Detoxification of Methylmercury by Hydrogen Sulfide-Producing

Sep 27, 2011 - (2) It has been believed that this process plays a central role in the detoxification process of MeHg. Hydrogen sulfide (H2S) is a gase...
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Detoxification of Methylmercury by Hydrogen Sulfide-Producing Enzyme in Mammalian Cells Eiko Yoshida,†,|| Takashi Toyama,†,‡,|| Yasuhiro Shinkai,† Tomohiro Sawa,§ Takaaki Akaike,§ and Yoshito Kumagai*,† †

Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan ‡ Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472, Japan § Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan

bS Supporting Information ABSTRACT: Methylmercury (MeHg) covalently modifies cellular proteins through their SH groups, resulting in cytotoxicity. We report that cystathionine β-synthase (CBS), which catalyzes the production of hydrogen sulfide, contributes to cellular protection against MeHg. Pretreatment with NaHS or overexpression of CBS reduced MeHg cytotoxicity, whereas transfection with CBS small interfering RNA enhanced MeHg toxicity in human neuroblastoma SH-SY5Y cells. Bismethylmercury sulfide ((MeHg)2S) was identified as a metabolite of MeHg in SH-SY5Y cells exposed to MeHg and in the livers of rats treated with MeHg. (MeHg)2S had little chemical protein modification capability and little cytotoxicity compared with MeHg in vitro and in vivo.

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ethylmercury (MeHg) is an environmental electrophile that can be covalently bound to protein sulfhydryls to form protein MeHg complexes. These covalent modifications, referred to as S-mercuration, are associated with the disruption of enzyme function and neurotoxicity.1 Although it is well recognized that Minamata disease was due to the intake of fish exposed to high concentrations of MeHg through biologically mediated concentration in Minamata, Japan, the risks of MeHg to human health from the accumulation of MeHg in the body through daily consumption of large predatory fish, such as tuna and swordfish, remains. Once humans are exposed to MeHg, this heavy metal undergoes S-conjugation by glutathione (GSH), an abundant low-molecular-weight thiol compound, in the absence and presence of GSH S-transferase (GST), to form a MeHg-SG, a polar nonelectrophilic adduct that is thought to be excreted into extracellular space by multidrug resistance-associated protein (MRP) transporters.2 It has been believed that this process plays a central role in the detoxification process of MeHg. Hydrogen sulfide (H2S) is a gaseous, weakly acidic molecule mainly produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in cells. Both enzymes are extensively localized in a variety of tissues and require pyridoxal phosphate (PLP) as a cofactor.3 Accumulated evidence has indicated that H2S is protective in neural damage and heart failure,4 but mechanistic details remain to be elucidated. Interestingly, the pKa of H2S is estimated at 6.76,5 suggesting that approximately 80% of H2S is dissociated into its anions (HS ) at physiological pH. This property raised the possibility that the HS , generated during the enzymatic reaction of CBS is a nucleophile that could react with MeHg to yield a MeHg-SH adduct with a pKa value of r 2011 American Chemical Society

7.5.6 The anion of MeHg-SH is capable of reacting with a second equivalent of MeHg, resulting in the formation of bismethylmercury sulfide ((MeHg)2S). In 1978, Craig and Bartlett7 had reported the possibility of (MeHg)2S production during the reaction of MeHg with H2S because there are numerous environmental sources of H2S. To our knowledge, however, no studies have examined the contribution of enzymatically generated H2S to the detoxification of MeHg in cultured cells or intact animals. Here, we report for the first time, that the enzyme-producing H2S is a catalyst for the suppression of MeHg toxicity through the production of (MeHg)2S. Exposure of human neuroblastoma SH-SY5Y cells to MeHg caused a concentration-dependent cytotoxicity (IC50 value = 1.46 ( 0.1 μM). Pretreatment with NaHS suppressed MeHg toxicity (Figure 1A), suggesting that H2S gas diminishes cytotoxicity caused by MeHg. Immunoblot analysis indicated that CBS was abundant in SH-SY5Y cells, whereas little appreciable expression of CSE was seen in the cells compared to human pulmonary A549 cells and human hepatoma HepG2 cells (Figure S1, Supporting Information). For this reason, we thought that SHSY5Y cells would be convenient for manipulating CBS-mediated production of H2S. Overexpression of CBS in SH-SY5Y cells significantly reduced the cytotoxicity of MeHg (Figure 1B,C), whereas knockdown of CBS enhanced the toxicity (Figure 1B,D). These results suggest that H2S produced by CBS in the cells represses MeHg-mediated cellular toxicity.

Received: September 9, 2011 Published: September 27, 2011 1633

dx.doi.org/10.1021/tx200394g | Chem. Res. Toxicol. 2011, 24, 1633–1635

Chemical Research in Toxicology

Figure 1. CBS producing H2S suppresses MeHg toxicity in SH-SY5Y cells. (A) Cells were pretreated with NaHS (500 μM) for 12 h and washed with PBS before exposure to the indicated concentrations of MeHg for 24 h. MTT assay was then performed. (B) Cells were transfected with pcDNA or pcDNA/CBS for 24 h and with control siRNA or CBS siRNA for 48 h. Then, total cell lysates were subjected to Western blot analysis using the indicated antibodies. (C) SH-SY5Y cells were transfected with pcDNA or pcDNA/CBS for 24 h; (D) Cells were transfected with control siRNA or CBS siRNA for 48 h and exposed to the indicated concentrations of MeHg for 24 h. MTT assay was then performed. *P < 0.05 and **P < 0.01 vs control. Each value is the mean ( SEM of three determinations.

MeHg-SG is not electrophilic and cannot covalently modify proteins; that is, it is an inactive metabolite of MeHg. With primary hepatocytes from wild- and Nrf2 knockout-mice, we found that deletion of Nrf2 increases susceptibility to MeHg because GCL, GST, and MRP, which are responsible for detoxification and excretion of MeHg into extracellular space, are coordinately regulated by Nrf2.8 In a separate experiment with Nrf2 knockout mice, we showed that CBS is not regulated by Nrf2 (Toyama, T., et al., unpublished observation). Thus, our findings suggest an Nrf2independent protection pathway against MeHg toxicity by H2S derived from CBS. We speculate that CBS will protect electrophilic modification of cellular proteins and cellular toxicity against electrophiles since H2S is a more reactive nucleophile than GSH. Although cysteine is the common substrate for H2S generation by CBS and GSH by GCL, cellular GSH levels were unaltered during overexpression or knockdown of CBS in SH-SY5Y cells (Figure S1, Supporting Information). Furthermore, mercury accumulation during MeHg exposure was unaffected by the pretreatment of NaHS, overexpression, or knockdown of CBS in SH-SY5Y cells (Figure S1, Supporting Information). These results suggest that increased sensitivity to MeHg caused by the decline of CBS expression in SH-SY5Y cells is not due to decreased GSH synthesis and increased mercury excretion. In other words, H2S produced by CBS is responsible for the blockage of MeHg cytotoxicity through presumably the formation of (MeHg)2S. To determine whether (MeHg)2S, produced during the reaction of MeHg with H2S, is an inactive metabolite of MeHg, we synthesized (MeHg)2S from MeHg and NaHS, and its ability to modify cellular proteins and its cytotoxicity were assessed. Electronionization mass spectrometry (EI-MS) indicated that the reaction product had a molecular mass of 464 and fragment masses of 449, 248, 216, and 202 (Figure S2, Supporting Information) with

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Figure 2. Effect of (MeHg)2S on S-mercuration of cellular proteins and toxicity in vitro and in vivo. (A) S-mercuration of cellular proteins by MeHg and (MeHg)2S. The cell lysates with RIPA buffer from SH-SH5Y cells were incubated with (MeHg)2S or MeHg in the absence or presence of NaHS (100 μM) for 30 min at 37 °C and then allowed to react with 10 μM BPM (30 min at 37 °C). The reaction mixture was subjected to Western blot analysis with avidin horseradish peroxidase conjugate. (B) Cytotoxicity of MeHg and (MeHg)2S in SH-SY5Y cells. The cells were exposed to mercury compounds at molar equivalent concentrations of MeHg. After a 24 h incubation, the MTT assay was performed. **P < 0.01 vs control. Each value is the mean ( SEM of three determinations. (C) Mortality of MeHg and (MeHg)2S in mice (n = 10 each). The mice were treated with MeHg (0.1 mmol/kg) or (MeHg)2S (0.05 mmol/kg) by intraperitoneal injection. (D) Mercury accumulation in the brain, heart, liver, and kidney (n = 3 each). The mice were intraperitoneally injected with MeHg (0.1 mmol/kg) or (MeHg)2S (0.05 mmol/kg). After 24 h, samples of each organ were subjected to atomic absorption spectroscopy.

broad isotope peaks characteristic of mercury.9 In the high-performance liquid chromatography (HPLC) conditions employed in the present study, retention times of MeHg and (MeHg)2S were 4 and 16 min, respectively (Figure S3, Supporting Information). Incubation of 1 mM MeHg with 1 mM NaHS at 37 °C for 30 min resulted in the appearance of a new product corresponding to (MeHg)2S on HPLC accompanied by the reduction of MeHg (Figure S3, Supporting Information). Next, we determined the enzymatic production of (MeHg)2S from MeHg by recombinant CBS. Purified human CBS incubated with cysteine, homocysteine, and PLP produces hydrogen sulfide. As expected, omission of cysteine blocked hydrogen sulfide formation (data not shown). Addition of MeHg to the H2Sproducing system produced a metabolite with a retention time (14 min) identical to that of authentic (MeHg)2S (Figure S4, Supporting Information). This metabolite was not observed in the absence of cysteine, consistent with the enzymatic formation of (MeHg)2S from MeHg by human CBS generated H2S. As expected, the molecular mass (m/z = 464) of the reaction product was identical to that of authentic (MeHg)2S on EI-MS (Figure S4, Supporting Information), supporting the fact that CBS catalyzes the formation of (MeHg)2S from MeHg. Furthermore, we attempted to determine whether (MeHg)2S was produced as a metabolite of MeHg in cells and in rats. We developed a procedure to extract (MeHg)2S from biological samples; with this procedure, the recovery of (MeHg)2S was 66.5% ( 12.7% (n = 3) from cell lysates of SH-SY5Y cells and 1634

dx.doi.org/10.1021/tx200394g |Chem. Res. Toxicol. 2011, 24, 1633–1635

Chemical Research in Toxicology

’ ASSOCIATED CONTENT

bS

Author Contributions

)

86.1% ( 4.9% (n = 3) from homogenates of rat liver. Exposure of SH-SY5Y cells to 20 μM MeHg and subcutaneous injection of rats with MeHg (0.04 mmol/kg for 8 days) produced a metabolite corresponding to (MeHg)2S in the livers of the exposed animals (Figure S3, Supporting Information). Quantitative analysis revealed that (MeHg)2S was formed at 1.04 pmol per g in the livers of rats given MeHg under these conditions. The production of (MeHg)2S in SH-SY5Y cells exposed to MeHg and in the liver of rats injected MeHg was confirmed by EI-MS with the authentic chemical (Figure S3, Supporting Information). To explore whether (MeHg)2S could cause S-mercuration of cellular proteins and thus cytotoxicity, we developed the biotinPEAC5-maleimide (BPM)-labeling assay (Figure S5, Supporting Information). As shown in Figure 2A, MeHg covalently modified numerous proteins in SH-SY5Y cells in a concentration-dependent fashion, whereas (MeHg)2S did not. This metabolite had a minimal cytotoxicity compared to that of MeHg in SHSY-5Y cells (Figure 2B). Consistent with this, intraperitoneal administration of (MeHg)2S to mice (0.05 mmol/kg) had no effect on the death rate, although the exposure to MeHg (0.1 mmol/kg) caused a time-dependent mortality (Figure 2C); under these conditions, mercury levels in a variety of tissues after the treatment with MeHg to mice were not significantly different from those with (MeHg)2S (Figure 2D). MeHg can cross the blood brain barrier, thereby affecting the central nervous system. The uptake of MeHg with different types of cultured cells is found to be temperature-independent, indicating that MeHg is readily incorporated into cells by passive diffusion.10 This observation suggests that the incorporation of MeHg is not the rate-limiting step. In our preliminary studies, we found that CBS is a molecular target for MeHg through covalent modification of its thiol groups in SH-SY5Y cells (Toyama, T. et al., unpublished observation). Because covalent modification of protein by MeHg potentially causes a conformational change, leading to substantial loss of the function,11 it seems likely that MeHg could affect intracellular H2S concentrations by the inhibition of CBS activity as well as scavenging of this gas. Since the current consensus is that H2S has a function for protection against neurodegradation,4 alteration in CBS activity caused by higher or prolonged exposure to MeHg is of interest to understand the neurotoxicity of this heavy metal. In conclusion, CBS catalyzes the formation of H2S, which exists predominantly as HS at physiological pH. Like GS , HS is also a nucleophile that readily attacks MeHg, resulting in substantial production of (MeHg)2S, thereby resulting in a reduction of S-mercuration of cellular proteins involved in cytotoxicity.

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These authors contributed equally to this work.

Funding Sources

This work was supported by the Mitsubishi Foundation, and by a grant-in-aid (#23117703 to Y.K.) for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

’ ACKNOWLEDGMENT We thank Dr. Daigo Sumi, University of Tsukuba, for helpful advice regarding this study. ’ REFERENCES (1) Clarkson, T. W. (1997) The toxicology of mercury. Crit. Rev. Clin. Lab. Sci. 34, 369–403. (2) Madejczyk, M. S., Aremu, D. A., Simmons-Willis, T. A., Clarkson, T. W., and Ballatori, N. (2007) Accelerated urinary excretion of methylmercury following administration of its antidote N-acetylcysteine requires Mrp2/Abcc2, the apical multidrug resistance-associated protein. J. Pharmacol. Exp. Ther. 322, 378–384. (3) Singh, S., Padovani, D., Leslie, R. A., Chiku, T., and Banerjee, R. (2009) Relative contributions of cystathionine beta-synthase and gammacystathionase to H2S biogenesis via alternative trans-sulfuration reactions. J. Biol. Chem. 284, 22457–22466. (4) Lu, M., Hu, L. F., Hu, G., and Bian, J. S. (2008) Hydrogen sulfide protects astrocytes against H(2)O(2)-induced neural injury via enhancing glutamate uptake. Free Radical Biol. Med. 45, 1705–1713. (5) Hughes, M. N., Centelles, M. N., and Moore, K. P. (2009) Making and working with hydrogen sulfide: The chemistry and generation of hydrogen sulfide in vitro and its measurement in vivo: a review. Free Radical Biol. Med. 47, 1346–1353. (6) Dyrssen, D., and Wedborg, M. (1991) The sulphur-mercury(II) system in natural waters. Water, Air, Soil Pollut. 56, 1669–1710. (7) Craig, P. J., and Bartlett, P. D. (1978) The role of hydrogen sulphide in environmental transport of mercury. Nature 275, 635–637. (8) Toyama, T., Sumi, D., Shinkai, Y., Yasutake, A., Taguchi, K., Tong, K. I., Yamamoto, M., and Kumagai, Y. (2007) Cytoprotective role of Nrf2/Keap1 system in methylmercury toxicity. Biochem. Biophys. Res. Commun. 363, 645–650. (9) Trumpler, S., Nowak, S., Meermann, B., Wiesmuller, G. A., Buscher, W., Sperling, M., and Karst, U. (2009) Detoxification of mercury species--an in vitro study with antidotes in human whole blood. Anal. Bioanal. Chem. 395, 1929–1935. (10) Heggland, I., Kaur, P., and Syversen, T. (2009) Uptake and efflux of methylmercury in vitro: comparison of transport mechanisms in C6, B35 and RBE4 cells. Toxicol. in Vitro 23, 1020–1027. (11) Kanda, H., Sumi, D., Endo, A., Toyama, T., Chen, C. L., Kikushima, M., and Kumagai, Y. (2008) Reduction of arginase I activity and manganese levels in the liver during exposure of rats to methylmercury: a possible mechanism. Arch. Toxicol. 82, 803–808.

Supporting Information. Detailed experimental section; figures showing the effect of CBS on intracellular GSH and mercury accumulation, EI-MS analysis of the product of the reaction of MeHg with NaHS, identification of (MeHg)2S in SH-SY5Y cells and rat liver following exposure to MeHg, and CBS-dependent enzymatic formation of (MeHg)2S from MeHg. This material is available free of charge via the Internet at http://pubs.acs.org.

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dx.doi.org/10.1021/tx200394g |Chem. Res. Toxicol. 2011, 24, 1633–1635