IPSIndian Journal of Pharmacology
Home  IPS  Feedback Subscribe Top cited articles Login 
Users Online : 11941 
Small font sizeDefault font sizeIncrease font size
Navigate Here
Resource Links
 »  Similar in PUBMED
 »  Search Pubmed for
 »  Search in Google Scholar for
 »Related articles
 »  Article in PDF (2,793 KB)
 »  Citation Manager
 »  Access Statistics
 »  Reader Comments
 »  Email Alert *
 »  Add to My List *
* Registration required (free)

In This Article
 »  Abstract
 » Introduction
 »  Materials and Me...
 » Results
 » Discussion
 » Conclusion
 »  References
 »  Article Figures
 »  Article Tables

 Article Access Statistics
    PDF Downloaded98    
    Comments [Add]    
    Cited by others 3    

Recommend this journal


 Table of Contents    
Year : 2021  |  Volume : 53  |  Issue : 2  |  Page : 132-142

Therapeutic potential of diosmin, a citrus flavonoid against arsenic-induced neurotoxicity via suppression of NOX 4 and its subunits

Department of Pharmacology, Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women's University), Tirupati, Andhra Pradesh, India

Date of Submission14-Jan-2020
Date of Decision09-May-2020
Date of Acceptance03-May-2021
Date of Web Publication26-May-2021

Correspondence Address:
Dr. Sujatha Dodoala
Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women's University) - 517 502, Andhra Pradesh
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijp.IJP_837_19

Rights and Permissions

 » Abstract 

Objectives: Water contaminated with arsenic affected millions of people worldwide and arsenic exposure is related to various neurological disorders. Hence, the current study was planned to investigate the neuroprotective activity of diosmin (DSN) against arsenic induced neurotoxicity as an attempt to identify therapeutic intervention to combat arsenicism.
Materials and Methods: Sodium arsenite an inducer of neurotoxicity was administered orally (13 mg/kg) and DSN treatment at two selected doses (50 and 100 mg/kg) was done for 21 days. Behavioral and biochemical variations were examined by various parameters. Furthermore, histopathological and immunohistochemistry studies were done with the brain sections.
Results: The behavioral studies evidenced that arsenic has suppressed the exploratory behavior and motor coordination in rats and DSN treatment has recovered the behavioral changes to normal. Arsenic administration has also found to induce oxidative stress and DSN co-treatment has ameliorated the oxidative stress markers. Interestingly, depleted levels of neurotransmitters were observed with the arsenic and it was restored back by the DSN treatment. Histopathological alterations like pyknosis of the neuronal cells were identified with arsenic exposure and subsided upon DSN co administration. Immunohistochemical studies have revealed the expression of NOX4 and its gp91phox and P47phox subunits and its suppression by DSN treatment may be the key therapeutic factor of it.
Conclusions: Treatment with DSN showed a beneficial effect in protecting against arsenic-induced neurotoxicity by suppressing the toxicity changes and the antioxidant effect of DSN might be attributed to its ability of suppressing NOX4 and its subunits.

Keywords: Arsenic, diosmin, neurotoxicity, NOX4

How to cite this article:
Peruru R, Dodoala S. Therapeutic potential of diosmin, a citrus flavonoid against arsenic-induced neurotoxicity via suppression of NOX 4 and its subunits. Indian J Pharmacol 2021;53:132-42

How to cite this URL:
Peruru R, Dodoala S. Therapeutic potential of diosmin, a citrus flavonoid against arsenic-induced neurotoxicity via suppression of NOX 4 and its subunits. Indian J Pharmacol [serial online] 2021 [cited 2023 Oct 4];53:132-42. Available from: https://www.ijp-online.com/text.asp?2021/53/2/132/316956

 » Introduction Top

Arsenic is a ubiquitous metalloid distributed widely in the environment and exists in three forms such as elemental, organic, and inorganic forms. Arsenic stands first in the toxicity profile of hazardous chemicals. Arsenic leaches from the bedrocks into the ground water leading to arsenic pollution in drinking water throughout many parts of India and across the world.[1] Apart from the typical skin lesions, chronic exposure to arsenic leads to innumerable malformations including cardiovascular, neuronal, metabolic, developmental disorders, and multiple cancers.[2]

Arsenic-induced toxicity mechanisms are not yet clear but the most accepted proposal is the augmentation of oxidative stress by arsenic via the production of reactive oxygen species (ROS). Arsenic-induced ROS mediates by altering signaling pathways, transcription factors, mitochondrial function, and enzyme activity. It was reported to activate few enzymes which accounts for the ROS generation including hemoxygenase reductase, thioredoxin reductase, and NADPH Oxidase (NOX).[3]

NOX enzyme acts as a key producer of ROS in many cells. The subunits of NOX include gp91phox, P22phox P40phox P47phox P67phox and Rac. NOX generates superoxide radicals by reduction of molecular oxygen using NADPH. Arsenic has demonstrated to activate p47phox subunit a key component of NOX in SVEC4-10 endothelial cells.[4] Arsenic also phosphorylates and translocates p47phox, Rac1 and p67phox in arsenic trioxide (As2O3) treated macrophages for generating superoxide radicals.[5] Hence, in the case of arsenic-induced neurotoxicity, excessive activation of NOX by arsenic has a crucial role in ROS production and in turn generation of oxidative stress.

Arsenic-induced neurotoxicity is a burning issue in people exposed to arsenic pollution and consequently, developing novel and cost-effective pharmacological interventions in this area has crucial importance. Flavonoids are the secondary plant components with extensive biological activities and these are the most important plant products employed in combating various disorders due to their high antioxidant profile. Diosmin (DSN) is a glycosylated polyphenolic compound abundantly available in citrus species. DSN has put forth for its multitude of pharmacological applications including antioxidant, anti-inflammatory, vaso-protective, anti-proliferative, anti-cancerous, and anti-diabetic activities. DSN exhibits anti-inflammatory activity by suppression of overexpressed nuclear factor κB, cyclooxygenase-2, tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase.[6] Its potential in treating diabetic neuropathy and neuropathic pain was reported in many studies recently and proved to have neuroprotective activity in peripheral neurons.[7],[8] Still a lack of research was found in the area of DSN activity on the central nervous system. In this regard, the study was planned to estimate the ability of DSN in combating neurobehavioral and neurotoxicity changes induced by arsenic in rats and to determine the molecular level targets on which it acts to exhibit neuroprotection.

 » Materials and Methods Top


DSN (purity >94%) was obtained from Sigma-Aldrich Chemical Co. Sodium arsenite from Hi media chemicals (Mumbai, India) and antibodies (NOX4, P47phoxand gp91phox) were procured from St. John's laboratory Ltd., London. Other chemicals were procured from Hi-media and Sigma-Aldrich.


Adult female rats of Wistar strain (180–220 g) were procured from the CPCSEA approved breeder. Animals were accommodated in sterile cages under standard 12 h light and dark cycle by providing water and food pellets ad libitum. Animals were allowed to acclimatize in the laboratory before conducting experiments for a week. The animal experimental designs were performed with the approval of the Institutional Animal Ethical Committee (Regd. No. 1677/PO/Re/2012/CPCSEA/07).

Preparation of drug solutions

Sodium arsenite was used as an inducing agent of neurotoxicity in the rats and its solution was prepared by dissolving it in physiological saline solution (0.9% NaCl). Apocynin was employed as a positive control as it is a specific inhibitor of NOX.[9] DSN and apocynin doses were prepared as 1% CMC suspension. Sodium arsenite and DSN dose selection were done based on previous publications and drugs were administered by oral route at doses of 13 mg/kg of sodium arsenite and 50 and 100 mg/kg DSN for 3 weeks.[10],[11]

Experimental design

A total of 30 rats were used for the experiment and animals divided into five groups (n = 6).

  • Group I – Normal control group (Received vehicle)
  • Group II – Disease/Arsenic control group given with 13mg/kg of sodium arsenite
  • Group III – Standard group treated with 13 mg/kg sodium arsenite and 10 mg/kg apocynin
  • Group IV – DSN treatment I group received 13 mg/kg sodium arsenite and 50 mg/kg DSN
  • Group V – DSN treatment II group received 13 mg/kg sodium arsenite and 100 mg/kg DSN.

Behavioral evaluation

All the behavioral observations as listed below were performed at an interval of 7 days throughout the study for 21 days. Every parameter was performed one time on each animal in a group.

Elevated plus maze

Elevated plus maze (EPM) is usually a simple parameter to validate the anxiety behavior in rodents. The apparatus was arranged with two open arms (50 cm × 10 cm), two closed arms (50 cm × 10 cm × 20 cm), and central platform (10 cm × 10 cm), and arranged 50 cm above the ground. The animal was kept on the central platform directing toward the closed field and their behavior was observed for 5 min. The number of entries to open and closed fields and time spent in each field were measured to assess the total exploratory activity. Along with that rearing activity of the animal was also determined and the maze was cleaned after each trail by 10% ethanol.[12]

Forced swim test (FST)

Forced swim test (FST) is an extensively used technique to assess the depressive behavior of animals.[13] The apparatus used for FST is an opaque Plexiglas cylinder (50 cm × 20 cm) filled with water to 30 cm height and maintained at room temperature. Animals were placed in the water and acclimatized for 5 min and the immobility and swimming time periods were noticed for the next 10 min.

Hole-board test

Hole-board test was performed on a hole-board unit 35 cm × 35 cm × 15 cm having 16 holes of diameter 3 cm which were symmetrically arranged in 4 rows. The animal was placed in one corner and allowed to move freely on the board the for 2 min. Then the number of head dipings and rearing moments was noted in the next 5 min.[14]

Beam walk test

To determine the coordination and balance of animals beam walk analysis was performed.[15],[16] The apparatus constitutes a beam (1–2 cm wide and 1 m long), arranged on a stand of 30 cm height. Animals were acclimatized on the beam by allowing it to move from one end to another end for 3 times. Then, the number of leg slips by the animal to walk along the beam to other end for one time was observed.

Biochemical evaluation

Animals were sacrificed on the 21st day of the study by cervical decapitation. The brains were carefully isolated, made into two transverse sections. One-half stored at −80°C for biochemical analysis and the other half stored in formalin for immunohistochemistry (IHC) and histopathological analysis. The homogenization (Remi RQT-127 homogenizer) was carried out with lysis buffer (pH 7.4). Then, homogenate centrifuged at 12,000 rpm for 10 min at 4°C and supernatant was employed to estimate the biochemical parameters.

Estimation of arsenic content in the brain tissue

Brain tissue was minced and dried at 90°C for 3 h and then it was digested in concentrated nitric acid using the heating medium. Then, samples were diluted with double distilled water and filtered using 40 μm membrane filters. Arsenic content was estimated by atomic absorption spectrophotometer (ICP-OES, Perkin Elmer, Optima 7300 DV).[17]

Estimation of neurotransmitter levels in the brain

The neurotransmitter levels in the brain were determined by the spectrofluorimetric method. The neurotransmitters including dopamine, noradrenaline, and serotonin were measured by following the method of Schlumpf et al. by slight modifications.[18]

Tissue extracts preparation

The brain was homogenized in Hcl and butanol solution (0.85 ml Conc. HCl in 1 lit n-butanol) and the supernatant was collected by centrifuging at 2000 rpm for 10 min. Then, 1 ml of supernatant was transferred to a reaction tube containing HCl and heptane (0.31 ml HCl of 0.1 M in 2.5 ml heptane). Then, the aqueous extract was employed for the assay.

Estimation of dopamine and noradrenaline

To the reaction tube 0.2 ml aqueous extract, 0.05 ml 0.4 M HCl, 0.1 ml of sodium acetate buffer (pH 6.9) and 0.1 ml of 0.1 M iodine solution were added and allowed to react for 2 min. Then, 0.1 ml of 0.2 M Na2SO3 solution was added to terminate the reaction process. Immediately, after 1.5 min, acetic acid 10 M was added and boiled for 5 min; then allowed to reach for room temperature. For dopamine, excitation and emission spectra were observed at 330–375 nm and for noradrenaline at 395–485 nm.

Estimation of serotonin

To estimate serotonin levels in a reaction tube aqueous extract 0.2 ml and O-phthaldialdehyde reagent (O-phthaldialdehyde 20 mg in 100 ml of Conc. HCl) 0.25 ml were taken and heated up to 100°C for 10 min. The excitation and emission absorbance were recorded at 360–470 nm in spectrofluorimeter.

Estimation of total protein levels

Total protein levels were estimated in the supernatant by following the Bradford assay method and the standard was 1 mg/ml bovine serum albumin.

Evaluation of antioxidant profile

Supernatant of brain homogenate was employed to estimate the antioxidant parameters such as malondialdehyde (MDA) assay, reduced glutathione (GSH) levels, superoxide dismutase (SOD) activity, and catalase (CAT) activity.[19] The parameters were estimated using ultraviolet (UV)-spectrophotometer (UV-1800, UV-Vis Spectrophotometer by Shimadzu).

Estimation of nitric oxide levels

To estimate the nitric oxide levels 150 μl of supernatant, 50 μl of 10 mM sodium nitroprusside solution was added and incubated for 150 min at room temperature and then 60 μl of griess reagent was added and incubated at 250C for 30 min and absorbance was taken at 546 nm with UV-spectrophotometer.[19]

Estimation of protein carbonyl levels

The supernatant was treated with 10% streptomycin sulfate to remove nucleic acids by centrifuged at 4000 rpm. Then, 2,4-dinitrophenylhydrazide was added which forms protein hydrazone derivatives and these derivatives were precipitated using 10% TCA, equal proportions of ethanol and ethyl acetate mixture and then centrifuged. Re suspended the pellet with guanidine hydrochloride (6 M concentration) and the absorbance taken at 370 nm. The calculation was done using the spectral difference between protein hydrazone in guanidine hydrochloride and a protein blank sample.[20]


Brain sections were prepared and stained using eosin and hematoxylin. Each brain section was examined a minimum of 10 fields and images were captured by optical microscope (Olympus BX51 fluorescent microscope) at ×10.[21]


Deparaffinized the sections with xylene and ethanol and then incubated with H2O2 and methanol for 10 min. Then, boiled the sections for 10 min with 10 mM citrate buffer using 1000W microwave oven. The tissue sections were then incubated for overnight at 4°C with the primary antibodies (NOX 4, P47phox and gp91phox in a dilution of 1: 5000, 1:2000, and 1:2000 respectively). Then the sections were treated with IHC reagent (PV 6001 and PV6002; Zhongshan Goldenbridge, Beijing, China) for 30 min at 37oC. Sections were turned brown after DAB (3,3'-diaminobenzidine) colorization. Few sections were incubated with primary antibody as a negative control and followed the above-mentioned steps and no staining was observed.[22]

Statistical analysis

Statistics were performed by using prism graph pad software (version 5.01) and statistical significance was considered at P < 0.05. Each value was a mean ± standard deviation analyzed by one-way or two-way ANOVA using Bonferroni's post hoc test.

 » Results Top

Diosmin treatment effect on elevated plus maze

Effect of DSN treatment on the open field time spent, % open field entries and rearing moments of animals are represented in [Figure 1]. The time spent in the open field by the arsenic control group animals was found to be less compared to the normal control animals. DSN treatment animals at both doses have shown a significant (P < 0.05) rise in the time spent in open field from the 7th day of the study. Arsenic treatment more over decreased % open field entries and rearing behavior of animals in the EPM. DSN at 100 mg/kg had significantly improved the % open field entries and the rearing moments from the 7th day onward, whereas DSN 50 mg/kg from the14th day onward compared to the arsenic control group.
Figure 1: Diosmin treatment effect on EPM test a) Open field time spent (S) b) % Open field entries c) Number of rearing moments *p<0.05 is considered significant in comparison with normal and # p<0.05 is considered significant in comparison with arsenic control. E sentence

Click here to view

Diosmin treatment effect on forced swim test

Regarding the arsenic control group a significantly shortened immobility period while swimming was observed in FST compared to normal group animals. [Figure 3]a represents data for the effect of DSN co-administration on the immobility time period of animals. DSN at both the doses the immobility time has significantly (P < 0.05) increased from the 7th day onward compared to arsenic control animals dose dependently. The DSN results were in comparison with apocynin treatment.
Figure 2: Diosmin treatment effect on hole-board test (Number of head dips and rearings) n = 6 (a) Number of head dips of hole-board test (b) Number of rearing moments of hole-board test. *P < 0.05 is considered significant in comparison with normal and #P < 0.05 is considered significant in comparison with arsenic control

Click here to view
Figure 3: Diosmin treatment effect on forced swim test (Immobility period; n = 6) and beam walk test (Number of leg slips; n = 6) (a) Immobility period of forced swim test (b) Number of leg slips during beam walk. *P < 0.05 is considered significant in comparison with normal and #P < 0.05 is considered significant in comparison with arsenic control

Click here to view

Diosmin treatment effect on hole-board test

The number of head dips and rearing moments of the animals were recorded in the hole-board test and the results are represented in [Figure 2]a and [Figure 2]b. Head dipping behavior of the animals treated with only arsenic was recorded as significantly less compared the normal group and the number of head dips upon DSN co-treatment was significantly (P<0.05) increased according to the dose from the 7th day onwards. In addition, the rearing activity of the animals on the hole-board which was decreased by the arsenic only treatment also improved by DSN administration at both doses.

Diosmin treatment effect on the beam walk test

In the beam walk test the number of leg slips from the beam while crossing it were calculated, arsenic control group animals have found to show more leg slips on the beam compared to the normal group. DSN at 50 and 100 mg/kg have shown a significant and dose dependent decrease in the number of leg slips in the animals. The results of DSN were comparable to that of apocynin and given in [Figure 3]b.

Diosmin treatment effect on arsenic content in the brain tissue

Arsenic in the brain is considered to be proportional to the damage induced by arsenic to the brain. The animals treated with only arsenic was found to have 15.49 ± 0.74 ng/mg of tissue of arsenic. This concentration of arsenic was decreased to 7.95 ± 0.96 and 6.03 ± 0.20 ng/mg of tissue, respectively, by DSN 50 and 100 mg/kg treatment on the 21st day of the study. The significant (P < 0.05) difference was found between the disease control group and treatment groups represented in [Table 1].
Table 1: Diosmin treatment effect on arsenic content in the brain tissue (n=6)

Click here to view

Diosmin treatment effect on neurotransmitter levels

A significant (P < 0.05) decrease in the levels of dopamine, noradrenaline and serotonin was noticed in the arsenic exposed animals upon 21 days of arsenic treatment. Simultaneously, treatment of arsenic and DSN at 50 and 100 mg/kg had restored dopamine, noradrenaline, and serotonin levels significantly according to the dose. Results are shown in [Table 2].
Table 2: Diosmin treatment effect on neurotransmitter levels (n=6)

Click here to view

Diosmin treatment effect on the antioxidant profile

Arsenic treatment had significantly (P < 0.05) brought down the enzymes GSH, CAT, and SOD level as compared to the normal control while DSN co-administration at doses 50 and 100 mg/kg reinstituted the diminished levels of antioxidant enzymes to normal. Moreover, a significant increase in the MDA and NO in arsenic only treated animals and the co-administration of DSN has dose dependently brought back the elevated levels to normal. The data are depicted in [Table 3].
Table 3: Diosmin treatment effect on antioxidant profile (n=6)

Click here to view

Diosmin treatment effect on total proteins and protein carbonyl levels

Total proteins and protein carbonyl levels were measured in the brain tissue samples to estimate the protein oxidative damage caused by arsenic. Arsenic-only treatment had significantly decreased the total protein in the animals than the normal control animals and additionally the protein carbonyl content was also measured to be high in the arsenic control group. On treatment with DSN, the total protein levels were significantly increased than the arsenic control animals and DSN also suppressed the protein carbonyl formation in the brain compared to the arsenic control group. It indicated the protective activity of DSN against oxidative damage of proteins. The data are given in [Table 4]. The results were shown in [Figure 4].
Figure 4: Diosmin treatment effect on brain histopathology (a) Normal control group and red arrow representing the normal brain architecture and cells (b) Arsenic group showing induction of pyknosis (Black arrow), hyperemic blood vessels (Yellow arrow), vaccuoles (Green arrow) and necrosis in brain cells (Blue arrow) (c) Apocynin treatment group (d) diosmin (50 mg/kg) showing both pyknotic cells (Black arrow) and normal neuronal cells (Red arrow) and (e) diosmin (100 mg/kg)

Click here to view
Table 4: Diosmin treatment effect on total proteins and protein carbonyl levels (n=6)

Click here to view

Diosmin treatment effect on brain histopathology

The normal control group had exhibited the clear glial and neuronal cell arrangement. The arsenic-only treatment had brought degenerative changes in the brain tissue; the preliminary observations indicated vacuolization, hyperemic blood vessels, and inflammatory infiltration. Arsenic was also found to induce karyosis, pyknosis, and necrosis in the neuronal cells. DSN 50 mg/kg treated group has shown less pyknotic and necrotic neuronal cells than the arsenic only treated animals and DSN at a dose of 100 mg/kg has shown good regenerative changes and the normal histology of the brain was retained.

Diosmin treatment effect on immunohistochemistry of brain sections

When the brain sections were treated with Anti NOX 4, gp91phox and p47phox the arsenic control animals were showed an increased expression of NOX 4 and its subunits gp91phox and p47phox when compared to the normal which are indicated in [Figure 5] [Figure 6] [Figure 7]. Integrated densities of NOX 4, gp91phox, P47phox expressions are represented in [Figure 8]. DSN treatment has significantly suppressed their expression and the results were comparable to that of apocynin treatment.
Figure 5: Diosmin treatment effect on NOX 4 expression (a) normal control (b) arsenic control group (c) apocynin 10 mg/kg (d) diosmin (50 mg/kg) and (e) diosmin (100 mg/kg)

Click here to view
Figure 6: Diosmin treatment effect on gp91phox expression (a) normal control (b) arsenic control group (c) apocynin 10 mg/kg (d) diosmin (50 mg/kg) and (e) diosmin (100 mg/kg)

Click here to view
Figure 7: Diosmin treatment effect on P47phox expression (a) normal control (b) arsenic control group (c) apocynin 10 mg/kg (d) diosmin (50 mg/kg) and (e) diosmin (100 mg/kg)

Click here to view
Figure 8: Integrated densities of NOX 4, gp91phox, P47phox expressions (a) indicating the expression of NOX 4 in different treatment groups (b) the expression of gp91phox in different treatment groups (c) the expression of P47phox in different treatment groups. *P < 0.05 is considered significant in comparison with normal and #P < 0.05 is considered significant in comparison with arsenic control

Click here to view

 » Discussion Top

Arsenic is a toxic metalloid and its exposure was reported for its various neurocognitive, neurobehavioral, and neurological toxicities in the earlier studies. The current study was focused on the neurobehavioral changes and neurotoxicities induced by arsenic upon exposure and to bring out a natural therapeutic option to combat the adverse neuronal effects of arsenic. Trivalent arsenic undergoes methylation in the biological system and produces methylated arsenicals in the presence of S-adenosylmethionine.[23] Diversified mechanisms were elucidated by various investigators for arsenic-initiated neuronal toxicities including elevated oxidative stress, depletion of biogenic amines in different brain regions, and augmentation of apoptosis in the brain cells. Arsenic was reported to promote neuronal apoptosis by activating of JNK3 and p38 MAP Kinases in a study with cerebral neurons and arsenic also, it involves in cytoskeletal framework disruption and in turn axonal degradation.[24] The present investigation furthermore dealt with the exploration of NOX 4 and its subunits expression in connection to oxidative stress produced by arsenic and to found the effect of DSN supplementation in suppressing the NOX 4 overexpression and oxidative stress as well.

DSN, a natural flavonoid illustrated for disparate pharmacological activities, but documented evidence suggest that it is most effective in relieving neuropathic pain. Analgesic effect of DSN in case of neuropathic pain is by activating NO-cGMP-PKG-ATP-sensitive potassium channels signaling pathway and anti-inflammatory activity by suppressing the mRNA expression of pro-inflammatory mediators.[7],[25] However, neuroprotective properties and its mechanisms of DSN remained unexplored. DSN was screened at two different doses of 50 and 100 mg/kg to predict the neuroprotective effectiveness in arsenic produced oxidative stress and neuronal damage.

Arsenic was employed at various doses by different routes in multiple studies to analyze its toxicity manifestations. Intake of drinking water constituting arsenic for 60 days induces nitrosative stress and DNA damage in the brain tissue.[26] Human fetal brain explants exposed to 0.3 mg/l of arsenic have evidenced apoptosis and necrosis in the neuronal cells after 18 days of exposure.[27] In the present investigation 13 mg/kg of sodium arsenite was administered for 21 days and evaluated neurobehavioral, biochemical, histopathological alterations and also the expression of NOX 4 in the brain.

The neurobehavioral and neurocognitive change upon arsenic exposure was previously showed by few earlier investigations. Here, in the present investigation, the neurobehavioral changes were monitored on every 7th day for 21 days and the changes found with arsenic only treated rats was a radical decrease in the time spent in open field and % entries to the open field of EPM, immobility period in FST, hole dipping behavior on hole-board. Moreover, the rearing moments on EPM and hole-board were also notably decreased upon arsenic treatment. These observed changes in the animal behavior clearly demarcated the property of inducing anxiety-related behavior by arsenic in the rats. These outcomes were in association with the previous demonstrations which reported that upon subchronic exposure to arsenic for 4 weeks in drinking water enhances anxiety-like behavior followed by depression after 8th week of exposure by acting on the BDNF-TrkB signaling pathway.[28] Furthermore, arsenic affects the locomotor activity which was previously illustrated in a study by Itoh et al., where mice treated with arsenic trioxide has exhibited biphasic locomotor activity.[29] This study outcomes also in concurrence with the previous results by representing an altered locomotor activity in the animals treated with arsenic. Co-administration of DSN has manifested the positive results by suppressing the anxiety-like behavior induced by arsenic and corrected the locomotor in-coordination and balance in the animals.

Biogenic amines are the essential elements in maintaining the behavioral and physiological functions of the biological system and alterations in the biogenic amine levels affect the neuronal signaling and behavioral responses.[30] Short-term and long-term exposure to arsenic in both developmental and adult rats have shown an alteration in the neurotransmitter levels. Adult rats exposed to 5 mg/kg of arsenic have evidenced diminished DA and 5HT levels in the striatum, whereas in the hippocampus decreased NA and 5HT was observed. In developing rats given with 5 mg/kg of arsenic for 60 days, DA levels were decreased in hypothalamus, cerebellum, hippocampus, and nucleusaccumbens, while those of increased in the motor cortex and brain stem. A selective change in the brain monogenic amine levels was observed when mice treated with arsenic trioxide, that is an increase in NA, 5HT, and DOPAC in the cerebral cortex and hypothalamus and decrease in DOPAC and HVA in the corpus striatum.[29] Here, the biogenic amines were tested in the whole brain which revealed a decrease in NA, DA, and 5HT levels. DSN has restored the values to the normal upon 21 days of co-administration with arsenic. An increase in dopamine levels was reported to be responsible for the behavioral changes including increase in anxiety-like behavior and impairment in sensorimotor gating in socially isolated rats.[31] In addition, increased levels of NA and serotonin were also accountable for the increase in anxious behavior.[32],[33],[34] Together these reports support that the restoration of these neurotransmitter levels to normal by DSN might be the underlying mechanism for improvement of arsenic-induced anxiety-like behavior in the experimental animals.

Oxidative stress generally refers to increased intracellular ROS, due to depletion of antioxidant enzymes and activation of few enzyme systems involves in the production of ROS. Cellular damage is one of the prime consequences of uncontrolled oxidative stress and ROS can bring out direct damage to the cellular lipids by causing lipid peroxidation. ROS reacts with varied polyunsaturated fatty acids and produces multiple bi-products including MDA which is considered as a valid biomarker for lipid peroxidation. Here, arsenic treatment has increased the levels of MDA indicating the induction of lipid peroxidation and DSN co-administration ameliorated the MDA levels significantly. The decrease in antioxidant enzyme levels such as GSH, SOD, and CAT was in consistent with the earlier reports of arsenic which depletes antioxidant enzyme levels. Arsenic has high affinity for binds toward GSH due to the presence of the thiol group and depletes its levels, arsenic withdraw the electron from GSH and converts from pentavalent to trivalent arsenicals.[3] GSH is a major antioxidant enzyme which is an important factor in protecting from oxidative stress and keeps redox homeostasis of the biological system. Previously, decreased levels of Gpx, CAT, and SOD in the brain were also reported with rats exposed to arsenic. DSN has restored the antioxidant enzyme levels on its concomitant administration with arsenic revealing its strong antioxidant potential and this can be considered as a key mechanism in suppressing arsenic induced oxidative stress. NO, a pro-inflammatory mediator activated by inflammatory signaling has been recognized as a key causative agent in the pathogenesis of various neurodegenerative disorders. Arsenic-only treatment in this study has augmented the NO production in the rat brain specifies the initiation of neuroinflammation. DSN has documented earlier for its anti-inflammatory mechanisms as it suppresses the activation of pro-inflammatory mediators like interleukin-6 (IL-6), IL-12, NO, PGE2, and TNF-α; also their mRNA expression.[25] Similarly, DSN in the present study also alleviated the NO levels responsible for neuroinflammation. The supplementation with DSN also reduced the formation of protein carbonyls, which has formed by oxidation of proteins due to excess of oxidative stress.

Histopathological changes were examined to evidence the arsenic-induced brain tissue damage. The study results revealed histological changes including vacuolization, formation of hyperemic blood vessels, and infiltration of lymphocytes and macrophages. In addition, karyorrhexis, pyknosis, and necrosis of the neuronal cells were also observed with arsenic treatment. DSN at 100 mg/kg has shown a recovered tissue with nearly normal histology indicating its protective role against the histological changes induced by arsenic.

NOX is mostly presenting on the plasma membranes of phagosomes helps in the transportation of electrons across it and has a prime part in the production of ROS. NOX 4 predominantly expresses in different regions of the brain and acts as a key source of oxidative stress generation in the brain in turn mediates apoptosis in the brain.[35] Arsenic was reported to activate NOX by phosphorylating the p47phox subunit of it and promotes the formation of superoxide radicals and also increases translocation of Rac1 and p67phox subunits in human macrophages.[4],[5],[36] However, induction of NOX 4 by the arsenic in the brain has not yet investigated and gives a new path in finding the prime mechanisms of arsenic-induced neurotoxicity. The IHC studies of the brain sections unfolded the results of over expression of NOX 4 and its subunits. These results were in concurrence with the previous demonstrations; phosphorylation of p47phox leading to activation of NOX. Apocynin a potent NOX inhibitor was employed as a positive control or standard for the study to compare the efficacy of DSN in inhibiting NOX. DSN treatment at high doses has significantly suppressed the expression of NOX4 and its subunits p47phox and gp91phox nearly similar to the apocynin and the results were comparable.

 » Conclusion Top

The toxicity results found with arsenic treatment were in correlation with the levels of arsenic reached to the brain. The arsenic content in the brain was high in the arsenic control group animals and DSN has dose dependently reduced the levels of arsenic in the brain. The molecular mechanisms of DSN in reducing the levels of arsenic in the brain were unclear and need to be studied in detail. In conclusion, the study illustrated that arsenic activates NOX4 and its subunits in the brain and in turn produce oxidative stress leading to neuronal damage. DSN exhibited a good neuroprotective activity by suppressing the expression of NOX4 and excessive oxidative stress. The study testifies that it can be developed as an effectual therapeutic agent in combating arsenic-induced neurotoxicity.


Authors would like to acknowledge DST-FIST, UGC-SAP of IPT and DST-CURIE, SSIE-TBI of Sri Padmavati Mahila Visvavidyalayam for facilitating the instrumentation required for the study.

Financial support and sponsorship

The authors are thankful to UGC-NFSC scheme for providing funding for Rupasree Peruru to carry out the study.

Conflicts of interest

There are no conflicts of interest.

 » References Top

Guha Mazumder DN, Haque R, Ghosh N, De BK, Santra A, Chakraborty D, et al. Arsenic levels in drinking water and the prevalence of skin lesions in West Bengal, India. Int J Epidemiol 1998;27:871-7.  Back to cited text no. 1
Kannan GM, Tripathi N, Dube SN, Gupta M, Flora SJ. Toxic effects of arsenic (III) on some hematopoietic and central nervous system variables in rats and guinea pigs. J Toxicol Clin Toxicol 2001;39:675-82.  Back to cited text no. 2
Flora SJ. Arsenic-induced oxidative stress and its reversibility. Free Radic Biol Med 2011;51:257-81.  Back to cited text no. 3
Smith KR, Klei LR, Barchowsky A. Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 2001;280:L442-9.  Back to cited text no. 4
Lemarie A, Bourdonnay E, Morzadec C, Fardel O, Vernhet L. Inorganic arsenic activates reduced NADPH oxidase in human primary macrophages through a Rho kinase/p38 kinase pathway. J Immunol 2008;180:6010-7.  Back to cited text no. 5
Tahir M, Rehman MU, Lateef A, Khan R, Khan AQ, Qamar W, et al. Diosmin protects against ethanol-induced hepatic injury via alleviation of inflammation and regulation of TNF-α and NF-kB activation. Alcohol 2013;47:131-9.  Back to cited text no. 6
Bertozzi MM, Rossaneis AC, Fattori V, Longhi-Balbinot DT, Freitas A, Cunha FQ, et al. Diosmin reduces chronic constriction injury-induced neuropathic pain in mice. Chem Biol Interact 2017;273:180-9.  Back to cited text no. 7
Jain D, Bansal MK, Dalvi R, Upganlawar A, Somani R. Protective effect of diosmin against diabetic neuropathy in experimental rats. J Integr Med 2014;12:35-41.  Back to cited text no. 8
Petrônio MS, Zeraik ML, Fonseca LM, Ximenes VF. Apocynin: Chemical and biophysical properties of a NADPH oxidase inhibitor. Molecules 2013;18:2821-39.  Back to cited text no. 9
Liu X, Zhang X, Zhang J, Kang N, Zhang N, Wang H, et al. Diosmin protects against cerebral ischemia/reperfusion injury through activating JAK2/STAT3 signal pathway in mice. Neuroscience 2014;268:318-27.  Back to cited text no. 10
Das J, Ghosh J, Manna P, Sinha M, Sil PC. Arsenic-induced oxidative cerebral disorders: Protection by taurine. Drug Chem Toxicol 2009;32:93-102.  Back to cited text no. 11
Lister RG. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology (Berl) 1987;92:180-5.  Back to cited text no. 12
Detke MJ, Rickels M, Lucki I. Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants. Psychopharmacology (Berl) 1995;121:66-72.  Back to cited text no. 13
File SE, Pellow S. The effects of triazolobenzodiazepines in two animal tests of anxiety and in the holeboard. Br J Pharmacol 1985;86:729-35.  Back to cited text no. 14
Goldstein LB, Davis JN. Beam-walking in rats: Studies towards developing an animal model of functional recovery after brain injury. J Neurosci Methods 1990;31:101-7.  Back to cited text no. 15
Luong TN, Carlisle HJ, Southwell A, Patterson PH. Assessment of motor balance and coordination in mice using the balance beam. JoVE (Journal of Visualized Experiments) 2011: 49: 2376.  Back to cited text no. 16
Vahidnia A, Romijn F, van der Voet GB, de Wolff FA. Arsenic-induced neurotoxicity in relation to toxicokinetics: Effects on sciatic nerve proteins. Chem Biol Interact 2008;176:188-95.  Back to cited text no. 17
Schlumpf M, Lichtensteiger W, Langemann H, Waser PG, Hefti F. A fluorometric micromethod for the simultaneous determination of serotonin, noradrenaline and dopamine in milligram amounts of brain tissue. Biochem Pharmacol 1974;23:2437-46.  Back to cited text no. 18
Alam MN, Bristi NJ, Rafiquzzaman M. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm J 2013;21:143-52.  Back to cited text no. 19
Dalle-Donne I, Rossi R, Giustarini D, Milzani A, Colombo R. Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta 2003;329:23-38.  Back to cited text no. 20
Zhang J, Xiong H. Brain tissue preparation, sectioning, and staining. Current Laboratory Methods in Neuroscience Research: Springer, new York: 2014: pp. 3-30.  Back to cited text no. 21
Goslin K, Schreyer DJ, Skene JH, Banker G. Changes in the distribution of GAP-43 during the development of neuronal polarity. J Neurosci 1990;10:588-602.  Back to cited text no. 22
Drobna Z, Styblo M, Thomas DJ. An overview of arsenic metabolism and toxicity. Current protocols in toxicology 2009; 42: 4.31.1- 4.31.6.  Back to cited text no. 23
Namgung U, Xia Z. Arsenic induces apoptosis in rat cerebellar neurons via activation of JNK3 and p38 MAP kinases. Toxicol Appl Pharmacol 2001;174:130-8.  Back to cited text no. 24
Berkoz M. Diosmin suppresses the proinflammatory mediators in lipopolysaccharide-induced RAW264.7 macrophages via NF-κB and MAPKs signal pathways. Gen Physiol Biophys 2019;38:315-24.  Back to cited text no. 25
Ma N, Sasoh M, Kawanishi S, Sugiura H, Piao F. Protection effect of taurine on nitrosative stress in the mice brain with chronic exposure to arsenic. J Biomed Sci 2010;17 Suppl 1:S7.  Back to cited text no. 26
Chattopadhyay S, Bhaumik S, Purkayastha M, Basu S, Nag Chaudhuri A, Das Gupta S. Apoptosis and necrosis in developing brain cells due to arsenic toxicity and protection with antioxidants. Toxicol Lett 2002;136:65-76.  Back to cited text no. 27
Chang CY, Guo HR, Tsai WC, Yang KL, Lin LC, Cheng TJ, et al. Subchronic arsenic exposure induces anxiety-like behaviors in normal mice and enhances depression-like behaviors in the chemically induced mouse model of depression. Biomed Res Int 2015;2015:159015.  Back to cited text no. 28
Itoh T, Zhang YF, Murai S, Saito H, Nagahama H, Miyate H, et al. The effect of arsenic trioxide on brain monoamine metabolism and locomotor activity of mice. Toxicol Lett 1990;54:345-53.  Back to cited text no. 29
Torda C. Effects of catecholamines on behavior. J Neurosci Res 1976;2:193-202.  Back to cited text no. 30
Yorgason JT, España RA, Konstantopoulos JK, Weiner JL, Jones SR. Enduring increases in anxiety-like behavior and rapid nucleus accumbens dopamine signaling in socially isolated rats. Eur J Neurosci 2013;37:1022-31.  Back to cited text no. 31
Bordukalo-Niksic T, Mokrovic G, Stefulj J, Zivin M, Jernej B, Cicin-Sain L. 5HT-1A receptors and anxiety-like behaviours: Studies in rats with constitutionally upregulated/downregulated serotonin transporter. Behav Brain Res 2010;213:238-45.  Back to cited text no. 32
Morilak DA, Barrera G, Echevarria DJ, Garcia AS, Hernandez A, Ma S, et al. Role of brain norepinephrine in the behavioral response to stress. Prog Neuropsychopharmacol Biol Psychiatry 2005;29:1214-24.  Back to cited text no. 33
Johnston AL. The implication of noradrenaline in anxiety. In: New Concepts in Anxiety. Springer; London; 1991. p. 347-65.  Back to cited text no. 34
Kleinschnitz C, Grund H, Wingler K, Armitage ME, Jones E, Mittal M, et al. Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration. PLoS Biol 2010;8:e1000479.  Back to cited text no. 35
Qian Y, Liu KJ, Chen Y, Flynn DC, Castranova V, Shi X. Cdc42 regulates arsenic-induced NADPH oxidase activation and cell migration through actin filament reorganization. J Biol Chem 2005;280:3875-84.  Back to cited text no. 36


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]

  [Table 1], [Table 2], [Table 3], [Table 4]

This article has been cited by
1 The protective effect of natural or chemical compounds against arsenic-induced neurotoxicity: Cellular and molecular mechanisms
Mersedeh Shayan, Samira Barangi, Hossein Hosseinzadeh, Soghra Mehri
Food and Chemical Toxicology. 2023; : 113691
[Pubmed] | [DOI]
2 Autophagy and apoptosis cascade: which is more prominent in neuronal death?
Rohan Gupta, Rashmi K. Ambasta, Pravir Kumar
Cellular and Molecular Life Sciences. 2021; 78(24): 8001
[Pubmed] | [DOI]
3 Pharmacology of Diosmin, a Citrus Flavone Glycoside: An Updated Review
Samar H. Gerges, Sara A. Wahdan, Doaa A. Elsherbiny, Ebtehal El-Demerdash
European Journal of Drug Metabolism and Pharmacokinetics. 2021;
[Pubmed] | [DOI]


Print this article  Email this article


Site Map | Home | Contact Us | Feedback | Copyright and Disclaimer | Privacy Notice
Online since 20th July '04
Published by Wolters Kluwer - Medknow