Glutathione Adduct of Methylmercury Activates the Keap1–Nrf2

Sep 18, 2014 - MRP inhibitor. As expected, the MeHg−SG adduct was able to modify cellular proteins in the SH-SY5Y cells and purified Keap1. We also ...
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Glutathione Adduct of Methylmercury Activates the Keap1−Nrf2 Pathway in SH-SY5Y Cells Eiko Yoshida,†,§ Yumi Abiko,†,‡,§ and Yoshito Kumagai*,†,‡ †

Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan ‡ Environmental Biology Section, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan S Supporting Information *

ABSTRACT: Methylmercury (MeHg) reacts readily with GSH, leading to the formation of a MeHg−SG adduct that is excreted into extracellular space through multidrug-resistance-associated protein (MRP), which is regulated by the transcription factor Nrf2. We previously reported that MeHg covalently modifies Keap1 and activates Nrf2 in human neuroblastoma SH-SY5Y cells. In the study presented here, we examined whether the MeHg−SG adduct could also modulate the Keap1−Nrf2 pathway because the formation of the Hg−S bond is believed to be reversible in the presence of a nucleophile. SH-SY5Y cells exposed to the synthetic ethyl monoester of the MeHg−SG adduct (which is hydrolyzed by cellular esterase(s) to give the MeHg−SG adduct) exhibited a concentration-dependent cellular toxicity that was enhanced by pretreatment with a specific MRP inhibitor. As expected, the MeHg−SG adduct was able to modify cellular proteins in the SH-SY5Y cells and purified Keap1. We also found that this prodrug, as well as MeHg, causes the cellular Keap1 in the cells to be modified, resulting in Nrf2 activation and, thereby, the upregulation of the downstream genes. These results suggest that the MeHg−SG adduct is not electrophilic but that it modifies protein thiols (including Keap1) through S-transmercuration and that rapid Nrf2-dependent excretion of the MeHg−SG adduct is essential in decreasing the cytotoxicity of MeHg.

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INTRODUCTION Methylmercury (MeHg) is an electrophile that is found in the environment and covalently bonds to cellular proteins through thiol groups because of its high association constant (10−15). MeHg-mediated toxicity is, at least in part, caused by protein modifications, which result in alterations to the structures and functions of the proteins. MeHg can extensively modify several cellular proteins, but some of the MeHg can undergo nucleophilic attack by GSH produced by glutamate cysteine ligase (GCL) to form an MeHg−SG adduct that is, in turn, excreted to the outside of the cell by multidrug-resistanceassociated protein (MRP).1,2 Interestingly, the gene expressions of GCL and MRP are cooperatively regulated by the transcription factor Nrf2.3−6 We previously reported that exposure of human neuroblastoma SH-SY5Y cells to MeHg resulted in the activation of Nrf2 that was negatively regulated by Keap1 in the cells.7 It has also been suggested that the covalent modification of Keap1 by MeHg (determined by atomic absorption spectrophotometry) is potentially involved in the activation of Nrf27 because modification of Keap1 is thought to play a role in Nrf2 activation.8 Subsequent studies using SH-SY5Y cells and mice revealed that Nrf2 plays a critical role in protecting against MeHg.7,9 This suggests that the excretion of the MeHg−SG adduct into the extracellular space © 2014 American Chemical Society

is a rate-limiting step in the decrease in the toxicity of MeHg and that this is coupled with a decrease in the covalent modification of cellular proteins by MeHg. It is well-known that the Hg−S bond is covalent but that it is not stable in the presence of nucleophiles, unlike the C−S bond.10 For example, using isoelectric focusing agarose gel electrophoresis and detection using synchrotron radiation X-ray fluorescence line analysis, we found that MeHg was covalently attached to Mn-superoxide dismutase. Adding GSH facilitated the dissociation of the MeHg from the Mn-superoxide dismutase.11 Therefore, we thought that an MeHg−SG adduct derived from MeHg would also interact with cellular proteins through thiol groups, forming protein adducts of MeHg. If the MeHg−SG adduct is able to modify Keap1, then it could also be postulated that Nrf2 would be activated by such a polar metabolite. To investigate this hypothesis, we synthesized an ethyl monoester of the MeHg−SG adduct (MeHg−SGEt) because it is fairly difficult for the MeHg−SG adduct to be transferred into cells. Received: June 13, 2014 Published: September 18, 2014 1780

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proteins when we used the BPM assay with SH-SY5Y cell lysates and purified Keap1 (see Figure 2). Determination of GSH. The GSH concentrations were determined following the method described by Vignaud et al.13 with slight modifications, using an HPLC instrument equipped with an electron capture detector (ECD). Briefly, a 20 μL aliquot of a sample was loaded onto an analytical HPLC system (equipped with a 17 mm long and 4.6 mm i.d. guard column and a YMC-Pack ODS-AM column (250 mm long, 4.6 mm i.d., 5 μm particle size; YMC Co., Ltd., Kyoto, Japan)). The mobile phase was 20 mM ammonium phosphate (pH 2.5) containing 1.8 v/v % acetonitrile, and the flow rate was 0.6 mL/min. LC−MS/MS Analysis. The Keap1−MeHg adducts formed during the S-transmercuration of Keap1 and MeHg−SG were analyzed using a nanoUPLC−MS/MS system (Waters Corp.) equipped with a BEH130 nanoAcquity C18 column (100 mm long, 75 μm i.d., 1.7 μm particle size) following the method described by Abiko et al.14 with slight modifications. Briefly, murine recombinant Keap1 (4.2 μM) was incubated with dimethyl sulfoxide (DMSO), MeHg (8.4 μM), or MeHg−SG (8.4 μM) in 20 mM Tris-HCl (pH 8.4) for 30 min at 25 °C. Native and MeHg-modified murine Keap1 was separated using SDS-PAGE, and the gel was stained with Coomassie dye. The gel containing the Keap1 band was removed, transferred into a tube, and then washed with a 1:1 (v/v) mixture of acetonitrile and 50 mM aqueous ammonium bicarbonate until the Coomassie dye was completely removed. The gel was then reduced by adding 10 mM TCEP in 50 mM aqueous ammonium bicarbonate (100 μL) and incubating the mixture for 1 h at 56 °C. The mixture was then alkylated by adding 55 mM 2-iodoacetamide in 50 mM aqueous ammonium bicarbonate (100 μL) and incubating it in the dark for 45 min at 25 °C; then, it was rinsed with a 1:1 (v/v) mixture of acetonitrile and 50 mM aqueous ammonium bicarbonate. The rinsed gel was then dehydrated using acetonitrile. The Keap1 was digested with 20 ng of MS-grade modified trypsin (Promega, Madison, WI, USA) for 16 h at 37 °C. The resulting peptide was extracted from the gel by adding 5 μL of a 50:0.1:49.9 (v/v) mixture of acetonitrile, trifluoroacetic acid, and water, and then the mixture was transferred to a sample vial. HPLC separation was performed using two mobile phases. Mobile phase A was DDW containing 0.1 v/v % formic acid, and mobile phase B was acetonitrile containing 0.1 v/v % formic acid. The flow rate was 0.3 μL/min, and the mobile phase program was 1 v/ v % B for 1 min, linearly increased over 90 min to 40 v/v % B, linearly increased over 1 min to 95 v/v % B for 4 min, and linearly decreased to 1 v/v % B over 1 min. The eluted peptides were transferred to a nanoElectroSpray MS instrument (Waters Corp.), in which the ESI capillary voltage was 2.8 kV and the sampling cone voltage was 35 V. A low (6 eV) or an elevated (using steps from 15 to 60 eV) collision energy was used to generate either intact peptide precursor ions (at low energy) or peptide product ions (at elevated energies). The source temperature was 100 °C, and the detector was operated in positive ion mode. Biopharmlynx version 1.2 software (Waters) was used to perform baseline subtraction and smoothing, deisotoping, de novo peptide sequence identification, and database searches. Western Blot Analysis. SH-SY5Y cells were treated with MeHg or MeHg−SGEt for 6, 12, or 24 h and then collected by scraping them into a 2% SDS solution. The cells were lysed at 95 °C for 20 min, and then the protein concentrations were determined using the bicinchoninic acid protein assay (Piece, Rockford, IL, USA). Each sample was incubated with an SDS-PAGE loading buffer (62.5 mM Tris-HCl, pH 6.8, 6% SDS, 24% glycerol, and 0.015% bromophenol blue) containing 50 mM TCEP or 5% 2-mercaptoethanol at 95 °C for 5 min. The cellular proteins were separated by SDS-PAGE and transferred to a poly(vinylidene difluoride) membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% skim milk in TTBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20), incubated with primary antibodies in TTBS, and then incubated with HRP-conjugated anti-rabbit IgG. Immunoreactive proteins were detected using a chemiluminescence system (ChemiLumi One L; Nacalai Tesque). Representative blots from three independent experiments are shown.

MATERIALS AND METHODS

Materials. MeHgCl and anti-actin antibodies were purchased from Sigma-Aldrich (San Diego, CA, USA). GSH, Tris(2-carboxyethyl)phosphine (TCEP), and biotin-PEAC5-maleimide (BPM) were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan), Nacalai Tesque Inc. (Kyoto, Japan), and Invitrogen (Carlsbad, CA, USA), respectively. The ethyl monoester of GSH was purchased from Calbiochem (Darmstadt, Germany). Anti-Nrf2 antibodies, anti-Keap1 antibodies, and anti-glutamate-cysteine ligase and its modifier subunit (GCLM) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-heme oxygenase-1 (HO-1) and horseradish peroxidase (HRP)-linked anti-rabbit IgG were purchased from Stressgen (Victoria, Canada) and Cell Signaling Technology (Beverly, MA, USA), respectively. All other reagents were of the highest grade available. Cell Culture. SH-SY5Y cells were kindly gifted by Prof. Akira Naganuma, Tohoku University, Japan. Cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10% fetal bovine serum, 2 mM L-alanyl-L-glutamine (Invitrogen), and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) in an incubator at 37 °C, supplied with a 5% CO2 atmosphere. When extracting the total protein, 1 × 106 cells were seeded in 35 mm dishes. The cells were preincubated in serum-free medium for 12 h before they were treated with the test compounds. Synthesis and Isolation of the MeHg−SG and MeHg−SGEt Adducts. MeHg (125 mg or 20 mg) was reacted with GSH (153 mg) or the ethyl monoester of GSH (13.7 mg) for 30 min with stirring. The products of a reaction were applied to an Ultra-Pack ODS-A-40A column (300 mm long, 11 mm i.d., 40 μm particle size, Yamazen Science Inc., Osaka, Japan), which was eluted with deionized distilled water (DDW) at a flow rate of 2 mL/min. The contents of the eluate were monitored using atomic absorption spectrometry (Nippon Instruments Corp., Osaka, Japan), and the fractions containing MeHg−SG and MeHg−SGEt were collected. The collected fractions were evaporated in vacuo and then freeze-dried. The purified products were identified using ultra-high-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS; Waters Corp., Milford, MA, USA) and NMR spectroscopy (500 MHz 1H NMR; Bruker, Rheinstetten, Germany) and are described below. MeHg−SG Adduct. Electrospray ionization (ESI)-MS [M − H]−; m/z = 522.07. 1H NMR (D2O, 500 MHz): δ 4.52−4.54 (t, 1H), 3.91 (s, 2H), 3.74−3.77 (t, 1H), 3.34−2.38 (m, 2H), 3.26−3.30 (m, 2H), 2.48−2.53 (m, 2H), 2.08−2.13 (m, 2H), 0.68 (t, 3H) (Figure S2, Supporting Information). MeHg−SGEt Adduct. ESI-MS [M − H]−; m/z = 550.08. 1H NMR (D2O, 500 MHz): δ 4.48−4.50 (t, 1H), 4.10−4.14 (m, 2H), 3.93 (t, 2H), 3.66−3.69 (t, 1H), 3.32 (m, 1H), 3.25−3.26 (m, 1H), 2.43−2.48 (m, 2H), 2.05−2.07 (m, 2H), 1.16−1.18 (t, 3H), 0.66 (t, 3H) (Figure S4, Supporting Information). Detection of the Covalent Modification of Proteins. The BPM labeling assay was performed following the method described by Toyama et al.12 with a slight modification. Briefly, Keap1 (1 μg) was incubated with MeHg (100 or 500 μM) or its GSH adduct (100 or 500 μM) in 20 mM Tris-HCl (pH 8.5) for 30 min at 25 °C and then added to BPM to give a final BPM concentration of 25 μM. The reaction mixture was then subjected to western blot analysis using an HRP-conjugated anti-biotin antibody (supplied by Cell Signaling Technology). A decrease in BPM-binding reflected a decrease in the availability of free thiol groups. In the cell system, MeHg− or MeHg− SGEt-treated cells were washed with PBS and then lysed with RIPA buffer (50 mM Tris-HCl, pH 8, 0.1% SDS, 150 mM NaCl, 1% NP-40, and 0.5% deoxycholic acid). The cell lysate was centrifuged, and the supernatant was incubated with 5 mM BPM (1 μL) for 30 min at 37 °C. The lysate was precipitated using streptavidin−agarose (SigmaAldrich) overnight at 4 °C and then washed. The precipitated proteins were eluted then subjected to western blot analysis. It should be noted that higher concentrations of MeHg and its GSH adduct were required to determine the degree of chemical modification of the cellular 1781

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Figure 1. Effect of MK571 on the cytotoxicity caused by the MeHg−SG adduct. (A) The MeHg−SGEt adduct was hydrolyzed by esterase, producing MeHg−SG. (B) SH-SY5Y cells were pretreated with 100 μM MK571 for 1 h and then treated with MeHg or the MeHg−SGEt adduct for 24 h. The cell viability was measured using an MTT assay. Each value is the mean ± the standard error from three determinations. **p < 0.01 vs control; ## p < 0.01 vs MgHg−SGEt.

Figure 2. S-Transmercuration reaction of purified Keap1 with the MeHg−SG adduct and the release of GSH from the adduct. (A) SH-SY5Y cell lysate was incubated with MeHg or the MeHg−SG adduct for 30 min at 25 °C and then reacted with 100 μM BPM for 30 min at 37 °C. The reaction mixture was subjected to western blot analysis with an avidin-HRP conjugate. (B) Recombinant murine Keap1 was incubated with MeHg or the MeHg−SG adduct in 20 mM Tris-HCl (pH 8.2) for 30 min at 25 °C. The reaction mixture was subjected to western blot analysis with the antibodies indicated. (C) HPLC-ECD chromatogram of authentic GSH. (C) HPLC-ECD chromatogram of the recombinant murine Keap1. (D−F) Recombinant murine Keap1 was incubated with MeHg or the MeHg−SG adduct in 20 mM Tris-HCl (pH 8.2) for 1 h at 25 °C, and then the Keap1 was removed by ultrafiltration. The reaction mixture was analyzed by HPLC-ECD, and GSH, indicated by the arrow, was detected. Real-Time Reverse-Transcription Polymerase Chain Reaction. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized from 2 μg of total RNA using a high-capacity RNA-to-cDNA kit (Applied Biosystems, Grand Island, NY, USA), following the protocols supplied by the manufacturers. An aliquot of the resulting cDNA (100 ng) was mixed with 2.5 μM of primers (0.8 μL), which are specified in Table S1 (Supporting Information), and SYBER green master mix (10 μL, Applied Biosystems) to give a total volume of 20 μL. Amplification was performed in a 7500 real-time PCR system (Applied Biosystems) using a thermal cycle of 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Emission data were quantified using the Ct values, which were normalized to values for β2-microgrobulin. Cell Viability. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Dojindo, Kumamoto, Japan) assay was used to determine the cell viability, as has been described previously.15 Briefly, SH-SY5Y cells were pretreated with MK571 (0 or 100 μM) for 1 h. The medium was removed, and the cells were treated with MeHg or

MeHg−SGEt for 24 h and then incubated with 5 mg/mL MTT solution (1:10, v/v) for 30 min in a CO2 incubator at 37 °C. The medium was removed, and then DMSO was added to dissolve the MTT formazan. The absorbance at a wavelength of 540 nm was measured using a spectrophotometer.

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RESULTS Cellular Toxicity of the MeHg−SG Adduct. In our preliminary study, we found that MeHg caused a concentration-dependent death of SHSY5Y cells, but we found that the MeHg−SG adduct did not cause cell death even at 1 μM (data not shown), suggesting that it was difficult for the cells to incorporate the MeHg−SG adduct. As shown in Figure 1A, however, the MeHg−SGEt adduct, which was taken into the cells easily, underwent enzymatic hydrolysis by the cellular esterase(s) to give the MeHg−SG adduct. MeHg was cytotoxic to the SH-SY5Y cells in a concentration-dependent manner, 1782

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Figure 3. Identification of MeHg-modified cysteine residues in Keap1 using UPLC−MS/MS. Recombinant murine Keap1 was incubated with (A) MeHg or (B) the MeHg−SG adduct in 20 mM Tris-HCl (pH 8.2) for 30 min at 25 °C. After the reaction, Keap1 protein was digested with trypsin and analyzed using UPLC−MS/MS under the conditions described in the Materials and Methods. The corresponding MS/MS data are shown in Tables 1 and S2 (Supporting Information).

Table 1. Sites at Which Keap1 Is Modified by MeHg and Its GSH Adduct (MeHg−SG)a compd

position

peptide sequenceb

calculated MS

observed MS

Cys

MeHg MeHg−SG

151−169 299−320

C*VLHVMNGAVMYQIDSVVR DYLVQIFQELTLHKPTQAVPC*R

2349.04 2814.35

2349.13 2814.40

Cys151 Cys319

The recombinant murine Keap1 was reacted with MeHg or MeHg−SG for 30 min at 25 °C. Keap1 was digested with trypsin and analyzed by UPLC−MS/MS, as described in the Materials and Methods. The table shows the amino acid sequences of the tryptic peptides in the MeHgmodified murine Keap1. The position is the relevant portion of the amino acid sequence in murine Keap1. bMeHg-modified Cys.

a

SG adduct. Consistent with this notion, a product corresponding to the authentic GSH (with the same retention time) was detected by HPLC analysis (Figure 2C−E), and increasing the amount of MeHg available to react with Keap1 increased the amount of GSH released (Figure 2F). Modification of Keap1 and the Activation of Nrf2 by the MeHg−SG Adduct. As shown in Figure 3 and Tables 1 and S2 (Supporting Information), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed that MeHg modified Keap1 through Cys151.8 However, MeHg−SG was found to attack nucleophilic Keap1, leading to a Keap1−MeHg adduct being formed through Cys319. These Keap1 modifications were also observed when SH-SY5Y cells were exposed to MeHg and its GSH adduct (Figure 4), suggesting that the MeHg−SG adduct could activate Nrf2. Consistent with this, the polar metabolites activated Nrf2 in a time- and concentration-dependent manner (Figure 5A,B) through the transactivation of the ARE (data not shown). As a result, the Nrf2-dependent downstream genes for

and the MeHg−SGEt adduct was also toxic to the cells, but to a lower degree (Figure 1B). Interestingly, the MeHg−SGEtmediated cytotoxicity was enhanced by pretreating the cells with MK571, which is a specific inhibitor of MRP (Figure 1B), suggesting that the prolonged retention of MeHg−SG (caused by its excretion into extracellular space being blocked) increased its toxicity. This implies that the MeHg−SG adduct could interact with protein thiols, leading to the covalent modification of the proteins with MeHg, a process that is called S-transmercuration. To confirm that S-transmercuration was possible, we used the BPM assay to determine the chemically modified cellular proteins during incubation with the MeHg−SG adduct.12 As expected, numerous proteins modified by both MeHg and the MeHg−SG adduct were found in the lysate of the SH-SY5Y cells (Figure 2A). Purified Keap1 was also modified by these mercurial chemicals (Figure 2B). If the MeHg−SG adduct causes the S-transmercuration of Keap1, forming the Keap1− MeHg adduct, then GSH should be released from the MeHg− 1783

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(Figure 3). However, MeHg was also found to modify Keap1 and activate Nrf2 in cells in our previous studies.7,9 The Nrf2 activation mediated by the MeHg−SG adduct was also confirmed by the transactivation of ARE and the substantial upregulation in the expression of Nrf2-dependent genes, as shown in Figures 5 and 6. There are 27 cysteine residues in human Keap1. Of these, three have been found to be Cys-based stress sensors, and these are Cys151, Cys273/Cys288, and Cys226/Cys613. These Cysbased stress sensors have been found to be highly reactive thiols that can be chemically modified by electrophiles involved in Nrf2 activation. Another group of thiols, Cys257, Cys297, Cys319, Cys434, Cys489, Cys583, and Cys624, have also been found to be targets for electrophiles.16−19 As is shown in Figure 3, Cys151 and Cys319 were found to be the sites at which Keap1 was modified by MeHg and its GSH adduct under the conditions used in this study. It has been reported that biotinylated iodoacetamide and nitrated oleic acid can covalently bond to Cys319.16,20 This cysteine residue is in the intervening region (IVR), which is important for the ability of Keap1 to maintain ubiquitin E3 ligase activity and promote the degradation of Nrf2.16 The covalent bond between Keap1 and MeHg produced through the S-transmercuration reaction with the MeHg−SG adduct may interfere with the activation of Cul3, causing the accumulation of a substantial amount of Nrf2. However, Kansanen et al. suggested that the covalent modification of the IVR, including Cys319, by a nitrated fatty acid can be attributed to substantial structural changes in the double glycine repeat and the C-terminal region of Keap1 (DC domain), resulting in the inhibition of Nrf2 ubiquitination.20 We speculate that Cys319 might be a sensor, like Cys151, Cys226, Cys273, Cys 288, and Cys613, although the significance of its modification is unclear at present. Further study will be required to show the mechanistic details of the activation of Nrf2 coupled with the MeHg-dependent modification of Keap1. Higgins et al.21 found that pretreating Nrf2+/+ mouse embryonic fibroblasts (MEFs) with sulforaphane protected them against subsequent exposure to HgCl2 but that treating Nrf2−/− MEFs in the same way did not protect them against exposure to HgCl2. The Nrf2−/− MEFs were actually more sensitive to HgCl2 than were the Nrf2+/+ MEFs. Unfortunately, they did not study MeHg. We have previously found that pretreating SH-SY5Y cells with sulforaphane, which activates Nrf2 and upregulates the phase-II detoxification enzymes and phase-III transporters, protects the cells against MeHg-induced cytotoxicity via an Nrf2-dependent mechanism.9 In that paper, we compared the effect on the MeHg-induced cytotoxicity of treating cells with MK571 and buthionine sulfoximine or ethacrynic acid.9 The results suggested that Nrf2 would protect SH-SY5Y cells against MeHg and its GSH adduct. As shown in Figures 1 and 2, it seems likely that MeHg−SG adduct-mediated modifications of several proteins in SH-SY5Y cells are linked to concentration-dependent cytotoxicity when the cells are exposed to the MeHg−SGEt adduct. More importantly, the cell viability was decreased further when the cells were pretreated with the specific MRP inhibitor. From these observations, we speculate that the rapid excretion of MeHg−SG (after the conjugation of MeHg with GSH produced by GCL) into the extracellular space through MRP is a crucial step in decreasing MeHg toxicity. This is because the MeHg−SG adduct can still interact with cellular proteins, resulting in the formation of protein adducts that alter the

Figure 4. Modification of cellular Keap1 by MeHg and the MeHg− SGEt adduct. SH-SY5Y cells were treated with MeHg (0, 10, or 20 μM) or the MeHg−SGEt adduct (0, 10, or 20 μM) for 3 h and then lysed with RIPA buffer. The cell lysates were incubated with biotinPEAC5-maleimide and then precipitated with streptavidin−agarose. The precipitated proteins were subjected to western blot analysis with an anti-Keap1 antibody. The band intensity was quantified using ImageJ software. Each value is the mean ± the standard error from three determinations. *p < 0.05 vs 0 μM.

Figure 5. MeHg− and MeHg−SGEt-mediated activation of Nrf2/ antioxidant responsive element (ARE) signaling. (A, B) SH-SY5Y cells were exposed to DMSO, MeHg (1, 2, or 4 μM), or the MeHg−SGEt adduct (1, 2, or 4 μM) for (A) 6 h and (B) 12 h. The cell lysates were subjected to western blot analysis using the antibody indicated. The band intensity was quantified using ImageJ software. Each value is the mean ± the standard error from three determinations. *p < 0.05 vs control.

the GCL catalytic subunit, GCLM, HO-1, and xCT, were upregulated during the period of exposure to the MeHg−SGEt adduct (Figure 6A). We also found that the presence of the MeHg−SG adduct increased the HO-1 and GCLM protein concentrations (Figure 6B).

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DISCUSSION The MeHg−SG adduct was shown to become covalently bound to a variety of cellular proteins regardless of its hydrophilicity and lack of electrophilicity. This is first evidence for the S-transmercuration of proteins by a MeHg−SG adduct using its ethyl ester derivative. The BPM assay and LC−MS/ MS analysis revealed that the MeHg−SG adduct is a metabolite of MeHg that causes the activation of Nrf2, presumably through covalent attachment to Keap1 during the exposure of the SH-SY5Y cells to the MeHg−SGEt adduct, which was deduced because recombinant Keap1 was found to be modified by the MeHg−SG adduct, as determined by LC−MS/MS 1784

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Figure 6. MeHg− and MeHg−SGEt-mediated upregulation of the downstream Nrf2 genes. (A) SH-SY5Y cells were exposed to DMSO, MeHg (1, 2, or 4 μM), or the MeHg−SGEt adduct (1, 2, or 4 μM) for 6 h. The mRNA concentrations were determined using real-time reverse-transcription polymerase chain reaction analysis using the primers indicated, as described in the Materials and Methods. All mRNA expression concentrations were normalized to the β-2-microgrobulin mRNA concentration. Each value is the mean ± the standard error from three determinations. *p < 0.05 vs 0 μM. (B) SH-SY5Y cells were exposed to DMSO, MeHg (1, 2, or 4 μM), or the MeHg−SGEt adduct (1, 2, or 4 μM) for 24 h. The cell lysates were subjected to western blot analysis using the antibody indicated. The band intensity was quantified using ImageJ software. Each value is the mean ± the standard error from three determinations. *p < 0.05 vs 0 μM.

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ABBREVIATIONS ARE, antioxidant responsive element; BPM, biotin-PEAC5maleimide; BSO, L-buthionine-(S,R)-sulfoximine; DDW, deionized distilled water; GCLM, glutamate-cysteine ligase, modifier subunit; GCLC, glutamate-cysteine ligase, catalytic subunit; HO-1, heme oxygenase-1; Keap1, Kelch-like ECHassociated protein 1; MeHg−SGEt, ethyl monoester of MeHg− SG adduct; MRP, multidrug resistance-associated protein; Nrf2, NF-E2-related factor 2; TCEP, tris(2-carboxyethyl)phosphine; UPLC, ultrahigh performance LC

protein’s structure and enzymatic function. However, HPLC analysis (Figure 2C−F) indicated that GSH is indeed released from the MeHg−SG adduct during the S-transmercuration of purified Keap1 by the MeHg−SG adduct. This suggests that dissociated GSH is used to conjugate unreacted MeHg (see the Abstract graphic). Taken together, our results suggest that there is a reaction cycle between the protein thiols, MeHg, and GSH within cells. In summary, we found that both MeHg and its GSH adduct activate Nrf2, thereby increasing the expression of its downstream genes. This suggests that even the polar metabolites of MeHg play roles in initial environmental responses to the presence of MeHg.

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ASSOCIATED CONTENT

* Supporting Information S

Experimental procedure for LC−MS/MS analysis, UPLC−MS and −MS/MS analyses of the authentic MeHg−SG and MeHg−SGEt adducts, 500 MHz 1H NMR spectra of the authentic MeHg−SG and MeHg−SGEt adducts, and human gene-specific real-time reverse-transcription polymerase chain reaction primers. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Ballatori, N. (2002) Transport of toxic metals by molecular mimicry. Environ. Health Perspect. 110, 689−694. (2) Madejczyk, M. S., Aremu, D. A., Simmons-Willis, T. A., Clarkson, T. W., and Ballatori, N. (2007) Accelerated urinary excretion of methylmercury following administration of its antidote N-acetylcysteine requires Mrp2/Abcc2, the apical multidrug resistance-associated protein. J. Pharmacol. Exp. Ther. 322, 378−384. (3) Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M., and Nabeshima, Y. (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun. 236, 313−322. (4) Moinova, H. R., and Mulcahy, R. T. (1999) Up-regulation of the human gamma-glutamylcysteine synthetase regulatory subunit gene involves binding of Nrf-2 to an electrophile responsive element. Biochem. Biophys. Res. Commun. 261, 661−668. (5) Hayashi, A., Suzuki, H., Itoh, K., Yamamoto, M., and Sugiyama, Y. (2003) Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts. Biochem. Biophys. Res. Commun. 310, 824− 829. (6) Vollrath, V., Wielandt, A. M., Iruretagoyena, M., and Chianale, J. (2006) Role of Nrf2 in the regulation of the Mrp2 (ABCC2) gene. Biochem. J. 395, 599−609. (7) Toyama, T., Sumi, D., Shinkai, Y., Yasutake, A., Taguchi, K., Tong, K. I., Yamamoto, M., and Kumagai, Y. (2007) Cytoprotective role of Nrf2/Keap1 system in methylmercury toxicity. Biochem. Biophys. Res. Commun. 363, 645−650.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-29-853-3297; Fax: +81-29-853-3259; E-mail: [email protected]. Author Contributions §

E.Y. and Y.A. contributed equally to this work.

Funding

This work was supported by a grant-in-aid (no. 25220103 to Y.K.) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Notes

The authors declare no competing financial interest. 1785

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Chemical Research in Toxicology

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(8) Kumagai, Y., Kanda, H., Shinkai, Y., and Toyama, T. (2013) The role of the Keap1/Nrf2 pathway in the cellular response to methylmercury. Oxid. Med. Cell. Longevity 2013, 848279. (9) Toyama, T., Shinkai, Y., Yasutake, A., Uchida, K., Yamamoto, M., and Kumagai, Y. (2011) Isothiocyanates reduce mercury accumulation via an Nrf2-dependent mechanism during exposure of mice to methylmercury. Environ. Health Perspect. 119, 1117−1122. (10) Rabenstein, D. L., Reid, R. S., and Isab, A. A. (1983) H NMR study of the effectiveness of various thiols for removal of methylmercury from hemolyzed erythrocytes. J. Inorg. Biochem. 18, 241−251. (11) Shinyashiki, M., Kumagai, Y., Homma-Takeda, S., Nagafune, J., Takasawa, N., Suzuki, J., Matsuzaki, I., Satoh, S., Sagai, M., and Shimojo, N. (1996) Selective inhibition of the mouse brain Mn-SOD by methylmercury. Environ. Toxicol. Pharmacol. 2, 359−366. (12) Toyama, T., Shinkai, Y., Kaji, T., and Kumagai, Y. (2013) Convenient method to assess chemical modification of protein thiols by electrophilic metals. J. Toxicol. Sci. 38, 477−484. (13) Vignaud, C., Rakotozafy, L., Falguieres, A., Potus, J., and Nicolas, J. (2004) Separation and identification by gel filtration and high-performance liquid chromatography with UV or electrochemical detection of the disulphides produced from cysteine and glutathione oxidation. J. Chromatogr. A 1031, 125−133. (14) Abiko, Y., and Kumagai, Y. (2013) Interaction of Keap1 modified by 2-tert-butyl-1,4-benzoquinone with GSH: evidence for Stransarylation. Chem. Res. Toxicol. 26, 1080−1087. (15) Shinkai, Y., Sumi, D., Toyama, T., Kaji, T., and Kumagai, Y. (2009) Role of aquaporin 9 in cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes. Toxicol. Appl. Pharmacol. 237, 232−236. (16) Taguchi, K., Motohashi, H., and Yamamoto, M. (2011) Molecular mechanisms of the Keap1−Nrf2 pathway in stress response and cancer evolution. Genes Cells. 16, 123−140. (17) Dinkova-Kostova, A. T., Holtzclaw, W. D., Cole, R. N., Itoh, K., Wakabayashi, N., Katoh, Y., Yamamoto, M., and Talalay, P. (2002) Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc. Natl. Acad. Sci. U.S.A. 99, 11908− 11913. (18) Fourquet, S., Guerois, R., Biard, D., and Toledano, M. B. (2010) Activation of NRF2 by nitrosative agents and H2O2 involves KEAP1 disulfide formation. J. Biol. Chem. 285, 8463−8471. (19) McMahon, M., Lamont, D. J., Beattie, K. A., and Hayes, J. D. (2010) Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals. Proc. Natl. Acad. Sci. U.S.A. 107, 18838−18843. (20) Kansanen, E., Bonacci, G., Schopfer, F. J., Kuosmanen, S. M., Tong, K. I., Leinonen, H., Woodcock, S. R., Yamamoto, M., Carlberg, C., Yla-Herttuala, S., Freeman, B. A., and Levonen, A. L. (2011) Electrophilic nitro-fatty acids activate NRF2 by a KEAP1 cysteine 151independent mechanism. J. Biol. Chem. 286, 14019−14027. (21) Higgins, L. G., Kelleher, M. O., Eggleston, I. M., Itoh, K., Yamamoto, M., and Hayes, J. D. (2009) Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents. Toxicol. Appl. Pharmacol. 237, 267−280.

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