evaluation of comparative effect of pre- and posttreatment of selenium on mercury-induced oxidative...

J BIOCHEM MOLECULAR TOXICOLOGYVolume 24, Number 2, 2010

Evaluation of Comparative Effect of Pre- andPosttreatment of Selenium on Mercury-InducedOxidative Stress, Histological Alterations, andMetallothionein mRNA Expression in RatsRakhi Agarwal,1,2 S. Raisuddin,2 Shikha Tewari,1 Sudhir K. Goel,1 R. B. Raizada,1

and Jai Raj Behari1

1Indian Institute of Toxicology Research (Formerly: Industrial Toxicology Research Centre) [Council of Scientific and Industrial Research,India], Mahatma Gandhi Marg, Lucknow 226 001, India; E-mail: [email protected]. in, [email protected] of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar,New Delhi 110 062, India

Received 2 June 2009; accepted 12 July 2009

ABSTRACT: To evaluate the effect of pre- or post-treatment of selenium (6 μμmol/kg b.w., single in-traperitoneal injection) in mercury intoxication, ratswere exposed to mercury (12 μμmol/kg b.w., single in-traperitoneal injection). Exposure to mercury resultedin induced oxidative stress in liver, kidney, and braintissues. Marked changes in serum biochemical parame-ters together with alterations in histopathology and aninduction in metallothionein-I and metallothionein-II mRNA expression in the liver and kidney wereobserved. Pretreatment with selenium to mercury-exposed animals had protective effect on the liver,whereas posttreatment had partial protection onrestoration of altered oxidative stress parameters. Inthe kidney, pretreatment with selenium showed par-tial protection on restoration of altered biochemical pa-rameters, whereas no protection was observed in post-treatment. The pretreatment with selenium resulted inrestoration of mercury-induced metallothionein-I andmetallothionein-II mRNA expression, which was com-pletely restored in the liver whereas partial restorationwas observed in the kidney. Posttreatment with sele-nium resulted in further induction in metallothionein-I and metallothionein-II mRNA expression in the liverand kidney. In the brain, selenium showed partial pro-tection on alerted biochemical parameters. Results in-dicate that pretreatment with selenium is beneficial incomparison to posttreatment in mercury intoxication.Thus, dietary intake of selenium within safe limit may,

Correspondence to: Jai Raj Behari.Present address of Rakhi Agarwal: Division of Toxicology,

Central Drug Research Institute (Council of Scientific and IndustrialResearch, India), Chattar Manzil Palace, Lucknow 226 001, India.c© 2010 Wiley Periodicals, Inc.

therefore, enable us in combating any foreseen effectsdue to mercury exposure. C© 2010 Wiley Periodicals, Inc.J Biochem Mol Toxicol 24:123–135, 2010; Published on-line in Wiley InterScience (www.interscience.wiley.com).DOI 10:1002/jbt.20320

KEYWORDS: Mercury; Selenium; Oxidative Stress;Histopathology; Metallothionein Expression

INTRODUCTION

Mercury is a nonessential element that exhibits ahigh degree of toxicity to humans and animals. Ex-posure to mercury has been associated with adversehealth effects especially nephrotoxicity and neurobe-havioral disorders. It has been observed that probablyit affects the inherent protein structure, which may in-terfere with functions relating to protein production[1]. Mercury is also known to induce reactive oxygenspecies (ROS) in animals. Furthermore, mercury has astrong affinity for sulfhydryl, amine phosphoryl, andcarboxyl groups, and thus inactivates a wide range ofenzyme systems. Metal exposure including mercury isfrequently associated with the induction of metalloth-ioneins (MTs). Role of MTs in metal detoxification andsequestration is reported extensively in mammalianand nonmammalian species [2]. Increased MT expres-sion has been observed in mercury-exposed animalsas an indicator of toxicity together with its subsequentprotection [3–5].

Selenium is a structural component of severalenzymes with physiological antioxidant properties,

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including glutathione peroxidases and thioredoxin re-ductase [6]. It has been well documented that mercuryand selenium interact biologically, and the coadminis-tration of both reduces the toxicity of each other [7].The mechanism underlying the protective effects of se-lenite against mercury toxicity is still unsolved. Thereis evidence that selenide (produced from selenite inthe presence of glutathione) forms a mercury–seleniumcomplex, which then binds to selenoprotein P to forma ternary complex [8]. The ability of selenium to reducemercury toxicity is well reported [7,8]. Selenium is alsoreported to affect mercury elimination by reducing itsurinary and fecal excretion, the main pathway for theelimination of inorganic mercury from the body [9].Thus, Hg–Se interactions have been experimentally re-ported to counteract mercury toxicity [10,11]. Simulta-neous administration of sodium selenite with mercuricchloride is reported to protect against mercury toxic-ity in vivo [11,12]. The present work was, therefore,undertaken to evaluate the effect of pre- or posttreat-ment with selenium to mercury-exposed animals interms of hepatic, renal, and cerebral oxidative damage,histological changes, serum biochemical parameters,and metallothionein-I and metallothionein-II mRNAexpression in liver and kidney tissues of the rats.

MATERIALS AND METHODS

Chemicals, Reagents, Kits, and Primers

Mercuric chloride and sodium selenite fromMerck, Mumbai (India), trizol reagent from Gibco BRL,Gaithersburg, MD and other chemicals of the purestgrade available were obtained from Sigma ChemicalCo. (St. Louis, MO), or other standards suppliers.Activity/levels of serum biomarkers were determinedusing commercial kits, procured from Spinreact, SA,Sant Esteve de Bas 17176 (Spain). For cDNA synthesis,RevertAid H Minus First cDNA Synthesis Kit wasprocured from Fermentas Life Sciences, Leon-Rot(Germany). SYBR Green JumpStart Taq ReadyMixfor quantitative polymerase chain reaction (PCR)was procured from Sigma-Aldrich (St. Louis, MO).Primers of specific sequence for rat metallothionein-I(MT-I) and metallothionein-II (MT-II) were procuredfrom Metabion International AG, Martinsried 82152(Germany), whereas those of GAPDH from MWGBiotech AG, Ebersberg (Germany) were used for PCRamplification.

Animals, Treatment, and Collectionof Tissues

Thirty adult male Wistar rats (150 ± 10 g) wereprocured from Indian Institute of Toxicology Research,

Lucknow breeding colony maintained under con-trolled temperature (20–22◦C) and 12 h alternate lightand dark cycle, with free access to water and pelletfood (Lipton India Ltd, Mumbai, India) were used inthe study. Mercuric chloride (12 μmol/kg b.w.) andsodium selenite (6 μmol/kg b.w.) were dissolved inphysiological saline solution. Animals were randomlydivided into five groups (n = 6). They were adminis-tered n-saline (group-I), mercury (group-II), selenium(group-III), selenium + mercury (selenium pretreat-ment, group-IV), and mercury + selenium (seleniumposttreatment, group-V). Group I (control), received in-traperitoneal (ip) injection of normal saline. Groups IIand III received one ip injection of mercury or selenium,respectively. Group IV was first administered with sele-nium and then mercury with a time gap of 2 h, whereasgroup V was injected with mercury and after 2 h givenselenium.

Rats were sacrificed 24 h after the last treatment un-der ether anesthesia. Blood was drawn from the heartin two separate vials, i.e. heparinized (for mercurydetermination) and nonheparinized (for biochemicalassays, viz., alkaline phosphatase [ALP], lactate dehy-drogenase [LDH], blood urea nitrogen [BUN], and cre-atinine), and stored at 4◦C until assay. A portion of liverand kidney (50–70 mg) was removed immediately andstored at −20◦C for total RNA isolation. The remainingtissues from each organ and whole brain were quicklyremoved, trimmed of extraneous materials, washedwith chilled saline solution, blotted, and weighed. Forhistopathology, one lobe of the liver and right kidneywas fixed in 10% formalin. Remaining part of the liverand kidney and the whole brain were minced andhomogenized (10% w/v) in ice-cold 0.15% KCl 0.1 Mphosphate buffer (pH 7.4) in a Potter–Elvehjem homog-enizer. The tissue homogenates were stored at −20◦Cfor the assays of lipid peroxidation (LPO), reduced glu-tathione (GSH) and activities of antioxidant enzymes.Total mercury content was also determined in liver,kidney, brain tissue homogenates, and whole blood.

Biochemical Assays

Malondialdehyde (MDA), an end product of lipidperoxidation, was measured with the absorbancecoefficient of the MDA–TBA (Thio Barbituric Acid)complex at 532 nm on Thermo Scientific Spectronic,Spectrophotometer Genesys 10UV, Waltham, MAUSA, using 1,1,3,3-tetraethoxypropane as standard[13]. Glutathione levels were determined using5,5′-dithio-bis(2-nitrobenzoic acid) for color de-velopment spectrophotometrically at 412 nm [14].Superoxide dismutase (SOD) activity was measuredspectrophotometrically by the modified method of the

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Volume 24, Number 2, 2010 EFFECT OF SELENIUM IN MERCURY INTOXICATION 125

NADH-phenazinemethosulfate-nitroblue tetrazoliumformazan inhibition reaction at 550 nm by using anELISA plate reader [15]. Glutathione peroxidase (GPx)activity was measured by the method of Flohe and Gun-zler [16], and readings were taken at 420 nm. Catalase(CAT) activity was measured, spectrophotometricallyat 570 nm by the method of Sinha [17]. Protein wasassayed according to the method of Lowry et al. [18]using bovine serum albumin as standard. The activitiesof ALP and LDH and levels of BUN and creatinine weredetermined photometrically, according to the manu-facturer’s protocol with a diagnostic automated labo-ratory analyzer (ChemWell-Biological Auto-Analyzer,Awareness Technology, Palm City, FL 34990, USA).

Mercury Estimation

Mercury was analyzed in tissue homogenates andwhole blood by the method of Skare [19] using atomicabsorption spectrophotometer equipped with vaporgeneration assembly (GBC Avanta-�). Briefly, 1 mL tis-sue homogenates (10% w/v) or heparinized blood wasdigested with 7 mL of nitric acid and perchloric acid(6:1) mixture by heating on a water bath maintainedat 85◦C for 18–20 h. Clear homogeneous digest wasobtained, which was allowed to cool at room temper-ature, and 2 mL of 20% hydroxyl ammonium chloridesolution was added. Finally, the volume was made upto 20 mL with distilled water. Samples were analyzedagainst standards within the linear range of the calibra-tion curve.

Histopathology

Small pieces of the liver or half part of kidney tis-sue were taken into metal sieves and left overnightfor washing under running tap water. Washed tissueswere then placed in progressively concentrated ethanolfor dehydration, then left overnight in toluene andmounted in paraffin wax. Sections of 5 μm thicknesswere cut using rotary microtome (Leica MicrosystemsNussloch GmbH, Nussloch, Germany), fixed on themicroscopic slides by thin layer of Mayer’s Albumin,stained with haematoxylin–eosin (H&E), mounted inDPX resin by using glass coverslips and examinedunder light microscope (Nikon Eclipse E600, Tokyo,Japan).

Metallothionein Gene Expression Study

Total RNA Isolation and cDNA Synthesis(Reverse Transcription)

The total RNA was isolated using trizol reagentas per manufacturer’s instructions (Gibco BRL). The

OD260/OD280 ratio of all RNA samples used was morethan 1.8. Total RNA (0.5 μg) from each individual rattissue (n = 3) of every group was reverse transcribed tocomplementary DNA (cDNA) with kit according to themanufacturer’s protocol using Peltier Thermal Cycler,Bio-Rad Life Sciences, Select Science Ltd., BA2 9AP, UK.

Real-Time Polymerase Chain Reaction(Real-Time PCR)

PCR amplification and quantitation of gene ex-pression was performed for MT-I (forward primer: 5′-TGGACCCCAACTGCTCCTG-3′ and reverse primer:5′-TCAGGCACAGCACGTGCAC-3′) and MT-II (for-ward primer: 5′-TGGACCCCAACTGCTCCTG-3′ andreverse primer: 5′-TCAGGCGCAGCAGCTGCAC-3′)together with housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [forward primer:5′-CAGCAAGGATACTAGGAG-3′ and reverse primer:5′-GGATGGAATTGTGAGGGAGATG-3′] cDNA fromliver and kidney tissues. Real-Time PCR, usingSYBR Green reagent, was performed on StratageneMx3000P® QPCR System, USA. The expected lengthof the PCR product of rat MT-I, MT-II, and GAPDHgene was 200–400 bp. All reactions were done ina 25-μL reaction mixture in triplicate following themanufacturer’s protocol. The real-time PCR protocolused was denaturation program (10 min @ 95◦C forMT-I and MT-II, whereas 2 min @ 94◦C for GAPDH),amplification and quantification program repeated40 times (15 s @ 95◦C; 10 s @ 60◦C; 30 s @ 72◦C witha single fluorescence measurement), melting curveprogram (60–99◦C with a heating rate of 0.1◦C/s andcontinuous fluorescence measurements), and coolingprogram down to 40◦C [20].

Data Analysis

The data obtained from real-time PCR is presentedas threshold cycle (Ct), the cycle at which fluorescencewas determined to be statistically significant abovebackground signal contributed by the labeled oligonu-cleotides within the PCR reaction [20]. The results ofCt values were represented as mean ± SE. Lowering ofCt values in comparison to control shows induction inthe expression of the gene levels as the Ct is inverselyproportional to the log of the initial copy number ofthe PCR product. For the calculation of fold changesin a target gene, relative quantification “delta–deltaCt method” was used for comparing relative expres-sion between treatments in real-time PCR [20]. Themethod is based on the relative expression of a tar-get gene versus a reference gene. In the present work,we used GAPDH as housekeeping gene for relative



quantification of MT-I and MT-II gene expression inliver and kidney tissues.

Statistical Analysis

Statistical significance of mean value of differ-ent observations was analyzed using one-way analy-sis of variance (ANOVA). The homogeneity of vari-ance between treatment groups was ascertained usingBartlett’s test [21]. Post hoc analysis was carried outto compare the mean values between the pair of treat-ments using Scheffe’s multiple comparison procedure[22]. A probability of less than or equal to 0.05 wasconsidered to be significant.

RESULTS

Lipid Peroxidation

Significant enhancement was observed in LPO asmeasured by MDA levels in all the three tissues ex-amined among the mercury-exposed animals whencompared with control animals (Figure 1A). The en-hancement in MDA levels, however, was minimized inliver, kidney, and brain tissues in selenium pretreatedmercury-exposed animals. However, in posttreatmentwith selenium, no restoration was observed in the liverwhereas kidney and brain tissues showed significantrestoration in MDA levels.

Glutathione

A marked decrease was observed in GSH levelsin mercury-exposed animals when compared with thecontrol group in liver and kidney tissues (Figure 1B),whereas no change was observed in brain tissue. Treat-ment with selenium alone showed significant enhance-ment in GSH levels in liver and brain tissues, whereaskidney tissue showed some depletion in GSH levels.Pretreatment with selenium to the mercury-exposedanimals showed complete restoration in GSH levels inliver, which was not observed in posttreatment of sele-nium. In kidney tissue, selenium did not produce anychange either in pre- or posttreatment whereas in brainGSH levels were enhanced significantly when com-pared with control as well as mercury-exposed animals.

Superoxide Dismutases Activity

Exposure with mercury resulted in significant re-duction in SOD activity in liver, kidney, and braintissues (Figure 1C). Treatment with selenium aloneshowed significant enhancement in SOD activity in

liver tissue. Pre- or post-treatment of selenium tomercury-exposed animals showed significant recoveryin SOD activity in liver and brain tissues when com-pared with mercury-exposed animals; however, similareffect was not observed in the kidney.

Glutathione Peroxidase Activity

GPx activity was significantly increased in liverand brain tissues, whereas it was significantly de-creased in kidney tissue of mercury-exposed animals(Figure 1D). A similar effect was also observed in liverand kidney tissues of selenium-treated animals. Pre-treatment with selenium showed significant restorationin liver and brain tissues, but no restoration was ob-served in kidney tissue. In selenium posttreatment, norestoration was observed in liver GPx activity, whereaskidney showed further depletion and complete restora-tion was found in the brain.

Catalase Activity

CAT activity was significantly reduced in liver,kidney, and brain tissues in mercury-exposed animals(Figure 1E). Selenium-treated animals also showed sig-nificant reduction in catalase activity in kidney andbrain tissues. Pretreatment with selenium showed sig-nificant restoration in catalase activity in liver tissueonly, whereas no effect of selenium pretreatment wasobserved in kidney and brain tissues. Selenium post-treatment, however, showed further depletion in cata-lase activity in all the three tissues examined.

Mercury Accumulation

The highest accumulation of mercury was ob-served in the kidney followed by the liver and blood(Figure 1F). Mercury was not detectable in brain tis-sue at the dose we used. Pre- or posttreatment withselenium to the mercury-exposed animals significantlyenhanced the accumulation of mercury in liver andkidney tissues when compared with mercury-alone-treated group. Posttreatment with selenium, however,showed more accumulation in liver and kidney tissues.In blood tissue, no significant effect was observed inselenium pretreatment whereas posttreatment with se-lenium enhanced mercury concentration significantlywhen compared with mercury-exposed animals.

Serum Biochemical Investigations

The ALP activity was found to be decreased inmercury- as well as selenium-treated groups when



Lipid peroxidation

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FIGURE 1. Effect of selenium treatment (pre or post) on mercury-induced oxidative stress parameters ((A)–(E)) and mercury concentration(F) in rat tissues. The results are presented as mean ± SE (n = 6). Difference between groups were considered to be significant when p < 0.05.∗Values significantly different from the control group. #Values significantly different from the mercury-treated group.

compared with the control group (Figure 2A). Signif-icant recovery was observed both in selenium pre- orposttreatment to the mercury-exposed animals. Activ-ity of LDH was enhanced in mercury-exposed animals

(Figure 2B), which was significantly restored by thetreatment with selenium. The levels of creatinine andBUN were enhanced in mercury- as well as in selenium-treated groups when compared with the control group



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FIGURE 2. Effect of selenium treatment (pre or post) on mercury-induced alterations on rat serum. The results are presented as mean ±SE(n = 6). Difference between groups were considered to be significant when p < 0.05. ∗Values significantly different from the control group.#Values significantly different from the mercury-treated group.

(Figures 2C and 2D). The restoration in the creatininelevel was observed only in the selenium pretreatedgroup, whereas no effect was observed in the sele-nium posttreated group. Selenium treatment (either preor post) had no protective effect on the BUN level inmercury-exposed animals.

Histopathological Observations

Photomicrographs of transverse section (TS) ofliver tissue are shown in Figure 3. In control animals,the liver showed a normal structure of hepatocytes withgranular cytoplasm, centrally placed nuclei, and opensinusoidal spaces (A). The animals exposed with mer-cury showed mild degeneration in cytoplasm, loss ofnuclei, occasional presence of vacuoles, and dilatationof sinusoidal spaces (B). Photomicrographs of TS of thekidney (cortex) are shown in Figure 4. Kidney of controlanimals showed well-developed glomerulus with nor-mal tubular cells (A), whereas in mercury-exposed an-imals’ kidney (cortex) showed atrophy of glomerulus,dilatation of the Bowman capsule, and degeneration

of tubular cells with picnotic nuclei (B). Figure 5 rep-resents photomicrographs of TS of kidney (medulla).It showed well-developed collecting tubules and dis-tal convoluted tubules with distinct cell boundaries incontrol animals (A), whereas mercury-exposed animalsshowed degeneration of tubular cells with picnotic nu-clei and loss of nucleus in the kidney medulla region(B). Alterations in liver and kidney due to mercury ex-posure were not restored by the pre- or post-treatmentwith selenium as shown in Figures 3–5 (C and D) forthe liver, kidney (cortex), and kidney (medulla), respec-tively. Treatment of selenium alone also showed a nor-mal structure of liver and kidney tissues.

Effect on Metallothionein (MT-I and MT-II)mRNA Expression

Real-Time SYBR Green fluorescence history versuscycle number for MT-I and MT-II mRNA in rat liver andkidney is shown in Figures 6 and 7, respectively. Mer-cury exposure caused an induction in the MT mRNAexpression in the liver (MT-I: 19.29-fold and MT-II:



FIGURE 3. TS of the liver of male rat treated with mercuric chloride (Hg) alone, and in combination with sodium selenite (Se) (singleintraperitoneal injection of both compounds). (A) Control (H&E ×254): showing normal structure of hepatocytes with granular cytoplasm,centrally placed nuclei, and open sinusoidal spaces. (B) Mercury treatment (H&E ×254): showing mild degeneration in cytoplasm (1), loss ofnuclei (2), occasional presence of vacuoles (3), and dilatation of sinusoidal spaces (4). (C) Selenium treatment prior to mercury administration(H&E ×254): showing degeneration in hepatocytes with fatty infiltration (1), picnotic nuclei (2), and cytoplasmic vacuolation (3), with lossof nuclei (4). (D) Selenium treatment after mercury administration (H&E ×254): showing degeneration of hepatocytes (1), loss of nuclei (2),dilatation of sinusoidal spaces (3), and occasional presence of vacuoles (4).

6.68-fold) (Figure 8A) and the kidney (MT-I: 12.64-foldand MT-II: 27.28-fold) (Figure 8B). Selenium alone didnot exert any effect on the MT mRNA expression in theliver (Figures 8C), whereas slight induction in expres-sion of both isoforms was observed in the kidney, i.e.,3.03-fold for MT-I and 4.56-fold for MT-II (Figures 8Cand 8D). The pretreatment with selenium amelioratesinduced MT mRNA expression in both liver and kidneytissues in mercury-exposed animals (Figures 8C and8D). Complete restoration was observed in liver tissue,whereas the kidney showed 7–20-fold lowering whencompared with mercury-exposed animals. However,selenium posttreatment to the mercury-exposed ani-mals showed synergistic effect, i.e., further inductionin MT-I and MT-II mRNA expression in the liver (MT-I:171.25-fold and MT-II: 24.76-fold) and the kidney (MT-I:80.44-fold and MT-II: 294.06-fold) (Figures 8C and 8D).

DISCUSSION

A single dose of mercuric chloride resulted in en-hanced LPO and accumulation of mercury in tissues.

Similar findings were reported in mercury intoxica-tion by Huang et al. [23] and El-Demerdash [11] also.Mercuric chloride increases the production of manyendogenous oxidants such as H2O2 [24], which causeLPO in tissues [23]. Alterations observed in the activ-ity of SOD, GPx, and CAT in liver, kidney, and braintissues of mercury-exposed animals indicate the gen-eration of ROS (O−

2 or H2O2). Inhibition in the activ-ity of renal SOD, GPx, and CAT in addition to de-pletion of GSH levels was also reported earlier [25].Depletion in ALP and enhancement in LDH activityshowed liver and kidney injury as these two enzymesare reported as a biomarkers of liver and kidney dis-eases [26,27]. Enhanced creatinine and BUN levels in-dicate nephrotoxicity as also reported by Rumbeihaet al. [28]. Histopathological alterations were reportedby Carmichael and Fowler [29] and Al-Saleh et al. [30]in liver tissue after mercury exposure. Rumbeiha et al.[28], Al-Saleh et al. [30], Alam et al. [31], and Augustiet al. [32] have also reported similar histopathologicalalterations in mercury-induced nephrotoxicity.

Induction in MT-I and MT-II mRNA expression inmercury-exposed animals may be for scavenging the



FIGURE 4. TS of the kidney (cortex) of male rat treated with mercuric chloride (Hg) alone, and in combination with sodium selenite (Se)(single intraperitoneal injection of both compounds). (A) Control (H&E ×254): showing well-developed glomerulus with normal tubular cells.(B) Mercury treatment (H&E ×254): showing atrophy of glomerulus (1), dilatation of the Bowman capsule (2), and degeneration of tubular cells(3) with picnotic nuclei (4). (C) Selenium treatment prior to mercury administration (H&E ×254): showing hemorrhages beneath the Bowmancapsule (1), degeneration of glomerulus (2) and tubular cells (3) with picnotic nuclei (4). (D) Selenium treatment after mercury administration(H&E ×254): showing hemorrhages (1), degeneration of glomerulus (2) and tubular cells (3) with picnotic nuclei (4).

ROS generated due to mercury exposure. Induced MT-I and MT-II mRNA expression upon treatment withvarious metal ions (especially group IIB elements) hasbeen reported by Durnam et al. [33] and Koropatnickand Leibbrandt [34]. MTs provide an increased bindingcapacity for both toxic metals such as mercury (protec-tive) as well as essential metals like zinc (functional).The toxicity of mercury is primarily associated withits cationic state (Hg2+), and the increased content ofMT induced in both the liver and kidney acts as anintracellular ligand to bind this cation. The induced MTexpression got rapidly lowered in the liver, whereasit persisted in the kidney. This is consistent withprogressive loss of hepatic mercury soon after acuteexposure, whereas renal content of mercury remainedelevated. High intracellular levels of MT are knownto be capable of sequestering mercury and preventingtoxic effects of mercury in different organs [35–38].

The antioxidative role of selenium in organisms re-sults from the fact that it is built into the catalytic centerof GPx, the main enzyme that deactivates free radicalsand protects against excessive LPO [39]. No inductionon MT mRNA expression was observed in liver tissue

after selenium treatment. Slight induction in kidney MTmRNA expression after the selenium treatment alonemay be due the depletion in GSH levels and GPx andCAT activities. In the case of selenium administration tomercury-exposed animals, the enhanced accumulationof mercury in liver, kidney, and blood tissues was ob-served. There is, therefore, some mechanism that facil-itates the accumulation of mercury, which may be dueto the formation of mercury selenium complex withplasma protein [8,40]. The complex may increase thetendency of mercury to accumulate rather than elimi-nate from the body. This also gets support from the factthat selenium blocks the effects of chelating agents inmercury intoxication, and the process may hold goodfor elimination of mercury from the body in naturalcourse leading to higher accumulation of mercury inthe body organs [9].

Selenium pretreatment had protective effect onliver tissue in terms of restoration of oxidative stress pa-rameters altered due to mercury exposure when com-pared with the control group. Restoration of altered bio-chemical parameters also gets support from the resultsof induced MT mRNA expression in liver tissue due



FIGURE 5. TS of kidney (medulla) of male rat treated with mercuric chloride (Hg) alone, and in combination with sodium selenite (Se) (singleintraperitoneal injection of both compounds). (A) Control (H&E ×254): showing well-developed collecting tubules and distal convoluted tubules,having distinct cell boundaries. (B) Mercury treatment (H&E ×635): showing degeneration of tubular cells (1) with picnotic nuclei (2) and loss ofnucleus (3). (C) Selenium treatment prior to mercury administration (H&E ×254): showing degeneration of tubular cells (1) with large quantityof picnotic nuclei (2) and loss of nucleus (3). (D) Selenium treatment after mercury administration (H&E ×254): showing complete degenerationof tubular cells (1) with picnotic nuclei (2) and loss of nucleus (3).

to mercury exposure in selenium pretreated mercury-exposed animals. However, an enhanced level (64%)of mercury was detected in liver tissue after seleniumpretreatment compared to mercury-exposed animals.Selenium pretreatment, however, had no protective ef-fect on the altered activity of SOD, GPx, and CAT inkidney, which shows the presence of ROS in kidney tis-sue. Partial protection was also observed in kidney MTmRNA expression as there is slight induction in MTmRNA expression compared to the control group. Thismay be due to the enhanced mercury level in kidney(121% higher than that of mercury-exposed animals).No morphological protection at the cellular level in thetissues was observed by the pretreatment of seleniumto mercury-exposed animals. This may be due to thefact that repairing at the cellular level may require moretime.

The posttreatment with selenium to mercury-exposed animals showed restoration in some biochem-ical parameters in liver tissue, but no protection wasobserved in LPO. We also detected 337% enhancedmercury content in liver tissue and no protection inhistopathological changes when compared with the

mercury-treated group. Posttreatment with seleniumalso showed no protection in altered biochemical pa-rameters except restoration in LPO in kidney tis-sue, whereas significantly enhanced mercury content(384%) was observed compared to mercury-exposedanimals. Further induction in MT mRNA expressionwas also observed in liver and kidney tissues after theposttreatment of selenium to the mercury-exposed an-imals. While correlating the MT levels with content ofmercury in liver and kidney tissues, it was observedthat selenium posttreatment showed higher mercuryaccumulation over and above the mercury levels in thegroup treated with mercury alone. Similarly, no pro-tection was observed in histopathological alterationsin kidney tissue as well. In the brain, pre- or post-treatment with selenium to the mercury-exposed an-imals showed partial protection in altered biochemi-cal parameters and complete restoration in LPO levels.However, no mercury was detected in brain tissue.

The principal toxic effects of mercury involve in-teractions with a number of cellular processes includ-ing the formation of complexes with thiol groups,which may lead to oxidative stress. Mercuric chloride



FIGURE 6. Real-time PCR SYBR Green florescence history versus cycle number of MT-I (A) and MT-II (B) gene in cDNA of the rat liver tissueafter single intraperitoneal administration of mercuric chloride (Hg) and/or sodium selenite (Se).

is known to act as a prooxidant as it exerts oxidativestress [11]. The potential role of antioxidant miner-als like selenium has been extensively studied in theprevention of numerous degenerative diseases includ-ing tumor growth and carcinogenesis [41]. Seleniumpresent in the active site of GPx is essential for its cat-alytic activity [42]. One of the major roles of this essen-tial trace element within the body is, therefore, to actas a cofactor of this key antioxidant enzyme in whichit contributes both toward catalytic activity and spa-tial conformation. Also, selenium has ability to protectmembrane lipids from oxidative damage [11]. Presentstudy reveals that selenium restored LPO levels in all

FIGURE 7. Real-time PCR SYBR Green florescence history versus cycle number of MT-I (A) and MT-II (B) gene in cDNA of the rat kidney tissueafter single intraperitoneal administration of mercuric chloride (Hg) and/or sodium selenite (Se).

the three tissues in pretreatment and also in kidney andbrain tissues in posttreatment to the mercury-exposedanimals. Thus prior administration of selenium to themercury-exposed animals showed its protective effectmore on mercury toxicity in all the tissues examined,whereas partial protection was observed after the post-treatment with selenium to mercury-exposed animals.Although selenium increased mercury accumulation inliver and kidney tissues in our experiment, this accu-mulated mercury may need more time to be eliminated.

In conclusion, the results of this study indicate thatdietary intake of selenium within safe limit may, there-fore, enable to antagonize the toxic manifestations of



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FIGURE 8. Relative abundance (fold increase) of MT-I and MT-II mRNA expression in cDNA of rat liver and kidney tissues after singleintraperitoneal administration of mercuric chloride (Hg) and/or sodium selenite (Se). The fold increase is expressed as treated groups versuscontrol group in comparison of reference gene (GAPDH).

mercury; thus, it is suggested that pretreatment of sele-nium in mercury intoxication is more effective than itsposttreatment.

ACKNOWLEDGMENTS

Authors are grateful to the Director, Indian In-stitute of Toxicological Research for his interest andencouragement in the work. Thanks are also due toShri Ram Chandra, technical officer, for his overalltechnical assistance in the work. Rakhi Agarwal isgrateful to the University Grants Commission, New

Delhi, India for the award of junior research and seniorresearch fellowships.

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evaluation of comparative effect of pre- and posttreatment of selenium on mercury-induced oxidative...

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