Reversal of Methylmercury-Induced Oxidative Stress, Lipid ...

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Deepmala Joshi,a,* Mittal Deepak Kumar,a Shakya Arvind Kumar,b & Shukla Sangeetaa. aReproductive Biology and Toxicology Laboratory, UNESCO Satellite ...
Journal of Environmental Pathology, Toxicology and Oncology, 33(2):167–182 (2014)

Reversal of Methylmercury-Induced Oxidative Stress, Lipid Peroxidation, and DNA Damage by the Treatment of N-Acetyl Cysteine: A Protective Approach Deepmala Joshi,a,* Mittal Deepak Kumar,a Shakya Arvind Kumar,b & Shukla Sangeetaa Reproductive Biology and Toxicology Laboratory, UNESCO Satellite center of Trace Element Research & School of Studies in Zoology, Jiwaji University, Gwalior, Madhya Pradesh, India bIndira Gandhi National Open University, Biochemistry Department, School of Sciences, New Delhi, India a

*Address all correspondence to: Deepmala Joshi, Reproductive Biology and Toxicology Laboratory, UNESCO Satellite center of Trace Element Research & School of Studies in Zoology, Jiwaji University, Gwalior, Madhya Pradesh, India; Tel.: + 91-751-4036868; Fax: + 91-751-2341450; [email protected]

ABSTRACT: This study was designed to evaluate the protective effect of N-acetyl cysteine in reducing methylmercury (MeHg)–induced oxidative stress, lipid peroxidation, DNA damage in liver, kidney, and brain, and their ability to restore altered hepatic, renal, and other biochemical variables. Male Sprague-Dawley rats (150 ± 10 g) were randomly divided into three groups. Group 1 served as the control. Groups 2 and 3 were administered methylmercury (1 mg kg–1 orally, 5 days/week) for 12 weeks, and group 2 served as the experimental control. Group 3 received N-acetyl cysteine (0.6 mg kg–1 intraperitoneally, two days/week) for 12 weeks after methylmercury exposure. Methylmercury exposure caused a significant rise in bilirubin, gamma-glutamyl transpeptidase, protein, triglycerides, cholesterol, urea, creatinine, uric acid, and blood urea nitrogen, with a concomitant decrease in albumin content, reduced glutathione level and acetyl cholinesterase activity, antioxidant enzymes such as glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, and adenosine triphosphatase. However, lipid peroxidation level, metallothionein expression, and DNA damage with increment of tail length were observed after methylmercury intoxication. N-acetyl cysteine, a widely available, nontoxic amino acid derivative, is a promising antioxidant with a wide spectrum of biological functions. The ability of N-acetyl cysteine to enhance mercury excretion and its wide availability in clinical use indicate that it may be an ideal therapeutic agent against methylmercury poisoning. KEY WORDS: methylmercury, N-acetyl cysteine, lipid peroxidation, dlutathione, DNA damage

I. INTRODUCTION Human exposure to methylmercury occurs mostly through consumption of fish and other seafood, although several major inadvertent exposures have also occurred, including epidemics in Japan (Minamata disease), Iraq, Pakistan, and Guatemala. Thus, it is listed by the International Program of Chemical Safety as one of the most dangerous chemicals present in the environment.1–4 Exposure may also occur in research laboratories, as evidenced by the recent fatal poisoning of a chemist at Dartmouth College.5 Among three forms of mercury (elemental, inorganic, 0731-8898/14/$35.00  © 2014 by Begell House, Inc.

and organic), organic mercury (MeHg) is considered the most toxic form of mercury and has a half-life of 70 days in humans as it passes the blood brain barrier due to its lipid solubility.6 The damage has vast implications, with human beings at the top of food chain getting the worst of the deal due to biomagnifications. Transport mechanisms of MeHg result in systemic distribution, which explains its high rate of deposition in both hematopoietic, hepatic, renal, and neural tissues.7 MeHg binds to sulfhydryl molecules (–SH) such as L-cysteine, glutathione, hemoglobin, albumin, and other cysteine-containing polypeptides in tissues. It is transported across the endothelium, 167

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through the blood-brain barrier and into cells via the L-type large neutral amino-acid transporters (LATs).8,9 Mercury causes lipid peroxidation and depletion of sulfhydryl contents, resulting in DNA damage. Several chelating agents have been studied as potential MeHg antidotes and recently used for mercury elimination from the body. Of the agents that have been examined, meso-2,3-dimercaptosuccinic acid (DMSA, succimer) and 2,3-dimercapto-1-propanesulfonic acid (DMPS, dimaval) are the most efficient at enhancing methylmercury excretion and in preventing toxicity in both experimental animals10–13 and humans.14,15 However, these agents have limited stability in solution, limited availability for human use, and a propensity to mobilize other essential and nonessential metals.10,16–18 Thus, there is an urgent need to develop a more effective antidote that will be associated with less or ideally no toxic side effects. N-acetyl cysteine (NAC) is also shown to be remarkably effective for enhancing MeHg excretion in mice.19 Unlike other chelating agents, NAC is a potent antioxidant and does not alter tissue distribution of essential metals. Surprisingly, the possible use of NAC as a chelating agent for chronic methylmercury toxicity has not been previously evaluated. N-acetyl cysteine, the acetylated variant of the amino acid L-cysteine, is an excellent source of sulfhydryl (SH) groups and is converted in the body to metabolites capable of stimulating glutathione (GSH) synthesis, promoting detoxification, and acting directly as free radical scavengers.20 We previously reported the protective effect of NAC against acute toxicity of dimethylmercury.21–23 Recently, NAC has gained considerable attention as a potential antioxidant in different pharmacological intervention studies. It appears that NAC exerts its beneficial effects in conditions where oxidative stress plays a critical role in the induction of cellular damage/target organ toxicity. Hence, it was considered for the present study and an attempt has been made to evaluate the possible protective effects of NAC against methylmercury-induced toxicity in the liver, kidney, and brain. Our results clearly indicate that NAC treatment reduced methylmercury-induced oxidative stress and cytotoxicity. Furthermore, NAC treatment led to decreased DNA damage and associated cellular toxicity.

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II. MATERIALS AND METHODS A. Animals and Chemicals Adult male Sprague-Dawley rats (150 ± 10 g body wt) were obtained from a departmental animal facility where they were housed under standard husbandry conditions (25±2°C temperature, 60–70% relative humidity, and 12-h photoperiod) with standard rat feed (Pranav Agro Industries, India) and water ad libitum. Experiments were conducted in accordance with guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA; Chennai, India) and experimental protocols were approved by the Institutional Ethical Committee (CPCSEA/501/01/A). Methylmercury (MeHg) and thiol chelator as N-acetyl cysteine (NAC) were procured from Sigma Chemical Co. (St Louis, MO) and Himedia Laboratories Ltd. (India) and all other experimental chemicals were analytical grade and stored as per recommendations. B. Experimental Design and Preparation of Doses Methylmercury (1 mg 5 ml–1 kg–1) was dissolved in olive oil and administered orally with an intragastric catheter. A chelating agent, N-acetyl cysteine (0.6 mg 2 ml−1 kg−1), was dissolved in tripledistilled water, administered intraperitoneally. Equal amounts of olive oil (5 ml–1 kg−1) and triple-distilled water (2 ml–1 kg−1) were given as a vehicle to control animals. Selection of the toxicant (MeHg) and therapeutic agent (NAC) dose and duration of treatments were based on earlier works24,25 as well as our previous studies. Twenty-four adult male albino rats were divided into three groups of eight animals (n = 8) each. The treatment schedule is summarized as follows: group 1: control, received vehicle (olive oil and triple-distilled water) for 12 weeks (once daily for 7 days, Monday through Sunday); group 2: experimental control, received methylmercury for 12 weeks (once daily for 5 days, Monday through Friday in a week plus vehicle once daily for 2 days, Saturday through Sunday after methylmercury administration); and Journal of Environmental Pathology, Toxicology and Oncology

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group 3: received methylmercury for 12 weeks (once daily, 5 days, Monday through Friday in a week, followed by the treatment of chelating agent as NAC once daily for 2 days, Saturday through Sunday in a week after methylmercury administration). On completion of the experimental period, animals from all groups (groups 1–3) were euthanized after 48 h of last treatment. Blood was collected in heparinized tubes (for mercury determination), and nonheparinized (for biochemical assays) and serum was isolated to assess biochemical variables. C. Isolation of Serum and Homogenate Preparation After keeping the blood for 1 h at room temperature, serum was isolated by centrifugation at 1000 × g for 15 min and stored at –20°C until analyzed. Samples of liver, kidney, and brain were homogenized in 0.1 M potassium phosphate buffer (pH 7.4) and in 0.25 M sucrose to determine the level of lipid peroxidation26 and glutathione content.27 Remaining tissues of liver, kidney, and brain were homogenized with ice-cold 1.15% KCl for the determination of GR, GPx, and G-6-PDH activities. The brain tissues were homogenized respectively in 0.25 M sucrose solution for determination of acetyl cholinesterase activity. The homogenates (10%, w/v) liver, kidney, and brain were prepared in chilled hypotonic solution for the estimation of adenosine triphosphatase (ATPase) activity. D. Blood Biochemical Analysis Protein contents,28 serum albumin, bilirubin, gammaglutamyl transpeptidase (γ-GT), cholesterol, triglycerides (TG), urea, creatinine, uric acid, and blood urea nitrogen (BUN) in serum were measured using E-Merck’s kit according to the manufacturer’s instructions. E. Determination of Lipid Peroxidation Level and Glutathione Content Malondialdehyde (MDA) was formed by reaction with thiobarbituric acid (TBA) and used as an index Volume 33, Number 2, 2014

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of LPO. About 1 ml of homogenate prepared in KCl (0.15 M) was incubated at 37°C for 30 min, and proteins were precipitated by adding 1 ml chilled trichloroacetic acid (TCA) (10%) and then centrifuged at 450 × g for 15 min. Supernatant and TBA solution (0.67%) of 1 ml each were kept in a boiling water bath for 10 min, and after cooling, optical density was noted at λ535 nm. Glutathione (GSH) was estimated using 5,5dithiobis- 2-nitrobenzoic acid (DTNB). About 0.1 ml of homogenate and 0.9 ml of distilled water was added with 1.0 ml sulfosalicylic acid (10%) followed by centrifugation at 1000 × g for 10 min. Blank and standards were prepared by taking 0.5 ml of distilled water and 0.5 ml of GSH standard, respectively. About 0.5 ml of supernatant was added with 4.5 ml of tris buffer (pH 8.23). Color was developed by adding 0.5 ml of DTNB solution, and optical density was recorded at λ 412 nm. F. Analysis of Acetyl Cholinesterase and Adenosine Triphosphatase Activities Acetyl cholinesterase (AChE) activity in fore, mid, and hind brain was measured using dithionitrobenzoic (DTNB) acid29 and optical density was recorded immediately at λ 412 nm. For ATPase activity,30 0.2 ml of tris buffer (pH 7.4) and 0.2 ml of ATP (0.1 M) were added into 0.05 ml homogenate and the volume was brought up to 2 ml with distilled water followed by incubation at 37°C for 15 min. The reaction was stopped by adding 1 ml of TCA (10%). Supernatants were collected after centrifugation at 2000 rpm for 15 min. About 2 ml of distilled water and NaKPO4 were used for blank and standard, respectively. About 2 ml supernatant, 6.6 ml distilled water, and 2 ml ammonium molybdate were added with 0.4 ml ANSA solution, and absorbance was noted at λ620 nm against the blank after 10 min of incubation. G. Antioxidant Defense System Enzymes Glutathione reductase (GR),31 Glutathione peroxidase (GPx),32 and glucose-6-phosphate dehydrogenase

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(G6PDH)33 activities were measured in vital organs (liver, kidney, and brain) with a spectrophotometer. The reaction mixture contained 0.2 mM oxidized glutathione (GSSG) as a substrate, 0.12 mM NADPH, and protein sample in phosphate buffer (pH 7.0) for GR activity estimation. The reaction mixture contained 0.15 M GSH, 2.25 M sodium azide, 0.011 M H2O2, 0.84 mM NADPH, and 2 U/ml of glutathione reductase as a substrate for GPx activity determination. The reaction mixture contained 0.1 M glucose 6-phosphatase, 2.7 mM NADPH, and 0.1 M phosphate buffer (pH 7.0) for G6PDH activity observation. The decrease in absorbance at 340 nm in terms of NADPH oxidation was measured. Specific activity was expressed as μmol NADPH min–1 mg–1 protein. H. Estimation of Metallothionein Level and Mercury Ion Concentration The assay of the metallothionein (MT) was performed according to the procedure given by Eaton and Toal.34 For this assay, 10,000 × g supernatant was analyzed using radiolabeled cadmium (Cd) as a substrate. Briefly, 0.2 ml of carrier-free 109Cd solution was incubated with the appropriate amount of sample for 10 min. This was followed by the addition of 0.1 ml of 2% bovine hemoglobin and subsequent boiling of the reaction tubes at 80°C for 2 min. The tubes were centrifuged, 0.2 ml of clear supernatant was transferred to disposable gamma counting tubes, and activity amount was measured on a gamma counter. Blank samples without tissue homogenate were counted with each set of test reactions, the total amount of Cd-binding MT was calculated, and concentrations were expressed as micrograms per gram (μg/g). Mercury35 concentrations in the liver, kidney, and brain were determined by cold vapour atomic absorption spectrophotometry. In brief, 0.2 g of each tissue was dissolved in 5 ml of nitric acid and left for 12 h. 5.0 ml of H2SO4 (1:1 with distilled water) and 30 ml of saturated solution of KMnO4 and was left for 3 to 4 days at 37°C. A 4.0 ml amount of hydroxyl ammonium acetate (40%), 1.0 ml of SnCl2 was added, and finally the solution was brought up

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to 70 ml with the addition of deionized water and analyzed on an atomic absorption spectrophotometer. I. Measurement of DNA Damage DNA damage36,37 was evaluated by single-cell gel electrophoresis (comet assay). Lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, 1% sodium sarcosinate, 1% Triton X-100, and 10% DMSO, Tris–HCl buffer [0.4 M, pH 7.5], and electrophoresis buffer [300 mM NaOH, 1 mM EDTA) were prepared. 0.2 g of tissues was placed in 1 ml chilled mincing solution (HBSS with 20 mM EDTA and 10% DMSO) and chopped into pieces and centrifuged at 400 × g for 10 min. The supernatant containing the single cells was taken for comet assay. The cells were suspended in 110 μL of low-melting agarose (LMA) (pH 7.4) and pipetted on a glass microscope slide coated with 140 μL of 1% normal melting point agarose (pH 7.4). The agarose was allowed to set for 10 min at 4°C, and thereafter, the cover slip was removed and exposed for 24 h to lysis solution, and the slides were rinsed. These slides were subjected to electrophoresis for 20 min (300 mA, 25 mV). Then, the alkali was neutralized with tris-HCl buffer and stained with ethidium bromide. Images of 50 randomly selected cells were analyzed from each sample, and the comet moment was calculated in microns. J. Statistics Statistical analysis38 was carried out using one-way ANOVA followed by the Student’s t-test. Percent protection (%) was also used for the protective activity by treatment groups. The protective activity by treatment groups, expressed as protective percentage (H), was calculated as follows: H = [1– (T – V/C–V)] × 100, where T is mean value of N-acetyl cysteine and methylmercury exposed group, C is the mean value of methylmercury exposed group alone, and V is the mean value of control animals. Journal of Environmental Pathology, Toxicology and Oncology

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FIG. 1: Protective effect of N-acetyl cysteine on the liver function tests in chronic methylmercury-exposed rats. Mean ± S.E., n = 6. #Significant difference between MeHg per se versus control at p ≤ 0.05. *Significant difference between MeHg+ NAC versus MeHg per se at p ≤ 0.05. % = percent protection of N-acetyl cysteine against methylmercury-induced toxicity.

III. RESULTS A. Estimation of Liver and Kidney Functions Figure 1 represents the protective effect of N-acetyl cysteine on methylmercury-induced alterations in liver function tests. The concentrations of bilirubin, GGT, protein, cholesterol, and triglycerides in serum were increased significantly (P ≤ 0.05), whereas Volume 33, Number 2, 2014

albumin was found to be decreased after chronic methylmercury intoxication. N-acetyl cysteine supplementation recovered these parameters significantly (P ≤ 0.05) toward control. About 70–85% recovery was found in the above-mentioned parameters. Figure 2 demonstrates the recovery pattern by N-acetyl cysteine through kidney markers. The release of urea, creatinine, uric acid, and blood urea nitrogen was significantly (P ≤ 0.05) increased in circulation after methylmercury toxicity. Treatment with

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FIG. 2: Protective effect of N-acetyl cysteine on the kidney function tests in chronic methylmercuryexposed rats. Mean ± S.E., n = 6. #Significant difference between MeHg per se versus control at p ≤ 0.05. *Significant difference between MeHg+ NAC versus MeHg per se at p ≤ 0.05. % = percent protection of N-acetyl cysteine against methylmercury-induced toxicity.

NAC significantly (P ≤ 0.05) recouped all contents toward control and established maximum protection (70–84%). B. Estimation of Markers of Oxidative Stress Figure 3 demonstrates that chronic administration of methylmercury provoked a significant (P ≤ 0.05) increased the lipid peroxidation level with a concomitant decline in glutathione content in liver, kidney, and brain tissues, as evidenced by increased production of malondialdehyde (MDA) level and reduction in nonprotein sulfhydryl (–SH) contents. The highest level of MDA was found in kidney, fol-

lowed by brain and liver (Fig. 3, A–C). Glutathione content also showed maximum reduction in kidney followed by liver and brain (Fig. 3, D–F). Treatment with N-acetyl cysteine diminished the production of MDA in liver (71%), kidney (77%), and brain (75%) and restored GSH levels showing maximum percent protection in kidney (93%), liver (82%), and brain (79%) toward control. Figure 4 represents chronic administration of methylmercury caused a significant (P ≤ 0.05) depletion in enzymatic activity of acetyl cholinesterase in forebrain, midbrain, and hind brain (Fig. 4, A–C) and adenosine triphosphate (ATPase) in liver, kidney, and brain (Fig. 4, D–F) when compared to the respective control group. Post-treatment with N-acetyl cysteine restored the Journal of Environmental Pathology, Toxicology and Oncology

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FIG. 3: Protective effect of N-acetyl cysteine of on lipid peroxidation level and reduced glutathione contents in chronic methylmercury-exposed rats. Mean ± S.E., n = 6. #Significant difference between MeHg per se versus control at p ≤ 0.05. *Significant difference between MeHg+ NAC vs MeHg per se at p ≤ 0.05. % = Percent protection of N-acetyl cysteine against methylmercury-induced toxicity.

depleted values of both parameters and prevented the release of enzyme activities significantly (P ≤ 0.05) as is evident by percent protection. C. Analysis of Enzymatic Activities (GR, GPx, and G6PDH) The protective effect of N-acetyl cysteine on chronic methylmercury-induced disturbance in antioxidant Volume 33, Number 2, 2014

status is shown in Figs. 5 and 6. Methylmercury administration decreased enzymatic activities of GR (Fig. 5, A–C), GPx (Fig. 5, D–F), and G6PDH (Fig. 6, A–C) (P ≤ 0.05) in liver, kidney, and brain. Therapy with N-acetyl cysteine recovered all enzymatic activities toward control. About 55–73% recovery was found in GR and GPx enzyme activity, whereas 57–69% improvement was observed in G6PDH enzyme activity.

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FIG. 4: Protective effect of N-acetyl cysteine of on the enzymatic activities of acetyl cholinesterase in different regions of brain and adenosine triphosphatase activity in liver, kidney, and brain in chronic methylmercury-exposed rats. Mean ± S.E., n = 6. #Significant difference between MeHg per se versus control at p ≤ 0.05. *Significant difference between MeHg+ NAC versus MeHg per se at p ≤ 0.05. % = percent protection of N-acetyl cysteine against methylmercury induced toxicity.

D. Determination of Mercury Ion Concentration and Metallothionein Expression Changes in mercury ion concentration in liver, kidney, brain, and blood after chronic methylmercury administration are summarized in Fig. 7, A–D. Statistical analyses showed that methylmercury administration led to a significant (P ≤ 0.05) increase in the retention of Hg ion concentration in liver, kidney, brain,

and blood. Post-treatment with N-acetyl cysteine significantly (P ≤ 0.05) reduced the accumulation of mercury in these tissues and blood. The metallothionein expression in liver and kidney were significantly (P≤0.05) increased in methylmercury exposed rats in comparison to the control animals (Fig. 7, E–F). The values were found to exceed after chronic methylmercury exposure when compared to the control group, respectively. Posttreatment with N-acetyl cysteine downregulated/ Journal of Environmental Pathology, Toxicology and Oncology

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FIG. 5: Protective effect of N-acetyl cysteine of on the enzymatic activities of glutathione reductase and glutathione peroxidase in liver, kidney, and brain in chronic methylmercury-exposed rats. Mean ± S.E., n = 6. #Significant difference between MeHg per se versus control at p ≤ 0.05. *Significant difference between MeHg+ NAC vs MeHg per se at p ≤ 0.05, % = percent protection of N-acetyl cysteine against methylmercury-induced toxicity.

arrested the metallothionein expressions in liver and kidney tissues and showed 55–71% recovery. E. Measurement of Mean DNA Damage by Tail Length The alkaline comet assay was used to measure the DNA single-strand breaks in liver, kidney, and brain of control, methylmercury-exposed, and N-acetyl cysteine–treated groups (Fig. 8, A–C). A significant increase was observed in the mean DNA damage Volume 33, Number 2, 2014

that was calculated by the tail migration and tail length in methylmercury-administered animals when compared to respective controls of liver (C1), kidney (C2), and brain (C3). The tail length was increased in liver (C4), kidney (C5), and brain (C6) by an order of magnitude after chronic methylmercury exposure. The measurement of tail length was found to be high in liver, followed by kidney and brain. Therapy with N-acetyl cysteine markedly lowered the DNA damage with protection of 80% for liver (C7), 70% for kidney (C8), and 84% for brain (C9).

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IV. DISCUSSION At present, no effective strategy exists to promote the rapid clearance of methylmercury from poisoned individuals. A number of sulfhydryl-containing compounds have been evaluated extensively and found to be only marginally effective.11–15 In contrast, from the present findings, it can be demonstrated that N-acetyl cysteine may be an ideal therapeutic agent for chronic methylmercury poisoning. N-acetyl cysteine is an amino acid derivative that is currently used in clinical medicine, not toxic to humans, and rapidly excreted in urine.19 The chronic toxicity produced by methylmercury is associated with a variety of alterations. Albumin, bilirubin, triglycerides, and cholesterol are some of the most frequent clinical tests to evaluate the extent of chemically induced hepatotoxicity. By contrast, gamma-glutamyl transpeptidase is the cholangiocyte-specific protein, found on the cell surface, with particularly high concentrations in the liver, bile ducts, and kidney.1 Increase in serum cholesterol after methylmercury exposure directly indicates the hepatorenal injury and formation of severe steatosis after methylmercury administration, which may be due to the accumulation of triglycerides. These changes are almost completely restored with treatment of N-acetyl cysteine. Treatment of N-acetyl cysteine recouped alterations of the above liver function tests. A significant rise was found in urea, creatinine, uric acid, and blood urea nitrogen concentration after long-term methylmercury administration, which indicated renal failure. Several in vivo and in vitro studies have demonstrated that methylmercury induced oxidative stress39,40 by increasing the intracellular levels of ROS and modifying enzyme activities,41,42 which may be due to high concentration of TBARS in methylmercury-exposed rats. Endogenous glutathione (GSH) is one of the most abundant and essential thiol tripeptide present in mammalian cells for scavenging reactive oxygen species.43 It is well known that the most important mechanism for mercury toxicity is oxidative damage and its strong reactivity with –SH components,44,45 which can lead to diminishing the antioxidant reserves, contributing to the increase in reactive species production, and damaging membranes, proteins, and DNA.46 Fur-

FIG. 6: Protective effect of N-acetyl cysteine of on the enzymatic activities of glucose-6-phosphate dehydrogenase in liver, kidney, and brain in chronic methylmercury-exposed rats. Mean ± S.E., n = 6. #Significant difference between MeHg per se versus control at p ≤ 0.05. *Significant difference between MeHg+ NAC versus MeHg per se at p ≤ 0.05. % = percent protection of N-acetyl cysteine against methylmercury-induced toxicity.

thermore, biliary excretion is the major pathway for MeHg elimination and this mechanism appears to be dependent on GSH bioavailability,47 which is bound in MeHg by a direct chemical interaction, forming GS-Hg-CH3 complexes.48 In a study carried out by Journal of Environmental Pathology, Toxicology and Oncology

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FIG. 7: Protective effect of N-acetyl cysteine of on mercury ion concentration in liver, kidney, and brain and metallothionein expression in liver and kidney in chronic methylmercury-exposed rats. Mean ± S.E., n = 6. #Significant difference between MeHg per se versus control at p ≤ 0.05. *Significant difference between MeHg+ NAC versus MeHg per se at p ≤ 0.05. % = percent protection of N-acetyl cysteine against methylmercury-induced toxicity.

Thompson et al,49 it was observed that exposure to high levels of MeHg throughout the gestation resulted in GSH depletion in the fetus.43 Reduced GSH is an antioxidant and free radicals are detoxified by various antioxidants enzymes, such as GR, GPx, CAT, and SOD. We observed decreased GR, GPx, and G-6PDH activities in the present study when rats were treated with chronic MeHg, which could Volume 33, Number 2, 2014

be explained by a direct inhibition of these enzymes by the MeHg. It has been reported that GR plays a pivotal role in the maintenance of GSH concentration by reduction of GSSG to GSH, which is NADPH dependent. However, G6PDH activity may also be reduced by oxidative stress that can be generated due to mercury toxicity; thus, GR activity might be limited by the G6PD-dependent supply of NADPH.

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FIG. 8: Level of mean DNA damage, liver, kidney and brain of control, methylmercury-exposed, and N-acetyl cysteine–treated rats. Each value is expressed as Mean ± S.E., n = 6. #Significant difference between MeHg per se vs control at p ≤ 0.05. *Significant difference between MeHg+ NAC versus MeHg per se at p ≤ 0.05. % = percent protection of N-acetyl cysteine against methylmercury-induced toxicity. Micrographs of a comet images of liver, kidney, and brain (C1–C9) of control, MeHg-exposed group, and MeHg+ NAC treated group.

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It has been suggested that besides GSH/GSSG, the activities of GPx and GR are important in predicting tissue redox state.50 Glucose-6-phosphatse dehydrogenase catalyses is the first step of oxidation in the hexose monophosphate pathway by which glucose may enter into the pentose monophosphate shunt and produce NADPH, which is required as hydrogen donor for reactions of various biochemical pathways. This enzyme plays an important role in the regulation of sugar metabolism and determines whether glucose undergoes glycolysis or is utilized via the pentose phosphate pathway. The decrease in the activity of glucose-6-phosphate dehydrogenase due to mercury intoxication might indicate decreased ability to generate reducing power in the form of NADPH due to oxidative stress, which is induced by mercury toxicity. The therapeutic agent as N-acetyl cysteine provides protection against the deleterious effects of methylmercury toxicity by increasing the values of this enzyme near the control group. Ho et al.51 and Mukherje et al.52 also reported the protective effects of N-acetyl cysteine on glucose-6-phosphate dehydrogenase deficient individuals. Metallothionein is a low molecular weight metal-binding protein that plays a key role in the homeostasis of essential metals, such as zinc and copper.53 It may be involved in the detoxification of heavy metals and scavenge free radicals. In the present study, chronic exposure of methylmercury-induced elevation in metallothionein concentration in liver and kidney, which may be due to the oxidative stress caused by reactive oxygen species (ROS) generated by mercury toxicity. Peixoto et al.54 reported that mercury induced metallothionein synthesis. Treatment with N- acetyl cysteine showed protection against chronic methylmercury toxicity. DNA damage has been recognized as the onset of many diseases and could be a useful indicator of the oxidative stress and antioxidant defense system of an organism. It is reported that methylmercury is known to induce DNA single-strand breaks and inhibit DNA repair mechanism by free radical generation.55 In the present study, methylmercury-exposed rats showed increased mean DNA damage in liver, kidney, and brain. Some reported studies showed that methylmercury caused oxidative DNA base Volume 33, Number 2, 2014

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damage in mammalian cells,56,57 and such oxidative DNA damage, if not repaired, can lead to mutation and cancer.58 A dose-dependent effect of monomethylmercury on the human cells assessed by using the comet assay has been reported by Betti and Nigro,59 Bucio et al.,60 and Taddei et al.,61 who noticed that methylmercury exposure to dolphin leucocytes induced dose-dependent DNA single-strand breaks. Present findings clearly support, in agreement, that post-treatment with N-acetylcysteine reverses all these alterations when compared to the control rats. The thiol N-acetyl cysteine is readily deacetylated in cells to yield L-cysteine, thereby promoting intracellular GSH synthesis. Besides this activity as a GSH precursor, NAC is responsible for protective effects in the extracellular environment, mainly due to its nucleophilc and antioxidant properties, which influence the toxicokinetics of xenobiotics. Collectively, our findings provide reasonable evidence on the protective effect of N-acetyl cysteine in liver, kidney, brain, and blood, which is suggestive of the broad therapeutic potential of this antidote in mitigating oxidative stress-mediated cellular dysfunctions. V. CONCLUSIONS In conclusion, our results show that the beneficial effects of N-acetyl cysteine against methylmercury-induced chronic toxicity in rats, as is evident from the decreased oxidative stress, mean DNA damage, and increment in antioxidant defense system. These results provide evidence for the first time that N-acetyl cysteine exerts significant protection against MeHg-induced toxicity. Although its precise site of action remains unclear v, it can be presumed that the modulatory properties of N-acetyl cysteine are likely to be responsible for the greater part of its protection against MeHg-induced toxicity. However, these issues need further investigation. ACKNOWLEDGMENTS This work was supported by the University Grants Commission (New Delhi, India; Project No. F/ DEV/2007/1277) and Jiwaji University (Gwalior, India). The authors declare that this manuscript

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was consistent with the guidelines and principles of Institutional Ethical Committee of Jiwaji University, Gwalior for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) Chennai, India (CPCSEA/501/01/A).

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Zimmer W. Carter DE. The efficacy of 2,3-dimercaptopropanol and D-penicillamine on methyl mercury induced neurological signs and weight loss. Life Sci. 1978;23:1025–34.

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