Detoxification and antioxidant effects of curcumin in rats experimentally exposed to mercury

Detoxification and antioxidant effects ofcurcumin in rats experimentally exposedto mercuryRakhi Agarwal,a† Sudhir K. Goelb* and Jai Raj Beharia

ABSTRACT: Curcumin, a safe nutritional component and a highly promising natural antioxidant with a wide spectrum ofbiological functions, has been examined in several metal toxicity studies, but its role in protection against mercury toxicity hasnot been investigated. Therefore, the detoxification and antioxidant effects of curcumin were examined to determine itsprophylactic/therapeutic role in rats experimentally exposed to mercury (in the from of mercuric chloride-HgCl2, 12 mmol kg-1

b.w. single intraperitoneal injection). Curcumin treatment (80 mg kg-1 b.w. daily for 3 days, orally) was found to have aprotective effect on mercury-induced oxidative stress parameters, namely, lipid peroxidation and glutathione levels andsuperoxide dismutase, glutathione peroxidase and catalase activities in the liver, kidney and brain. Curcumin treatment wasalso effective for reversing mercury-induced serum biochemical changes, which are the markers of liver and kidney injury.Mercury concentration in the tissues was also decreased by the pre/post-treatment with curcumin. However, histopathologicalalterations in the liver and kidney were not reversed by curcumin treatment. Mercury exposure resulted in the induction ofmetallothionein (MT) mRNA expressions in the liver and kidney. Metallothionein mRNA expression levels were found todecrease after the pre-treatment with curcumin, whereas post-treatment with curcumin further increased MT mRNAexpression levels. Our findings suggest that curcumin pretreatment has a protective effect and that curcumin can be used as atherapeutic agent in mercury intoxication. The study indicates that curcumin, an effective antioxidant, may have a protectiveeffect through its routine dietary intake against mercury exposure. Copyright © 2010 John Wiley & Sons, Ltd.

Keywords: mercury; curcumin; oxidative stress; antioxidant defense system; histopathology; MT expression

INTRODUCTION

Although mercury has been recognized as a hazardous environ-mental pollutant, inorganic mercury is widely used in certaintypes of batteries and continues to be an essential component offluorescent light bulbs (Clarkson, 1997). Inorganic mercury accu-mulates preferentially in the kidneys and may cause acute renalfailure (Tanaka-Kagawa et al., 1998). The uptake, accumulationand toxicity of inorganic mercury in the kidney have been relatedto its binding to endogenous thiol-containing molecules (Zalups,2000). Thiol-containing enzymes have been identified as thetargets of inorganic mercury (Emanuelli et al., 1996; Nogueiraet al., 2003). Additionally, binding of mercuric ions to sulfhydrylgroups may result in decreased glutathione levels, leading toincreases in levels of reactive oxygen species, such as superoxideanion radicals, hydrogen peroxide and hydroxyl radicals (Stohsand Bagchi, 1995). Metallothioneins (MTs), which are metallopro-teins, are actively involved in the transport and detoxification ofmercury (Olanow and Arendash, 1994; Johnson, 2000) by inhibit-ing sulfur ligands, which inactivates metalloproteins that nor-mally bind metal ions such as copper (Ladner and Lindestron,1999). This leads to toxic levels of copper in many types of mem-brane. Exposure to mercury also results in changes in metallopro-tein compounds that affect gene expression (Boot, 1995). Someof the processes affected by this control of gene expressioninclude cellular respiration, metabolism, enzymatic processes,metal-specific homeostasis and adrenal stress response systems(Matts et al., 1991). Considering that oxidative stress and endog-

enous thiol depletion are involved in inorganic mercury toxicity, ithas been suggested that antioxidants could contribute to thetreatment of mercury poisoning (Patrick, 2002).

Curcumin from turmeric, a well known biologically active com-pound, has been shown to ameliorate oxidative stress and it isconsidered to be a potent antioxidant (Eybl et al., 2006). Curcuminis a potent inducer of detoxifying enzymes and thereby countersthe toxicity induced by chemical carcinogens (Singletary et al.,1998). It is already used as a drug to control various diseases,including inflammatory disorders, and is expected to be used as adrug to prevent for carcinogenesis and oxidative stress-inducedpathogenesis in the future. Curcumin prevents paraquat-induced

*Correspondence to: S. K. Goel, Petroleum Toxicology Division, Indian Institute ofToxicology Research, Post Box 80, M. G. Marg, Lucknow-226001, India.E-mail: [email protected]

aToxicokinetics Section, Indian Institute of Toxicology Research (formerly: Indus-trial Toxicology Research Centre), Council of Scientific and Industrial Research,India, Post Box 80, Mahatma Gandhi Marg, Lucknow 226001, India.

bPetroleum Toxicology Division, Indian Institute of Toxicology Research (formerly:Industrial Toxicology Research Centre), Council of Scientific and IndustrialResearch, India, Post Box 80, Mahatma Gandhi Marg, Lucknow 226001, India.

†Present address: Forensic Science Institute, Gujarat Forensic Sciences University,DFS Head Quarter, Sector 18/A, Near Police Bhavan, Gandhinagar-382007,Gujarat, India.

Research Article

Received: 29 August 2009, Revised: 29 January 2010, Accepted: 1 February 2010 Published online in Wiley InterScience: 12 March 2010

(www.interscience.wiley.com) DOI 10.1002/jat.1517

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J. Appl. Toxicol. 2010; 30: 457–468 Copyright © 2010 John Wiley & Sons, Ltd.

lung toxicity and protects the cell membrane (Venkatesan, 2000).Having a polyphenolic structure and diketone functional groups,curcumin is a stronger antioxidant inhibitor of lipid peroxidationthan other flavonoids, which have a single phenolic hydroxylgroup (Phan et al., 2001). The effective antioxidant property ofcurcumin inhibits the utilization of vitamins C and E in the liver,thus maintaining their levels (Rukkumani et al., 2003). Curcuminhas been reported to protect hepatocytes against alcohol andpolyunsaturated fatty acid (PUFA) induced liver toxicity (Rukku-mani et al., 2002). It has been used as an antioxidant in toxicitystudies of several metals including cadmium (Daniel et al., 2004),copper (Nair et al., 2005), iron (Manjunatha and Srinivasan, 2006),lead (Dairam et al., 2007) and selenium (Padmaja and Raju, 2004).However, the efficacy of curcumin treatment for mercury toxicityhas not been investigated so far. We therefore carried out thiswork to evaluate the efficacy of curcumin in amelioratingmercury toxicity and the antioxidant potential of curcumin treat-ment before or after mercury exposure in rats. The effect ofmercury toxicity on the expressions of metallothionein (MT-I andMT-II) mRNAs in liver and kidney tissues and the role of pre- orpost-treatment with curcumin on metallothionein mRNA expres-sion levels were also studied by real-time RT-PCR analysis.

MATERIALS AND METHODS

Chemicals and Reagents

Mercuric chloride was obtained from Merck, India, curcumin fromSigma Chemical Co. (St Louis, MO USA) and trizol reagent fromGibco BRL (USA). Other chemicals of the purest grade availablewere obtained from Sigma Chemical Co. or other standardsuppliers.

Kits and Primers

Activities/levels of serum biomarkers were determined usingcommercial kits procured from Spinreact, Spain. For cDNA syn-thesis, a RevertAid H Minus First cDNA Synthesis kit was procuredfrom Fermentas Life Sciences, Germany. SYBR Green JumpStartTaq ReadyMix for quantitative PCR was procured from Sigma-Aldrich, USA. Primers of specific sequences for rat MT-I and MT-IIwere procured from Metabion International AG, Germanywhereas those for GAPDH from MWG Biotech., AG, Germany wereused for PCR amplification.

Animals, Treatment and Collection of Tissues

Thirty six adult male Wistar rats (150 � 10 g) procured from theIndian Institute of Toxicology Research (IITR), Lucknow breedingcolony maintained under controlled temperature (22–25 °C) anda 12 h alternate light and dark cycle and with free access to waterand pellet food (Lipton India Ltd, Mumbai) were used in thestudy. Approval from the Institutional Animal Ethics Committeeof IITR, Lucknow was obtained before the experiment was initi-ated. A single dose of mercury (Hg) in the form of mercuric chlo-ride (12 mmol kg-1 b.w.) was administered intraperitoneally(dissolved in n-saline) and three doses of curcumin (Cur80 mg kg-1 b.w.) were given orally after an interval of 24 h (dis-solved in groundnut oil, GNO) to the animals. The animals wererandomly divided into six groups (six animals each) namely

control (n-saline + GNO; group I), GNO + Hg (group II), Hg + GNO(group III), Cur (group IV), Cur + Hg (curcumin pre-treatment,group V) and Hg + Cur (curcumin post-treatment, group VI). Thetime interval between the treatments with mercury and cur-cumin was 2 h in both pre/post treated groups.

Rats were sacrificed under anesthesia 24 h after the last treat-ment. Blood was drawn from the heart into two separate vials, i.e.heparinized (for mercury determination) and nonheparinized [forserum biochemical assays of alkaline phosphatase (ALP), lactatedehydrogenase (LDH), blood urea nitrogen (BUN) and creatinine],and stored at 4 °C until assay. Portions of the liver and kidney(50–70 mg) were removed immediately and stored at -20 °C fortotal RNA isolation.The remaining tissues from each organ and thewhole brain were immediately removed, trimmed of extraneousmaterials, washed with chilled saline solution, blotted andweighed. For histopathological analysis, one lobe of the liver andright kidney was fixed in 10% formalin. The remaining parts of theliver and kidney and the whole brain were minced and homog-enized (10% w/v) in ice-cold 0.15% KCl–0.1 M phosphate buffer(pH 7.4) in a Potter–Elvehjem homogenizer. The tissue homoge-nates were stored at -20 °C for the assays of lipid peroxidation(LPO), level of reduced glutathione (GSH) and activities of antioxi-dant enzymes. Total mercury concentration was also determinedin liver, kidney, brain tissue homogenates and whole blood.

Biochemical Assays

The level of malondialdehyde (MDA), an end product of lipidperoxidation, was measured with the absorbance coefficientof the MDA-TBA complex at 532 nm on SpectrophotometerGenesys 10UV, USA, using 1,1,3,3-tetraethoxypropane as thestandard (Ohkawa et al., 1979). Glutathione level was deter-mined using 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) forcolor development spectrophotometrically at 412 nm (Ellman,1959). Superoxide dismutase (SOD) activity was measuredspectrophotometrically by the modified method of NADH-phenazinemethosulfate nitroblue tetrazolium formazan inhibi-tion reaction at 550 nm using an ELISA plate reader (Kakkar et al.,1984). Glutathione peroxidase (GPx) activity was measured by themethod of Flohe and Gunzler (1984) at 420 nm. Catalase (CAT)activity was measured spectrophotometrically at 570 nm by themethod of Sinha (1972). Protein level was assayed in accordancewith the method of Lowry et al. (1951) using bovine serumalbumin (BSA) as the standard.

Serum Biochemical Assays

The activities of ALP and LDH and the levels of BUN and creati-nine were determined photometrically, in accordance with themanufacturer’s protocol using a diagnostic automated labora-tory analyzer (ChemWell-Biological Auto-Analyzer, USA).

Mercury Level Estimation

Mercury was analyzed in tissue homogenates and whole bloodby the method of Skare (1972) using an atomic absorption spec-trophotometer (AAS) equipped with a vapor generation assem-bly (GBC Avanta-S). Samples were analyzed against standardswithin the linear range of the calibration curve.

Histopathology

Small pieces of the liver or half of the kidney were used for his-topathological analysis. Sections of 5 mm thickness were cut

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using a rotary microtome (Leica Microsystems Nussloch GmbH,Germany), stained with hematoxylin–eosin (H&E) and examinedunder a light microscope (Nikon Eclipse E600, Japan).

Total RNA Extraction and Reverse Transcription

Total RNA was isolated using trizol reagent in accordance withthe manufacturer’s instructions (Gibco BRL, USA). The OD260/OD280 ratio of all the RNA samples used was over 1.8. Total RNA(0.5 mg) from each individual rat tissue (n = 3) of every group wasreverse-transcribed to complementary DNA (cDNA) with a kit inaccordance with the manufacturer’s protocol using a PeltierThermal Cycler, USA.

Real-time RT-PCR

PCR amplification and quantitation of gene expression were per-formed for MT-I (forward primer, 5’-TGGACCCCAACTGCTCCTG-3’; reverse primer, 5’-TCAGGCACAGCACGTGCAC-3’) and MT-II(forward primer, 5’-TGGACCCCAACTGCTCCTG-3; reverse primer,5’-TCAGGCGCAGCAGCTGCAC-3’) together with the housekeep-ing gene glyceraldehyde-3-phosphate dehydrogenase (GAPDHforward primer, 5’-CAGCAAGGATACTAGGAG-3’; reverse primer,5’-GGATGGAATTGTGAGGGAGATG-3’) cDNA from liver and kidneytissues. Real-time RT-PCR by SYBR Green detection kit was per-formed using a Stratagene Mx3000P® QPCR System, USA. Theexpected lengths of the PCR products of rat MT-I, MT-II andGAPDH genes were 200–400 bp. All the reactions were carriedout in a 25 mL reaction mixture in triplicate following the manu-facturer’s protocol. The real time RT-PCR protocol used was asfollows: denaturation (10 min at 95 °C for MT-I and MT-II, and2 min at 94 °C for GAPDH), amplification and quantificationrepeated 40 times (15 s at 95 °C; 10 s at 60 °C; 30 s at 72 °C with asingle fluorescence measurement), melting curve (60–99 °C at aheating rate of 0.1 °C s-1 and continuous fluorescence measure-ments), cooling to 40 °C (Pfaffl et al., 2003).

Data Analysis

The data obtained by real time RT-PCR are presented as thresholdcycle (Ct), which is the cycle at which fluorescence intensity wasdetermined to be statistically significantly higher than the back-ground signal contributed by the labeled oligonucleotides withinPCR (Pfaffl et al., 2003). A Ct lower than the control Ct indicatesthat an increase in the expression level of a gene at Ct is inverselyproportional to the log of the initial copy number of a PCRproduct. For the calculation of fold changes in target geneexpression level, relative quantification by the ‘delta-delta CtMethod’ was used for comparing relative expression levelsbetween treatments in real-time RT-PCR (Pfaffl et al., 2003). Themethod is based on the relative expression of a target gene vsthat of a reference gene. In this work, we used GAPDH as thehousekeeping gene for relative quantification of MT-I and MT-IIgene expressions in the liver and kidney tissues.

Statistical Evaluation

The statistical significance of mean value of different observa-tions was analyzed by one-way analysis of variance (ANOVA). Thehomogeneity of variance between treatment groups was ascer-

tained using Bartlett’s test (Sendercor and Cochran, 1967). Posthoc analysis was carried out to compare the mean valuesbetween a pair of treatments using Scheffe’s multiple compari-son procedure (Zar, 1984). A probability of less than or equal to0.05 was considered to be significant.

RESULTS

Lipid Peroxidation

In the GNO + Hg group, significant enhancement of LPO wasobserved in the tissues examined compared with those of controlanimals (Fig. 1A). While in the other mercury-exposed group(Hg + GNO), significant enhancement of LPO was observed inthe kidney and brain tissues. The enhancement of LPO was sig-nificantly reversed by curcumin pre- or post-treatment.

Glutathione

Significant decreases in GSH level were observed in the liver andkidney tissues of animals exposed to mercury (GNO + Hg; Fig. 1B).These decreases were significantly reversed in the group pre-treated with curcumin. The animals of the Hg + GNO group didnot show any change in GSH level compared with those in thecontrol group.

Superoxide Dismutase Activity

In the GNO + Hg group, exposure to mercury significantlyincreased SOD activity in the liver, kidney and brain tissues(Fig. 1C), which was completely reversed in the liver and braintissues of animals pretreated with curcumin, whereas in thekidney, the extent of reversal of SOD activity increase was lowerthan that in the control group. In the Hg + GNO group, only thebrain tissue showed an increased SOD activity compared with thecontrol group, which was reversed by the post-treatment withcurcumin. However, the kidney tissue of the curcumin-post-treated group showed a decrease in SOD activity compared withthe control as well as the Hg + GNO group. Treatment of theanimals with curcumin alone resulted in a significant decrease inSOD activity in the kidney and brain tissues.

Glutathione Peroxidase Activity

The activity of GPx was increased in the liver, kidney and braintissues of the GNO + Hg treated group (Fig. 1D). The enhance-ment was significantly reversed in the liver and brain tissues ofanimals with curcumin pretreatment. However, in the kidneytissue, the extent of reversal of increased GPx activity was lowerthan that in the kidney tissue of the control group. The Hg + GNOgroup showed decreased GPx activity in the kidney tissue,whereas no changes were observed in the liver and brain tissues.Post-treatment with curcumin had no effect on decreased GPxactivity in the kidney tissue.

Catalase Activity

CAT activity was significantly decreased in the liver and kidneytissues of the GNO + Hg and Hg + GNO groups compared with the

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control group, which was completely reversed by the pre- orpost-treatment with curcumin (Fig. 1E). In the brain, no changewas observed after mercury exposure (both in the GNO + Hg andHg + GNO groups). However, the curcumin-treated (pre- or post-)group showed decreased catalase activity compared with thecontrol group.

Mercury Accumulation

The highest concentration of mercury was observed in thekidney followed by the liver and blood (Fig. 1F). Mercury was notdetected in the brain tissue at the dose we used. The concentra-tion of mercury was higher in the GNO + Hg group than in theHg + GNO group. Pretreatment with curcumin did not signifi-cantly reduce mercury accumulation in the liver, kidney andblood tissues, whereas post-treatment with curcumin signifi-cantly reduced the accumulation of mercury in these tissues.

Serum Biochemical Parameters

In serum, the activity of ALP was found to be decreased in theGNO + Hg and Hg + GNO groups (Fig. 2A), whereas the activity ofLDH was increased significantly by mercury exposure (Fig. 2B).Pre- or post-treatment with curcumin was significantly effectivein restoring the altered ALP and LDH activities. The levels of crea-tinine and BUN were significantly increased after mercury expo-sure (GNO + Hg and Hg + GNO; Fig. 2C and 2D, respectively). Norestoration of creatinine levels was observed for curcumin pre-treatment, whereas post-treatment with curcumin significantlyrestored creatinine levels. The BUN levels were restored com-pletely by curcumin pre- and post-treatments.

Histopathological Observations

Photomicrographs of transverse sections (TS) of the liver areshown in Fig. 3. In the control animals, the liver showed a normal

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Figure 2. Effect of curcumin treatment (pre- or post-) on mercury-induced alterations in rat serum. The results are presented as mean � SE (n = 6).Differences between groups were considered significant when P < 0.05. *Values significantly different from control group. #Values significantly differentfrom mercury-treated group.

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structure of hepatocytes with a granular cytoplasm, centrallyplaced nuclei and open sinusoidal spaces (Fig. 3A). The liver ofmercury-exposed animals showed degeneration of the cyto-plasm, picnotic nuclei, occasional presence of vacuoles and dila-tation of sinusoidal spaces (Fig. 3B). Curcumin pretreated animalsshowed degeneration of hepatocytes with fatty infiltration, cyto-plasmic vacuolation and loss of nuclei (Fig. 3C), whereas cur-cumin post-treated animals showed severe degeneration ofhepatocytes with picnotic nuclei, dilatation of sinusoidal spacesand occasional presence of vacuoles (Fig. 3D). Animals treatedwith curcumin alone showed a normal structure of the livertissue. Figure 4 shows TS of the kidney (cortex); control animalsshowed well-developed glomerulus with normal tubular cells(Fig. 4A), whereas in the mercury-exposed group, the kidneyshowed atrophy of glomeruli, dilatation of the Bowman capsuleand degeneration of tubular cells (Fig. 4B). In the pre- or post-treatment with curcumin the kidney showed lesions as repre-sented by the hemorrhages beneath the Bowman capsule,degeneration of glomerulus and loss of tubular cells with pic-notic nuclei (Fig. 4C, D). TS of the kidney (medulla) (Fig. 5) showedwell-developed collecting tubules and distal convoluted tubuleswith distinct cell boundaries in control animals (Fig. 5A), whereasin mercury-exposed animals, the medulla region showed degen-eration of tubular cells with picnotic nuclei and loss of nucleus(Fig. 5B). Animals pre- or post-treated with curcumin showeddegeneration of tubular cells with fatty infiltration, large numberof picnotic nuclei, presence of vacuoles and loss of nuclei in themedulla (Fig. 5C, D). Animals treated with curcumin aloneshowed a normal structure of kidney tissues.

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

Real-time SYBR Green fluorescence intensity history vs cyclenumber and Ct values (threshold cycle) for MT-I and MT-II mRNAexpressions in the rat liver are shown in Fig. 6(A, B), whereas forthe kidney they are shown in Fig. 7(A, B). Mercury exposureinduced metallothionein mRNA expressions in the liver andkidney (Fig. 8A, B). Treatment with curcumin alone induced MTmRNA expressions in the liver and kidney tissues (Fig. 8C, D).Pretreatment with curcumin downregulated/arrested theinduced MT mRNA expressions in the liver and kidney tissues,whereas in the curcumin post-treated group, MT-I and MT-IImRNA expressions were further induced compared with theircorresponding mercury-exposed group (Fig. 8C, D).

DISCUSSION

The toxicity of HgCl2 is attributed to the high affinity of Hg(II) tothiol groups owing to which it can deplete cellular GSH anddamage proteins and thiol enzymes (Augusti et al., 2007). Wefound decreases in GSH levels in liver and kidney tissues as aresult of mercury exposure (Fig. 1B). GSH level may also decreaseduring neutralization of oxidative stress generated by HgCl2

(Lund et al., 1993). Hg(II) enhances H2O2 production because of itseffect on the inner mitochondrial membrane. H2O2 so producedis speculated to induce LPO, which plays a crucial role in Hg(II)-induced nephrotoxicity (Huang et al., 1996). In this study, wefound that HgCl2 enhanced LPO in tissues (Fig. 1A). Oxidative

Figure 3. TS of liver of male rat treated with mercuric chloride [(Hg) single intraperitoneal injection] alone and in combination with curcumin (threeoral doses). (A) Control (H&E X 254), showing normal structure of hepatocytes with granular cytoplasm, centrally placed nuclei and open sinusoidalspaces. (B) Mercury treatment (H&E X 254), showing degeneration of cytoplasm (1) with picnotic nuclei (2), occasional presence of vacuoles (3) anddilatation of sinusoidal spaces (4). (C) Treatment with curcumin prior to mercury administration (H&E X 254), showing degeneration of hepatocytes withfatty infiltration (1) and cytoplasmic vacuolation (2) with loss of nuclei (3). (D) Treatment with curcumin after mercury administration (H&E X 254),showing degeneration of hepatocytes (1) with picnotic nuclei (2), dilatation of sinusoidal spaces (3) and occasional presence of vacuoles (4).

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stress and the production of free radicals have been suggested tobe involved in Hg-induced tissue injury (Stohs and Bagchi, 1995;Zalups, 2000), and the high affinity between mercury and endog-enous thiol-containing molecules, such as GSH and d-ALA-D,seems to contribute to this processes. Girardi and Elias (1995)suggested that changes in GPx and CAT activities do not underlieHgCl2-induced lipid peroxidation or tissue damage. Our resultscorroborate this suggestion, since we observed lipid peroxida-tion along with histopathological damage with alterations inSOD, GPx and CAT activities in the liver, kidney and brain tissues.These findings also indicate that free radicals generated by HgCl2

altered endogenous antioxidant activity and induced oxidativedamage in tissues. The increases in SOD and GPx activitiesoccurred probably as a defense response against hydrogen per-oxide generated by HgCl2, as mercury was also detected in theliver and kidney tissues (Fig. 1F). However, mercury was notdetected in the brain tissues, which may be due to the fact thatinorganic mercury can hardly penetrate the blood–brain barrier.The enhancement of LPO and decrease in GSH level showed thatthe generation of ROS in the brain is due to mercury toxicity. It iswell known that the brain tissues are more sensitive to oxidativedamage because of their high concentration of unsaturatedlipids and high rate of oxidative metabolism (Goering et al., 2002).This is also supported by our findings of the highest MDA level inthe brain among the tissues examined in mercury-treatedanimals and by our previous works (Agarwal and Behari,2007a, b).

Interestingly, we also found SOD depletion in the kidneytissues, in both curcumin pre- and post-treated mercury-exposedanimals compared with the control as well as the GNO + Hg and

Hg + GNO exposed animals. SOD inhibition may be related to thecovalent attachment of mercury ions to SOD active site reactivecysteine residues (Shimojo et al., 2002) or to a decreased avail-ability of Cu and Zn as a result of their binding to metallothion-eins, which are involved in the detoxification of metals such asmercury (Brzoska et al., 2002). Alternatively, SOD inhibition mayalso be a consequence of excessive reactive oxygen species gen-eration, which would affect enzyme structure (Salo et al., 1988).SOD catalyzes superoxide anion radical dismutation into hydro-gen peroxide. Therefore, regardless of the underlying mecha-nism, SOD inhibition may contribute to the enhanced oxidationobserved in mercury-exposed rats. A similar pattern was alsoobserved for GPx activity as it was increased in the kidney of theGNO + Hg group, but in pre- or post-treatment with curcuminGPx activity was lower than that of the control group. This may bedue to the decreased SOD activity in curcumin treatment alone.The decreased CAT activity comes to normal level in both the pre-and post-treatment with curcumin. Similarly, we also found thecomplete restoration of LPO levels in all the tissues examinedafter curcumin treatment of the mercury-exposed animals.

Mercury was also detected in whole blood (Fig. 1F), which indi-cates the circulation of mercury in the body after mercury admin-istration. ALP is a sensitive biomarker and is related to thetransport of various metabolites (El-Demerdash, 2001). A signifi-cant decrease was observed in serum ALP activity after mercurytreatment, which may be due to a breakdown of the mem-brane transport system, an inhibitory effect on cell growth andproliferation in mercury toxicity (Lakshmi et al., 1991). Theincreased activity of LDH (Fig. 2B) may be due to hepatic damageinduced by environmental pollutants (Fernandez et al., 1994).

Figure 4. TS of kidney (cortex) of male rat treated with mercuric chloride [(Hg) single intraperitoneal injection] alone and in combination withcurcumin (three oral doses). (A) Control (H&E X 254), showing well developed glomerulus with normal tubular cells. (B) Mercury treatment (H&E X 254),showing atrophy of glomerulus (1), dilatation of the Bowman capsule (2) and degeneration of tubular cells (3). (C) Treatment of curcumin prior tomercury administration (H&E X 254), showing hemorrhages beneath the Bowman capsule (1), degeneration of glomerulus (2), and tubular cells (3) withpicnotic nuclei (4). (D) Treatment with curcumin after mercury administration (H&E X 254), showing hemorrhages (1), degeneration of glomerulus (2) andtubular cells (3) with picnotic nuclei (4).

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El-Demerdash (2001) also found decrease in ALP activity and anincrease in LDH activity after mercury exposure. The increasedlevels of creatinine and BUN indicate mercury-induced nephro-toxicity (Fig. 2C, D), which is also reported by Rumbeiha et al.(2000). ALP and LDH are the best biomarkers of liver and kidneydiseases (Pesce, 1984; Wenger, 1984), whereas alterations in BUNand creatinine levels indicate renal toxicity (Murray, 1984; Kaplan,

1984). Histopathological alterations also supported these findingsin the liver and kidney tissues associated with mercury toxicity. Wefound hepatocyte damage, loss of nuclei and picnotic nuclei in theliver tissue (Fig. 3), which is due to mercury toxicity as alsoreported by Carmichael and Fowler (1979) and Al-Saleh et al.(2005). In kidney tissue, the sign of toxicity was also observed(Figs 4 and 5). Rumbeiha et al. (2000), Al-Saleh et al. (2005), Alam

Figure 5. TS of kidney (medulla) of male rat treated with mercuric chloride [(Hg) single intraperitoneal injection] alone and in combination withcurcumin (three oral doses). (A) Control (H&E X 254), showing well developed collecting tubules and distal convoluted tubules, having distinct cellboundaries. (B) Mercury treatment (H&E X 635): showing degeneration of tubular cells (1) with picnotic nuclei (2) and loss of nuclei (3). (C) Treatment withcurcumin prior to mercury administration (H&E X 635): degeneration of tubular cells (1) with large quantity of picnotin nuclei (2) and fatty infiltration(3). (D) Treatment with curcumin after mercury administration (H&E X 635), showing complete degeneration of tubular cells (1) with the presence ofvacuoles (2), picnotic nuclei (3) and loss of nuclei (4).

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Figure 8. Relative abundance (fold increase) of MT-I and MT-II mRNA expressions in cDNA of rat liver and kidney tissues after single intraperitonealadministration of mercuric chloride (Hg) and/or three oral doses of curcumin. The fold increase is presented as the expression levels of MT-I and MT-IIgenes in treated groups versus control group in comparison with the expression level of the reference gene (GAPDH).

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et al. (2007) and Augusti et al. (2007) have also reported similarchanges due to mercury-induced nephrotoxicity. The high MT-Iand MT-II mRNA expression levels in the liver tissues of the GNO +Hg group may be due to the oxidative stress caused by ROSgenerated by mercury toxicity. This is also shown by alteration inLPO and GSH levels and in the activities of SOD, GPx, CAT, ALP andLDH in the liver tissue. Similarly, the kidney tissue also showedinduction in MT-I and MT-II mRNA expressions. However, theexpression levels were lower than those in the liver tissue. Thereason for this may be that the hepatic burden of mercury is asmuch as 7–8% of the dose 1 h after exposure (Zalups et al., 1999).Thus, the maximal hepatic burden of mercury is reached rapidlyafter exposure, and hepatic elimination of mercury begins withinhours. In the Hg + GNO group, induction of MT-I and MT-II mRNAexpressions was also observed in both the liver and kidney tissues.However, the kidney tissue showed higher expression levels thanliver tissue. This may be due to only about 5–6% of the dosepresent in the liver by the end of the first day after exposure(Zalups and Cherian, 1992; Zalups, 1995). Numerous studies(Zalups and Cherian, 1992; Zalups, 1995, 2000) indicate that thekidneys of rats exposed to a low dose of inorganic mercury accu-mulate approximately one-half the dose during the initial 24 hafter exposure. Approximately 75% of mercury accumulated inthe kidneys is retained by tubular epithelial cells over the subse-quent weeks after exposure (Zalups and Koropatnick, 2000).

Curcumin has a long history as a food additive and herbalmedicine in India and is also a potent polyphenolic antioxidant. Itis a yellow curry spice derived from turmeric. Curcumin is severaltimes more potent than vitamin E as a free radical scavenger,protects the brain from lipid peroxidation and scavengesNO-based radicals. It is a non-toxic, highly promising natural anti-oxidant compound with a wide spectrum of biological functions.The antioxidant activity of curcumin was reported as early as1975 (Sharma, 1976). Studies have consistently shown thatcurcumin is relatively nontoxic. Studies using curcumin at2000 mg kg-1 b.w. revealed no mortalities in mice. Curcumin wasadministered to wistar rats, guinea pigs and monkeys of bothsexes at a dose of 300 mg kg-1 b.w. No pathological or behaviouralabnormalities or lethality was observed. No adverse effects wereobserved on growth, counts of erythrocytes and leucocytes andlevels of blood constituents such as hemoglobin, total serumprotein and alkaline phosphatase. Human clinical trials also indi-cate that curcumin is non toxic when administered at doses of 1–8and 10 g/day (Chattopadhyay et al. 2004). Curcumin acts asa scavenger of oxygen free radicals (Subramaniam et al., 1994;Ruby et al., 1995) and can protect hemoglobin from oxidation(Unnikrishnan and Rao, 1995). Curcumin also decreases the pro-duction of ROS in vivo (Joe and Lokesh, 1994) and inhibits lipidperoxidation in rat liver microsomes, erythrocyte membranes andbrain homogenates (Pulla and Lokesh, 1992). Interestingly, cur-cumin not only exhibits antioxidative and free radical scavengingproperties, but also increases the activities of other antioxidantenzymes, such as SOD, CAT and GPx (Pulla and Lokesh, 1994).

To examine the role of curcumin in mercury toxicity, we carriedout curcumin pre- or post-treatments to evaluate the prophylac-tic or therapeutic effects of curcumin. From our findings, it is clearthat curcumin prevents mercury toxicity in terms of attenuatedLPO, decreased GSH, creatinine and BUN levels and alteredendogenous enzyme activities in the liver, kidney, brain andblood tissues. Groups with pre- or post-treated with curcuminalso showed reduced mercury concentration in the liver, kidneyand blood compared with their corresponding mercury-treated

groups. However, statistically significant mercury concentrationreductions in all the tissues were found only in curcumin post-treatment. Curcumin treatment showed no protective effectagainst histopathological changes in the liver and kidney tissues.This may be due to the fact that the time required for the repair ofdamage at the cellular level may be short, during which reversalof histopathological changes is not possible. We found someinduction of MT-I and MT-II mRNA expressions in the liver andkidney tissues after curcumin treatment alone, which may be dueto ROS normally present in the animals that might be scavengedfrom the body by curcumin treatment. This elucidated the anti-oxidative property of curcumin. Pretreatment with curcuminresulted in complete protection of the liver and kidney tissues. Inthe curcumin-post-treated group, further induction of metal-lothionein mRNA expression was observed in the liver and kidneytissues. The kidney tissue showed greater induction than the livertissue. This may be due to the slow elimination of mercury fromthe kidney tissue.

Thus we observed that curcumin pretreatment resulted in thecomplete protection against mercury exposure in terms of oxida-tive stress. In conclusion, the present work suggests that cur-cumin intake should be helpful in the prevention of mercurytoxicity and that curcumin can be used as a therapeutic agent formercury intoxication.

Acknowledgments

We thank Professor Y. K. Gupta, Ex-Director, IITR for his encour-agement in the work. We are also grateful to Dr R.B. Raizada,Ex-Scientist F, IITR for his guidance and support in histopathologi-cal studies. We also thank Shri Ram Chandra, TO for his overalltechnical assistance in the work. This work was supported bythe Junior and Senior Research fellowships awarded to RakhiAgarwal by University Grants Commission, New Delhi, India.

REFERENCES

Agarwal R, Behari JR. 2007a. Effect of selenium pretreatment in chronicmercury intoxication in rats. Bull. Environ. Contam. Toxicol. 79: 306–310.

Agarwal R, Behari JR. 2007b. Role of selenium in mercury intoxication inmice. Ind. Health 45: 388–395.

Alam MS, Kaur G, Jabbar Z, Javed K, Athar M. 2007. Eruca sativa seedspossess antioxidant activity and exert a protective effect on mercuricchloride induced renal toxicity. Food Chem. Toxicol. 45(6): 910–920.

Al-Saleh I, El-Doush I, Shinwari N, Al-Baradei R. 2005. Does low mercurycontaining skin lightening cream (fair & lovely) affect the kidney, liver,and brain of female mice? Cutan. Ocul. Toxicol. 24: 11–29.

Augusti PR, Conterato GMM, Somacal S, Einsfeld L, Ramos AT, HosomiFYM, Grace DL, Emanuelli T. 2007. Effect of lycopene on nephrotoxic-ity induced by mercuric chloride in rats. Basic Clin. Pharmacol. Toxicol.100(6): 398–402.

Boot JH. 1995. Effects of SH-blocking compounds on the energymetabolism in isolated rat hepatocytes. Cell Struct. Funct. 20(3):233–238.

Brzoska MM, Moniusko-Jakoniuk J, Jurczuk M, Galazyn-Sidorczuk M. 2002.Cadmium turnover and changes of zinc and copper body statusof rats continuously exposed to cadmium and ethanol. Alcohol 37:213–221.

Carmichael NG, Fowler BA. 1979. Effects of separate and combinedchronic mercuric chloride and sodium selenite administration in rats:histological, ultrastructural and X-ray microanalytical studies of liverand kidney. J. Environ. Pathol. Toxicol. 3: 399–412.

Chattopadhyay I, Biswas K, Bandyopadhyay U, Banerjee RK. 2004. Tur-meric and curcumin: biological actions and medicinal applications.Curr. Sci. 87(1): 44–53.

Clarkson TW. 1997. The toxicology of mercury. Crit. Rev. Clin. Lab. Sci. 34:369–403.

466

R. Agarwal et al.


Dairam A, Limson JL, Watkins GM, Antunes E, Daya S. 2007. Curcuminoids,curcumin and demethoxycurcumin reduce lead-induced memorydeficits in male Wistar rats. J. Agic. Food Chem. 55(3): 1039–1044.

Daniel S, Limson JL, Dairam A, Watkins GM, Daya S. 2004. Through metalbinding, curcumin protects against lead- and cadmium-induced lipidperoxidation in rat brain homogenates and against lead-inducedtissue damage in rat brain. J. Inorg. Biochem. 98: 266–275.

El-Demerdash RM. 2001. Effects of selenium and mercury on the enzy-matic activities and lipid peroxidation in brain, liver and blood of rats.J. Environ. Sci. Health B 36: 489–499.

Ellman GL. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70–77.

Emanuelli T, Rocha JBT, Pereira ME, Porciuncula LO, Morsch VM, MartinsAF, Souza DO. 1996. Effect of mercuric chloride intoxication anddimercaprol treatment on delta-aminolevulinate desidratase frombrain, liver and kidney of adult mice. Pharmacol. Toxicol. 79: 136–143.

Eybl V, Kotyzova D, Koutensky J. 2006. Comparative study of naturalantioxidants – curcumin, resveratrol and melatonin – in cadmium-induced oxidative damage in mice. Toxicology 225: 150–156.

Fernandez A, Verde MT, Gascon M, Ramo J, Gomez J, Luco DF, Chavez G.1994. Variations of clinical biochemical parameters of laying hens andbroiler chickens fed aflatoxin-contaminated feed. Avian Pathol. 23:37–47.

Flohe L, Gunzler WA. 1984. Assays of glutathione peroxidase. MethEnzymol. 105: 114–121.

Girardi G, Elias MM. 1995. Mercuric chloride effects on rat renal redoxenzymes activities: SOD protection. Free Radic. Biol. Med. 18: 61–66.

Goering PL, Morgan DL, Ali SF. 2002. Effect of mercury vapor inhalation onreactive oxygen species and antioxidant enzymes in rat brain andkidney are minimal. J. Appl. Toxicol. 22: 167–172.

Huang YL, Cheng SL, Lin TH. 1996. Lipid peroxidation in rats adminis-trated with mercuric chloride. Biol. Trace Elem. Res. 52: 193–206.

Joe B, Lokesh BR. 1994. Role of capsaicin, curcumin and dietary n-3 fattyacids in lowering the generation of reactive oxygen species in ratperitoneal macrophages. Biochim. Biophys. Acta 1224: 255–263.

Johnson S. 2000. The possible role of gradual accumulation of copper,cadmium, lead, and iron depletion of zinc, magnesium, selenium,vitamins B2, B6, D, and E and essential fatty acids in multiple sclerosis.Med. Hypotheses 55(3): 239–241.

Kakkar P, Das B, Viswanathan PN. 1984. A modified spectrophotometricassay of superoxide dismutase. Ind. J. Biochem. Biophys. 21: 130–132.

Kaplan A. 1984. Blood urea nitrogen. In Clinical Chemistry, Kaplan A (ed.).Mosby: St Louis; 1257–1260.

Ladner L, Lindestron L. 1999. Copper in Society and the Environment, 2ndrevised edition. Environmental Research Group (MFG).

Lakshmi R, Kundu R, Thomas E, Mansuri AP. 1991. Mercuric chlorideinduced inhibition of acid and alkaline phosphatase activity in thekidney of mudskipper; Boleophthalmus dentatus. Acta Hydrochim.Hydrobiol. 3: 341–344.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measure-ment with folin phenol reagent. J. Biol. Chem. 193: 265–275.

Lund BO, Miler DM, Woods JS. 1993. Studies on Hg(II)- induced H2O2formation and oxidative stress in vivo and in vitro in rat kidney mito-chondria. Biochem. Pharmacol. 45: 2017–2024.

Manjunatha H, Srinivasan K. 2006. Protective effect of dietary curcuminand capsaicin on induced oxidation of low-density lipoprotein, iron-induced hepatotoxicity and carrageenan-induced inflammation inexperimental rats. FEBS 273(19): 4528–4537.

Matts RL, Schatz JR, Hurst R, Kagen R. 1991. Toxic heavy metal ions inhibitreduction of disulfide bonds. J. Biol. Chem. 266(19): 12695–12703.

Murray R. 1984. Creatinine. In Clinical Chemistry, Kaplan A (ed.). Mosby: StLouis; 1261–1266.

Nair J, Strand S, Frank N, Knauft J, Wesch H, Galle PR, Bartsch H. 2005.Apoptosis and age-dependant induction of nuclear and mitro-chondrial etheno-DNA adducts in Long–Evans Cinnamon (LEC) rats:enhanced DNA damage by dietary curcumin upon copper accumula-tion. Carcinogenesis 26(7): 1307–1315.

Nogueira CW, Soares FA, Nascimento PC, Muller DA, Rocha JBT.2003. 2,3-Dimercaptopropane-1-sulfonic acid and meso-2,3-dimercaptosuccninic acid increase mercury and cadmium-inducedinhibition of d-aminolevulinic acid dehydratase. Toxicology 184:85–95.

Ohkawa H, Ohishi N, Yagi K. 1979. Assay for lipid peroxides inanimal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351–358.

Olanow CW, Arendash GW. 1994. Metals and free radicals in neurodegen-eration. Curr. Opin. Neurol. 7(6): 548–558.

Padmaja S, Raju TN. 2004. Antioxidant effect of curcumin in seleniuminduced cataract of Wistar rats. Ind. J. Exp. Biol. 42(6): 601–603.

Patrick L. 2002. Mercury toxicity and antioxidants: part I: role of glu-tathione and a-lipoic acid in the treatment of mercury toxicity. Altern.Med. Rev. 7: 456–471.

Pesce A. 1984. Lactate dehydrogenase. In Clinical Chemistry, Kaplan A(ed.). Mosby: St Louis; 1124–1127.

Pfaffl MW, Gerstmayerb B, Bosiob A, Windisch W. 2003. Effect of zincdeficiency on the mRNA expression pattern in liver and jejunum ofadult rats: monitoring gene expression using cDNA microarrays com-bined with real-time RT-PCR. J. Nutr. Biochem. 14: 691–702.

Phan TT, See P, Lee ST, Chan SY. 2001. Protective effects of curcuminagainst oxidative damage on skin cells in vitro: its implication forwound healing. J. Trauma 51: 927–931.

Pulla A, Lokesh BR. 1994. Effect of dietary turmeric (Curcuma longa) oniron-induced lipid peroxidation in the rat liver. Food Chem. Toxicol. 32:279–283.

Pulla A, Lokesh BR. 1992. Studies on spice principles as antioxidant in theinhibition of lipid peroxidation of rat liver microsomes. Mol. CellBiochem. 111: 117–124.

Ruby AJ, Kuttan G, Dinesh BK, Rajasekharan KN, Kuttan R. 1995. Antitumorand antioxidant activity of natural curcuminoids. Cancer Lett. 94:79–83.

Rukkumani R, Balasubashini S, Menon VP. 2002. Comparative effects ofcurcumin and photo-irradiated curcumin on alcohol and polyunsatu-rated fatty acid induced toxicity. Pharmacol. Res. 46: 257–264.

Rukkumani R, Balasubashini S, Menon VP. 2003. Protective effects of cur-cumin and photoirradiated curcumin on circulatory lipids and lipidperoxidation products in alcohol and polyunsaturated fatty acid-induced toxicity. Phytother. Res. 17: 925–929.

Rumbeiha WK, Fitzgerald SD, Braselton WE, Roth RA, Kaneene JB. 2000.Potentiation of mercury-induced nephrotoxicity by endotoxin in theSprague–Dawley rat. Toxicology 149: 75–87.

Salo DC, Pacifini RE, Davies KJN. 1988. Superoxide dismutase is preferen-tially degraded by a proteolytic system from red blood cells followingoxidative modification by hydrogen peroxide. Free Radic. Biol. Med. 5:335–339.

Sendercor GW, Cochran WG. 1967. Statistical Methods, 6th edn. IBH:Oxford; 296–298.

Sharma OP. 1976. Antioxidant activity of curcumin and related com-pounds. Biochem. Pharmacol. 25: 1811–1812.

Shimojo N, Kumagai Y, Nagafune J. 2002. Difference between kidney andliver in decreased manganeso superoxide dismutase activity causedby exposure of mice to mercuric chloride. Arch. Toxicol. 76: 383–387.

Singletary K, MacDonald C, Lovinelli M, Fisher C, Wallig M. 1998. Effect ofthe diketones diferuloylmethane (curcumin) and dibenzoylmeth-ane on rat mammary DNA adducts and tumor induced by 7,12-dimethylbenz(a)anthracene. Carcinogenesis 19: 1039–1043.

Sinha AK. 1972. Colorimetric assay of catalase. Anal. Biochem. 47: 389–394.

Skare I. 1972. Microdetermination of mercury in biological samples partIII: automated determination of mercury in urine, fish and bloodsamples. Analyst 97: 148–155.

Stohs SJ, Bagchi D. 1995. Oxidative mechanisms in the toxicity of metalions. Free Radical Biol. Med. 18: 321–336.

Subramaniam M, Sreejayan-Rao MNA, Devasagayam TPA, Singh BB. 1994.Diminution of singlet oxygen induced DNA damage by curcumin andrelated antioxidants. Mutat. Res. 311: 249–255.

Tanaka-Kagawa T, Suzuki M, Naganuma A, Yamanaka N, Imura N. 1998.Strain difference in sensitivity of mice to renal toxicity of inorganicmercury. J. Pharmacol. Exp. Ther. 285: 335–341.

Unnikrishnan MK, Rao MN. 1995. Inhibition of nitric-induced oxidation ofhemoglobin by curcuminoids. Pharmazie 50: 490–492.

Venkatesan N. 2000. Pulmonary protective effects of curcumin againstparaquat toxicity. Life Sci. 66: 21–28.

Wenger C. 1984. Alkaline phosphatase. In Clinical Chemistry, Kaplan A(ed.). Mosby: St Louis; 1094–1098.

Zalups RK. 1995. Progressive losses of renal mass and the renal andhepatic disposition of administered inorganic mercury. Toxicol. Appl.Pharmacol. 130: 121–131.

Zalups RK. 2000. Molecular interactions with mercury in the kidney. Phar-macol. Rev. 52: 113–143. 467



Zalups RK, Cherian MG. 1992. Renal metallothionein metabolism after areduction of renal mass. II. Effect of zinc pretreatment on the renaltoxicity and intrarenal accumulation of inorganic mercury. Toxicology71: 103–117.

Zalups RK, Koropatnick J. 2000. Temporal changes in metallothioneingene transcription in rat kidney and liver: relationship to content ofmercury and metallothionein protein. J. Pharmacol. Exp. Ther. 295:74–82.

Zalups RK, Barfuss DW, Lash LH. 1999. Disposition of inorganic mercuryfollowing biliary obstruction and chemically induced glutathionedepletion: dispositional changes one hour after the intravenousadministration of mercuric chloride. Toxicol. Appl. Pharmacol. 154:135–144.

Zar JH. 1984. Biostatistical Analysis, 2nd edn. Prentice Hall: EnglewoodCliffs, NJ; 195–198.

468

R. Agarwal et al.


Detoxification and antioxidant effects of curcumin in rats experimentally exposed to mercury

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