Exposure to Electrophiles Impairs Reactive Persulfide-Dependent

Article pubs.acs.org/crt

Exposure to Electrophiles Impairs Reactive Persulfide-Dependent Redox Signaling in Neuronal Cells Hideshi Ihara,*,†,∇ Shingo Kasamatsu,†,‡,∇ Atsushi Kitamura,†,∇ Akira Nishimura,‡ Hiroyasu Tsutsuki,§ Tomoaki Ida,‡ Kento Ishizaki,† Takashi Toyama,∥ Eiko Yoshida,∥ Hisyam Abdul Hamid,‡,⊥ Minkyung Jung,‡ Tetsuro Matsunaga,‡ Shigemoto Fujii,‡ Tomohiro Sawa,§ Motohiro Nishida,# Yoshito Kumagai,∥ and Takaaki Akaike*,‡ †

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka 599-8531, Japan Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan § Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan ∥ Environmental Biology Section, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan ⊥ Department of Pharmaceutical Pharmacology and Chemistry, Faculty of Pharmacy, Universiti Teknologi MARA Puncak Alam Campus, 42300 Puncak Alam, Selangor, Malaysia # Division of Cardiocirculatory Signaling, National Institute for Physiological Sciences (Okazaki Institute for Integrative Bioscience), National Institutes of Natural Sciences, Aichi 444-8787, Japan ‡

S Supporting Information *

ABSTRACT: Electrophiles such as methylmercury (MeHg) affect cellular functions by covalent modification with endogenous thiols. Reactive persulfide species were recently reported to mediate antioxidant responses and redox signaling because of their strong nucleophilicity. In this study, we used MeHg as an environmental electrophile and found that exposure of cells to the exogenous electrophile elevated intracellular concentrations of the endogenous electrophilic molecule 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitrocGMP), accompanied by depletion of reactive persulfide species and 8-SHcGMP which is a metabolite of 8-nitro-cGMP. Exposure to MeHg also induced S-guanylation and activation of H-Ras followed by injury to cerebellar granule neurons. The electrophile-induced activation of redox signaling and the consequent cell damage were attenuated by pretreatment with a reactive persulfide species donor. In conclusion, exogenous electrophiles such as MeHg with strong electrophilicity impair the redox signaling regulatory mechanism, particularly of intracellular reactive persulfide species and therefore lead to cellular pathogenesis. Our results suggest that reactive persulfide species may be potential therapeutic targets for attenuating cell injury by electrophiles.

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long-term exposure to MeHg are now of worldwide concern.4 Because of a strong electrophilic property, electrophiles form covalent bonds with endogenous sulfhydryl groups of biomolecules and proteins, which leads to enzymatic dysregulation and impaired cell phyiology.5−7 Electrophiles reportedly exert not only cytotoxicity but also redox signaling functions.8,9 The high dose exposure to strong electrophiles may cause nonspecific disruption of various endogenous molecules, which may impair redox homeostasis causing various disorders. In contrast, low doses of electrophiles can activate signaling pathways via modifications of specific redox sensor and effector proteins.

INTRODUCTION Electrophiles such as 1,2-naphthoquinone and methylmercury (MeHg) occur naturally in the environment. The chemical compounds 1,2-naphthoquinone and MeHg are known as an atmospheric contaminant and an aquatic pollutant, respectively.1,2 For example, Minamata disease is caused by the intake of fish exposed to high levels of MeHg that has accumulated through biological concentration.3 Researchers now increasingly recognize, however, that not only acute but also long-term environmental exposure to electrophiles can have deleterious effects on human health and may be indirectly involved in the pathogenesis of various diseases, e.g., atherosclerosis, cardiac dysfunction, neurodegenerative diseases, and even cancers. People who consume considerable amounts of large predatory fish in their diet are exposed to low doses of MeHg over the long-term. The risks to human health of such low-dose but © 2017 American Chemical Society

Received: May 4, 2017 Published: August 24, 2017 1673

DOI: 10.1021/acs.chemrestox.7b00120 Chem. Res. Toxicol. 2017, 30, 1673−1684

Article

Chemical Research in Toxicology

moto, Japan). 8-Nitro-cGMP and 8-SH-cGMP were synthesized as previously described.15,22 PEGylated superoxide dismutase (PEGSOD), a membrane-permeable SOD, was prepared as previously described.30 All other chemicals and reagents were from common suppliers and were of the highest grade commercially available. Cell Culture and Stimulation. PC12 and NPC1231 cells were maintained in RPMI 1640 medium supplemented with 5% FBS, 5% HS, and 1% penicillin−streptomycin in a humidified atmosphere at 37 °C. Human neuroblastoma SH-SY5Y cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin−streptomycin. CGNs were prepared in compliance with the Guideline for Animal Experimentation at Osaka Prefecture University, with an effort to minimize the number of animals used and their suffering. CGNs were obtained from Wistar rats 7−10 days old, as described previously.31 CGNs were maintained in MEM containing 25 mM K+ (HK-MEM) with 5% FBS, 5% HS, and 1% penicillin−streptomycin. After 12 days in vitro, CGNs were used for each experiment. Cells were plated at a density of 3.0 × 104 cells/well in 96-well plates for the MTS assay, 1.0 × 107 cells/60 mm culture dish for Western blotting, and 3.0 × 107 cells/100 mm culture dish for liquid chromatography−electrospray ionization−tandem mass spectrometry (LC-ESI-MS/MS) and HPLC atomic absorption spectrophotometry (HPLC-AAS) analyses. For fluorescence detection of intracellular ROS and polysulfide generation and immunocytochemistry, cells were seeded on circular coverslips at a density of 2.0 × 105 cells/well in 4-well plates (Nalge Nunc International, Naperville, IL). To compare MeHg sensitivities of different cells, cells were incubated in culture medium containing MeHg (156 nM-4 mM) for 24 h at 37 °C. To study the formation of ROS, reactive persulfide species, 8-nitro-cGMP, and 8-SH-cGMP in CGNs by fluorescence microscopy, cells were stimulated with MeHg (2 μM) for 2, 4, and 8 h in a humidified atmosphere at 37 °C. To investigate detailed mechanisms of MeHg-induced cytotoxicity, several experiments were performed, including pretreatment with PD98059 or Na2S4, followed MeHg exposure. Fluorescence Imaging for Intracellular ROS and Reactive Persulfide Species Formation. Fluorescence imaging with the ROS-sensitive probe DHE and the reactive persulfide species-sensitive probe SSP4, an improved version of SSP2, was performed as described previously.10 Fluorescence intensity in the cells was detected by using an inverted fluorescence microscope (Ti-S/L100; Nikon Corporation, Tokyo, Japan). Images were captured and processed via Nikon Ti-S/ L100 software. Adobe Photoshop Elements v. 7.0 was used for additional image processing and quantification. Analysis for Reactive Persulfide Species by LC-ESI-MS/MS. Endogenous formation of reactive sulfide and persulfide species were examined by LC-ESI-MS/MS with β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM, Molecular Bioscience, Boulder, CO) as a trapping agent for hydrosulfide/persulfide species, according to our previous paper.32 CGNs were exposed to 2 μM MeHg for 4 and 8 h, washed with ice-cold HK-MEM twice, homogenized in ice-cold 80% methanol containing 1 mM HPE-IAM, and incubated at 37 °C for 1 h. The homogenate was centrifuged at 15,000g at 4 °C for 10 min, and the resultant supernatant was diluted 10 times with 0.1% formic acid containing known amounts of isotope-labeled internal standards, which were synthesized as described in our previous work.32 HPEIAM adducts formed via the reaction of HPE-IAM with various reactive sulfide species were analyzed with a Waters Alliance HPLC system coupled to a Waters Xevo TQD ESI triple-stage quadrupole mass spectrometer. Separation was achieved by using a Mightysil RP18 column (50 mm long ×2 mm inner diameter, Kanto Chemical) with a linear 20−80% methanol gradient for 10 min in 0.1% formic acid at 40 °C. The total flow rate was 0.3 mL/min. Mass spectrometric analyses were performed online using ESI-MS/MS in the positive ion mode with multiple reaction monitoring (MRM) mode. The MRM parameters are summarized in Table S1. Preparation of 8-NEM-S-cGMP Antibodies. Rat polyclonal 8NEM-S-cGMP antibodies were obtained from Wistar rats immunized with 8-NEM-S-cGMP-conjugated BSA, according to the method of Sawa, T. et al. with slight modifications.15 Briefly, 2′-O-succinyl-8-SHcGMP was derivatized with NEM to form 2′-O-succinyl-8-NEM-S-

We recently demonstrated that reactive persulfide species, such as cysteine hydropersulfide (CysSSH), are produced endogenously in cells and tissues10 and possess significant nucleophilic properties for the modulation of electrophiles. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) clearly mediate signal transduction, via a mechanism called redox signaling, during both basal physiological metabolism and pathological responses.11−14 Redox signaling is itself mediated by endogenous electrophilic byproducts of diverse chemical reactions among biomolecules, ROS, and RNS, such as the nitrated nucleotide 8-nitroguanosine 3′,5′cyclic monophosphate (8-nitro-cGMP).8,15−20 8-Nitro-cGMP is an electrophilic second messenger of redox signaling that can form a protein-S-cGMP adduct in a process known as Sguanylation.15,21−28 Various S-guanylated proteins, such as HRas and Kelch-like ECH-associated protein 1 (Keap1), have been shown to be involved in regulating different biological events. For example, S-guanylation of H-Ras simultaneously activates the protein and induces cell senescence in cardiac cells, which leads to chronic heart failure.22 Our recent studies confirmed that reactive persulfide species play critical roles in the metabolism of endogenous and exogenous electrophiles including 8-nitro-cGMP, nitro and keto derivatives of unsaturated fatty acids, and even MeHg via the formation of sulfhydrated derivatives of electrophiles.10,29 In fact, nucleophilic reactive persulfide species may mediate both redox signaling and metabolism of electrophiles, such that we expect that the high nucleophilicity of reactive persulfides will lead to detoxification and modulation of the electrophilic toxicity of many environmental chemicals and contaminants. However, the exact molecular mechanisms of the electrophilic toxicity and ROS stress signaling that are regulated by reactive persulfide species are not fully understood. In this study, we utilized MeHg as an environmental electrophile to elucidate the molecular mechanism of electrophilic toxicity from the viewpoint of reactive persulfideregulated redox signaling. Our results demonstrated that exposure of cerebellar granule neurons (CGNs) to MeHg depleted reactive persulfide species formed in the cells, presumably by degradation of persulfides via electrophilic conversion to bismethylmercury sulfide [(MeHg)2S] with MeHg, which led to substantial accumulation of the endogenous electrophile 8-nitro-cGMP. The result of these changes was extensive activation of H-Ras cell signaling induced by H-Ras S-guanylation, which finally caused neuronal cell damage.

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EXPERIMENTAL PROCEDURES

Materials. Mouse monoclonal anti-nNOS (clone C7), anti-8-nitrocGMP (clone 1G6) antibodies and rabbit polyclonal anti-S-guanylated protein (8-RS-cGMP) antibodies were obtained as described previously.15 PC12 cells were a generous gift from Professor Masami Takahashi, Kitasato University School of Medicine, Kanagawa, Japan. RPMI 1640 medium, penicillin−streptomycin, MeHg, minimum essential medium (MEM), Dulbecco’s modified MEM (DMEM), proteinase inhibitor cocktail, phosphatase inhibitor cocktail, Blocking One, Na2S4, SSP4, N-(6-maleimidocaproyloxy)succinimide (EMCS), dihydroethidium (DHE), nitro-L-arginine methyl ester (L-NAME), PD98059, Block Ace, Can Get Signal Immunoreaction Enhancer Solution, MagneGST Glutathione Particles, MTS assay, FBS, and horse serum (HS) were obtained from Sigma-Aldrich (St. Louis, MO), Wako Pure Chemical Industries (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), and other commercial suppliers. 2,3-Diaminonaphthalene (DAN) was purchased from Dojindo Laboratories (Kuma1674


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sensitive fluorescent probes DHE.33 CGNs were exposed to 2 μM MeHg for 2, 4, and 8 h, washed with ice-cold HK-MEM twice, and incubated with 20 μM DHE in HK-MEM at 37 °C for 30 min. The cells were then washed with ice-cold HK-MEM twice, and 2hydroxyethidium (2-OH-E+), a product produced from the reaction of superoxide anion (O2−) with DHE, in cells was extracted with icecold 80% methanol. The homogenate was centrifuged at 15,000g at 4 °C for 10 min, and the resultant supernatant was dried in vacuo and dissolved in 0.1% formic acid. 2-OH-E+ were analyzed with a Waters Alliance HPLC system coupled to a Waters Xevo TQD electrospray ionization triple-stage quadrupole mass spectrometer. Separation was achieved by using a COSMOSIL Cholester column (150 mm long ×2 mm inner diameter, Nacalai Tesque) with a linear 10−25% acetonitrile gradient for 20 min in 0.1% formic acid at 40 °C. The total flow rate was 0.3 mL/min. Mass spectrometric analyses were performed online using ESI-MS in the positive ion mode with single ion resolution mode (m/z 330.1, cone potential, 40 eV). LC-ESI-MS/MS Analysis for NO Generation. NO generation in CGNs was assessed by the DAN assay described previously34 with minor modifications. CGNs were exposed to 2 μM MeHg for 2, 4, and 8 h, and 0.1 mL of medium was removed and mixed with 10 μL of fresh DAN (0.5 mg/mL in 0.62 M HCl) for 10 min at room temperature in the dark. Reactions were terminated with 5 μL of 2.8 M NaOH. 2,3-Naphthotriazole (NAT), an end product of DAN with nitrogen oxide, was measured by a Waters Alliance HPLC system coupled to a Waters Xevo TQD electrospray ionization triple-stage quadrupole mass spectrometer. Separation was achieved by using a Mightysil RP-18 column (50 mm long × 2 mm inner diameter, Kanto Chemical, Tokyo, Japan) with a linear 20−80% methanol gradient for 10 min in 0.1% formic acid at 40 °C. The total flow rate was 0.3 mL/ min. Mass spectrometric analyses were performed online using ESIMS/MS in the positive ion mode with the MRM mode (cone potential, 45 eV; collision energy, 25 eV). The MRM parameter was as follows: m/z 170.0 → 115.0. Detection of (MeHg)2S by HPLC-AAS. (MeHg)2S formed in the cells after MeHg treatment was determined by HPLC-AAS as described previously.29 In brief, CGNs were exposed to 10 nM MeHg for 24 h and were then sonicated in 50 mM potassium phosphate buffer (pH 7.4) and centrifuged at 9,000g for 5 min 4 °C. The resulting supernatant was mixed with trichloroacetic acid (to give a final concentration of 5% v/v). The mixture was centrifuged at 9,000g for 5 min, and the supernatant was added to three volumes of 1 M Tris−HCl buffer (pH 8.5) and was then applied to a Sep-Pak C18 cartridge (Waters). The cartridge was washed with 20 mL of water, after which the metabolites were eluted with 1.5 mL of methanol. The eluted solution was evaporated to dryness and then dissolved in 10% methanol. An aliquot (10 μL) of the sample was subjected to HPLC analysis. Separation was achieved by using a Zorbax Eclipse XDB-C18 column (50 mm long, 2.1 mm i.d., 5-μm particle size; Agilent Technologies). The mobile phase was a 9:1 (v/v) mixture of 0.1% formic acid and methanol, and the total flow rate was 0.5 mL/min. A fraction was collected every 1 min after the injection. To determine the (MeHg)2S concentrations in the eluate fractions, aliquots (10 μL) of each fraction were analyzed with an AAS instrument (MA-3000; Nihon Instruments, Osaka, Japan). Cell Viability Assay. Cell viability was determined by means of the MTS assay according to the manufacturer’s instructions. The cell viability of all treatment groups was normalized to that of untreated cells. Ras Pulldown Assays. Ras pulldown assays were performed as described previously.22 Briefly, after MeHg treatment, CGNs were washed with ice-cold PBS and lysed with pulldown buffer containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10 mM sodium pyrophosphate, proteinase inhibitor cocktail, 50 mM NaF, and 1 mM Na3VO4. The cell lysate was incubated in the presence of 5 μg of the GST-Rasbinding domain of Raf-1, with rotation, for 2 h at 4 °C. The lysate was further incubated in the presence of MagneGST Glutathione Particles, with rotation, for 2 h at 4 °C. After the supernatant was removed, beads were washed with pulldown buffer and suspended in SDS

cGMP. 8-NEM-S-cGMP was conjugated to BSA via a succinyl coupling between the 2′-OH group of 2′-O-succinyl-8-NEM-S-cGMP and the lysine residues in BSA. Rats were immunized by using a subcutaneous injection of 8-NEM-S-cGMP conjugated with BSA plus TiterMax Gold adjuvant (TiterMax USA Inc., Norcross, GA). After the removal of contaminated anti-S-guanylated protein antibody via an S-guanylated BSA-conjugated HiTrap column (GE Healthcare), the specific 8-NEM-S-cGMP antibody was affinity purified from antiserum by using 8-NEM-S-cGMP-conjugated Toyopearl AF-Amino-650 M (particle size, 65 μm; Tosoh, Kyoto, Japan), which was prepared according to the manufacturer’s instructions with 2′-O-succinyl-8NEM-S-cGMP. Antibody specificity was confirmed with a competitive ELISA with a general protocol by using cGMP, cAMP, 8-nitro-GMP, 8-SH-cGMP, or 8-NEM-S-cGMP. 8-NEM-S-cGMP conjugated with chicken egg albumin was used as an antigen for the competitive ELISA (see Figure S1). Immunocytochemistry. Immunocytochemical analysis for intracellular 8-nitro-cGMP in rat CGNs was performed by using a previously described method.31 For the detection of cellular 8-SHcGMP levels, the cells were washed three times with ice-cold PBS and were fixed with methanol containing 5 mM EMCS, a cross-linking reagent with maleimide and N-hydroxysuccinimide groups, at room temperature for 10 min. After cells were washed three times with icecold PBS and incubated in PBS containing 5 mM EMCS at 4 °C overnight, they were incubated with BlockAce overnight at 4 °C to block nonspecific antigenic sites. After the cells were washed three times with TBS containing 0.05% Tween 20 (TBST), they were incubated in Can Get Signal Immunoreaction Enhancer Solution 1 containing anti-8-NEM-cGMP polyclonal antibody, which was prepared as described above, at 4 °C overnight. After the cells were rinsed three times with TBST, they were incubated at room temperature for 1 h with Alexa488-labeled goat anti-rat IgG antibody (Cell Signaling Technology Inc., Tokyo). The cells were washed three times with TBST, mounted in a mounting solution, covered with coverslips, and examined with a fluorescence microscope (FV1200; Olympus, Tokyo, Japan). The signals were detected with a confocal laser scanning microscope (FV1200; Olympus, Tokyo, Japan). Additional image processing and quantification were performed with Adobe Photoshop v. 7.0 (Adobe Systems, Waltham, MA). Quantification of 8-SH-cGMP and 8-Nitro-cGMP by LC-ESIMS/MS. 8-SH-cGMP and 8-nitro-cGMP were extracted from CGNs by methanol precipitation as reported previously.21,24 Briefly, cells were treated with 10 nM MeHg for 24 h, washed twice with ice-cold PBS, and homogenized in ice-cold methanol containing 5 mM NEM and 1 μmol 13C-labeled authentic 8-nitro-c[13C10]GMP for 8-nitrocGMP quantification, or were homogenized in ice-cold methanol containing 2% acetic acid and 1 μmol 13C-labeled authentic 8-SHc[13C10]GMP for 8-SH-cGMP quantification. The homogenate was centrifuged at 5,000g at 4 °C for 15 min, and the resultant supernatant was dried in vacuo and dissolved in distilled water for 8-nitro-cGMP analysis. For 8-SH-cGMP analysis, the supernatant was collected and subjected to anion-exchange purification with an Oasis WAX cartridge (Waters, Milford, MA). After the cartridge was washed with methanol, 8-SH-cGMP was collected in the elution with 1 mL of methanol containing 15% aqueous ammonia. The eluted sample was dried in vacuo and then dissolved in distilled water. Endogenous levels of 8-SHcGMP and 8-nitro-cGMP were quantified by means of LC-ESI-MS/ MS and a stable-isotope dilution method.10,22 A TSQ Vantage Electrospray Ionization Triple-Stage Quadrupole Mass Spectrometer (Thermo Fisher Scientific) was used after HPLC separation with a YMC-Triart C18 column (50 mm long ×2.0 mm inner diameter; YMC, Kyoto, Japan). The observed ion masses (parent → daughter ions) were m/z 391 → 197 and m/z 401 → 202 for endogenous 8nitro-cGMP and spiked 8-nitro-c[13C10]GMP, respectively, and m/z 378 → 184 and m/z 388 → 189 for endogenous 8-SH-cGMP and spiked 8-SH-c[13C10]GMP, respectively. The signals of the endogenous cGMP derivatives were identified simultaneously with the respective 13C-derivatives. LC-ESI-MS/MS Analysis for ROS Production. Intracellular ROS generation was analyzed by using LC-ESI-MS/MS using the oxidant1675


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Figure 1. nNOS worsens MeHg toxicity. (A) Analysis of the viability of PC12 cells, CGNs, and SH-SY5Y cells after MeHg exposure. Cell viability was determined after 24 h of incubation with the indicated concentrations of MeHg. The values are the means ± SD of four independent experiments. (B) nNOS protein expression in untreated PC12, CGNs, and SH-SY5Y cells. (C) Viability of PC12 and NPC12 cells after exposure to MeHg. The values are the means ± SD of four independent experiments. (D) nNOS protein expression in untreated PC12 and NPC12 cells. analysis of variance (ANOVA), two-way ANOVA, or unpaired t test via GraphPad Prism software (GraphPad, La Jolla, CA). p < 0.05 was considered significant.

sample buffer without 2-mercaptoethanol. After the samples underwent heat denaturing, they were subjected to Western blot analysis. Western Blot Analysis. To prepare cell lysate samples, cells were washed with ice-cold PBS three times and solubilized with ice-cold PBS containing 1% Triton X-100, proteinase inhibitor cocktail, and phosphatase inhibitor cocktail. Cell lysate proteins were heat denatured, separated by using SDS−PAGE, and then transferred to nitrocellulose membranes (Hybond-C; GE Healthcare, Little Chalfont, England). After membranes were blocked with Blocking One, they were incubated with primary antibodies. Primary antibodies used in the Western blot analysis were anti-nNOS,31 anti-S-guanylated protein,15 anti-ERK1/2 (Cell Signaling, Beverly, MA), anti-phosphoERK1/2 (Cell Signaling), anti-β-actin (Sigma-Aldrich), and anti-H-Ras (Santa Cruz Biotechnology, Dallas, TX). The membranes were incubated again for 1 h with HRP-conjugated secondary antibodies. The immunoreactive bands were detected by using a chemiluminescence reagent (SuperSignal Reagent; Thermo Fisher Scientific, Osaka, Japan) and a luminescent image analyzer (LAS-1000 mini; Fujifilm, Tokyo, Japan). The optical density of the film was scanned and quantified with ImageJ software. Animal Experiments. We used male Wistar rats (6−7 weeks old) in this study. Treatment of the animals followed the method of Shinyashiki et al. with a slight modification.6 Rats were housed in plastic cages and given free access to food and water. Rats received MeHg [10 mg/(kg·day)] by intraperitoneal injection for 8 days. All animal protocols were approved by the University of Tsukuba Animal Care and Use Committee and were performed with strict adherence to its guidelines for alleviation of suffering. Rats were killed and perfused with 4% paraformaldehyde in PBS at days 2, 5, 8, and 11 after MeHg injections, and whole brains were removed. To detect 8-nitro-cGMP and S-guanylated proteins in rat cerebellum, tissue was fixed in buffered 4% paraformaldehyde for 4 h and then embedded in paraffin and analyzed by using the immunohistochemical procedures described below. Immunohistochemistry. Immunohistochemical analysis of paraffin-embedded tissue sections was performed as previously described,6 with antibodies against or recognizing 8-nitro-cGMP15 and Sguanylated proteins,15 followed by HRP-labeled secondary antibodies. Reactions were visualized with a diaminobenzidine substrate system (Nichirei, Tokyo, Japan). Immunostained samples were counterstained with hematoxylin and mounted with SUPER Mount (Matsunami Glass Industry, Osaka, Japan). Statistical Analysis. All experiments were performed at least three times. The values for individual experiments are presented as the means ± SD. Statistical significance was determined by using one-way

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RESULTS Neuronal NOS (nNOS) Exacerbates MeHg Toxicity. Although involvement of nNOS in MeHg-induced toxicity has been reported,5,35,36 the mechanism of the cytotoxicity is still unclear. To examine whether nNOS can participate in MeHg cytotoxicity, we compared MeHg sensitivity in rat pheochromocytoma PC12 cells, human neuroblastoma SH-SY5Y cells, and rat primary CGNs, all of which express different levels of nNOS (Figure 1). MeHg sensitivity was higher in CGNs, SHSY5Y cells, and PC12 cells, in that order, in parallel with the intracellular nNOS protein levels (Figure 1A and B), with LC50 values of 1.94 μM, 2.83 μM, and 14.25 μM, respectively. To clarify the association of nNOS with MeHg toxicity, we studied nNOS-expressing PC12 (NPC12) cells (Figure 1D). Sensitivity of NPC12 cells to MeHg (LC50 = 2.34 μM) was much higher than that of PC12 cells (Figure 1C), which indicated that nNOS worsens the MeHg cytotoxicity. Because nNOS produces not only NO but also ROS via uncoupling reactions,31,37 nNOS-derived NO and/or ROS signals may be involved in enhancing the MeHg toxicity. In fact, the brain region most susceptible to MeHg cytotoxicity was reportedly the cerebellum, in which nNOS is highly expressed.38,39 Therefore, we believed that the primary cultured CGNs were an appropriate model for our MeHg exposure experiments as described below. MeHg Disrupts Reactive Persulfide-Dependent Regulation of Redox Signaling. Because reactive persulfide species possess potent antioxidant and redox-related signaling functions as a result of strong nucleophilicity, these species are likely involved in the electrophilic cytotoxicity of MeHg. In our studies here, we investigated the relationship of reactive persulfide species (e.g., hydrogen persulfide and cysteine persulfide) to MeHg toxicity in CGNs. We analyzed the intracellular reactive persulfide species status in MeHg-treated CGNs by using the sulfane sulfur-specific fluorescent probe SSP4. The levels of intracellular reactive persulfide species in MeHg-treated CGNs significantly decreased in a time-depend1676


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Figure 2. MeHg modulates the metabolism of reactive persulfide species. (A) Fluorescence microscopic analysis of the formation of reactive persulfide species in MeHg-treated rat CGNs. The graph at the right shows quantification of this formation. Scale bars, 500 μm. (B) Immunocytochemistry of 8-SH-cGMP formation in MeHg-treated rat CGNs. The graph at the right shows quantification of 8-SH-cGMP formation. Scale bars, 100 μm. (C) LC-ESI-MS/MS quantification of 8-SH-cGMP formation in MeHg-treated CGNs. Representative LC-ESI-MS/MS chromatograms of spiked 13C-labeled authentic 8-SH-c[13C10]GMP and endogenous 8-SH-cGMP are shown at the left. Methanol extracts were obtained from CGNs without MeHg treatment (control) and CGNs treated with 10 nM MeHg for 24 h. Results are presented as the means ± SD (n = 3). *p < 0.001 compared with control (0 h); one-way ANOVA with Tukey’s multiple comparisons test or an unpaired t test.

ROS in a primary culture of CGNs. Excess formation of superoxide anion in MeHg-treated CGN cells was confirmed not only by the DHE fluorescence imaging but also by truly quantitative LC-ESI-MS/MS analysis for the DHE and superoxide reaction (Figures 3A and S4), a result that is consistent with the findings of previous reports.43 Moreover, LC-ESI-MS/MS analysis with the DAN assay indicated that intracellular NO levels increased after exposure to MeHg (Figure S5). Certain studies revealed that ROS-derived redox signaling is a key modulator of oxidative stress.12,14 Such signaling is precisely regulated by endogenous electrophilic substances such as 8-nitro-cGMP, which are generated in a manner dependent on ROS and NO production.15,21,31,46 We therefore studied the effects of MeHg on redox signaling and formation of 8-nitrocGMP by using immunofluorescence staining and LC-ESI-MS/ MS. Both analyses showed that MeHg treatment increased 8nitro-cGMP formation in CGNs (Figure 3B and C). We also observed MeHg-induced enhancement of 8-nitro-cGMP production in NPC12 cells but not PC12 cells (Figure S6). These results indicate that 8-nitro-cGMP formation, stimulated by MeHg in CGNs, depended on nNOS expression. Additionally, we examined the effects of NOS inhibitor L-NAME and PEG-SOD (a membrane-permeable superoxide scavenger) on 8-nitro-cGMP accumulation and cell death induced by MeHg. The treatment of L-NAME or PEG-SOD significantly reduced MeHg-induced 8-nitro-cGMP accumulation (Figure 4A). We also found that the treatment of L-NAME or PEG-SOD

ent manner (Figure 2A). Further rigorous analysis by LC-ESIMS/MS revealed that the cellular amounts of reactive persulfide species including cysteine persulfide, glutathione persulfide, and dihydrogen persulfide were reduced appreciably after exposure to MeHg (Figure S2). Reactive persulfide species react quite effectively with 8-nitrocGMP to form 8-SH-cGMP, with the release of a nitrite anion.10,17,18,22 In view of this unique 8-nitro-cGMP reaction with persulfides, we analyzed intracellular 8-SH-cGMP, which provides evidence of the 8-nitro-cGMP and persulfide reaction. Reactive persulfide species and 8-SH-cGMP signals were both reduced by MeHg treatment (Figure 2A and B). LC-ESI-MS/ MS analysis confirmed that exposure to MeHg reduced 8-SHcGMP formation (Figure 2C). Our previous study found that MeHg reacted readily with reactive persulfide species to form (MeHg)2S, a detoxified metabolite of MeHg.29,40 We thus investigated the intracellular formation of (MeHg)2S in CGNs exposed to MeHg by using HPLC-AAS. As Figure S3 shows, exposure of CGNs to MeHg resulted in intracellular (MeHg)2S formation, which suggests that the lower levels of reactive persulfide species and 8-SHcGMP formation mediated by MeHg exposure in CGNs are associated with the capture of reactive persulfide species by this environmental electrophile. Endogenous Electrophilic Signaling Is Activated by MeHg Exposure. Several papers reported that ROS and NO are critically involved in MeHg-induced electrophilic toxicity.6,35,36,41−45 We first determined the level of intracellular 1677


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Figure 3. MeHg modulates redox signaling. (A) Fluorescence microscopic analysis of ROS formation in MeHg-treated rat CGNs. The graph at the right shows the quantification of ROS formation. Scale bars, 100 μm. (B) Immunocytochemistry of 8-nitro-cGMP formation in MeHg-treated rat CGNs. The graph at the right shows quantification of 8-nitro-cGMP formation. Scale bars, 100 μm. (C) LC-ESI-MS/MS quantification of intracellular 8-nitro-cGMP in MeHg-treated CGNs. Representative LC-ESI-MS/MS chromatograms of spiked 13C-labeled authentic 8-nitroc[13C10]GMP and endogenous 8-nitro-cGMP are shown at the left. Methanol extracts were obtained from CGNs without MeHg treatment (control) and CGNs treated with 10 nM MeHg for 24 h. Results are presented as the means ± SD (n = 3). *p < 0.001 compared with control (0 h); one-way ANOVA with Tukey’s multiple comparisons test or an unpaired t test.

canceled the cell death induce by MeHg (Figure 4B). These results suggest that 8-nitro-cGMP accumulation and cell death induced by MeHg were mediated by the NO/ROS-dependent signal pathway. In light of the present findings from our in vitro experiments, we used immunohistochemistry to investigate the level of 8nitro-cGMP (Figure 5A) and S-guanylated proteins (Figure 5B) in animal models of MeHg intoxication. MeHg-exposed rats demonstrated a gradual increase in 8-nitro-cGMP and Sguanylated protein signals in granule cells and Purkinje cells by 5 days after MeHg administration. However, the signals decreased in day 8 and day 11 samples. Also, on day 8 and day 11, both granule and Purkinje cells manifested morphological changes. MeHg-Induced Cell Impairment Is Mediated by H-Ras and ERK Signaling. Our previous report demonstrated that 8nitro-cGMP simultaneously S-guanylated and activated H-Ras, which promoted the phosphorylation of MEK and ERK and led to cellular senescence in cardiac tissue after myocardial infarction.22 The finding that 8-nitro-cGMP accumulated in CGNs after MeHg treatment, as just mentioned, prompted us to see whether MeHg intoxication would trigger such a pathway in CGNs. We thus studied the activation of H-Ras by performing an active Ras pulldown assay (Figure 6A). Exposure to MeHg led to S-guanylation and activation of H-Ras protein. The S-guanylated H-Ras then activated downstream phosphorylation signals including MEK and ERK. In this study, we also used Western blotting to investigate the phosphorylation of ERK in CGNs exposed to MeHg. As Figure 6B shows, MeHg

exposure significantly enhanced ERK phosphorylation. To determine whether H-Ras signaling is involved in MeHginduced cell damage, we treated CGNs with an MEK inhibitor (PD98059) and then evaluated its cytoprotective effects after MeHg exposure. As Figure 6C illustrates, pretreatment of the cells with the MEK inhibitor partially attenuated MeHg toxicity in CGNs, which indicates that MeHg toxicity to CGNs is, at least in part, due to the activation of the H-Ras/ERK signaling pathway. Reactive Persulfide Species Attenuate MeHg Toxicity. To confirm the relationship of reactive persulfide species to MeHg-induced cell damage, we studied the effect of a synthetic reactive persulfide species donor, sodium tetrasulfide (Na2S4), on electrophilic toxicity induced by MeHg in CGNs. After Na2S4 treatment of the cells, we removed Na2S4 by washing and then performed MeHg treatment, followed by assessment of the intracellular reactive persulfide species, phosphorylated ERK, and cell viability. Pretreatment with Na2S4 attenuated the MeHg-induced reduction of intracellular reactive persulfide species (Figure 7A). We then examined the effect of the reactive persulfide species donor on ERK phosphorylation after MeHg treatment. As Figure 7B illustrates, Na2S4 pretreatment suppressed the increase in ERK phosphorylation in MeHgexposed CGNs. We also studied the effect of this reactive persulfide species donor on MeHg-induced cell damage and determined that Na2S4 pretreatment lessened the MeHg cytotoxicity (Figure 7C). These results suggest that intracellular reactive persulfide species play a crucial role in ameliorating electrophilic damage induced by MeHg. 1678


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NO production, but it also produces ROS via an uncoupling reaction.31,37 A previous study demonstrated that MeHg increased the intracellular Ca2+ concentration via voltagedependent Ca2+ channels in CGNs, which presumably led to the activation of nNOS to produce NO and ROS. ROS and RNS are well-known to cause nonspecific destructive modifications of biological molecules.13,51 Indeed, various experiments with brain tissue and in vitro neuronal models have demonstrated increased ROS production after MeHg exposure.41,42,45 Moreover, numerous studies implicated mitochondria as an MeHg target organelle, and such an interaction between mitochondria and MeHg results in mitochondrial respiratory failure, with consequent excessive amounts of ROS.43,44,47,52−54 Taken together, these data suggest that ROS formation is crucial to cell damage and cell death mediated by MeHg and that ROS in concert with RNS are responsible for the enhanced MeHg-induced cell damage. Our group discovered that the reactive persulfide species produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are probably associated with certain critical regulatory biological functions of antioxidant and ROS stress signaling.10 Reactive persulfide species exhibited stronger nucleophilicity compared with that of monothiols such as GSH and cysteine (CysSH) and may have been involved in the negative feedback mechanism of redox signaling by readily reacting with electrophiles including 8-nitro-cGMP.10 Nucleophiles such as hydropersulfides were vital as regulatory intermediates in converting 8-nitro-cGMP into the stable derivative 8-SH-cGMP.10,17,18,22 Thus, the presence of 8-SHcGMP is important for identifying 8-nitro-cGMP metabolism mediated by reactive persulfide species. In fact, our data (Figure 2) clearly showed that both reactive persulfide species and 8SH-cGMP levels were significantly reduced in MeHg-treated CGNs, which suggests that the MeHg-induced reduction in reactive persulfide species levels inhibits conversion of 8-nitrocGMP to 8-SH-cGMP. Reactive persulfide species react readily with MeHg to give (MeHg)2S, which is a detoxified metabolite of MeHg.29,40 Our previous cell-free experiments with MeHg and reactive persulfide species indicated that (MeHg)2S is the common product after reactions with sodium hydrogen sulfide (NaHS), glutathione hydropersulfide, Na2S4, and even protein-bound reactive persulfide species,29 which suggests that MeHg is an environmental electrophile that can capture reactive persulfide species produced in cells and in vivo. In other words, formation of (MeHg)2S in CGNs after MeHg exposure, as Figure S3 shows, occurred as a result of the interaction of MeHg with reactive persulfide species that regulate the metabolism of 8nitro-cGMP. Overall, our present study suggests that MeHgrelated elimination of endogenously formed and stored reactive persulfide species leads to substantial accumulation of 8-nitrocGMP in MeHg-treated CGNs. Accumulating evidence has indicated that ROS and NO mediate redox signaling during both physiological and pathological responses.11−13 Redox signaling is fine-tuned by endogenous electrophilic substances, such as 8-nitro-cGMP, that are generated from ROS and NO during oxidative stress responses.8,15−20 In fact, we previously discovered that the nitrated cyclic nucleotide 8-nitro-cGMP, as a second messenger of redox signaling, formed from NO and ROS.15,21,31,46 We observed herein a clear increase in ROS, NO, and 8-nitrocGMP formation after MeHg exposure (Figures 3 and S5). In this regard, it is important to mechanistically understand how

Figure 4. Effects of NO and ROS suppression on MeHg-induced 8nitro-cGMP accumulation and cytotoxicity. (A) The effects of an NOS inhibitor (0.2 mM L-NAME) and a membrane-permeable SOD (200 units/ml PEG-SOD) on 8-nitro-cGMP formation in MeHg-treated rat CGNs. CGNs were exposed to 2 μM MeHg for 4 h in the presence or absence of L-NAME or PEG-SOD. 8-Nitro-cGMP formation was analyzed by immunocytochemistry. Scale bars, 100 μm. (B) The effect of L-NAME (0.2 mM) or PEG-SOD (200 units/mL) treatment on cell viability after MeHg exposure. CGNs were exposed to 2 μM MeHg for 24 h in the presence or absence of L-NAME or PEG-SOD. *p < 0.01, # p < 0.01 compared with the control and MeHg treatment, respectively; one-way ANOVA with Tukey’s multiple comparisons test.

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DISCUSSION In this study, we used MeHg as an environmental electrophile and investigated the molecular mechanism of environmental electrophile-induced cell impairment from the viewpoint of redox signaling regulated by reactive persulfide species. The main findings in this study are the following: (i) MeHg disrupted reactive persulfide species-dependent metabolism of endogenous redox signaling molecules derived from nNOS. (ii) MeHg caused intracellular accumulation of 8-nitro-cGMP, which resulted in activation of the downstream H-Ras/ERK pathway and led to the injury of CGNs. (iii) Pretreatment with the reactive persulfide species donor Na2S4 attenuated the MeHg-induced cell damage. These data therefore suggest that reactive persulfide species may be applied to the prevention and treatment of exogenous and endogenous electrophile-related diseases, such as inflammation, infection, cancer, atherosclerosis, and neurodegeneration. In the past two decades, several studies showed that MeHg toxicity leads to the loss of CGN function and cell death.38,43,47 To date, several mechanisms were suggested as underlying the MeHg toxicity in CGNs, including inhibition of RNA and protein synthesis, inhibition of insulin-like growth factor I, translocation of apoptosis-inducing factor from mitochondria to the nucleus, increased membrane lipoperoxidation, and intracellular Ca2+ channel dysregulation.48,49 Although MeHg toxicity was also reportedly associated with increased nNOS activity and even oxidative stress,6,35,36,50 the molecular mechanisms of the association of nNOS with MeHg have not been fully elucidated. In this study, we first demonstrated that nNOS enhanced MeHg toxicity in several cultured cells (Figure 1). nNOS is a Ca2+-dependent enzyme that is responsible for 1679


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Figure 5. 8-Nitro-cGMP and S-guanylated protein formation in the cerebellum of a model animal exposed to MeHg. Immunohistochemical analysis of 8-nitro-cGMP generation (A) and S-guanylated protein expression (B) in the brains of MeHg-exposed samples. Scale bars, 100 μm.

Figure 6. MeHg activated H-Ras signaling. (A) Western blot analysis of MeHg-induced H-Ras S-guanylation and activation. Densitometric analysis of the band intensity appears below. (B) Western blot analysis of MeHg-induced ERK phosphorylation. Densitometric analysis of the band intensity appears below. (C) Viability assay of MeHg-treated CGNs after the use of the MEK inhibitor PD98059. CGNs were pretreated with the indicated concentrations of PD98059 for 1 h, followed by MeHg treatment (0 or 2 μM) for 24 h. Results are presented as the means ± SD (n = 3). *p < 0.001, # p < 0.001 compared with the control and MeHg treatment, respectively; one-way ANOVA with Tukey’s multiple comparisons test.

reactive hydropersulfides that are decomposed readily by environmental chemicals, e.g., MeHg. In fact, we clarified earlier that the amounts of 8-nitro-cGMP formed in the cells,

the endogenous weak electrophile 8-nitro-cGMP is regulated physiologically, in terms of its effective intracellular concentrations, as affected by endogenous potent nucleophiles like 1680


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Figure 7. Effects of reactive persulfide species on MeHg cytotoxicity. (A) Fluorescence microscopic analysis of the effects of Na2S4 treatment on reactive persulfide species formation in MeHg-exposed rat CGNs. CGNs were treated with 50 μM Na2S4 for 10 min and then washed to remove Na2S4, followed by exposure to MeHg (0 or 2 μM) for 24 h. The graph at the right shows quantification of reactive persulfide species-related signal intensities. Scale bars, 500 μm. (B) Western blot analysis of the effect of reactive persulfide species treatment on MeHg-induced ERK phosphorylation. Densitometric analysis appears right. CGNs treated with Na2S4 similar to that in A were exposed to MeHg (0 or 2 μM) for 24 h. (C) Viability assay of Na2S4-treated CGNs after MeHg exposure. CGNs were treated with 25 μM Na2S4 for 30 min and washed to remove Na2S4, followed by exposure to the indicated concentrations (0.25 and 0.5 μM) of MeHg for 24 h. Results are presented as the means ± SD (n = 3). *p < 0.01, #p < 0.01 compared with the control and MeHg treatment, respectively; one-way ANOVA with Tukey’s multiple comparisons test.

adducts rather than CysS-electrophile adducts.10,57 Such polysulfide adducts are reportedly reduced by the thioredoxin system.58 It may be possible that MeHg with strong electrophilicity disrupts polysulfide moieties on protein CysSH and directly alkylates the CysSH residues, which may modulate redox signal functions of the proteins. As for the HRas redox signaling, however, this model is not feasible. In fact, our previous study showed that only endogenous electrophile, i.e., 8-nitro-cGMP can activate the H-Ras signaling pathway via S-guanylation of Cys184 but that other electrophiles such as MeHg cannot do the same activation, suggesting that 8-nitrocGMP may function as a specific ligand for H-Ras signaling.22 Therefore, the present model for the electrophilic H-Ras activation is only valid for the 8-nitro-cGMP signaling, which may be regulated in a manner independent of the protein polysulfidation potentially affected by MeHg. Reactive persulfide species precisely regulate redox signaling mediated by 8-nitro-cGMP.10,17,18,22 We previously reported that long-term treatment of mice with NaHS suppressed Sguanylation and activation of H-Ras, myocardial cell senescence, and left ventricular dysfunction in hearts after myocardial infarction by eliminating the accumulation of 8nitro-cGMP.22 While NaHS barely eliminated electrophiles in vitro, a novel metabolic pathway producing nucleophilic reactive persulfide species identified recently is likely to contribute to the electrophile elimination.59 We demonstrated here that MeHg causes toxicity by the disruption of reactive persulfide species metabolism and activation of 8-nitro-cGMP-mediated redox signaling. Therefore, we expected that an improvement in reactive persulfide species metabolism would alleviate electrophilic toxicity by decreasing the redox signal. In this study, we pretreated cells with the reactive persulfide species donor Na2S4, and we found significant improvements in reactive persulfide species conditions, ERK activation, and cell viability in MeHg-exposed cells (Figure 7). These data suggest that reactive persulfide species serve as valuable therapeutic targets

which can specifically activate cellular signaling, ranged from hundreds fmol/mg protein.21,22,55 In the present study, 8-nitrocGMP was found to be produced at about 100 fmol/mg protein after exposure to MeHg, indicating that 8-nitro-cGMP accumulated after MeHg treatment was considered to be physiologically relevant because of its strong activating potential for the downstream H-Ras signal pathway. Furthermore, we performed in vivo experiments to determine 8-nitro-cGMP and S-guanylated protein levels by using a rat model of MeHg toxication. As Figure 5 shows, 8-nitro-cGMP and S-guanylated protein signals increased moderately in both CGNs and Purkinje cells by day 5, after which these signals disappeared, as did the cells. Loss of Purkinje cells has been observed as a typical pathological feature in people with MeHg poisoning.38,39 Our results strongly suggest a contribution of 8nitro-cGMP to MeHg toxicity in in vitro neuronal models and in vivo as well. 8-Nitro-cGMP was reportedly involved in post-translational modification of several proteins including Keap1,15,21 H-Ras,22 heat shock proteins,23 synaptosomal-associated protein 25,24 cGMP-dependent protein kinase,25 and tau protein26 through a process known as S-guanylation. Protein S-guanylation in bacteria was also observed.27,28 In this study, we focused mainly on H-Ras, which is an 8-nitro-cGMP-specific protein target. We found that the levels of S-guanylated and activated H-Ras in MeHg-exposed CGNs increased in a time-dependent manner (Figure 6A). We also observed markedly increased ERK phosphorylation after exposure of CGNs to low levels of MeHg, in agreement with several earlier findings.56 However, as Figure 6C illustrates, the major cell loss was reduced if cells were pretreated with an MEK inhibitor (PD98059), which indicates that MeHg exerted its toxicity, at least in part, via HRas signaling. It was recently proposed that polysulfidation of protein CysSH residues remarkably affects electrophile-induced CysSH modification through the formation of CysS-Sn-electrophile 1681


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Figure 8. Possible mechanism of MeHg-induced cell impairment in neuronal cells. Under physiological conditions, CBS/CSE-derived reactive persulfide species regulate redox signaling via the metabolism of endogenous electrophiles (e.g., 8-nitro-cGMP), which results in inactivation of the 8-nitro-cGMP-dependent signaling pathway via metabolic conversion to form 8-SH-cGMP. MeHg diffuses through cytoplasmic membranes and causes increased NO/ROS production several ways, such as mitochondrial dysfunction and effects on several types of Ca2+ channels to induce Ca2+ influx that triggers nNOS activation. In addition, MeHg interacts with reactive persulfide species to disrupt the mechanism for regulating electrophilic signaling, which results in the accumulation of intracellular 8-nitro-cGMP. Finally, increased 8-nitro-cGMP activates the H-Ras/ERK cascade via Sguanylation of H-Ras, which causes the impairment of neuronal cells.

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for MeHg and other environmental electrophilic toxicants as well. In conclusion, our findings reveal a novel mechanism of electrophilic cell impairment induced by an environmental electrophile MeHg (Figure 8). MeHg extensively degrades endogenously produced reactive persulfide species and activates NO and ROS signals via nNOS and/or mitochondria, which results in the accumulation of the endogenous electrophile 8nitro-cGMP. Consequently, accumulated 8-nitro-cGMP activates the H-Ras/ERK pathway and leads to neuronal cell impairment. Therefore, environmental electrophiles as antioxidants and redox signal disruptors may contribute to the pathogenesis and promotion of many diseases that affect humans who have long-term exposure to environmental electrophilic stress.

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AUTHOR INFORMATION

Corresponding Authors

*(H.I.) Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka 599-8531, Japan. Phone: +81-72-254-9753. Fax: +81-72-254-9753. E-mail: [email protected]. *(T.A.) Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Miyagi Prefecture, Aoba-ku, Seiryo-cho, 2-1, Sendai 980-8575, Japan. Phone: +81-22-717-8164. Fax: +81-22-7178219. E-mail: [email protected]. ORCID

Yoshito Kumagai: 0000-0003-4523-8234 Takaaki Akaike: 0000-0002-0623-1710 Author Contributions ∇

H.I. and S.K. contributed equally to this work. H.I., S.K., and T.A. designed the study; H.I., S.K., A.K., A.N., H.T., T.I., K.I., T.T., E.Y., H.A.H., M.J., and T.M. performed the research; H.I., S.K., S.F., T.S., M.N., Y.K., and T.A. analyzed the data; and H.I., S.K., and T.A. wrote the paper.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00120. MRM parameters for various HPE-IAM adducts; competitive ELISA of the rat polyclonal antibody and 8-NEM-S-cGMP; reactive polysulfide species in MeHgtreated CGNs; detection of (MeHg)2S formation in MeHg-exposed CGNs by means of HPLC-AAS; LC-ESIMS/MS analysis of ROS formation in MeHg-treated rat CGNs; LC-ESI-MS/MS analysis of NO formation in MeHg-treated rat CGNs; and dependence of nNOS on MeHg-induced 8-nitro-cGMP production in PC12 and NPC12 cells (PDF)

Funding

This work was supported, in part, by Grants-in-Aid for Young Scientists B (to S.K., [15K20855]), Scientific Research C (to S.F., [15K08456]), Scientific Research B (to H.I., [16H04674]; to T.S., [15H03115]), Scientific Research A (to T.A., [25253020]), Scientific Research S (to Y.K., [25220103]), and Challenging Exploratory Research (to T.A., [16K15208]); a grant from the Japan Science and Technology Agency Precursory Research for Embryonic Science and Technology program (to T.S., [10104025]); and Grants-in-Aid for Scientific Research on Innovative Areas (Research in a Proposed Area) (to T.A., [26111008, 26111001]; to Y.K., [15H01392]) from the Ministry of Education, Sciences, Sports, and Technology, 1682


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Japan, and Nagase Science and Technology Foundation (to H.I.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank J. B. Gandy for her excellent editing of the manuscript before submission. We also thank Y. Fujiwara and M. Takeya for their technical assistance.

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ABBREVIATIONS MeHg, methylmercury; ROS, reactive oxygen species; RNS, reactive nitrogen species; 8-nitro-cGMP, 8-nitroguanosine 3′,5′-cyclic monophosphate; 8-SH-cGMP, 8-mercaptoguanosine 3′,5′-cyclic monophosphate; Keap1, Kelch-like ECHassociated protein 1; CGNs, cerebellar granule neurons; (MeHg)2S, bismethylmercury sulfide; nNOS, neuronal NOS; NPC12, nNOS-expressing PC12; LC-ESI-MS/MS, liquid chromatography−electrospray ionization−tandem mass spectrometry; AAS, atomic absorption spectrometry; DHE, dihydroethidium; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; MEM, minimum essential medium; EMCS, N-(6-maleimidocaproyloxy)succinimide; HS, horse serum; NEM, N-ethylmaleimide; ANOVA, analysis of variance

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