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Cyanate causes depletion of ascorbate in organisms
Biochimica et Biophysica Acta 1336 Ž1997. 566–574
Cyanate causes depletion of ascorbate in organisms Ichiro Koshiishi, Yoshie Mamura, Toshio Imanari
)
Faculty of Pharmaceutical Sciences, Chiba UniÕersity, 1-33 Yayoi, Inage, Chiba-shi, Chiba 263, Japan Received 5 May 1997; accepted 19 May 1997
Abstract Ascorbate–dehydroascorbate redox cycle plays a key role in protecting organisms from an excess of oxidants. Recently, we found a novel reaction of dehydroascorbate with cyanate under the conditions of neutral pH and ordinary temperature. In this report, we demonstrated that through this irreversible reaction, cyanate causes the depletion of ascorbate in the matrix, where the ascorbate–dehydroascorbate redox cycle revolves. When the leaves of weed Ž Erigeron canadensis. were soaked in sodium cyanate solution generally used as a herbicide, the depletion of ascorbate as well as dehydroascorbate in them was observed, followed by the change in color from green to brown. These results suggest that a possible way of cyanate toxicity is to inflict oxidative stress on organisms. q 1997 Elsevier Science B.V. Keywords: Cyanate; Ascorbate; Dehydroascorbate; Carbamylation; Oxidative stress
1. Introduction In general, organisms are exposed to oxidative stress, so these organisms have some antioxidant systems. The ascorbate Ž AscHy. –dehydroascorbate ŽDHA. redox cycle is one of these systems w1–3x. After AscHy is oxidized to DHA, the resultant DHA is mostly reduced to AscHy by DHA reductases using glutathione or NADŽ P.H as a substrate w4–9x. Therefore, a substance that can hinder this redox cycle system will cause a damage to organisms. Cyanate is widely known to prevent the physiological reactions in organisms including plants and Abbreviations: AscHy, L-ascorbate; DHA, L-dehydroascorbate; Cb-DHA, carbamylated dehydroascorbate derivative; CbAscHy, 3-O-carbamyl ascorbate; Cb-isoAscHy, 3-O-carbamyl iso-ascorbate; EDTA, ethylenediaminetetraacetic acid; HPLC, high-performance liquid chromatography ) Corresponding author. Fax: q81-43-255-1574.
mammals as a herbicide w10,11x and an uremic toxin w12–14x. Cyanate is electrophilic, and thus it has been studied as an agent for carbamylation of amino groups and sulfhydryl groups w15–20x. If a biological active substance involves amino group or sulfhydryl group in its active site, cyanate could inhibit its activity. On the basis of the earlier in vitro studies, it seemed that the toxicity of cyanate was attributed to the carbamylation of these groups w21–28x. Recently, we found that cyanate irreversibly reacts with both AscHy and DHA in neutral solution at ordinary temperature, resulting in the production of carbamyl ascorbate Ž Cb-AscHy. and carbamylated dehydroascorbate derivative ŽCb-DHA. , respectively ŽScheme 1.. This finding indicates that, if the extents of these reactions are greatly increased in vivo, organisms would be significantly damaged through a declining of the antioxidant system. The objective of our study reported here is to clarify whether cyanate affects the AscHy–DHA redox cycle.
0304-4165r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 6 5 Ž 9 7 . 0 0 0 7 3 - 1
I. Koshiishi et al.r Biochimica et Biophysica Acta 1336 (1997) 566–574
2. Materials and methods 2.1. Materials Ascorbic acid, sodium cyanate and ascorbate oxidase ŽEC 1.10.3.3, from Cucurbita species. were purchased from Wako Pure Chemicals Ž Japan. . Dehydroascorbic acid was purchased from Aldrich Chem. Co. ŽUSA. . All other chemicals were of reagent grade. TSKgel SAX was obtained from Tosoh Co. ŽJapan.. A standard solution of cyanate was prepared from sodium cyanate that had been recrystallized from ethanol. 2.2. Apparatus The post-column high-performance liquid chromatography ŽHPLC. was assembled with a HPLC pump ŽHitachi, L-6000., a sample injector Ž Rheodyne, 7725. , a double-plunger pump wShimamura Instrument Co. ŽJapan. , PSU-2.5Wx, a dry reaction bath ŽShimamura Instrument Co., DB-5. , a UV-Vis detector ŽHitachi, L-4200., a fluorescence spectrophotometer ŽHitachi, F-1050. , chromato-integratorŽ Hitachi, D-2500. and Teflon tubing Ž 0.5 mm i.d. and 0.25 mm i.d... 2.3. Syntheses of carbamylated deriÕatiÕes 2.3.1. Carbamylated dehydroascorbate deriÕatiÕe Dehydroascorbate was prepared from ascorbic acid by oxidation using cupric ion. Ascorbic acid powder Ž52 g. was added to 2 liters of 0.15 M copper Ž II. acetate solution, and then the reaction solution was stirred vigorously at room temperature. After stand-
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ing 10 min, 1 liter of 0.75 M sodium cyanate solution was added dropwise to the solution, and the reaction mixture was stood overnight with stirring. The reaction mixture was filtered through filter paper ŽAdvantec Toyo, 51A. , and the filtrate was passed through Dowex 1-X8 ŽCly-form, 50–100 mesh, 5 cm i.d. = 10 cm.. The eluate was concentrated to 200 ml by evaporation, and the solution was added dropwise to 800 ml of 99% ethanol. The Cb-DHA precipitate was recovered by filtration. The precipitate was dissolved in water, and the volume was adjusted to 200 ml. Cb-DHA in the solution was crystallized by mixing with 800 ml of 99% ethanol. Cb-DHA was recrystallized four times from ethanol–water. Cb-DHA has the molecular formula, C 7 H 8 NO 8 Na, and contains two water molecules of crystallization. The result of the elemental analysis was as follows; calculated for C 7 H 8 NO 8 Na P 2H 2 O: C, 28.70%; H, 4.09%; N, 4.78%; O, 54.61%; Na, 7.85%. Found: C, 28.70%; H, 3.89%; N, 4.55%; Na, 7.70%; other elements, 55.16%. 2.3.2. Carbamyl ascorbate Ascorbic acid powder Ž 52 g. and sodium cyanate Ž48.8 g. were dissolved in 1.0 liter of water, and then the solution was adjusted pH to 6.0 by acetic acid. The mixture solution was maintained pH in the range of 5.5 to 6.0 by acetic acid for 3 h at room temperature with stirring by magnetic stirrer. The reaction solution was concentrated to 150 ml by evaporation, and then the solution was stood in an ice-water bath overnight. The solution was filtered through filter paper ŽAdvantec Toyo, 51A. , and the filtrate was added dropwise to 300 ml of 80% ethanol. Furthermore, the mixture solution was added dropwise to 1.2
Scheme 1. Structures of carbamylated dehydroascorbate derivative and 3-O-carbamyl ascorbate.
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liter of acetone with stirring. Carbamyl ascorbate was separated from solvent as a colorless oily liquid. The oily phase was washed with 500 ml of acetone three times, and then dried in vacuo. Carbamyl iso-ascorbate was synthesized from iso-ascorbic acid and sodium cyanate in the similar manner as above. 2.4. Determination of carbamylated dehydroascorbate deriÕatiÕe and carbamyl ascorbate Cb-DHA and Cb-AscHy were determined simultaneously by a post-column HPLC equipped with a UV-Vis detector. The chromatographic conditions are as follows: column, TSKgel SAX Ž 4.6 mm i.d. = 250 mm.; eluent, 0.15 M NaCl Ž 0.6 mlrmin.; column temperature, 608C; reagent 1, 0.16 M acetic acid containing 2% phenol and 0.01% sodium nitroprusside; reagent 2, 0.3 M NaOH containing 0.1% sodium hypochlorite. The post-column derivatization conditions are as follows: eluate was mixed with 0.1 M NaOH Ž 0.1 mlrmin. and the mixture was passed through a Teflon tube Ž0.5 mm i.d. = 10 m. . The reaction solution was mixed with reagent 1 Ž0.35 mlrmin. and the mixture was passed through a Teflon tube Ž0.5 mm i.d. = 15 m.. The reaction solution was mixed with reagent 2 Ž0.35 mlrmin. and the mixture was passed through a Teflon tube Ž 0.5 mm i.d. = 15 m.. Each tube was heated at 808C. The resultant indophenol was monitored by UV-Vis detector Ž635 nm.. Carbamyl iso-ascorbate was spiked with sample solution as an internal standard. The sample solution was passed through a Dowex 1-X8 column Ž600 m l.. The column was washed with 1 ml of H 2 O, and then Cb-DHA and Cb-AscHy were eluted with 0.5 M NaCl. First 300 m l of eluate was discarded and the next 1 ml was collected. A portion of the fraction was submitted to HPLC. 2.5. Other determination methods Ascorbate and dehydroascorbate were determined by a post-column HPLC equipped with fluorescence spectrophotometer w29x. Cyanate was determined sensitively and specifically by a post-column HPLC w30x. S-Carbamyl groups were determined indirectly by converted to cyanate w19x.
2.6. Preparation of glutathione-dependent dehydroascorbate reductase from rat liÕer Liver from Wistar male rat was used as a starting material w8x. The liver was washed of as much blood as possible with 0.13 M NaCl containing 20 mM EDTAP 2Na and minced into small pieces. The same solution was used to give 20% Žwrv. solution, and the solution was homogenized with a Potter–Elvehjem type homogenizer under cooling in an ice-water bath. The homogenate was passed through two layers of gauze, and then centrifuged at 10 000 = g for 30 min at 48C. The supernatant was heated by placing in a water bath, maintained at 708C. When the supernatant reached 628C, the suspension was immediately placed on ice and then centrifuged at 10 000 = g for 30 min at 48C. Ammonium sulfate was slowly added to the supernatant to give a final concentration of 40% saturated ammonium sulfate. After standing on ice for 10 min, the supernatant was centrifuged at 10 000 = g for 30 min. Ammonium sulfate was added to the supernatant to give a final concentration of 65% saturated ammonium sulfate, and the resulting suspension was centrifuged at 10 000 = g for 30 min after standing on ice for 15 min. The supernatant was dialyzed against 50 mM Tris-HCl buffer Ž pH 6.8., and then the dialysate was submitted to the DHA-reductase assay according to the convenient method w8x with a minor modification.
3. Results 3.1. Reaction of cyanate with DHA and AscH y Cyanate reacts with DHA in neutral buffered solution at 308C, producing Cb-DHA irreversibly as shown in Fig. 1. The structure of Cb-DHA was identified by spectroscopic ŽNMR and negative FABMS. and X-ray analyses w31x. Similarly, cyanate reacts irreversibly with AscHy in neutral buffered solution at 308C, producing Cb-AscHy as shown in Fig. 2. The NMR spectroscopic study Ž NOESY. indicated that the carbamylation had occurred on the C 3 hydroxyl group Ždata not shown. . In neutral solution, C 3 hydroxyl group of AscHy exists in alcoholate ion form, R–Oy. This reaction proceeds under the conditions of the high concentration of cyanate, and its
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reaction rate is significantly lower than that with DHA under the same conditions. 3.2. HPLC for the determination of Cb-DHA and Cb-AscH y Both Cb-DHA and Cb-AscHy are commonly alkaline labile, releasing cyanate. This allowed us to determine Cb-DHA and Cb-AscHy simultaneously and specifically by HPLC with post column derivatization. After separating Cb-DHA from Cb-AscHy on an anion exchange column ŽTSKgel SAX, 4.6 mm i.d. = 250 mm., they were degraded to cyanate by heating in alkaline solution. Successively, the cyanate was hydrolyzed to ammonia by heating in acidic solution, and then the resultant ammonia was de-
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tected by the indophenol reaction. These serial reactions were performed in Teflon tubes. The chromatogram of synthetic Cb-DHA and Cb-AscHy is shown in Fig. 3. 3-O-Carbamyl iso-ascorbate ŽCb-isoAscHy. is an internal standard. 3.3. Effect of cyanate on the DHA reductase actiÕity In organisms, DHA produced from AscHy by oxidation under oxidative stress is reduced to AscHy by DHA reductases using glutathione or NADŽ P.H as a substrate. These enzymes are playing a key role in maintaining AscHy at high level by recycling DHA. The activity of DHA reductases are attributed to sulfhydryl groups Žcysteine residues.. Since cyanate reversibly reacts with sulfhydryl groups to S-carba-
Fig. 1. Reaction of cyanate with dehydroascorbate in neutral solution at ordinary temperature. Tris-HCl buffer Ž10 mM, pH 6.8. containing 1 mM sodium cyanate and 1 mM dehydroascorbic acid was incubated at 308C. At scheduled time, a portion of the reaction mixture was taken out, and submitted to the determination methods for cyanate and Cb–DHA as described in Section 2.
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myl groups, it may be a possible inhibitor for these enzymes. From rat liver, a glutathione-dependent DHA reductase was isolated and submitted to the DHA-reducing activity assay as follows; the rat hepatic glutathione-dependent DHA reductase Ž 6.58 = 10y3 units. was incubated with 0.5 mM DHA in 50 mM Tris-HCl buffer Ž pH 6.8. containing 1 mM glutathione at 308C for 5 min. Fig. 4 shows the effect of cyanate on the activity. Accompanied with an increase of cyanate concentration, a slight inhibition of the activity was observed. This phenomenon was caused by the decrease of the DHA concentration. Approximately 20–30% of added cyanate irreversibly reacted with DHA during the period of incubation. In addition, a part of residual cyanate was reversibly transformed to S-carbamyl glutathione. As shown in Fig. 5, when 0.2 mM cyanate was incubated with 1.0
mM glutathione in 50 mM Tris-HCl buffer Ž pH 6.8. at 308C for 5 min, approximately 10% of cyanate was transformed to S-carbamyl glutathione. Taken together, these results suggested that the extent of inhibition of glutathione-dependent DHA reductase by the residual cyanate Ž 60–70% of added cyanate. is comparatively low. 3.4. Effect of cyanate on the AscH y–DHA redox cycle in Õitro When AscHy was incubated with glutathione-dependent DHA reductase and glutathione in neutral buffered solution under oxidative stress, equilibrium was established between the oxidation of AscHy and reduction of DHA Ž AscHy–DHA redox cycle. . In this experiment, AscHy oxidase Ž EC 1.10.3.3, from
Fig. 2. Reaction of cyanate with ascorbate and iso-ascorbate in neutral solution at ordinary temperature. Acetate buffer solution Ž0.5 M, pH 6.2. containing 200 mM ascorbic acid and 300 mM sodium cyanate was incubated at 308C. Cb–AscHy was determined by the HPLC as described in Section 2.
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Fig. 3. Chromatogram of synthetic Cb–DHA and Cb–AscHy. Column, TSKgel SAX Ž4.6 mm i.d. = 250 mm.; eluent, 0.15 M NaCl Ž0.6 mlrmin.; column temperature, 608C. The post-column derivatization conditions were described in Section 2. A portion Ž20 m l. of a standard mixture solution Ž20 m M Cb–DHA, 20 m M Cb–AscHy and 20 m M Cb– isoAscHy . was applied on column. Cb– isoAscHy was added to sample solution as an internal standard.
Cucurbita sp.. was used to oxidize AscHy to DHA instead of oxidants. By using this model system, the effect of cyanate on the AscHy–DHA redox cycle was studied. The results are shown in Fig. 6. When 200 m M AscHy was incubated with 6.58 = 10y3 units rat hepatic glutathione-dependent DHA reductase and 7.5 = 10y3 units AscHy oxidase in 50 mM Tris-HCl buffer ŽpH 6.8. containing 5 mM glutathione, at least 45 min were required to reach equilibrium. Thereafter, both AscHy and DHA concentrations decreased gradually by the spontaneous cleavage of lactone ring of DHA, producing 2,3-diketogulonate. In contrast, in the presence of cyanate in this system, the resultant DHA quickly and irreversibly reacted with cyanate, and thus the concentration of AscHy decreased with first-order kinetics. However, production of Cb-AscHy in this system was not observed Ždata not shown..
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Fig. 4. Effect of cyanate on the rat hepatic glutathione-dependent dehydroascorbate reductase activity. 50 mM Tris-HCl buffer ŽpH 6.8. containing 6.58=10y3 units rat hepatic glutathione-dependent dehydroascorbate reductase, 1 mM glutathione, 0.5 mM DHA and cyanate Žconcentration is indicated in figure. was incubated at 308C for 5 min. A portion of reaction mixture was submitted to HPLC for the determination of AscHy and DHA.
Fig. 5. Time-courses of cyanate and S-carbamyl glutathione levels in neutral buffered solution. Sodium cyanate Ž200 m M. was incubated with 1.0 mM glutathione in 50 mM Tris-HCl buffer ŽpH 6.8. at 308C for indicated times. Portions of the reaction mixture were submitted to the determination method for cyanate and that for S-carbamyl groups.
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3.5. Disappearance of AscH y in cyanate-treated plants Effect of cyanate on the AscHy–DHA redox cycle in leaves of Erigeron canadensis, most popular weed in our campus, was studied. In the leaves of Erigeron canadensis exposed to sun-light, the concentration of DHA was higher than that of AscHy. The leaves of Erigeron canadensis were soaked in 0.5 M sodium cyanate solution Ž 0.5 M sodium cyanate solution is generally sprinkled on plants as herbicide. at 308C for 30 min w10x. After washed well with water, they were kept in a dry incubator at 308C. Within 2 h, the leaves changed in color from green to brown Ž picture is not shown.. The time-courses of AscHy, DHA, Cb-DHA and Cb-AscHy levels in the leaves of
Fig. 7. Time-courses of AscHy, DHA, Cb–DHA and Cb–AscHy levels in cyanate-treated leaves of Erigeron canadensis. Lower, the leaves of Erigeron canadensis were soaked in 0.5 M sodium cyanate solution at 308C for 30 min, and then washed with water. Upper, the leaves of Erigeron canadensis were washed with water without a cyanate-treatment. The leaves were allowed to settle in a dry incubator at 308C. The leaves were homogenized in 1% metaphosphoric acid, and the homogenate was centrifuged at 10 000= g for 10 min. The supernatant was submitted to the determination methods for AscHy, DHA, Cb–DHA and Cb– AscHy.
Fig. 6. Effect of cyanate on the AscHy –DHA redox cycle in vitro. Tris-HCl buffer Ž350 m l. containing 6.58=10y3 units rat hepatic glutathione-dependent DHA reductase was mixed with 10 m l of 8 mM ascorbic acid, 20 m l of 100 mM glutathione, 10 m l of AscHy oxidase Ž0.752 unitsrml. and sodium cyanate Žconcentration is indicated in figure., successively. After standing at 308C for 5 min, the reaction solution was centrifuged at 10 000= g for 5 min, and then the supernatant was submitted to HPLC for the determination of AscHy and DHA.
Erigeron canadensis treated with cyanate during the period of this entire test were shown in Fig. 7. By soaking the leaves in the cyanate solution, the DHA level was lowered less than 30% of the initial one with producing equivalent amounts of Cb-DHA, whereas no alteration of AscHy in concentration was observed. Thereafter, the exposure of leaves to air resulted in the oxidation of AscHy to DHA, and then DHA was spontaneously degraded to 2,3-diketogulonate. Both AscHy and DHA had disappeared
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within 3 h. This phenomenon appears to be responsible for the unbalance between the excess oxidants and the less antioxidant activity.
4. Discussion An excess of cyanate present in the intercellular or intracellular matrix, where oxidants are deactivated by AscHy–DHA redox cycle, can prevent this cycle by transforming DHA to Cb-DHA as shown in Fig. 8. Proceeding of this reaction results in the depletion of AscHy in the matrix. The resultant Cb-DHA no longer reverts to AscHy. These results suggest that, when organisms are exposed to cyanate, they are damaged by an excess of oxidants. It has been known that cyanate quickly blights plants just after attaching to leaves and stems, and thus sodium cyanate has been widely used as a herbicide w10,11x. In spite of the popularity of cyanate as a herbicide, the mechanism of this phenomenon is
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unclear. In general, it is known that the depression of antioxidant activity under oxidative stress is responsible for withering of plants w32,33x. This fact coupled with our findings that cyanate depletes AscHy in plants, suggests that the depletion of AscHy in the cyanate-treated plants is the principal cause of the withering of plants. It has been reported that the long-term administration of cyanate as a therapeutic drug for sickle-cell disease was accompanied by side-effects such as cataract w34x. In addition, cyanate produced from urea is an uremic toxin, so it is thought to be involved in cataract, one of the symptoms of uremia w35x. So far, the mechanism for this phenomenon was postulated as follows: cyanate reacts with amino groups on crystallins, and thus the resultant N-carbamylated crystallins aggregate in a lens w36x. However, in our earlier observations, an in vitro treatment of a bovine lens with cyanate did not result in the turbidity of the lens Žunpublished data.. From this finding, it was speculated that an additional factor as well as N-
Fig. 8. Possible mechanism for the depletion of AscHy caused by cyanate.
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carbamylation of crystallins may be attributed to the induction of cataract. In general, cataract is induced by oxidative stress. The turbidity of a lens is caused by the formation of mixed disulfide bonds on crystallins and the cross-linking Ž disulfide bonds. among crystallin molecules under oxidative stress. These facts suggest that the depletion of AscHy in the cyanate-treated lens may be a principal cause of cataract. Previous studies have noted that the depression of antioxidant system by cyanate is due to the carbamylation of amino groups andror sulfhydryl groups on enzymes concerning with this system. Furthermore, in the present study, we present evidence that cyanate hinders AscHy–DHA redox cycle, by which oxidants were directly deactivated.
Acknowledgements The present work was supported by a Grant-in-Aid for Scientific Research Ž C. Ž No. 08672470. from the Ministry of Education, Science, Sports and Culture, Japan. We thank Dr. Toshihiko Toida at Chiba University Ž Japan. for NMR spectroscopic analyses.
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Cyanate causes depletion of ascorbate in organisms Ichiro Koshiishi, Yoshie Mamura, Toshio Imanari
)
Faculty of Pharmaceutical Sciences, Chiba UniÕersity, 1-33 Yayoi, Inage, Chiba-shi, Chiba 263, Japan Received 5 May 1997; accepted 19 May 1997
Abstract Ascorbate–dehydroascorbate redox cycle plays a key role in protecting organisms from an excess of oxidants. Recently, we found a novel reaction of dehydroascorbate with cyanate under the conditions of neutral pH and ordinary temperature. In this report, we demonstrated that through this irreversible reaction, cyanate causes the depletion of ascorbate in the matrix, where the ascorbate–dehydroascorbate redox cycle revolves. When the leaves of weed Ž Erigeron canadensis. were soaked in sodium cyanate solution generally used as a herbicide, the depletion of ascorbate as well as dehydroascorbate in them was observed, followed by the change in color from green to brown. These results suggest that a possible way of cyanate toxicity is to inflict oxidative stress on organisms. q 1997 Elsevier Science B.V. Keywords: Cyanate; Ascorbate; Dehydroascorbate; Carbamylation; Oxidative stress
1. Introduction In general, organisms are exposed to oxidative stress, so these organisms have some antioxidant systems. The ascorbate Ž AscHy. –dehydroascorbate ŽDHA. redox cycle is one of these systems w1–3x. After AscHy is oxidized to DHA, the resultant DHA is mostly reduced to AscHy by DHA reductases using glutathione or NADŽ P.H as a substrate w4–9x. Therefore, a substance that can hinder this redox cycle system will cause a damage to organisms. Cyanate is widely known to prevent the physiological reactions in organisms including plants and Abbreviations: AscHy, L-ascorbate; DHA, L-dehydroascorbate; Cb-DHA, carbamylated dehydroascorbate derivative; CbAscHy, 3-O-carbamyl ascorbate; Cb-isoAscHy, 3-O-carbamyl iso-ascorbate; EDTA, ethylenediaminetetraacetic acid; HPLC, high-performance liquid chromatography ) Corresponding author. Fax: q81-43-255-1574.
mammals as a herbicide w10,11x and an uremic toxin w12–14x. Cyanate is electrophilic, and thus it has been studied as an agent for carbamylation of amino groups and sulfhydryl groups w15–20x. If a biological active substance involves amino group or sulfhydryl group in its active site, cyanate could inhibit its activity. On the basis of the earlier in vitro studies, it seemed that the toxicity of cyanate was attributed to the carbamylation of these groups w21–28x. Recently, we found that cyanate irreversibly reacts with both AscHy and DHA in neutral solution at ordinary temperature, resulting in the production of carbamyl ascorbate Ž Cb-AscHy. and carbamylated dehydroascorbate derivative ŽCb-DHA. , respectively ŽScheme 1.. This finding indicates that, if the extents of these reactions are greatly increased in vivo, organisms would be significantly damaged through a declining of the antioxidant system. The objective of our study reported here is to clarify whether cyanate affects the AscHy–DHA redox cycle.
0304-4165r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 6 5 Ž 9 7 . 0 0 0 7 3 - 1
I. Koshiishi et al.r Biochimica et Biophysica Acta 1336 (1997) 566–574
2. Materials and methods 2.1. Materials Ascorbic acid, sodium cyanate and ascorbate oxidase ŽEC 1.10.3.3, from Cucurbita species. were purchased from Wako Pure Chemicals Ž Japan. . Dehydroascorbic acid was purchased from Aldrich Chem. Co. ŽUSA. . All other chemicals were of reagent grade. TSKgel SAX was obtained from Tosoh Co. ŽJapan.. A standard solution of cyanate was prepared from sodium cyanate that had been recrystallized from ethanol. 2.2. Apparatus The post-column high-performance liquid chromatography ŽHPLC. was assembled with a HPLC pump ŽHitachi, L-6000., a sample injector Ž Rheodyne, 7725. , a double-plunger pump wShimamura Instrument Co. ŽJapan. , PSU-2.5Wx, a dry reaction bath ŽShimamura Instrument Co., DB-5. , a UV-Vis detector ŽHitachi, L-4200., a fluorescence spectrophotometer ŽHitachi, F-1050. , chromato-integratorŽ Hitachi, D-2500. and Teflon tubing Ž 0.5 mm i.d. and 0.25 mm i.d... 2.3. Syntheses of carbamylated deriÕatiÕes 2.3.1. Carbamylated dehydroascorbate deriÕatiÕe Dehydroascorbate was prepared from ascorbic acid by oxidation using cupric ion. Ascorbic acid powder Ž52 g. was added to 2 liters of 0.15 M copper Ž II. acetate solution, and then the reaction solution was stirred vigorously at room temperature. After stand-
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ing 10 min, 1 liter of 0.75 M sodium cyanate solution was added dropwise to the solution, and the reaction mixture was stood overnight with stirring. The reaction mixture was filtered through filter paper ŽAdvantec Toyo, 51A. , and the filtrate was passed through Dowex 1-X8 ŽCly-form, 50–100 mesh, 5 cm i.d. = 10 cm.. The eluate was concentrated to 200 ml by evaporation, and the solution was added dropwise to 800 ml of 99% ethanol. The Cb-DHA precipitate was recovered by filtration. The precipitate was dissolved in water, and the volume was adjusted to 200 ml. Cb-DHA in the solution was crystallized by mixing with 800 ml of 99% ethanol. Cb-DHA was recrystallized four times from ethanol–water. Cb-DHA has the molecular formula, C 7 H 8 NO 8 Na, and contains two water molecules of crystallization. The result of the elemental analysis was as follows; calculated for C 7 H 8 NO 8 Na P 2H 2 O: C, 28.70%; H, 4.09%; N, 4.78%; O, 54.61%; Na, 7.85%. Found: C, 28.70%; H, 3.89%; N, 4.55%; Na, 7.70%; other elements, 55.16%. 2.3.2. Carbamyl ascorbate Ascorbic acid powder Ž 52 g. and sodium cyanate Ž48.8 g. were dissolved in 1.0 liter of water, and then the solution was adjusted pH to 6.0 by acetic acid. The mixture solution was maintained pH in the range of 5.5 to 6.0 by acetic acid for 3 h at room temperature with stirring by magnetic stirrer. The reaction solution was concentrated to 150 ml by evaporation, and then the solution was stood in an ice-water bath overnight. The solution was filtered through filter paper ŽAdvantec Toyo, 51A. , and the filtrate was added dropwise to 300 ml of 80% ethanol. Furthermore, the mixture solution was added dropwise to 1.2
Scheme 1. Structures of carbamylated dehydroascorbate derivative and 3-O-carbamyl ascorbate.
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liter of acetone with stirring. Carbamyl ascorbate was separated from solvent as a colorless oily liquid. The oily phase was washed with 500 ml of acetone three times, and then dried in vacuo. Carbamyl iso-ascorbate was synthesized from iso-ascorbic acid and sodium cyanate in the similar manner as above. 2.4. Determination of carbamylated dehydroascorbate deriÕatiÕe and carbamyl ascorbate Cb-DHA and Cb-AscHy were determined simultaneously by a post-column HPLC equipped with a UV-Vis detector. The chromatographic conditions are as follows: column, TSKgel SAX Ž 4.6 mm i.d. = 250 mm.; eluent, 0.15 M NaCl Ž 0.6 mlrmin.; column temperature, 608C; reagent 1, 0.16 M acetic acid containing 2% phenol and 0.01% sodium nitroprusside; reagent 2, 0.3 M NaOH containing 0.1% sodium hypochlorite. The post-column derivatization conditions are as follows: eluate was mixed with 0.1 M NaOH Ž 0.1 mlrmin. and the mixture was passed through a Teflon tube Ž0.5 mm i.d. = 10 m. . The reaction solution was mixed with reagent 1 Ž0.35 mlrmin. and the mixture was passed through a Teflon tube Ž0.5 mm i.d. = 15 m.. The reaction solution was mixed with reagent 2 Ž0.35 mlrmin. and the mixture was passed through a Teflon tube Ž 0.5 mm i.d. = 15 m.. Each tube was heated at 808C. The resultant indophenol was monitored by UV-Vis detector Ž635 nm.. Carbamyl iso-ascorbate was spiked with sample solution as an internal standard. The sample solution was passed through a Dowex 1-X8 column Ž600 m l.. The column was washed with 1 ml of H 2 O, and then Cb-DHA and Cb-AscHy were eluted with 0.5 M NaCl. First 300 m l of eluate was discarded and the next 1 ml was collected. A portion of the fraction was submitted to HPLC. 2.5. Other determination methods Ascorbate and dehydroascorbate were determined by a post-column HPLC equipped with fluorescence spectrophotometer w29x. Cyanate was determined sensitively and specifically by a post-column HPLC w30x. S-Carbamyl groups were determined indirectly by converted to cyanate w19x.
2.6. Preparation of glutathione-dependent dehydroascorbate reductase from rat liÕer Liver from Wistar male rat was used as a starting material w8x. The liver was washed of as much blood as possible with 0.13 M NaCl containing 20 mM EDTAP 2Na and minced into small pieces. The same solution was used to give 20% Žwrv. solution, and the solution was homogenized with a Potter–Elvehjem type homogenizer under cooling in an ice-water bath. The homogenate was passed through two layers of gauze, and then centrifuged at 10 000 = g for 30 min at 48C. The supernatant was heated by placing in a water bath, maintained at 708C. When the supernatant reached 628C, the suspension was immediately placed on ice and then centrifuged at 10 000 = g for 30 min at 48C. Ammonium sulfate was slowly added to the supernatant to give a final concentration of 40% saturated ammonium sulfate. After standing on ice for 10 min, the supernatant was centrifuged at 10 000 = g for 30 min. Ammonium sulfate was added to the supernatant to give a final concentration of 65% saturated ammonium sulfate, and the resulting suspension was centrifuged at 10 000 = g for 30 min after standing on ice for 15 min. The supernatant was dialyzed against 50 mM Tris-HCl buffer Ž pH 6.8., and then the dialysate was submitted to the DHA-reductase assay according to the convenient method w8x with a minor modification.
3. Results 3.1. Reaction of cyanate with DHA and AscH y Cyanate reacts with DHA in neutral buffered solution at 308C, producing Cb-DHA irreversibly as shown in Fig. 1. The structure of Cb-DHA was identified by spectroscopic ŽNMR and negative FABMS. and X-ray analyses w31x. Similarly, cyanate reacts irreversibly with AscHy in neutral buffered solution at 308C, producing Cb-AscHy as shown in Fig. 2. The NMR spectroscopic study Ž NOESY. indicated that the carbamylation had occurred on the C 3 hydroxyl group Ždata not shown. . In neutral solution, C 3 hydroxyl group of AscHy exists in alcoholate ion form, R–Oy. This reaction proceeds under the conditions of the high concentration of cyanate, and its
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reaction rate is significantly lower than that with DHA under the same conditions. 3.2. HPLC for the determination of Cb-DHA and Cb-AscH y Both Cb-DHA and Cb-AscHy are commonly alkaline labile, releasing cyanate. This allowed us to determine Cb-DHA and Cb-AscHy simultaneously and specifically by HPLC with post column derivatization. After separating Cb-DHA from Cb-AscHy on an anion exchange column ŽTSKgel SAX, 4.6 mm i.d. = 250 mm., they were degraded to cyanate by heating in alkaline solution. Successively, the cyanate was hydrolyzed to ammonia by heating in acidic solution, and then the resultant ammonia was de-
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tected by the indophenol reaction. These serial reactions were performed in Teflon tubes. The chromatogram of synthetic Cb-DHA and Cb-AscHy is shown in Fig. 3. 3-O-Carbamyl iso-ascorbate ŽCb-isoAscHy. is an internal standard. 3.3. Effect of cyanate on the DHA reductase actiÕity In organisms, DHA produced from AscHy by oxidation under oxidative stress is reduced to AscHy by DHA reductases using glutathione or NADŽ P.H as a substrate. These enzymes are playing a key role in maintaining AscHy at high level by recycling DHA. The activity of DHA reductases are attributed to sulfhydryl groups Žcysteine residues.. Since cyanate reversibly reacts with sulfhydryl groups to S-carba-
Fig. 1. Reaction of cyanate with dehydroascorbate in neutral solution at ordinary temperature. Tris-HCl buffer Ž10 mM, pH 6.8. containing 1 mM sodium cyanate and 1 mM dehydroascorbic acid was incubated at 308C. At scheduled time, a portion of the reaction mixture was taken out, and submitted to the determination methods for cyanate and Cb–DHA as described in Section 2.
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myl groups, it may be a possible inhibitor for these enzymes. From rat liver, a glutathione-dependent DHA reductase was isolated and submitted to the DHA-reducing activity assay as follows; the rat hepatic glutathione-dependent DHA reductase Ž 6.58 = 10y3 units. was incubated with 0.5 mM DHA in 50 mM Tris-HCl buffer Ž pH 6.8. containing 1 mM glutathione at 308C for 5 min. Fig. 4 shows the effect of cyanate on the activity. Accompanied with an increase of cyanate concentration, a slight inhibition of the activity was observed. This phenomenon was caused by the decrease of the DHA concentration. Approximately 20–30% of added cyanate irreversibly reacted with DHA during the period of incubation. In addition, a part of residual cyanate was reversibly transformed to S-carbamyl glutathione. As shown in Fig. 5, when 0.2 mM cyanate was incubated with 1.0
mM glutathione in 50 mM Tris-HCl buffer Ž pH 6.8. at 308C for 5 min, approximately 10% of cyanate was transformed to S-carbamyl glutathione. Taken together, these results suggested that the extent of inhibition of glutathione-dependent DHA reductase by the residual cyanate Ž 60–70% of added cyanate. is comparatively low. 3.4. Effect of cyanate on the AscH y–DHA redox cycle in Õitro When AscHy was incubated with glutathione-dependent DHA reductase and glutathione in neutral buffered solution under oxidative stress, equilibrium was established between the oxidation of AscHy and reduction of DHA Ž AscHy–DHA redox cycle. . In this experiment, AscHy oxidase Ž EC 1.10.3.3, from
Fig. 2. Reaction of cyanate with ascorbate and iso-ascorbate in neutral solution at ordinary temperature. Acetate buffer solution Ž0.5 M, pH 6.2. containing 200 mM ascorbic acid and 300 mM sodium cyanate was incubated at 308C. Cb–AscHy was determined by the HPLC as described in Section 2.
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Fig. 3. Chromatogram of synthetic Cb–DHA and Cb–AscHy. Column, TSKgel SAX Ž4.6 mm i.d. = 250 mm.; eluent, 0.15 M NaCl Ž0.6 mlrmin.; column temperature, 608C. The post-column derivatization conditions were described in Section 2. A portion Ž20 m l. of a standard mixture solution Ž20 m M Cb–DHA, 20 m M Cb–AscHy and 20 m M Cb– isoAscHy . was applied on column. Cb– isoAscHy was added to sample solution as an internal standard.
Cucurbita sp.. was used to oxidize AscHy to DHA instead of oxidants. By using this model system, the effect of cyanate on the AscHy–DHA redox cycle was studied. The results are shown in Fig. 6. When 200 m M AscHy was incubated with 6.58 = 10y3 units rat hepatic glutathione-dependent DHA reductase and 7.5 = 10y3 units AscHy oxidase in 50 mM Tris-HCl buffer ŽpH 6.8. containing 5 mM glutathione, at least 45 min were required to reach equilibrium. Thereafter, both AscHy and DHA concentrations decreased gradually by the spontaneous cleavage of lactone ring of DHA, producing 2,3-diketogulonate. In contrast, in the presence of cyanate in this system, the resultant DHA quickly and irreversibly reacted with cyanate, and thus the concentration of AscHy decreased with first-order kinetics. However, production of Cb-AscHy in this system was not observed Ždata not shown..
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Fig. 4. Effect of cyanate on the rat hepatic glutathione-dependent dehydroascorbate reductase activity. 50 mM Tris-HCl buffer ŽpH 6.8. containing 6.58=10y3 units rat hepatic glutathione-dependent dehydroascorbate reductase, 1 mM glutathione, 0.5 mM DHA and cyanate Žconcentration is indicated in figure. was incubated at 308C for 5 min. A portion of reaction mixture was submitted to HPLC for the determination of AscHy and DHA.
Fig. 5. Time-courses of cyanate and S-carbamyl glutathione levels in neutral buffered solution. Sodium cyanate Ž200 m M. was incubated with 1.0 mM glutathione in 50 mM Tris-HCl buffer ŽpH 6.8. at 308C for indicated times. Portions of the reaction mixture were submitted to the determination method for cyanate and that for S-carbamyl groups.
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3.5. Disappearance of AscH y in cyanate-treated plants Effect of cyanate on the AscHy–DHA redox cycle in leaves of Erigeron canadensis, most popular weed in our campus, was studied. In the leaves of Erigeron canadensis exposed to sun-light, the concentration of DHA was higher than that of AscHy. The leaves of Erigeron canadensis were soaked in 0.5 M sodium cyanate solution Ž 0.5 M sodium cyanate solution is generally sprinkled on plants as herbicide. at 308C for 30 min w10x. After washed well with water, they were kept in a dry incubator at 308C. Within 2 h, the leaves changed in color from green to brown Ž picture is not shown.. The time-courses of AscHy, DHA, Cb-DHA and Cb-AscHy levels in the leaves of
Fig. 7. Time-courses of AscHy, DHA, Cb–DHA and Cb–AscHy levels in cyanate-treated leaves of Erigeron canadensis. Lower, the leaves of Erigeron canadensis were soaked in 0.5 M sodium cyanate solution at 308C for 30 min, and then washed with water. Upper, the leaves of Erigeron canadensis were washed with water without a cyanate-treatment. The leaves were allowed to settle in a dry incubator at 308C. The leaves were homogenized in 1% metaphosphoric acid, and the homogenate was centrifuged at 10 000= g for 10 min. The supernatant was submitted to the determination methods for AscHy, DHA, Cb–DHA and Cb– AscHy.
Fig. 6. Effect of cyanate on the AscHy –DHA redox cycle in vitro. Tris-HCl buffer Ž350 m l. containing 6.58=10y3 units rat hepatic glutathione-dependent DHA reductase was mixed with 10 m l of 8 mM ascorbic acid, 20 m l of 100 mM glutathione, 10 m l of AscHy oxidase Ž0.752 unitsrml. and sodium cyanate Žconcentration is indicated in figure., successively. After standing at 308C for 5 min, the reaction solution was centrifuged at 10 000= g for 5 min, and then the supernatant was submitted to HPLC for the determination of AscHy and DHA.
Erigeron canadensis treated with cyanate during the period of this entire test were shown in Fig. 7. By soaking the leaves in the cyanate solution, the DHA level was lowered less than 30% of the initial one with producing equivalent amounts of Cb-DHA, whereas no alteration of AscHy in concentration was observed. Thereafter, the exposure of leaves to air resulted in the oxidation of AscHy to DHA, and then DHA was spontaneously degraded to 2,3-diketogulonate. Both AscHy and DHA had disappeared
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within 3 h. This phenomenon appears to be responsible for the unbalance between the excess oxidants and the less antioxidant activity.
4. Discussion An excess of cyanate present in the intercellular or intracellular matrix, where oxidants are deactivated by AscHy–DHA redox cycle, can prevent this cycle by transforming DHA to Cb-DHA as shown in Fig. 8. Proceeding of this reaction results in the depletion of AscHy in the matrix. The resultant Cb-DHA no longer reverts to AscHy. These results suggest that, when organisms are exposed to cyanate, they are damaged by an excess of oxidants. It has been known that cyanate quickly blights plants just after attaching to leaves and stems, and thus sodium cyanate has been widely used as a herbicide w10,11x. In spite of the popularity of cyanate as a herbicide, the mechanism of this phenomenon is
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unclear. In general, it is known that the depression of antioxidant activity under oxidative stress is responsible for withering of plants w32,33x. This fact coupled with our findings that cyanate depletes AscHy in plants, suggests that the depletion of AscHy in the cyanate-treated plants is the principal cause of the withering of plants. It has been reported that the long-term administration of cyanate as a therapeutic drug for sickle-cell disease was accompanied by side-effects such as cataract w34x. In addition, cyanate produced from urea is an uremic toxin, so it is thought to be involved in cataract, one of the symptoms of uremia w35x. So far, the mechanism for this phenomenon was postulated as follows: cyanate reacts with amino groups on crystallins, and thus the resultant N-carbamylated crystallins aggregate in a lens w36x. However, in our earlier observations, an in vitro treatment of a bovine lens with cyanate did not result in the turbidity of the lens Žunpublished data.. From this finding, it was speculated that an additional factor as well as N-
Fig. 8. Possible mechanism for the depletion of AscHy caused by cyanate.
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carbamylation of crystallins may be attributed to the induction of cataract. In general, cataract is induced by oxidative stress. The turbidity of a lens is caused by the formation of mixed disulfide bonds on crystallins and the cross-linking Ž disulfide bonds. among crystallin molecules under oxidative stress. These facts suggest that the depletion of AscHy in the cyanate-treated lens may be a principal cause of cataract. Previous studies have noted that the depression of antioxidant system by cyanate is due to the carbamylation of amino groups andror sulfhydryl groups on enzymes concerning with this system. Furthermore, in the present study, we present evidence that cyanate hinders AscHy–DHA redox cycle, by which oxidants were directly deactivated.
Acknowledgements The present work was supported by a Grant-in-Aid for Scientific Research Ž C. Ž No. 08672470. from the Ministry of Education, Science, Sports and Culture, Japan. We thank Dr. Toshihiko Toida at Chiba University Ž Japan. for NMR spectroscopic analyses.
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