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Environmental Toxicology and Pharmacology 29 (2010) 97–103

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Role of micronutrients against dimethylmercury intoxication in male rats

Deepmala Joshi ∗, Deepak Kumar Mittal, Monika Bhadauria, Satendra Kumar Nirala,Sadhana Shrivastava, Sangeeta ShuklaReproductive Biology and Toxicology Laboratory, UNESCO Satellite center of Trace Element Research & School of Studies in Zoology, Jiwaji University, Gwalior, (M.P.) 474011, India

a r t i c l e i n f o

Article history:Received 17 May 2009Received in revised form 22 October 2009Accepted 14 November 2009Available online 31 December 2009

Keywords:N-acetyl cysteineZincSeleniumAcetyl cholinesterase

a b s t r a c t

Mercury is one of the most toxic non-radioactive heavy metals. Chelation therapy has been the basisfor the medical treatment of mercury poisoning. Male albino rats were administered dimethylmercury(1.5 mg/kg) orally for 21 days. Chelation therapy with N-acetyl cysteine along with combination of antiox-idants viz. zinc and selenium was given for 5 days after 24 h of toxicant administration. All animals weresacrificed after 48 h of last treatment and various blood biochemical parameters were performed. Toxi-cant caused rise in bilirubin, �-GT, cholesterol, triglycerides, urea, creatinine, the uric acid content with adecline in albumin. A significant elevation was observed in LPO content and mercury concentration, alongwith concomitant decline in GSH levels after toxicant administration in liver, kidney and brain. Noticeablefall was also observed in AChE enzyme. Histopathological analysis was consistent with the biochem-ical observations and led to conclude that combination therapy provided protection against mercurytoxicity.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

The Industrial revolution has changed the face of the worldforever, but it has also brought environmental pollution as abyproduct, which has increasingly become a burden on mankind.Mercury pollution is one such major area of concern and itstoxicity first came to public attention by way of the Minamataand Iraqi tragedies. Mercury is a toxic heavy metal, which pro-duced by natural degassing, earth crust and combustion of fossilfuels, plants (Zhang et al., 2002). The WHO has reported theindustrial use of mercury and its general toxic effects on humanand animal systems (WHO, 1990). Organic mercury is completelyabsorbed in gastrointestinal tract (90–95%) accumulates brain, kid-ney, liver, hair and skin (WHO, 1991). Thus, it exhibits the abilityto produce reactive oxygen species, resulting in lipid peroxida-tion, DNA damage, depletion of sulfhydryls status and alteredcalcium homeostasis. The first target organ of methylmercuryis CNS.

Sulfur-containing nutrients play an important role as detoxifica-tion and protecting cells and cellular components against oxidativestress. N-acetyl cysteine is the derivative of naturally occurringamino acid cysteine and is an excellent source of sulfhydryl (-SH) groups. It can stimulate reduced glutathione (GSH) synthesis,

∗ Corresponding author. Tel.: +91 751 2442750/+91 9893499190 (M);fax: +91 751 2341450.

E-mail address: deepmalajoshi953@gmail.com (D. Joshi).

enhance glutathione-S-transferase activity, promote detoxifica-tion and act directly on reactive oxidant radicals (Ballatori etal., 1998). It has shown the ability to help prevent the damagethat comes with aging to mitochondria (Miquel, 2002; Ceballoset al., 2001; Studer et al., 2001). These dual properties helprepair oxidative damage in the body. Several nutrients have beenfound to affect the toxicity of mercury. Recently, our group hasreported that NAC as well as Zn and Se reverses dimethylmer-cury induced acute toxicity (Singh et al., 2007; Shukla et al., 2007).Based on our previous findings and those reported in other lab-oratories, we further investigated the combined effect of NACwith Zn and Se against dimethylmercury induced subchronic tox-icity considering the measurement of liver, kidney and brainfunction tests, GSH, thiobarbituric reactive substances (TBARS),adenosine triphosphatase and glucose-6-phosphatase dehydro-genase enzymes, distribution study as well as histopathologicalanalysis.

2. Materials and methods

2.1. Animals and chemicals

Adult male Sprague–Dawley rats (150 ± 10 g body wt) were obtained from thedepartmental animal facility where they were housed under standard husbandryconditions (25 ± 2 ◦C temp., 60–70% relative humidity and 12 h photoperiod) withstandard rat feed (Pranav Agro Industries, India) and water ad libitum. Experimentswere conducted in accordance with the guidelines set by the Committee for thePurpose of Control and Supervision of Experiments on Animals (CPCSEA), India andexperimental protocols were approved by the institutional animal ethics Commit-tee (CPCSEA/501/01/A). Dimethylmercury (CH3–Hg–CH3) and the chelating agentsviz. N-acetyl cysteine (NAC), zinc acetate [(CH3COO)2 Zn·H2O] and sodium selenite

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Fig. 1. Effect of conjoint treatment on the liver function tests in subchronic dimethylmercury-treated rats. Mean ± S.E., N = 6, #significant difference between DMM per se vscontrol at P ≤ 0.05; *significant difference between DMM + NAC + Zn + Se vs. DMM per se at P ≤ 0.05.

(Na2SeO4) were procured from Sigma–Aldrich and Himedia Laboratories Ltd., Indiaand all other experimental chemicals were analytical grade and stored as per rec-ommendations. All doses of therapeutic agents were prepared fresh before use andstored at 4 ◦C after preparation.

2.2. Experimental design

Dimethylmercury (1.5 mg/5 ml−1 kg−1) (RTECS, 1986) was suspended in olive oiland administered orally with the help of an intragastric rubber catheter. Therapeuticagents as N-acetyl cysteine (2 mM/kg, 2 ml−1 kg−1) intraperitoneally by Giradi andElias (1993), zinc (2 mM/kg p.o.) by Fukino et al. (1984) and selenium (0.5 mg/kg,p.o.) according to Johri et al. (2002). Equal amount of olive oil was given as vehicleto control animals. Animals were divided into three groups consisting of six animals(N = 6) each and groups 2–3 received DMM for 21 days and treated as follows:

• Group 1: control, received vehicle only (for 26 days).• Group 2: experimental control, received DMM only for 21 days + vehicle (for 5

days after DMM administration).• Group 3: received DMM (21 days) followed by NAC + Zn + Se (for 5 days after DMM

administration).

Animals of all the groups were euthanized after 48 h of last administration; bloodwas collected, serum was isolated to assess various biochemical variables. Tissuesamples (liver, kidney and brain) of each animal were immediately processed forthe biochemical analysis and histological preparations.

2.3. Isolation of serum and homogenate preparation

After keeping the blood for 1 h at room temperature, serum was isolated bycentrifugation at 1000 × g for 15 min and stored at −20 ◦C until analyzed. Tissuesamples of liver kidney and brain were homogenized with ice-cold 1.15% KCl for thedetermination of G-6-PDH activity. The brain tissues were homogenized respec-tively, in 0.25 M sucrose solution for the determination of acetyl cholinesteraseactivity. The homogenates (10%, w/v) liver, kidney and brain were prepared in chilledhypotonic solution for total proteins estimation, adenosine triphosphatase (ATPase)activity.

2.4. Blood biochemical analysis

Serum protein contents (Lowry et al., 1951), serum albumin, bilirubin, �-glutamyl transpeptidase (�-GT), cholesterol, triglycerides, urea, creatinine and uric

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Fig. 2. Effect of conjoint treatment kidney function tests in subchronic dimethylmercury treated rats. Mean ± S.E., N = 6, #significant difference between DMM per se vs controlat P ≤ 0.05; *significant difference between DMM + NAC + Zn + Se vs. DMM per se at P ≤ 0.05.

acid were measured using E-Merck’s kit according to the manufacturer’s instruc-tions.

2.5. Assessment of AChE in fore, mid and hind brain

Enzymatic activity of acetyl cholinesterase in different regions of brain (fore,mid and hind brain) was measured using dithionitrobenzoic (DTNB) acid (Ellman etal., 1961) and optical density was recorded immediately at � 412 nm.

2.6. Tissue biochemical analysis

Fresh tissues of liver, kidney and brain were immediately processed for the esti-mation of total protein. Total proteins were measured using BSA as standard byLowry et al. (1951). Estimation of metabolic enzymatic activities included adeno-sine triphosphatase (ATPase) (Seth and Tangri, 1966) and glucose-6-phosphatasedehydrogenase (G-6-PDH) (Askar et al., 1996).

The levels of mercury contamination in liver, kidney and brain were analyzed.They were digested according to the method of Jacobs et al. (1960). 0.2 g of eachtissue was dissolved in 5 ml of nitric acid and was left for 12 h. 5.0 ml of H2SO4 (1:1with distilled water) and 30 ml of saturated solution of KMnO4 and was left for 3to 4 days at room temperature. 4.0 ml of hydroxyl ammonium acetate (40%), 1.0 mlof SnCl2 was added and finally solution was raised up to 70 ml volume with theaddition of deionized water and analyzed on Atomic Absorption Spectrophotometer(Thermo, Japan).

2.7. Histopathological analysis

For light microscopic observations, liver, kidney and brain tissues were fixed inBouin’s fixative and processed routinely for embedding in parafin. Tissue sections

of 5 �m thickness were stained with hematoxylin and eosin (H&E) and examinedunder photomicroscope.

2.8. Statistics

Statistical analysis was carried out using one-way analysis of variance (ANOVA)taking significant at P ≤ 0.05 followed by Student’s t-test taking significant at P ≤ 0.05(Snedecor and Cochran, 1994). Percent protection was also used for comparison fordifferent treatment groups.

3. Results

3.1. Assessment of liver functions

Fig. 1(a–f) represents the effect of NAC + Zn + Se on DMM inducedblood biochemical alterations. The observations listed were fromthe animals received mercury (DMM), the serum bilirubin (P ≤ 0.05)concentration, �-GT (P ≤ 0.05) activity, serum protein (P ≤ 0.05),cholesterol (P ≤ 0.05) and triglycerides (P ≤ 0.05) contents weresignificantly increased, where as albumin (P ≤ 0.05) was found tobe depleted. Therapy with chelating agent and antioxidants sup-plementation for 5 days recovered these parameters significantlytowards control (P ≤ 0.05). More than 65% recovery was found inabove mentioned parameters however, cholesterol showed only52% protection and ANOVA was found to be significant at 5%

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Fig. 3. Effect of conjoint treatment on the acetyl cholinesterase activity and mercury ion concentration in subchronic dimethylmercury treated rats. Mean ± S.E., N = 6,#significant difference between DMM per se vs control at P ≤ 0.05; *significant difference between DMM + NAC + Zn + Se vs. DMM per se at P ≤ 0.05.

level. No significant change was observed between both exper-imental and control groups, when analyzed by Student’s t-test(P ≤ 0.05).

3.2. Assessment of kidney functions

Fig. 2(a–d) demonstrates the recovery pattern by combina-tion therapy of NAC + Zn + Se through kidney marker enzymes.The release of urea, creatinine, uric acid and blood urea nitro-gen was significantly increased in the circulation after DMMintoxication (P ≤ 0.05). Five days of combination therapy ofNAC + Zn + Se significantly recouped all these contents towardscontrol (P ≤ 0.05) and established more than 75% protection increatinine and uric acid concentration except urea and bloodurea nitrogen, which showed more than 50% protection in itsrelease.

3.3. Acetyl cholinesterase and mercury concentration

Fig. 3(a–c) shows drastic decline in the of AChE activity infore, mid and hind brain. Subchronic administration of DMMsignificantly inhibited the activity of AChE (P ≤ 0.05). Statisticalanalysis clearly demonstrates that 5 days treatment of NAC + Zn + Seimproved the activity of AChE (P ≤ 0.05) and ANOVA was found tobe significant at 5% level in reviving the mercury induced inhibi-tion in above mentioned regions of brain and bringing the levelvery near to normal.

Fig. 3(d–f) depicts the protective effect of NAC + Zn + Se on tissuedistribution of mercury in liver, kidney and brain. The distribu-tion study by atomic absorption spectrophotometer reveals the factthat administration of DMM per se for 21 days regimen caused anultimate rise in the concentration of mercury ion (P ≤ 0.05). Com-bined treatment with NAC + Zn + Se for 5 days showed significantprotection (P ≤ 0.05) in all the organs.

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Fig. 4. Effect of conjoint treatment on the adenosine triphosphatase and glucose-6-phosphatase dehydrogenase activity in subchronic dimethylmercury treated rats.Mean ± S.E., N = 6, #significant difference between DMM per se vs control at P ≤ 0.05; *significant difference between DMM + NAC + Zn + Se vs. DMM per se at P ≤ 0.05.

3.4. Adenosine triphosphatase and glucose-6-phosphatasedehydrogenase activities

Fig. 4(a–f) represents the toxic effect of DMM on metabolicenzymes. Activities of hepatic renal and neuronal ATPase andG-6-PDH were decreased significantly (P ≤ 0.05). Treatment withNAC + Zn + Se significantly recouped the ATPase (P ≤ 0.05) and G-6-PDH activity (P ≤ 0.05) towards control. More than 75% recoverywas found in both the enzymes in all organs with combined therapyexcept kidney ATPase activity.

3.5. Histopathological analysis

Fig. 5 presents the histology of liver, kidney and brain respec-tively after different treatments. Light microscopic evaluationshowed regular morphology of the liver parenchyma with welldesigned hepatic cells and sinusoids in control group (Fig. 5a). Atlower magnification the DMM treated group showed severe liverinjury with significant degeneration in hepatic cords, swollen hepa-

tocytes, prominent congestion in sinusoidal space and central veinfilled with fluid (Fig. 5b). Normal hepatocytes were observed inmost areas after NAC + Zn + Se treatment as well arranged cordswith clear sinusoidal spaces (Fig. 5c). Kidney of control groupshowed normal features of renal tubules and Bowman’s capsules(Fig. 5d). The DMM group illustrated degenerated renal tubuleswith obstructed lumen, swollen glomerulus with transparent fluid(Fig. 5e); whereas with the treatment of NAC + Zn + Se clearlydepicted well formed renal tubules. Glomerulus appeared to benormal in most of the areas after combined therapy (Fig. 5f). Brain(cerebral hemispheres) which consist of a convoluted cortex of graymatter overlying the central medullary mass of white matter whichconveys fibers between different parts of the cortex. Numerouswell formed neurons, nerve fillaments, and microglial cells wereclearly seen (Fig. 5g). The DMM treated group showed significantreduction in number of nerve fibers, neuronal degeneration, loss ofnerve filaments, large number of fusion of nerve bundles (Fig. 5h).Combination therapy of NAC + Zn + Se provoked well formed nervefilaments and recovered glial cells. The architecture of cerebrum

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Fig. 5. (a) - Photomicrographs of control rat liver showing maintained chord arrangement with well formed hepatocytes (X120), (b) - Mercury led to disorganization inhepatic plates with congestion in sinuses, massive peripheral necrosis as well as severe mononuclear infiltration and central vein filled with eosinophilic fluid (X120), (c) -Treatment with NAC + Zn + Se showed recouped hepatocytes with maintained sinusoidal spaces with normal appearance chord arrangement (X120), (d) - Photomicrographsof control rat kidney showing normal features, well formed renal tubules and Bowman’s capsule with compact glomeruli (X120), (e) - Administration of mercury showingsevere degeneration in renal tubules, degenerated Bowman’s capsule filled with transparent fluid (X120), (f) - Conjoint treatment with NAC + Zn + Se showed recoupedBowman’s capsule along with normal glomerular tuft. Well formed renal corpuscles were observed (X120), (g) - Photomicrograph of control rat cerebrum showing normalappearance of layers in cortex region. Well formed fascicles, neurons, nerve filaments were noticed (X120), (h) - Mercury caused significant loss of nerve fibers, disruptionin all cell layers were noticed. Irregular shaped neurons were noted. Nerve tangles were clearly seen. Massive neurofibrillary degeneration was clear (X120), (i) - Cerebrumtreated with NAC + Zn + Se showed almost normal feature in cortex and medullary region. Well-arranged neurons and nerve fibeers were observed and nerve tangles wererecouped (X120).

was still intact. Number of neurons and nerve fibers were restored(Fig. 5i).

4. Discussion

Mercury exhibits its toxicity by interacting with sulfhydrylgroups of proteins and alters enzymes involved in biochemicalreaction (Shireen et al., 2004). In the present study we investi-gated that dimethylmercury caused severe alterations in the bloodand biochemical parameters. Serum albumin level has been usedas a test for liver function. Albumin and bilirubin is one of themost frequent clinical tests to evaluate the extent of chemicallyinduced hepatotoxicity (Zimmerman, 1973) and its accumulationis a measure of binding, conjugating and excretory capacity of hepa-tocytes direct related to erythrocyte degeneration rate. �-glutamyltranspeptidase is the colengiocyte specific protein, found on thecell surface, with particularly high concentrations in the liver, bileducts and kidney (Anilkumar et al., 2004). Combined therapy ofNAC + Zn + Se recouped the activity. Thus this indicates and reflectsthe membrane stabilizing activity and improvement of liver func-tion. These changes are reported with the metals like beryllium(Nirala et al., 2007), aluminium (Yousef, 2004). In the present inves-tigation there was a significant rise in urea concentration aftertoxicant administration, this may be due to dysfunctional and dys-

trophic changes in the liver and kidney. Accumulation of the ureahas been reported in liver diseases like cirrhosis and encephalopa-thy. Prasada Rao et al. (1989) also observed kidney damage withelectron microscopy the treatments of lead and mercury. Increasedlevel of urea and creatinine as a result of heavy metals toxicitywere also noticed with cobalt and lead (Hanafy and Soltan, 2004).Increase in serum cholesterol after mercury exposure directly indi-cates the hepatorenal injury. Thus formation of fatty lipid steatosisafter acute mercury administration is mainly due to the accu-mulation of triglycerides. These changes in the lipid metabolismare almost completely restored with the combination ofNAC + Zn + Se.

The result suggests that mercury induced elevation in lipid per-oxidation might be because of the lower level of glutathione asobserved in this study, which is in agreement with our earlierreports (Shukla et al., 2007). Treatment with NAC + Zn + Se affordedbetter protection through decreased production of free radicalsderivatives, as is evident from the decreased level of TBARS. Dueto its high lipophilicity, MeHg by itself is capable of diffusingthrough the cell membrane. Altered levels of acetylcholinesterasehave been reported in brain tissues of rabbits with neurofibrillarydegeneration induced by intracisternal injection of metal (Sharmaand Mishra, 2006). The combination therapy was far too superiorin neutralizing the toxic potential of mercury, by extracting the

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metal. A number of investigators have previously demonstrated theantioxidative effect of magnesium (Hans et al., 2002) and lipoic acid(Maritim et al., 2003) against mercury induced toxicity. Any alter-ation in membrane lipid leads to changes in membrane fluidity,which in turns alters the ATPase activity and cellular function. Con-joint treatment with chelating agents and adjuvant/antioxidantsin rats exposed to mercury resulted in the reversal of the levelof ATPase in liver, kidney and brain. Histological observationsbasically support the results obtained from biochemical assays.We found several different histopathological changes, specificallymononuclear cell infiltrations. When necrosis occurs in hepato-cytes, the associated plasma membrane leakage can be detectedbiochemicaly as done in our study. The present study showed theeffect of conjoint treatment on the mobilization of dimethylmer-cury in the tissues. Animals showed significantly higher levels ofmercury in liver, kidney followed by brain. NAC can stimulateGSH synthesis, enhance glutathione-S-transferase activity, pro-mote detoxification and act directly on reactive oxidant radicals.The cysteine (-SH) group is responsible for a great deal of metabolicactivity of NAC, while the acetyl-substituted amino group makesthe molecule more stable against oxidation (Aremu et al., 2008).Therapeutic role of Zn and Se in the present study against deleteri-ous metal mediated free radical attacks was clear. Zn prevents freeradical generation, protecting biological membranes from damageinduced by mercury (Stefanidou et al., 2006). Increased metal-lothionien induction (Zn-MT) by zinc also helped detoxification.Selenium is an essential component of several enzymes such asglutathione peroxidase, thioredoxin reductase and selenoprotein P,which contains Se as selenocysteine. The mechanism of protectionincludes the capability of Se to alter the distribution of mercuryin tissues and induces binding of the Hg-Se complexes to pro-teins (Toufektsian et al., 2000). Recently, our group has reportedthat NAC as well as Zn and Se reverses dimethylmercury inducedacute toxicity (Singh et al., 2007; Shukla et al., 2007). Zinc pre-vented free radical generation, protecting biological membranesfrom damage induced by mercury. Selenium is a powerful antiox-idant and it is as an important element in glutathione peroxidase.The Selenium administration combats oxidative stress, thus mini-mizing the metal intoxication. It played a major role in maintainingthe integrity of cell membrane and keeping it intact (Agarwal etal., 2007). Present observations of this study led us to concludethat combination therapy may be provided therapeutic potentialagainst dimethylmercury induced enzymatic and cellular dysfunc-tions.

Conflict of interest

None.

Acknowledgements

The authors feel indebted to Prof. R. Mathur, School of Studies inZoology, Jiwaji University, Gwalior for their invaluable suggestions.We are grateful to UGC, New Delhi-India, Jiwaji University, Gwaliorfor financial assistance.

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