The Uptake and Release of Polysulfur Cysteine Species by Cells

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The Uptake and Release of Polysulfur Cysteine Species by Cells: Physiological and Toxicological Implications Joseph Lin, Masahiro Akiyama, Iris Bica, Faith T. Long, Catherine F. Henderson, Robert N. Goddu, Valeria Suarez, Blaine Baker, Tomoaki Ida, Yasuhiro Shinkai, Péter Nagy, Takaaki Akaike, Jon M. Fukuto, and Yoshito Kumagai Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00340 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 28, 2019

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Chemical Research in Toxicology

The Uptake and Release of Polysulfur Cysteine Species by Cells: Physiological and Toxicological Implications Joseph Lin‡*, Masahiro Akiyama#, Iris Bica‡, Faith T. Long‡, Catherine F. Henderson‡, Robert N. Goddu‡, Valeria Suarez†, Blaine Baker†, Tomoaki Ida§, Yasuhiro Shinkai#, Peter Nagy¶, Takaaki Akaike§, Jon M. Fukuto†* and Yoshito Kumagai#* ‡Department

of Biology, Sonoma State University, Rohnert Park, CA 94928, #Environmental Biology Section,

Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan, †Department of Chemistry, Sonoma State University, Rohnert Park, CA 94928, §Department of Environmental Medicine and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan, ¶Department of Molecular Immunology and Toxicology, National Institute of Oncology, Budapest, Hungary

*To whom correspondence can be addressed: Joseph Lin, [email protected]; Jon M. Fukuto, [email protected]; Yoshito Kumagai, [email protected]

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Abstract Hydropersulfides and related polysulfides have recently become topics of significant interest due to their physiological prevalence and proposed biological functions. Currently, examination of the effects of hydropersulfide treatment on cells has been difficult due to their lack of inherent stability with respect to disproportionation. Herein, it is reported that the treatment of a variety of cell types with cysteine trisulfide (also known as thiocystine; Cys-SSS-Cys), results in an increase in intracellular hydropersulfide levels (e.g. cysteine hydropersulfide; Cys-SSH, and glutathione hydropersulfide; GSSH). Thus, Cys-SSS-Cys represents a possible pharmacological agent for examining the effects of hydropersulfides on cell function/viability. It has also been found that cells with increased intracellular hydropersulfide levels can export Cys-SSH into the extracellular media. Interestingly, the Cys-SSH is the major hydropersulfide exported by cells, although GSSH is the predominant intracellular species. The possible implications of cellular export are discussed.

Keywords: Hydropersulfides, cysteine hydropersulfide, glutathione hydropersulfide, hydrogen sulfide.


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Introduction Recent work has reported the prevalence and alluded to the likely physiological importance of cysteinebased polysulfur species such as cysteine hydropersulfide (Cys-SSH), glutathione hydropersulfide (GSSH), cysteine trisulfide (or thiocystine; Cys-SSS-Cys) as well as polysulfur species in cysteine proteins1-5. Although the function and/or biochemical utility of per- and poly-sulfides in cells remains unestablished, based on the chemistry of these species it has been proposed that they may serve a protective function1,6 among other possible roles. In a previous report, our lab found that HEK293T cells were capable of taking up Cys-SSSCys, leading to increased intracellular polysulfides, especially Cys-SSH and GSSH7. Consistent with the idea that hydropersulfides can be protective, increased intracellular levels of Cys-SSH and GSSH from the pretreatment of cells with Cys-SSS-Cys correlated with an increase in resistance to electrophilic toxicity. This is not an unexpected result since hydropersulfides are superior nucleophiles and reductants compared to the corresponding thiol species and thus predicted to be capable of protection against electrophilic/oxidative stress1,2,6. Cell viability in these studies was monitored using the WST8 assay, which measures the reductive capacity of the cell (as a correlate to viability) based on the levels and/or availability of intracellular reduced nicotinamides (NADH or NADPH)8,9. In this assay, a substituted extracellular tetrazolium compound, WST-8, can be reduced by viable cells to a water-soluble formazan compound that strongly absorbs at 570 nm. The assay requires a second component, a 1-methoxy phenazenium salt (1-methoxy PMS), to serve as a membrane-associated redox molecule capable of shuttling electrons from the intracellular compartment (from NADH/NADPH) to the extracellular milieu containing the reduceable and cell impermeable tetrazolium WST8 compound (Scheme 1).

O 3S

O 3S

SO3

SO3 N HN N N

N

WST 8

N

(yellow)

N

NO2

N

CH3O

CH3O

NO2

WST 8 formazan (orange) NO2

NO2 H N

OCH3

OCH3

N N

N CH3

e-

electron transfer mediator (1-methoxy PMS)

CH3

NADH/NADPH

Cell Scheme 1: WST-8 assay for cell viability.


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Interestingly, when using the WST-8 assay it was found that treatment of cells with Cys-SSS-Cys in the absence of any cellular stressor resulted in an apparent increase in “viability”7. That is, cells pre-treated with Cys-SSS-Cys exhibited an increase in WST8 reduction compared to untreated cells. Since hydropersulfides are superior reductants compared to the corresponding thiol10,11 and levels of hydropersulfides were increased in Cys-SSS-Cys treated cells, it was surmised that the increased ability of cells to reduce extracellular WST-8 was a result of an increase in the intracellular reducing capacity of the cells. Moreover, since hydropersulfides have been proposed to be important to maintaining mitochondrial energetics12, it was also proposed that an increase in the reductive capacity of cells may be a result of effects on mitochondria. Although these remain possible explanations for at least part of this effect, it remained possible that a reductant is exported by CysSSS-Cys treated cells that is capable of reducing WST-8 to the formazan product directly in the extracellular media. Herein, the source of the hyper-reductive capacity of Cys-SSS-Cys treated cells, as measured by the WST8 assay, is more closely examined. Experimental Procedures Cells HEK293T, HeLa, A549, and 3T3 cells were culture in DMEM (Gibco) whereas Jurkat and THP-1 cells were cultured in RPMI (Gibco). Both media were supplemented with 10% FBS (JRH Biosciences) and Pen/Strep/Glu (Gibco) and cells grown in a humidified incubator at 5% CO2. Reagents Cystine trisulfide13 and methoxycarbonyl penicillamine disulfide (MCPD)14 were synthesized as previously described. WST8 assay kit (volumes are given since concentrations are not known) was from Cayman Chemical, 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) from ThermoFisher, L-Cysteine from Sigma-Aldrich, β-(4hydroxyphenyl) ethyl iodoacetamide (HPE-IAM) from Funakoshi Co., and NaHS from Strem Chemical. The use of sulfasalazine and xCT siRNA were previously described15. All other chemicals and reagents were purchased from commercial suppliers and were of the highest purity available. Cellular WST-8 assay For adherent cells, 5 x 104 cells were plated in triplicate on a 96 well plate the day before. For suspension cells, 105 cells/well were used the day of. Media was changed prior to treatment to DMEM without Cys and Met (Gibco). Cells were then treated with 1 mM cysteine trisulfide for 3 hours with 5 μl of WST-8 and the electron mediator added during the final hour. For the time course, WST-8 with or without the electron mediator was added at the time of cysteine trisulfide treatment. Absorbance at 450 nm was then read on a Synergy HT plate reader at the indicated time points (BioTek Instruments).


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Conditioned media WST8 and DTNB assay Cells were plated (4 x 105 cells/well in a 6 well plate) and left untreated or treated with 1 mM cysteine trisulfide in DMEM without Cys and Met. As a control, media only or cells killed with 0.01% Triton X-100 or MeOH were used. After 3 hours, media was collected and centrifuged at 17,000 x g for 5 minutes. Media (100 μl) was then assayed with WST-8 (2.5 μl) with or without the electron mediator (2.5 μl) or DTNB (1 mM) for 30 min. Absorbance at 450 nm (WST8) or 412 nm (DTNB) was then determined. Direct WST-8 and DTNB assay MCPD, Cysteine, and NaHS were each solubilized in PBS. 100 μl of PBS, 0.5 mM MCPD, 0.5 mM Cysteine, or 0.5 mM NaHS were reacted with WST8 without the electron mediator (2.5 μl) or DTNB (1 mM) for 30 min at room temp in triplicate. Absorbance at 450 nm (WST-8) or 412 nm (DTNB) was then determined. Mass spectrometry Samples were treated and analyzed as previously described15. In brief, for mass spectrometry of cell lysates, HEK293T cells were treated in DMEM without Cys and Met for 3 hours. Cells were harvested in PBS and cell pellets sonicated 10x (Output 2, duty 20) in the presence of HPE-IAM (1 mM in MeOH). Samples were then incubated for 30 min at 37 °C, centrifuged (14,000 x g) for 10 min, and then an equal volume of 0.1% formic acid added. For mass spectrometry of cultured media, media samples were centrifuged at 17,000 x g for 5 minutes. HPE-IAM (5 mM) was then added to the supernatant for 30 min at 37 °C, followed by an equal volume of 0.1% formic acid. Results The effect of Cys-SSS-Cys pre-treatment on WST-8 reduction: Our lab previously reported that treatment of HEK293T cells with Cys-SSS-Cys results in an increase in apparent viability as measured by the WST-8 assay7. That is, cells treated with Cys-SSS-Cys were capable of reducing WST-8 to the formazan analog to a greater extent than cells that were not exposed to Cys-SSS-Cys. Since this assay views WST-8 reduction as a measure the reducing capacity of a cell (which is correlated with cell health or viability), it was deduced that Cys-SSS-Cys pre-treatment results in an increase in cellular reducing capacity. In order to determine whether this phenomenon is unique to HEK293T cells or whether it is more general, other cells lines were also examined. As shown in Figure 1A, treatment of a variety of cells with Cys-SSS-Cys results in a variable and cell-dependent effect with regards to WST-8 reduction. Clearly, Cys-SSS-Cys-treated HEK293T cells demonstrate the greatest ability to reduce WST-8 while Jurkat cells show little, if any effect under the


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conditions of these experiments. The other cell types exhibit activities intermediate between the HEK293T and Jurkat cells. As mentioned previously, the treatment of HEK293T cells with Cys-SSS-Cys results in an increase in intracellular hydropersulfide levels7. In order to determine whether this phenomenon is unique to HEK293T cells or is more general, other cell types were also examined. As shown in Figure 1B, treatment of HeLa and A549 cells with Cys-SSS-Cys also results in increased intracellular hydropersulfide levels (Cys-SSH, GSSH) as well as H2S, similar to what occurs in HEK293T cells. Thus, the ability for Cys-SSS-Cys treatment to cause an increase in intracellular hydropersulfide levels appears not to be unique to HEK293T cells.

Figure 1. Cellular responses to cysteine trisulfide occur in multiple cell types. (A) The indicated cell types were plated and then left untreated or treated with 1 mM cysteine trisulfide for 3 hours. WST8 was added during the last hour. Absorbance was then measure at 450 nm. The graph shows the averages of triplicate samples with error bars denoting standard deviation. (B) HeLa, HEK293T, and A549 cells were treated with 0.2 mM cystine or cysteine trisulfide for 3 hrs. Cells were then harvested, reacted with HPE-IAM, and assayed by mass spectrometry for the presence of cysteine hydropersulfide (CysSSH), glutathione hydropersulfide (GSSH), and H2S.


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xc- (xCT) and cellular uptake of Cys-SSS-Cys: It has been previously shown that pre-treatment of cells with Cys-SSS-Cys results in an increase in intracellular levels of a variety of polysulfide species such as CysSSH and GSSH7. It was speculated at that time that the Cys-SS-Cys transporter xc- could be responsible for Cys-SSS-Cys uptake and that Cys-SSS-Cys serves as the precursor to the generation of intracellular polysulfide species. The xc- transporter is made up of two proteins, xCT, which is a glutamate antiporter and ultimately responsible for Cys-SS-Cys transport, and 4F2hc, which localizes xCT to the membrane16. In order to determine whether xc- is involved, the effects of sulfasalazine (an inhibitor of xCT17) and xCT knockdown, via siRNA, on Cys-SSS-Cys-dependent increases in intracellular per- and poly-sulfide levels was examined. As shown in Figure 2A, the presence of sulfasalazine did not decrease the ability of Cys-SSS-Cys treatment to result in increased intracellular levels of Cys-SSH or GSSH in HEK293T cells. Moreover, inorganic polysulfides such as H2S or H2S2 (as measured by the trapped HPE-AM species, bis-S-HPE-AM and bis-SSHPE-AM, respectively) were also not decreased. Qualitatively similar results were also found with siRNA knock down of xCT, Figure 2B. That is, siRNA knock down did not decrease Cys-SSS-Cys-mediated increases in intracellular levels of per- and poly-sulfide species. Thus, xCT is not mediating Cys-SSS-Cys import into cells and does not participate in the observed increases in intracellular polysulfides. It is notable that sulfasalazine treatment appears to result in an increase in Cys-SSS-Cys-dependent intracellular Cys-SSH, GSSH and H2S2 levels (Figure 2A). xCT knock down, on the other hand, did not exhibit this effect. This implies that sulfasalazine treatment may somehow induce an increase in intracellular persulfide levels (vide infra).


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Figure 2. xCT is not required for cysteine trisulfide-induced changes in sulfur-containing molecules in the cytoplasm. (A) HEK293T cells were treated with 0, 0.1 mM, or 1 mM of the xCT inhibitor sulfasalazine for 3 hrs followed by incubation with 0.5 mM cystine or 0.5 mM cysteine trisulfide for another 3 hrs. Cells were then harvested, reacted with 1 mM HPE-IAM, and analyzed by mass spectrometry for the indicated sulfur-containing molecules. (B) HEK293T cells were treated with siRNA targeting xCT. After 30 hrs, the cells were incubated with 0.5 mM cystine or 0.5 mM cysteine trisulfide for an additional 3 hrs. Cells were then harvested and analyzed as in (A). Possible WST-8 reduction by media and cell-based species: As mentioned above, a previous report from this lab proposed that the increase in “reducing capacity” by cells treated with Cys-SSS-Cys (as measured by the increase in extracellular WST-8 reduction) could be the result of an increase in intracellular reducing equivalents7. That is, an increase in WST-8 reduction could occur via a Cys-SSS-Cys-dependent increase in intracellular NADH/NADPH levels (as shown in Scheme 1). However, it is also possible that the


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increase in WST-8 reduction observed could be due to the export of a potent reductant from Cys-SSS-Cystreated cells into the cellular media. To test this possibility, the isolated media from cells was examined directly. As shown in Figure 3A, samples of media with and without Cys-SSS-Cys, media from live HEK293T cells with and without Cys-SSS-Cys treatment and media from dead HEK293T cells with and without Cys-SSSCys treatment were examined for WST-8 reactivity. Only the media from live HEK293T cells treated with CysSSS-Cys gave significant WST-8 reduction. Importantly, a significant amount of WST-8 reduction in media from Cys-SSS-Cys-treated HEK293T cells was found even when the electron-transfer mediator 1-methoxy PMS (Scheme 1) was omitted. These results are consistent with the idea that Cys-SSS-Cys-treated HEK293T cells release a potent reductant into the media that is capable of reducing WST-8. It is also worth noting that media-mediated WST-8 reduction occurs to a greater extent in the presence of the electron-transport mediator, indicating that the media-induced reduction of WST8 can in part be reduced indirectly through the electron mediator in the absence of cells. In an attempt to further characterize the nature of the released reductant, sample conditions examined for Figure 3A were also tested for their ability to react with the thiol-reactive 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB or Ellman’s reagent). As shown in Figure 3B, the only sample with significant DTNB reactivity was the same sample that gave significant WST-8-reactivity, media from live Cys-SSS-Cys-treated HEK293T cells. This result is consistent with the idea that the exported reductant is a reactive sulfur species.

Figure 3. Conditioned media from cysteine trisulfide-treated cells can reduce WST8 directly. (A,B) HEK293T cells were treated with 1 mM cysteine trisulfide and incubated for 3 hours. Media was then collected and treated with WST8 with and without the electron mediator


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(A) or DTNB (B). Absorbance was measure at 450 nm or 412nm respectively. The results with DTNB are representative of several separate experiments. Cellular WST-8 reduction by Cys-SSS-Cys-treated HEK293T cells: It appears that HEK293T cells treated with Cys-SSS-Cys are capable of exporting a potent reductant capable of reducing WST-8. This was further examined by monitoring the rate of WST-8 reduction by cells under a variety of conditions. As shown in Figure 4, control cells (not treated with Cys-SSS-Cys) exhibited WST-8 reduction that required the electrontransfer mediator (1-methoxy PMS). However, cells treated with Cys-SSS-Cys were capable of WST-8 reduction to a greater extent than control cells and, importantly, reduction of WST-8 occurred even in the absence of the electron-transfer mediator. These results are consistent with the idea that Cys-SSS-Cys treatment leads to an increase in overall “reductive capacity” of HEK293T cells (as proposed previously7) but also leads to the export of a reducing species (since the electron-transfer mediator is not required).

Figure 4. Time course of WST8 reduction following treatment of cells with cysteine trisulfide in the presence or absence of the electron mediator. HEK293T cells were plated and then treated with 1 mM cysteine trisulfide and WST8 with or without the electron mediator. WST8 reduction was then monitored at the indicated time points for a total of 2 hours. Shown


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are averages of triplicate samples at each time point with error bars denoting standard deviation. WST-8 reduction and DTNB reactivity by sulfur species: The finding that HEK293T cells treated with Cys-SSS-Cys are capable of releasing/exporting a potent reductant capable of reducing WST-8 and, presumably, capable of reacting with the thiol reactive reagent DTNB is novel and presents several potential possibilities. As described previously by this lab, HEK293T cells treated with Cys-SSS-Cys leads to an intracellular increase in per- and poly-sulfide species such as Cys-SSH and GSSH7. An increase in, for example, Cys-SSH could also lead to an increase in other sulfur species such as H2S and Cys-SH, via a dynamic equilibrium between these species that may exist within the cell18. Export of any of these species may, therefore, be responsible for the observed WST-8 and DTNB reactivity. Thus, in order to determine which sulfur species is capable of the observed reaction profile (WST-8 reduction and DTNB reactivity), H2S, Cys-SH and the hydropersulfide donor methoxycarbonyl penicillamine disulfide6,14 (MCPD) were examined. As shown in Figure 5A, only the hydropersulfide donor species MCPD exhibited significant WST-8 reduction under the conditions of the experiment (0.5 mM, 30 minutes). Both Cys-SH and H2S showed no significant reducing activity under these conditions. However, as expected, all species exhibited DTNB reactivity Figure 5B. It is important to note that the comparison of MCPD, cysteine and H2S with respect to DTNB and WST-8 reactivity is only qualitative since MCPD is a persulfide donor and cannot be kinetically compared to cysteine and NaHS. Moreover, the hydropersulfide made via MCPD decomposition can react with undegraded MCPD10 as well as DTNB or WST-8, thus the reactivity of the persulfide may be underestimated compared to the other sulfur species. Finally, NaHS (HS-) is in equilibrium with H2S in solution which is volatile and capable of leaving the system, thus the reactivity of H2S may also be underestimated. It is also important to note that some reactivity of WST-8 with, for example, cysteine can be seen after an extended time (data not shown). However, over the course of these experiments (30 minutes), little reactivity is observed. Regardless, it is clear that the reactivity profile of the exported cellular reductant appears to be that of a hydropersulfide (represented by the donor MCPD). This result is not necessarily surprising since hydropersulfides are reported to be superior reductants compared the


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corresponding thiol1,2,10,11.

Figure 5. WST8 reduction by persulfides. PBS (control), 0.5 mM MCPD, 0.5 mM Cysteine, or 0.5 mM NaHS were reacted with WST8 (A) or DTNB (B) for 30 min at RT. Absorbance was then read at 450 nm or 412 nm respectively. Shown are averages of triplicate samples with error bars denoting standard deviation. Chemical identification of exported cellular WST-8-reactive species: Thus far it appears that cellular export of a hydropersulfide after Cys-SSS-Cys treatment can occur. In order to test this further, the isolated media from experiments with HEK293T cells were specifically examined for a variety of sulfur species (i.e. H2S, H2S2, H3S3, Cys-SH, Cys-SSH, Cys-SSSH, GSH, GSSH and GSSSH). Cells were incubated for 3 hours with 1 mM Cys-SSS-Cys followed by analyte trapping and analysis. Cells were not washed to remove any excess Cys-SSS-Cys in order to maximize the levels and thus detectability of possible exported species. As shown in Figure 6A, significant levels of Cys-SSH were found in the media of Cys-SSS-Cys treated HEK293T cells. Low levels of Cys-SH and Cys-SSSH were also found. Importantly, media or untreated cells did not result in significant levels of Cys-SSH (or any other sulfur species). Interestingly, media with Cys-SSS-Cys alone resulted in some Cys-SSH, possibly via the reaction of a component of the media with Cys-SSS-Cys to give Cys-SSH. Regardless, the greatest amount of Cys-SSH was observed when HEK293T cells were present. As shown in Figure 6B, little if any GSSH is found in media from Cys-SSS-Cys-treated cells. The levels of other sulfur-based species (GSH, GSSH, Cys-SH, etc.) in media from Cys-SSS-Cys-treated cells are also considerably lower than that observed for Cys-SSH (approximately 1200 micromolar for Cys-SSH versus, for example, 3-5 micromolar for GSH and GSSH). Finally, H2S was also found in the extracellular media of Cys-SSS-Cys-treated HEK293T cells (Figure 6B). This result may be not surprising since H2S is in equilibrium with Cys-SSH and Cys-SH (as well as other thiols)7,18 and is not easily compartmentalized.


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However, the levels of H2S detected are also considerably lower (approximately 90 micromolar) than those found for Cys-SSH (approximately 1.2 millimolar).

Figure 6. Cysteine trisulfide treatment results in extracellular sulfur-containing molecules. HEK293T cells were incubated with 1 mM cysteine trisulfide for 3 hours. Culture media was then harvested, centrifuged, and the supernatant reacted with 5 mM HPE-IAM for 30 min at 37 °C. Samples were analyzed by mass spectrometry for cysteine-related species (A) and glutathione and inorganic sulfur-related species (B). Shown is a representative dataset from two independent treatments.

Discussion It is clear that HEK293T cells are capable of interacting with Cys-SSS-Cys in culture media resulting in increases in intracellular levels of hydropersulfides such as GSSH and Cys-SSH7. Importantly, this phenomenon is not unique to HEK293T cells and can occur with other cell types as well (i.e. HeLa and A549, Figure 1B). However, it is not known how Cys-SSS-Cys treatment leads to higher intracellular levels of


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hydropersulfides. It was originally thought that the well-characterized importer of extracellular cystine (Cys-SSCys) xc- 16,19 could be responsible for Cys-SSS-Cys uptake as well but this was found not to be the case. In fact, inhibition of xc- activity by sulfasalazine or siRNA knockdown of xCT (the actual transporter protein component of xc-) did not decrease the ability for Cys-SSS-Cys to lead to increases in intracellular Cys-SSH or GSSH levels (Figure 2). Indeed, in some cases higher levels could be observed with xc- inhibition (vide infra). Moreover, it is not known whether intact Cys-SSS-Cys is transported into the cell or whether only the sulfane sulfur atom is somehow transposed, leading to hydropersulfide generation on intracellular thiols. If Cys-SSSCys were transported into the cell intact, it may be expected that it could be reduced enzymatically to generate Cys-SSH, analogous to the reduction of glutathione trisulfide (GSSSG) by glutathione reductase20. Also, TRP14, a thioredoxin-related protein has been shown to be capable of cystine (Cys-SS-Cys) reduction21 and it would not be surprising if Cys-SSS-Cys were also a substrate, especially since it has been shown that TRP-14 can reduce inorganic polysulfides and protein hydropersulfides22. These possibilities are under current investigation. Regardless, it is clear that cells treated with Cys-SSS-Cys possess higher levels of intracellular polysulfur species, which results in protection against electrophilic toxicity7. Thus, Cys-SSS-Cys can be a useful pharmacological tool for examining the effects increased intracellular hydropersulfides have on cell function and viability. As mentioned above, sulfasalazine appears to actually increase Cys-SSS-Cys-dependent levels of intracellular Cys-SSH, GSSH and even H2S2 (opposite of what would be expected if Cys-SSS-Cys was taken up by xc-) (Figure 2A). Although sulfasalazine is used in this study as an xc- inhibitor, it is important to note that it is also used clinically to treat inflammation. The pharmacological utility of sulfasalazine is not considered to be related to its ability to inhibit xc- but rather a result of its anti-inflammatory properties and as such is often used for the treatment of rheumatoid arthritis and inflammatory bowel disease23. The mechanism(s) of the anti-inflammatory actions of sulfasalazine is(are) not fully established, although at least part of its activity may be a result of adenosine release and/or NFB activation inhibition24. Although it is premature to speculate on the relationship between sulfasalazine and intracellular levels of per- and poly-sulfide species, it may be noteworthy that a drug that acts as an anti-inflammatory leads to increased levels of intracellular hydropersulfides. This is especially provocative since hydropersulfides have been hypothesized to be important cellular protectants against both oxidative and electrophilic stress2, both of which can lead to an inflammatory response. The results presented herein show that cells treated with Cys-SSS-Cys appear to export thiol-based reductants into the media. This is evidenced by noting that Cys-SSS-Cys pretreatment results in the extracellular generation of species that can reduce WST8 and are DTNB-reactive (Figure 3). Importantly, these effects are not necessarily limited to only HEK293T cells as other cells appear to be capable of releasing WST-8 reducing species as well when treated with Cys-SSS-Cys, albeit to varying levels (Figure 1A).


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Importantly, all of the WST-8 reduction did not require 1-methoxy PMS (Figure 4), indicating that the reducing equivalents were not exclusively coming from intracellular sources as some reductants must have been exported by cells into the extracellular media. Although numerous reductants including H2S and other biological thiol species (e.g. cysteine and GSH) may be DTNB reactive, only a hydropersulfide reacts significantly with WST-8 under the conditions of these experiments (Figure 5). Further chemical characterization of sulfur species in the media of Cys-SSS-Cys treated cells (Figure 6) shows that hydropersulfides, particularly the highly nucleophilic and reducing Cys-SSH, are present in the media. Since hydropersulfides are both highly reducing and nucleophilic (superior to thiols in both cases), they are likely the predominant DTNB- and WST8-reactive species released by cells. It is intriguing to speculate on the reasons for the presumed cellular export of Cys-SSH. As mentioned previously, it has been proposed that hydropersulfides such as Cys-SSH and GSSH may be beneficial to a cell since they can react with and neutralize toxic electrophiles and can act as antioxidants1,2,6. However, it is also worth noting that in spite of the fact that hydropersulfides are good reductants, superior to the corresponding thiol11, they are oxidized thiol species and, as such, have the potential to react with crucial cellular thiols possibly adversely affecting their function (Reaction 1 or 2). RSSH + Protein-SH  Protein-SSR + H2S

(1)

RSSH + Protein-SH  Protein-SSH + RSH

(2)

GSSG + Protein-SH  Protein-SSG + GSH

(3)

That is, the deprotonated RSS- form is reducing and nucleophilic, however the protonated RSSH form is electrophilic and capable of reacting with reduced thiol species. Thus, abnormally high levels of hydropersulfides in cells may be deleterious, akin to how high levels of GSSG can be deleterious to a cell via generation of protein mixed disulfides which often inhibit protein function25 (Reaction 3). Indeed, export and/or compartmentalization of GSSG is required to maintain healthy cellular redox homeostasis26. Another possible reason for Cys-SSH export is that these highly nucleophilic and reducing species may act to intercept cellular toxins (e.g. electrophiles or heavy metals) in the extracellular space before they have the opportunity to enter the cell. For example, Abiko and coworkers27 and Akiyama et al.28 have reported that organic and inorganic hydropersulfides are capable of reacting with and detoxifying methylmercury and cadmium via formation of bismethylmercury sulfide and cadmium sulfide, respectively. If this were to occur in extracellular space, methylmercury or cadmium would not have any opportunity to disrupt intracellular thiol proteins. This phenomenon has been tentatively referred to as “phase zero” metabolism29. Finally, it is possible that


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extracellular Cys-SSH may have cell-to-cell, paracrine-like signaling functions. That is, cells producing extracellular Cys-SSH are not only protected against oxidative/electrophilic stress but are also capable of communicating the presence of this stress to other neighboring cells as well as provide a level of protection to these otherwise naïve cells. Clearly other possible explanations exist for Cys-SSH export and it is possible that, depending on the cell type and environment, all of the above scenarios are relevant. It is important to note that the levels of extracellular Cys-SSH are much greater than the levels of GSSH in spite of the fact that GSSH has been found to be the predominant intracellular hydropersulfide species15. This may indicate that significant levels of intracellular GSSH (or other hydropersulfides) are not particularly deleterious to cells, even though intracellular levels of an analogous oxidized GSH species, GSSG, are carefully monitored and regulated and either compartmentalized or exported to limit its toxicity26. Thus, although GSSG and GSSH can be considered to be at the same oxidation state (in both cases the two sulfur atoms are in the -1 oxidation state), GSSH may be distinct with respect to its biological chemistry and significant levels of GSSH may not be as deleterious as high levels of GSSG since, as mentioned above, GSSH is a good reductant and nucleophile (better than GSH) whereas GSSG can only act as an oxidant or electrophile under biological conditions. It is also worthwhile to consider that GSSH can exist in its deprotonated anionic state (GSS-), a form that will not be electrophilic or oxidizing like GSSG. To be sure, it remains possible that intracellular compartmentalization of GSSH also occurs, limiting its possible deleterious effects, similar to what occurs with GSSG. It is important to note that the levels of extracellular hydropersuflides (i.e. Cys-SSH, GSSH, etc.) were measured after several hours of cellular incubation with Cys-SSS-Cys. Thus, the presence of high levels of Cys-SSH in the media could be a result of chemical processes leading to extracellular Cys-SSH and not necessarily direct export of Cys-SSH, as proposed above. For example, export of GSSH (which is known to be the predominant intracellular hydropersulfide7,15) could result in a reaction with extracellular Cys-SSS-Cys (Reaction 4), leading to extracellular Cys-SSH. GSSH + Cys-SSS-Cys  Cys-SSS-G + Cys-SSH

(4)

Although this remains a possibility, this mechanism for the formation of extracellular Cys-SSH seems unlikely since Reaction 4 would be an equilibrium process7 that would be expected to generate similar levels of GSSH and Cys-SSH under the conditions of these experiments. However, measured levels of extracellular Cys-SSH versus GSSH are approximately 1200 to 3-4 micromolar, respectively. Thus, a more reasonable mechanism, at this time, appears to be selective Cys-SSH export and intracellular GSSH retention.


17


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It is important to mention that although the results presented herein are consistent with Cys-SSH export, it is also possible that release of H2S into the media and subsequent reaction with extracellular CysSSS-Cys can be responsible for at least some of the observed extracellular Cys-SSH (Reaction 5). H2S + Cys-SSS-Cys  2Cys-SSH

(5)

This mechanism is clearly a possibility, especially since an increase in Cys-SSS-Cys-dependent H2S is also observed (Figure 6B). Although the increased extracellular levels of Cys-SSH via Reaction 5 cannot be currently ruled out, it may be significant that conditions leading to increased levels of intracellular Cys-SSH7 appear to correlate with increased levels of extracellular Cys-SSH and measurable extracellular levels of H2S are at significantly lower levels than that found for Cys-SSH. Of course, it remains possible that the equilibrium of Reaction 5 lies to the right (as shown) and that H2S is efficiently trapped by Cys-SSS-Cys. Regardless, characterization of a possible Cys-SSH transport system, if it exists, becomes an important factor in determining the possible mechanism(s) of extracellular Cys-SSH generation. A scheme that possibly explains the results of this study is shown below (Scheme 2). The reasons for hydropersulfide export are currently a matter of speculation and further work is required to establish the purpose of this phenomenon. Clearly, much can be gleaned from the determination of the mechanisms of both uptake and export of poly/per-sulfides and how these processes are regulated. It will also be important to characterize the specificity of transport (i.e., is uptake specific for Cys-SSS-Cys and are other polysulfides species also exported?) as well as the fate of the exported species.


18

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WST8(red) TNB (colored) RSS-TNB

DTNB

WST8(ox) Cys-SSSS-Cys/RSSn-SnSR Cys-SSH/RSn

extracellular protection?

Cys-SSS-Cys H 2S

I

? ?

? RSSn

Cys-SSS-Cys + RSH

RSS

Cys-SSH + Cys-SSR

H N O HPE-IAM

OH

H N O MS analysis

OH

intracellular protection, redox function

Scheme 2: Import and export of per/polysulfide species.

Funding Information: P. N. received funding from the Hungarian National Research, Development and Innovation Office, grants No. KH17 126766 and K18 129286 for support. T. A. received support from Grantsin-Aid for Scientific Research (18H05277) and a Grant-in-Aid for scientific research on Innovative Areas (26111008) from the Ministry of Education, Sciences, Sports and Technology (MEXT), Japan. Y. K. received support from a Grant-in-Aid (#18H05293) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Acknowledgments: None Abbreviations: Cysteine hydropersulfide (Cys-SSH), glutathione hydropersulfide (GSSH), cysteine trisulfide (thiocystine; Cys-SSS-Cys), glutathione (GSH), cysteine (Cys-SH), methoxycarbonyl penicillamine disulfide (MCPD), 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), β-(4-hydroxyphenyl) ethyl iodoacetamide (HPE-IAM).


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References 1. 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) The redox chemistry and chemical biology of H2S, hydropersulfides and derived species: Implications of their possible biological activity and utility, Free Radic. Biol. Med., 77, 82-94. 2. Alvarez, L., Bianco, C. L., Toscano, J. P., Lin, J., Akaike, T. and Fukuto, J. M. (2017) The chemical biology of hydropersulfides and related species: Possible roles in cellular protection and redox signaling, Antiox. Redox Sign., 27(10), 622-633. 3. Filipovic, M. R., Zivanovic, J., Alvarez, B. and Banerjee, R. (2018) Chemical Biology of H2S Signaling through Persulfidation, Chem. Rev., 118, 1253-1337. 4. Cuevasanta, E., Moller, M. N. and Alvarez, B (2017) Biological chemistry of hydrogen sulfide and persulfides, Arch. Biochem. Biophys., 617, 9-25. 5. Kasamatsu, S., Nishimura, A. Morita, M., Matsunaga, T. Hamid, H. A. and Akaike, T. (2016) Redox Regulation regulated by cysteine persulfide and protein polysulfidation, Molecules, 21, 1721. 6. Millikin, R., Bianco, C. L., White, C., Saund, S. S., Henriquez, S., Sosa, V., Akaike, T., Kumagai, Y., Soeda, S., Toscano, J. P., Lin, J. and Fukuto, J. M. (2016) The chemical biology of protein hydropersulfides: Studies of a possible protective function of biological hydropersulfide generation, Free Rad. Biol. Med., 97, 136-147. 7. Bianco, C. L., Akaike, T., Ida, T., Nagy, P., Bogdandi, V., Toscano, J. P., Kumagai, Y., Henderson, C. F., Goddu, R. N., Lin, J. and Fukuto, J. M. (2018) The reaction of hydrogen sulfide with disulfides: Formation of a stable trisulfide and implications to biological systems, Brit. J. Pharmacol., manuscript published online May 29, 2018. 8. Ishiyama, M., Miyazono, Y., Sasamoto, K., Ohkura, Y. and Ueno, K. (1997) A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability, Talanta, 44, 12991305. 9. Tominaga, H., Ishiyama, M., Ohseto, F., Sasamoto, K., Hamamoto, T., Suzuki, K. and Watanabe, M. (1999) A water-soluble terazolium salt useful for colorimetric cell viability assay, Anal. Commun., 36, 47-50. 10. Everett, S. A. and Wardman, P. (1995) Perthiols as antioxidants: Radical-scavenging and prooxidative mechanisms, Meth. Enzymol., 251, 55-69. 11. Bianco, C. L., Chavez, T. A., Sosa, V., Nguyen, Q. N. N., Tantillo, D. J., Ichimura, A. S., Toscano, J. P. and Fukuto, J. M. (2016) The chemical biology of the persulfide (RSSH)/perthiyl (RSS·) redox couple and possible role in biological redox signaling, Free Rad. Biol. Med., 101, 20-31. 12. Akaike, T., Ida, T., Wei, F.-W., Nishida, M., Kumagai, Y., Alam, M., Ihara, H., Sawa, T., Matsunaga, T., Kasamatsu, S., Nishimura, A., Morita, M., Tomizawa, K., Nishimura, A., Watanabe, S., Inaba, K., Shima, H., Tanuma, N., Jung, M., Fujii, S., Watanabe, Y., Ohmuraya, M., Nagy, P., Feelisch, M., Fukuto, J. M. and Motohashi, H., (2017) Cysteinyl-tRNA synthase governs cysteine polysulfidation and mitochondrial bioenergetics, Nature Comm., 8, 1177, 1-15. 13. Savige, W. E., Eager, J. A., Maclaren, J. A. and Roxburgh, C. M. (1964). The S-monoxides of cystine, cystamine and homocysteine, Tetrahedron Lett, 5, 3289–3293.


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Page 21 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


14. Artaud I and Galardon E (2014). A persulfide analogue of the nitrosothiol SNAP: formation, characterization and reactivity. ChemBioChem 15: 2361-2364. 15. Ida, T., Sawa, T., Ihara, H., Kasamatsu, S., Kunieda, K., Tsuchiya, Y., Watanabe, Y., Kumagai, Y., Nishida, M., Suematsu, M., Motohashi, H., Fujii, S., Matsunaga, T., Yamamoto, M., Ono, K., Devarie-Baez, N. O., Xian, M., Fukuto, J. M. and Akaiake, T. (2014) Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling, Proc. Natl. Acad. Sci., USA, 111, 7606-7611. 16. Conrad, M. and Sato, H. (2012) The oxidative stress-inducible cystine/glutamate antiporter, system xc-: Cystine supplier and beyond, Amino Acids 42, 231-246. 17. Gout, P. W., Buckley, A. R. and Bruchovsky, N. (2001) Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the xc- cystine transporter: a new action of an old drug, Leukemia, 15, 1633-1640. 18. Bogdandi, V., Ida, T., Sutton, T., Bianco, C., Koster, G., Henthron, H., Toscano, J. P., van der Vliet, A., Pluth, M. D., Feelisch, M., Fukuto, J. M., Akaike, T, and Nagy, P. (2018) Speciation of reactive sulfur species and their reactions with alkylating agents: Have we got a clue of what is inside the cell? Brit. J. Pharmacol., manuscript published online, June 17, 2018. 19. Lewerenz, J., Hewett, S. J., Huang, Y., Lambros, M., Gout, P. W., Kalivas, P. W., Massie, A., Smolders, I., Methner, A., Pergande, M., Smith, S. B., Ganapathy, V. and Maher, P. (2013) The cystine/glutamate antiporter system xc- in health and disease: From molecular mechanisms to novel therapeutic opportunities, Antiox. Redox Signal. 18, 522-555. 20. Moutiez, M., Aumercier, M., Teissier, E., Parmentier, B., Tartar, A. and Sergheraert, C. (1994) Reduction of a trisulfide derivative of glutathione by glutathione reductase, Biochem. Biophys. Res. Commun., 202, 1380-1386. 21. Pader, I., Sengupta, R., Cebula, M., Xu, J., Lundberg, J. O., Holmgren, A., Johansson, K. and Arner, E. S. J. (2014) Thioredoxin-related protein of 14 kDa is an efficient L-cysteine reductase and S-denitrosylase, Proc. Natl. Acad. Sci., USA, 111, 6964-6969. 22. Doka, E., Pader, I., Biro, A., Johansson, K., Cheng, Q., Ballago, K., Prigge, J. R., Pastor-Flores, D., Dick, T. P., Schmidt, E. E., Arner, E. S. J. and Nagy, P. (2016) A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems, Sci. Adv., 2, e1500968. 23. Plosker, G. L. and Croom, K. F. (2005) Sulfasalzine, A Review of its Use in the Management of Rheumatoid Arthritis, Drugs 65, 1825-1849. 24. Wahl, C., Liptay, S., Adler, G. and Schmid, R. M. (1998) Sulfasalazine: A potent inhibitor of nuclear factor kappa B, J. Clin. Invest., 101, 1163-1174. 25. Ghezzi, P. (2013) Protein glutathionylation in health and disease, Biochim. Biophys. Acta, 1830, 31653172. 26. Morgan, B., Ezerina, D., Amoako, T. N., Riemer, J., Seedorf, M. and Dick, T. P. (2013) Multiple glutathione disulfide removal pathways mediate cytosolic redox homeostasis, Nature Chem. Biol., 9, 119-125.


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27. Abiko, Y., Yoshida, E., Ishii, I., Fukuto, J. M., Akaike, T., Kumagai, Y. (2015) Involvement of reactive persulfides in biological bismethylmercury sulfide formation, Chem. Res. Toxicol., 28, 1301-1306. 28. Akiyama, M., Shinkai, Y., Unoki, T., Shim, I., Ishii, I., and Kumagai, Y. (2017) Capture of cadmium by reactive polysulfides attenuates cadmium-induced adaptive response and hepatotoxicity, Chem. Res. Toxicol., 30, 2209-2217. 29. Kumagai, Y., and Abiko, Y. (2017) Environmental electrophiles: protein adducts, modulation of redox signaling and interaction with persulfides/polysulfides, Chem. Res. Toxicol., 30: 203-219.


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The Uptake and Release of Polysulfur Cysteine Species by Cells

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