± standard error (n = 3 each). The 'F' and 'P ' values are by one way analysis of variance with Student Newman Keuls's multiple comparison test,aSignificantly different from the control group,bSignificantly different from the fluoride group. GBE: Gingko biloba extract

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Effect of Ginkgo biloba extract on delta aminolevulinic acid synthatase in fluoride-induced toxicity

The fluoride group showed an elevated levels of DALS compared to control. It was found to be statistically significant (P < 0.001). GBE 50 mg reduced the DALS level compared to fluoride, and it was found to be statistically significant. GBE 100 mg/kg further reduced the DALS levels compared to fluoride, and it was found to be statistically significant. GBE 200 mg/kg reduced the DALS level compared to fluoride, it was found to be statistically significant. 100 mg/kg GBE provided effective protection in fluoride toxicity in the present study [Figure 1]: Bottom panel].

Effect of Ginkgo biloba extract on gluatathione in fluoride-induced toxicity

The fluoride group showed elevated levels of GSH compared to control. It was found to be statistically significant (P < 0.001). In all GBE treatment groups (50, 100, and 200 mg/kg), the GSH levels were significantly reduced than that of fluoride group. However, it was found that GBE 100 mg/kg had better protective and antioxidant effect than other doses tested [Figure 2]: Top panel].
Figure 2: The effect of GBE on serum gluatathione and oxidized gluatathione in fluoride intoxicated rats. GBE-50, GBE-100 and GBE-200 are the doses (mg/kg, p. o). The values are mean ± standard error (n = 3 each). The 'F' and 'P ' values are by one-way analysis of variance with Student Newman Keuls's multiple comparison test,aSignificantly different from the control group,bSignificantly different from the fluoride group. GBE: Gingko biloba extract

Click here to view


Effect of GBE on oxidized gluatathione in fluoride-induced toxicity

The fluoride group showed reduced levels of GSSG as compared to control and it was statistically significant (P < 0.001). GBE drug treatments (50 and 100 mg/kg) significantly elevated GSSG levels as compared to fluoride group. However, GBE 200 mg/kg although elevated the GSSG levels compared to fluoride, it was statistically not significant. Results showed GBE 100 mg/kg had rendered better protective effects from fluoride toxicity [Figure 2]: Bottom panel].

Effect of Ginkgo biloba extract on acetylcholinesterase activity in fluoride-induced toxicity of hippocampus

The fluoride group showed reduced levels of AChE activity compared to control. It was found to be statistically significant (P < 0.001). GBE 50 mg elevated the AChE levels compared to fluoride group and it was found to be statistically significant. Notably, treatment with GBE at 100 mg/kg and 200 mg/kg significantly decreased the AChE activity than those of the fluoride intoxicated group [Figure 3] which is interesting.
Figure 3: The effect of GBE on hippocampal acetylcholinesterase activity in fluoride intoxicated rats. GBE-50, GBE-100 and GBE-200 are the doses (mg/kg, p. o). The values are mean ± standard error (n = 3 each). The 'F' and 'P ' values are by one way analysis of variance with Student Newman Keuls's multiple comparison test,aSignificantly different from the control group,bSignificantly different from the fluoride group. GBE: Gingko biloba extract

Click here to view


Effect of Ginkgo biloba extract on reverse transcription polymerase chain reaction mGluR5 gene expression in fluoride-induced toxicity

The fluoride group showed a decreased gene expression compared to control. It was found to be statistically significant (P < 0.001). GBE 50 and GBE 200 mg/kg groups showed decreased mGluR5 gene expression when compared to fluoride, it was found to be statistically significant. GBE 100 mg/kg showed decreased gene expression when compared to fluoride and it was not statistically significant. GBE drug treatment at various dose levels (50, 100, and 200 mg/kg) to fluoride exposed rats did not revert back the considerable loss in the expression levels of mGluR5. The present findings indicate that GBE did not ameliorate the fluoride toxicity through group I metabotropic glutamate receptor. In fact, higher dose GBE 200 mg treatment caused further reduction of mGluR5 expression levels [Figure 4]a and b].
Figure 4: (a) The effect of Gingko biloba extract on mGluR5 in fluoride intoxicated rats (n = 3 rats each group) by reverse transcription polymerase chain reaction technique. Panels (A: MGluR B: β-actin) represents mGluR5 and β-actin mRNA expression in the hippocampus of rats (M: Marker, F: Fluoride, C: Control, F + 50 GBE: Fluoride + 50 mg GBE, F + 100 GBE: Fluoride + 100 mg GBE, F + 200 GBE: Fluoride + 200 mg GBE). (b) Representative bar graph showing the fold-change expression of mGluR5 gene in various groups. GBE-50, GBE-100 and GBE-200 are the doses (mg/kg, p. o). The values are mean ± standard error (n = 3 each). The 'F' and 'P ' values are by one way analysis of variance with Student Newman Keuls's multiple comparison test,aSignificantly different from the control group,bSignificantly different from the fluoride group. GBE: Gingko biloba extract

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Effect of Ginkgo biloba extract on histopathology of liver and kidney in fluoride-induced toxicity

Histopathological assessment of the liver of rats treated with fluoride showed hepatocellular necrosis, degenerative changes, hepatic hyperplasia and alterations in liver architecture [Figure 5]. Fluoride-induced rat kidney showed glomerular necrosis and atrophy, glomerular capsule tubules dilatation, shrunken glomeruli and vacuolation of cytoplasm in renal tubules. But GBE treatment to fluoride intoxicated rats reverted all those changes and reduced the damage considerably [Figure 6].
Figure 5: The effect of GBE on liver histopathology in fluoride intoxicated rats (n = 3 each group). Photomicrograph showing rat liver. (a) Control, (b) Fluoride, (c) F +50 mg GBE, (d) F +100 mg GBE, (e) F +200 mg GBE (H and E, ×100) CV: Central vein, H: Hepatocyte, HN: DCV: Dilated central vein, Hepatocyte necrosis, IC: Inflammatory cells, VA: Vacuolization, CS: Congested Sinusoids, NS: Normal hepatocyte, NS: Normal sinusoids, GBE: Gingko biloba extract

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Figure 6: The effect of Gingko biloba extract on kidney histopathology in fluoride intoxicated rats (n = 3 each group). Photomicrograph showing rat kidney: (a) Control, (b) Fluoride, (c) F +50 mg GBE, (d) F +100 mg GBE, (e) F +200 mg GBE (H and E, ×100) GN: Glomerular necrosis and atrophy, RD: Renal tubules dilatation, VC: Vacuolation, NG: Normal glomerulus

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   Discussion Top


Several lines of research have shown the importance of testing plant based drugs to overcome fluoride toxicity. In the present work, the efficacy of GBE was tested in a rat model of fluorosis by the assessment of serum biochemical parameters, histopathology, brain AChE activity and mGluR5 gene expression. Increased serum DALS activity was noticed in NAF toxicity and in toxicity caused by other metals.[31],[32],[33],[34] DALD activity was found elevated in heavy metals toxicity like iron[35] and lead.[36],[37],[38] In addition, plasma delta-aminolevulinic acid concentrations were also found increased in lead poisoned cattle.[39] The present study results showed significantly elevated levels of both DALD and DALS in fluoride treated rats compared with the control group. GBE drug treatment ameliorated the fluoride-induced toxicity by significantly altering the DALD and DALS levels. Increased DALS activity after arsenic and fluoride co-exposure implicates increased oxidative stress.[31] While several studies depicted decreased delta-aminolevulinate dehydratase activity during metal and fluoride toxicity associated oxidative stress condition,[40],[41],[42],[43] we indent to report elevated delta-aminolevulinate dehydratase activity from the present study findings. Quite interestingly, elevated δ-aminolevulinic acid dehydratase was reported in rat erythrocytes due to lead poisoning.[38] It was suggested that increased activity of DALD in blood could be due to the counter effect of anaemia implicating heme synthesis.[44] As fluorosis is connected with anemia based on some studies[45],[46] our findings on elevated serum DALD levels could be justified clearly. However, it is interesting to notice that the striatal DALD activity was increased in MPTP-induced PD mice implicating the role of heme groups.[47]

Reduced GSH is one of the main scavengers of ROS and along with oxidised glutathione (GSSG) ratio it may be considered as a good marker of oxidative stress. GSH is one of the most abundant non-enzymatic antioxidant bio-molecules present in tissues.[48] The ratio between GSH: GSSG is considered as main marker to diagnose the cellular toxicity.[49] Previous studies conducted on GSH levels indicate the severity of lipid peroxidation. Study results showed increased GSH levels,[50] decreased GSH levels[13] and also unaltered GSH levels in fluoride exposure.[51] Chronic fluoride toxicity in sheep resulted in increased levels of serum MDA, red blood cells and levels of GSH due to free radical mediated oxidative stress and increased lipid peroxidation.[52] Female mice treated with 50 mg/L fluoride for 30 days showed significant elevation in brain GSH levels, which was reversed and ameliorated by treatment with 20 mg/kg GBE.[53] Present study results revealed elevated GSH levels in rats exposed to 100 ppm of fluoride and showed decreased levels of GSSG. The present results were found to be similar to previous works showing increased GSH levels after fluoride exposure. The antioxidant functions of GBE have been reported suggesting its effect against fluoride-induced oxidative stress and lipid peroxidation.[54] Antioxidant functions of GBE against hydrogen peroxide-induced lipoperoxidation were also noticed in normal human erythrocyte membrane samples.[55]

AChE is an enzyme that takes part in cholinergic neurotransmission. AChE activity is inhibited by several compounds which include drugs, pesticides and some chemical toxins[56],[57] although many studies support beneficial neuroprotective functions following AChE activity inhibition.[58] In fact, reduced AChE activity was reported in several neurological and neurodegenerative disorders.[59],[60],[61],[62] Similar to present findings, inhibition of brain AChE activities in fluoride ingested rats were reported by several works.[63],[64] By contrast, GBE treatment at 50 mg/kg to fluoride intoxicated rats increased the brain AChE activity whereas other doses (100 and 200 mg/kg) caused greater inhibition of AChE activity than the fluoride group. While reduced brain AChE activity in hypercholesterolemic mice was accounted for inflammation and mitochondrial dysfunctions perse leading to cognitive dysfunctions in neurodegenerative diseases.[65] Mice treated with sodium fluoride for 30 days showed reduced brain AChE enzyme activity.[66] Female rats treated with 500 ppm of sodium fluoride in drinking water for 60 days revealed decreased AChE activity.[67] Taken together, the present study results are in agreement with previous works showing fluoride mediated reduction of AChE activity in brain tissues. Previous research findings revealed that administration of certain phytochemical drugs could alter the activity of AChE in brain to ameliorate the toxicity of several agents including metals.[68],[69],[70] But it is important to notice that inhibition of AChE activity after GBE treatment has proven neuroprotective effects in animal models of neurotoxicity.[71],[72] Therefore, it suggested that studies that would explore in details the mechanistic insights of inhibition of AChE activity during fluorosis would better indicate the sequel of brain associated pathogenesis.

In earlier research work, GB treatment in AD patients along with ChEIs provided additional cognitive benefits.[73]G.biloba leaf extract rich in flavonol compounds promoted dopaminergic and cholinergic neurotransmission in the prefrontal cortex of rats.[74]

G.biloba protected the brain from beta-amyloid neurotoxicity and reversed the memory deficits through its effect on the cholinergic system.[75] Ginkgolides A and B administration inhibited the effect of β-amyloid on the release of acetylcholine from hippocampal neurons.[76] It was reported that G.biloba improved the cognitive ability through interactions with the antioxidant and cholinergic systems.[77] In the present work, treatment with various doses of GBE (50, 100, and 200 mg/kg) ameliorated fluoride toxicity by altering hippocampal AChE activity.

Glutamate is the primary excitatory neurotransmitter in the brain and activates both ionotropic glutamate receptors and G protein coupled metabotropic glutamate receptors mGluRs.[78] mGluR5 signaling changes were noticed in various neurodegenerative diseases like Parkinson's disease, Huntington's disease, and Alzheimer's disease.[79] mGluR5 are found in the neurons and glia cells all over the CNS including the cortex and the hippocampus.[80],[81] Inhibition of mGluR5 expression impaired the spatial learning in experimental rats[82] Present study showed inhibition of mGluR5 expression in fluoride-induced toxicity. However, treatment of GBE at various dose levels (50, 100, and 200 mg/kg) to fluoride exposed rats did not reveal any considerable changes in the mGluR5 expression levels when compared with fluoride group. The present findings indicate that GBE did not ameliorate the fluoride toxicity through group I metabotropic glutamate receptor.

Based on previous study, accumulation of fluoride diminished the aerobic metabolism and distorted the free radical metabolism in liver and kidney.[83] Chronic fluoride exposure leads to critical degenerative changes in rat kidney like glomerular necrosis and vascular congestion.[84] Hepatoprotective activity of G.biloba may be due to presence of compounds like flavonoid (ginkgo-flavone glycosides) and terpenoid (ginkgolides and bilobalides) which have antioxidant effect.[85] Present study results confirmed that rats treated with GBE showed amelioration in structure of liver and kidney against fluoride toxicity.


   Conclusion Top


The present study investigated the therapeutic efficacy of GBE in experimental model of fluorosis. Fluoride-induced serum enzyme parameter alterations, AChE activity, liver and kidney histopathological changes were reversed by GBE. Results of the present study concluded beneficial effects of GBE in experimental model of fluorosis.

Acknowledgements

We thank Dr. R. Vijayaraghavan, Director-Research, Saveetha Institute of Medical and Technical Sciences, Chennai for providing his support on statistical analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Bera I, Sabatini R, Auteri P, Flace P, Sisto G, Montagnani M, et al . Neurofunctional effects of developmental sodium fluoride exposure in rats. Eur Rev Med Pharmacol Sci 2007;11:211-24.  Back to cited text no. 1
    
2.
Dhar V, Bhatnagar M. Physiology and toxicity of fluoride. Indian J Dent Res 2009;20:350-5.  Back to cited text no. 2
[PUBMED]  [Full text]  
3.
Blaylock RL. Excitotoxicity a possible central mechanism in fluoride neurotoxicity. Fluoride 2004;37:301-14.  Back to cited text no. 3
    
4.
Dong YT, Wang Y, Wei N, Zhang QF, Guan ZZ. Deficit in learning and memory of rats with chronic fluorosis correlates with the decreased expressions of M1 and M3 muscarinic acetylcholine receptors. Arch Toxicol 2015;89:1981-91.  Back to cited text no. 4
    
5.
Zheng X, Sun Y, Ke L, Ouyang W, Zhang Z. Molecular mechanism of brain impairment caused by drinking-acquired fluorosis and selenium intervention. Environ Toxicol Pharmacol 2016;43:134-9.  Back to cited text no. 5
    
6.
Long YG, Wang YN, Chen J, Jiang SF, Nordberg A, Guan ZZ. Chronic fluoride toxicity decreases the number of nicotinic acetylcholine receptors in rat brain. Neurotoxicol Teratol 2002;24:751-7.  Back to cited text no. 6
    
7.
Ekambaram P, Paul V. Modulation of fluoride toxicity in rats by calcium carbonate and by withdrawal of fluoride exposure. Pharmacol Toxicol 2002;90:53-8.  Back to cited text no. 7
    
8.
Süzen HS, Duydu Y, Aydın A, Işımer A, Vural N. Influence of the delta-aminolevulinic acid dehydratase (ALAD) polymorphism on biomarkers of lead exposure in Turkish storage battery manufacturing workers. Am J Ind Med 2003;43:165-71.  Back to cited text no. 8
    
9.
Arulkumar M, Vijayan R, Penislusshiyan S, Sathishkumar P, Angayarkanni J, Palvannan T. Alteration of paraoxonase, arylesterase and lactonase activities in people around fluoride endemic area of Tamil Nadu, India. Clin Chim Acta 2017;471:206-15.  Back to cited text no. 9
    
10.
Niu Q, He P, Xu S, Ma R, Ding Y, Mu L, et al . Fluoride-induced iron overload contributes to hepatic oxidative damage in mouse and the protective role of Grape seed proanthocyanidin extract. J Toxicol Sci 2018;43:311-9.  Back to cited text no. 10
    
11.
Nicotera P, Orrenius S. Role of thiols in protection against biological reactive intermediates. Adv Exp Med Biol 1986;197:41-51.  Back to cited text no. 11
    
12.
Younes M, Siegers CP. Mechanistic aspects of enhanced lipid peroxidation following glutathione depletion in vivo . Chem Biol Interact 1981;34:257-66.  Back to cited text no. 12
    
13.
Shivarajashankara YM, Shivashankara AR, Rao SH, Bhat PG. Oxidative stress in children with endemic skeletal fluorosis. Fluoride 2001;34:103-7.  Back to cited text no. 13
    
14.
Palomero J, Galán AI, Muñoz ME, Tuñón MJ, González-Gallego J, Jiménez R. Effects of aging on the susceptibility to the toxic effects of cyclosporin A in rats. Changes in liver glutathione and antioxidant enzymes. Free Radic Biol Med 2001;30:836-45.  Back to cited text no. 14
    
15.
Zhao XL, Wu JH. Actions of sodium fluoride on acetylcholinesterase activities in rats. Biomed Environ Sci 1998;11:1-6.  Back to cited text no. 15
    
16.
Mukherjee S, Manahan-Vaughan D. Role of metabotropic glutamate receptors in persistent forms of hippocampal plasticity and learning. Neuropharmacology 2013;66:65-81.  Back to cited text no. 16
    
17.
Jiang S, Su J, Yao S, Zhang Y, Cao F, Wang F, et al . Fluoride and arsenic exposure impairs learning and memory and decreases mGluR5 expression in the hippocampus and cortex in rats. PLoS One 2014;9:e96041.  Back to cited text no. 17
    
18.
Sun Z, Zhang Y, Xue X, Niu R, Wang J. Maternal fluoride exposure during gestation and lactation decreased learning and memory ability, and glutamate receptor mRNA expressions of mouse pups. Hum Exp Toxicol 2018;37:87-93.  Back to cited text no. 18
    
19.
Takagi M, Shiraki S. Acute sodium fluoride toxicity in the rat kidney. Bull Tokyo Med Dent Univ 1982;29:123-30.  Back to cited text no. 19
    
20.
Hassan HA, Yousef MI. Mitigating effects of antioxidant properties of black berry juice on sodium fluoride induced hepatotoxicity and oxidative stress in rats. Food Chem Toxicol 2009;47:2332-7.  Back to cited text no. 20
    
21.
Li BY, Gao YH, Pei JR, Yang YM, Zhang W, Sun DJ. ClC-7/Ostm1 contribute to the ability of tea polyphenols to maintain bone homeostasis in C57BL/6 mice, protecting against fluorosis. Int J Mol Med 2017;39:1155-63.  Back to cited text no. 21
    
22.
Dubey AK, Shankar PR, Upadhyaya D, Deshpande VY. Ginkgo biloba – An appraisal. Kathmandu Univ Med J (KUMJ) 2004;2:225-9.  Back to cited text no. 22
    
23.
Li L, Zhang QG, Lai LY, Wen XJ, Zheng T, Cheung CW, et al . Neuroprotective effect of ginkgolide B on bupivacaine-induced apoptosis in SH-SY5Y cells. Oxid Med Cell Longev 2013;2013:159864.  Back to cited text no. 23
    
24.
Raju S, Sivanesan S, Gudemalla K, Mundugaru R, Swaminathan M. Effect of Ginkgo biloba extract on hematological and biochemical alterations in fluoride intoxicated Wistar rats. Res J Pharm Tech 2019;12:3843-6.  Back to cited text no. 24
    
25.
Raju S, Sivanesan SK, Gudemalla K. Cognitive enhancement effect of Ginkgo biloba extract on memory and learning impairments induced by fluoride neurotoxicity. Int J Res Pharm Sci 2019;10:129-34.  Back to cited text no. 25
    
26.
Jetti R, Raghuveer CV, Mallikarjuna RC. Protective effect of ascorbic acid and Ginkgo biloba against learning and memory deficits caused by fluoride. Toxicol Ind Health 2016;32:183-7.  Back to cited text no. 26
    
27.
Sassa S. Delta-aminolevulinic acid dehydratase assay. Enzyme 1982;28:133-45.  Back to cited text no. 27
    
28.
Marver HS, Tschudy DP, Perlroth MG, Collins A. δ-Aminolevulinic acid synthetase I. Studies in liver homogenates. J Biol Chem 1966;241:2803-9.  Back to cited text no. 28
    
29.
Ellman GL, Courtney KD, Andres V Jr., Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88-95.  Back to cited text no. 29
    
30.
Srikumar BN, Ramkumar K, Raju TR, Shankaranarayana RB. Assay of acetylcholinesterase activity in the brain. Brain Behavior 2004;25:142-4.  Back to cited text no. 30
    
31.
Mittal M, Chatterjee S, Flora SJ. Combination therapy with Vitamin C and DMSA for arsenic-fluoride co-exposure in rats. Metallomics 2018;10:1291-306.  Back to cited text no. 31
    
32.
Gupta R, Kannan GM, Sharma M, S Flora SJ. Therapeutic effects of Moringa oleifera on arsenic-induced toxicity in rats. Environ Toxicol Pharmacol 2005;20:456-64.  Back to cited text no. 32
    
33.
Modi M, Mittal M, Flora SJ. Combined administration of selenium and meso-2, 3-dimercaptosuccinic acid on arsenic mobilization and tissue oxidative stress in chronic arsenic-exposed male rats. Ind J Pharm 2007;39:107.  Back to cited text no. 33
    
34.
Agrawal ND, Nirala SK, Bhadauria M, Srivastava S, Shukla S. Protective potential of Moringa oleifera Lam. along with curcumin and piperine against beryllium-induced alterations in hepatorenal biochemistry and ultramorphology in rats. Ind J Biochem and Biophysics 2019;56:70-80.  Back to cited text no. 34
    
35.
Labbé RF, Finch CA. Effects of iron status on δ-aminolevulinic acid dehydratase activity. Biochem Med 1977;18:323-9.  Back to cited text no. 35
    
36.
Maes J, Gerber GB. Increased ALA dehydratase activity and spleen weight in lead-intoxicated rats. A consequence of increased blood cell destruction. Experientia 1978;34:381-2.  Back to cited text no. 36
    
37.
Fujita H, Orii Y, Sano S. Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats. Biochim Biophys Acta 1981;678:39-50.  Back to cited text no. 37
    
38.
Kajimoto M, Kondo M, Niwa M, Suzuki T, Kimura H, Sasaki A, et al . Increase of delta-aminolevulinic acid dehydratase (ALAD) in rat erythrocytes in lead poisoning. Arch Toxicol 1983;52:1-11.  Back to cited text no. 38
    
39.
Kang HG, Bischoff K, Ebel JG, Cha SH, McCardle J, Choi CU. Comparison of blood lead and blood and plasma δ-aminolevulinic acid concentrations as biomarkers for lead poisoning in cattle. J Vet Diagn Invest 2010;22:903-7.  Back to cited text no. 39
    
40.
Baierle M, Charão MF, Göethel G, Barth A, Fracasso R, Bubols G, et al . Are delta-aminolevulinate dehydratase inhibition and metal concentrations additional factors for the age-related cognitive decline Int J Environ Res Public Health 2014;11:10851-67.  Back to cited text no. 40
    
41.
do Nascimento SN, Charão MF, Moro AM, Roehrs M, Paniz C, Baierle M, et al . Evaluation of toxic metals and essential elements in children with learning disabilities from a rural area of southern Brazil. Int J Environ Res Public Health 2014;11:10806-23.  Back to cited text no. 41
    
42.
Chouhan S, Lomash V, Flora SJ. Fluoride-induced changes in haem biosynthesis pathway, neurological variables and tissue histopathology of rats. J Appl Toxicol 2010;30:63-73.  Back to cited text no. 42
    
43.
Mittal M, Flora SJ. Vitamin E supplementation protects oxidative stress during arsenic and fluoride antagonism in male mice. Drug Chem Toxicol 2007;30:263-81.  Back to cited text no. 43
    
44.
França RT, Da Silva AS, Wolkmer P, Oliveira VA, Pereira ME, Leal ML, et al . Delta-aminolevulinate dehydratase activity in red blood cells of rats infected with Trypanosoma evansi . Parasitology 2011;138:1272-7.  Back to cited text no. 44
    
45.
Eren E, Ozturk M, Mumcu EF, Canatan D. Fluorosis and its hematological effects. Toxicol Ind Health 2005;21:255-8.  Back to cited text no. 45
    
46.
Susheela AK, Mondal NK, Gupta R, Sethi M, Pandey RM. Fluorosis is linked to anaemia. Curr Sci 2018;4:692-700.  Back to cited text no. 46
    
47.
Sampaio TB, Marcondes Sari MH, Pesarico AP, Nogueira CW. δ-Aminolevulinate dehydratase activity is stimulated in a MPTP mouse model of Parkinson's disease: Correlation with myeloperoxidase activity. Cell Mol Neurobiol 2017;37:911-7.  Back to cited text no. 47
    
48.
Ji LL, Stratman FW, Lardy HA. Antioxidant enzyme systems in rat liver and skeletal muscle: Influences of selenium deficiency, chronic training and acute exercise. Arch Biochem Biophys 1988;263:150-60.  Back to cited text no. 48
    
49.
Townsend DM, Tew KD, Tapiero H. The importance of glutathione in human disease. Biomed Pharmacother 2003;57:145-55.  Back to cited text no. 49
    
50.
Jeji J, Sharma R, Jolly SS, Pamnani S. Implication of glutathione in endemic fluorosis. Fluoride 1985;18:117-9.  Back to cited text no. 50
    
51.
Wei ZD, Li F, Zhou L, Chen X, Dai G. Studies on fluoride-aluminum combined toxicosis. Fluoride 1995;28:37.  Back to cited text no. 51
    
52.
Güven A, Kaya N. Effect of fluoride intoxication on lipid peroxidation and reduced glutathione in Tuj sheep. Fluoride 2005;38:139-42.  Back to cited text no. 52
    
53.
Atmaca N, Yıldırım E, Güner B, Kabakçı R, Bilmen FS. Effect of resveratrol on hematological and biochemical alterations in rats exposed to fluoride. BioMed Res Int 2014;2014:698628.  Back to cited text no. 53
    
54.
Bridi R, Crossetti FP, Steffen VM, Henriques AT. The antioxidant activity of standardized extract of Ginkgo biloba (EGb 761) in rats. Phytother Res 2001;15:449-51.  Back to cited text no. 54
    
55.
Kose K, Dogan P. Lipoperoxidation induced by hydrogen peroxide in human erythrocyte membranes. J Int Med Res 1995;23:1-18.  Back to cited text no. 55
    
56.
Colović MB, Krstić DZ, Lazarević-Pašti TD, Bondžić AM, Vasić VM. Acetylcholinesterase inhibitors: Pharmacology and toxicology. Curr Neuropharmacol 2013;11:315-35.  Back to cited text no. 56
    
57.
Schwarz M, Glick D, Loewenstein Y, Soreq H. Engineering of human cholinesterases explains and predicts diverse consequences of administration of various drugs and poisons. Pharmacol Ther 1995;67:283-322.  Back to cited text no. 57
    
58.
Sramek JJ, Cutler NR. RBC cholinesterase inhibition: A useful surrogate marker for cholinesterase inhibitor activity in Alzheimer disease therapy Alzheimer Dis Assoc Disord 2000;14:216-27.  Back to cited text no. 58
    
59.
Kuhl DE, Koeppe RA, Minoshima S, Snyder SE, Ficaro EP, Foster NL, et al .In vivo mapping of cerebral acetylcholinesterase activity in aging and Alzheimer's disease. Neurology 1999;52:691-9.  Back to cited text no. 59
    
60.
Mufson EJ, Counts SE, Perez SE, Ginsberg SD. Cholinergic system during the progression of Alzheimer's disease: Therapeutic implications. Expert Rev Neurother 2008;8:1703-18.  Back to cited text no. 60
    
61.
Méndez M, Méndez-López M, López L, Aller MA, Arias J, Arias JL. Acetylcholinesterase activity in an experimental rat model of Type C hepatic encephalopathy. Acta Histochem 2011;113:358-62.  Back to cited text no. 61
    
62.
Ruberg M, Rieger F, Villageois A, Bonnet AM, Agid Y. Acetylcholinesterase and butyrylcholinesterase in frontal cortex and cerebrospinal fluid of demented and non-demented patients with Parkinson's disease. Brain Res 1986;362:83-91.  Back to cited text no. 62
    
63.
Bharti VK, Srivastava RS, Anand AK, Kusum K. Buffalo (Bubalus bubalis ) epiphyseal proteins give protection from arsenic and fluoride-induced adverse changes in acetylcholinesterase activity in rats. J Biochem Molecular Toxicol 2012;26:10-5.  Back to cited text no. 63
    
64.
Basha PM, Rai P, Begum S. Fluoride toxicity and status of serum thyroid hormones, brain histopathology, and learning memory in rats: A multigenerational assessment. Biol Trace Elem Res 2011;144:1083-94.  Back to cited text no. 64
    
65.
Paul R, Borah A. Global loss of acetylcholinesterase activity with mitochondrial complexes inhibition and inflammation in brain of hypercholesterolemic mice. Sci Rep 2017;7:17922.  Back to cited text no. 65
    
66.
Shah SD, Chinoy NJ. Adverse effects of fluoride and/or arsenic on the cerebral hemisphere of mice and recovery by some antidotes. Fluoride 2004;37:162-71.  Back to cited text no. 66
    
67.
Ekambaram P, Paul V. Effect of Vitamin D on chronic behavioral and dental toxicities of sodium fluoride in rats. Fluoride 2003;36:189-97.  Back to cited text no. 67
    
68.
Klimaczewski CV, Ecker A, Piccoli B, Aschner M, Barbosa NV, Rocha JB. Peumus boldus attenuates copper-induced toxicity in Drosophila melanogaster. Biomed Pharmacother 2018;97:1-8.  Back to cited text no. 68
    
69.
Bortoli PM, Alves C, Costa E, Vanin AP, Sofiatti JR, Siqueira DP, et al . Ilex paraguariensis: Potential antioxidant on aluminium toxicity, in an experimental model of Alzheimer's disease. J Inorg Biochem 2018;181:104-10.  Back to cited text no. 69
    
70.
Al Omairi NE, Radwan OK, Alzahrani YA, Kassab RB. Neuroprotective efficiency of Mangifera indica leaves extract on cadmium-induced cortical damage in rats. Metab Brain Dis 2018;33:1121-30.  Back to cited text no. 70
    
71.
Abd-Elhady RM, Elsheikh AM, Khalifa AE. Anti-amnestic properties of Ginkgo biloba extract on impaired memory function induced by aluminum in rats. Int J Dev Neurosci 2013;31:598-607.  Back to cited text no. 71
    
72.
Gong QH, Wu Q, Huang XN, Sun AS, Nie J, Shi JS. Protective effect of Ginkgo biloba leaf extract on learning and memory deficit induced by aluminum in model rats. Chin J Integr Med 2006;12:37-41.  Back to cited text no. 72
    
73.
Canevelli M, Adali N, Kelaiditi E, Cantet C, Ousset PJ, Cesari M, et al . Effects of Gingko biloba supplementation in Alzheimer's disease patients receiving cholinesterase inhibitors: Data from the ICTUS study. Phytomedicine 2014;21:888-92.  Back to cited text no. 73
    
74.
Kehr J, Yoshitake S, Ijiri S, Koch E, Nöldner M, Yoshitake T. Ginkgo biloba leaf extract (EGb 761®) and its specific acylated flavonol constituents increase dopamine and acetylcholine levels in the rat medial prefrontal cortex: Possible implications for the cognitive enhancing properties of EGb 761®. Int Psychogeriatr 2012;24 Suppl 1:S25-34.  Back to cited text no. 74
    
75.
Tang F, Nag S, Shiu SY, Pang SF. The effects of melatonin and Ginkgo biloba extract on memory loss and choline acetyltransferase activities in the brain of rats infused intracerebroventricularly with β-amyloid 1–40. Life Sci 2002;71:2625-31.  Back to cited text no. 75
    
76.
Lee TF, Chen CF, Wang LC. Effect of ginkgolides on β-amyloid-suppressed acetylocholine release from rat hippocampal slices. Phytother Res 2004;18:556-60.  Back to cited text no. 76
    
77.
Nathan P. Can the cognitive enhancing effects of Ginkgo biloba be explained by its pharmacology Med Hypotheses 2000;55:491-3.  Back to cited text no. 77
    
78.
Dhami GK, Ferguson SS. Regulation of metabotropic glutamate receptor signaling, desensitization and endocytosis. Pharmacol Ther 2006;111:260-71.  Back to cited text no. 78
    
79.
Thathiah A, De Strooper B. The role of G protein-coupled receptors in the pathology of Alzheimer's disease. Nat Rev Neurosci 2011;12:73-87.  Back to cited text no. 79
    
80.
Romano C, Sesma MA, McDonald CT, O'Malley K, Van den Pol AN, Olney JW. Distribution of metabotropic glutamate receptor mGluR5 immunoreactivity in rat brain. J Comp Neurol 1995;355:455-69.  Back to cited text no. 80
    
81.
Kerner JA, Standaert DG, Penney JB Jr, Young AB, Landwehrmeyer GB. Expression of group one metabotropic glutamate receptor subunit mRNAs in neurochemically identified neurons in the rat neostriatum, neocortex, and hippocampus. Brain Res Mol Brain Res 1997;48:259-69.  Back to cited text no. 81
    
82.
Balschun D, Wetzel W. Inhibition of mGluR5 blocks hippocampal LTPin vivo and spatial learning in rats. Pharmacol Biochem Behav 2002;73:375-80.  Back to cited text no. 82
    
83.
Mathur M. Sodium fluoride induced toxic effect on tumor protein 53 forming gene in Swiss albino mice. Cibtech J Zool. 2013;2:24-9.  Back to cited text no. 83
    
84.
Karaoz E, Oncu M, Gulle K, Kanter M, Gultekin F, Karaoz S, et al . Effect of chronic fluorosis on lipid peroxidation and histology of kidney tissues in first- and second-generation rats. Biol Trace Elem Res 2004;102:199-208.  Back to cited text no. 84
    
85.
Itil T, Martorano D. Natural substances in psychiatry (Ginkgo biloba in dementia). Psychopharmacol Bull 1995;31:147-58.  Back to cited text no. 85
    


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