Biol Trace Elem Res (2014) 159:332–345 DOI 10.1007/s12011-014-9977-8
Preventive Effects of Dextromethorphan on Methylmercury-Induced Glutamate Dyshomeostasis and Oxidative Damage in Rat Cerebral Cortex Shu Feng & Zhaofa Xu & Wei Liu & Yuehui Li & Yu Deng & Bin Xu
Received: 25 January 2014 / Accepted: 10 April 2014 / Published online: 13 May 2014 # Springer Science+Business Media New York 2014
Abstract Methylmercury (MeHg) is a well-known environmental pollutant leading to neurotoxicant associated with aberrant central nervous system (CNS) functions, but its toxic mechanisms have not yet been fully recognized. In the present study, we tested the hypothesis that MeHg induces neuronal injury via glutamate (Glu) dyshomeostasis and oxidative damage mechanisms and that these effects are attenuated by dextromethorphan (DM), a low-affinity and noncompetitive Nmethyl-D-aspartate receptor (NMDAR) antagonist. Seventytwo rats were randomly divided into four groups of 18 animals in each group: control group, MeHg-treated group (4 and 12 μmol/kg), and DM-pretreated group. After the 4-week treatment, we observed that the administration of MeHg at a dose of 12 μmol/kg significantly increased in total mercury (Hg) levels, disrupted Glu metabolism, overexcited NMDARs, and led to intracellular calcium overload in the cerebral cortex. We also found that MeHg reduced nonenzymatic and enzymatic antioxidants, enhanced neurocyte apoptosis, induced reactive oxygen species (ROS), and caused lipid, protein, and DNA peroxidative damage in the cerebral cortex. Moreover, glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) appeared to be inhibited by MeHg exposure. These alterations were significantly prevented by the pretreatment with DM at a dose of 13.5 μmol/kg. In conclusion, these findings strongly implicate that DM has potential to protect the brain from Glu dyshomeostasis and oxidative damage resulting from MeHginduced neurotoxicity in rat.
S. Feng : Z. Xu (*) : W. Liu : Y. Li : Y. Deng : B. Xu Department of Environmental Health, School of Public Health, China Medical University, Shenyang, People’s Republic of China e-mail: [email protected]
Keywords Methylmercury . Neurotoxicity . Glutamate dyshomeostasis . Oxidative damage . Dextromethorphan
Introduction Methylmercury (MeHg) is an environmental contaminant that has been established to cause neurological deficits in both experimental animals and humans [1, 2]. Although several studies have contributed to the understanding of the critical phenomena that mediate MeHg-induced neurotoxicity, the accurate molecular mechanisms related to its toxicity are not completely understood. A number of molecular mechanisms have been proposed to be implicated in its neurotoxic effects including impairment of intracellular calcium homeostasis [3, 4], alteration of Glutamate (Glu) homeostasis [5–7], and oxidative damage [8–10]. Understanding the precise molecular mechanisms of MeHg toxicity is therefore of fundamental interest and will contribute to the development of new treatment strategies. One of the most widely documented effects caused by MeHg on the central nervous system (CNS) is associated with Glu-mediated excitotoxicity. Glu, the main excitatory neurotransmitter, plays a crucial role in development, learning, memory, and response to injury [11]. However, excessive amounts of Glu in the synaptic space could overstimulate Glu receptors, in which N-methyl-D-aspartate receptors (NMDARs) are one of the subtypes playing important roles in physiological processes in the CNS [12], leading to increased Na+ and Ca2+ influx into neurons [13, 14]. The rising level of intracellular Ca2+ is associated with the generation of oxidative damage and neurotoxicity [15, 16]. Accordingly, the extracellular Glu is taken up by the action of Glu transporters located on astrocytic cell membranes, which remove extracellular Glu from the synaptic cleft, keeping its extracellular
Preventive Effects of Dextromethorphan
concentrations below toxic levels [17, 18]. In accordance, several studies have demonstrated that the Glu over activation of NMDA receptor induced by MeHg inhibition of Glu uptake can raise intracellular Ca2+ influx and reactive oxygen species (ROS) overproduction [19]. Oxidative damage has also been known to contribute to MeHg-induced CNS damage [20]. MeHg could induce oxidative damage in mitochondria directly, which results in the overproduction of ROS. ROS are important mediators of damage to cell structures, including lipids, proteins, and nucleic acids [21, 22]. In addition, many studies have reported that MeHg-induced oxidative damage can also cause a decrease in the endogenous nonenzymatic antioxidants as well as an inhibition of the antioxidant enzymes [23–29]. Moreover, MeHg-induced ROS (mainly H2O2) production appears to directly inhibit astrocyte Glu transporters, leading to increased Glu concentrations in the extracellular fluid [30, 31], suggesting that Glu dyshomeostasis and oxidative damage appear to be connected, affecting each other. However, little data exist on the in vivo effects of MeHg poisoning on the interaction between Glu dyshomeostasis and oxidative damage, information that is critical for more fully evaluating the MeHg-induced neurotoxicity. Despite massive efforts in the search for new drugs that counteract mercurial toxicity, there are no effective treatments available that completely abolish its toxic effects. Low-affinity NMDAR antagonists have been the focus recently because of their ability that preferentially blocks excessive NMDAR activity without disrupting physiological synaptic activity [32, 33]. Dextromethorphan (DM), a low-affinity, noncompetitive NMDA antagonist, has been used as an antitussive agent. In addition to its NMDAR antagonistic and antiinflammatory effect is the potential of DM to reduce the production of both extracellular superoxide free radicals and intracellular ROS [34]. The present study was carried out to examine the effects of MeHg on Glu dyshomeostasis and oxidative damage and postulated that they are interacting with each other in MeHg poisoning. To test this hypothesis, we developed a rat model of MeHg subchronic poisoning to evaluate its neurotoxic effects. We also tested that DM could effectively attenuate the toxicity of MeHg in vivo, which may contribute to understanding the mechanisms of MeHg neurotoxicity.
Materials and Methods Chemicals Methylmercury chloride was purchased from Dr. Ehrenstorfer (purity 97 %; Augsburg, Germany). Analysis kits of Glu, glutamine (Gln), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), protein sulfhydryl, and carbonyl were
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provided by Jiancheng Bioengineering Institute (Nanjing, China). Annexin V-FITC/PI reagent kit was purchased from KeyGen Biotech Corporation (Nanjing, China). Real-time PCR analysis kits were purchased from TaKaRa Biotechnology Company (Dalian, China). Rabbit polyclonal antibodies developed against a peptide mapping at the C-terminus of NR1, NR2A, NR2B, glutamate/aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), and β-actin were obtained from Santa Cruz Biotechnology Company (Santa Cruz, CA, USA). Mouse monoclonal antibody developed against 8′-hydroxy-2′-deoxyguanosine (8-OHdG) and goat anti-rabbit/mouse secondary antibodies were obtained from Santa Cruz. DM, fura-2 acetoxymethyl (Fura-2 AM), 2′,7′dichlorofluorescin-diacetate (DCFH-DA), streptavidin–biotin–peroxidase complex (SABC), and 3′,3′-diaminobenzidine (DAB) were purchased from Sigma (St. Louis, MO, USA), while other chemicals of analytical grade were provided by local chemical suppliers. Animal Grouping and Treatment Adult (6 weeks) Wistar rats with an initial body weight of 160–180 g were provided by Laboratory Animal Center of China Medical University (SPF grade, certificate number SCXK2008-0005, n=72). They were housed under conventional conditions at a room temperature of 23–27 °C, with a 12:12-h light/dark cycle, humidity of 30–40 %, and free access to food and water. All animal procedures were conformed to the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). Rats were randomly divided into four groups of 18 animals (nine males and nine females) in each group: control group, MeHg-treated groups (4 and 12 μmol/kg), and DM-pretreated group. The control group rats were subcutaneously (s.c.) injected with 0.9 % NaCl, 2 h before intraperitoneally (i.p.) administrated with 0.9 % NaCl. MeHg-treated rats were s.c. injected with 0.9 % NaCl, and 2 h later, i.p. injected with 4 and 12 μmol/kg MeHg in sterile deionized water, respectively. DM-pretreated group rats were s.c. injected with 13.5 μmol/kg DM in sterile deionized water, 2 h before i.p. administrated with 12 μmol/kg MeHg. The amount of MeHg delivered was adjusted for the molar concentration in the chloride form so as to achieve a precise dose of 12 μmol/kg. This dose was selected based upon pilot studies indicating that 12 μmol/kg was the sufficient dose to induce significant neurologic deficits in exposed rats during the course of 4-week MeHg exposure. In addition, a dose of 4 μmol/kg was selected to observe the possible dosedependent effects of MeHg exposure in the present study. As DM, which crosses the blood–brain barrier, readily produces a noncompetitive blockade of the NMDA receptor, it was subcutaneously injected first in order to be fully absorbed and reach a high concentration in the brain, 2 h before MeHg
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administration, which ensured that DM pretreatment could not be interpreted by MeHg treatment. The volume of injection was 5 ml/kg body weight, five times per week, for up to 4 weeks. Sample Collections At 24 h after the last injection, rats were killed by decapitation after anesthetized. The head was immersed in running cold physiological saline for 30 s to clear blood contamination. First, the brain capsules of six rats (three males and three females) in each group were removed, and the cerebral cortex was isolated on ice bath. One hundred milligrams of the cortex was digested by nitric acid for 12 h in order to determine the total mercury (Hg) levels; other cortex was prepared for 5 or 10 % homogenate in order to determine Glu, Gln, GS, phosphate-activated glutaminase (PAG), GSH, SOD, GSHPx, malondialdehyde (MDA), protein sulfhydryl, and carbonyl levels. Second, the cerebral cortex of four rats (two males and two females) in each group was prepared for single-cell suspension in order to measure intracellular free calcium levels, ROS formation, and apoptosis levels. Third, total RNA and protein of the cerebral cortex of four rats (two males and two females) in each group were extracted for the determination of messenger RNA (mRNA) and protein expressions of NMDAR subunits and Glu transporters. The remaining four rats (two males and two females) of each group were perfused into the left ventricular with physiological saline followed by buffered 4 % paraformaldehyde for 8-OHdG determination. All the substance contents and enzyme activities were normalized to the protein amount that was measured according to the method of Lowry et al. [35], using bovine serum albumin (BSA) as standard. Determination of Total Mercury Levels in the Cerebral Cortex Hg concentrations were detected with cold vapor atomic fluorescence spectrometry as described by Stockwell and Corns [36]. One hundred milligrams of cerebral cortex was digested in 2.0 ml of nitric acid for 12 h. After digestion, 2.0 ml of 50 % sulfuric acid and 3.0 ml of equilibrium KMnO4 were added in, at 90 °C for 1 h. After cooling, the oxidant excess was reduced by the addition of 50 % hydroxylamine. One milliliter of sample was added in 1.0 ml of dehydrated alcohol and 2.0 ml of 20 % stannous chloride and then analyzed by F732 mercury analyzer immediately. Values were obtained from a standard curve prepared with HgCl2. Determination of Glu Metabolism in the Cerebral Cortex Glu and Gln contents were determined with analysis kits and operated according to the methods recommended by the manufacturers. In brief, the cerebral cortex was made into 10 %
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homogenate with 0.9 % NaCl. For Glu measurement, 0.2 ml of homogenate was mingled with 0.6 ml reagent added into each tube for centrifugation (1,000×g for 10 min at 4 °C). Then, 0.5 ml of supernatant was mixed with other reagents and incubated at 37 °C for 40 min. For Gln determination, homogenate was mixed with other reagents and was incubated at 37 °C for 15 min. Absorbance was recorded at 340 and 630 nm for Glu and Gln, respectively. GS Activity was determined based on the reaction of glutamine and hydroxylamine, forming γglutamylhydroxamate and NH3 as described by Allen et al. [31]. Five percent homogenate was added into the reaction mixture containing 110 mM glutamine, 0.45 mM ATP, 9 mM Na2HAsO4, 110 mM hydroxylamine, 1.8 mM MnCl2, and 0.1 mM dithiothreitol in 50 mM imidazole buffer, pH 7.2, and enzymatic extract with 100 μg of protein, all in a final volume of 1.5 ml. The reaction mixture was incubated at 37 °C for 30 min and stopped by 0.75 ml 0.6 M Fe(NO3)3 in 40 % trichloroacetic acid. The tubes were centrifuged at 800×g for 5 min in order to remove the protein precipitate. The absorbance was determined in the supernatant at 535 nm, and the values were obtained from a standard curve prepared with L-γ-glutamylhydroxamate. PAG activity was measured according to the method of Curi [37]. The cerebral cortex was homogenized in an extraction medium containing 150 mM potassium phosphate, 1 mM ethylenediaminetetraacetic acid (EDTA), and 50 mM Tris– HCl at pH 8.6. The assay medium consisted of 50 mM phosphate buffer, 0.2 mM EDTA, 50 mM Tris–HCl, 20 mM Glu, and 0.05 % Triton X-100, to which 100 μl of homogenate was added. The total volume was 1.0 ml at pH 8.6. Assay media, in duplicate, were incubated at 37 °C. The reaction was initiated by addition of freshly prepared glutamine, promoting a 10min linear reaction time course, and stopped by the addition of 0.2 ml of 25 % perchloric acid, and then neutralized. The amount of Glu was determined, and the absorbance was recorded at 340 nm.
Determination of Nonenzymatic and Enzymatic Antioxidants in the Cerebral Cortex Nonprotein sulfhydryl (NPSH) content was measured in accordance with the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) colorimetric method [38]. In brief, the cerebral cortex was homogenized to the concentration of 10 % in phosphatebuffered saline (PBS). The PBS was mixed with 0.1 M Na2HPO4 and 0.1 M NaH2PO4, and the final pH was 8.0. Following centrifugation (1,000×g for 10 min at 4 °C), a mixture of 0.1 ml of the supernatant and 0.9 ml of 5 % trichloroacetic acid (TCA) was mingled and centrifuged (2,300×g for 15 min at 4 °C). Then, 0.5 ml of the supernatant was added into 1.5 ml of 0.01 % DTNB, and the reaction was
Preventive Effects of Dextromethorphan
monitored at 412 nm after standing at room temperature for 5 min. SOD and GSH-Px activities were determined with analysis kits according to the manufacturer’s instructions. SOD estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltet-razolium chloride to form a red formazan dye. The SOD activity was measured by the degree of inhibition of this reaction. GSH-Px determination was based on the principle that GSH-Px catalyzes the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione reductase and reduced nicotinamide adenine dinucleotide phosphate, the oxidized glutathione is immediately converted to the reduced form with a contaminant oxidation of NADPH to NADP+. Absorbance differences were recorded at 560 and 340 nm for SOD and GSH-Px, respectively. Measurement of Oxidative Damage in the Cerebral Cortex MDA was measured as the production of lipid peroxide, which combines with thiobarbituric acid (TBA) to form a pink chromogen compound [39]. In brief, 10 % homogenate was mingled with the reaction mixture consisting of 0.1 M ice-cold PBS and 0.1 M FeCl2 in a total volume of 1.0 ml (pH 7.4). The reaction was stopped by 1.0 ml of 10 % TCA followed by 1.0 ml of 0.67 % TBA addition. After centrifugation at 2,500×g for 10 min, the absorbance was recorded at 530 nm using tetramethoxypropane as standard. Protein sulfhydryl and carbonyl contents were measured as the indicators of protein oxidative damage. Protein sulfhydryl was detected by the DTNB method according to the manufacturer’s instructions. The colorimetric reaction was measured at 412 nm. Protein sulfhydryl contents were calculated from total sulfhydryl by subtracting nonprotein sulfhydryl. Protein carbonyl contents were detected by the 2,4dinitrophenylhy-drazine (DNPH) method according to the manufacturer. In brief, proteins were precipitated with an equal volume of 20 % TCA and washed three times with 2 ml of a 1:1 ethanol/ethyl acetate mixture. Finally, the precipitates were dissolved in 6-M guanidine HCl solution, and the absorbance was measured at 370 nm. 8-OHdG was measured as the indicator of DNA oxidative damage, and the immunostaining was performed by SABC method recommended by the manufacturer. Animals were deeply anesthetized followed by transcardial perfusion with the aid of a peristaltic pump. Initial perfusion consisted of 50 ml of saline and heparin (1 %) at room temperature, followed by 250 ml of 4 % paraformaldehyde in 0.1 M PBS (pH 7.4). The cerebral cortex was removed and post-fixed in 4 % paraformaldehyde solution for 12 h at 4 °C. Then, tissues were transferred in PBS with 30 % sucrose for 48 h and were frozen and sectioned in a cryostat (Leica, Heidelberg,
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Germany) at 12-μm thickness after dehydrated in a graded series of alcohol. Sections were incubated in boiling 0.01 M citrate buffer (3×30 min), hydrogen peroxide (0.3 % in methanol, 10 min), and 5 % normal serum for 1 h, followed by 8OHdG mouse monoclonal antibody (1:500) overnight at 4 °C. The primary antibody was detected using a biotinylated secondary antibody goat anti-mouse IgG (1:100) for 2 h, followed by incubation with SABC (1:100) for 3 h at room temperature. After incubation in 0.05 % DAB for 15 min, dehydrated processes were performed through alcohols and xylenes. The sections were mounted on gelatin–chromalum-coated slides for light microscope examination. To evaluate the morphologic characteristics and the relative cell immunoreactivity for 8OHdG in the cerebral cortex, according to the process of Morello [40], we used a light microscope equipped with a ×40 objective. Analysis of 8-OHdG-immunoreactive positive cells was performed in three immunostained sections per immunoreactive marker per animal (n=4). The intensity of the immunoreactive signal was calculated from the integrated optical density (IOD) of the immunoreactive positive cells using a microcomputer-based image display system (Image ProPlus, Media Cybernetics 5.0). Preparation of Dissociated Cerebral Cortex Cells The cerebral cortex (100 mg) was dissected and washed by phosphate-buffered saline (PBS, pH 7.2–7.4) for three to five times. The tissues were minced in 10 ml PBS and supplemented with 0.125 % trypsin for 10–15 min at 37 °C with vigorous shaking. The subsequent mechanical dissociation with Pasteur pipettes and nylon mesh screens was carried out. Cells were finally suspended in DMEM supplemented with 10 % BSA. The concentration of cells was evaluated by viable cell count (trypan blue stained). It was diluted to 1×106 cells/ml for intracellular free calcium level, ROS formation, and apoptosis level detection. Determination of Intracellular Free Calcium Levels Using the Ca2+ Indicator Fura-2 in the Cerebral Cortex The intracellular free calcium ([Ca2+]i) assay was performed by a method described previously [41]. Briefly, for fura-2 experiments, absolute values of [Ca2+]i in neurocyte were calibrated from the measurement of fluorescence signals by the use of F-4500 fluorescence spectrophotometer (HICATHI, Japan). The calibration equation used was [Ca2+]i =Kd[(R− Rmin) / (Rmax −R)]×(Sf380 / Sb380), where Kd is the dissociation constant of the dye, R is the ratio at excitation wavelengths 340/380 nm, Rmin is the ratio at zero [Ca2+]i, and Rmax is the ratio at saturated [Ca2+]i. Procedures for obtaining Rmax and Rmin caused damage to cells and were, therefore, carried out at the end of the experiments. Rmax was obtained first by adding 0.2 % Triton X-100, making the cell membrane permeable to
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Ca2+, and allowing the extracellular and intracellular Ca2+ to equilibrate. Following this, Rmin was obtained by adding the chelator EGTA to chelate all Ca2+ inside and outside the cells. The present experiments were carried out at pH 7.4 and at a temperature of 37 °C. A Kd value of 224 nM was used. Measurement of Intracellular ROS Level Using Flow Cytometry in the Cerebral Cortex The intracellular ROS level was monitored by using the peroxide-sensitive fluorescence probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (Molecular Probes) as described previously [42]. In brief, cells (1×106 cells/ml) were incubated with 10 μM DCFH-DA at 37 °C for 30 min. The fluorescent dye DCFH-DA passes through the cell membrane and undergoes deacetylation by intracellular esterases to produce the nonfluorescent compound DCFH that is trapped inside the cells. Oxidation of DCFH by ROS produces the highly fluorescent DCF, which was measured and monitored at 488 nm (excitation)/525 nm (emission) by a FACScan flow cytometer (Becton–Dickinson, Germany). Apoptosis Assays Using Flow Cytometry in the Cerebral Cortex The apoptosis levels were measured by flow cytometry according to annexin V-FITC/PI double-stained method. Annexin V is a Ca2+-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine; annexin VFITC is a sensitive probe for identifying cells that are undergoing apoptosis, while propidium iodide (PI) is a nonspecific DNA dye that is excluded from living cells with intact plasma membranes but incorporated into the nonviable cell. The cerebral cortex cells (1×106 cells/ml) were mingled with 200 μl ice-cold binding buffer followed by 10 μl horseradish peroxidase FITC-labeled annexin V and 5 μl PI adding. After a 15-min incubation in darkness, the apoptosis rate was determined immediately. In the present study, annexin V+/PI− populations were considered as the early apoptotic cells. Real-Time PCR Analysis Total RNA was extracted using the RNAiso Plus according to the manufacturer and finally dissolved in RNase-free H2O. OD260/OD280 of total RNA was between 1.6 and 1.8. Semiquantitative reverse transcription was performed on Applied Biosystems 2720 Thermal Cycler (Applied Biosystems, Singapore) with PrimeScript RT reagent Kit and gDNA Eraser, using β-actin as an endogenous control. The gene-specific primers were designed by TaKaRa Company. NR1 forward primer 5′-GCCTACAAGCGACACAAGGATG-3′, reverse primer 5′-TTAGGGTCGGGCTCTGCTCTAC-3′; NR2A forward primer 5′-ACCTCGCTCTGCTCCAGTTTG-3′, reverse
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primer 5′-GTTGTGGCAGAGCCCGTAA-3′; NR2B forward primer 5′’-CGCCTAGAGGTTTGGCGTCTAC-3′, reverse primer 5′-GAACGAGCTTTGCTGCCTGA-3′; GLAST forward primer 5′-TCGCTGCACTGGATTCCAAC-3′, reverse primer 5′-ACCAGGCTTGATGCTCACAACTAAC-3′; GLT1 forward primer 5′-GTTCAAGGACGGGATGAATGTC TTA-3′, reverse primer 5′-CATCAGCTTGGCCTGCTCAC3′; β-actin forward primer 5′-GGAGATTACTGCCCTGGC TCCTA-3′, reverse primer 5′-GACTCATCGTACTCCTGC TGCTG-3′. For the quantitative PCR reaction, the reaction mixture contained 25-μl buffer (2×), 2-μl PCR forward primer (10 μM), 2-μl PCR reverse primer (10 μM), 1-μl ROX reference dye II (50×), 4-μl template DNA, and 16 μl dH2O up to a final volume of 50 μl. The initial denaturation was carried out at 95 °C for 30 s, followed by amplification in 40 cycles, 95 °C for 3 s, and 60 °C for 34 s, using the 7500 real-time PCR system. The relative expression analysis was assessed by the method of 2−△△CT. Western Blot Analysis Protein extraction and immunoblot analysis were conducted as described by Guerguerian et al. [43]. Total protein was extracted from the cerebral cortex using RIPA buffer (10 mM Na2HPO4, 150 mM NaCl, 1 % sodium deoxicolate, 1 % Nonidet P-40, and 0.1 % SDS) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.2 mM 1,10phenanthroline, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 mM benzamidine) at 4 °C for 30 min. Protein concentrations were quantified with the BCA reagent. Equal amounts of protein (30 μg per lane) were separated by 8 or 10 % SDSpolyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). PVDF membranes were subsequently blocked overnight at 4 °C in Tween 20 tris-buffered saline (TBST) containing 5 % nonfat powdered milk, followed by briefly rinsing in TBST and incubated with NR1 (1:1,000), NR2A (1:1,000), NR2B (1:1,000), GLAST (1:500), GLT-1 (1:300), and β-actin (1:500) primary antibody in TBST at 4 °C overnight. Specific protein expression was then detected by incubating the membranes with HRP-conjugated secondary antibody (1:5,000). Protein bands were visualized by using ECL Western blotting chemiluminescent detection reagents and autoradiography. The intensity of bands was evaluated semiquantitatively by densitometry using an imageanalyzing software (FluorChem v2.0) and normalized by the internal control (β-actin). The changes of intensity of NR1, NR2A, NR2B, GLAST, and GLT-1 proteins after MeHg treatment were normalized using the intensity obtained in the internal control bands (β-actin).
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Statistical Analysis All statistical analyses were performed using the SPSS software, version 11.5. Assays were performed three times in duplicate for each rat, and the mean of duplicate was used for statistical analysis. Data were analyzed using analysis of variance (ANOVA) after confirmation of normal distributions, and group means were compared by Fischer’s least significant difference (LSD) procedure. Data were expressed as mean ± standard deviation. The difference at either P
Preventive Effects of Dextromethorphan on Methylmercury-Induced Glutamate Dyshomeostasis and Oxidative Damage in Rat Cerebral Cortex Shu Feng & Zhaofa Xu & Wei Liu & Yuehui Li & Yu Deng & Bin Xu
Received: 25 January 2014 / Accepted: 10 April 2014 / Published online: 13 May 2014 # Springer Science+Business Media New York 2014
Abstract Methylmercury (MeHg) is a well-known environmental pollutant leading to neurotoxicant associated with aberrant central nervous system (CNS) functions, but its toxic mechanisms have not yet been fully recognized. In the present study, we tested the hypothesis that MeHg induces neuronal injury via glutamate (Glu) dyshomeostasis and oxidative damage mechanisms and that these effects are attenuated by dextromethorphan (DM), a low-affinity and noncompetitive Nmethyl-D-aspartate receptor (NMDAR) antagonist. Seventytwo rats were randomly divided into four groups of 18 animals in each group: control group, MeHg-treated group (4 and 12 μmol/kg), and DM-pretreated group. After the 4-week treatment, we observed that the administration of MeHg at a dose of 12 μmol/kg significantly increased in total mercury (Hg) levels, disrupted Glu metabolism, overexcited NMDARs, and led to intracellular calcium overload in the cerebral cortex. We also found that MeHg reduced nonenzymatic and enzymatic antioxidants, enhanced neurocyte apoptosis, induced reactive oxygen species (ROS), and caused lipid, protein, and DNA peroxidative damage in the cerebral cortex. Moreover, glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) appeared to be inhibited by MeHg exposure. These alterations were significantly prevented by the pretreatment with DM at a dose of 13.5 μmol/kg. In conclusion, these findings strongly implicate that DM has potential to protect the brain from Glu dyshomeostasis and oxidative damage resulting from MeHginduced neurotoxicity in rat.
S. Feng : Z. Xu (*) : W. Liu : Y. Li : Y. Deng : B. Xu Department of Environmental Health, School of Public Health, China Medical University, Shenyang, People’s Republic of China e-mail: [email protected]
Keywords Methylmercury . Neurotoxicity . Glutamate dyshomeostasis . Oxidative damage . Dextromethorphan
Introduction Methylmercury (MeHg) is an environmental contaminant that has been established to cause neurological deficits in both experimental animals and humans [1, 2]. Although several studies have contributed to the understanding of the critical phenomena that mediate MeHg-induced neurotoxicity, the accurate molecular mechanisms related to its toxicity are not completely understood. A number of molecular mechanisms have been proposed to be implicated in its neurotoxic effects including impairment of intracellular calcium homeostasis [3, 4], alteration of Glutamate (Glu) homeostasis [5–7], and oxidative damage [8–10]. Understanding the precise molecular mechanisms of MeHg toxicity is therefore of fundamental interest and will contribute to the development of new treatment strategies. One of the most widely documented effects caused by MeHg on the central nervous system (CNS) is associated with Glu-mediated excitotoxicity. Glu, the main excitatory neurotransmitter, plays a crucial role in development, learning, memory, and response to injury [11]. However, excessive amounts of Glu in the synaptic space could overstimulate Glu receptors, in which N-methyl-D-aspartate receptors (NMDARs) are one of the subtypes playing important roles in physiological processes in the CNS [12], leading to increased Na+ and Ca2+ influx into neurons [13, 14]. The rising level of intracellular Ca2+ is associated with the generation of oxidative damage and neurotoxicity [15, 16]. Accordingly, the extracellular Glu is taken up by the action of Glu transporters located on astrocytic cell membranes, which remove extracellular Glu from the synaptic cleft, keeping its extracellular
Preventive Effects of Dextromethorphan
concentrations below toxic levels [17, 18]. In accordance, several studies have demonstrated that the Glu over activation of NMDA receptor induced by MeHg inhibition of Glu uptake can raise intracellular Ca2+ influx and reactive oxygen species (ROS) overproduction [19]. Oxidative damage has also been known to contribute to MeHg-induced CNS damage [20]. MeHg could induce oxidative damage in mitochondria directly, which results in the overproduction of ROS. ROS are important mediators of damage to cell structures, including lipids, proteins, and nucleic acids [21, 22]. In addition, many studies have reported that MeHg-induced oxidative damage can also cause a decrease in the endogenous nonenzymatic antioxidants as well as an inhibition of the antioxidant enzymes [23–29]. Moreover, MeHg-induced ROS (mainly H2O2) production appears to directly inhibit astrocyte Glu transporters, leading to increased Glu concentrations in the extracellular fluid [30, 31], suggesting that Glu dyshomeostasis and oxidative damage appear to be connected, affecting each other. However, little data exist on the in vivo effects of MeHg poisoning on the interaction between Glu dyshomeostasis and oxidative damage, information that is critical for more fully evaluating the MeHg-induced neurotoxicity. Despite massive efforts in the search for new drugs that counteract mercurial toxicity, there are no effective treatments available that completely abolish its toxic effects. Low-affinity NMDAR antagonists have been the focus recently because of their ability that preferentially blocks excessive NMDAR activity without disrupting physiological synaptic activity [32, 33]. Dextromethorphan (DM), a low-affinity, noncompetitive NMDA antagonist, has been used as an antitussive agent. In addition to its NMDAR antagonistic and antiinflammatory effect is the potential of DM to reduce the production of both extracellular superoxide free radicals and intracellular ROS [34]. The present study was carried out to examine the effects of MeHg on Glu dyshomeostasis and oxidative damage and postulated that they are interacting with each other in MeHg poisoning. To test this hypothesis, we developed a rat model of MeHg subchronic poisoning to evaluate its neurotoxic effects. We also tested that DM could effectively attenuate the toxicity of MeHg in vivo, which may contribute to understanding the mechanisms of MeHg neurotoxicity.
Materials and Methods Chemicals Methylmercury chloride was purchased from Dr. Ehrenstorfer (purity 97 %; Augsburg, Germany). Analysis kits of Glu, glutamine (Gln), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), protein sulfhydryl, and carbonyl were
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provided by Jiancheng Bioengineering Institute (Nanjing, China). Annexin V-FITC/PI reagent kit was purchased from KeyGen Biotech Corporation (Nanjing, China). Real-time PCR analysis kits were purchased from TaKaRa Biotechnology Company (Dalian, China). Rabbit polyclonal antibodies developed against a peptide mapping at the C-terminus of NR1, NR2A, NR2B, glutamate/aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), and β-actin were obtained from Santa Cruz Biotechnology Company (Santa Cruz, CA, USA). Mouse monoclonal antibody developed against 8′-hydroxy-2′-deoxyguanosine (8-OHdG) and goat anti-rabbit/mouse secondary antibodies were obtained from Santa Cruz. DM, fura-2 acetoxymethyl (Fura-2 AM), 2′,7′dichlorofluorescin-diacetate (DCFH-DA), streptavidin–biotin–peroxidase complex (SABC), and 3′,3′-diaminobenzidine (DAB) were purchased from Sigma (St. Louis, MO, USA), while other chemicals of analytical grade were provided by local chemical suppliers. Animal Grouping and Treatment Adult (6 weeks) Wistar rats with an initial body weight of 160–180 g were provided by Laboratory Animal Center of China Medical University (SPF grade, certificate number SCXK2008-0005, n=72). They were housed under conventional conditions at a room temperature of 23–27 °C, with a 12:12-h light/dark cycle, humidity of 30–40 %, and free access to food and water. All animal procedures were conformed to the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). Rats were randomly divided into four groups of 18 animals (nine males and nine females) in each group: control group, MeHg-treated groups (4 and 12 μmol/kg), and DM-pretreated group. The control group rats were subcutaneously (s.c.) injected with 0.9 % NaCl, 2 h before intraperitoneally (i.p.) administrated with 0.9 % NaCl. MeHg-treated rats were s.c. injected with 0.9 % NaCl, and 2 h later, i.p. injected with 4 and 12 μmol/kg MeHg in sterile deionized water, respectively. DM-pretreated group rats were s.c. injected with 13.5 μmol/kg DM in sterile deionized water, 2 h before i.p. administrated with 12 μmol/kg MeHg. The amount of MeHg delivered was adjusted for the molar concentration in the chloride form so as to achieve a precise dose of 12 μmol/kg. This dose was selected based upon pilot studies indicating that 12 μmol/kg was the sufficient dose to induce significant neurologic deficits in exposed rats during the course of 4-week MeHg exposure. In addition, a dose of 4 μmol/kg was selected to observe the possible dosedependent effects of MeHg exposure in the present study. As DM, which crosses the blood–brain barrier, readily produces a noncompetitive blockade of the NMDA receptor, it was subcutaneously injected first in order to be fully absorbed and reach a high concentration in the brain, 2 h before MeHg
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administration, which ensured that DM pretreatment could not be interpreted by MeHg treatment. The volume of injection was 5 ml/kg body weight, five times per week, for up to 4 weeks. Sample Collections At 24 h after the last injection, rats were killed by decapitation after anesthetized. The head was immersed in running cold physiological saline for 30 s to clear blood contamination. First, the brain capsules of six rats (three males and three females) in each group were removed, and the cerebral cortex was isolated on ice bath. One hundred milligrams of the cortex was digested by nitric acid for 12 h in order to determine the total mercury (Hg) levels; other cortex was prepared for 5 or 10 % homogenate in order to determine Glu, Gln, GS, phosphate-activated glutaminase (PAG), GSH, SOD, GSHPx, malondialdehyde (MDA), protein sulfhydryl, and carbonyl levels. Second, the cerebral cortex of four rats (two males and two females) in each group was prepared for single-cell suspension in order to measure intracellular free calcium levels, ROS formation, and apoptosis levels. Third, total RNA and protein of the cerebral cortex of four rats (two males and two females) in each group were extracted for the determination of messenger RNA (mRNA) and protein expressions of NMDAR subunits and Glu transporters. The remaining four rats (two males and two females) of each group were perfused into the left ventricular with physiological saline followed by buffered 4 % paraformaldehyde for 8-OHdG determination. All the substance contents and enzyme activities were normalized to the protein amount that was measured according to the method of Lowry et al. [35], using bovine serum albumin (BSA) as standard. Determination of Total Mercury Levels in the Cerebral Cortex Hg concentrations were detected with cold vapor atomic fluorescence spectrometry as described by Stockwell and Corns [36]. One hundred milligrams of cerebral cortex was digested in 2.0 ml of nitric acid for 12 h. After digestion, 2.0 ml of 50 % sulfuric acid and 3.0 ml of equilibrium KMnO4 were added in, at 90 °C for 1 h. After cooling, the oxidant excess was reduced by the addition of 50 % hydroxylamine. One milliliter of sample was added in 1.0 ml of dehydrated alcohol and 2.0 ml of 20 % stannous chloride and then analyzed by F732 mercury analyzer immediately. Values were obtained from a standard curve prepared with HgCl2. Determination of Glu Metabolism in the Cerebral Cortex Glu and Gln contents were determined with analysis kits and operated according to the methods recommended by the manufacturers. In brief, the cerebral cortex was made into 10 %
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homogenate with 0.9 % NaCl. For Glu measurement, 0.2 ml of homogenate was mingled with 0.6 ml reagent added into each tube for centrifugation (1,000×g for 10 min at 4 °C). Then, 0.5 ml of supernatant was mixed with other reagents and incubated at 37 °C for 40 min. For Gln determination, homogenate was mixed with other reagents and was incubated at 37 °C for 15 min. Absorbance was recorded at 340 and 630 nm for Glu and Gln, respectively. GS Activity was determined based on the reaction of glutamine and hydroxylamine, forming γglutamylhydroxamate and NH3 as described by Allen et al. [31]. Five percent homogenate was added into the reaction mixture containing 110 mM glutamine, 0.45 mM ATP, 9 mM Na2HAsO4, 110 mM hydroxylamine, 1.8 mM MnCl2, and 0.1 mM dithiothreitol in 50 mM imidazole buffer, pH 7.2, and enzymatic extract with 100 μg of protein, all in a final volume of 1.5 ml. The reaction mixture was incubated at 37 °C for 30 min and stopped by 0.75 ml 0.6 M Fe(NO3)3 in 40 % trichloroacetic acid. The tubes were centrifuged at 800×g for 5 min in order to remove the protein precipitate. The absorbance was determined in the supernatant at 535 nm, and the values were obtained from a standard curve prepared with L-γ-glutamylhydroxamate. PAG activity was measured according to the method of Curi [37]. The cerebral cortex was homogenized in an extraction medium containing 150 mM potassium phosphate, 1 mM ethylenediaminetetraacetic acid (EDTA), and 50 mM Tris– HCl at pH 8.6. The assay medium consisted of 50 mM phosphate buffer, 0.2 mM EDTA, 50 mM Tris–HCl, 20 mM Glu, and 0.05 % Triton X-100, to which 100 μl of homogenate was added. The total volume was 1.0 ml at pH 8.6. Assay media, in duplicate, were incubated at 37 °C. The reaction was initiated by addition of freshly prepared glutamine, promoting a 10min linear reaction time course, and stopped by the addition of 0.2 ml of 25 % perchloric acid, and then neutralized. The amount of Glu was determined, and the absorbance was recorded at 340 nm.
Determination of Nonenzymatic and Enzymatic Antioxidants in the Cerebral Cortex Nonprotein sulfhydryl (NPSH) content was measured in accordance with the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) colorimetric method [38]. In brief, the cerebral cortex was homogenized to the concentration of 10 % in phosphatebuffered saline (PBS). The PBS was mixed with 0.1 M Na2HPO4 and 0.1 M NaH2PO4, and the final pH was 8.0. Following centrifugation (1,000×g for 10 min at 4 °C), a mixture of 0.1 ml of the supernatant and 0.9 ml of 5 % trichloroacetic acid (TCA) was mingled and centrifuged (2,300×g for 15 min at 4 °C). Then, 0.5 ml of the supernatant was added into 1.5 ml of 0.01 % DTNB, and the reaction was
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monitored at 412 nm after standing at room temperature for 5 min. SOD and GSH-Px activities were determined with analysis kits according to the manufacturer’s instructions. SOD estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltet-razolium chloride to form a red formazan dye. The SOD activity was measured by the degree of inhibition of this reaction. GSH-Px determination was based on the principle that GSH-Px catalyzes the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione reductase and reduced nicotinamide adenine dinucleotide phosphate, the oxidized glutathione is immediately converted to the reduced form with a contaminant oxidation of NADPH to NADP+. Absorbance differences were recorded at 560 and 340 nm for SOD and GSH-Px, respectively. Measurement of Oxidative Damage in the Cerebral Cortex MDA was measured as the production of lipid peroxide, which combines with thiobarbituric acid (TBA) to form a pink chromogen compound [39]. In brief, 10 % homogenate was mingled with the reaction mixture consisting of 0.1 M ice-cold PBS and 0.1 M FeCl2 in a total volume of 1.0 ml (pH 7.4). The reaction was stopped by 1.0 ml of 10 % TCA followed by 1.0 ml of 0.67 % TBA addition. After centrifugation at 2,500×g for 10 min, the absorbance was recorded at 530 nm using tetramethoxypropane as standard. Protein sulfhydryl and carbonyl contents were measured as the indicators of protein oxidative damage. Protein sulfhydryl was detected by the DTNB method according to the manufacturer’s instructions. The colorimetric reaction was measured at 412 nm. Protein sulfhydryl contents were calculated from total sulfhydryl by subtracting nonprotein sulfhydryl. Protein carbonyl contents were detected by the 2,4dinitrophenylhy-drazine (DNPH) method according to the manufacturer. In brief, proteins were precipitated with an equal volume of 20 % TCA and washed three times with 2 ml of a 1:1 ethanol/ethyl acetate mixture. Finally, the precipitates were dissolved in 6-M guanidine HCl solution, and the absorbance was measured at 370 nm. 8-OHdG was measured as the indicator of DNA oxidative damage, and the immunostaining was performed by SABC method recommended by the manufacturer. Animals were deeply anesthetized followed by transcardial perfusion with the aid of a peristaltic pump. Initial perfusion consisted of 50 ml of saline and heparin (1 %) at room temperature, followed by 250 ml of 4 % paraformaldehyde in 0.1 M PBS (pH 7.4). The cerebral cortex was removed and post-fixed in 4 % paraformaldehyde solution for 12 h at 4 °C. Then, tissues were transferred in PBS with 30 % sucrose for 48 h and were frozen and sectioned in a cryostat (Leica, Heidelberg,
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Germany) at 12-μm thickness after dehydrated in a graded series of alcohol. Sections were incubated in boiling 0.01 M citrate buffer (3×30 min), hydrogen peroxide (0.3 % in methanol, 10 min), and 5 % normal serum for 1 h, followed by 8OHdG mouse monoclonal antibody (1:500) overnight at 4 °C. The primary antibody was detected using a biotinylated secondary antibody goat anti-mouse IgG (1:100) for 2 h, followed by incubation with SABC (1:100) for 3 h at room temperature. After incubation in 0.05 % DAB for 15 min, dehydrated processes were performed through alcohols and xylenes. The sections were mounted on gelatin–chromalum-coated slides for light microscope examination. To evaluate the morphologic characteristics and the relative cell immunoreactivity for 8OHdG in the cerebral cortex, according to the process of Morello [40], we used a light microscope equipped with a ×40 objective. Analysis of 8-OHdG-immunoreactive positive cells was performed in three immunostained sections per immunoreactive marker per animal (n=4). The intensity of the immunoreactive signal was calculated from the integrated optical density (IOD) of the immunoreactive positive cells using a microcomputer-based image display system (Image ProPlus, Media Cybernetics 5.0). Preparation of Dissociated Cerebral Cortex Cells The cerebral cortex (100 mg) was dissected and washed by phosphate-buffered saline (PBS, pH 7.2–7.4) for three to five times. The tissues were minced in 10 ml PBS and supplemented with 0.125 % trypsin for 10–15 min at 37 °C with vigorous shaking. The subsequent mechanical dissociation with Pasteur pipettes and nylon mesh screens was carried out. Cells were finally suspended in DMEM supplemented with 10 % BSA. The concentration of cells was evaluated by viable cell count (trypan blue stained). It was diluted to 1×106 cells/ml for intracellular free calcium level, ROS formation, and apoptosis level detection. Determination of Intracellular Free Calcium Levels Using the Ca2+ Indicator Fura-2 in the Cerebral Cortex The intracellular free calcium ([Ca2+]i) assay was performed by a method described previously [41]. Briefly, for fura-2 experiments, absolute values of [Ca2+]i in neurocyte were calibrated from the measurement of fluorescence signals by the use of F-4500 fluorescence spectrophotometer (HICATHI, Japan). The calibration equation used was [Ca2+]i =Kd[(R− Rmin) / (Rmax −R)]×(Sf380 / Sb380), where Kd is the dissociation constant of the dye, R is the ratio at excitation wavelengths 340/380 nm, Rmin is the ratio at zero [Ca2+]i, and Rmax is the ratio at saturated [Ca2+]i. Procedures for obtaining Rmax and Rmin caused damage to cells and were, therefore, carried out at the end of the experiments. Rmax was obtained first by adding 0.2 % Triton X-100, making the cell membrane permeable to
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Ca2+, and allowing the extracellular and intracellular Ca2+ to equilibrate. Following this, Rmin was obtained by adding the chelator EGTA to chelate all Ca2+ inside and outside the cells. The present experiments were carried out at pH 7.4 and at a temperature of 37 °C. A Kd value of 224 nM was used. Measurement of Intracellular ROS Level Using Flow Cytometry in the Cerebral Cortex The intracellular ROS level was monitored by using the peroxide-sensitive fluorescence probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (Molecular Probes) as described previously [42]. In brief, cells (1×106 cells/ml) were incubated with 10 μM DCFH-DA at 37 °C for 30 min. The fluorescent dye DCFH-DA passes through the cell membrane and undergoes deacetylation by intracellular esterases to produce the nonfluorescent compound DCFH that is trapped inside the cells. Oxidation of DCFH by ROS produces the highly fluorescent DCF, which was measured and monitored at 488 nm (excitation)/525 nm (emission) by a FACScan flow cytometer (Becton–Dickinson, Germany). Apoptosis Assays Using Flow Cytometry in the Cerebral Cortex The apoptosis levels were measured by flow cytometry according to annexin V-FITC/PI double-stained method. Annexin V is a Ca2+-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine; annexin VFITC is a sensitive probe for identifying cells that are undergoing apoptosis, while propidium iodide (PI) is a nonspecific DNA dye that is excluded from living cells with intact plasma membranes but incorporated into the nonviable cell. The cerebral cortex cells (1×106 cells/ml) were mingled with 200 μl ice-cold binding buffer followed by 10 μl horseradish peroxidase FITC-labeled annexin V and 5 μl PI adding. After a 15-min incubation in darkness, the apoptosis rate was determined immediately. In the present study, annexin V+/PI− populations were considered as the early apoptotic cells. Real-Time PCR Analysis Total RNA was extracted using the RNAiso Plus according to the manufacturer and finally dissolved in RNase-free H2O. OD260/OD280 of total RNA was between 1.6 and 1.8. Semiquantitative reverse transcription was performed on Applied Biosystems 2720 Thermal Cycler (Applied Biosystems, Singapore) with PrimeScript RT reagent Kit and gDNA Eraser, using β-actin as an endogenous control. The gene-specific primers were designed by TaKaRa Company. NR1 forward primer 5′-GCCTACAAGCGACACAAGGATG-3′, reverse primer 5′-TTAGGGTCGGGCTCTGCTCTAC-3′; NR2A forward primer 5′-ACCTCGCTCTGCTCCAGTTTG-3′, reverse
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primer 5′-GTTGTGGCAGAGCCCGTAA-3′; NR2B forward primer 5′’-CGCCTAGAGGTTTGGCGTCTAC-3′, reverse primer 5′-GAACGAGCTTTGCTGCCTGA-3′; GLAST forward primer 5′-TCGCTGCACTGGATTCCAAC-3′, reverse primer 5′-ACCAGGCTTGATGCTCACAACTAAC-3′; GLT1 forward primer 5′-GTTCAAGGACGGGATGAATGTC TTA-3′, reverse primer 5′-CATCAGCTTGGCCTGCTCAC3′; β-actin forward primer 5′-GGAGATTACTGCCCTGGC TCCTA-3′, reverse primer 5′-GACTCATCGTACTCCTGC TGCTG-3′. For the quantitative PCR reaction, the reaction mixture contained 25-μl buffer (2×), 2-μl PCR forward primer (10 μM), 2-μl PCR reverse primer (10 μM), 1-μl ROX reference dye II (50×), 4-μl template DNA, and 16 μl dH2O up to a final volume of 50 μl. The initial denaturation was carried out at 95 °C for 30 s, followed by amplification in 40 cycles, 95 °C for 3 s, and 60 °C for 34 s, using the 7500 real-time PCR system. The relative expression analysis was assessed by the method of 2−△△CT. Western Blot Analysis Protein extraction and immunoblot analysis were conducted as described by Guerguerian et al. [43]. Total protein was extracted from the cerebral cortex using RIPA buffer (10 mM Na2HPO4, 150 mM NaCl, 1 % sodium deoxicolate, 1 % Nonidet P-40, and 0.1 % SDS) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.2 mM 1,10phenanthroline, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 mM benzamidine) at 4 °C for 30 min. Protein concentrations were quantified with the BCA reagent. Equal amounts of protein (30 μg per lane) were separated by 8 or 10 % SDSpolyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). PVDF membranes were subsequently blocked overnight at 4 °C in Tween 20 tris-buffered saline (TBST) containing 5 % nonfat powdered milk, followed by briefly rinsing in TBST and incubated with NR1 (1:1,000), NR2A (1:1,000), NR2B (1:1,000), GLAST (1:500), GLT-1 (1:300), and β-actin (1:500) primary antibody in TBST at 4 °C overnight. Specific protein expression was then detected by incubating the membranes with HRP-conjugated secondary antibody (1:5,000). Protein bands were visualized by using ECL Western blotting chemiluminescent detection reagents and autoradiography. The intensity of bands was evaluated semiquantitatively by densitometry using an imageanalyzing software (FluorChem v2.0) and normalized by the internal control (β-actin). The changes of intensity of NR1, NR2A, NR2B, GLAST, and GLT-1 proteins after MeHg treatment were normalized using the intensity obtained in the internal control bands (β-actin).
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Statistical Analysis All statistical analyses were performed using the SPSS software, version 11.5. Assays were performed three times in duplicate for each rat, and the mean of duplicate was used for statistical analysis. Data were analyzed using analysis of variance (ANOVA) after confirmation of normal distributions, and group means were compared by Fischer’s least significant difference (LSD) procedure. Data were expressed as mean ± standard deviation. The difference at either P
