Efficacy of some antioxidants supplementation in reducing oxidative stress post sodium tungstate exposure in male wistar rats

Journal of Trace Elements in Medicine and Biology xxx (2014) xxx–xxx

G ModelJTEMB 25499 No. of Pages 7

Toxicology

Efficacy of some antioxidants supplementation in reducing oxidativestress post sodium tungstate exposure in male wistar rats

S. Sachdeva, S.J.S. Flora *Division of Regulatory Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474 002, India

A R T I C L E I N F O

Article history:Received 13 November 2013Accepted 28 January 2014

Keywords:Sodium tungstate toxicityOxidative stressAntioxidantFlavonoidsRats

A B S T R A C T

This study aimed to evaluate the protective efficacy of some antioxidants against sodium tungstateinduced oxidative stress in male wistar rats. Animals were sub-chronically exposed to sodium tungstate(100 ppm in drinking water) for three months except for control group. In the same time, many rats weresupplemented orally with different antioxidants (alpha-lipoic acid (ALA), n-acetylcysteine (NAC),quercetin or naringenin (0.30 mM)) for five consecutive days a week for the same mentioned periodbefore. Exposure to sodium tungstate significantly (P < 0.05) inhibit blood d-aminolevulinic aciddehydratase (ALAD) activity, liver and blood reduced glutathione (GSH) levels and an increase in oxidizedglutathione (GSSG) and thiobarbituric acid reactive species (TBARS) levels in tissues. ALA acid and NACsupplementation post sodium tungstate exposure increased GSH and also, was beneficial in the recoveryof altered superoxide dismutase and catalase activity, besides, significantly reducing blood and tissuereactive oxygen species and TBARS levels. The results suggest a more pronounced efficacy of ALA acid andNAC supplementation than quercetin or naringenin supplementation post sodium tungstate exposure inpreventing induced oxidative stress in rats.

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1. Introduction

Tungsten, a well known transition metal, belongs to group VIand d-block of the periodic table. Due to the unusual and peculiarproperties of tungsten, it is nowa days preferred as a replacement tolead for the manufacture of depleted uranium bullets andammunitions in military [1]. Apart from its military applicationsusage of tungsten-based products has grown rapidly ranging fromdaily household necessities to modern science and technology [2].Due to its wide applications, the persistence of tungsten is graduallyincreasing in the environment, thereby posing a serious environ-ment concern. Major exposure to tungsten occurs through drinkingwater, although U.S. Environmental Protection Agency (EPA) has noguidelines for the permissible load of tungsten in water. The toxicprofile of tungsten has yet to be established in comparison to otherheavy metals [3]. There also exists less knowledge about thechemical and physiological behaviour of the element. Underoxidising and acidic conditions, tungsten salt gets rapidlymetabolised to tungstate. Sodium tungstate is thus thermodynam-ically stable form [4]. Several pharmacokinetic studies indicate therapid absorption of tungstate through oral route, followed by

* Corresponding author. Tel.: +91 751 2378196; fax: +91 751 2341148.E-mail address: [email protected] (S. Flora).

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Please cite this article in press as: S. Sachdeva, S.J.S. Flora, Efficacy of sosodium tungstate exposure in male wistar rats, J Trace Elem Med Biol (2

metabolization and rapid elimination via urine [4,5]. Sodiumtungstate is known for its therapeutic value and oral administrationof sodium tungstate has been shown to be effective in the treatmentof hyperglycemic conditions in animal model [6,7]. Recently itstoxicity has been investigated and also been reported that it ismediated through oxidative stress [8,9].

Flavonoids are natural antioxidants, categorized as a group ofpolyphenolic phytochemicals, actively involved in the defensemechanism against oxidative stress. The presence of hydroxylgroups and other unique features in the chemical structure offlavonoids are responsible for their function as a potent antioxidantand free radical scavenger [10]. Antioxidants such as ALA, NAC etc.,inhibit the oxidation of other biologically relevant molecules eitherby specifically quenching free radicals or by chelation of redoxmetals [10,11]. ALA is a thiol antioxidant, used for the treatment ofvarious metal associated pathophysiologies due to its efficientantioxidant and therapeutic potential [12,13]. It is producednaturally in plants and animals and exists both in reduced (DHLA)and oxidised (ALA) form. Both of these form may act synergisticallyin scavenging free radicals from both aqueous and lipid domains.Antioxidant properties of ALA as earlier been reported againstarsenic and lead toxicity [14,15]. Also, NAC is also a thiol-containingantioxidant that has been used to combat various conditions ofoxidative stress. The antioxidant activity of NAC originates fromits ability to stimulate GSH synthesis, thereby maintaining

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intracellular GSH levels, scavenging reactive oxygen species and itsmetal chelating properties. Co-administration of NAC along withMiADMSA produced significant recovery in oxidative stressvariables and the removal of lead from soft organs [16]. Treatmentwith NAC alone generated a moderate response during arsenictoxicity [17]. Quercetin, has most compatible chemical formulationand acts as a potent antioxidant. Due to the presence of threechelating sites, quercetin also acts as an excellent chelator.Quercetin administration has been useful in the treatment ofarsenic poisoning [18]. Naringenin is a natural flavonoid, a glyconeof naringenin and has been frequently investigated for itspharmacological actions, including antitumor [19], anti-inflamma-tory [20], and hepato-protective effects [21]. Similarity in itsstructural position of functional groups with quercetin raised thepossibility of its potential use as a protective agent against arsenictoxicity [22].

Keeping in view the potential supplementation of thesecompounds led to study their effects and protection againstsodium tungstate-induced oxidative stress. The present study wasplanned to elucidate whether oral supplementation of theseantioxidants, can ameliorate toxic effects of sodium tungstate interms of recovery in biochemical and hematological variablesindicative of oxidative stress and altered heme synthesis pathway.

2. Materials and methods

2.1. Animals, chemicals and diets

Sodium tungstate dihydrate (Na3WO4�2H2O; Product No.20684; lot # 2178 6802-1, purity – 96%) was procured from FischerScientific (Qualigens Fine Chemicals, Mumbai, India). All otherlaboratory chemicals and reagents were purchased from Merck(Germany), Sigma (USA) or BDH chemicals (Mumbai, India). Tripledistilled water prepared by Millipore (New Delhi, India) was usedthroughout the experiment to avoid contamination, and for thepreparation of reagents and buffers used for various biochemicalassays in the study.

2.2. Experimental protocol

Experiments were performed on healthy, male wistar rats,weighing approximately 100–120 g. Animals were obtained fromthe animal house facility of the defence research and developmentestablishment (DRDE), Gwalior. All animals received humane carein compliance with the guidelines of the Committee for the Purposeof Control and Supervision of Experiments on Animals (CPCSEA).The Animal Ethical Committee of DRDE, Gwalior, India alsoapproved the protocols for the experiments. Prior to dosing, theywere acclimatized for 7 days to light from 06:00 to 18:00 h,alternating with 12 h darkness. The animals were housed instainless steel cages in an air-conditioned room with temperaturemaintained at 25 � 2 �C. Rats were allowed standard chow diet(Amrut feeds, Pranav Agro, New Delhi, India).

Cu, Zn, Mn, Co and Fe levels in diet were 10.0, Zn 45.0, 55.0, 5.0and 75.0 ppm dry weight throughout the experiment, respectively,and water ad libitum.

Fifty rats were divided into ten groups of 5 rats each as follows:Group I: control (received normal water) for three months.Group II: rats treated with sodium tungstate, 100 ppm in

drinking water, daily for threemonths.Group III: rats treated with quercetin (0.30 mM) alone, orally,

once, daily for fiveconsecutive days a week for three months.Group IV: rats treated with NAC (0.30 mM) alone, orally, once,

daily for fiveconsecutive days a week for three months.Group V: rats treated with naringenin (0.30 mM) alone, orally,

once, daily for fiveconsecutive days a week for three months.


Group VI: rats treated with ALA (0.30 mM) alone, orally, once,daily for fiveconsecutive days a week for three months.

Group VII: rats treated with sodium tungstate and quercetin (asin group II and group III, respectively) for the same period.

Group VIII: rats treated with sodium tungstate and NAC (as ingroup II + group IV, respectively) for the same period.

Group IX: rats treated with sodium tungstate and naringenin (asin group II + group V, respectively) for the same period.

Group X: rats treated with sodium tungstate and ALA (as ingroup II + group VI, respectively) for the same period.

The doses for sodium tungstate [23] and equimolar doses ofantioxidants ALA, NAC, quercetin and Naringenin were selectedbased on earlier publications [16,21,24,25]. At the end of theexperiment all rats were anesthetized by light ether and blood wascollected by cardiac puncture in heparinized vials. Liver and spleenwere rinsed in cold saline, blotted, weighted and used immediatelyfor various biochemical variables as indicated later. We carried outbiochemical analysis of sodium tungstate’s effects only in liver andspleen for the reason, (i) liver being the organ responsible for thebiotransformation of a toxicant/xenobiotic. Thus we consideredassessment of oxidative stress in liver to be important and (ii)spleen is the another organ in the body which acts as sieve or bloodfilter and plays an important roles in removing old red blood cellsand holds a reserve of blood. Above all, in one of our recent study [8],we reported involvement of oxidative stress as an early toxic eventin liver and spleen following sodium tungstate exposure based onsignificant alteration in lipid per-oxidation Thus we selected liverand spleen in order to check the efficacy of some antioxidants in thepresent study.

2.3. Separation of red blood cells

Heparinized blood was used for RBCs isolation using the methodof Steck and Kant [26]. Briefly, heparinized blood was centrifuged at1500 � g for 10 min at 4 �C and through aspiration plasma and buffycoat were removed. The RBCs were washed three times inphosphate buffer saline (0.1 M). The packed cell volume (PCV)obtained was divided into two part, one part of which was dilutedwith chilled distilled water and kept for the analysis of reactiveoxygen species (ROS), catalase, TBARS and GSH, while the other partwas used for the estimation of other antioxidant enzyme i.e.superoxide dismutase (SOD). In order to carry out the SOD analysis,hemoglobin was precipitated in PCV by means of chloroform andethanol, which was further centrifuged at 3000 � g for 10 min at4 �C. The supernatant obtained was used for all above mentionedenzymatic assays.

2.4. Blood d-aminolevulinic acid dehydratase (ALAD) assay

The activity of blood ALAD was determined using method ofBerlin et al. [27]. The assay system consisted of 0.2 ml of heparinizedblood and 1.3 ml of distilled water. After complete hemolysis, forwhich incubation was done for 10 min at 37 �C for, 1 ml of standardd-aminolevulinic acid was added to the tubes and incubated for60 min at 37 �C. The reaction was stopped after 1 h by adding 1 ml of10% TCA. Equal volume of Ehrlich reagent was added to thesupernatant, and the absorbance was recorded at 555 nm after5 min.

2.5. Reactive oxygen species (ROS) assay

ROS estimation in blood was measured using 20, 70-dichlro-fluorescein diacetate (DCF-DA) which further gets converted intohighly fluorescent DCF by cellular peroxides (including hydrogenperoxide). The assay was performed as described by Socci et al. [28].As per the procedure, for blood analysis, 5% RBC hemolysate was



S. Sachdeva, S.J.S. Flora / Journal of Trace Elements in Medicine and Biology xxx (2014) xxx–xxx 3


prepared and diluted to 1.5% with ice-cold 40 mM tris–HCl buffer(pH 7.4). Similarly for tissues, 10% tissue homogenate was preparedby homogenizing 10 mg of tissue in 1 ml of ice-cold 40 mM tris–HClbuffer (pH 7.4). This was further diluted to 0.25% with the samebuffer and placed on ice. Then after, 40 ml of 1.25 mM DCF-DA inmethanol was added for ROS estimation. All samples wereincubated for 15 min in a 37 �C water bath. Fluorescence wasdetermined at 488 nm excitation and 525 nm emission using afluorescence plate reader (Tecan Spectra Fluor Plus).

2.6. Glutathione (GSH) assay

Method of Ellman [29], modified by Jollow et al. [30] wasfollowed for the analysis of blood GSH concentration. As per theprotocol, 0.2 ml of packed cell volume was added to 1.8 ml ofdistilled water and incubated for 10 min at 37 �C, for completehemolysis. 3 ml of 4% sulfosalicylic acid was further added andtubes were centrifuged at 2500 rpm for 15 min. To the supernatantobtained, 0.2 ml of 10 mM solution of 5,50-dithiobis-(2-nitro-benzoic acid), DTNB, was added in presence of phosphate buffer(0.1 M, pH 7.4). Absorbance recorded at 412 nm was used forcalculation of GSH concentration.

2.7. Blood thiobarbituric acid reactive substances (TBARS) assay

Method by Stocks and Dormandy [31] was followed todetermine TBARS in red blood cells. RBCs were washed with ice-cold buffered saline (pH 7.4, 0.1 M) and the PCV was adjusted to2.5%. To this was added sodium azide (2 mmol/l), followed by H2O2

(0.068%). The mixture was incubated at 37 �C for 1 h after which thereaction was stopped by the addition of 2 ml trichloroacetic acid(TCA) arsenite solution (28% TCA + 0.1 M sodium arsenite). Centri-fugation was done at 3000 � g for 15 min at 4 �C, to obtainsupernatant, to which 1% TBA was added. The tubes were placedon boiling water bath for 15 min. The absorbance was read at532 nm using a spectrophotometer. Results were expressed asnmol/min/gm of hemoglobin.

2.8. Tissue thiobarbituric acid reactive substances (TBARS) assay

Measurement of lipid peroxidation in tissue was done by themethod of Ohkawa et al. [32]. Tissue homogenate was prepared(10% homogenate (w/v) in 150 mM KCl for liver, 5% homogenate (w/v) in 150 mM KCl for kidney and spleen) and further incubated for30 min at 37 �C. The incubation was interrupted by adding 0.1 ml of10% TCA. After centrifugation, 1 ml of the supernatant was mixedwith 1 ml of 0.65% thiobarbituric acid and kept in a boiling waterbath for 15 min to get the red color of thiobarbituric acidmalondialdehyde complex, the absorbance of which was recordedat 535 nm. The amount of TBARS was calculated using a molarextinction coefficient of 1.56 � 105M�1 cm�1.

2.9. Tissue reduced glutathione (GSH) and oxidized glutathione (GSSG)assay

Tissue GSH and GSSG levels were measured by the method ofHissin and Hilf [33]. 0.25 g of tissue sample was homogenized on icewith 3.75 ml of phosphate-EDTA buffer and 1 ml of 25% HPO3whichwas used as a protein precipitant. The total homogenate was ofH2O2 (1 mM) and 0.3 ml of tissue supernatant. Incubation was doneat 37 �C for 15 min, after which the reaction was terminated byaddition of 0.5 ml of 5% TCA. Tubes were further centrifuged at1500 � g for 5 min and supernatant was collected. To 0.1 ml ofsupernatant, 0.2 ml of phosphate buffer (0.1 M, pH 7.4) and 0.7 ml ofDTNB (0.4 mg/ml) were added and absorbance was recorded at420 nm after proper mixing.


2.10. Superoxide dismutase (SOD) assay

Superoxide dismutase (SOD) activity in liver and blood wasassayed spectrophotometrically by the method of Kakkar et al. [34].Reaction mixture was prepared which contained 1.2 ml (0.052 mM)sodium pyrophosphate buffer, 0.1 ml (186 mM) phenazine metho-sulphate, 0.3 ml (300 mM) nitro blue tetrazolium. 5% hemolysate/homogenate was centrifuged at 1500 � g, 10 min followed by10,000 � g, 15 min and 0.2 ml of the supernatant obtained. To thisreaction mixture was added. Reaction was initiated by adding0.2 ml NADH (780 mM) and stopped by adding 1 ml glacial aceticacid. Color intensity of the chromogen was measured at 560 nm andthe activity was expressed as units/min/mg of protein.

2.11. Catalase assay

Procedure of Sinha [35] was used to determine catalase activityin tissue and blood at room temperature. In the presence of 0.01 Mphosphate buffer (pH 7.4), 0.1 ml of 5% RBC hemolysate/tissuehomogenate was incubated with 0.5 ml of H2O2 (0.2 M) at 37�C for90 s precisely. Reaction was stopped by adding 5% dichromatesolution and samples were further incubated at 100 �C for 15 min inboiling water bath. Amount of H2O2 consumed was determined byrecording absorbance at 570 nm.

2.12. Statistical analysis

The results are expressed as the mean � (SE) of number ofobservations. Comparisons of means were carried out using oneway analysis of variance (ANOVA) followed by Bonferroni multiplecomparison test to compare means between the differenttreatment groups using InStat (GraphPad Software, San Diego,USA). Differences were considered significant at P < 0.05 unlessotherwise stated in the footnote of table and figures.

3. Results

3.1. Effects of antioxidants on body weight in post sodium tungstateexposed rats

Table 1 shows the effect of Sodium tungstate exposure on bodyweight in rats. Significant (P < 0.05) reduction in body weight wasobserved in rats exposed to sodium tungstate. However no changeswere observed in groups exposed to antioxidants alone. Significantchanges were observed in groups receiving both sodium tungstateand antioxidants simultaneously.

3.2. Effects of antioxidants on blood ALAD activity in post sodiumtungstate exposed rats

Fig. 1 shows the effect of sodium tungstate exposure andprotective value of antioxidants on blood ALAD activity at theend of the experiment. The blood ALAD activity decreased onsodium tungstate exposure compared to normal. All the fourantioxidants were may prevent sodium tungstate inducedinhibition of ALAD activity. Among the antioxidants ALAexhibited more pronounced recovery followed by NAC. Nobeneficial effects of quercetin and naringenin were notedagainst blood ALAD activity (Fig. 1).

3.3. Effects of antioxidants on blood oxidative stress variables in postsodium tungstate exposed rats

Table 2 shows the effects of antioxidants supplementation onsodium tungstate induced blood oxidative stress variables. BloodROS increased significantly (P < 0.05) on sodium tungstate



Fig. 1. Effects of different antioxidants on blood d-aminolevulinic acid dehydratase (ALAD) activity in post sodium tungstate exposed rats.Abbreviations used and units – ALAD as d-aminolevulinic acid dehydratase as nmole/min/mg protein. Values expressed as mean � (S. E). yP < 0.001 compared to normalcontrol; *P < 0.05 compared to sodium tungstate exposed (control).

Table 1Effects of different antioxidants on body weight in post sodium tungstate exposed rats.

Groups Initial body weight (in g) Final body weight (in g) Body weight gain or loss (in g)

Normal 116.9 � 1.9 165 � 0.9 48.1 � 0.58Quercetin 115.8 � 1.6 156 � 1.9 40.2 � 0.45Naringenin 112 � 1.4 158 � 2.3 46 � 0.85N-aceytlcysteine 114.5 � 0.8 163 � 1.4 48.5 � 2.02Alpha-lipoic acid (ALA) 119 � 1.2 161 � 1.3 42 � 1.04Sodium tungstate (Na3WO4) 117.5 � 1.7 97 � 1.2 20.5 � 1.08a

Na3WO4 + quercetin 111.3 � 0.10 141.2 � 1.01 29.9 � 1.4*

Na3WO4 + naringenin 119.5 � 0.8 139 � 1.4 20.5 � 1.8Na3WO4 + NAC 116 � 1.5 143.5 � 1.21 27.5 � 1.4*

Na3WO4 + ALA 120 � 1.6 146.5 � 1.11 26.5 � 0.81*

Values are mean � (S.E).a P < 0.05 compared to normal animals, *P < 0.05 compared to sodium tungstate exposed (control).



exposure compared to control. ALA and NAC supplementationprovided significant (P < 0.05) reduction of blood ROS levels. Also,quercetin supplementation led to slight reduction in blood ROSlevels compared to naringenin. Sodium tungstate decreased bloodGSH level which responded favourable to ALA and NAC

Table 2Effects of antioxidants on blood oxidative stress and antioxidant variables in post sodiu

Groups Blood ROS

Normal 448.6 � 9.7

Quercetin 472.2 � 2.05a

Naringenin 458.2 � 3.6

N-acetylcysteine (NAC) 470.6 � 1.8a

Alpha-lipoic acid (ALA) 452.8 � 4.5

Sodium tungstate (Na3WO4) 475.6 � 5.4a

Na3WO4 + quercetin 455.2 � 7.3*

Na3WO4 + naringenin 446.2 � 5.6**

Na3WO4 + NAC 455.4 � 5.3**

Na3WO4 + ALA 447.8 � 2.9**

Abbreviations used and units – ROS: reactive oxygen species as FIU (Fluorescent Intensity

substances as mg/ml of RBC. Values are mean � (S.E).a P < 0.05 compared to normal animals, **P < 0.01, *P < 0.05 compared to sodium tung


supplementation. No beneficial effects of quercetin as well as innaringenin were noted. Sodium tungstate exposure inducedincrease in TBARS, also showed partial protection with ALAsupplementation followed by NAC. No effects of quercetin andnaringenin were noted.

m tungstate (100 ppm in drinking water) exposed rats.

Blood GSH Blood TBARS

0.241 � .006 18.06 � 1.020.269 � .09 19.79 � 0.560.234 � .013 17.1 � 0.750.249 � .004 19.54 � 0.910.247 � .004 19.35 � 1.40.222 � .003a 21.82 � 2.20.254 � .006** 21.92 � 1.10.233 � .001* 21.95 � 0.530.227 � .007 20.62 � 0.430.229 � .007 19.87 � 0.94

unit); GSH, reduced glutathione as mg/ml of RBC; TBARS: thiobarbituric acid reactive

state exposed (control).



Fig. 2. Effects of different antioxidants on catalase and superoxide dismutase (SOD) activity in post sodium tungstate exposed rats.Abbreviations used and units – SOD: superoxide dismutase as units min�1mg protein�1; Catalase as nmoles of H2O2 consumed min�1mg protein�1. Values are mean � (S.E).yP < 0.05 compared to normal control; *P < 0.05 compared to sodium tungstate exposed (control).

Table 3Effects of different antioxidants on liver oxidative stress and antioxidant variables in post sodium tungstate exposed rats.

Groups Liver ROS Liver GSH/ GSSG Liver TBARS

Normal 349.4 � 14.7 1.06 � 0.06 5.17 � 0.15Quercetin 348.2 � 15.9 0.93 � 0.03 4.63 � 0.18Naringenin 352 � 23.05 0.89 � 0.02 5.76 � 0.60N-acetylcysteine (NAC) 355.4 � 15.3 0.97 � 0.04 4.53 � 0.27Alpha-lipoic acid (ALA) 347.8 � 12.9 0.99 � 0.05 4.71 � 0.19Sodium tungstate (Na3WO4) 423.6 � 15.7a 0.77 � 0.01a 6.52 � 0.10a

Na3WO4+ quercetin 372.2 � 12.05** 0.90 � 0.02* 4.51 � 0.14Na3WO4+ naringenin 358.2 � 23.6** 0.88 � 0.02* 4.71 � 0.27Na3WO4+ NAC 370.0 � 10.8** 0.86 � 0.02* 5.14 � 0.15*

Na3WO4+ ALA 352.8 � 24.5** 0.88 � 0.02* 5.01 � 0.42**

Abbreviations used and units – ROS: reactive oxygen species as FIU (Fluorescent Intensity unit); GSH: GSSG ratio in rat liver. GSH, reduced glutathione as mg/gm tissue; GSSG,oxidized glutathione as mg/gm tissue; TBARS: thiobarbituric acid reactive substances as mg/gm of tissue weight. FIU: values are mean � (S.E).

a P < 0.05 compared to normal animals, **P < 0.01, *P < 0.05 compared to sodium tungstate exposed (control).



3.4. Effects of antioxidants on blood antioxidant enzymes in postsodium tungstate exposed rats

Both SOD and Catalase activities showed marginal (non-significant) (P < 0.05) decrease on sodium tungstate exposure.ALA and NAC supplementation provided recovery while quercetinand naringenin were ineffective (Fig. 2).

3.5. Effects of antioxidants on liver oxidative stress and antioxidantvariables in post sodium tungstate exposed rats

Hepatic ROS levels increased significantly (P < 0.05) on sodiumtungstate exposure. Elevated ROS level was significantly (P < 0.05)restored by ALA supplementation and to the same extent by NAC

Table 4Effects of different antioxidants on spleen oxidative stress and antioxidant variables in

Groups Spleen ROS

Normal 251.2 � 14.8

Quercetin 254.4 � 16.09

Naringenin 266.4 � 17.4

N-acetylcysteine(NAC) 267.4 � 24.1

Alpha-lipoic acid (ALA) 275.2 � 11.4

Sodium tungstate(Na3WO4) 283.4 � 12.5a

Na3WO4+ quercetin 280.6 � 17.7

Na3WO4+ naringenin 265.2 � 18.1

Na3WO4+ NAC 259.6 � 10.1*

Na3WO4+ ALA 261.2 � 12.08*

Abbreviations used and units – ROS: reactive oxygen species as FIU (Fluorescent Intensity uoxidized glutathione as mg/gm tissue; TBARS: thiobarbituric acid reactive substances a

a P < 0.05 compared to normal animals, *P < 0.05 compared to sodium tungstate expo


supplementation. Also, it was noted depletion in GSH level and anincrease in GSSG and TBARS levels in liver leading to a decreasedGSH/GSSG ratio on exposure to sodium tungstate. Significant(P < 0.05) recovery was noted in groups treated with ALA and NACsupplementation compared to quercetin and naringenin supple-mentation (Table 3).

3.6. Effects of antioxidants on spleen oxidative stress and antioxidantvariables in post sodium tungstate exposed rats

The effects of tungstate exposure and supplementation ofantioxidants on biochemical variables indicative of spleen oxida-tive stress are shown in Table 4. A moderate increase in ROS levels(non-significant) depletion of GSH and slight increase in GSSG and

post sodium tungstate exposed rats.

Spleen GSH/ GSSG Spleen TBARS

1.11 � 0.04 9.2 � 0.241.18 � 0.08 10.59 � 0.410.91 � 0.04 9.13 � 0.321.03 � 0.02 9.26 � 0.701.01 � 0.01 9.21 � 0.300.94 � 0.04a 11.59 � 0.45a

1.20 � 0.04* 9.51 � 0.14*

1.05 � 0 .03 10.24 � 0.211.20 � 0.05* 9.51 � 0.18*

1.15 � 0.03* 9.56 � 0.16*

nit); GSH: GSSG ratio in rat spleen. GSH, reduced glutathione as mg/gm tissue; GSSG,s mg/gm of tissue weight. Values are mean � (S.E).sed (control).





TBARS levels were noted. ALA, NAC and quercetin supplementationalong with sodium tungstate exposure showed non-significant(P > 0.05) recovery of the altered variables to control (Table 4).

4. Discussion and conclusion

Tungsten is a widely used rare heavy metal for which verylimited information on environmental and toxicological effects areknown. Of particular interest is the lack of information regarding itstoxicity [36]. Some of the toxic effects of sodium tungstate exposureare mediated through oxidative stress and its exposure may lead tothe disruption of pro-oxidant and antioxidant in vivo [8]. Sodiumtungstate exposure induced disruption in the balance of the pro-oxidant and antioxidants levels prompted us to explore if co-administration of antioxidants may be beneficial in preventingsome of these adverse effect. It is well known that oxidative injuriescan be prevented by simultaneous supplementation of variousantioxidants [10]. ALA, NAC, quercetin and naringenin supplemen-tation are well known for their effective free radical scavenging andmetal chelating properties [10,14,37]. These antioxidants, individ-ually and in combination with sodium tungstate exposure areselected for their efficacy against sodium tungstate induced toxicmanifestations.

Tungsten interferes with heme synthesis pathway, by inhibitingd-ALAD activity in tungstate exposed group [8]. d-ALAD, a zincdependent metalloenzyme, known to play a key role in hemesynthesis pathway and highly susceptible to the alteration inducedby metals [38,39]. Inhibition of d-ALAD results in the accumulationof ALA metabolite, which releases Fe2+ from ferritin and thusinduces oxidative damage [40]. ALA possess three distinctantioxidant actions: (a) reactive oxygen species scavenging activity(b) capacity to regenerate endogenous antioxidants, such asglutathione and vitamins and, (c) metal chelating activity, and inaddition, presence of sulfhydryl group [10]. NAC act directly byscavenging reactive radicals and replenishing glutathione. Itsnucleophilic nature results in direct trapping of free radicalscausing mutagenicity, blockage of metabolites of promutagens andinhibition of nitrosation reactions [41]. Also, quercetin acts as achelating agent in the therapy of acute heavy metal poisoning [10].Balcerzak et al. [42] reported three possible chelating sites in itsstructure; the 3-hydroxychromone, the 5-hydroxychromone andthe 30,40-dihydroxyl group which may attribute to its scavengingactivity. Blood d-ALAD activity has restored to normal in animals co-exposed with ALA and NAC. This may be attributed to the fact thatALA and NAC tend to chelate metal ions more effectively thenquercetin and naringenin. As a result of metal toxicity elevatedlevels of ROS occurs which, in turn, trigger lipid peroxidation whichis known to overcome by few antioxidants such as ALA [43]. TBARSlevel provides insight into the peroxidation process, therebyindicating toxic manifestations. Tungsten did not elicit any changesin blood TBARS level which probably suggest no impairment in themembrane of the RBC. On the other hand, results showed that liverand spleen TBARS levels increased significantly which respondedfavourably on adminstration of ALA, NAC and quercetin. All thesealterations such as increased ROS and TBARS, leads to impairedcellular antioxidant defense system [10]. This can further besupported by depleted blood GSH level. Glutathione is one of thekey constituent in the antioxidative system, with a significantfunction in ROS scavenging and acts as a redox buffer to keep thecellular redox state in balance [44]. Either reduced (GSH) oroxidized (GSSG) forms. Liver is the major target organ of tungstatetoxicity [8]. A decreased GSH: GSSG ratio may be considered as acrucial indicator of tungstate induced oxidative stress [45]. Thedepletion of GSH: GSSG ratio was more pronounced in the liver thanin spleen. These alterations might be due to the possiblemodulation of GSH status either to GSSG or tungstateglutathione


(W–SG) through enzymatic or non enzymatic processes [46]. Theseresults can be attributed to the free radical scavenging activity ofALA as well as NAC [11]. The activity of two main antioxidantenzymes SOD and catalase were also studied in order to understandthe basic mechanism behind sodium tungstate induced oxidativestress. Significant changes in enzyme systems along with elevationin reactive oxygen species level are also observed. During oxidativestress activity of catalase decreases, thereby favouring theperoxidation of lipids. In this study, significant alteration incatalase activity wasw noted, however moderate changes wereobserved in SOD activity either on exposure or following co-administration of antioxidants. There was more pronouncedrestoration of altered enzyme activity upon ALA and NACsupplementation.

To the best of our knowledge, this is the first study including theprotective efficacy of these antioxidants against sodium tungstateinduced alterations. Interestingly, more pronounced significantprotective efficacy was noted with ALA and NAC supplementation,followed by quercetin and naringenin supplementation in reducingthe oxidative stress of sodium tungstate exposure. The probablemechanism by which these antioxidants ameliorate toxic mani-festations induced by sodium tungstate might be the presence ofsulfhydryl group (��SH) in both of these antioxidants. Tungstategroup may have an affinity towards the ��SH group causing lesserbioavailability of toxic tungstate group in the body. Apart from thisreplenishment of endogenous antioxidants such as glutathione andfree radicals scavenging may also be a contributing fact towardsprotective potential of these antioxidants. Presence of ��OH group[47] in flavonoids (quercetin and naringenin) is reported but theremight not be any interaction between tungstate ions and flavonoidsdue to lesser electronegativity of tungstate group [36]. Therefore,no significant protective efficacy was observed with two flavonoids,quercetin and naringenin supplementation.

The present study provides a useful strategy to protect fromsodium tungstate induced toxic manifestations. The results fromthe present study suggest beneficial role of flavanoids, ALA and NACsupplementation in restoring some of the altered biochemicalvariables in blood and tissues (liver and spleen) suggestive ofoxidative stress. Quercetin and naringenin supplementation werecomparative less effective as only marginal recovery was seen upontheir administration to rats, compared to ALA and NAC supplemen-tation. These antioxidants may be easily incorporated into the dietor could also be co-supplemented during exposure for granting anaffordable protective efficacy against tungstate-induced oxidativeinjury.

Acknowledgements

The authors thank Dr M.P. Kaushik, Director of the DefenseResearch and Development Establishment for his support andencouragement. Sherry Sachdeva thanks Department of Scienceand Technology (DST) for the award of Junior Research Fellowship.

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Efficacy of some antioxidants supplementation in reducing oxidative stress post sodium tungstate exposure in male wistar rats