Capture of cadmium by reactive polysulfides attenuates cadmium

stress response proteins, through activation of signal transduction pathways triggered ..... induction of adaptive response proteins is an indicator o...
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The Capture of Cadmium by Reactive Polysulfides Attenuates Cadmium-Induced Adaptive Responses and Hepatotoxicity Masahiro Akiyama,† Yasuhiro Shinkai,† Takamitsu Unoki,† Ilseob Shim,‡ Isao Ishii,§ and Yoshito Kumagai*,† †

Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan Department of Environmental Health Research, National Institute of Environmental Research (NIER), Environmental Complex, Gyungseodong, Seogu, Incheon 22689, Korea § Laboratory of Health Chemistry, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan ‡

S Supporting Information *

ABSTRACT: Cadmium (Cd) is an environmental electrophile that modifies protein nucleophiles, thereby modulating cellular signaling and toxicity. While reactive persulfides/ polysulfides exhibit relatively high nucleophilic properties, their roles in the altered gene expression and toxicity caused by Cd remain unclear. Exposing primary mouse hepatocytes to Cd caused heat shock protein 70 (HSP70) and metallothionein (MT)-I/II to be upregulated and cytotoxicity to occur. These effects were blocked in the presence of polysulfide sodium tetrasulfide (Na2S4). Electrospray ionization mass spectrometry analysis indicated that cadmium sulfide (CdS) and cadmium thiosulfate (CdS2O3) were produced when Cd reacted with Na2S4. Authentic CdS did not cause cellular signaling responses to be activated or hepatotoxic effects, while CdS2O3 had effects similar to those of Cd. HSP70 and MT-I/II upregulation and hepatotoxicity caused by exposure to Cd were significantly enhanced by the deletion of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive persulfides/polysulfides. Deleting CSE also exacerbated Cd-mediated liver injury, whereas little hepatic damage was found when CdS or Na2S4 along with Cd was administered. Overall, the results suggest that the persulfide/polysulfide-mediated formation of sulfur adducts of Cd such as CdS rather than CdS2O3 is, at least in part, involved in decreasing the level of Cd-mediated activation of cellular signaling and toxicity.

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INTRODUCTION Cadmium (Cd), an environmental electrophile, is a toxic metal present in tobacco smoke and in foods such as rice. It can enter the human body through inhalation and ingestion.1 Because of its electrophilic reactivity, Cd reacts readily with protein nucleophiles, resulting in protein adduct formation. Effects of such modifications on disruption of cellular homeostasis are, at least in part, considered to cause Cd toxicity.2 On the other hand, the electrophilic properties of Cd are also involved in induction of stress response proteins, through activation of signal transduction pathways triggered by electrophilic modification of sensor proteins. For example, heat shock protein 70 (HSP70) acts as a chaperone for protein folding to prevent irreversible aggregation of proteins in non-native conformations, thereby protecting cells from injury.3 We recently reported that Cd binds to thiol groups in HSP90, which acts as a negative regulator for heat shock factor 1 (HSF1), leading to weakening of the interaction of HSP90 with HSF1, thereby substantially activating HSF1 to induce expression of HSP70.4 The induction of metallothionein (MT) expression is also regarded as an initial protective response to Cd, because MT can capture this heavy metal by allowing it to displace zinc on its thiol groups. Cd was reported to cause the release of zinc from cellular proteins, thereby © 2017 American Chemical Society

activating metal-responsive transcription factor 1 (MTF1) to induce downstream MT genes.5−8 In addition to cellular response systems using sensor proteins, there is another type of defense system for inactivating electrophiles, through interaction with sulfur-containing lowmolecular weight nucleophiles such as cysteine (CysSH) and glutathione (GSH).9−11 We recently found that cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) contributed to production of CysSH persulfide (CysSSH) and its related reactive persulfide/polysulfides (e.g., GSSH, CysSSSH, GSSSH, HSSH, and HSSSH).12 These reactive species have high nucleophilicity, compared with those of their corresponding thiol-containing compounds. Such reactive persulfides/polysulfides contain mobilized sulfur atom(s) bound to the thiol groups of CysSH or GSH, and this sulfane sulfur in persulfides/ polysulfides can react with electrophilies to form sulfur adducts.13−15 For example, we showed that methylmercury (MeHg), another electrophilic metal, reacted with hydrogen sulfide16 and reactive persulfides/polysulfides, such as sodium tetrasulfide (Na2S4) and GSSSG, to form bismethylmercury sulfide [(MeHg)2S], which is non-electrophilic and less toxic Received: October 9, 2017 Published: November 8, 2017 2209

DOI: 10.1021/acs.chemrestox.7b00278 Chem. Res. Toxicol. 2017, 30, 2209−2217

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Chemical Research in Toxicology than MeHg.17 Furthermore, (MeHg)2S was identified as a novel detoxified MeHg metabolite in SH-SY5Y cells exposed to MeHg, as well as in liver tissues of rats to which MeHg had been administered.16 Low appreciable levels of (MeHg)2S were found in a variety of tissues of CSE knockout (KO) mice exposed to MeHg, suggesting that CSE plays an important role in the formation of sulfur adducts with electrophiles in vivo.17 We also demonstrated that 1,4-naphthoquinone (1,4-NQ), an atmospheric electrophile, reacted with Na2S4 to form a sulfur adduct that is non-electrophilic and less toxic than 1,4-NQ.18 With regard to Cd, we recently reported that knockdown of CSE and CBS decreased cellular reactive persulfide/polysulfide levels and increased the level of induction of HSP70 and cytotoxicity, caused by Cd exposure in bovine aortic endothelial cells.4 These results suggest that reactive persulfides/polysulfides may capture Cd, leading to formation of its inert sulfur adducts, although such adducts were not identified. Here, we examine the ability of Na2S4 to protect against Cd-induced acute hepatotoxicity in vitro and in vivo and identify the products of the reaction between Cd and Na2S4. We also investigate the role CSE plays in the induction of stress proteins such as HSP70 and MT and in the acute hepatotoxicity of Cd in wild-type and CSE KO mice.

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(WT) mice were comparatively analyzed. Genotypes were confirmed by polymerase chain reaction using tail DNA. All animal experiments were performed humanely with the approval of the Institutional Animal Experiment Committee of the University of Tsukuba and in accordance with the University of Tsukuba’s Regulation for Animal Experiments and the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions, under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan. Preparation of Primary Mouse Hepatocytes. Primary hepatocytes were isolated from C57BL/6 background adult WT and CSE KO male (Figure 5) or female (except Figure 5) mice, as previously described.21 Cells were seeded at a density of 8 × 104 cells/ cm2 on type I collagen-coated 12-well plates (Corning Inc., Corning, NY) and cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2 in William’s Medium E containing 10% fetal bovine serum, 2 mM L-alanyl-L-glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL). Forty-eight hours after being isolated, cells were cultured in serum-free medium for 24 h and then treated with each chemical in serum-free medium. Western Blot Analysis. After treatments, cells were washed twice with ice-cold PBS (−). Total cell protein samples were prepared by lysis of cells in sodium dodecyl sulfate (SDS) sample buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, and 10% glycerol], followed by incubation at 95 °C for 10 min. Protein concentrations were determined using a BCA protein assay reagent kit (Pierce Biotechnology, Waltham, MA) before 2-mercaptoethanol and bromophenol blue were added to each sample. The cellular proteins were separated by SDS−PAGE, and bands were then electrotransferred onto a poly(vinyl difluoride) (PVDF) membrane (BioRad Laboratories, Hercules, CA) at 2 mA/cm2 for 1 h, as described previously (Kyhse-Andersen, 1984). Membranes were blocked with 5% skim milk in TTBS [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] and then incubated with primary antibodies for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (Chemi-Lumi One L, Nacalai Tesque, Kyoto, Japan) and detected with a LAS 3000 instrument (Fujifilm, Tokyo, Japan). Signal intensities were quantified with Multi Gauge software (Fujifilm). Cell Viability. The 3-(4,5-dimethylthiazol-2-yl)-2,5-triphenyltetrazolium bromide (MTT) assay was used to estimate cell viability, as described previously.22 Briefly, primary mouse hepatocytes in 96-well plates were exposed to CdCl2, with or without Na2S4 (10 or 100 μM), administered for 1 h before exposure or simultaneously with Cd. After 24 h, cells were treated with 5 mg/mL MTT for 4 h at 37 °C. After the medium had been removed, DMSO (100 μL/well) was added to dissolve the formazan precipitate. The absorbance at 540 nm was read with an iMark microplate reader (Bio-Rad Laboratories). Measurement of Cd Concentrations. Cd concentrations in primary mouse hepatocytes and mouse liver tissue samples were determined as previously described.23 Briefly, primary mouse hepatocytes were exposed to CdCl2 (5 μM), with or without Na2S4 (10 or 100 μM), or to CdS (5 μM). After the culture medium had been removed, 0.6 mL of nitric acid and 0.2 mL of H2O2 were added to each well, and samples were incubated overnight at room temperature. The solutions were then transferred to test tubes and digested at 130 °C for 2 days in an aluminum dry heating block. WT mice were intraperitoneally injected with CdCl2 (5 mg/kg), with or without Na2S4 (10 mg/kg), or with CdS (3.16 mg/kg). After 24 h, 50 mg liver tissue pieces were each transferred to a test tube and digested in 1.8 mL of nitric acid and 0.6 mL of H2O2 at 50 °C for 2 h and then at 130 °C for 3 days in an aluminum dry heating block. After being dried, samples were suspended to 0.1 N nitric acid, and Cd concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS) (ELAN DRC-e, PerkinElmer). Acute Hepatotoxicity Induction and Treatment. Adult CSE KO and WT mice were used, and acute hepatotoxicity was induced by intraperitoneal injection of CdCl2. CdCl2-injected WT mice were divided into three groups, receiving CdCl2 alone (5 mg of CdCl2/kg as a single ip dose), CdCl2 and Na2S4 (5 mg of CdCl2/kg and 10 mg of

EXPERIMENTAL PROCEDURES

Materials. Cadmium chloride (CdCl2), cadmium sulfide (CdS), cadmium sulfate (3CdSO4·8H2O), and barium thiosulfate (BaS2O3· H2O) were from Wako Pure Chemical Industries (Osaka, Japan). Sodium tetrasulfide (Na2S4) was from Alfa Aesar (Ward Hill, MA). Biotin-PEAC5-maleimide (BPM) was from Dojindo (Kumamoto, Japan). The anti-β-actin antibody was from Cell Signaling Technology (Beverly, MA). The anti-GAPDH antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-HSP70 antibody was from Stressgen (Collegeville, PA). The anti-MT-I/II antibody was from Dako (Carpinteria, CA). Recombinant human HSP90 (ADI-SPP-777) was from Enzo Life Sciences (Farmingdale, NY, USA). All other reagents and chemicals were of the highest grade available. Synthesis of Cadmium Thiosulfate. Cadmium thiosulfate (CdS2O3·2H2O) was obtained by reacting cadmium sulfate with barium thiosulfate. Briefly, an aqueous solution (30 mL) of cadmium sulfate (2.1 mmol) was added to an aqueous solution (270 mL) of barium thiosulfate (2.1 mmol). The mixture was stirred at room temperature for 10 min and then centrifuged at 5000g for 10 min at 4 °C. The supernatant was lyophilized to give a light-yellow-tinged powder. Elemental analysis using a PerkinElmer (Waltham, MA) 2400 II CHNS/O analyzer showed that the hydrogen and sulfur contents of the product were 1.4 and 19.2% (by mass), respectively, and that the carbon and nitrogen contents were negligible. ESI-MS. To analyze sulfur adducts of Cd, ESI-MS analysis (Synapt HDMS system, Waters, Milford, MA) was performed. ESI was used with a capillary voltage of 3.5 kV, a sampling cone voltage of 40 V, and a transfer cone voltage of 4 V. The source temperature was 100 °C, and the detector was operated in negative ion mode. Data were collected from m/z 50 to 1000 and analyzed with MassLynx, version 4.1, and MassFragment, version 1.1. BPM Labeling Assay. The BPM labeling assay was performed using a previously described method.19 Briefly, recombinant human HSP90 (1 μM) was reacted with CdCl2, CdS, or CdS2O3 (1, 10, or 100 μM) in 50 mM Tris-HCl (pH 7.5) at 25 °C for 30 min, and then the mixture was incubated with BPM (25 μM) at 25 °C for 30 min. The resulting sample was mixed with sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) loading buffer containing 50 mM tris(2-carboxyethyl)phosphine, and then the mixture was subjected to Western blotting analysis using anti-biotin horseradish peroxidase-conjugated antibodies. Mice. CSE KO mice were previously generated on a C57BL/6J background,20 and sex- and age-matched (12−16 weeks old) wild-type 2210

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Figure 1. Protective effects of Na2S4 on Cd-induced toxicity in vitro. (A) Primary mouse hepatocytes were treated with Na2S4 (100 μM) for 1 h before being exposed to CdCl2 (5 μM) with fresh medium for 6, 12, or 24 h (pretreat) or simultaneously exposed to CdCl2 and Na2S4 (100 μM) for 6, 12, or 24 h (co-treat). Total cell lysates were subjected to Western blotting using the indicated antibodies. Each value is the mean ± SE of three independent experiments. *p < 0.05 and **p < 0.01, compared with 0 h. (B) Primary mouse hepatocytes were treated with Na2S4 (10 or 100 μM) for 1 h before being exposed to CdCl2 with fresh medium at the indicated concentrations for 24 h (pretreat) or simultaneously exposed to CdCl2 and Na2S4 (10 or 100 μM) for 24 h (co-treat). The cell viability in each control sample was defined as 100%. Each value is the mean ± SE of three independent experiments. *p < 0.05 and **p < 0.01, compared with the control. (C) Primary mouse hepatocytes were simultaneously exposed to CdCl2 (5 μM), with or without Na2S4 (10 or 100 μM), for the indicated times. The cellular Cd content was determined by ICP-MS. Each value is the mean ± SE of three independent experiments. *p < 0.01. Na2S4/kg as a single ip dose), or CdS (the same Cd dose as the CdCl2 as a single ip dose). The control group received only equal volumes of distilled water under similar conditions. Mice were euthanized by pentobarbital injection at 24 h to collect blood plasma with heparin and liver tissue for alanine transaminase (ALT) and aspartate transaminase (AST) activity assays and histopathological analysis, respectively. AST and ALT Activity Assays. Blood was obtained by cardiac puncture for analysis of AST and ALT, as an index of hepatotoxicity. The AST and ALT assays were performed using the Transaminase CII Test Wako kit, according to the manufacturer’s instructions (Wako Pure Chemical Industries). Histopathological Analysis. Mice were intracardially perfused with 4% paraformaldehyde (PFA) in PBS. Livers were dissected and postfixed in the same solution at 4 °C overnight. The postfixed tissues were embedded in paraffin and sectioned at 3 μm using a microtome (REM-710, Yamato Kohki, Saitama, Japan) and then stained with hematoxylin and eosin (HE) by standard techniques. Statistical Analysis. Statistical significance was assessed with a Student’s t test or by one-way ANOVA, followed by Tukey’s or Holm’s post hoc tests, using Graphpad Prism (Graphpad Software, San Diego, CA) or KaleidaGraph (Synergy Software, Reading, PA). p < 0.05 or p < 0.01 was considered significant.

treatment with Na2S4 also suppressed the concentrationdependent cytotoxicity of CdCl2, although such a protective effect of pretreatment with Na2S4 was only modest (Figure 1B). These results indicate that both intracellular polysulfide and extracellular polysulfide contribute to protection against Cdinduced toxicity on one level or another. Significantly less intracellular Cd accumulation occurred in cells co-treated with Na2S4 and CdCl2 than in cells treated with only CdCl2 (Figure 1C). Formation of Sulfur Adducts of Cd by Reaction with Polysulfides. To examine whether Cd could interact with reactive polysulfides, leading to formation of its sulfur adducts, we used Na2S4 as a model polysulfide in this study. A reaction mixture containing CdCl2 and Na2S4 was analyzed by ESI-MS in negative ion mode. As shown in Figure 2, with CdCl2 alone, the chloride adduct ion (m/z 218.8) was detected, with characteristic Cd isotopes, whereas at least two signals, together with the characteristic Cd isotopes, were found when Na2S4 was added to the incubation mixture. One major peak had a m/z ratio of 180.8, presumably corresponding to Cd sulfide (CdS) adducts with chloride, and the other peak had a m/z ratio of 260.8, presumably corresponding to Cd thiosulfate (CdS2O3) adducts with chloride (Figure 2). The formation of CdS2O3 was confirmed by the same peaks being detected when CdCl2 was incubated with Na2S2O3 (Figure S1), indicating that aqueous Na2S4 partly decomposed to give S2O3 through the formation of H2S and the subsequent oxidation of the H2S.24 Consistent with this, both H2S and its oxidation product, S2O3, were detected when only Na2S4 was incubated [data not shown (see Scheme 1)]. Characterization of CdS and CdS2O3 in Vitro. Sulfur adducts of Cd, such as CdS and CdS2O3, appear to be slightly electrophilic compared to CdCl2, so CdS and CdS2O3 are less

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RESULTS Suppression of Cd-Induced Stress Protein Expression and Cytotoxicity by Na2S4 in Vitro. We examined the effects of Na2S4 on Cd-induced stress protein expression and cytotoxicity in primary mouse hepatocytes. Our rationale is that pretreatment with Na2S4 reflects the effect of intracellular polysulfide and that co-treatment with Na2S4 reflects the effect of both intracellular and extracellular polysulfide. As shown in Figure 1A, Cd-induced protein upregulation was blocked by pretreatment with Na2S4 for HSP70 and by co-treatment with Na2S4 for both HSP70 and MT-I/II. Pretreatment or co2211

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icity (Figure 4A,B). In addition, CdS (the same Cd dose as that of CdCl2) was less hepatotoxic than CdCl2 (Figure 4A,B), whereas CdS2O3 was hepatotoxic like CdCl2 (data not shown). On the basis of measurements of Cd accumulation, the Cd content in the liver was slightly decreased in mice treated with Na2S4 and CdCl2 and was markedly decreased in mice treated with CdS, compared with the Cd content of those receiving only Cd (Figure 4C). Suppression of Cd-Induced Stress Protein Expression and Cytotoxicity by CSE in Vitro. To examine the physiological role of endogenous reactive persulfides/polysulfides in Cd-induced stress protein induction and cytotoxicity, we used CSE KO mice. CSE is an enzyme that generates reactive persulfides/polysulfides by catalyzing the biosynthesis of CysSSH from cystine,12 while also catalyzing production of cysteine from cystathionine.29 We found that CSE deletion significantly enhanced induction of HSP70 and MT protein expression by Cd in primary mouse hepatocytes (Figure 5A). Also, CSE deletion resulted in a significant enhancement of the concentration-dependent cytotoxicity caused by CdCl2 exposure (Figure 5B). Under these conditions, intracellular Cd accumulation was not different in primary hepatocytes from WT and CSE KO mice (Figure S2A) . Role of CSE in Protection against Cd-Induced Hepatotoxicity in Vivo. We next examined the participation of CSE in protecting against Cd-induced acute hepatotoxicity in vivo. As shown in Figure 6A, treatment of WT mice with CdCl2 (4 mg/kg) did not cause liver damage, as evaluated by HE staining. However, treating CSE KO mice with the same dose of CdCl2 caused a liver damage as indicated by the dotted line (Figure 6A). Consistent with these results, plasma ALT and AST levels in CSE KO mice were higher than in WT mice after injection of CdCl2 (Figure 6B). Under these conditions, the amounts of Cd that accumulated in the livers of WT mice and CSE KO mice were not significantly different (Figure S2B).

Figure 2. Product analysis of the reaction mixture of Cd with Na2S4. Cadmium chloride (100 μM) was incubated with or without Na2S4 (100 μM) in water at 25 °C for 5 min. Aliquots of the reaction mixture were subjected to ESI-MS in direct injection mode.

toxic and less able to cause chemical modifications than CdCl2. To address such a possibility, a model sensor protein HSP904 was incubated with CdCl2, CdS, and CdS2O3, and then the BPM labeling assay was performed. As expected, CdS was not reactive with HSP90, but CdCl2 modified HSP90 proteins in a concentration-dependent manner (Figure 3A). However, CdS2O3 modified HSP90 in the same way that CdCl2 did, indicating that, unlike CdS, CdS2O3 is not a stable sulfur adduct of Cd (Figure 3A). CdS was less cytotoxic than CdCl2 in primary mouse hepatocytes as evaluated by the MTT assay (Figure 3B). Cd is known to induce expression of stress response proteins HSP70 and MT.25−27 CdCl2 increased the level of expression of HSP70 and MT, but CdS did not increase the level of expression of HSP70 and MT in primary mouse hepatocytes (Figure 3C). However, CdS2O3 had toxicity patterns similar to those of CdCl2 in the cells (Figure 3D). Adding a large amount of Na2S2O3 (1 or 5 mM) protected against CdS2O3-induced cytotoxicity (Figure 3E). This protection against Cd toxicity at a high Na2S2O3 concentration has been described previously.28 These results further support the notion that CdS2O3 reversibly forms adducts and that S2O3 needs to be added to allow sulfur adducts to be formed from unstable CdS2O3 (see Scheme 1). Na2S4-Mediated Protection against Cd-Induced Hepatotoxicity in Vivo. We also tested the protective effects of Na2S4 on Cd-induced acute hepatotoxicity in vivo by measuring plasma levels of ALT and AST, indicators of hepatotoxicity, because Na2S4 is an effective polysulfide, presumably through the capture of Cd to form CdS without activating cellular signaling and causing cytotoxicity in vitro (Figure 1). In mice treated with CdCl2 alone, plasma ALT and AST levels were significantly elevated and liver damage was also present, as indicated by HE staining. In contrast, co-treatment with Na2S4 and CdCl2 conferred protection against Cd-induced hepatotox-

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DISCUSSION The results indicated that the strong nucleophile Na2S4 suppresses Cd-induced toxicity in primary mouse hepatocytes and mice and that the protective effect of the polysulfide could be caused by the formation of sulfur adducts of Cd. In previous studies using cultured cells, CdS was found to be less toxic than CdCl2,30,31 but we found that Na2S4 captured Cd, resulting in the formation of at least two sulfur adducts, CdS and CdS2O3. These adducts were detected by ESI-MS in negative ion mode (Figure 2). However, biochemical effects of these sulfur adducts were fairly varied. For example, CdS modified proteins little, poorly activated cellular signaling, and was not hepatotoxic at all in vitro or in vivo under the conditions that were used. In contrast, CdS2O3 had effects almost as strong as the effects of CdCl2, as shown in Figures 3 and 4 (no studies of cellular signaling activation and cytotoxicity were performed in vitro). The marked differences in the cellular toxicities of CdS and CdS2O3 and their abilities to modify proteins appear to be caused by the stabilities of these sulfur adducts (see Scheme 1). As shown in Figure 1C, co-treatment of CdCl2 with Na2S4, leading to CdS production, decreased intracellular Cd levels in primary mouse hepatocytes, in a concentration-dependent manner. We observed similar results in liver tissue from mice (Figure 4C). Cd is incorporated into cells via specific membrane transporters, such as divalent metal transporter 1 (DMT1), ZIP8, and ZIP14.32 DMT1 transports divalent cations, such as Fe2+, Cd2+, Co2+, and Mn2+, in exchange for 2212

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Figure 3. Comparison of reactivity and toxicity between Cd and its sulfur adducts in vitro. (A) Recombinant HSP90 (1 μg) was incubated with the indicated concentrations of cadmium compounds in 100 mM Tris-HCl (pH 7.5) for 30 min, and then the BPM labeling assay was performed. (B) Primary mouse hepatocytes were exposed to the indicated concentrations of CdCl2 or CdS for 24 h, and then the MTT assay was performed. (C) Primary mouse hepatocytes were exposed to 5 μM CdCl2 or CdS for the indicated times. Total cell lysates were subjected to Western blotting using the indicated antibodies. (D) Primary mouse hepatocytes were exposed to the indicated concentrations of CdCl2 or CdS2O3 for 24 h, and then the MTT assay was performed. (E) Primary mouse hepatocytes were exposed to the indicated concentrations of CdS2O3 with or without Na2S2O3 (1 or 5 mM) for 24 h, and then the MTT assay was performed. Each value is the mean ± SE of three or four independent experiments. *p < 0.05 and **p < 0.01, compared with CdCl2.

Figure 4. Protective effects of Na2S4 on Cd-induced hepatotoxicity in vivo. WT mice were divided into four groups: control, CdCl2 treatment alone, CdCl2 and Na2S4 treatment, and CdS treatment. Liver tissue and plasma samples were collected 24 h after treatments. (A) Representative HEstained liver sections. The liver damage area is outlined by a dashed line. (B) Plasma AST and ALT levels. (C) Accumulation of Cd in liver. Each value is the mean ± SE of three independent experiments, *p < 0.05 and **p < 0.01, compared with the control. The scale bar is 100 μm.

a single proton.33 ZIP8 and ZIP14, primarily zinc transport proteins, are involved in Fe2+, Cd2+, and Mn2+ transport.34

Because CdS is no longer a divalent cation, we postulate that these Cd transporters are not involved in the uptake of the 2213

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sulfur adduct of Cd into cells. Simultaneous administration of CdCl2 and Na2S4 to mice had an effect similar to that of CdS alone (Figure 4), further supporting our rationale that CdS is an abundant product when Cd interacts with Na2S4. Our results therefore suggest that Na2S4 can effectively capture Cd in the extracellular space, preventing the uptake of Cd by cells. Cd-induced toxicity depends on not only uptake rates but also electrophilic characteristics. It is well considered that an electrophilic property is crucial for Cd-induced toxicity, because Cd can enter the body, thereby targeting nucleophilic centers in various proteins or nitrogen atoms in DNA and forming adducts. Such modifications lead to the disruption of cellular homeostasis, which is related to toxicity.35−39 However, the current consensus is that environmental electrophiles, at levels lower than toxic concentrations, can also activate a variety of cellular redox signal transduction pathways, consisting of sensor proteins with reactive thiols that can capture electrophiles and effector molecules (e.g., kinases and transcription factors) through covalent modification of the sensor proteins, leading to upregulation of their downstream proteins.40−42 For example, we recently reported that Cd modified HSP90 at Cys462 and Cys564, leading to dissociation of HSF1 from HSP90, thereby facilitating the nuclear location of HSF1 associated with an increased level of HSE target genes such as HSP70.4 Thus, induction of adaptive response proteins is an indicator of cellular stress conditions as well as being a cytoprotective action against electrophiles. In our study, HSP70 and MT-I/II25−27 were examined as examples of downstream proteins induced by Cd. CdS nanocrystals are mainly synthesized by physical and chemical processes.43−45 However, some studies reported biosynthesis of CdS nanoparticles from Cd in bacteria and mammals.46−51 In bacteria, intracellular biosynthesis of CdS nanocrystals in Escherichia coli and Klebsiella pneumoniae and extracellular biosynthesis of the sulfur adduct in Rhodopseudomonas palustris, Gluconoacetobacter xylinus, and Klebsiella aerogenes were observed. The ability of some microorganisms to resist Cd toxicity is believed to be attributable to CdS

Figure 5. Protective role of CSE in Cd-induced toxicity in vitro. (A) Primary hepatocytes from WT and CSE KO mice were exposed to CdCl2 at the indicated concentrations for 24 h. Total cell lysates were subjected to Western blotting using the indicated antibodies. (B) Primary hepatocytes from WT and CSE KO mice were exposed to CdCl2 at the indicated concentrations for 24 h, and then the MTT assay was performed. Each value is the mean ± SE of three independent experiments. *p < 0.05 and **p < 0.01, compared with the control.

Figure 6. Protective role of CSE on Cd-induced hepatotoxicity in vivo. WT and CSE KO mice were treated with CdCl2 (1, 2, 3, or 4 mg/kg). Liver tissue and plasma samples were collected 24 h after treatment. (A) Representative HE-stained liver sections from WT and CSE KO mice treated with CdCl2 (4 mg/kg). The liver damage area is outlined by with a dashed line. (B) Plasma AST and ALT levels. The scale bar is 100 μm. Each value is the mean ± SE of three independent experiments. *p < 0.05 and **p < 0.01, compared with the control. 2214

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decreased CysSH and GSH levels may also be a cause of high Cd sensitivity in CSE KO mice, because previous studies reported that the Cd−SG adduct was involved in the excretion of Cd into extracellular spaces.55,56 However, few appreciable differences in intracellular Cd content in vitro and hepatic Cd content in vivo were observed between WT and CSE KO mice (data not shown), suggesting that CSE primarily contributes to Cd capture, resulting in CdS formation, but not by affecting cellular efflux of Cd as its GSH adduct. Similar to those of MeHg, CysSH and GSH adducts of Cd, through their Cd−S bonds, are relatively unstable, thereby potentially modifying cellular protein thiols, leading to increased cytotoxicity. In fact, the CysSH adduct of Cd, as well as CdCl2, induced stress proteins and toxicity in the rat kidney and liver,57 strongly supporting this concept. Interestingly, we found that, unlike that in CdS, the Cd−S bond in CdS2O3 is relatively unstable, allowing CdS2O3 to modify protein thiols and cause toxicity (Figure 3). Adding Na2S2O3 protected cells from CdS2O3induced cytotoxicity, so S2O3 can clearly trap Cd to form CdS2O3. However, this complex is formed reversibly, as shown in Scheme 1. Taking these results together, we speculate that the capture of electrophilic metal species such as MeHg and Cd by reactive persulfides/polysulfides is critical to the suppression of the health risks posed. In summary, we demonstrated, in vivo and in vitro, that Na2S4 conferred protection against Cd-induced acute hepatotoxicity, via formation of CdS but not CdS2O3, and that CSE KO mice given Cd were more susceptible than Cd-treated WT mice. As shown in Scheme 1, CSE-mediated reactive persulfides/ polysulfides, Na2S4, and the H2S byproduct appear to be involved in the stable formation of CdS from Cd, whereas the oxidized H2S metabolite S2O3 can capture Cd to reversibly form the CdS2O3 adduct. It was also shown that Cd-mediated upregulation of HSP70 and MT proteins was regulated by Na2S4 and CSE. The study presented here therefore suggests that reactive persulfides/polysulfides are powerful small molecules for regulating activation of adaptive responses by Cd and protecting against Cd-induced hepatotoxicity, through formation of Cd sulfur adducts.

Scheme 1. Possible Pathways through Which Cd Is Deactivated by Reactive Persulfides/Polysulfides

biosynthesis.52,53 Using X-ray diffraction, Trabelsi et al.46 detected CdS nanoparticles in the liver and kidneys of rats, following CdCl2 injection. However, the biosynthetic mechanisms of Cd sulfur adducts are unknown. Our recent study showed that CSE and CBS knockdown significantly decreased levels of cellular reactive persulfides/polysulfides in bovine aortic endothelial cells and enhanced Cd-induced HSP70 expression and cytotoxicity.4 Consistent with these findings, levels of endogenous reactive persulfides/polysulfides in primary hepatocytes from CSE KO mice were lower than in those from WT mice (T. Unoki et al., unpublished observations). Under these conditions, Cd-induced hepatotoxicity and/or the levels of HSP70 and MT expression were increased (Figures 5 and 6) without affecting the intracellular accumulation of Cd in vitro and hepatic accumulation of Cd in vivo. These results suggested that endogenous reactive persulfides/polysulfides could also capture Cd, producing its CdS sulfur adduct within cells, not only to regulate the activation of electrophilic signaling but also to reduce toxicity caused by Cd exposure. While the MeHg−SG adduct is recognized as a detoxified MeHg metabolite, the Hg−S bond is not as stable as the C−S bond. In support of this, we previously showed that exposure of SH-SY5Y cells to the synthetic monoester of a MeHg−SG adduct led to activation of transcription factor Nrf2 and concentration-dependent cytotoxicity, caused by S-mercuration of Keap1 and of cellular proteins, respectively.54 This implied that the monoester of the MeHg−SG adduct underwent hydrolysis by cellular esterase(s) to yield a MeHg−SG adduct that could covalently modify cellular proteins through Stransmercuration, resulting in increased cytotoxicity. By contrast, an alternative detoxified metabolite, (MeHg)2S, produced by the reaction of MeHg with H2S, GSSH, and GSSSG, was fairly stable.17,54 It has also been found that cellular proteins have minimal covalent interactions with (MeHg)2S, unlike with the MeHg−SG adduct.42 It is therefore clear that (MeHg)2S is irreversibly produced but that the MeHg−SG adduct is reversibly produced. CSE is well-known to have a low substrate specificity. With cystathionine as a substrate, CSE produced CysSH as the major product, whereas CySSH was the predominant reaction product when cystine was used as a substrate for the enzyme.12,29 In our preliminary findings, we detected a substantial decline in levels of CysSH, GSH, and their reactive persulfides/polysulfides in primary hepatocytes from CSE KO mice, compared with the levels of those from WT mice (T. Unoki et al., unpublished observations). In this context,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00278. Formation of the CdS2O3 complex after incubation of CdCl2 with Na2S2O3 in vitro and comparison of hepatic accumulation of Cd between WT and CSE KO mice in vitro and in vivo (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 3058575, Japan. E-mail: [email protected]. Telephone: +81-29-853-3133. Fax: +81-29-853-3259. ORCID

Yoshito Kumagai: 0000-0003-4523-8234 Author Contributions

M.A., Y.S., and T.U. contributed equally to this work. 2215

DOI: 10.1021/acs.chemrestox.7b00278 Chem. Res. Toxicol. 2017, 30, 2209−2217

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Chemical Research in Toxicology Funding

(15) Ono, K., Akaike, T., Sawa, T., Kumagai, Y., Wink, D. A., Tantillo, D. J., Hobbs, A. J., Nagy, P., Xian, M., Lin, J., and Fukuto, J. M. (2014) Redox chemistry and chemical biology of H2S, hydropersulfides, and derived species: Implications of their possible biological activity and utility. Free Radical Biol. Med. 77, 82−94. (16) Yoshida, E., Toyama, T., Shinkai, Y., Sawa, T., Akaike, T., and Kumagai, Y. (2011) Detoxification of Methylmercury by Hydrogen Sulfide-Producing Enzyme in Mammalian Cells. Chem. Res. Toxicol. 24, 1633−1635. (17) Abiko, Y., Yoshida, E., Ishii, I., Fukuto, J. M., Akaike, T., and Kumagai, Y. (2015) Involvement of Reactive Persulfides in Biological Bismethylmercury Sulfide Formation. Chem. Res. Toxicol. 28, 1301− 1306. (18) Abiko, Y., Shinkai, Y., Unoki, T., Hirose, R., Uehara, T., and Kumagai, Y. (2017) Polysulfide Na2S4 regulates the activation of PTEN/Akt/CREB signaling and cytotoxicity mediated by 1,4naphthoquinone through formation of sulfur adducts. Sci. Rep. 7, 4814. (19) 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. (20) Ishii, I., Akahoshi, N., Yamada, H., Nakano, S., Izumi, T., and Suematsu, M. (2010) Cystathionine gamma-Lyase-deficient Mice Require Dietary Cysteine to Protect against Acute Lethal Myopathy and Oxidative Injury. J. Biol. Chem. 285, 26358−26368. (21) 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. (22) Denizot, F., and Lang, R. (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89, 271−277. (23) Hirano, S., Cui, X., Li, S., Kanno, S., Kobayashi, Y., Hayakawa, T., and Shraim, A. (2003) Difference in uptake and toxicity of trivalent and pentavalent inorganic arsenic in rat heart microvessel endothelial cells. Arch. Toxicol. 77, 305−312. (24) Lowicka, E., and Beltowski, J. (2007) Hydrogen sulfide (H2S) the third gas of interest for pharmacologists. Pharmacol. Rep. 59, 4−24. (25) Shinkai, Y., Kimura, T., Itagaki, A., Yamamoto, C., Taguchi, K., Yamamoto, M., Kumagai, Y., and Kaji, T. (2016) Partial contribution of the Keap1-Nrf2 system to cadmium-mediated metallothionein expression in vascular endothelial cells. Toxicol. Appl. Pharmacol. 295, 37−46. (26) Souza, V., del Carmen Escobar, Ma., Bucio, L., Hernandez, E., Gomez-Quiroz, L. E., and Gutierrez Ruiz, M. C. (2009) NADPH oxidase and ERK1/2 are involved in cadmium induced-STAT3 activation in HepG2 cells. Toxicol. Lett. 187, 180−186. (27) Wang, Y. Y., Gibney, P. A., West, J. D., and Morano, K. A. (2012) The yeast Hsp70 Ssa1 is a sensor for activation of the heat shock response by thiol-reactive compounds. Mol. Biol. Cell 23, 3290− 3298. (28) Oh, S. R., Kim, J. K., Lee, M. J., and Choi, K. (2008) Dechlorination with sodium thiosulfate affects the toxicity of wastewater contaminated with copper, cadmium, nickel, or zinc. Environ. Toxicol. 23, 211−217. (29) Steegborn, C., Clausen, T., Sondermann, P., Jacob, U., Worbs, M., Marinkovic, S., Huber, R., and Wahl, M. C. (1999) Kinetics and inhibition of recombinant human cystathionine gamma-lyase. Toward the rational control of transsulfuration. J. Biol. Chem. 274, 12675− 12684. (30) L’Azou, B., Passagne, I., Mounicou, S., Treguer-Delapierre, M., Puljalte, I., Szpunar, J., Lobinski, R., and Ohayon-Courtes, C. (2014) Comparative cytotoxicity of cadmium forms (Cdcl(2), CdO, CdS micro- and nanoparticies) in renal cells. Toxicol. Res. 3, 32−41. (31) Zapor, L. (2014) Evaluation of the Toxic Potency of Selected Cadmium Compounds on A549 and CHO-9 Cells. Int. J. Occup. Saf. Ergon. 20, 573−581. (32) Vergauwen, L., Hagenaars, A., Blust, R., and Knapen, D. (2013) Temperature dependence of long-term cadmium toxicity in the

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants 25220103 to Y.K. and 15K08042 to Y.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of the manuscript. ABBREVIATIONS CSE, cystathionine γ-lyase; CBS, cystathionine β-synthase; Nrf2, NF-E2-related factor 2; MT, metallothionein; HSP70, heat shock protein 70; Na2S4, sodium tetrasulfide; BPM, BiotinPEAC5-maleimide

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