Chemico-Biological Interactions 110 (1998) 189–202

The glutathione dependence of inorganic sulfateformation from L- or D-cysteine in isolated rat

hepatocytes

James Huang, Sumsullah Khan, Peter J. O’Brien *

Faculty of Pharmacy, Uni6ersity of Toronto, Toronto, Ontario M5S 2S2, Canada

Received 16 September 1997; received in revised form 19 January 1998; accepted 29 January 1998

Abstract

The GSH dependence of the metabolic pathways involved in the conversion of cysteine tosulfate in intact cells has been investigated. It was found that hepatocyte-catalysed sulfateformation from added L-cysteine did not occur if hepatocyte GSH was depleted beforehand,but was restored when GSH levels recovered. Furthermore, sulfate formation did not recoverin GSH-depleted hepatocytes if GSH synthesis was prevented with buthionine sulfoximine.Thiosulfate formation was, however, markedly enhanced in GSH-depleted hepatocytes.These results suggest that thiosulfate is an intermediate in the formation of inorganic sulfatefrom L-cysteine and that GSH was required for the conversion of thiosulfate to inorganicsulfate. Much less sulfate was formed if the cysteine was replaced with cysteinesulfinate.Furthermore, sulfate formation from L-cysteine was markedly inhibited by the addition ofthe transaminase inhibitor DL-cycloserine or the g-cystathionase inhibitor DL-propargyl-glycine. The major routes of sulfate formation from L-cysteine therefore seems to involvepathways that do not involve L-cysteinesulfinate. Similar amounts of sulfate were formedfrom D-cysteine as L-cysteine. Thiosulfate instead of sulfate was also formed in GSH-de-pleted hepatocytes. However, sulfate formation from D-cysteine differed from L-cysteine inthat it was inhibited by the D-aminoacid oxidase inhibitor sodium benzoate and was notaffected by transaminase or g-cystathionase inhibitors. These results suggest that thiosulfateis an intermediate in sulfate formation from D-cysteine and involves the oxidation of

Abbre6iations: L-CSA; L-cysteinesulfinic acid; BSO, buthionine sulfoximine; CN, cyanide; GSH,reduced glutathione; TCA, trichloroacetic acid; PAPS, 3%-phosphoadenosine 5% phosphosulfate.

* Corresponding author. Tel.: +1 416 9782716; fax: +1 416 9788511.

0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.

PII S0009-2797(98)00015-5

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202190

D-cysteine by D-amino acid oxidase to form b-mercaptopyruvate. © 1998 Elsevier ScienceIreland Ltd. All rights reserved.

1. Introduction

Sulfation is an important biotransformation pathway. It is one of the mostcommon phase II reactions for xenobiotic metabolism [1]. The reaction is catalysedby cytosolic sulfotransferases which generally increase the water solubility of thexenobiotic for easier excretion in the urine or bile [2,3]. Many important biologi-cally active endogenous compounds are also substrates for sulfation, includingsteroid hormones, thyroid hormones and monoamine neurotransmitters [2,4]. Oneof the regulatory factors for sulfate conjugation is the synthesis of the active formof inorganic sulfate, PAPS [5,6]. The synthesis of PAPS in turn depends on theavailability of inorganic sulfate [2,7–9]. In mammals, most of the inorganic sulfaterequirements are derived from the oxidation of dietary L-cysteine [2,10]. Dietaryinorganic sulfate is of only minor importance, since 80–90% of dietary sulfates areexcreted in the urine [11].

It has been suggested that inorganic sulfate is mostly formed following thesulfoxidation of cysteine to cysteinesulfinate in the cytosol which is then transami-nated by the mitochondria to produce sulfinopyruvate. The latter decomposesspontaneously to sulfite which could be oxidised to sulfate by mitochondrial sulfiteoxidase [12–14]. However, Coloso et al. [15] concluded that cysteinesulfinate-inde-pendent pathways were more important since sulfate formation from cysteine wasfound to be decreased by the g-cystathionase inhibitor, PPG (DL-propargylglycine).Rat liver mitochondria also converted L-cysteine to sulfate without forming L-cys-teinesulfinate [16].

One way of determining whether the cysteinesulfinate-independent pathway wasa major pathway in the formation of sulfate from cysteine would be to determineif prior GSH depletion prevented sulfate formation. This is because thiosulfatereductase, a rate limiting step in the cysteinesulfinate-independent pathway requiresGSH to reduce thiosulfate to sulfate [17]. Gregus et al. [14] showed that depletionof hepatic GSH in vivo decreased serum inorganic sulfate levels. Stipanuk et al. [18]also found a decrease in [35S]sulfate and [35S]taurine formation from [35S]L-cysteinein GSH depleted hepatocytes, but suggested that this resulted from the decrease inL-cysteine availability as a result of compensatory GSH resynthesis.

In addition, we were also interested in the metabolic pathways involved in sulfateformation from D-cysteine because in vivo studies carried out by Krijgsheld et al.[13] and Glazenburg et al. [19] have shown D-cysteine to be a better sulfate sourcethan L-cysteine due to the fact that D-cysteine is not utilized for protein and GSHsynthesis and is hence more available for sulfate formation.

In this paper, we have shown for the first time that sulfate formation from eitherL- or D-cysteine did not occur in GSH depleted hepatocytes even when GSHresynthesis was prevented and cysteine availability was in excess. Furthermore, the

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202 191

transaminase and/or g-cystathionase-dependent pathways may be the main routesfor sulfate formation from L-cysteine whereas the main pathway for sulfateformation from D-cysteine involved the D-amino acid oxidase-catalyzed conversionof D-cysteine to b-mercaptopyruvate. The GSH requirement for sulfate formationfrom L-cysteine could best be explained if thiosulfate were the preferred precursorof sulfate.

2. Materials and methods

2.1. Materials

L-Cysteine, D-cysteine, reduced glutathione, L-CSA, b-mercaptopyruvate, sodiumhydrosulfide, BSO, DL-cycloserine, DL-propargylglycine, sodium benzoate, bariumchloride, polyethylene glycol, ferric nitrate, iodoacetic acid, fluoro-2,4-dinitroben-zene were purchased from Sigma (St. Louis, MO). 1-Bromoheptane was purchasedfrom Aldrich (Milwaukee, WI). Potassium cyanide was purchased from FisherScientific (Fair Lawn, NJ). Sodium thiosulfate was purchased from J.T. Baker(Phillipsburg, NJ). Cupric sulfate and sodium sulfate were purchased from BDHChemicals (Toronto, ON). Collagenase (from Clostridium histolyticum), Hepes andbovine serum albumin were obtained from Boehringer-Mannheim (Montreal, PQ).HPLC grade solvents were purchased from Calden (Georgetown, ON). All otherchemicals used were of analytical grade.

2.2. Isolation and incubation of hepatocytes

Freshly isolated hepatocytes were prepared from adult male Sprague-Dawley rats(280–300 g) fed ad libitum. The cells were isolated by collagenase perfusion of theliver as described by Moldeus et al. [20]. The cells (1×106 or 4×106 cells/ml) werepreincubated in Krebs–Henseleit bicarbonate buffer (pH 7.4) supplemented with12.5 mM Hepes for 30 min in an atmosphere of 20% O2/5% CO2/75% N2 at 37°Cin continuously rotating 50 ml round bottom flasks before the addition of chemi-cals. Stock solutions of chemicals were made either in incubation buffer of inDMSO and added to the hepatocyte suspensions at the indicated time points.Hepatocytes were incubated in an atmosphere of 20% O2/5% CO2/75% N2 at 37°Cfor the duration of the experiment.

2.3. GSH depleted and/or BSO inhibited hepatocytes

GSH depleted hepatocytes were prepared by pre-incubating hepatocytes with 200mM 1-bromoheptane per 106 cells/ml for 30 min [21]. g-Glutamyl-cysteine syn-thetase activity was inhibited where indicated by incubating the hepatocytes withBSO (0.5 mM).

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202192

2.4. Sulfate determination

The concentration of inorganic sulfate formed in isolated hepatocytes wasmeasured in deproteinised samples (5% TCA) by the turbidimetric proceduredescribed by Sorbo [22]. Cysteine (10 mM) or cysteine metabolites were added tothe hepatocytes (4×106 cells/ml) at the beginning of the incubation and sulfatelevels were measured at fixed intervals. Enzyme inhibitors or cyanide were added tothe hepatocytes 30 min prior to the addition of the substrates.

2.5. Thiosulfate determination

The concentration of thiosulfate formed in isolated hepatocytes were measured indeproteinised samples (5% TCA) by the colorimetric procedure described byWestley [23]. Cysteine (10 mM) was added to the hepatocytes (4×106 cells/ml) atthe beginning of the incubation and thiosulfate levels were measured at fixedintervals. Enzyme inhibitors were added to the hepatocytes 30 min prior to theaddition of cysteine.

2.6. Glutathione determination

The total amount of GSH in isolated hepatocytes was measured in deproteinisedsamples (5% meta-phosphoric acid) after derivatization with iodoacetic acid andfluoro-2,4-dinitrobenzene by HPLC as described by Reed et al. [24].

2.7. Statistics

Statistically significant differences between control and experimental groups wereobtained using Student t-test. The minimal level of significance chosen was PB0.05.

3. Results

3.1. Dependence of L-cysteine con6ersion to sulfate on intracellular GSH le6els

As shown in Fig. 1A, sulfate was formed over a 3-h incubation period in thepresence of L-cysteine after an initial lag period of approximately 30 min. However,with GSH-depleted hepatocytes, the rate of sulfate formation in the presence ofL-cysteine was almost completely inhibited for the first hour of the incubationperiod. The rate of sulfate formation from L-cysteine began to increase between60–90 min and continued to increase till the end of the 3-h incubation period.When GSH resynthesis was prevented in GSH-depleted hepatocytes using theg-glutamyl-cysteine synthetase inhibitor BSO, the conversion of L-cysteine tosulfate was almost completely inhibited throughout the entire 3-h incubationperiod.

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202 193

Fig. 1. The dependence of inorganic sulfate formation from L-cysteine on hepatocyte GSH levels. Rathepatocytes (4×106 cells/ml) were incubated at 37°C, pH 7.4 and 20% O2/5% CO2/75% N2. GSHdepleted and g-glutamyl-cysteine synthetase inhibited hepatocytes were prepared as described in Section2. (A) Inorganic sulfate formation from L-cysteine. Reactions were stopped by the addition of 5% (w/v)TCA to hepatocyte samples taken at 30, 60, 90, 120 and 180 min after the addition of L-cysteine. Theconcentrations of inorganic sulfate were determined as described under Section 2. Each symbolrepresents the mean9S.E.M. of 3–9 incubations. Legends: (�) 10 mM L-cysteine+control hepato-cytes; (�) 10 mM L-cysteine+GSH depleted hepatocytes; (") 10 mM L-cysteine+GSH depleted/g-glu-tamyl-cysteine synthetase inhibited hepatocytes. (B) The restoration of GSH levels in GSH depletedhepatocytes by cysteine. Reactions were stopped by the addition of 5% (w/v) meta-phosphoric acid tohepatocyte samples taken at 30, 60, 90, 120 and 180 min after the addition of L-cysteine. The levels ofGSH were determined as described under Section 2. Legends: (�) control hepatocytes; (�) 10 mML-cysteine+GSH depleted hepatocytes; (�) GSH-depleted hepatocytes; (�) 10 mM L-cysteine+GSHdepleted/g-glutamyl-cysteine synthetase inhibited hepatocytes.

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202194

As shown in Fig. 1B, when GSH depleted rat hepatocytes were incubated withL-cysteine, 25% of normal intracellular GSH levels recovered to control levels by 90min whereas much less recovery of GSH occurred in the absence of L-cysteine.Hepatocyte GSH did not recover with L-cysteine if GSH synthesis was preventedwith BSO (Fig. 1B).

3.2. Formation of sulfate from D-cysteine in rat hepatocytes

As shown in Fig. 2A, sulfate was formed after a lag period when rat hepatocyteswere incubated with 10 mM D-cysteine. However, when intracellular GSH wasdepleted beforehand, no sulfate formation was observed. Hepatocyte GSH levelwas not restored.

3.3. Dependence of sulfate formation from thiosulfate on hepatocyte GSH le6els

As shown in Fig. 2B, sulfate was formed in rat hepatocytes with 1 mMthiosulfate. However, when intracellular GSH levels were depleted beforehand, noconversion of thiosulfate to sulfate occurred.

3.4. Dependence of thiosulfate formation from L-cysteine on hepatocyte GSH le6els

As shown in Fig. 3A, thiosulfate was formed rapidly without a lag period whenrat hepatocytes were incubated with 10 mM L-cysteine. The initial rate of thiosul-fate formation by 1 h was approximately 2-fold greater in GSH-depleted hepato-cytes. Total thiosulfate formation at 2 h was also increased 65% if GSH resynthesiswas prevented with BSO, a g-glutamyl-cysteine synthetase inhibitor.

3.5. Formation of thiosulfate from D-cysteine in rat hepatocytes

As shown in Fig. 3B, thiosulfate formation in hepatocytes incubated withD-cysteine was somewhat slower than with L-cysteine. The initial rate of thiosulfateformation by 1 h was also 2-fold higher in GSH-depleted hepatocytes.

3.6. Formation of sulfate from cysteine metabolites in rat hepatocytes and li6erhomogenates

As shown in Table 1, L-cysteinesulfinate formed about 7% of the sulfate as thatformed with L-cysteine in hepatocytes but was much more effective in rat liverhomogenates. This may not be attributed to differences in cell permeability ofL-CSA and L-cysteine as cysteinesulfinate was metabolised by isolated hepatocytesto CO2 more readily than L-cysteine [25]. By contrast, the other cysteine metabolitesb-mercaptopyruvate, thiosulfate and hydrosulfide at 1 mM formed sulfate aseffectively as L- or D-cysteine at 10 mM.

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202 195

3.7. The effect of aminotransferase, g-cystathionase and D-amino acid oxidaseinhibitors on the formation of sulfate from L- and D-cysteine in rat hepatocytes

As shown in Table 2, the transaminase inhibitor DL-cycloserine inhibited sulfateformation from L-cysteine by 43% but had no significant effect on sulfate formationfrom D-cysteine or mercaptopyruvate. Similarly, the g-cystathionase inhibitor DL-propargylglycine inhibited sulfate formation from L-cysteine by 62%, but had only

Fig. 2. The effect of hepatocyte GSH depletion on inorganic sulfate formation from D-cysteine andthiosulfate. Experimental conditions and methods used were as described in the legends to Fig. 1. Eachsymbol represents the mean9S.E.M. of 3–9 incubations. (A) Inorganic sulfate formation fromD-cysteine. Legends: ( ) 10 mM D-cysteine+control hepatocytes; () 10 mM D-cysteine+GSHdepleted hepatocytes. (B) Inorganic sulfate formation from thiosulfate. Legends: (�) 1 mM thiosul-fate+control hepatocytes; (�) 1 mM thiosulfate+GSH depleted hepatocytes.

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202196

Fig. 3. The effect of hepatocyte GSH depletion on thiosulfate formation from L- and D-cysteine.Experimental conditions and methods used were as described in the legends to Fig. 1. The concentra-tions of thiosulfate were determined as described under Section 2. Each symbol represents themean9S.E.M. of 3–9 incubations. (A) Thiosulfate formation from L-cysteine. Legends: (�) 10 mML-cysteine+control hepatocytes; (�) 10 mM L-cysteine+GSH depleted hepatocytes; (") 10 mML-cysteine+GSH depleted/g-glutamyl-cysteine synthetase inhibited hepatocytes. (B) Thiosulfate forma-tion from D-cysteine. Legends: (�) 10 mM D-cysteine+control hepatocytes; (�) 10 mM D-cysteine+GSH depleted hepatocytes.

little effect on sulfate formation from D-cysteine or mercaptopyruvate. On the otherhand, the D-amino acid oxidase inhibitor sodium benzoate [26] did not have anyeffect on sulfate formation from L-cysteine or mercaptopyruvate but markedly

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202 197

inhibited sulfate formation from D-cysteine by 93%. Sulfate formation from L-cys-teine sulfinate was not affected by these inhibitors (results not shown).

3.8. The effect of cyanide on sulfate formation from thiosulfate, L-cysteine andD-cysteine in rat hepatocytes

As shown in Table 2, cyanide, a potent ‘sulfane trap’ decreased sulfate formationfrom L-cysteine by 40% and from D-cysteine by 31% but increased sulfate formationfrom thiosulfate by 42%.

4. Discussion

It has been suggested that the major pathway for sulfate formation fromL-cysteine involves cysteinesulfinate transamination which results in the formationof sulfite that could be oxidised to sulfate by mitochondrial sulfite oxidase [12]. Ifso, then sulfate formation would not be expected to depend on GSH levels.However, our data shows that sulfate formation from L-cysteine was greatlydelayed when hepatocyte GSH was depleted and recommenced once the GSH levelsrecovered. One explanation for the GSH requirement for sulfate formation fromL-cysteine could be if the GSH dependent thiosulfate reductase was involved [17]and therefore that thiosulfate is an important metabolic intermediate. The notionthat thiosulfate is an intermediate in inorganic sulfate formation from L-cysteinehas also been suggested by others [25,27]. Furthermore, our data shows thatthiosulfate accumulated when GSH-depleted hepatocytes were incubated with

Table 1Hepatocyte-catalysed inorganic sulfate formation from L-cysteine or D-cysteine and their metabolites

Inorganic sulfate formedAddition to incubation

HomogenateHepatocytes(nmol/4×106 cells) (nmol/mg protein)

549.4937.9 104.1911.2L-Cysteine (10 mM)339.5932.0 88.399.1D-Cysteine (10 mM)

L-Cysteinesulfinate (10 mM) 77.0912.339.594.1†567.3934.1 135.2911.2b-Mercaptopyruvate(1 mM)425.0932.8 103.6917.6Hydrosulfide (1 mM)585.1931.2 127.7913.4Thiosulfate (1 mM)

Compounds were added to isolated rat hepatocytes (4×106 cells/ml) or liver homogenates (8 mgprotein/ml) incubated at 37°C, pH 7.4 and 20% O2/5% CO2/75% N2.Reactions were stopped at 90 min after the addition of the compounds with 5% (w/v) TCA. Theconcentrations of inorganic sulfate formed within the 90 min incubation period were measured asdescribed under Section 2.Each value represents the mean9S.E.M. of 3–9 incubations.† Significantly different from L-cysteine alone (PB0.001).

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202198

Table 2The involvement of thiosulfate, g-cystathionase, transaminase and D-amino acid oxidase in thehepatocyte-catalysed sulfate formation from L-cysteine, D-cysteine or thiosulfate

Inorganic sulfate formed (nmol/4×106 cells)Inhibitors

MercaptopyruvateThiosulfateD-CysteineL-Cysteine(10 mM)(10 mM) (1 mM) (1 mM)

796.2962.3 567.3934.1None 339.5932.0549.4937.9324.8918.1DL-Cycloserine 746.8958.2311.5926.5† 536.9951.1

(50 mM)326.4916.3DL-Propargylglycine 753.7953.1210.4916.4† 1543.2952.2

(350 mM)732.8971.2 561.2942.0543.6925.1Sodium benzoate 24.893.7***

(500 mM)nd1129.0978.4‡Cyanide (400 mM) 234.3918.4*326.9924.8†

Inhibitors were added to the incubations 30 min prior to the addition of L-cysteine, D-cysteine andthiosulfate.Rat hepatocytes (4×106 cells/ml) were incubated at 37°C, pH 7.4, and 20% O2/5% CO2/75% N2.Reactions were stopped at 90 min after the addition of cysteine and thiosulfate with 5% (w/v) TCA. Theconcentrations of inorganic sulfate formed within the 90 min were measured as described under Section2.Each value represents the mean9S.E.M. of 3–9 incubations.† Significantly different from L-cysteine alone (PB0.05).‡ Significantly different from thiosulfate alone (PB0.05).* Significantly different from D-cysteine alone (PB0.05).*** Significantly different from D-cysteine alone (PB0.001).

cysteine thereby confirming that the metabolism of L-cysteine to thiosulfate seemedto be dependent on GSH levels.

Sulfate synthesis recommenced as GSH levels recovered in the GSH-depleted rathepatocytes. The recovery of GSH levels in GSH-depleted rat hepatocytes incu-bated with L-cysteine is not unexpected. L-Cysteine is the limiting amino acid inglutathione synthesis [28,29] and g-glutamyl-cysteine synthetase is normally underfeedback inhibition by GSH [30]. Others have also found a significant increase inthe GSH levels when hepatocytes were incubated with L-cysteine [31–33].

Sulfate formation from D-cysteine, which does not participate in GSH synthesis,was also markedly inhibited in GSH-depleted rat hepatocytes but did not recover asoccurred with L-cysteine. Thiosulfate also accumulated thereby indicating thatthiosulfate is also a metabolic intermediate in the formation of sulfate fromD-cysteine.

To confirm that GSH is a co-substrate in sulfate formation from L-cysteine at thethiosulfate reductase step, the metabolic conversion of thiosulfate to sulfate wasmeasured in both control and GSH-depleted hepatocytes. Sulfate was readilyformed from thiosulfate in control hepatocytes but not in GSH-depleted hepato-cytes. Previously, Szczepkowski et al. [27] showed that thiosulfate was converted tosulfate in intact rats. Sulfate formation from thiosulfate in rat liver homogenates or

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202 199

mitochondria, respectively, was also markedly increased by the addition of GSH[34–36].

The four major pathways which are known to be involved in inorganic sulfateformation from cysteine are summarized in Scheme 1. It is clear from Table 1 thatL-cysteinesulfinate can only give rise to about 7% of the sulfate formed from thesame concentration of L-cysteine. This result may not be attributed to the poorentry of L-cysteinesulfinate into hepatocytes in comparison with that of L-cysteineas Drake et al. [37] also showed that 14C-L-cysteinesulfinate was metabolised byisolated hepatocytes to 14CO2 more readily than L-cysteine. This suggests that in the

Scheme 1.

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202200

intact hepatocyte most of the cysteinesulfinate is metabolised by the cytosolicdecarboxylase to form hypotaurine. L-Cysteinesulfinate has also been shown topermeate rat liver mitochondria via the glutamate-aspartate carrier [38].

Sulfate was readily formed from the cyst(e)ine metabolites b-mercaptopyruvateor sulfide. Furthermore, it is likely that the L-cysteine aminotransferase- or cys-tathionine b-synthase-dependent pathways that form b-mercaptopyruvate orsulfide respectively are involved as sulfate formation from cysteine was readilyinhibited by the transaminase inhibitor DL-cycloserine. This suggests that up to 43%of sulfate formation from L-cysteine is dependent on the transamination of L-cys-teine to b-mercaptopyruvate. L-Cycloserine has previously been shown to inhibitL-alanine aminotransferase activity with little effect on g-cystathionase activity [37].Similarly, the inhibition of g-cystathionase by DL-propargylglycine suggests that upto 62% of sulfate formation from L-cysteine is derived from L-cystine with subse-quent b-cleavage by g-cystathionase.

Sulfate formation from L-cysteine was inhibited 41% by cyanide whereas sulfateformation from thiosulfate was increased 42%. The latter increase could be at-tributed to rhodanese and cyanide catalyzing sulfite formation from thiosulfate(Scheme 1). The inhibition of sulfate formation from L-cysteine by cyanide on theotherhand suggests that at least 41% of the sulfate is formed from thiocystine andmercaptopyruvate which can transfer their sulfur to cyanide to form thiocyanatewith rhodanese or b-mercaptopyruvate sulfurtransferase, respectively. Hepatocytecatalyzed thiocyanate formation from L-cysteine and cyanide was also recentlyattributed to mercaptopyruvate and thiocystine rather than thiosulfate as it was notaffected by GSH depletion [39].

The formation of sulfate from D-cysteine in contrast to L-cysteine, was notinhibited by DL-cycloserine or DL-propargylglycine but was prevented by benzoate,a D-amino acid oxidase inhibitor [25]. This rules out the involvement of D-cystinein sulfate formation and indicates that the major pathway for sulfate formationfrom D-cysteine involves the D-amino acid oxidase-catalyzed conversion of D-cys-teine to b-mercaptopyruvate which is converted via a sulfurtransferase to formthiosulfate. The similar decrease in sulfate formation from D-cysteine as L-cysteinewith cyanide therefore probably also results from the transfer of sulfur fromb-mercaptopyruvate to cyanide by b-mercaptopyruvate sulfurtransferase.

We can therefore conclude that GSH may be required for the metabolism of L-and D-cysteine to sulfate as thiosulfate accumulates without sulfate formation ifGSH levels are depleted. This suggests that the GSH dependent thiosulfate reduc-tase may be involved in sulfate formation from L- and D-cysteine.

References

[1] R.T. Williams, Detoxication Mechanisms, 2nd ed., Chapman and Hall, London, 1959.[2] G.J. Mulder, Sulfation of Drugs and other Compounds, CRC Press, Boca Raton, FL, 1981.[3] G.J. Mulder, W.B. Jakoby, Sulfation, in: G.J. Mulder (Ed.), Conjugation Reactions in Drug

Metabolism, Taylor and Francis, London, 1990, pp. 107–161.

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202 201

[4] R. Hobkirk, Steroid sulfotransferases and steroid sulfate sulfatases: characteristics and biologicalroles, Can. J. Biochem. Cell. Biol. 63 (1985) 1127–1144.

[5] I.H. Segel, F. Renosto, P.A. Seubert, Sulfate activating enzymes, Methods Enzymol. 143 (1987)334–349.

[6] J.R. Dawson, K. Norbeck, P. Moldeus, The effectiveness of different sulfate precursors insupporting extra-hepatic sulfate conjugation, Biochem. Pharmacol. 32 (1983) 1789–1791.

[7] P. Moldeus, B. Anderson, V. Gergely, Regulation of glucuronidation and sulfate conjugation inisolated hepatocytes, Drug Metab. Dispos. 7 (1979) 416–419.

[8] K.R. Krijgsheld, E. Scholtens, G.J. Mulder, The dependence of the rate of sulphate conjugation onthe plasma concentration of inorganic sulphate in the rat in vivo, Biochem. Pharmacol. 31 (1982)3997–4000.

[9] G. Levy, Sulfate conjugation in drug metabolism: role of inorganic sulfate, Fed. Proc. 45 (1986)2235–2240.

[10] O.W. Griffith, Mammalian sulfur amino acid metabolism: an overview, Methods Enzymol. 143(1987) 366–375.

[11] D.B. Papadopoulou, Urinary sulfur partition in normal men and in cancer patients, Clin. Chem. 3(1957) 257–262.

[12] J.C. Jocelyn, The metabolism of thiols and disulphides, in: Biochemistry of the SH group, AcademicPress, London, 1972, pp. 163–189.

[13] K.R. Krijgsheld, E.J. Glazenburg, E. Scholtens, G.J. Mulder, The oxidation of L- and D-cysteine toinorganic sulfate and taurine in the rat, Biochim. Biophys. Acta. 677 (1981) 7–12.

[14] Z. Gregus, C. White, S. Howell, C.D. Klaassen, Effect of glutathione depletion on sulfate activationand sulfate ester formation in rats, Biochem. Pharmacol. 37 (1988) 4307–4312.

[15] R.M. Coloso, M.R. Drake, M.H. Stipanuk, Effect of bathocuproine disulfonate, a copper chelator,on cyst(e)ine metabolism by freshly isolated rat hepatocytes, Am. J. Physiol. 259 (1990) E443–3450.

[16] A. Wainer, The production of sulfate from cysteine without the formation of free cysteinesulfinicacid, Biochem. Biophys. Res. Comm. 16 (1964) 141–144.

[17] T.R. Chauncey, L.C. Uhteg, J. Westley, Thiosulfate reductase, Methods Enzymol. 143 (1987)350–354.

[18] M.H. Stipanuk, P.J. Bagley, Y.C. Hou, D.L. Bella, M.F. Banks, L.L. Hirschberger, Hepaticregulation of cysteine utilization for taurine synthesis, in: R. Huxtable, D.V. Michalk, (Eds.),Taurine in Health and Disease, Plenum Press, New York, 1994, pp. 79–89.

[19] E.J. Glazenburg, I.M.C. Jekel-Halsema, A. Baranczyk-Kuzma, K.R. Krijgsheld, G.J. Mulder,D-Cysteine as a selective precursor for inorganic sulfate in the rat in vivo, Biochem. Pharmacol. 33(1984) 625–628.

[20] P. Moldeus, J. Hogberg, S. Orrenius, Isolation and use of liver cells, Methods Enzymol. 52 (1978)60–71.

[21] S. Khan, P.J. O’Brien, 1-Bromoalkanes as new potent nontoxic glutathione depletors in isolated rathepatocytes, Biochem. Biophys. Res. Commun. 179 (1991) 436–441.

[22] B. Sorbo, Sulfate: turbidimetric and nephelometric methods, Methods Enzymol. 143 (1987) 3–6.[23] J. Westley, Thiocyanate and thiosulfate, Methods Enzymol. 143 (1987) 22–25.[24] D.J. Reed, J.R. Babson, P.W. Beatty, A.E. Brodie, W.W. Ellis, D.W. Potter, High performance

liquid chromatography analysis of nanomole levels of glutathione, glutathione distilfides, relatedthiols and distilfides, Anal. Biochem. 106 (1980) 55–62.

[25] M.H. Stipanuk, J.D. Rosa, L.L. Hirschberger, Catabolism of cysteine by rat renal cortical tubules,J. Nutr. 120 (1990) 450–458.

[26] A. Mattevi, M.A. Vanoni, F. Todone, M. Rizzi, A.Teplyakov, A. Coda, M. Bolognesi, B. Curti,Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolutionwith flavocytochrome b2, Proc. Natl. Acad. Sci. USA 93 (1996) 7496–7501.

[27] T.W. Szczepkowski, B. Skarzynski, M. Weber, The metabolic state of thiosulphate, Nature 189(1961) 1007–1008.

[28] M.E. Anderson, A. Meister, Intracellular delivery of cysteine, Methods Enzymol. 143 (1987)313–325.

J. Huang et al. / Chemico-Biological Interactions 110 (1998) 189–202202

[29] N. Tateishi, T. Higashi, S. Shinya, A. Naruse, Y. Sakamoto, Studies on the regulation ofglutathione level in rat liver, J. Biochem. 75 (1974) 93–103.

[30] P.G. Richman, A. Meister, Regulation of g-glutamyl-cysteine synthetase by non-allosteric feedbackinhibition by glutathione, J. Biol. Chem. 250 (1975) 1422–1426.

[31] M. Banks, M. Stipanuk, The utilization of N-acetylcysteine and 2-oxothiazolidine-4-carboxylate byrat hepatocytes is limited by their rate of uptake and conversion to cysteine, J. Nutr. 124 (1994)378–387.

[32] M. Stipanuk, R.M. Coloso, R.A. Garcia, M.F. Banks, Cysteine concentration regulates cysteinemetabolism to glutathione, sulfate and taurine in rat hepatocytes, J. Nutr. 122 (1992) 420–427.

[33] P. Beatty, D. Reed, Influence of cysteine upon the glutathione status of isolated rat hepatocytes,Biochem. Pharmacol. 30 (1981) 1227–1230.

[34] B. Sorbo, Mechanism of oxidation of inorganic thiosulfate and thiosulfate esters in mammals, Acta.Chem. Scand. 18 (1964) 821–823.

[35] A. Koj, J. Frendo, Z. Janik, 35S]Thiosulfate oxidation by rat liver mitochondria in the presence ofglutathione, Biochem. J. 103 (1967) 791–795.

[36] T. Ubuka, S. Yuasa, J. Ohta, N. Masuoka, K. Yao, M. Kinuta, Formation of sulfate fromL-cysteine in rat liver mitochondria, Acta. Med. Okayama 44 (1990) 55–64.

[37] M.R. Drake, J. De La Rosa, M.H. Stipanuk, Metabolism of cysteine in rat hepatocytes: evidencefor cysteinesulphinate-independent pathways, Biochem. J. 244 (1987) 279–286.

[38] F. Palmieri, I. Stipani, V. Iacobazzi, The transport of L-cysteinesulfinate in rat liver mitochondria,Biochim. Biophys. Acta 555 (1979) 531–546.

[39] J. Huang, H. Niknahad, S. Khan, P.J. O’Brien, Hepatocytes catalysed detoxification of cyanide byL- and D-cysteine, Biochem. Pharmacol. (in press).

..