Detoxification of Selenite to Form Selenocyanate in Mammalian Cells

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Chemical Research in Toxicology 1

2 Detoxification of selenite to form selenocyanate in mammalian cells Yasumi Anan†, Momoko Kimura†, Marina Hayashi†, Ren Koike† and Yasumitsu Ogra‡,* †

Laboratory of Chemical Toxicology and Environmental Health, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan

‡

Department of Toxicology and Environmental Health, Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675, Japan

To whom correspondence should be addressed: Yasumitsu Ogra, Ph.D. Professor, Department of Toxicology and Environmental Health, Graduate School of Pharmaceutical Sciences, Chiba University 1-8-1 Inohana, Chuo, Chiba 260-8675, Japan Tel./Fax.: +81 43 226 2944 E-mail: [email protected]

Keywords: selenium; selenoprotein; spectroscopy; glutathione peroxidase; mass spectrometry; toxicology; metal homeostasis; selenocyanate; speciation

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Chemical Research in Toxicology

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TOC GRAPHIC

ABSTRACT When human hepatoma HepG2 cells were exposed to sodium selenite, an unknown selenium metabolite was detected in the cytosolic fraction by HPLC-inductively coupled plasma mass spectrometry (ICP-MS). The unknown selenium metabolite was also detected in the mixture of HepG2 homogenate and sodium selenite in the presence of exogenous glutathione (GSH). The unknown selenium metabolite was identified as selenocyanate by electrospray ionization mass spectrometry (ESI-MS) and ESI quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Because exogenous cyanide increased the amount of selenocyanate in the mixture, selenocyanate seemed to be formed by the reaction between selenide or its equivalent, the product of the reduction of selenite, and endogenous cyanide. Rhodanase, an enzyme involved in thiocyanate synthesis, was not required for the formation of selenocyanate. Selenocyanate was less toxic to HepG2 cells than selenite or cyanide, suggesting that it was formed to reduce the toxicity of selenite. On the other hand, selenocyanate could be assimilated into selenoproteins and selenometabolites in rats in the same manner as selenite. Consequently, selenite was metabolized to selenocyanate to temporarily ameliorate its toxicity, and selenocyanate acted as an intrinsic selenium pool in cultured cells exposed to surplus selenite.


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INTRODUCTION Selenium is an essential element in animals. The sources of nutritional selenium include both naturally occurring inorganic (selenite and selenate) and organic (proteinaceous amino acids, such as selenocysteine

and

selenomethionine;

Se-methylselenocysteine, selenocompounds.

1

All

and

free

selenoamino

γ-glutamyl-Se-methylselenocysteine, of

the

selenocompounds

are

acid and

involved

derivatives,

such

as

selenohomolanthionine) in

the

synthesis

of

selenoproteins/selenoenzymes in which selenium is present as selenocysteinyl residues that participate in the redox reaction of selenoenzymes as the active center.2, 3 Such diverse sources of nutritional selenium have to be converted into the same metabolic intermediate, i.e., selenide or its equivalent form of selenium such as GSSeH.4, 5 Selenide or its equivalent is initially converted into selenophosphate by selenophosphate synthetases, and then, the selenium in selenophosphate is transferred to selenocysteinyl-tRNA to be incorporated into selenoenzymes according to the UGA codon that encodes the selenocysteinyl residue.6-8 Surplus selenium from a large dose of selenium and selenium liberated by degraded selenoproteins are excreted mostly into urine as 1β-methylseleno-N-acetyl-D-galactosamine (selenosugar), and in the case of exceedingly large doses, as selenosugar and trimethylselenonium (TMSe).9-12 These observations indicate that selenium-containing molecules, such as the nutritional selenium sources and the degradation products of selenium from selenoproteins, are transformed initially into selenide or its equivalent, and subsequently into the urinary metabolites for excretion. 1β-Glutathionylseleno-N-acetyl-D-galactosamine (GS-selenosugar), a precursor of selenosugar, is detected in the liver of experimental animals.10 Contrary to selenoproteome, we have to directly detect selenium to access selenometabolome. Due to its chemical properties, selenium can form a carbon-selenium bond through the metabolic pathway in animals and plants, and its biological and toxicological effects are very much dependent on its chemical form. Hence, the identification of selenium-containing metabolites is expected to provide important clues to elucidate the metabolic pathway of selenium. As selenium is essential but exists in a trace amount, the analyses of selenometabolome require massive amounts of samples, such as urine, tissues/organs, and blood. Indeed, studies of selenometabolome in cultured cells are limited. Gene modification is easier to perform in cultured cells than in whole animals. In this regard, the utilization of cultured cells is expected to provide new insights into the analyses of selenometabolome. Hence, we attempted to examine selenometabolome in cultured cells, in particular, during the detoxification of inorganic selenium compounds, by speciation analysis. Speciation analysis involves the analysis of the distribution of an element among defined chemical species in a system. In other words, it is the quantitative and qualitative detection of elements.13 Inductively coupled plasma mass spectrometry (ICP-MS) is a superior technique for elemental speciation



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owing to its high sensitivity and specificity when used in combination with HPLC.14, 15 LC-ICP-MS is the technique of choice for the speciation analysis of selenium, in particular, for the screening of selenium compounds in samples.16, 17 The assignment of selenium compounds by LC-ICP-MS is accomplished by matching the retention times of samples with those of authentic standards. However, this technique is inappropriate and insufficient for the assignment of selenium compounds whose standards are not available.

18, 19

Electrospray ionization mass spectrometry (ESI-MS) is another technique to identify

unknown selenium compounds/metabolites in biological samples.20-22 The present study was conducted to determine the metabolism, in particular, the detoxification of selenium in cultured cells, and to identify selenium-containing metabolite(s) in cultured cells by mass spectrometry. In addition, to clarify the biological and toxicological roles of the identified selenium metabolites, the metabolism of selenium was also evaluated in whole animals by mass spectrometry.

EXPERIMENTAL PROCEDURES Chemicals. Sodium selenite, elemental form of selenium, potassium cyanide, sodium thiosulfate pentahydrate,

potassium

thiocyanate,

sodium

sulfite,

2-[4-(2-hydroxyl-ethyl)-1-piperazinyl]ethane-sulfonic acid (HEPES), and L-glutamine were purchased from Wako Pure Chemical Industries (Osaka, Japan). The elemental form of

82

Se (99.9% enriched) was

purchased from Isoflex USA (San Francisco, CA, USA). Glutathione (reduced form, GSH), potassium selenocyanate, and rhodanase from bovine liver were purchased from Sigma-Aldrich (St. Louis, NJ, USA). GSSeH and GSSeSG were synthesized according to the literatures.23, 24

Cell culture. The minimal deviation human hepatoma cell line, HepG2, was obtained from RIKEN Cell Bank (Tsukuba, Japan). HepG2 cells were grown and maintained in DMEM (Dulbecco's Modified Eagle Medium; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Wako, Osaka, Japan), 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA), and 100 µg/mL streptomycin (Invitrogen) at 37 °C under 5% CO2 atmosphere. The cells were passaged after reaching confluence using 0.025% (v/v) trypsin/EDTA in PBS. Other cells, including mouse hepatoma cells (hepa1-6), African green monkey kidney fibroblasts (COS7), human embryonic kidney cells (HEK293), rat adrenal pheochromocytoma cells (PC12), and rat liver cells (RL34), were also obtained from RIKEN and maintained under respective standard conditions indicated by RIKEN. Exposure of living cells and cell homogenate to selenite. HepG2 cells were exposed to 10 µM sodium selenite for 24 h. The selenite-exposed cells were collected and suspended in the same volume of 20 mM HEPES-KOH, pH 8.0, containing 0.4 mM phenylmethylsulfonyl fluoride (PMSF) as the packed cell volume. Then, the suspended cells were disrupted with an ultrasonic homogenizer (Bioruptor® UCD-200, Cosmo Bio Co., Ltd., Tokyo, Japan) on ice at 200 W, 20 kHz three times each for 30 s at 30-s intervals.


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The cytosolic fraction for selenium speciation was obtained by ultracentrifugation of the homogenate at 105,000xg for 60 min at 4 °C in a 250-µL ultracentrifuge tube (Hitachi Koki Co., Tokyo, Japan). This supernatant was termed the in vivo sample. Homogenates (33%v/v) were also prepared from HepG2, hepa1-6, COS7, HEK293, PC12, and RL34 cells that were not exposed to selenite. The homogenates were mixed with 100 µM sodium selenite plus 10 mM GSH to yield a 25% homogenate, and the mixtures were incubated at 37 °C for 1 h. Then, the cytosolic fractions were obtained by using the same procedures as those mentioned above. The supernatants were termed in vitro samples. Speciation analysis of selenium metabolites by LC-ICP-MS. A 20 µL aliquot of the sample was applied to an HPLC coupled with an ICP-MS (Agilent 7500ce or Agilent 7700, Agilent Technologies, Hachioji, Japan) to analyze the distribution of selenium. The HPLC system (Prominence, Shimadzu, Kyoto, Japan) consisted of an on-line degasser, an HPLC pump, a Rheodyne six-port injector, and a multi-mode size exclusion column (Shodex GS-520HQ, exclusion size >300,000 kDa, 7.5 i.d. x 300 mm with a guard column; Showa Denko, Tokyo, Japan). The multi-mode size exclusion column (GS-520HQ) was eluted with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min. The eluate was introduced directly into the ICP-MS nebulizer to detect selenium at m/z 82. Identification of unknown selenium compound by LC-ESI-MS, ESI-Q-TOF-MS, and LC-ICP-MS. A 5 µL aliquot of the supernatant of HepG2 homogenate treated with 100 µM sodium selenite plus 10 mM GSH, or selenocyanate authentic standard was applied to the HPLC coupled with an ESI-MS (Quattro micro, Waters Corporation, Milford, MA, USA). The multi-mode gel filtration column (Shodex GS-320A-2E, 2.0 i.d. x 250 mm; Showa Denko) was eluted with 10 mM ammonium acetate, pH 6.5, at the flow rate of 0.04 mL/min. The MS spectra were obtained in the negative ion mode. A 5 µL aliquot of the supernatant of HepG2 homogenate treated with sodium selenite and GSH was applied to an ESI quadrupole time-of-flight mass spectrometer (ESI-Q-TOF-MS, Agilent 6450, Agilent Technologies) with 0.3% ammonia solution at the flow rate of 0.1 mL/min. The MS spectrum was obtained in the negative ion mode. The supernatant of HepG2 homogenate mixed with selenite and GSH was spiked or not spiked with selenocyanate authentic standard for the identification of the unknown selenium metabolite based on its chromatographic behavior by LC-ICP-MS. A 20 µL aliquot of the mixture was applied to LC-ICP-MS equipped with the multi-mode size exclusion column of normal bore size (GS-520HQ). The column was eluted under the same conditions as those mentioned above, and the eluate was introduced directly into the ICP-MS nebulizer to detect selenium at m/z 82. Synthesis of selenosulfate, [82Se]-labeled selenocyanate, and [82Se]-labeled selenite. Sodium selenosulfate was synthesized according to the literature with slight modifications.25 Sodium selenosulfate in the solution form was prepared as follows: 395 mg of elemental Se powder was added to 10 mL of 1.0



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M sodium sulfite, and the mixture was stirred and refluxed for 3 h at 90 °C under nitrogen atmosphere. [82Se]-labeled selenocyanate was synthesized via selenosulfate. [82Se]-labeled selenosulfate was synthesized in 1/10 scale as mentioned above using [82Se]-enriched elemental Se instead of naturally occurring elemental Se. Then, a 0.5 mL aliquot of 1.0 M potassium cyanide was added. The reaction mixture was refluxed for an additional hour at 90 °C under nitrogen atmosphere. [82Se]-labeled selenite was prepared by dissolving elemental 82Se in nitric acid, followed by adjusting to neutral pH with NaOH. The three solutions containing selenosulfate, [82Se]-labeled selenocyanate or [82Se]-labeled selenite were filtered and diluted with an appropriate buffer to the required concentration. Effects of cyanide and rhodanase on the formation of selenocyanate. The HepG2 homogenate was mixed with sodium selenite and GSH at the final concentrations of 100 µM and 10 mM, respectively, in the presence or absence of potassium cyanide at the final concentration of 10 µM. The homogenates were incubated at 37 °C for 1 h. Then, the cytosolic fraction was obtained by following the same procedures as those mentioned above. A 20 µL aliquot of the supernatant was applied to LC-ICP-MS equipped with the multi-mode size exclusion column of normal bore size (GS-520HQ). The column was eluted under the same conditions as those mentioned above, and the eluate was introduced directly into the ICP-MS nebulizer to detect selenium at m/z 82. The effect of rhodanase on the formation of selenocyanate was also evaluated by LC-ICP-MS. The reaction mixture consisted of 100 µM sodium selenite, 10 mM GSH, 200 µM potassium cyanide, and 5 x 10-3 unit/µL rhodanase in 100 mM phosphate buffer, pH 8.6. The reaction mixture containing 0.005% albumin instead of rhodanase served as the negative control. The reaction mixture containing 10 mM thiosulfate or 10 mM selenosulfate instead of selenite and GSH served as the positive control. A 20 µL aliquot of the mixture was applied to LC-ICP-MS equipped with the GS-520HQ column. The column was eluted under the same conditions as those mentioned above, and the eluate was introduced directly into the ICP-MS nebulizer to detect selenium and sulfur at m/z 82 and 34, respectively. Measurement of cytotoxicity. HepG2 cells were seeded on a 96-well plate at 5.0 x 104 cells/well and pre-incubated for 24 h. The pre-incubated cells were exposed to 5.0, 10, 50, 100, and 500 µM sodium selenite, and 5.0, 10, 50, 100, 500, 1000, and 5000 µM potassium cyanide or potassium selenocyanate in FBS-free HEPES-buffered DMEM for 24 h. After the treatment, the culture medium was exchanged with fresh DMEM containing 10% FBS and MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; Dojindo Laboratories, Kumamoto, Japan), and then incubated for another 4 h at 37 °C. After the medium was removed, dimethyl sulfoxide (Wako Pure Chemical Industries) was added to the wells to extract MTT formazan. Absorbance at 560 nm was measured with a microplate reader (Molecular Devices, Tokyo, Japan). The experiments were repeated four times, and each datum is an average of data from the experiments.


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Animal experiments 1: bolus dose for selenium-deficient rats. All animal experiments were carried out according to the “Principles of Laboratory Animal Care” (NIH version, revised 1996) and the Guidelines of the Animal Investigation Committee, Showa Pharmaceutical University, Japan. Specific pathogen free (SPF) male Wistar rats (5 weeks of age; Sankyo Labo Service Corporation, Inc., Tokyo, Japan) were housed in a humidity-controlled room maintained at 22–25 °C with a 12 h light–dark cycle. The rats were fed a commercial diet (CE-2; Clea Japan Inc., Tokyo, Japan) and tap water ad libitum. After a five-day acclimation period, rats weighing 160–180 g were selected and divided into four groups. One group of four rats were continuously fed the commercial diet, and served as the control. The other groups of rats were fed a selenium-deficient diet (selenium concentration, 0.02 µg/g diet; Oriental Yeast) for 22 days. The rats fed the selenium-deficient diet were divided further into three groups. The rats of the first group and the control group were intravenously injected with saline. The rats of the second and third groups were intravenously injected with selenite and selenocyanate, respectively, at the dose of 0.5 mg selenium/kg body weight. The rats were individually housed in a metabolic cage to collect urine and feces over a 12-h period. All the rats in the four groups were sacrificed 24 h after the injection by exsanguination under anesthesia. Non-heparinized blood was collected and clotted blood was centrifuged at 1600xg for 10 min to obtain serum. Then, the liver and kidneys were excised. An approximately 1.0 g portion of liver or kidney was homogenized with fourfold volume of 50 mM Tris-HCl, pH 7.4, under nitrogen atmosphere. The homogenate was separated by ultracentrifugation at 105,000xg for 60 min at 4 °C to obtain the supernatant. The LC-ICP-MS instrument equipped with the Shodex GS-520HQ column was used for the speciation of selenium in the serum, tissue supernatants, and urine. A 200 µL aliquot of the serum and tissue supernatant samples was applied to the column, and then, elution was carried out with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min. A 20 µL aliquot of the urine sample was also applied to the same column and then eluted with 50 mM Tris-HCl, pH 8.0, at the flow rate of 0.6 mL/min. The eluate was introduced directly into the ICP-MS nebulizer and selenium in the eluate was monitored at m/z 82. Determination of selenium concentration in organs. A 0.1 g portion of the excised liver and kidney was wet-ashed with concentrated nitric acid (HNO3) and 30% H2O2, and then the ashed samples were diluted with Milli-Q water. Selenium concentration in the samples was determined by ICP-MS at m/z 82. Assay for GPx activity. Extracellular GPx (eGPx, GPx3) in the serum and cellular GPx (cGPx, GPx1) activities were determined according to the method of Lawrence and Burk with slight modification, i.e., tert-butylhydroperoxide was used as the substrate instead of hydrogen peroxide or cumene hydroperoxide.26 Animal experiments 2: nutritional dose for selenium-adequate rats. After a five-day acclimation



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period, rats weighing 160–180 g were selected and divided into two groups. A group of four rats were orally administered [82Se]-labeled selenite or [82Se]-labeled selenocyanate at the dose of 20 µg selenium/kg body weight. The rats were individually housed in a metabolic cage to collect urine over a 12-h period. All the rats were sacrificed 24 h after the administration by exsanguination under anesthesia. Blood serum was obtained with the same procedure as that mentioned above. The LC-ICP-MS instrument equipped with the Shodex GS-520HQ column was used for the speciation of selenium in the serum. To obtain a well-separated elution profile of selenium in the urine sample that contains a trace amount of selenium metabolites and large amounts of matrices, the GS-320HQ column (7.5 i.d. x 300 mm with a guard column; Showa Denko), which had a smaller exclusion size (>40,000) than the GS-520HQ column, was used for the analysis of urine sample by LC-ICP-MS. A 200 µL aliquot of serum sample was applied to the GS-520HQ column, and then, elution was carried out with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min. A 20 µL aliquot of the urine sample was applied to the GS-320HQ column, and then elution was carried out with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min. The eluate was introduced directly into the ICP-MS nebulizer and selenium in the eluate was monitored at m/z 77 and 82. The signal of exogenous Se expressed in counts per second (CPS) was calculated with the following equation: [CPS of exogenous Se] = [CPS of 82Se] – [CPS of 77Se] x (0.0873/0.0763). Statistics. Data are presented as means ± S.D. Statistical analysis involved the one-way analysis of variance followed by the Student’s t-test.

RESULTS Detection and identification of novel selenium metabolite in HepG2 cells. The major peak appearing at the retention time of 35.6 min was assigned to an unknown selenium metabolite present in the supernatant of HepG2 cells exposed to 10 µM sodium selenite (Fig. 1A). The retention time of the selenium metabolite did not match the retention times of any of the authentic standards of selenium metabolites reported previously, such as selenosugars, selenoamino acids, and methylated metabolites (e.g., trimethylselenonium ion, dimethylselenoxide and monomethylselenous acid). As the metabolite could be a novel selenium metabolite, we attempted to identify it by molecular mass spectrometry. To reduce the sample preparation time for the molecular mass spectrometry, we designed an in vitro sample preparation method. As mentioned in the Experimental Procedures, a tenfold higher concentration of sodium selenite (100 µM) and 10 mM GSH were mixed with the HepG2 cell homogenate to obtain the selenium metabolite. The selenium metabolite prepared in vitro showed the same retention time as the selenium metabolite in the in vivo sample (Fig. 1B). Although the tenfold higher concentration of sodium


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selenite was toxic to HepG2 cells (in vivo), that concentration of sodium selenite plus GSH yielded the selenium metabolite after incubation for 1 h. Hence, the in vitro sample was used for further identification. Se peaks corresponding to GSH-conjugated selenides present in excess, such as GSSeH and GSSeSG, were not well separated under the chromatographic conditions employed, and were eluted around 17 min (Fig. 1B). We also evaluated the in vitro experiment using other cell lines. The novel selenium metabolite was biosynthesized not only in the homogenate of HepG2 cells but also in the homogenates of hepa1-6, COS7, HEK293, PC12, and RL34 cells, although its amount varied among those cell lines (Figs. 2A-2F). Selenium consists of six isotopes: (49.6%), and

82

74

Se (0.89%),

76

Se (9.36%),

77

Se (7.63%),

78

Se (23.8%),

80

Se

Se (8.73%). Signals showing the isotope patterns of monomeric and trimeric selenium

were observed for the in vitro sample at m/z 106 and 316 as 80Se-containing molecular ions, respectively, in the negative ion mode (Fig. 3A). As the molecular mass seemed to be too small for MS-MS analysis as a precursor ion, we compared the mass spectrum of the in vitro sample with that of possible authentic standards, and used ESI-Q-TOF-MS to determine the elemental composition of the novel selenium metabolite based on its exact molecular mass. We speculated that the negative ion at m/z 106 was [80SeCN]-, i.e., selenocyanate; thus, the mass spectrum of commercially available selenocyanate was measured. As expected, an identical mass spectrum to that of the in vitro sample was obtained (Fig. 3B). In addition, the results of ESI-Q-TOF-MS were in agreement with the results mentioned above. The observed mass containing

80

Se in the sample was 105.92055, and the theoretical mass of

80

SeCN- is

105.91959 (Fig. 3C). Thus, the difference between the observed and theoretical masses was 9.06 ppm. To provide concrete proof of the identification, chromatographic behavior was compared between the unknown selenium metabolite and the authentic standard. When selenocyanate authentic standard was added to the sample, the peak at the retention time of 35.6 min was specifically increased (Fig. 4). This indicated that the unknown selenium metabolite was selenocyanate.

Biological pathway for selenocyanate synthesis. The addition of cyanide, which might be the substrate for selenocyanate synthesis, increased the amount of selenocyanate (Fig. 5). This suggested that selenocyanate was formed by the reaction between selenide and endogenous cyanide. Rhodanase is known to catalyze the formation of thiocyanate from thiosulfate and cyanide. Thus, we speculated that the enzyme also catalyzes the conversion of selenium, which belongs to the same group as sulfur. Rhodanase was required to convert thiosulfate into thiocyanate (Figs. 6A and 6B); however, it had no effect on the formation of selenocyanate from GSSeH and cyanide (Figs. 6C and 6D). In addition, selenosulfate was synthesized and subjected to the rhodanase-catalyzed reaction as another substrate for the formation of selenocyanate. Selenosulfate is an analog of thiosulfate wherein one of the sulfur atoms is replaced with selenium. Thus, selenosulfate seems to be more preferable than GSSeH for the evaluation of the involvement of rhodanase. Synthesized selenosulfate gave the major selenium peak at the retention time



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of 16.5 min (Fig. 6E). The peak position was shifted to the retention time corresponding to selenocyanate upon the addition of cyanide in the absence of rhodanase (Fig. 6E). No further effect of rhodanase on the formation of selenocyanate was observed (Fig. 6F). Comparison of cytotoxicity among selenite, cyanide, and selenocyanate. The cytotoxicity of selenocyanate was lower than those of selenite and cyanide (Fig. 7). The calculated IC50 values of selenite, cyanide, and selenocyanate were 42, 1850, and 6200 µM, respectively, under our experimental conditions. Bioavailability of selenocyanate in selenium-deficient rats. To evaluate the bioavailability of selenocyanate, we performed two sets of animal experiments. In the first animal experiment, we prepared selenium-deficient rats and fed them selenocyanate and selenite at the bolus dose, the latter of which served as the positive control. The nutritional status of selenium well reflects the activities/amounts of selenoenzymes/selenoproteins in blood plasma and the amounts of selenium metabolites in urine.11, 27-29 Two major selenoproteins were detected in control rat serum by LC-ICP-MS analysis, and their retention times were 11.7 and 14.3 min (Fig. 8A). The former and latter peaks were assignable to eGPx and SelP, respectively, according to our previous work.30 By feeding rats a selenium-deficient diet for three weeks, selenium deficiency was induced, as confirmed by the reduction in the amount of selenium in the two serum selenoproteins and the suppression of the activity of eGPx (Figs. 8B and 8E). The amount of selenium in SelP was fully recovered to the control level when selenite or selenocyanate was administered (Figs. 8C and 8D). Contrary to SelP, the amount of selenium in eGPx was partially recovered, and the recovery reflected the recovery of eGPx activity (Figs. 8C-8E). It has been reported that the recovery of SelP occurs faster than that of eGPx.31 This finding agrees with the previous literature. No apparent differences in the recovery of the activity and the amount of serum selenoproteins were noted between the selenocyanate- and selenite-replete groups at the bolus dose. The amount of selenium excreted into urine is another biological index of the nutritional status of selenium.32, 33 The chemical species of urinary selenium metabolites also reflect the status.11 Hence, the speciation analysis of selenium in urine would yield vital information regarding the nutritional status of selenium in the body. In the case of urine sampled from selenium-adequate rat (control), the selenium peak was detected at the retention time of 20.1 min (Fig. 9A). This urinary metabolite was identified as 1ß-methylseleno-N-acetyl-D-galactosamine (selenosugar) on the basis of its chromatographic behavior.9, 10 The amount of the urinary metabolite was considerably reduced when the rat was fed the selenium-deficient diet (Fig. 9B). However, the amount of selenosugar in rat urine collected over a period of 12 h after the administration of selenocyanate and selenite showed a slight recovery (Figs. 9C and 9D). In addition to the selenosugar peak, another large peak of selenium was detected at the retention time of 24.4 min when the

urine sample of rat administered either selenite or selenocyanate was measured. That peak was assigned to trimethylselenonium ion (TMSe) on the basis of its chromatographic behavior. It is known that in


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addition to selenosugar, TMSe is excreted into urine when an animal ingests selenium in an amount that exceeds the adequate level.34 The amounts of both selenium metabolites were smaller in the selenocyanate-replete rats than in the selenite-replete rats (Fig. 9C and 9D). An additional selenium urinary metabolite that was eluted at the retention time of 18.1 min was detected in the urine samples of both selenocyanate- and selenite-replete rats. However, the urinary metabolite could not be identified. In the enlarged elution profiles shown in Figs. 9E and 9F, selenocyanate was detected even when the animals were injected with selenite. This indicated that selenocyanate was also biosynthesized and excreted into urine when the rat was administered an extremely high dose of selenite. Then, the selenium concentrations in the liver and kidneys of the rats were determined. By feeding rats the selenium-deficient diet, selenium concentrations in the liver and kidneys were significantly decreased (Fig. 10A). The selenium concentrations in the liver and kidneys of the selenite-replete rats were recovered 24 h after the injection. In addition, the selenium concentrations in the selenocyanate-replete rats were significantly higher than those in the selenite-replete rats. Contrary to the selenium concentrations in the organs, cellular GPx (GPx1) activity showed a tendency to increase in the liver, and a significant increase in the kidney, with the injection of either selenium compound (Fig. 10B). The results suggested that the selenium incorporated into the organs had not been fully utilized for selenoprotein synthesis, at least for GPx synthesis, 24 h after the injection. The recovery of GPx1 activity seemed to be comparable to that of eGPx activity, i.e., GPx3 in serum (Fig. 8E). We performed the second experiment to evaluate the bioavailability of selenocyanate at the nutritional level in normal (selenium-adequate) rats. We administered [82Se]-labeled selenite or [82Se]-labeled selenocyanate to distinguish these exogenous (labeled) selenium compounds from endogenous selenium in selenium-adequate rats. The distributions of exogenous selenium in serum and urine were monitored by LC-ICP-MS. Typical elution profiles of exogenous selenium in the serum of rats administered [82Se]-labeled selenite or [82Se]-labeled selenocyanate are shown in Figs. 11A and 11B, respectively. Either selenium compound was proportionally incorporated into both serum selenoproteins, i.e., eGPx and SelP, in the selenium-adequate rats. Exogenous selenium was mainly excreted into urine as selenosugar, and approximately 10% of exogenous selenium in the urine was TMSe when either selenite or selenocyanate was administered at the nutritional level (Figs. 12A-12C).

DISCUSSION The newly detected selenium metabolite in the cultured cells was assigned to selenocyanate on the basis of three reasons. First, the mass spectrum of the unknown selenium metabolite was in good



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agreement with that of commercially available selenocyanate used as the authentic standard (Fig. 3B). Second, the observed accurate mass of the unknown selenium metabolite showed good agreement with the theoretical mass of selenocyanate (Fig. 3C). Third, the chromatographic behavior of the unknown selenenium metabolite was identical to that of an authentic standard (Fig. 4). Compared with the previous methods for the identification of novel selenium metabolites in animals, plants, and yeast,9, 20, 35 the identification methods used in this study are robust from the viewpoint of analytical chemistry.18 Hence, it can be concluded that inorganic selenium is transformed into selenocyanate in vivo and in vitro. Then, we determined the concentrations of cyanide in the culture medium and the reagents used because it was reported that exogenous cyanide reacted with selenide in the form of selenodiglutathione.36 However, no exogenous cyanide was supplied owing to the formation of selenocyanate in vivo and in vitro in this study. We speculate that selenocyanate is formed from selenide or its equivalent and endogenous cyanide, i.e., endogenous cyanide is generated from other biomolecule(s). It was reported that cyanide was de novo synthesized from glycine by myeloperoxidase in leukocytes and rat pheochromocytoma cells,37-39 namely, the N-chlorination of glycine, which is catalyzed by myeloperoxidase, results in the conversion of glycine into N-monochloroglycine. The acid-catalyzed dismutation of N-monochloroglycine results in N,N-dichloroglycine, an unstable compound that decomposes into 2-cyanoacetic acid. A further degradation product of 2-cyanoacetic acid in cells is cyanide. This is one of the feasible pathways to produce cyanide in HepG2 cells. Although endogenously generated cyanide reacted with selenide or its equivalent in the absence of an enzyme (Fig. 6), the amount of selenocyanate varied with the cell type (Fig. 2). This variation could be explained by the difference in endogenous cyanide producing capacity among the cells. This effect of endogenous cyanide seems to be reactive cyanogen species likened to NOand HS- of reactive nitrogen and sulfur species, respectively. Why did the cultured cells transform selenite into selenocyanate? We offer two explanations. It was reported that the administration of cyanide ameliorated the toxicity of selenite and vice versa, indicating that exogenous selenide, which is the reduced form of selenite in vivo, and cyanide antagonized each other.40 Likewise, endogenous cyanide could act as a detoxicant of selenide or its equivalent in the cultured cells. Previous studies have indicated that the major pathway for the detoxification of selenium involves methylation to form selenosugar and TMSe in whole animals,10,

34

i.e., these methylated

metabolites are much less toxic than selenite.41 Our first explanation is that selenide or its equivalent was transformed into the alternate metabolite, selenocyanate, to reduce the toxicity of selenite because cultured cells could have a smaller selenium methylating capacity than the organs of whole animals. Indeed, a trace amount of selenocyanate was detected in the urine of rats injected with a bolus dose of selenite. Although the methyltransferase responsible for the methylation of selenium is still unknown, the expression of methyltransferase and/or the amount of S-adenosylmethionine may define the selenium


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methylating capacity.42 In the present study, no methylated metabolites of selenium were detected in the in vivo and in vitro samples of cultured cells. Further studies are needed to estimate the selenium methylating capacity of the cells. Selenocyanate was less toxic than selenite in the cultured cells. In addition, the nutritional availability of selenocyanate was almost equivalent to that of selenite in the rats, in terms of the biosynthesis of selenoproteins, such as GPx and SelP (Fig. 8). Selenocyanate was transformed into the selenoproteins as efficiently as selenite in the selenium-deficient rats. This clearly indicates that selenocyanate is not the final metabolite of selenium in whole animals. Compared with selenocyanate, the methylated metabolites of selenium, such as selenosugar and TMSe, are poorly assimilated even in selenium-deficient animals.43, 44 Thus, as the second explanation, the cultured cells preferably transform excess selenium into selenocyanate to utilize it as the selenium pool. In other words, as selenocyanate is efficiently metabolized, it should be treated not as an inert metabolite of selenium but as a novel form of intracellular selenium pool. Selenium was predominantly incorporated into SelP in the selenium-deficient rats that received a bolus dose of selenium compounds (Fig. 8). eGPx and SelP contain 1 and 10 SeCys residues, respectively, in their molecules. Thus, the increase of selenium in the SelP peak is faster than that in the eGPx one during selenium repletion in the selenium-deficient condition. Contrary to the selenium-deficient rats, selenium originating from either selenite or selenocyanate was equally incorporated into eGPx and SelP in the selenium-adequate rats (Fig. 11). In other words, selenium in either selenite or selenocyanate is proportionally substituted to endogenous Se in the selenium-adequate rats. This suggests that selenocyanate is assimilated into selenoproteins in rats in the same manner as selenite. It has been shown that selenite is incorporated into red blood cells where it is reduced to selenide.33 Then, selenide is released by the red blood cells for it to bind to albumin in bloodstream, and finally, selenide is incorporated into organs.45 To de novo synthesize selenoproteins, selenocysteine (SeCys) should be formed.2 Selenium in SeCys is donated by selenophosphate to O-acetylcysteine, and selenophosphate is synthesized by the reaction between ATP and selenide with selenophosphate synthase 2 (SPS2) as the catalyst.46 In addition, urinary selenium metabolites, such as selenosugar and TMSe, are also synthesized from the common metabolic intermediate, selenide.47 Hence, to utilize selenium in selenocyanate for the synthesis of selenoproteins and selenium metabolites, selenocyanate should be decomposed to selenide or its equivalent, and then, selenide or its equivalent should be utilized to synthesize primarily selenoproteins and secondarily urinary selenium metabolites. It was reported that the cyanide moiety resulting from the decomposition of selenocyanate was excreted into urine as thiocyanate.48 This suggested that selenocyanate was incorporated in an intact form and then decomposed in the organs. Consequently, selenocyanate acts as a selenium pool.



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In conclusion, selenocyanate was identified as the novel selenium metabolite in cultured cells by inorganic (ICP) and organic (ESI) mass spectrometry. Selenocyanate was found to be less toxic than selenite when the selenium compounds were administered to the cultured cell lines. In the same manner as selenite, selenocyanate was metabolized and assimilated into selenoproteins in the selenium-deficient rats. In addition, selenocyanate showed the same behavior as selenite in terms of selenium incorporation into serum selenoproteins, when the selenium-adequate rats were administered the selenium compounds at the nutritional level. Taken together, selenite is metabolized to selenocyanate to temporarily ameliorate its toxicity, and selenocyanate acts as an intrinsic selenium pool.

FUNDING INFORMATION This study was supported by JSPS KAKENHI Grant Numbers 23390032, 24659022, 25870739, 26293030 and 15K14991, and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (2013-2017). We also thank Takeda Science Foundation, Japan for financial support.

ACKNOWLEDGEMENTS The authors thank Mr. Daichi Ikeda and Ms. Saki Hasegawa for technical assistance in animal experiments.

ABBREVIATIONS eGPx, extracellular glutathione peroxidase; ICP-MS, inductively coupled plasma-mass spectrometry; SeCys,

selenocysteine;

SelP,

selenoprotein

P;

SPS2,

selenophosphate

trimethylselenonium ion


synthase

2;

TMSe,

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FIGURE LEGENDS Fig. 1. Elution profiles of selenium in the supernatant of HepG2 cells in vivo and in vitro. HepG2 cells were exposed to 10 µM sodium selenite for 24 h. The exposed cells were collected and disrupted with an ultrasonic homogenizer on ice to obtain 50% homogenate. The cytosolic fraction for selenium speciation was obtained by ultracentrifugation of the homogenate. This supernatant was termed the in vivo sample. Another homogenate was prepared from HepG2 cells without the selenite exposure. The homogenate was mixed with 100 µM sodium selenite plus 10 mM GSH, and the mixture was incubated at 37 °C for 1 h. Then, the cytosolic fraction was obtained by ultracentrifugation of the homogenate. This supernatant was termed the in vitro sample. A 20 µL aliquot of the in vivo (A) and in vitro (B) samples was applied to LC-ICP-MS to analyze the distribution of selenium. The multi-mode size exclusion column (Shodex GS-520HQ) was eluted with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min. The eluate was introduced directly into the ICP-MS nebulizer to detect selenium at m/z 82. ukSe: unknown selenium metabolite.

Fig. 2. Elution profiles of selenium in supernatants of cultured cell lines in vitro. Homogenates (33%) were prepared from HepG2, hepa1-6, COS7, HEK293, PC12, and RL34 cells without selenite exposure. The homogenates were mixed with 100 µM sodium selenite plus 10 mM GSH, and the mixtures were incubated at 37 °C for 1 h to yield 25% homogenate as the final concentration. Then, the cytosolic fractions were obtained by ultracentrifugation of the homogenates. A 20 µL aliquot of the supernatants from HepG2 (A), hepa1-6 (B), COS7 (C), HEK293 (D), PC12 (E), and RL34 cells (F) was applied to LC-ICP-MS to analyze the distribution of selenium. The column (Shodex GS-520HQ) was eluted with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min. The eluate was introduced directly into the ICP-MS nebulizer to detect selenium at m/z 82. ukSe: unknown selenium metabolite.

Fig. 3. Mass spectra of unknown selenium metabolite in the supernatant of HepG2 homogenate and selenocyanate authentic standard. A 5 µL aliquot of the supernatant of HepG2 homogenate treated with 100 µM sodium selenite plus 10 mM GSH (A), or selenocyanate authentic standard (B) was subjected to LC-ESI-MS on a Shodex GS-320A-2E multi-mode gel filtration column, and the column was eluted with 10 mM ammonium acetate, pH 6.5, at the flow rate of 0.04 mL/min. The MS spectra were obtained in the negative ion mode. A 5 µL aliquot of the supernatant of HepG2 homogenate treated with sodium selenite plus GSH was subjected to ESI-Q-TOF-MS with 0.3% ammonia solution at the flow rate of 0.1 mL/min (C). The MS spectrum was obtained in the negative ion mode. ukSe: unknown selenium metabolite.

Fig. 4. Elution profiles of selenium in the supernatant of selenite-treated HepG2 unspiked or spiked



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with selenocyanate authentic standard. A 20 µL aliquot of the supernatant of selenite-treated HepG2 unspiked (A) or spiked (B) with selenocyanate authentic standard was subjected to LC-ICP-MS on a Shodex GS-520HQ column, and the column was eluted with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min. The eluate was introduced directly into the ICP-MS nebulizer to detect selenium at m/z 82. ukSe: unknown selenium metabolite.

Fig. 5. Effect of cyanide on selenocyanate formation in HepG2 homogenate. The HepG2 homogenate was mixed with sodium selenite and GSH at the final concentrations of 100 µM and 10 mM, respectively, in the absence (A) or presence (B) of potassium cyanide at the final concentration of 10 µM. The homogenates were incubated at 37 °C for 1 h. Then, the cytosolic fraction was obtained by ultracentrifugation of the homogenate. A 20 µL aliquot of the supernatant was applied to LC-ICP-MS equipped with a Shodex GS-520HQ column. The column was eluted under the same conditions as those mentioned above, and the eluate was introduced directly into the ICP-MS nebulizer to detect selenium at m/z 82.

Fig. 6. Effect of rhodanase on selenocyanate formation. The reaction mixture consisted of 100 µM sodium selenite (C and D) or sodium selenosulfate (F and G), 10 mM GSH, 200 µM potassium cyanide, and 5 x 10-3 units/µL rhodanase in 100 mM phosphate buffer, pH 8.6. The reaction mixture containing 0.005% albumin instead of rhodanase served as the negative control (A, C, and F). The solution of sodium selenosulfate in 100 mM phosphate buffer served as the standard to check the retention time of selenosulfate (E). The reaction mixture containing 10 mM thiosulfate instead of selenite and GSH served as the positive control (A and B). A 20 µL aliquot of the mixture was applied to LC-ICP-MS equipped with a Shodex GS-520HQ column. The column was eluted with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min, and the eluate was introduced directly into the ICP-MS nebulizer to detect selenium and sulfur at m/z 82 and 34, respectively.

Fig. 7. Comparison of cytotoxicity of selenite, cyanide, and selenocyanate in HepG2 cells. HepG2 cells were seeded on a 96-well plate and pre-incubation was carried out for 24 h. The pre-incubated cells were exposed to sodium selenite (triangles), potassium cyanide (circles) or potassium selenocyanate (squares) in HEPES-buffered DMEM not containing FBS for 24 h. After the treatment, the culture medium was exchanged with fresh DMEM containing 10% FBS and MTT, and then, incubation was carried out for another 4 h at 37 °C. After removing the medium, dimethyl sulfoxide was added to the wells to extract MTT formazan. Absorbance at 560 nm was measured with a microplate reader. The experiments were repeated four times.


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Fig. 8. Effect of selenocyanate or selenite injection into selenium-deficient rats on selenium distribution and GPx activity in serum. A 200 µL aliquot of the serum of control (A), selenium-deficient (B), selenite-replete (C) or selenocyanate-replete (D) rats was subjected to LC-ICP-MS on a Shodex GS-520HQ column. The column was eluted with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min. The eluate was introduced directly into the ICP-MS nebulizer to detect selenium at m/z 82. Extracellular GPx (eGPx, GPx3) activity in the serum was determined (E). The experiments were repeated three times. Data are presented as means ± S.D. Different letters on the bars (a, b, and c) represent the level of significant difference set at p < 0.05.

Fig. 9. Effect of selenocyanate or selenite injection into selenium-deficient rats on selenium distribution in urine. A 20 µL aliquot of the urine of control (A), selenium-deficient (B), selenite-replete (C and E) or selenocyanate-replete (D and F) rats was subjected to LC-ICP-MS on a Shodex GS-520HQ column. The column was eluted with 50 mM Tris-HCl, pH 8.0, at the flow rate of 0.6 mL/min. The eluate was introduced directly into the ICP-MS nebulizer and selenium in the eluate was monitored at m/z 82. Panels E and F are enlarged images of panels C and D, respectively.

Fig. 10. Effect of selenocyanate or selenite injection into selenium-deficient rats on selenium concentration and GPx activity in liver and kidneys. A 0.1 g portion of the excised liver and kidney was wet-ashed with concentrated nitric acid (HNO3) and 30% H2O2, and then the ashed samples were diluted with Milli-Q water. Selenium concentration in the samples was determined by ICP-MS at m/z 82 (A). Cellular GPx (cGPx, GPx1) activity in the liver and kidney was determined (B). The experiments were repeated three times. Data are presented as means ± S.D. Different letters on the bars (a, b, and c) represent the level of significant difference set at p < 0.05.

Fig. 11. Effect of selenocyanate or selenite administration to selenium-adequate rats on selenium distribution in serum. A group of four rats without selenium deficiency were orally administered [82Se]-labeled selenite or [82Se]-labeled selenocyanate at the dose of 20 µg selenium/kg body weight. Blood serum was obtained 24 h after the administration. A 200 µL aliquot of the serum of selenite-administered (A) or selenocyanate-administered (B) rats was subjected to LC-ICP-MS on a Shodex GS-520HQ column. The column was eluted with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min. The eluate was introduced directly into the ICP-MS nebulizer to detect selenium at m/z 77 and 82. The distribution of exogenous selenium determined on the basis of the calculation indicated in Experimental Procedures was plotted.



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Fig. 12. Effect of selenocyanate or selenite administration to selenium-adequate rats on selenium distribution in urine. A group of four rats without selenium deficiency were orally administered [82Se]-labeled selenite or [82Se]-labeled selenocyanate at the dose of 20 µg selenium/kg body weight. Urine was collected during the 24 h period after the administration. A 20 µL aliquot of the urine of selenite-administered (A) or selenocyanate-administered (B) rats was subjected to LC-ICP-MS on a Shodex GS-320HQ column. The column was eluted with 50 mM Tris-HCl, pH 7.4, at the flow rate of 0.6 mL/min. The eluate was introduced directly into the ICP-MS nebulizer to detect selenium at m/z 77 and 82. The distribution of exogenous selenium determined on the basis of the calculation indicated in Experimental Procedures was plotted.

Scheme 1. Structures of selenium compounds used in this study.


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Figure 1.



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Figure 2.


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Figure 3.



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Figure 4.


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Figure 5.



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Figure 6.


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Figure 7.



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Figure 8.


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Figure 9.



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Figure 10.


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Figure 11.



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Figure 12.


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Scheme 1.


Detoxification of Selenite to Form Selenocyanate in Mammalian Cells

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