http://informahealthcare.com/txmISSN: 1537-6516 (print), 1537-6524 (electronic)

Toxicol Mech Methods, Early Online: 1–8! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2014.945108

ORIGINAL ARTICLE

Hesperidin restores experimentally induced neurotoxicity in Wistar rats

Mehar Naseem and Suhel Parvez

Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi, India

Abstract

Carbon tetrachloride (CCl4) is a highly toxic industrial solvent with pronounced systemic toxicityincluding brain. Neurotoxicity may be a direct result of hepatic dysfunction from CCl4intoxication. Over the years CCl4 has been used as an excellent model for studyingexperimentally induced neurotoxicity in murine models. Hesperidin (HP) is a knowncytoprotectant with comprehensive anti-oxidant and neuroprotective properties. The aim ofthe present study was to evaluate experimentally induced neurotoxicity by CCl4 and itsabrogation by using antioxidant potential of HP. CCl4 caused a significant enhancement in thelipid peroxidation (LPO) levels and protein carbonyl (PC) content. HP supplementationsignificantly restored the LPO levels and PC content. It also replenished the altered enzymaticand non-enzymatic antioxidants in brain tissues of rats. The neurotoxicity markers were alsorestored to normalcy with HP treatment. It is suggested that HP, by attenuating neuronaloxidative stress, holds promise that can ameliorate CCl4-induced neurotoxicity. HP has thepotential to be explored as a universal neuroprotectant in xenobiotically induced neurotoxicitymediated by oxidative stress.

Keywords

Biomarkers, brain, carbon tetrachloride,natural compound, oxidative stress,rodents

History

Received 14 April 2014Revised 15 June 2014Accepted 11 July 2014Published online 30 July 2014

Introduction

A large increase in reactive oxygen species (ROS) which are

produced during various cellular metabolism leads to several

damaging effects like tissue damage, DNA damage, oxidative

stress, lipid peroxidation (LPO), and cell death (Saquib et al.,

2012). ROS is implicated in pathogenesis of a number of

neurodegenerative diseases in brain, cognitive dysfunction,

cancer, and a variety of other conditions (Kovacic &

Somanathan, 2012). Brain also contains high level of

ascorbate and iron, which may be essential during brain

development involved in the production of ROS. Numerous

studies have indicated that oxidative stress is responsible for

the disruption of blood brain barrier (BBB) integrity

associated with neurodegenerative disorders as it is prone to

ROS (Freeman & Keller, 2012). Brain may deal in a different

way with oxidative stress as compared with liver or kidney

(Seminotti et al., 2012).

Carbon tetrachloride (CCl4) is a well-known hepatotoxic

agent which has also been implicated in inhibiting mitochon-

drial respiratory chain in the brain (Boer et al., 2009). Natural

compounds have been used for various purposes in medicines,

cosmetics, and also as dietary supplements (Albarracin et al.,

2012). Flavonoids are compounds, which are antioxidants

from natural origin and are present in a number of

natural sources (Kamaraj et al., 2009). Hesperidin

(HP, 3,5,7-trihydroxy flavanone-7-rhamnoglucoside) is a

biologically and pharmacologically active citrus bioflavonoid

by-product of citrus cultivation, abundantly found in sweet

orange and lemon (Gaur et al., 2011). It has been previously

reported that HP is having free radical scavenging as well as

anti-LPO properties and has been shown to be powerful

against ROS and it has also been assayed for its neuropro-

tective efficacy in diabetic rat model (Ibrahim, 2008).

It has attracted particular interest due to its several health

beneficial effects like anti-inflammatory, anticarcinogenic,

antimicrobial, and immunomodulatory effects (Nones et al.,

2010).

HP has been shown to be protective against various

neurobehavioral alterations caused (Viswanatha et al., 2012).

It has been well documented that HP is a potential

neuroprotective antioxidant, it may because of being lipo-

philic in nature, crosses the BBB easily (Salem et al., 2012).

HP deals with neurotoxicity due to its antioxidative and anti-

inflammatory properties and also protects the dopamine

depletion which enhances it neuroprotective efficiency

(Tamilselvam et al., 2013). However, protective role of HP

against CCl4 induced neurotoxicity has not been investigated

in a rodent model. The aim of the present study was to explore

modulatory effects of HP against CCl4-induced neurotoxicity

in Wistar rats.

Address for correspondence: Dr. Suhel Parvez, Department of MedicalElementology and Toxicology, Jamia Hamdard (Hamdard University),New Delhi 110062, India. Tel: +91 11 26059688x5573. Fax:+91 11 26059663. E-mail: drsuhelparvez@yahoo.com, sparvez@jamiahamdard.ac.in

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Materials and methods

Chemicals

Sulfosalicylic acid and thiobarbituric acid (TBA) were

obtained from Hi-Media Labs (Mumbai, India). Butylated

hydroxytoluene (BHT), o-phosphoric acid (OPA), trichloro-

acetic acid (TCA), 1-Amino-2-naphthol-4-sulfonic acid

(ANSA), 2,4-dinitrophenylhydrazine (DNPH), and perchloric

acid (PCA) were purchased from Merck Limited (Mumbai,

India). Bovine serum albumin, reduced glutathione (GSH),

oxidized glutathione (GSSG), and benzylamine hydrochloride

(BAHC) were purchased from Sigma Chemicals Co.

(St. Louis, MO). Reduced nicotinamide adenine dinucleotide

phosphate (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB),

5,5-dithiobis-2-nitrobenzoic acid (DTNB), ethylenediamine-

tetraacetic acid (EDTA), and acetylthiocholine iodide (ATC)

were obtained from SRL (Mumbai, India). Other routine

chemicals were obtained from Hi-Media Labs (Mumbai,

India), Merck Limited (Mumbai, India), and SRL

(Mumbai, India). CCl4 and HP were obtained from Merck

Limited (Mumbai, India) and SRL Labs (Mumbai, India),

respectively.

Animals

Adult male Wistar rats weighing 250–300 g were used for the

study. The experimental rats were acclimatized for a week

prior to the commencement of the dose regime. The animals

were housed under standard laboratory conditions where

temperature was maintained at 22 ±2 �C with a relative

humidity of 65 ± 10% and a photoperiod of 12 h light/dark

cycle. All the experiments were carried out according to the

standard guidelines of Institutional Animal Ethics Committee

(IAEC) of Jamia Hamdard (Hamdard University).

Experimental design

The animals were randomly divided into four groups, and

each group consisted of seven animals. The description about

the different groups is as follows:

Group 1: Control group. The animals were treated with a

vehicle for HP (i.e. normal saline) by oral route for eight

consecutive days and a single subcutaneous (s.c.) injection of

olive oil on the 7th day.

Group 2: HP group. In this group, rats received HP (200 mg/

kg b.w., oral) treatment for a period of 8 d.

Group 3: CCl4 group. The animals were administered

normal saline for 8 d and were exposed to CCl4(2 ml/kg b.w., s.c., 40% v/v in olive oil) on the 7th day of

the treatment.

Group 4: HP (200 mg/kg b.w., oral) + CCl4 (2 ml/kg

b.w., s.c.). Rats were given HP treatment for 8 d and a

single injection of CCl4 on the 7th day along with HP.

The dose of CCl4 and HP used in our study was based on

previously published research reports (El-Sayed et al., 2008;

Jayakumar et al., 2008). At the end of the experimental period

of 8 d, the animals were anesthetized and sacrificed by

decapitation. The brain was immediately dissected, and

washed in chilled phosphate buffer to remove blood. The

effect of CCl4 and role of HP on the oxidative stress

biomarkers were studied in the brain tissue.

Sample preparation

Post-mitochondrial supernatant (PMS) and homogenate

preparation

The brain of the animals was promptly excised and washed

with chilled phosphate-buffered saline for the removal of

blood. The brain was homogenized 1:10 w/v in 0.1 M sodium

phosphate buffer (pH 7.4) with a Potter–Elvehjem homogen-

izer. The homogenates obtained were used to estimate brain

TBARS. The homogenates were centrifuged at 10 500 g for

20 min at 4 �C to get the PMS, using a REMI C-24 centrifuge

(Remi Sales and Engineering, Ltd., Mumbai, India). The

PMS supernatant obtained was used for the analysis of

oxidative stress biomarkers, non-enzymatic antioxidants,

enzymatic antioxidants, and neurotoxicity biomarkers.

Biochemical estimation

Oxidative stress biomarkers

Determination of LPO. The extent of LPO was measured

using the procedure of Tabassum et al. (2007). The concen-

tration of thiobarbituric acid reactive substances (TBARS)

was determined using an extinction coefficient of

1.56� 105 M�1 cm�1 and results were expressed as nmol

TBARS formed/h/mg protein.

Determination of protein oxidation. Protein oxidation in brain

PMS was measured as a concentration of protein carbonyls

(PC) formed. PC level was determined using DNPH assay

according to the procedure of Chaudhary & Parvez (2012).

The results were expressed as nmol DNPH incorporated/mg

protein based on the molar extinction coefficient of

2.1� 104 M�1 cm�1.

Non-enzymatic antioxidants

Estimation of non-protein thiol (NP-SH). The NP-SH was

measured according to the method described by Govil et al.

(2012). The molar extinction coefficient of 13 100M�1 cm�1 at

412 nm was used for the determination of the NP-SH content.

The values were expressed as nmol of the NP-SH/mg protein.

Estimation of reduced glutathione (GSH). The GSH content

was estimated according to the method of Chaudhary &

Parvez (2012). The GSH concentration was calculated as

nmol GSH/mg protein using a molar extinction coefficient of

1.36� 104 M�1 cm�1.

Enzymatic antioxidants

Activity of glutathione peroxidase (GPx). The activity of GPx

was assayed according to the method of Haque et al. (2003).

The assay mixture consisted of sodium phosphate

buffer (0.1 M, pH 7.4), EDTA (1 mM), sodium azide (1 mM),

GSH (1 mM), NADPH (0.02 mM), H2O2 (0.25 mM), and 0.1 ml

of PMS of brain in a total volume of 2 ml. Oxidation of NADPH

was recorded kinetically at 340 nm. The enzyme activity

was calculated as nmol NADPH oxidized/min/mg protein,

using a molar extinction coefficient of 6.22� 103 M�1 cm�1.

Activity of glutathione reductase (GR). The activity of GR was

assayed by the method of Haque et al. (2003). The assay

2 M. Naseem & S. Parvez Toxicol Mech Methods, Early Online: 1–8

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system consisted 0.1 M phosphate buffer (pH 7.4), 0.5 mM

EDTA, 1 mM GSSG, 0.1 mM NADPH, and 0.3 ml super-

natant of brain tissue in a total volume of 2 ml. The enzyme

activity was quantitated at 25 �C by measuring the disappear-

ance of NADPH at 340 nm, and was calculated as nmol

NADPH oxidized/min/mg protein using a molar extinction

coefficient of 6.223� 103 M�1 cm�1.

Activity of glutathione-s-transferase (GST). The activity of

GST was assayed using the method of Haque et al. (2003) by

the catalytic reaction of CDNB with the sulfhydryl group of

glutathione and was measured kinetically at 340 nm. The

assay system consisted of 0.1 M phosphate buffer (pH 7.4),

10 mM GSH, 10 mM CDNB, and 0.1 ml of PMS of rat brain.

The enzymatic activity was calculated as nmol CDNB

conjugate formed/min/mg protein using a molar coefficient

of 9.6� 103 M�1 cm�1.

Neurotoxicity biomarkers

Activity of monoamine oxidase (MAO). The activity of MAO

was measured by using the method of Chaudhary & Parvez

(2012). The enzyme activity was calculated as mmol BAHC

hydrolyzed/min/mg protein using a molar extinction coeffi-

cient of 7.6925 M�1 cm�1.

Activity of acetylcholinesterase (AChE). The activity of AChE

was estimated by using the method of Chaudhary & Parvez

(2012). The enzyme activity was calculated as nmol ATC

hydrolyzed/min/mg protein using a molar extinction coeffi-

cient of 1.36� 104 M�1 cm�1.

Activity of total ATPase. The activity of total ATPase was

measured as the release of inorganic phosphate (Pi) by the

method of Chaudhary & Parvez (2012). The activity was

measured as mg Pi liberated/min/mg protein.

Determination of protein

Estimation of protein contents in biological fractions of brain

was done using bovine serum albumin as standard by the

method of Lowry et al. (1951).

Statistical analysis

The results obtained were expressed as mean ± standard error

(SE). All data were analyzed using analysis of variance

(ANOVA) followed by Tukey’s test. Values of p50.05 were

considered as significant. All the statistical analyses were

performed using graph pad prism 5 software (Graph Pad

Software, Inc., San Diego, CA).

Results

No mortality and significant alteration in the body weight of

different groups of subject/animals were documented during

and after the treatment (data not shown).

Oxidative stress biomarkers

Effect on LPO

Treatment of animals with CCl4 (2 ml/kg b.w.) showed a

significant (p50.001) elevation in the LPO content of brain

homogenate when compared with the control group

(Figure 1A). HP (200 mg/kg, oral) pre-treatment revealed a

significant (p50.01) depletion in the LPO content of brain

homogenate as compared with the CCl4-treated group. HP

alone did not show any significant change in the LPO content

of animals beyond control values.

Effect on protein oxidation (PC)

Exposure to CCl4 resulted in a significant (p50.001)

enhancement in the activity of protein oxidation in the brain

PMS of CCl4-treated animals as compared with the control

group (Figure 1B). In HP pre-treated group, there was a

significant (p50.01) depletion in the PC content of brain

PMS. However, no significant effect in the PC content of HP

treated group was observed when compared with control.

Non-enzymatic antioxidant status

Effect on NP-SH

The level of NP-SH in brain PMS significantly (p50.001)

increased in CCl4-administered group of animals when

compared with the animals of the control group

(Figure 2A). CCl4-exposed HP pre-administered animals

showed a significant (p50.001) decrease in the NP-SH

level as compared with that of CCl4-administered animals.

Figure 1. Effect of CCl4 and HP on (A) LPO and (B) PC in brain of rat.Values were expressed as nmoles of TBARS formed/h/mg protein andnmoles of DNPH incorporated/mg protein, respectively. Each valuerepresented as mean ± SE (n¼ 6). Significant differences were indicatedby ***p50.001 when compared with control and significant differencewere indicated by ##p50.01 when compared with CCl4-treated animals.

DOI: 10.3109/15376516.2014.945108 Hesperidin as natural antioxidant 3

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HP-treated animals showed no any significant alteration in the

NP-SH level in brain PMS when compared with control group

of animals.

Effect on GSH

Significant (p50.001) higher level of the GSH content was

observed in brain PMS of CCl4-exposed animals when

compared with the control group (Figure 2B). HP pre-

treatment prior to CCl4 injection significantly (p50.001)

declines the GSH content and prevented these elevations.

Animals treated with HP did not show any significant change

when compared with the control group.

Enzymatic antioxidants activities

Effect on glutathione-metabolizing enzymes

There was a significant (p50.001) elevation in the activity of

GPx in brain PMS in the CCl4-treated group as compared

with the control group (Figure 3A). HP pretreatment showed a

significant (p50.001) decline in the activity of GPx as

compared with the CCl4-treated group. HP-treated group did

not change the GPx level in the brain PMS when compared

with the control group.

CCl4 significantly increased (p50.001) the glutathione

reductase level (GR) in CCl4-treated group as compared with

the control group (Figure 3B). A single dose of CCl4combined with pretreatment of HP showed a significant

(p50.01) decrease in GR activity as compared with the CCl4-

intoxicated group. HP treatment showed no significant change

in the activity of GR in the PMS of brain.

CCl4 exposure showed a significant (p50.001) rise in the

activity of GST when compared with the control group

(Figure 3C). In a single dose of CCl4 in the HP pre-treated

group revealed a significant (p50.001) decline in the GST

activity in brain PMS as compared with the CCl4-treated

group. Group treated with HP showed no effect in the activity

of GST.

Effect on neurotoxicity biomarkers

There was a significant (p50.001) increase in the activity of

MAO in the CCl4-treated group of animals (Figure 4A). HP

supplementation along with a single dose of CCl4 showed a

significant (p50.001) decline in the activity of MAO as

compared with that of the CCl4-treated group. No effect in the

activity of MAO was seen in the HP-treated group.

The CCl4-treated group showed significant (p50.001) rise

in the activity of AChE as compared with the control group

(Figure 4B). The HP pre-exposed group significantly

(p50.001) decreased the activity of AChE when compared

with the CCl4-treated group and prevented such elevations.

The HP-treated group exhibited no remarkable difference

when compared with control.

A single dose of CCl4 caused a significant (p50.01)

increase in the total ATPase content in brain PMS of animals

when compared with control (Figure 4C). HP pre-treatment in

CCl4-treated group caused significant (p50.01) decline in the

activity of ATPase. The HP-treated group did not show

significant change in the activity.

Discussion

Rats intoxicated with CCl4 leads to the generation of ROS,

ultimately resulting in neurotoxicity in rats (Samudram et al.,

2009). These highly reactive free radicals of CCl4 generated

by the CYP are capable of covalently binding to macromol-

ecules and cause intense LPO leading to the degeneration of

cell membrane of endoplasmic reticulum which is eminently

rich in PUFA (Altas et al., 2011). HP possesses highest

reducing power and exhibits anti-oxidative properties (Salem

et al., 2012). TBARS is a by-product of LPO, considered as a

reliable index of tissue damage (Tabassum et al., 2007).

In the present study, increased levels of TBARS in brain

tissue may be due to increased production of these free

radicals which are unstable and highly active, leading to fatty

acid damage following CCl4 intoxication which will lead to

enhanced LPO. The potential amelioration of free radicals

which leads to fatty acid damage by HP, which is a well-

known antioxidant reducing the severity of LPO by decreas-

ing TBARS concentration. It suggested that HP exerts its

protective effect by regulating LPO of membranes by

scavenging the debilitating consequences of free radicals

and augmenting the antioxidant defence system (Pradeep

et al., 2008).

Figure 2. Effect of CCl4 and HP on level of (A) NP-SH and (B) GSH inbrain tissue of rat. Values were expressed as nmoles NP-SH/mg proteinand nmoles GSH/mg protein, respectively. Each value represented asmean ± SE (n¼ 6). Significant differences were indicated by***p50.001 when compared with control. Significant differences###p50.001 were shown when compared with CCl4-treated animals.

4 M. Naseem & S. Parvez Toxicol Mech Methods, Early Online: 1–8

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PC is considered as an excellent biomarker of oxidative

protein damage as proteins are susceptible to oxidative

modification (Dalle-Donne et al., 2003). Oxidative modifica-

tion of proteins changes its configuration via aggregation,

fragmentation, and formation of cross linkages in the

polypeptide chain and also leads to secondary impairment

of biomolecules with loss of protein biological function, leads

to the generation of superoxide radical (Chen et al., 2005).

Addition of carbonyl group in proteins is directly proportional

to the protein subjected to oxidative damage and an increase

in carbonyl content is associated with various pathological

disorders (Almroth et al., 2005). A significant increase in

protein oxidation was seen in the brain of animals with the

single-dose administration of CCl4 as compared with the

control group. It might be due to increased oxidatively

modified protein in brain with the administration of CCl4. Our

result is in agreement with the findings of previous result

performed with liver (Srivastava & Shivanandappa, 2010).

Interestingly, pre-administration of HP attenuated the effect of

CCl4-induced protein damage, indicating that HP has a

protective action against CCl4-induced neurotoxicity. Our

results are in corroboration with the recent finding where HP

has been shown to be a good natural antioxidant against

cisplatin-induced oxidative damage (Sahu et al., 2013). HP

has been found to reduce the PC content in our study thereby

reducing oxidative stress and oxidative damage of protein by

scavenging the free radicals causing modification in the

protein structure.

Dietary antioxidant supplementation such as bioflavonoids

mainly protects cells and organs against ROS-induced oxida-

tive damage (Tsai et al., 2013). GSH scavenges free radicals

and provides a defence system against ROS as well as

detoxifies various xenobiotic compounds, H2O2, lipid hydro-

peroxides, and electrophilic compounds (Manna et al., 2011).

It plays crucial role in defence mechanism and also acts as a

co-enzyme for numerous enzymes involved in the cellular

defence system (Masella et al., 2005). GPx constitutes a

family of enzyme which reduces H2O2 and lipid hydroper-

oxides to the corresponding stable hydroxy compounds and

H2O utilizing GSH as a reducing substrate (Dickinson &

Forman, 2002). GR is a flavoenzyme and is represented by a

single copy gene in humans which takes part in the reduction

of GSSG (oxidized glutathione) to GSH with the help of

NADPH (Li et al., 2010). The activity of GR is very

important to maintain the level of GSH/GSSG ratio. GST is a

well-known phase-2 enzyme which plays a very crucial role in

the detoxification process of electrophilic compounds, favor-

ing the elimination from the body of the organ by conjugation

with glutathione (Afzal et al., 2013). GSH oxidizes to GSSG

by peroxide and GPx. GSH is restored from GSSG by the

enzyme GR using NADPH as an electron donor and the ratio

of GSH/GSSG within a cell is considered as the measure of

Figure 3. Effect of CCl4 and HP on (A) GPx, (B) GR and (C) GST activity in brain of rat. GPx and GR activities were expressed as nmoles of NADPHoxidized/min/mg protein whereas GST activity was expressed as nmoles of CDNB conjugate formed/min/mg protein. Each value represented asmean ± SE (n¼ 6). Significant differences were shown by ***p50.001 when compared with control. Significant differences were indicated by##p50.01 and ###p50.001 when compared with CCl4-treated animals.

DOI: 10.3109/15376516.2014.945108 Hesperidin as natural antioxidant 5

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cellular toxicity (Board & Menon, 2013). An increase in the

activity of GR, GPx, and GST was found in the CCl4-

intoxicated group of animals. An increase in the activity of

glutathione-dependent enzymes reveals the primary response

of the cell to the exposure to oxidative stress-inducing toxic

compounds with the potential of inducing oxidative stress and

damage caused in the cells and the cells may reduce the

severity of oxidative damage by maintaining a reduced state in

the cell. Induction in the activity of GPx on CCl4 adminis-

tration suggests that there might be a rapid oxidation of GSH

mediated by peroxide. This result is in agreement with the

findings of the effect of cadmium (Cd) on different organs,

liver, kidney, and gills of freshwater murrel Channa punctatus

(Dabas et al., 2012). The increased level of GST in CCl4-

intoxicated animals suggests that it might be due to the

conjugation between trichloromethyl radical and GSH to

mitigate the effect of increased oxidative stress. Our result is

in agreement with the result obtained earlier in the brain of

the CCl4-treated group of animals (Soliman & Fahmy, 2011).

HP administration restored the increased level of GSH which

suggests the reversal of the increased oxidative stress. Our

result corroborates with the findings of antioxidative and anti-

inflammatory role of HP on the rat air pouch model of

inflammation (Jain & Parmar, 2011). It may be due to the

depletion in the level of cysteine with the pretreatment of HP

as cysteine which is one of the major constituents of tripeptide

antioxidant GSH which may affect the level of GSH in the HP

pre-treated CCl4-intoxicated group of animals.

MAO, AchE, and Na+/K+-ATPase are considered as

important neurotoxicity biomarkers. MAO is a flavin-

containing brain-specific enzyme which plays an important

role in oxidative deamination of endogenous monoamine

neurotransmitters, regulates the oxidative deamination of

endogenous and exogenous monoamines in the central

nervous system, and converts biogenic amines to their

corresponding aldehydes (Secci et al., 2011). Alteration in

the function of MAO may lead to age-related disorder and a

number of neurological disorders and depression (Meyer

et al., 2006). MAO takes part in the metabolism of

catecholamine like adrenaline, noradrenaline, and dopamine

into free radicals, abundance of which may be responsible for

serotonin syndrome (Ghareeb et al., 2011). An increase in the

activity of MAO suggests that there is a high level of

oxidative stress. In the present assessment, a single dose

of CCl4 was found to be effective in increasing the activity of

MAO. Pretreatment of animals with HP inhibited the activity

of MAO. HP was found to be an effective inhibitor of MAO as

it inhibited the metabolism of the monoamines metabolization

of which form hydrogen peroxide and ammonia which is the

potential toxic products of MAO.

AchE is a key enzyme in the nervous system which is

responsible for the degradation of neurotransmitter,

Figure 4. Effect of CCl4 and HP on (A) MAO, (B) AChE, and (C) total ATPase activity in brain of rat. MAO activity was expressed as mmol BAHChydrolyzed/min/mg protein, AChE activity as nmol ATC hydrolyzed/min/mg protein and total ATPase activity as mg Pi liberated/min/mg protein.Each value represented as mean ± SE (n¼ 6). Significant differences were shown by **p50.01 and ***p50.001 when compared with control.Significant differences were indicated by ##p50.01 and ###p50.001 when compared with CCl4-treated animals.

6 M. Naseem & S. Parvez Toxicol Mech Methods, Early Online: 1–8

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acetylcholine (Ach), during neurotransmission which is

necessary for learning and memory deficits (Parent &

Baxter, 2004) and also plays a very important role in the

cholinergic activity (Antoniades et al., 2002). Our result is in

accordance with a recent finding in which an increase in the

activity of AchE in the region of brain has been reported with

streptozotocin treatment (Biasibetti et al., 2013). An increase

in the activity of AchE in the brain is responsible for the low

availability of acetylcholine and, therefore, it may lead to the

alteration in cholinergic transmission (Tiwari et al., 2009). HP

pre-treatment was able to reverse the altered activity of AchE

which deals with the cognitive defect by increasing cholin-

ergic neurotransmission as reported in a report with a natural

antioxidant curcumin (Agrawal et al., 2010).

The sodium potassium adenosine triphosphate (Na+/K+-

ATPase) is an enzyme which maintains neuronal excitability

and plays a major role in the export and import of three Na+

ions out and two K+ ions in of the cell by utilizing ATP across

the plasma membrane against their potential gradient in the

nervous system and Na+/K+-ATPase has also been reported as

a signal transducer (Lingrel, 2010). The Na+/K+-ATPase is an

integral membrane heterodimer belongs to a superfamily of

proteins collectively designated as P-type ATPase family

(Jaitovich & Bertorello, 2006). In the present study, a

remarkable elevation is shown in the activity of Na+/K+-

ATPase in the CCl4-intoxicated group of rats. HP pre-

treatment suppressed the elevated level of Na+/K+-ATPase

activity in brain due to its neuroprotective nature. Our result is

in agreement with the previous result reported with some

natural compounds, green tea and bamboo leaves on Na efflux

channel in ovariectomized rats (Ryou et al., 2012). Further

studies are required for deciphering cellular and molecular

mechanisms of experimentally induced neurotoxicity and the

role of HP in its attenuation.

Declaration of interest

The authors declare that they have no conflict of interest.

University Grants Commission (UGC), Government of India,

is gratefully acknowledged for providing funding under

Faculty Research Award to S.P. M.N. was supported by a

Junior Research fellowship from UGC – Maulana Azad

National Fellowship program.

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