Free Radical Research, 2014; Early Online: 1–15 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2014.916039

ORIGINAL ARTICLE

The protective role of tea polyphenols against methylmercury-induced neurotoxic effects in rat cerebral cortex via inhibition of oxidative stress W. Liu, Z. Xu, T. Yang, Y. Deng, B. Xu, S. Feng & Y. Li

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Department of Environmental Health, School of Public Health, China Medical University, Shenyang, Liaoning Province, P. R. China Abstract Methylmercury (MeHg) is a ubiquitous environmental contaminant that could induce oxidative stress and an indirect glutamate (Glu)mediated excitotoxicity. However, the underlying mechanisms through which MeHg affects the central nervous system have not been fully elucidated, and little has been known of the interaction between oxidative stress and Glu dyshomeostasis in MeHg neurotoxicity. Therefore, rats were administrated with different MeHg concentrations (0, 4, and 12 μmol/kg) to evaluate the neurotoxic effects in cerebral cortex. Moreover, we have investigated the neuroprotective role of tea polyphenols (TP), a natural antioxidant that has a formidable free radical scavenge ability, against MeHg-induced neurotoxicity. Eighty rats were randomly divided into five groups: control, TP control, MeHg-treated (4 and 12 μmol/kg), and TP pretreated (1 mmol/kg). Administration of MeHg at 12 μmol/kg for 4 weeks significantly increased total Hg and ROS levels in cerebral cortex. In addition, MeHg reduced non-enzymatic (non-protein sulfhydryl) and enzymatic (SOD and GSH-Px) antioxidants, up-regulated Nrf2, HO-1, and γ-GCS expression. Moreover, MeHg-induced ROS over-production appeared to inhibit the activities of GS, down-regulated GLAST and GLT-1 expression in cerebral cortex. Pretreatment with TP at a dose of 1 mmol/kg significantly prevented MeHg-induced oxidative stress and Glu uptake/metabolism disorders in cerebral cortex. In conclusion, the results suggested that oxidative stress resulting from excessive ROS formation plays a critical role in MeHg neurotoxicity. TP possesses the ability to attenuate MeHg-induced neurotoxic effects through its antioxidative properties. Keywords: methylmercury, oxidative stress, reactive oxygen species, glutamate uptake/metabolism, tea polyphenols

Introduction Methylmercury (MeHg), one of the most common environmental pollutants, has been shown to readily cross the blood–brain barriers and cause serious central nervous system damage in both animals and human beings. Chronic exposure to MeHg via consumption of fish and shellfish is still a major concern for human health. The explicit molecular mechanisms underlying MeHg neurotoxicity continue to be fully elucidated. There was no single mechanism that could explain the multitude of toxic effects observed in MeHg poisoning among the current literatures. The mechanisms involved in are mainly related to intracellular calcium homeostasis impairment [1,2]; glutamate (Glu) homeostasis disturbance and oxidative stress [3–9]. Though free radicals are known to play a physiological role in optimal cell function, oxidative stress resulting from excessive free radicals formation has been implicated in a variety of neurodegenerative diseases [6]. Oxidative stress is accelerated by a combination of reactive oxygen species (ROS) over-production and antioxidant capacity (mainly non-enzymatic antioxidants and antioxidant enzymes) impairment, which is associated with energy metabolism disruption, lipid peroxidation,

and mitochondrial membrane potential dissipation, implicated in multiple of MeHg neurotoxicity studies [6,10–13]. Mitochondrial dysfunctions may lead to a mitochondrial burst of ROS production that plays a critical role in mediating cell structures damage, including lipids, membranes, proteins, as well as nucleic acids peroxidation [6,14]. Nuclear factor-E2-related factor 2 (Nrf2) is a basic leucine zipper transcription factor involved in the induction of expression of genes encoding many cyto-protective proteins. Nrf2 is bound to Kelchlike ECH-associating protein 1 (Keap1) in the cytoplasm under physiological conditions. The interaction between Nrf2 and Keap1 is disrupted upon oxidative stress, resulting in the dissociation of Nrf2 from Keap1. Once in the nucleus, Nrf2 interacts with an antioxidant response element to initiate the transcription of target genes, and their encoded proteins serve to detoxify xenobiotics and endogenous reactive electrophiles [15,16]. Although the Nrf2 signaling pathway has been proved the existence of novel biological defense mechanisms contributing to antagonize MeHg-induced neurotoxicity [12,17,18], the protective mechanisms of Nrf2 pathway have not been yet clarified under in vivo conditions. On the other hand, MeHg-induced ROS over-production appears to directly inhibit astrocytic Glu uptake and metabolism, leading to

Correspondence: Zhaofa Xu, Department of Environmental Health, School of Public Health, China Medical University, Shenyang 110001, Liaoning Province, P. R. China. Tel: ⫹ 86-24-23256666-5395. Fax: ⫹ 86-24-23269025. E-mail address: [email protected] (Received date: 5 February 2014; Accepted date: 14 April 2014; Published online: 12 May 2014)

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2 W. Liu et al. increased Glu concentrations in the extracellular fluid, which might be the trigger of Glu-mediated neurotoxicity, referring to the consequence of increased calcium influx that could be detrimental to neuron survival. These suggested oxidative stress and Glu uptake/metabolism disorders appear to be a connected phenomenon playing in MeHg neurotoxicity [3,19,20]. However, as little data exist on MeHg poisoning under in vivo conditions, the possible relationships between oxidative stress and Glu dyshomeostasis have not been clearly understood, information that is critical for full evaluation of MeHg-induced neurotoxicity. Tea polyphenols (TP), extracted from fresh leaves of tea plants, which have possible health benefits and received a lot of attention in recent years. TP has a diverse of compounds with multiple active catechin components that have been reported to possess a variety of potent properties including anti-oxidation, anti-inflammation, and radical scavenging [21–25]. Furthermore, the properties of TP have been reported in preventing oxidative stress-related diseases such as cancer, cardiovascular, and neurodegenerative diseases [26–28]. They have been shown to prevent the metal-catalyzed formation of radical species, mainly superoxide anion and hydrogen peroxide, inhibit lipid peroxidation and modulate the activities of regulatory enzymes [21,22]. Previous studies indicated the green tea extracts especially epigallocatechin-3-gallate has a dosedependent radical species scavenge ability, and could activate Nrf2 target genes and their encoded proteins expression [29,30]. However, the exact molecular mechanisms of Nrf2 pathway activation have not been clearly elucidated. As there were only a few in vivo studies focused on the protective effects of TP on MeHg neurotoxicity, the possible protective mechanisms need to be deeply explored. We therefore postulated in the present study that oxidative stress and Glu homeostasis disruption may play important roles in MeHg-induced neurotoxicity. To test this hypothesis, we developed a rat model of MeHg subchronic poisoning to evaluate its neurotoxic effects in cerebral cortex focusing on oxidative stress and Glu uptake/metabolism disorders. In addition, we tested TP to verify whether it could antagonize MeHg-induced neurotoxicity via its anti-oxidation abilities, which may be helpful to fully evaluate the MeHg neurotoxicity mechanisms. Moreover, we designed a TP control group in order to confirm that TP is nontoxic in the dose (1 mmol/kg) in our present study. Materials and methods Chemicals Methylmercury chloride with a purity of 97.0% was provided by Laboratory of Dr. Ehrenstorfer-Schafers (Augsburg, Germany). TP with a purity of 95.0% was obtained from Lanwei Bioengineering Company (Changsha, China). Analysis kits of superoxide dismutase (SOD) and

glutathione peroxidase (GSH-Px) were obtained from Jiancheng Bioengineering Institute (Nanjing, China). RNAiso Plus, PrimeScript® RT reagent Kit with gDNA Eraser, and SYBR® Premix Ex TaqTM analysis kits were provided by TaKaRa Biotechnology Company (Dalian, China). Rabbit polyclonal antibodies developed against a peptide mapping at the C-terminus of Nrf2, heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCS), glutathione peroxidase-1 (Gpx-1), glutamine synthetase (GS), glutamate-aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1) against rat proteins were purchased from Santa Cruz Biotechnology Company (Santa Cruz, CA, USA). Mouse monoclonal antibody developed against β-actin was purchased from Santa Cruz. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies were obtained from Santa Cruz. The 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) and Folin phenol reagent were obtained from Sigma Chemical Corporation (Saint Louis, MO, USA). Other chemicals of analytical grade were provided by local chemical suppliers, and agents were prepared as stock solutions with sterile water. Animal grouping and treatment Adult Wistar rats (6 weeks) with an initial body weight ranged from 160 to 180 g were provided by Laboratory Animal Center of China Medical University (SPF grade, N ⫽ 80, equal numbers of male and female). Environmental controls for the animal room were maintained as a 12-h light: 12-h dark cycle, a controlled temperature of 21–24°C and a relative humidity of 30–40%. Diet and fresh water were provided ad libitum. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of China Medical University (Permit Number: SCXK2008-0005). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Rats were randomly divided into five groups by weight, with 16 animals (8 male and 8 female) in each group, including the control group, TP control group, 4 and 12 μmol/kg MeHg-treated groups, and TP pretreatment group. Rats in control and MeHg-treated groups were intragastrically (i.g.) administrated with 0.9% NaCl; rats in TP control and TP pretreatment groups were i.g. administrated with 1 mmol/kg TP in sterile deionized water. Two hours later, rats in the control and TP control groups were intraperitoneally (i.p.) injected with 0.9% NaCl; while rats in the 4 and 12 μmol/kg MeHgtreated groups were i.p. injected with 4 and 12 μmol/kg MeHg-chloride in sterile deionized water, respectively; rats in the TP pretreatment group were also i.p. injected with 12 μmol/kg MeHg-chloride to examine the effects of TP on MeHg neurotoxicity. 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.

Tea polyphenols against MeHg oxidative stress

This dose was selected based upon pilot studies indicating that 12 μmol/kg was the sufficient dose to induce measurable neurologic deficits in exposed rat during the course of 4-week subchronic MeHg exposure, lower doses required longer than 4 weeks to produce measurable deficits (data not shown). Administration was given every day at a volume of 5 ml/kg body weight, for up to 4 weeks.

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Sample collections At 24 h after the last administration, for biochemical analysis, 14 rats/group (7 male and 7 female) were sacrificed via decapitation after anesthetized. The head was immersed in running cold physiological saline for 30 seconds to clear blood contamination. Firstly, the brain capsules of 6 rats/ group (3 male and 3 female) were removed and the cerebral cortices were isolated on ice bath. Hundred milligrams of the cortex was digested by nitric acid for 12 h in order to determine total mercury levels, the cortex remained was prepared for 5% or 10% homogenate for determination of non-protein sulfhydryl (NPSH) contents as well as SOD and GSH-Px activities. Secondly, 4 rats/group (2 male and 2 female), the cerebral cortices were prepared for single-cell suspension in order to investigate the ROS levels. Thirdly, four rats/group (2 male and 2 female) were extracted the total RNA and protein in cerebral cortex for determination of Nrf2, HO-1, γ-GCS, Gpx-1, GS, GLAST, and GLT-1 mRNA and protein expressions. Two rats remained in each group (1 male and 1 female) were given left ventricular perfusion with physiological saline followed by buffered 4% paraformaldehyde, the cerebral cortices were rapidly separated on ice bath and then fixed in 4% paraformaldehyde followed by 2.5% glutaraldehyde for the ultra-structural changes observation in cerebral cortex. Determination of total mercury levels in cerebral cortex The mercury concentrations were detected with cold vapor atomic fluorescence spectrometry according to the method described previously [31]. Cerebral cortex tissue (100 mg) was digested with 2.0 ml of nitric acid for 12 h. Then 2.0 ml of 50% sulfuric acid and 3.0 ml of saturated KMnO4 were added in, then 90°C for an hour. After cooling, the oxidant excess was reduced by the addition of 50% hydroxylamine. This solution was raised up to 10 ml, homogenized and 1.0 ml taken to the instrument, to which 1.0 ml of dehydrated alcohol and 2.0 ml of 20% stannous chloride were added. Mercury concentrations were immediately analyzed using F732 Mercury Analyzer, via a standard calibration curve prepared by HgCl2. The levels of total mercury in cerebral cortex were expressed as μg/g wet cerebral cortex tissue. Preparation of dissociated cerebral cortex cells The brains were rapidly removed and the cerebral cortex (100 mg) was dissected for the preparation of dissociated cerebral cortex cells as described by the modified method

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of Villalba et al. [32]. The tissues were washed using phosphate-buffered saline (PBS, pH 7.2–7.4) for 3–5 times, and then 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 as described. Cells were finally suspended in DMEM supplemented with 10% bovine serum albumin until used. The concentration of cells was evaluated by viable cell count (Trypan blue stained). It was diluted to 1 ⫻ 106 cells/ ml for ROS formation detection. Quantification of ROS formation in cerebral cortex ROS formation was monitored using the oxidation-sensitive fluorescent dye DCFH-DA, which is a non-fluorescent compound that is freely taken up into cells, as described previously [33]. Briefly, cells (1 ⫻ 106) were incubated with 10 μM DCFH-DA at 37°C for 30 min. The fluorescence increase, which is due to the hydrolysis of DCFH-DA to DCF by nonspecific cellular esterase and its subsequent oxidation by peroxides, was measured and monitored at 488 nm (excitation)/525 nm (emission) using a FACScan flow cytometer (Becton-Dickinson, Germany). Measurement of NPSH contents in cerebral cortex NPSH contents were measured in accordance with the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) colorimetric method [34]. Briefly, the cerebral cortex was homogenized to the concentration of 10% in PBS. Following centrifugation (1000 ⫻ g for 10 min at 4°C), a mixture of 0.1 ml of the supernatant and 0.9 ml of 5% trichloroacetic acid was mingled and centrifuged (2300 ⫻ g for 15 min at 4°C). Then 0.5 ml of the supernatant was added into 1.5 ml of 0.01% DTNB. As DTNB reacts with NPSH to form a yellow product, the absorbance was measured at 412 nm. Determination of enzymatic antioxidants in cerebral cortex SOD and GSH-Px activities in cerebral cortex were measured with analysis kits according to the manufacturer instructions. SOD estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which reacts with 2-(4-iodophenyl)3-(4-nitrophenol)-5-phenyltetrazolium 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 could immediately convert to the reduced form with a contaminant oxidation of NADPH to NADP⫹. Absorbance differences were recorded at 560 nm for SOD and 340 nm for GSH-Px, respectively.

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4 W. Liu et al. Total RNA isolation and quantitative real-time RT-PCR

Western blotting

For total RNA isolation, tissues were homogenized in RNAiso Plus. After a 5-min room temperature incubation (20°C), chloroform was added for phase separation. The upper aqueous phase was collected and the RNA was precipitated by mixing with isopropyl alcohol. The RNA pellet was washed once with 75% ethanol and was airdried. It was finally redissolved in RNase-free water. The absorbance of the RNA solution was determined using NanoPhotometer (IMPIEN, Germany) at 260 and 280 nm, respectively. OD260/OD280 ratio was between 1.6 and 1.8. The first-strand cDNA was synthesized from 1 μg of total RNA by Reverse Transcriptase performed on Applied Biosystems 2720 Thermal Cycler (Applied Biosystems, Singapore) with PrimeScript® RT reagent Kit and oligo (dT) primers (TaKaRa) according to the manufacturer’s protocol. Real-time quantitative PCR (RTPCRq) was performed in SYBR® Premix Ex TaqTM II kit using ABI 7500 Real-Time PCR System (Applied Biosystems, USA). The reaction mixture containing 25 μl PCR 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. Real-time PCR cycle parameters included 30 s at 95°C followed by 40 cycles involving denaturation at 95 for 5 s, annealing at 60°C for 34 s and elongation at 72°C for 20 s. The sequences of the specific sets of primer for Nrf2, HO-1, γ-GCS, Gpx-1, GS, GLAST, GLT-1, and β-actin used in this study are given in Table I. Expressions of selected genes were normalized to β-actin, which was used as an internal housekeeping control. For relative quantification of the tested genes, we used the comparative CT method (ΔΔCT). All the real-time PCR experiments were performed in triplicate and data were expressed as the mean of at least three independent experiments.

Protein extraction and immunoblot analysis were conducted as described by Guerguerian et al. [35]. Total protein was extracted from cerebral cortex using RIPA buffer (10 mM Na2HPO4, 150 mM NaCl, 1% sodium deoxicolate, 1% NP-40, 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. Protein concentrations were quantified with the BCA reagent. Equal amounts of protein (30 μg per lane) was separated by 8% or 10% SDS-polyacrylamide 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 rinsed in TBST and incubated with Nrf2 (1:500), HO-1 (1:500), γ-GCS (1:300), Gpx-1 (1:500), GS (1:500), 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 HRPconjugated secondary antibody (1:5000). Protein bands were visualized using ECL Western Blot chemiluminescent detection reagents and autoradiography. The intensity of bands was evaluated semi-quantitatively through densitometry using image analyzing software (FluorChem v2.0). The changes of intensity of Nrf2, HO-1, γ-GCS, Gpx-1, GS, GLAST, and GLT-1 proteins were normalized using the intensity obtained in the internal control bands (β-actin). Ultra-structural changes observation The cerebral cortex was dissected after the rats were perfused with saline followed by buffered 4% paraformaldehyde solution. After fixed in 4% paraformaldehyde

Table I. Primer sequences used for the amplification of each gene in this study. Name

Oligo

Primer sequence

Nrf2

Sense prime Anti-sense primer Sense prime Anti-sense primer Sense prime Anti-sense primer Sense prime Anti-sense primer Sense prime Anti-sense primer Sense prime Anti-sense primer Sense prime Anti-sense primer Sense prime Anti-sense primer

5′-GCCTACAAGCGACACAAGGATG-3′ 5′-TTAGGGTCGGGCTCTGCTCTAC-3′ 5′-ACCTCGCTCTGCTCCAGTTTG-3′ 5′-GTTGTGGCAGAGCCCGTAA-3′ 5′-CGCCTAGAGGTTTGGCGTCTAC-3′ 5′-GAACGAGCTTTGCTGCCTGA-3′ 5′- CGGGGCCTGGTCGTGCTCGGCTTC-3′ 5′-GACAGCAGGGTTTCAATGTCAGGC-3′ 5′-ATCTTGCATCGGGTATGCGA-3′ 5′-AGTAACCCTTCTTCTCCTGG-3′ 5′-TCGCTGCACTGGATTCCAAC-3′ 5′-ACCAGGCTTGATGCTCACAACTAAC-3′ 5′-GTTCAAGGACGGGATGAATGTCTTA-3′ 5′-CATCAGCTTGGCCTGCTCAC-3′ 5′-GGAGATTACTGCCCTGGCTCCTA-3′ 5′-GACTCATCGTACTCCTGCTGCTG-3′

HO-1 γ-GCS Gpx-1 GS GLAST GLT-1 β-actin

β-actin was used as a constitutively expressed gene and all the data were normalized to β-actin expression.

Tea polyphenols against MeHg oxidative stress

followed by a buffered 2.5% glutaraldehyde in 0.1 M PBS (pH 7.2–7.4), one-millimeter thickness cortex slice was made, followed by prolonged fixation overnight. For electron microscopy, post-fixation in 1% OsO4 containing 1.25% potassium ferrocyanide was carried out. Finally, blocks were stained with uranyl acetate and lead citrate, and photographed in a JEM-1200EX transmission electron microscope equipped with an ultra-scan digital camera.

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Protein determination All the substance contents and enzyme activities were normalized to the protein amount measured according to the method of Lowry et al. [36], using bovine serum albumin as standard.

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Effects of MeHg exposure and TP pretreatment on total mercury levels in cerebral cortex Total mercury levels in cerebral cortex were measured after 4 weeks of MeHg administration and TP pretreatment. As were shown in Table III, the results illustrated that there was a concentration-dependent and significant elevation in total mercury levels in the 4 μmol/kg (P ⬍ 0.01) and 12 μmol/kg MeHg-treated groups (P ⬍ 0.01) when compared with those of the control group. However, mercury levels with TP pretreatment were not significantly different from that in the 12 μmol/kg MeHg-treated group. In addition, there was no significant difference between the control and TP control group in total mercury levels. Effects of MeHg exposure and TP pretreatment on NPSH contents in cerebral cortex

Statistical analysis All the grouped data were evaluated using SPSS13.0 software. One-way analysis of variance followed by Student-Newman-Keuls test was used to determine the statistical significance between groups. Data were shown as means ⫾ standard deviation. The difference at either P ⬍ 0.05 or P ⬍ 0.01 was regarded as statistically significant. Results Body weight and behavioral activity After being administrated for 4 weeks, there was a statistically significant decrease in body weight of MeHgtreated rats compared to that of control rats (P ⬍ 0.05 or P ⬍ 0.01, Table II). During this time, MeHg exposure caused the following behavioral changes: the appetite of the rats dropped to some extent, some of the rats were sedated and ataxic, and some of the rats enhanced locomotion in 12 μmol/kg MeHg-treated and TP pretreatment groups. Signs of abnormal behavioral activity of each rat were different. However, rats in 4 μmol/kg MeHg-treated group and TP control group were not observed for any signs of abnormal behavioral activity at the end of the fourth week. There was no behavioral activity difference between female and male rats.

After MeHg administration and TP pretreatment for 4 weeks, NPSH contents in cerebral cortex were determined (Table III). The results indicated that NPSH contents obviously decreased in a dose-dependent manner with the increase of administered MeHg concentrations, with the maximum decrease observed in 12 μmol/kg MeHg-treated group (P ⬍ 0.01), relative to that of control. On the contrary, as compared with the 12 μmol/kg MeHgtreated group, NPSH contents significantly elevated with TP pretreatment (P ⬍ 0.05). Meanwhile, there was no significant difference between the control and TP control groups in cerebral cortex NPSH contents. Effects of MeHg exposure and TP pretreatment on SOD and GSH-Px activities in cerebral cortex SOD and GSH-Px activities in cerebral cortex were determined after 4 weeks of MeHg exposure and TP pretreatment (Table III). The results showed that compared with those of the control group, SOD and GSH-Px activities decreased significantly in the 4 μmol/kg (P ⬍ 0.01 and P ⬍ 0.05) and 12 μmol/kg MeHg-treated groups (P ⬍ 0.01 and P ⬍ 0.01), respectively. On the contrary, there was a significant elevation of SOD and GSH-Px activities (P ⬍ 0.05, P ⬍ 0.01) in TP pretreatment group when compared with those in 12 μmol/kg MeHg-treated group, respectively. Moreover, there was no significant

Table II. Effects of MeHg exposure and TP pretreatment on body weight of rats. Body weight (g) Treated groups Control TP control 4 μmol/kg MeHg 12 μmol/kg MeHg TP pre-treatment

initial

after a week

after 2 weeks

after 3 weeks

after 4 weeks

173.2 ⫾ 5.4 175.0 ⫾ 7.2 171.3 ⫾ 6.2 176.4 ⫾ 4.5 172.2 ⫾ 5.5

214.3 ⫾ 11.0 211.1 ⫾ 11.7 210.3 ⫾ 13.3 204.1 ⫾ 11.2 206.4 ⫾ 12.0

236.1 ⫾ 14.7 232.1 ⫾ 16.2 227.2 ⫾ 14.6 216.0 ⫾ 13.0 221.2 ⫾ 13.3

248.1 ⫾ 18.7 246.2 ⫾ 14.3 236.1 ⫾ 15.3 224.2 ⫾ 15.4 232.3 ⫾ 16.4

258.3 ⫾ 20.9 260.2 ⫾ 18.2 245.3 ⫾ 18.3∗ 227.4 ⫾ 15.4∗∗ 237.1 ⫾ 17.1

∗ P ⬍ 0.05, ∗∗ P ⬍ 0.01 compared with the control group.

6 W. Liu et al. Table III. Effects of MeHg exposure and TP pretreatment on total mercury levels, NPSH contents, SOD and GSH-Px activities in rat cerebral cortex. Treated groups

Total mercury (μg/g tissue)

NPSH (μmol/g pro)

SOD (U/mg pro)

GSH-Px (U/mg pro)

Control TP control 4 μmol/kg MeHg 12 μmol/kg MeHg TP pre-treatment

1.02 ⫾ 0.30 0.79 ⫾ 0.16 4.98 ⫾ 1.16∗∗ 16.56 ⫾ 3.18∗∗ 16.18 ⫾ 2.56

32.32 ⫾ 3.11 33.06 ⫾ 4.35 30.41 ⫾ 5.25 21.55 ⫾ 4.31∗∗ 27.23 ⫾ 5.84▲

79.57 ⫾ 8.60 78.42 ⫾ 11.12 64.23 ⫾ 4.97∗∗ 32.61 ⫾ 7.63∗∗ 45.04 ⫾ 9.40▲

20.17 ⫾ 3.67 19.91 ⫾ 4.90 13.12 ⫾ 3.90∗ 10.53 ⫾ 3.10∗∗ 18.83 ⫾ 6.76▲▲

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∗P ⬍ 0.05, ∗∗P ⬍ 0.01 compared with the control group; 12 μmol/kg MeHg group.

difference between the control and TP control group in SOD and GSH-Px activities. Effects of MeHg exposure and TP pretreatment on ROS formation in cerebral cortex ROS formation in cerebral cortex cells was measured after 4 weeks of MeHg administration and TP pretreatment (Figure 1). Based on the findings from our study, the DCF fluorescence intensity elevated dosedependently with the increase of treated MeHg dosages. Exposed to 4 and 12 μmol/kg MeHg resulted in ROS formation 1.75-fold (P ⬍ 0.01) and 3.96-fold (P ⬍ 0.01) of the control group, respectively. However, as compared with the 12 μmol/kg MeHg-treated group, TP pretreatment significantly decreased the DCF fluorescence intensity by 24.65% (P ⬍ 0.01). Moreover, there was no significant difference between the control and TP control groups in ROS formation. Effects of MeHg exposure and TP pre-treatment on Gpx-1 mRNA and protein expressions in cerebral cortex After MeHg administration and TP pre-treatment for 4 weeks, Real-time PCR was performed for Gpx-1 mRNA expressions quantification. Results of evaluation and comparison after normalized with housekeeping gene β-actin

▲P ⬍ 0.05, ▲▲P ⬍ 0.01

compared with the

have been shown in Figure 2A. The data illustrated that the relative intensity of Gpx-1 mRNA decreased dosedependently with the increase of treated MeHg dosages. Compared with the control group, relative intensity of Gpx-1 mRNA in the 12 μmol/kg MeHg-treated group significantly down-regulated by 34.00% (P ⬍ 0.01). In contrast, there was an obvious increase of Gpx-1 mRNA expression (28.79% [P ⬍ 0.01]) in TP pretreatment group when compared with that in 12 μmol/kg MeHg-treated group. In addition, no significant difference between the control and TP control group was observed in Gpx-1 mRNA expression. After 4 weeks of MeHg exposure and TP pretreatment, semi-quantitative analyses of the results of Western Blotting experiments after normalized with β-actin have been shown in Figure 2B. The intensity of bands as well as densitometry evaluation and comparison after normalized with β-actin indicated that MeHg treatment induced a significant and dose-dependent downregulation of Gpx-1 protein expression. Gpx-1 protein levels were reduced by 20.13% (P ⬍ 0.05) and 42.19% (P ⬍ 0.01) after 4 and 12 μmol/kg MeHg exposure, respectively, relative to the control group. On the contrary, there was a significant elevation of Gpx-1 protein expression by 37.84% (P ⬍ 0.01) in TP pretreatment group when compared with those in 12 μmol/kg MeHg-treated group. Moreover, there was no significant

Figure 1. Effects of 4-week MeHg exposure (0, 4 and 12 μmol/kg) and TP pretreatment (1 mmol/kg) on ROS formation in cerebral cortex cells. Results are expressed as a percentage of control. Data are mean ⫾ S.D., n ⫽ 4. ∗∗P ⬍ 0.01 compared with the control group; ▲▲P ⬍ 0.01 compared with the 12 μmol/kg MeHg-treated group.

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Tea polyphenols against MeHg oxidative stress

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Figure 2. The alterations of Gpx-1 mRNA and protein expressions in cerebral cortex are quantified after 4 weeks of MeHg exposure (0, 4, and 12 μmol/kg) and TP pretreatment (1 mmol/kg). Relative intensity of Gpx-1 mRNA is normalized by the house-keeping gene β-actin (A). We used the comparative CT method (2⫺ΔΔCT) for relative quantification. The Western blotting products of Gpx-1 and β-actin as well as semi-quantitative analyses of the expression of Gpx-1 after Western blotting experiments were shown in (B). The results are expressed as relative intensity compared to those of β-actin. Data are mean ⫾ S.D., n ⫽ 4. ∗ P ⬍ 0.05, ∗∗ P ⬍ 0.01 compared with the control group; ▲▲P ⬍ 0.01 compared with the 12 μmol/kg MeHg-treated group.

difference between the control and TP control group in Gpx-1 protein expression. Effects of MeHg exposure and TP pretreatment on Nrf2, HO-1, and γ-GCS mRNA and protein expressions in cerebral cortex After MeHg administration and TP pretreatment for 4 weeks, Nrf2, HO-1, and γ-GCS mRNA expressions were quantified. Results of evaluation and comparison after normalized with β-actin have been shown in Figure 3A. Statistical comparisons revealed that there was a dosedependent and significant upregulation in the relative intensities of Nrf2, HO-1, and γ-GCS mRNA expressions in 4 and 12 μmol/kg MeHg-treated groups when compared

with those in control. Especially in the 12 μmol/kg MeHgtreated group, relative intensities of Nrf2, HO-1, and γ-GCS mRNA were 1.37-fold (P ⬍ 0.01), 1.82-fold (P ⬍ 0.01), and 1.55-fold (P ⬍ 0.01) of the control group, respectively. However, as compared with the 12 μmol/kg MeHg-treated group, relative intensities of Nrf2, HO-1, and γ-GCS mRNA expressions decreased by 10.79% (P ⬍ 0.05), 13.74% (P ⬍ 0.01), and 9.03% (P ⬍ 0.05) in TP pretreatment group, respectively. Moreover, no statistically significant differences were observed between the control and TP control group in Nrf2, HO-1, and γ-GCS mRNA expressions. After 4 weeks of MeHg exposure and TP pretreatment, semi-quantitative analyses of Nrf2, HO-1, and γ-GCS of Western Blotting experiments after normalized with

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8 W. Liu et al.

Figure 3. The alterations of Nrf2, HO-1, γ-GCS mRNA and protein expressions in cerebral cortex are quantified after 4 weeks of MeHg exposure (0, 4, and 12 μmol/kg) and TP pretreatment (1 mmol/kg). Relative intensity of Nrf2, HO-1, and γ-GCS mRNA are normalized by the house-keeping gene β-actin (A). We used the comparative CT method (2⫺ΔΔCT) for relative quantification. The Western blotting products of Nrf2, HO-1, γ-GCS, and β-actin as well as semi-quantitative analyses of the expression of Nrf2, HO-1, and γ-GCS after Western blotting experiments are shown in (B). The results are expressed as relative intensity compared to β-actin. Data are mean ⫾ S.D., n ⫽ 4. ∗∗P ⬍ 0.01 compared with the control group; ▲P ⬍ 0.05, ▲▲P ⬍ 0.01 compared with the 12 μmol/kg MeHg-treated group.

β-actin have been shown in Figure 3B. The intensity of bands as well as densitometry evaluation and comparison after normalized with β-actin indicated that MeHg treatment induced a concentration-dependent and significant upregulation of Nrf2, HO-1, and γ-GCS protein expressions, with the maximum alterations (2.25-fold [P ⬍ 0.01], 3.40-fold [P ⬍ 0.01] and 3.10-fold [P ⬍ 0.01] of control, respectively] observed in the 12 μmol/kg MeHg-treated group when compared with those in the control. On the contrary, compared with the 12 μmol/kg MeHg-treated group, relative expressions of Nrf2, HO-1, and γ-GCS proteins decreased by 17.77% (P ⬍ 0.05), 13.92% (P ⬍ 0.01),

and 15.63% (P ⬍ 0.01) in the TP pretreatment group, respectively. Meanwhile, there was no statistically significant difference between the control and TP control group in Nrf2, HO-1, and γ-GCS protein expressions. Effects of MeHg exposure and TP pretreatment on GS mRNA and protein expressions in cerebral cortex After 4 weeks of MeHg administration and TP pretreatment, GS mRNA expression in cerebral cortex was quantified, and results of evaluation and comparison after normalized with β-actin have been shown in Figure 4A.

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Tea polyphenols against MeHg oxidative stress

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Figure 4. The alterations of GS mRNA and protein expressions in cerebral cortex are quantified after 4 weeks of MeHg exposure (0, 4 and 12 μmol/kg) and TP pretreatment (1 mmol/kg). Relative intensity of GS mRNA is normalized by the house-keeping gene β-actin (A). We used the comparative CT method (2⫺ΔΔCT) for relative quantification. The Western blotting products of GS and β-actin as well as semi-quantitative analyses of the expression of GS after Western blotting experiments are shown in (B). The results are expressed as relative intensity compared to β-actin. Data are mean ⫾ S.D., n ⫽ 4. ∗P ⬍ 0.05, ∗∗P ⬍ 0.01 compared with the control group; ▲P ⬍ 0.05, ▲▲P ⬍ 0.01 compared with the 12 μmol/kg MeHg-treated group.

Statistical comparisons revealed that MeHg administration resulted in a dose-dependent downregulation of GS mRNA expression. Compared with that of the control group, the relative intensity of GS mRNA decreased by 13.73% (P ⬍ 0.05) and 29.41% (P ⬍ 0.01) in the 4 and 12 μmol/kg MeHg-treated groups, respectively. However, GS mRNA expression was significantly elevated in TP pretreatment group when compared with the 12 μmol/kg MeHg-treated group, a 20.83% (P ⬍ 0.01) increase has been observed in the present study. In addition, there was no significant difference between the control and TP control group in GS mRNA expression. GS protein expression in cerebral cortex was determined after 4 weeks of MeHg exposure and TP pretreatment

(Figure 4B). Based on the findings from the study, intensity of bands as well as densitometry evaluation and comparison after normalized with β-actin indicated that MeHg treatment induced a concentration-dependent downregulation of GS protein expression, 16.87% (P ⬍ 0.05) and 30.12% (P ⬍ 0.01) decrease in the 4 and 12 μmol/kg MeHg-treated groups when compared with that of the control group, respectively. On the contrary, there was a significant elevation of GS protein expression by 27.59% (P ⬍ 0.01) in TP pretreatment group, relative to that in 12 μmol/kg MeHg-treated group. Meanwhile, there was no statistically significant difference between the control and TP control group in GS protein expression.

10 W. Liu et al.

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Effects of MeHg exposure and TP pretreatment on GLAST and GLT-1 mRNA and protein expressions in cerebral cortex After MeHg administration and TP pretreatment for 4 weeks, GLAST and GLT-1 mRNA expressions in cerebral cortex were quantified. Results of evaluation and comparison after normalized with β-actin have been shown in Figure 5A. According to our present study, statistical comparisons revealed that there was a dosedependent downregulation in the relative intensities of GLAST and GLT-1 mRNA after different MeHg concentrations treatment. As compared with the control group, relative intensity of GLAST mRNA decreased significantly by 17.00% (P ⬍ 0.05) and 39.00% (P ⬍ 0.01) in the

4 and 12 μmol/kg MeHg-treated rats, respectively, while the intensity of GLT-1 mRNA expression decreased by 29.70% (P ⬍ 0.01) in the 12 μmol/kg MeHg-treated group. On the contrary, GLAST and GLT-1 mRNA expressions significantly elevated by 39.34% (P ⬍ 0.01) and 25.35% (P ⬍ 0.05) with TP pre-treatment when compared with the 12 μmol/kg MeHg-treated group. Moreover, there were no significant differences in GLAST and GLT-1 mRNA expressions between the control and TP control groups. GLAST and GLT-1 protein expressions in cerebral cortex were quantified after 4 weeks of MeHg administration and TP pretreatment (Figure 5B). The intensity of bands obtained from semi-quantitative analysis as well as densitometry evaluation and comparison after normalized

Figure 5. The alterations of GLAST and GLT-1 mRNA and protein expressions in cerebral cortex are quantified after 4 weeks of MeHg exposure (0, 4, and 12 μmol/kg) and TP pretreatment (1 mmol/kg). Relative intensity of GLAST and GLT-1 mRNA are normalized by the house-keeping gene β-actin (A). We used the comparative CT method (2⫺ΔΔCT) for relative quantification. The Western blotting products of GLAST, GLT-1 and β-actin as well as semi-quantitative analyses of the expression of GLAST and GLT-1 after Western blotting experiments are shown in (B). The results are expressed as relative intensity compared to β-actin. Data are mean ⫾ S.D., n ⫽ 4. ∗P ⬍ 0.05, ∗∗P ⬍ 0.01 compared with the control group; ▲P ⬍ 0.05, ▲▲P ⬍ 0.01 compared with the 12 μmol/kg MeHg-treated group.

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Tea polyphenols against MeHg oxidative stress

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with β-actin indicated that MeHg treatment induced a concentration-dependent downregulation of GLAST and GLT-1 protein levels with different extent. As compared with the control group, GLAST and GLT-1 protein expressions decreased by 40.23% (P ⬍ 0.01) and 24.00% (P ⬍ 0.05) in the 12 μmol/kg MeHg-treated group, respectively. On the contrary, compared with the 12 μmol/kg MeHg-treated group, GLAST protein expression significantly elevated by 30.77% (P ⬍ 0.01) with TP pretreatment. Meanwhile, there were no significant differences between the control and TP control group in GLAST and GLT-1 protein expressions.

mitochondrial cristae were partly lost (Figure 6C). And in the 12 μmol/kg MeHg-treated group, there were more serious damages appeared. Large amount of organelles were lost, the nuclear membrane was not intact with the heterochromatin condensed, but the mitochondrial swelling and vacuolar degeneration were observed (Figure 6D). In contrast, TP pretreatment significantly antagonized the ultra-structural damages induced by MeHg, as the nuclear membrane was clear, with more organelles and mitochondrial cristae appeared (Figure 6E).

Effects of MeHg exposure and TP pretreatment on ultrastructural changes in cerebral cortex

Discussion

As shown in Figure 6, the ultra-structural changes in neurons had been analyzed. Based on the finding in the present study, in the control and TP control groups, the nucleus was round-shaped with complete nuclear membranes, the chromatin was uniform, and the organelles were abundant including mitochondria, rough endoplasmic reticulum, lysosomal and so on (Figure 6A and B). In the 4 μmol/kg MeHg-treated group, the nuclear membrane was intact and the chromatin was abundant, but the cells presented some kind of early damages and the

Our present study had been attempted to evaluate the in vivo neurotoxic effects in rat cerebral cortex with MeHg administration. Although it was confirmed in several studies that MeHg may cause oxidative stress and an indirect Glu dyshomeostasis by altered Glu uptake/ metabolism [3,37,38], the molecular mechanisms have not been yet fully elucidated under in vivo conditions. In addition, we have investigated the effects of TP pretreatment to explore the neuroprotective roles against MeHg poisoning, which might be helpful to clarify the mechanisms of MeHg-induced neurotoxicity.

Figure 6. Electron microphotographs showed the ultra-structural changes in cerebral cortex after 4 weeks of MeHg exposure (0, 4, and 12 μmol/kg) and TP pretreatment (1 mmol/kg). The control group (A, ⫻ 4000), the TP group group (B, ⫻ 5000), the 4 μmol/kg MeHg-treated group (C, ⫻ 4000, arrow pointed to mitochondria with less cristae), the 12 μmol/kg MeHg-treated group (D, ⫻ 6000, arrows pointed to chromatin condensation and margination, not complete nuclear membrane, and vacuolated mitochondria), and the TP pretreated group (E, ⫻ 4000, arrow pointed to injured mitochondria) are shown, respectively. Observation of the changes in nucleus (n) and mitochondria (m) are focused.

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12 W. Liu et al. In our rat model of MeHg poisoning, compared with the control group, there was a statistically significant decrease in body weight and increase in total mercury levels of MeHg-treated rats. In addition, we observed the ultra-structural changes of neurons in cerebral cortex. The findings demonstrated the different doses of MeHg administration showed different degrees of neuron injuries, especially in the 12 μmol/kg MeHg-treated group, appreciable cellular ultra-structural damages appeared. The results also manifested that TP pretreatment could partially antagonize MeHg-induced neuron damages. However, TP did not attenuate total mercury levels in cerebral cortex. The mechanisms of MeHg-induced neurotoxicity might be related to oxidative stress induced by either ROS over-production or oxidative defense capacity deficiency. Mitochondrial was considered as the main intracellular site for ROS generation and one of the most susceptible targets for radical species. Excessive ROS formation may disrupt energy metabolism, impair activities of a number of mitochondrial enzymes that play critical roles in antagonizing oxidative damage. ROS formation in the present study showed a significant elevation with 12 μmol/kgMeHg administration, which was an important mediator of damage to cell structures and functions. The direct chemical interactions among MeHg and its high affinity for sulfhydryl groups from proteins and non-protein molecules play a crucial role in MeHg-induced neurotoxicity, resulting in the dysfunction of enzymatic and nonenzymatic antioxidants [39]. NPSH plays important roles in the protection against oxidative stress caused by the oxygen-derived ROS, and around 90% of NPSH is by reduced glutathione (GSH), the most abundant intracellular low molecular weight sulfhydryl compound. Previous literatures suggested MeHg-induced oxidative stress might be connected with alterations of antioxidant enzymes activities, such as SOD and GSH-Px, two of the central enzymes involved with detoxification of peroxides [5,40]. Gpx-1, one of the most important subtypes of GSH-Px, has been shown involved in the neuronal detoxification of ROS over-production [41,42]. In present study, reduction of NPSH levels after MeHg exposure indicated NPSH as one of the principal endogenous nonenzymatic antioxidants might be an important molecular target of MeHg poisoning, depletion of NPSH levels usually parallel increased oxidative stress caused by MeHg exposure [5,43,44]. Furthermore, binding to GSH has been reported to be responsible for the excretion of MeHg [45], corroborated with NPSH depletion in cerebral cortex of MeHg-treated rats. In addition, we obtained SOD and GSH-Px activities decreased obviously in MeHg poisoning rats, which may contribute to a high ROS levels in cerebral cortex. The high affinity of MeHg for sulfhydryl groups may play an important role in GSH-Px activity inhibition. Additional findings showed that expression of Gpx-1 was significantly downregulated with MeHg administration. This revealed MeHg might disrupt the activity of thiol- and selenol-containing proteins that are important components of the cellular antioxidant system in maintaining the normal redox balance.

In fact, decreased peroxide detoxification may induce lipoperoxyl radical generation in a chain reaction, aggravating lipid peroxidation and cellular antioxidants depletion [46]. So, of particular importance, it is notable that MeHg-induced decrease of brain Gpx activity also preceded NPSH depletion under in vivo conditions. The present findings together with other previously published results indicated MeHg exposure could be able to generate excessive ROS, aggravate endogenous antioxidants depletion, and also inhibit antioxidant enzymes activities. These factors might act independently or through a complex interaction to activate the cascade of events involved in oxidative stress induced by MeHg [47–50]. The activation of Nrf2 signaling pathway has been thought to be functional as a protective factor against MeHg-induced neurotoxicity via a major regulator of intracellular antioxidant response [12,17,18]. Once oxidative stress arise, Nrf2 was triggered to dissociate from Keap 1 and translocate into the nucleus, where it interacts with an antioxidant response element to initiate the transcription of target genes and their encoded proteins [51–53]. Notably, present data showed the expression of Nrf2 was upregulated obviously both in mRNA and protein levels with 12 μmol/kg MeHg administration. This indicated MeHg-induced oxidative stress could activate the dissociation of Nrf2 from Keap 1 protein, resulting in the induction of cytoprotective proteins. We also deduced the upregulation of Nrf2 expression might be related to the combination of MeHg and Keap 1 protein sulfhydryl, as well as disruption of the sulfhydryl groups via oxidative modifications. Additional findings showed a dose-dependent and significant upregulation of HO-1 and γ-GCS expressions in MeHg-treated rats, relative to control. These have suggested that MeHg-induced Nrf2 activation upregulated the expression of cellular antioxidant defenses. As a rate-limiting enzyme in GSH biosynthesis, upregulation of γ-GCS expression may promote cellular GSH synthesis for reduction of oxidative stress. Above all, these results corroborated with previous findings indicated the Nrf2 signaling pathway activation might play important roles in defending MeHg-induced oxidative stress [12,17,18,45,54]. Nevertheless, the exact molecular mechanisms of MeHg-induced Nrf2 signaling pathway activation under in vivo conditions have not been fully elucidated, additional studies are necessary for better evaluation. MeHg-induced oxidative stress and Glu dyshomeostasis have been suggested as connected phenomenon, affecting each other [3]. Because no extracellular enzymes exist for the breakdown of synaptic Glu, astrocytic Glu transporters, mainly GLAST and GLT-1, are exclusively responsible for the uptake of extracellular Glu and permit normal excitatory transmission [55]. Glu taken-up by astrocytes is amidated by GS, an enzyme localized almost in astrocytes, to form glutamine, thereby preventing synaptic Glu accumulation and Glu-dependent over-excitation [56]. Moreover, GS serves as a sensitive marker for the presence of ROS, oxidative stress aggravation may lead a severe inhibition of GS activity, contributing to a high

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Tea polyphenols against MeHg oxidative stress

concentration of synaptic Glu and the Glu-induced excitotoxicity. In our study, GS mRNA and protein expressions both were downregulated significantly after 4 weeks of 12 μmol/kg MeHg administration. This indicated MeHg exposure may lead to GS activity impairment under in vivo condition. The similar results have been reported by Swamy et al., suggesting that MeHg-induced excessive ROS accumulation to which GS is exquisitely sensitive [57]. Moreover, as compared with the control group, GLAST and GLT-1 mRNA and protein expressions were downregulated significantly in the 12 μmol/kg MeHgtreated group. Although the molecular mechanisms mediating decreased Glu transporter activities in MeHg neurotoxicity have not been yet completely understood, free radicals, especially hydrogen peroxide, could be a crucial molecule involved, aggravate Glu dyshomeostasis in cerebral cortex. Results of the present study supported the hypothesis ROS over-production act as a key role in mediating decreased astrocytic GS and Glu transporters activities of MeHg poisoning rats. This would contribute to Glu uptake/metabolism disorders and Glu-induced excitotoxicity. Previous documents have showed TP and its major active catechin components could react with free radicals because of their natural plant flavonoids, and possess a variety of protective properties [21,22,26,27]. However, there were only few studies designed to explore the protective roles of TP on MeHg-induced neurotoxicity under in vivo conditions. Present study showed ROS formation decreased obviously with TP pretreatment at a dose of 1 mmol/kg. It suggested that TP could remove ROS directly for mitigation of MeHg-induced oxidative stress under in vivo condition. The reduction of ROS levels with TP pretreatment would alleviate cellular NPSH depletion caused by MeHg administration. In addition, TP has an in vivo protective property on endogenous antioxidants such as GSH and cysteine that are rich in sulfhydryl. Additional findings showed TP could protect cellular antioxidant enzymes from MeHg-induced oxidative damage for the recovery of SOD and GSH-Px activities. The upregulation of Gpx-1 expression indicated TP could partially alleviate the inhibition of MeHg-induced selenol-containing protein activity. However, the exact protective molecular mechanisms need to be further explored. It is noteworthy that many dietary polyphenols could modify or oxidize Keap 1 cysteine thiols, resulting in conformational changes of Keap 1 protein that stimulate its separation from Nrf2. This phenomenon occurs possibly through spontaneous or enzymatic oxidation to form ROS [30]. Present study showed Nrf2 expression upregulated in TP control group to some extent, but no significant difference observed versus the saline control group. The molecular mechanisms of these alterations under in vivo conditions need further investigations. Compared with 12 μmol/kg MeHg-treated rats, both of the mRNA and protein levels of Nrf2, HO-1, and γ-GCS expression were downregulated with TP pretreatment. The reason might be illustrated as TP pretreatment mitigated oxidative

13

stress-induced Nrf2 signaling pathway activation by significantly scavenged ROS formation induced by MeHg administration. The similar results have been reported by Li et al. previously [58]. However, Nrf2 pathway activation in TP pretreatment group was still obviously higher than that in TP control, referring excessive ROS as well as the direct reaction of MeHg with Keap 1 sulfhydryl still significantly activate Nrf2 pathway, for antagonizing MeHg-induced oxidative stress. Nevertheless, additional researches are needed for clarification of the molecular mechanisms of Nrf2 signaling pathway activation under MeHg-induced oxidative stress or TP pretreatment. MeHg-induced disruption of Glu uptake/metabolism was partially antagonized with TP pretreatment. As an enzyme exquisitely sensitive to oxidative stress, recovered GS expression may relate to the low levels of free radicals with TP pretreatment. This phenomenon indicated TP could prevent Glu metabolism disorder via a pathway associated with GS activity protection. In addition, compared with 12 μmol/kg MeHg-treated rats, upregulation of GLAST and GLT-1 expressions with TP pretreatment well-supported Allen’s and Erikson’s views that Glu uptake/metabolism dysfunction could be alleviated via a low level of free radicals [19,59]. As there was no literature reported TP has the ability to directly antagonize Glu-mediated neurotoxicity, the present results supported the hypothesis that MeHg-induced Glu uptake/metabolism dysfunction and oxidative stress are connected phenomenon, and this dysfunction could be antagonized at least in part by TP pretreatment via its free radical scavenging abilities. The present study established a rat model of fourweek MeHg poisoning, which provided substantial evidence suggested oxidative stress play critical roles in MeHg neurotoxicity. TP pretreatment antagonized the oxidative stress significantly with a mitigation of the Glu uptake/metabolism disruption. This well supported the relationships between oxidative stress and Glu dyshomeostasis involved in MeHg neurotoxicity. On account of the diversity of possibilities and contradictions concerning the mechanisms of MeHg-induced neurotoxicity, additional studies are needed for clarifying the exact molecular mechanisms. Moreover, the neuroprotective roles of TP on MeHg neurotoxicity will be carried out a deeply research in our future studies. Acknowledgments In addition, we are grateful to Jingyi Sun (The first affiliated hospital of China Medical University) for his technical support. Declaration of interest The authors report no declarations interest. The authors alone are responsible for the content and writing of the paper.

14 W. Liu et al. This study was supported by the grants from the National Natural Science Foundation of China (No. 81172631).

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