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Redox regulation of homocysteine-dependent glutathione synthesis

Redox regulation of homocysteine-dependent glutathione synthesis Research article 1 2 2 1 2 Victor V itvitsky , Eugene Mosharov , Michael Tritt , Fazoil Ataullakhanov , Ruma Banerjee National Research Center for Hematology, Moscow, Russia Biochemistry Department, University of Nebraska, Lincoln, Nebraska, USA In certain tissues, glutathione biosynthesis is conn ected to methionine metabolism via the trans- sulfuration pathway. The latter condenses homocysteine and serine to cystathionine in a reaction catalyzed by cystathionine -synthase followed by cleavage of cystathionine to cysteine and - ketoglutarate by -cystathionase. Cysteine is t he limiting amino acid in glutathione biosynthesis, and studies in our laboratory have shown that approximately 50% of the cysteine in glutathione is derived from homocysteine in human liver cells. In this study, we have examined the effect of pro- and antioxidants on the flux of homocysteine through the trans-sulfuration pathway in the human hepatoma cell line, HepG2. Our studies reveal that pyrrolidine dithiocarbamate and butylated hydroxyanisole enhance the flux of homocysteine through the trans-sulfuration pathway as has been observed previously with the pro-oxidants, H O and tertiary butyl hydroperoxide. In contrast, 2 2 antioxidants such as catalase, superoxide dismutase and a water-soluble derivative of vitamin E elicit the opposite effect and result in diminished flux of homocysteine through the trans-sulfuration pathway. These studies provide the first evidence for the reciprocal sensitivity of the trans- sulfuration pathway to pro- and antioxidants, and demonstrate that the upstream half of the glutathione biosynthetic pathway ( i.e. leading to cysteine biosynthesis) is r edox sensitive as is t he regulation of the well-studied enzymes in the downstream half (leading from cysteine to glutathione), namely, -glutamyl-cysteine ligase and glutathione synthetase. INTRODUCTION uitously (Fig. 1). However, in tissues that have the largest concentrations of glutathione, and, therefore, the In aerobic organisms, a delicate balance exists between highest demand for cysteine (the limiting amino acid), oxygen-dependent metabolism and the generation of the trans-sulfuration pathway is p resent (Fig. 1). The lat- reactive oxygen species as side products. Glutathione, a ter presents an avenue for the conversion of the essential thiol-containing tripeptide plays a key role in intracellu- amino acid, methionine, to cysteine. Thus, the trans-sul- lar defense, and decreased levels of this antioxidant are furation pathway catalyzes the sequential conversion of correlated with the increased frequency of reactive oxy- homocysteine, a key junction metabolite in the methion- 1–3 gen species-mediated mitochondrial damage and ine cycle, to cystathionine and then cysteine. apoptosis. The intracellular glutathione pools are large Studies on the effect of pro-oxidants o n glutathione bio- 5–7 and vary from 1–10 mM depending on the cell type. synthesis have focused overwhelmingly on the last t wo 8–13 The final two steps in the biosynthesis of glutathione are enzymes in the biosynthetic pathway. A number of catalyzed by -glutamyl-cysteine ligase and glutathione conditions and agents elicit the transcriptional activation synthetase, enzymes that are believed to be present ubiq- of -glutamyl-cysteine ligase and some also activate glu- cis tathione synthetase. Three -acting elements that may be Received 18 Augu st 200 2 Abbreviations : ARE , antioxidant re sponse el ement; NF- kB, nuclear Accepted 28 Oc tober 20 02 factor kappa B; AP , ac tivator prot ein; t-B uOOH, te rtiary but yl hydroperoxide; PDT C, pyrr olidine dithiocarbamate; BH A, bu tylated Correspondence to: Prof. Ruma Banerjee, Biochemistry Department, University of Neb raska N13 3 Be adle Center, , L incoln, NE 68588- hydroxyanisole; MEM, Ea gle’s mi nimal essential me dium; PBS, 0664, USA phosphate bu ffered saline; FBS, fet al bov ine ser um; SOD, superoxide Tel : + 1 402 47 2 294 1; E-mail: rba [email protected] dismutase Redox Rep ort , V ol. 8, No. 1, 2003 © W . S. Maney & Son Lt d DOI 10.1179/135100003125001260 58 Vitvitsky, Mosharov, Trit t, Ataullakh anov, Banerjee AdoMet AdoHCy Cystathionine Cysteine -Glu-Cys GLUTA THION E Methionine HOMOCYSTEINE Propargylglycine Transmethylation pathway Glutathione synthesis pathway Transulfuration pathway Fig. 1. Metabolic pathway for glutathione biosynthesis in liver cells showing the upstream trans-sulfuration and the downstream glutathione synthesis reactions. AdoMet and AdoHcy denote S-adenosylmethionine and S-adenosylhomocysteine respectively. important in mediating this response have been identified pathway and on glutathione levels in HepG2 cells. We in the gene encoding the heavy subunit of -glutamyl-cys- found that catalase, a water-soluble form of vitamin E, and 14–16 teine ligase, which harbors the catalytic activity. These SOD elicit a small, but significant, reduction in the intracel- are the antioxidant response element (ARE-4), nuclear lular glutathione concentration. Furthermore, the decrease factor kappa B ( NF- B) and activator protein (AP-1). The in the glutathione pool size is p aralleled by a decrease in the promoter for the light subunit gene of -glutamyl-cysteine flux of homocysteine through the trans-sulfuration path- ligase also contains ARE and AP-1 elements. way. In contrast, PDTC and BHA increase flux of homo- In contrast, the responsiveness o f the trans-sulfuration cysteine through the trans-sulfuration pathway. These pathway enzymes t o changes i n redox status brought on by studies provide the first experimental evidence for recipro- pro- or antioxidants has received scant attention. This stems cal sensitiv ity of cystathionine -synthase activity to pro- largely from the lack of recognition of the importance of and antioxidants i n a model cell culture system. this pathway in the regulation of redox homeostasis. W e have recently demonstrated that the trans-sulfuration path- way plays a quantitatively major role in the maintenance of MATERIALS AND METHODS the intracellular glutathione pool in transformed human liver cells in culture. The two homocysteine junction Reagents and cell lines enzymes, methionine synthase and cystathionine -syn- thase, display reciprocal sensitiv ity to ambient redox tone Eagle’s m inimum essential medium, DL-propargyl- 18,19 in vitro under conditions. These observations led us to glycine, H O , t-BuOOH, PDTC, BHA, vitamin E (± - 2 2 hypothesize that redox changes may exert an additional tocopherol), superoxide dismutase, catalase, ascorbate, layer of regulation at this metabolic junction. The rationale thymol and trypan blue were purchased from Sigma. The for such a regulation, if it existed, could be that increased water-soluble analog of vitamin E (6-hydroxy-2,5,7,8- flux of homocysteine through the trans-sulfuration pathway tetramethylchroman-2-carboxylic acid) was purchased under oxid izing conditio ns would le ad to enhanced synthesi s from Aldrich. The antibiotic/antimycotic solution was of glu tathione and thus s er ve as a n autocorrective response to from Gibco BRL. Fetal bovine serum (FBS) was from an oxidative insult. Under th e opposite set of conditio ns, HyClone Company. Transformed human hepatocytes homocystei ne would be re-directed preferential ly to th e (HepG2) were from ATCC. [ S]-Methionine (>1000 tr ansmethyla tio n pathway leadin g to its r ecycling. Ci/m mol) was purchased from Amersham Pharmacia In a recent study using the human hepatoma cell line, Biotech. HepG2, we demonstrated that both H O and tertiary butyl 2 2 hydroperoxide (t-BuOOH) elicit a dose-dependent Cell culture increase in cystathionine production. This was accompa- nied by a corresponding increase in incorporation of radioactivity from [ S]-methionine into the glutathione Cells were grown until they reached 70–80% confluency pool. In this study, we have compared the effect of pro- in 60 x 15 mm Falcon 3004 tissue culture dishes in oxidants (H O and t-BuOOH) and substances that are MEM with 10% FBS, 2 mM L-glutamine, 2.2 g/l sodium 2 2 considered to be antioxidants (pyrrolidine dithiocarba- bicarbonate and 10 ml/l antibiotic/antimycotic solution. mate [PDTC], butylated hydroxyanisole [BHA], vitamin Each dish contained 4 ml of medium, which was E, catalase, ascorbate and superoxide dismutase [SOD]) changed every 4 days and 2 h before the start of the on the flux of homocysteine through the trans-sulfuration experiment. Experiments were initiated by adding the Redox sensitivity of homocysteine metabolism 59 desired reagents to the dishes. [ S]-Methionine solution was diluted in phosphate buffered saline (PBS) to give a specific activity of 1.25 mCi/ml and 1 ml of this solution was added per ml of cell culture medium to each dish. At the desired time intervals, cells were harvested and the protein concentration in samples was determined as described earlier. In experiments in which 1 mM H O 2 2 was used, cell viability was measured using the trypan blue exclusion method. Briefly, H O was added to a 2 2 concentration of 1 mM to HepG2 cells at 70–80% con- fluency and viability was determined after 2, 4, 10, and 20 h. The medium (4 ml) was removed and 0.5 ml of 0.4% trypan blue solution in 0.5 ml PBS was added. After 2 min, the cells were washed with 2 × 1 ml PBS, covered with 2 ml PBS an d the fraction of (dead) blue cells in a view area was determined. The average value from four view areas was calculated. Metabolite analysis Concentrations of glutathione and cystathionine in cell extracts were estimated using an HPLC method as described previously. The concentration of individual metabolites was determined by comparing integrated peak areas with previously generated calibration curves for each compound. To represent the thiol concentrations in units of mmol/l of cells, the amount of protein in HepG2 cells was taken to be 53 g protein/l of cells as estimated previously. Incorporation of [ S]-methionine into the glutathione pool was evaluated by measuring radioactivity in the relevant chromatographic fraction. Statistical significance was ana- Fig. 2. Kinetics of recovery of the intracellular glutathione pool following lyzed using the ANOVA test. oxidative challenge. The levels of glutathione and glutathione disulfide in untreated cells is represented by the open circles and triangles respectively. In cells treated with a single bolus of H O (1 mM), the glutathione (filled 2 2 circles) level rapidly declines before recovering, while the glutathione RESULT S AND DISCUSSION disulfide pool responds reciprocally (filled triangles). The culture dish contained ~4 x 10 cells in 4 ml medium which corresponds to a ratio of Kinetics of glutathione homeostasis following H O ~1 mmol H O /10 cells. Inset: comparison of viability o f cells treated with 2 2 2 2 a single bolus of 1 mM H O (hatched bars) versus untreated controls exposure 2 2 (solid bars) at fixed times following addition of peroxide or an equal volume of PBS to the culture medium. We have previously shown that relatively low concentra- tions of peroxides such as H O and t-BuOOH result in 2 2 enhanced flux of homocysteine through the trans-sulfura- the redox status of the cells, changes from an initial value tion pathway. However, at the oxidant concentrations of 400 ± 80 to 2–5 during the first few minutes following used in the earlier study (10–20 0 mM), there was no H O treatment. This is followed by a recovery phase dur- detectable change in the glutathione pool immediately fol- 2 2 ing which the glutathione level, as well as the glutathione- lowing exposure to the oxidants. In order to study the to-glutathione disulfide ratio, are restored over a period of kinetics of recovery of the intracellular glutathione pool 6–8 h. Glutathione disulfide displays a reciprocal response following oxidative stress, we examined the time-depen- and following an initial increase, diminishes to control dent changes in the concentration of glutathione and glu- levels within 2 h. The increase in glutathione concentra- tathione disulfide following exposure to 1 mM H O (Fig. 2 2 tion during the recovery phase is ref lected in the 2). Under these conditions, a decrease in the intracellular enhanced incorporation of [ S]-methionine into this glutathione concentration concomitant with an increase in pool (not shown), demonstrating the contribution of the glutathione disulfide is o bserved. The ratio of glutathione trans-sulfuration pathway in glutathione-linked redox to glutathione disulfide, which is k nown to be a marker for 60 Vitvitsky, Mosharov, Trit t, Ataullakh anov, Banerjee homeostasis. The glutathione levels finally overshoot the response to catalase was further characterized. The dose pool size in untreated cells. A similar observation has dependence of the catalase response is p resented in Table been reported in L2 cells (derived from type II pneumo- 2. Addition of 100 or 1000 U/ml of catalase to the cytes of adult rat lungs) treated with 4-hydroxy-2-none- medium resulted in an ~17% decrease in the intracellular nal, a major lipid peroxidation metabolite. glutathione concentration whereas 10 U/m l did not have a The kinetics of glutathione recovery as well as the corre- statistically significant effect. The decrease in glutathione spondence between our data set and that reported for L2 concentration was paralleled by a decrease in incorpora- cells, indicate that HepG2 cells treated with a single bolus tion of radioactivity. Since thymol is a dded as a preserva- of 1 mM H O remained viable during the time course of tive in the commercially available catalase that was 2 2 the experiment. This was confirmed by monitoring the via- initially employed in this study (as this preparation has a bility of HepG2 cells treated with a single bolus of 1 mM high specific activity (48,000 U/m g protein)), an addi- H O using the trypan blue exclusion assay. As seen in tional control was performed with thymol alone, which 2 2 Figure 2 (inset) there is n o difference in the number of was found to have no effect on intracellular glutathione viable cells in cells treated with 1 mM H O versus concentration and on S incorporation from methionine 2 2 untreated controls, and ! 1% of the cells were stained blue to glutathione (Table 2). We have also repeated these during the time course of the experiment. experiments with thymol-free catalase (specific activity, 15,500 U/m g protein). The intracellular glutathione con- centration and radioactivity incorporation were found to Effect of catalase, superoxide dismutase, ascorbate and be 82 ± 3% and 87 ± 6% ( n = 6), respectively, in the pres- vitamin E, on intracellular glutathione concentration ence of 100 U/m l catalase relative to untreated controls. In principle, the effect of catalase on the reduced glu- Addition of superoxide dismutase, a water-soluble form tathione pool size could be due to its e ffects on the upstream of vitamin E, and catalase resulted in a decrease in the trans-sulfuration pathway, the downstream glutathione syn- intracellular glutathione pool, which was reflected in the thesis pathway, or both. To determine whether or not cata- diminished incorporation of S from methionine into glu- lase affects the trans-sulfuration pathway, we examined the tathione (Table 1). In contrast, neither ascorbate nor flux of homocysteine through cystathionine -synthase in water-insoluble vitamin E had a statistically significant cells treated with propargylglycine. The latter is a suicide 21,22 effect. The limited solubility of vitamin E likely resulted inhibitor of -cystathionase, the second enzyme in the in its l ow availability in these experiments. Simultaneous trans-sulfuration pathway, and its a dministration leads to a addition of catalase, water-soluble vitamin E, and super- time-dependent increase in the intracellular cystathionine oxide dismutase did not have an additive effect. Since the pool which is t oo small to be readily detected under normal effect of catalase was more pronounced than that of super- conditions. Addition of 100 U/ml catalase to cells oxide dismutase and water-soluble vitamin E, the cellular treated with 2.5 mM propargylglycine resulted in a Table 1. E ffect of v arious pro- and antioxidants on intracellular glutathione concentration and o n inco rporation of rad ioactivity from [ S]-methionine Additions Relative intracellular Relative incorporation of glutathione concentration [ S] into glutathione Value n P value Value n P value Control 100 ± 1 37 100 ± 2 36 Catalase 83 ± 2 23 ! 0.001 86 ± 3 23 ! 0.001 SOD 92 ± 3 9 ! 0.02 92 ± 4 9 ! 0.1 Ascorbate 101 ± 5 4 NS 100 ± 2 3 NS Vitamin E 95 ± 4 8 NS 106 ± 7 8 NS Vitam in E w ater-soluble 90 ± 5 6 ! 0.05 81 ± 6 6 ! 0.01 Catalase + SOD + w ater-soluble vitamin E 87 ± 3 8 ! 0.001 77 ± 3 8 ! 0.001 t-BuOOH 110 ± 3 6 ! 0.01 131 ± 4 6 ! 0.001 PDTC 147 ± 9 7 ! 0.001 191 ± 18 6 ! 0.001 PDTC + t-B uOOH 160 ± 6 6 ! 0.001 257 ± 45 5 ! 0.01 BHA 111 ± 6 6 ! 0.1 153 ± 10 6 ! 0.001 The co ncentrations of the various additives employed were ca talase (100 U/ml of medium), SOD (100 U/m l of med ium), ascorbate (150 mM), vitam in E (2 00 mM), t-BuOOH (20 0 mM), PDTC (60 mM), B HA (20 0 mM), for 10 h fo llowing which the concentration of glutath ione and the label incorporation into glutathione were d etermined. n, num ber of independent determinations; NS, n ot significant. Redox sensitivity of homocysteine metabolism 61 Effect of PDTC and BHA on intracellular glutathione concentration PDTC is a relatively stable dithiocarbamate, a family of compounds that can chelate both free and protein-bound metals and is w idely used in medicine. The antioxidant behavior of dithiocarbamates results from their radical scavenging properties and cellular responses including 24,25 inhibition of lipid peroxidation, and inhibition of 26,27 oxidative activation of the transcription factor, NF- B. However, addition of 60 mM PDTC resulted in an increase in intracellular glutathione after 10 h that is similar to, but more pronounced than, the response observed with the oxidant, t-BuOOH (Table 1). Simultaneous addition of tBuOOH and PDTC had an additive effect resulting in a ~1.6-fold increase in glu- 5 h 1 0 h tathione concentration. The increase in incorporation of % D e c re a s e in 1 0 .7 ± 3 .3 1 9 .9 ± 3 .5 [ S]-methionine into glutathione was marginally greater c y s ta th io n in e (n = 1 2 ) (n = 1 0 ) w ith c a ta la s e than the fold-increase in glutathione concentration ( e.g. S ig n ific a n c e (P) 0 .0 2 0 .0 0 0 5 50 ~1.9-fold versus 1.5-fold with PDTC; Table 1) suggest- ing enhanced use of the trans-sulfuration pathway- derived cysteine in glutathione biosynthesis under these conditions. 0 2 4 6 8 10 BHA elicited a similar response under these condi- tions. Thus, addition of 200 mM BHA resulted in 1.1- Time (h) fold increase in intracellular glutathione after 10 h versus untreated controls (Table 1), and a 1.5-fold Fig. 3. Effect of extracellular catalase treatment on flux of homocysteine through the trans-sulfuration pathway. Cells were treated with 2.5 mM increase in incorporation of S from the methionine proparglycine only (open circles) or propargylglycine and catalase (100 pool. These results suggest that the effects of PDTC and U/ml; filled circles). The concentration of cystathionine was measured at BHA derive either from oxidation reactions or from the indicated times, and a representative data set is presented. The other presently unknown effects, rather than an antioxi- statistical analysis of the data is presented in the inset where n is the dant effect on the upstream trans-sulfuration pathway. number of independent determinations. While the antioxidant behavior of both these compounds 24,25 are well known, they have also been reported to have pro-oxidant effects. Specifically, PDTC has been shown decrease in cystathionine accumulation and corresponded to elicit time- and dose-dependent increases in the to a ~20% decrease after 10 h (Fig. 3) which is v irtually mRNAs encoding the catalytic and regulatory subunits of identical to the decrease in glutathione concentration -glutamyl-cysteine ligase in HepG2 cells. -Glutamyl- observed at the same time (Table 2). These results demon- cysteine ligase is b elieved to be the rate-limiting enzyme in strate, for the first time, the sensitivity of the upstream glutathione biosynthesis. BHA enhances the activity of trans-sulfuration pathway to antioxidants, and its e ffect on glutathione reductase and functions a s a transcriptional the pool size of the downstream product, glutathione. Table 2. E ffect of v arying concentrations of ex tracellular catalase on intracellular glutathione concentration and o n inc orporation of rad ioactivity from [ S]-methionine Additions Relative intracellular Relative incorporation of glutathione concentration [ S] into glutathione Value n P value Value n P value Control 100 ± 1 19 100 ± 3 19 Catalase (10 U /ml) 95 ± 3 9 NS 100 ± 10 9 NS Catalase (100 U/m l) 83 ± 2 23 ! 0.001 86 ± 3 23 ! 0.001 Catalase (1000 U /ml) 82 ± 3 9 ! 0.001 86 ± 8 9 ! 0.1 Thymol (150 nM ) 99 ± 4 8 NS 105 ± 10 8 NS n, num ber of independent determinations; NS, n ot significant. C y stath io n in e (mm o l/l c e lls) 62 Vitvitsky, Mosharov, Trit t, Ataullakh anov, Banerjee in a ~20% decrease in intracellular glutathione level and activator of -glutamyl-cysteine ligase. A 1.5–2- fold on flux through the trans-sulfuration pathway. Inter- increase in glutathione levels has been reported in mice 12 estingly, a combination of antioxidants did not have an days after feeding with BHA. The observed elevation in additive effect on glutathione metabolism. The effects of glutathione levels in response to PDTC and BHA in our PDTC and BHA on the trans-sulfuration pathway in studies is t hus consistent with previous r eports, while the HepG2 cells could be derived either from oxidation increased incorporation of radioactivity from the methion- reactions or from other, presently unknown, effects. ine to the glutathione pool additionally suggests enhanced flux through the trans-sulfuration pathway under these conditions. These results c onfirm and extend our previo us ACKNOWLEDGEMENTS obser vatio ns w it h peroxid e-based pro-oxid ants, which demonstr ated th at th e glutathione pool size is m odulated by This work was supported by grants from the National th e sen sitiv ity of the tr ans-sulfuratio n pathway to oxidative Institutes o f Health (ES011441 and HL58984) and by an stress. Established Investigator Award from the American Heart Recently, using a reporter construct containing one of Association to RB. the two functional promoters for cystathionine -syn- thase, decreased promoter activity was reported in HepG2 cells treated with " 50 mM H O . Although 2 2 these results are at odds with a published report of REFERENCES enhanced flux of homocysteine through cystathionine - synthase in HepG2 cells treated with H O and the 1. Ma rtensson J, Me ister A. 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Redox regulation of homocysteine-dependent glutathione synthesis

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Taylor & Francis
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© 2003 Maney Publishing
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1743-2928
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1351-0002
DOI
10.1179/135100003125001260
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Research article 1 2 2 1 2 Victor V itvitsky , Eugene Mosharov , Michael Tritt , Fazoil Ataullakhanov , Ruma Banerjee National Research Center for Hematology, Moscow, Russia Biochemistry Department, University of Nebraska, Lincoln, Nebraska, USA In certain tissues, glutathione biosynthesis is conn ected to methionine metabolism via the trans- sulfuration pathway. The latter condenses homocysteine and serine to cystathionine in a reaction catalyzed by cystathionine -synthase followed by cleavage of cystathionine to cysteine and - ketoglutarate by -cystathionase. Cysteine is t he limiting amino acid in glutathione biosynthesis, and studies in our laboratory have shown that approximately 50% of the cysteine in glutathione is derived from homocysteine in human liver cells. In this study, we have examined the effect of pro- and antioxidants on the flux of homocysteine through the trans-sulfuration pathway in the human hepatoma cell line, HepG2. Our studies reveal that pyrrolidine dithiocarbamate and butylated hydroxyanisole enhance the flux of homocysteine through the trans-sulfuration pathway as has been observed previously with the pro-oxidants, H O and tertiary butyl hydroperoxide. In contrast, 2 2 antioxidants such as catalase, superoxide dismutase and a water-soluble derivative of vitamin E elicit the opposite effect and result in diminished flux of homocysteine through the trans-sulfuration pathway. These studies provide the first evidence for the reciprocal sensitivity of the trans- sulfuration pathway to pro- and antioxidants, and demonstrate that the upstream half of the glutathione biosynthetic pathway ( i.e. leading to cysteine biosynthesis) is r edox sensitive as is t he regulation of the well-studied enzymes in the downstream half (leading from cysteine to glutathione), namely, -glutamyl-cysteine ligase and glutathione synthetase. INTRODUCTION uitously (Fig. 1). However, in tissues that have the largest concentrations of glutathione, and, therefore, the In aerobic organisms, a delicate balance exists between highest demand for cysteine (the limiting amino acid), oxygen-dependent metabolism and the generation of the trans-sulfuration pathway is p resent (Fig. 1). The lat- reactive oxygen species as side products. Glutathione, a ter presents an avenue for the conversion of the essential thiol-containing tripeptide plays a key role in intracellu- amino acid, methionine, to cysteine. Thus, the trans-sul- lar defense, and decreased levels of this antioxidant are furation pathway catalyzes the sequential conversion of correlated with the increased frequency of reactive oxy- homocysteine, a key junction metabolite in the methion- 1–3 gen species-mediated mitochondrial damage and ine cycle, to cystathionine and then cysteine. apoptosis. The intracellular glutathione pools are large Studies on the effect of pro-oxidants o n glutathione bio- 5–7 and vary from 1–10 mM depending on the cell type. synthesis have focused overwhelmingly on the last t wo 8–13 The final two steps in the biosynthesis of glutathione are enzymes in the biosynthetic pathway. A number of catalyzed by -glutamyl-cysteine ligase and glutathione conditions and agents elicit the transcriptional activation synthetase, enzymes that are believed to be present ubiq- of -glutamyl-cysteine ligase and some also activate glu- cis tathione synthetase. Three -acting elements that may be Received 18 Augu st 200 2 Abbreviations : ARE , antioxidant re sponse el ement; NF- kB, nuclear Accepted 28 Oc tober 20 02 factor kappa B; AP , ac tivator prot ein; t-B uOOH, te rtiary but yl hydroperoxide; PDT C, pyrr olidine dithiocarbamate; BH A, bu tylated Correspondence to: Prof. Ruma Banerjee, Biochemistry Department, University of Neb raska N13 3 Be adle Center, , L incoln, NE 68588- hydroxyanisole; MEM, Ea gle’s mi nimal essential me dium; PBS, 0664, USA phosphate bu ffered saline; FBS, fet al bov ine ser um; SOD, superoxide Tel : + 1 402 47 2 294 1; E-mail: rba [email protected] dismutase Redox Rep ort , V ol. 8, No. 1, 2003 © W . S. Maney & Son Lt d DOI 10.1179/135100003125001260 58 Vitvitsky, Mosharov, Trit t, Ataullakh anov, Banerjee AdoMet AdoHCy Cystathionine Cysteine -Glu-Cys GLUTA THION E Methionine HOMOCYSTEINE Propargylglycine Transmethylation pathway Glutathione synthesis pathway Transulfuration pathway Fig. 1. Metabolic pathway for glutathione biosynthesis in liver cells showing the upstream trans-sulfuration and the downstream glutathione synthesis reactions. AdoMet and AdoHcy denote S-adenosylmethionine and S-adenosylhomocysteine respectively. important in mediating this response have been identified pathway and on glutathione levels in HepG2 cells. We in the gene encoding the heavy subunit of -glutamyl-cys- found that catalase, a water-soluble form of vitamin E, and 14–16 teine ligase, which harbors the catalytic activity. These SOD elicit a small, but significant, reduction in the intracel- are the antioxidant response element (ARE-4), nuclear lular glutathione concentration. Furthermore, the decrease factor kappa B ( NF- B) and activator protein (AP-1). The in the glutathione pool size is p aralleled by a decrease in the promoter for the light subunit gene of -glutamyl-cysteine flux of homocysteine through the trans-sulfuration path- ligase also contains ARE and AP-1 elements. way. In contrast, PDTC and BHA increase flux of homo- In contrast, the responsiveness o f the trans-sulfuration cysteine through the trans-sulfuration pathway. These pathway enzymes t o changes i n redox status brought on by studies provide the first experimental evidence for recipro- pro- or antioxidants has received scant attention. This stems cal sensitiv ity of cystathionine -synthase activity to pro- largely from the lack of recognition of the importance of and antioxidants i n a model cell culture system. this pathway in the regulation of redox homeostasis. W e have recently demonstrated that the trans-sulfuration path- way plays a quantitatively major role in the maintenance of MATERIALS AND METHODS the intracellular glutathione pool in transformed human liver cells in culture. The two homocysteine junction Reagents and cell lines enzymes, methionine synthase and cystathionine -syn- thase, display reciprocal sensitiv ity to ambient redox tone Eagle’s m inimum essential medium, DL-propargyl- 18,19 in vitro under conditions. These observations led us to glycine, H O , t-BuOOH, PDTC, BHA, vitamin E (± - 2 2 hypothesize that redox changes may exert an additional tocopherol), superoxide dismutase, catalase, ascorbate, layer of regulation at this metabolic junction. The rationale thymol and trypan blue were purchased from Sigma. The for such a regulation, if it existed, could be that increased water-soluble analog of vitamin E (6-hydroxy-2,5,7,8- flux of homocysteine through the trans-sulfuration pathway tetramethylchroman-2-carboxylic acid) was purchased under oxid izing conditio ns would le ad to enhanced synthesi s from Aldrich. The antibiotic/antimycotic solution was of glu tathione and thus s er ve as a n autocorrective response to from Gibco BRL. Fetal bovine serum (FBS) was from an oxidative insult. Under th e opposite set of conditio ns, HyClone Company. Transformed human hepatocytes homocystei ne would be re-directed preferential ly to th e (HepG2) were from ATCC. [ S]-Methionine (>1000 tr ansmethyla tio n pathway leadin g to its r ecycling. Ci/m mol) was purchased from Amersham Pharmacia In a recent study using the human hepatoma cell line, Biotech. HepG2, we demonstrated that both H O and tertiary butyl 2 2 hydroperoxide (t-BuOOH) elicit a dose-dependent Cell culture increase in cystathionine production. This was accompa- nied by a corresponding increase in incorporation of radioactivity from [ S]-methionine into the glutathione Cells were grown until they reached 70–80% confluency pool. In this study, we have compared the effect of pro- in 60 x 15 mm Falcon 3004 tissue culture dishes in oxidants (H O and t-BuOOH) and substances that are MEM with 10% FBS, 2 mM L-glutamine, 2.2 g/l sodium 2 2 considered to be antioxidants (pyrrolidine dithiocarba- bicarbonate and 10 ml/l antibiotic/antimycotic solution. mate [PDTC], butylated hydroxyanisole [BHA], vitamin Each dish contained 4 ml of medium, which was E, catalase, ascorbate and superoxide dismutase [SOD]) changed every 4 days and 2 h before the start of the on the flux of homocysteine through the trans-sulfuration experiment. Experiments were initiated by adding the Redox sensitivity of homocysteine metabolism 59 desired reagents to the dishes. [ S]-Methionine solution was diluted in phosphate buffered saline (PBS) to give a specific activity of 1.25 mCi/ml and 1 ml of this solution was added per ml of cell culture medium to each dish. At the desired time intervals, cells were harvested and the protein concentration in samples was determined as described earlier. In experiments in which 1 mM H O 2 2 was used, cell viability was measured using the trypan blue exclusion method. Briefly, H O was added to a 2 2 concentration of 1 mM to HepG2 cells at 70–80% con- fluency and viability was determined after 2, 4, 10, and 20 h. The medium (4 ml) was removed and 0.5 ml of 0.4% trypan blue solution in 0.5 ml PBS was added. After 2 min, the cells were washed with 2 × 1 ml PBS, covered with 2 ml PBS an d the fraction of (dead) blue cells in a view area was determined. The average value from four view areas was calculated. Metabolite analysis Concentrations of glutathione and cystathionine in cell extracts were estimated using an HPLC method as described previously. The concentration of individual metabolites was determined by comparing integrated peak areas with previously generated calibration curves for each compound. To represent the thiol concentrations in units of mmol/l of cells, the amount of protein in HepG2 cells was taken to be 53 g protein/l of cells as estimated previously. Incorporation of [ S]-methionine into the glutathione pool was evaluated by measuring radioactivity in the relevant chromatographic fraction. Statistical significance was ana- Fig. 2. Kinetics of recovery of the intracellular glutathione pool following lyzed using the ANOVA test. oxidative challenge. The levels of glutathione and glutathione disulfide in untreated cells is represented by the open circles and triangles respectively. In cells treated with a single bolus of H O (1 mM), the glutathione (filled 2 2 circles) level rapidly declines before recovering, while the glutathione RESULT S AND DISCUSSION disulfide pool responds reciprocally (filled triangles). The culture dish contained ~4 x 10 cells in 4 ml medium which corresponds to a ratio of Kinetics of glutathione homeostasis following H O ~1 mmol H O /10 cells. Inset: comparison of viability o f cells treated with 2 2 2 2 a single bolus of 1 mM H O (hatched bars) versus untreated controls exposure 2 2 (solid bars) at fixed times following addition of peroxide or an equal volume of PBS to the culture medium. We have previously shown that relatively low concentra- tions of peroxides such as H O and t-BuOOH result in 2 2 enhanced flux of homocysteine through the trans-sulfura- the redox status of the cells, changes from an initial value tion pathway. However, at the oxidant concentrations of 400 ± 80 to 2–5 during the first few minutes following used in the earlier study (10–20 0 mM), there was no H O treatment. This is followed by a recovery phase dur- detectable change in the glutathione pool immediately fol- 2 2 ing which the glutathione level, as well as the glutathione- lowing exposure to the oxidants. In order to study the to-glutathione disulfide ratio, are restored over a period of kinetics of recovery of the intracellular glutathione pool 6–8 h. Glutathione disulfide displays a reciprocal response following oxidative stress, we examined the time-depen- and following an initial increase, diminishes to control dent changes in the concentration of glutathione and glu- levels within 2 h. The increase in glutathione concentra- tathione disulfide following exposure to 1 mM H O (Fig. 2 2 tion during the recovery phase is ref lected in the 2). Under these conditions, a decrease in the intracellular enhanced incorporation of [ S]-methionine into this glutathione concentration concomitant with an increase in pool (not shown), demonstrating the contribution of the glutathione disulfide is o bserved. The ratio of glutathione trans-sulfuration pathway in glutathione-linked redox to glutathione disulfide, which is k nown to be a marker for 60 Vitvitsky, Mosharov, Trit t, Ataullakh anov, Banerjee homeostasis. The glutathione levels finally overshoot the response to catalase was further characterized. The dose pool size in untreated cells. A similar observation has dependence of the catalase response is p resented in Table been reported in L2 cells (derived from type II pneumo- 2. Addition of 100 or 1000 U/ml of catalase to the cytes of adult rat lungs) treated with 4-hydroxy-2-none- medium resulted in an ~17% decrease in the intracellular nal, a major lipid peroxidation metabolite. glutathione concentration whereas 10 U/m l did not have a The kinetics of glutathione recovery as well as the corre- statistically significant effect. The decrease in glutathione spondence between our data set and that reported for L2 concentration was paralleled by a decrease in incorpora- cells, indicate that HepG2 cells treated with a single bolus tion of radioactivity. Since thymol is a dded as a preserva- of 1 mM H O remained viable during the time course of tive in the commercially available catalase that was 2 2 the experiment. This was confirmed by monitoring the via- initially employed in this study (as this preparation has a bility of HepG2 cells treated with a single bolus of 1 mM high specific activity (48,000 U/m g protein)), an addi- H O using the trypan blue exclusion assay. As seen in tional control was performed with thymol alone, which 2 2 Figure 2 (inset) there is n o difference in the number of was found to have no effect on intracellular glutathione viable cells in cells treated with 1 mM H O versus concentration and on S incorporation from methionine 2 2 untreated controls, and ! 1% of the cells were stained blue to glutathione (Table 2). We have also repeated these during the time course of the experiment. experiments with thymol-free catalase (specific activity, 15,500 U/m g protein). The intracellular glutathione con- centration and radioactivity incorporation were found to Effect of catalase, superoxide dismutase, ascorbate and be 82 ± 3% and 87 ± 6% ( n = 6), respectively, in the pres- vitamin E, on intracellular glutathione concentration ence of 100 U/m l catalase relative to untreated controls. In principle, the effect of catalase on the reduced glu- Addition of superoxide dismutase, a water-soluble form tathione pool size could be due to its e ffects on the upstream of vitamin E, and catalase resulted in a decrease in the trans-sulfuration pathway, the downstream glutathione syn- intracellular glutathione pool, which was reflected in the thesis pathway, or both. To determine whether or not cata- diminished incorporation of S from methionine into glu- lase affects the trans-sulfuration pathway, we examined the tathione (Table 1). In contrast, neither ascorbate nor flux of homocysteine through cystathionine -synthase in water-insoluble vitamin E had a statistically significant cells treated with propargylglycine. The latter is a suicide 21,22 effect. The limited solubility of vitamin E likely resulted inhibitor of -cystathionase, the second enzyme in the in its l ow availability in these experiments. Simultaneous trans-sulfuration pathway, and its a dministration leads to a addition of catalase, water-soluble vitamin E, and super- time-dependent increase in the intracellular cystathionine oxide dismutase did not have an additive effect. Since the pool which is t oo small to be readily detected under normal effect of catalase was more pronounced than that of super- conditions. Addition of 100 U/ml catalase to cells oxide dismutase and water-soluble vitamin E, the cellular treated with 2.5 mM propargylglycine resulted in a Table 1. E ffect of v arious pro- and antioxidants on intracellular glutathione concentration and o n inco rporation of rad ioactivity from [ S]-methionine Additions Relative intracellular Relative incorporation of glutathione concentration [ S] into glutathione Value n P value Value n P value Control 100 ± 1 37 100 ± 2 36 Catalase 83 ± 2 23 ! 0.001 86 ± 3 23 ! 0.001 SOD 92 ± 3 9 ! 0.02 92 ± 4 9 ! 0.1 Ascorbate 101 ± 5 4 NS 100 ± 2 3 NS Vitamin E 95 ± 4 8 NS 106 ± 7 8 NS Vitam in E w ater-soluble 90 ± 5 6 ! 0.05 81 ± 6 6 ! 0.01 Catalase + SOD + w ater-soluble vitamin E 87 ± 3 8 ! 0.001 77 ± 3 8 ! 0.001 t-BuOOH 110 ± 3 6 ! 0.01 131 ± 4 6 ! 0.001 PDTC 147 ± 9 7 ! 0.001 191 ± 18 6 ! 0.001 PDTC + t-B uOOH 160 ± 6 6 ! 0.001 257 ± 45 5 ! 0.01 BHA 111 ± 6 6 ! 0.1 153 ± 10 6 ! 0.001 The co ncentrations of the various additives employed were ca talase (100 U/ml of medium), SOD (100 U/m l of med ium), ascorbate (150 mM), vitam in E (2 00 mM), t-BuOOH (20 0 mM), PDTC (60 mM), B HA (20 0 mM), for 10 h fo llowing which the concentration of glutath ione and the label incorporation into glutathione were d etermined. n, num ber of independent determinations; NS, n ot significant. Redox sensitivity of homocysteine metabolism 61 Effect of PDTC and BHA on intracellular glutathione concentration PDTC is a relatively stable dithiocarbamate, a family of compounds that can chelate both free and protein-bound metals and is w idely used in medicine. The antioxidant behavior of dithiocarbamates results from their radical scavenging properties and cellular responses including 24,25 inhibition of lipid peroxidation, and inhibition of 26,27 oxidative activation of the transcription factor, NF- B. However, addition of 60 mM PDTC resulted in an increase in intracellular glutathione after 10 h that is similar to, but more pronounced than, the response observed with the oxidant, t-BuOOH (Table 1). Simultaneous addition of tBuOOH and PDTC had an additive effect resulting in a ~1.6-fold increase in glu- 5 h 1 0 h tathione concentration. The increase in incorporation of % D e c re a s e in 1 0 .7 ± 3 .3 1 9 .9 ± 3 .5 [ S]-methionine into glutathione was marginally greater c y s ta th io n in e (n = 1 2 ) (n = 1 0 ) w ith c a ta la s e than the fold-increase in glutathione concentration ( e.g. S ig n ific a n c e (P) 0 .0 2 0 .0 0 0 5 50 ~1.9-fold versus 1.5-fold with PDTC; Table 1) suggest- ing enhanced use of the trans-sulfuration pathway- derived cysteine in glutathione biosynthesis under these conditions. 0 2 4 6 8 10 BHA elicited a similar response under these condi- tions. Thus, addition of 200 mM BHA resulted in 1.1- Time (h) fold increase in intracellular glutathione after 10 h versus untreated controls (Table 1), and a 1.5-fold Fig. 3. Effect of extracellular catalase treatment on flux of homocysteine through the trans-sulfuration pathway. Cells were treated with 2.5 mM increase in incorporation of S from the methionine proparglycine only (open circles) or propargylglycine and catalase (100 pool. These results suggest that the effects of PDTC and U/ml; filled circles). The concentration of cystathionine was measured at BHA derive either from oxidation reactions or from the indicated times, and a representative data set is presented. The other presently unknown effects, rather than an antioxi- statistical analysis of the data is presented in the inset where n is the dant effect on the upstream trans-sulfuration pathway. number of independent determinations. While the antioxidant behavior of both these compounds 24,25 are well known, they have also been reported to have pro-oxidant effects. Specifically, PDTC has been shown decrease in cystathionine accumulation and corresponded to elicit time- and dose-dependent increases in the to a ~20% decrease after 10 h (Fig. 3) which is v irtually mRNAs encoding the catalytic and regulatory subunits of identical to the decrease in glutathione concentration -glutamyl-cysteine ligase in HepG2 cells. -Glutamyl- observed at the same time (Table 2). These results demon- cysteine ligase is b elieved to be the rate-limiting enzyme in strate, for the first time, the sensitivity of the upstream glutathione biosynthesis. BHA enhances the activity of trans-sulfuration pathway to antioxidants, and its e ffect on glutathione reductase and functions a s a transcriptional the pool size of the downstream product, glutathione. Table 2. E ffect of v arying concentrations of ex tracellular catalase on intracellular glutathione concentration and o n inc orporation of rad ioactivity from [ S]-methionine Additions Relative intracellular Relative incorporation of glutathione concentration [ S] into glutathione Value n P value Value n P value Control 100 ± 1 19 100 ± 3 19 Catalase (10 U /ml) 95 ± 3 9 NS 100 ± 10 9 NS Catalase (100 U/m l) 83 ± 2 23 ! 0.001 86 ± 3 23 ! 0.001 Catalase (1000 U /ml) 82 ± 3 9 ! 0.001 86 ± 8 9 ! 0.1 Thymol (150 nM ) 99 ± 4 8 NS 105 ± 10 8 NS n, num ber of independent determinations; NS, n ot significant. C y stath io n in e (mm o l/l c e lls) 62 Vitvitsky, Mosharov, Trit t, Ataullakh anov, Banerjee in a ~20% decrease in intracellular glutathione level and activator of -glutamyl-cysteine ligase. A 1.5–2- fold on flux through the trans-sulfuration pathway. Inter- increase in glutathione levels has been reported in mice 12 estingly, a combination of antioxidants did not have an days after feeding with BHA. The observed elevation in additive effect on glutathione metabolism. The effects of glutathione levels in response to PDTC and BHA in our PDTC and BHA on the trans-sulfuration pathway in studies is t hus consistent with previous r eports, while the HepG2 cells could be derived either from oxidation increased incorporation of radioactivity from the methion- reactions or from other, presently unknown, effects. ine to the glutathione pool additionally suggests enhanced flux through the trans-sulfuration pathway under these conditions. These results c onfirm and extend our previo us ACKNOWLEDGEMENTS obser vatio ns w it h peroxid e-based pro-oxid ants, which demonstr ated th at th e glutathione pool size is m odulated by This work was supported by grants from the National th e sen sitiv ity of the tr ans-sulfuratio n pathway to oxidative Institutes o f Health (ES011441 and HL58984) and by an stress. Established Investigator Award from the American Heart Recently, using a reporter construct containing one of Association to RB. the two functional promoters for cystathionine -syn- thase, decreased promoter activity was reported in HepG2 cells treated with " 50 mM H O . Although 2 2 these results are at odds with a published report of REFERENCES enhanced flux of homocysteine through cystathionine - synthase in HepG2 cells treated with H O and the 1. Ma rtensson J, Me ister A. 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Redox Report (Communications in Free Radical Research) Online – Taylor & Francis

Published: Feb 1, 2003

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