Radioprotective effect of cysteamine in glutathione synthetase-deficient cells

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect.     

Reduced repair of potentially lethal radiation damage in glutathione synthetase-deficient human fibroblasts after X-irradiation

Using a human fibroblast strain deficient in glutathione synthetase and a related proficient control strain, the role of glutathione (GSH) in repair of potentially lethal damage (PLD) has been investigated in determining survival by plating cells immediately or 24 h after irradiation. After oxic or hypoxic irradiation, both cell strains repair radiation-induced damage. However, under hypoxic conditions, the proficient cells repair PLD as well as under oxic conditions while the deficient cells repair less PLD after irradiation under hypoxic than under oxic conditions. Therefore, the oxygen enhancement ratio (o.e.r.) for proficient cells is similar whether the cells are plated immediately or 24 h later (2.0 and 2.13, respectively). In contrast, the o.e.r. for deficient cells is lower when the cells are plated 24 h after irradiation than when they are plated immediately thereafter (1.16 as compared to 1.55). The results indicate that GSH is involved in PLD repair and, in particular, in the repair of damage induced by radiation delivered under hypoxic conditions.     

The influence of oxygen on the induction of radiation damage in DNA in mammalian cells after sensitization by intracellular glutathione depletion

Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and non-protein-bound SH (NPSH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10-20 per cent for the three types of radiation damage. Also under severely hypoxic conditions (0.01 microM oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0-30 per cent). At somewhat higher concentrations of oxygen (0.5-10 microM) however, the sensitization amounted to about 90 per cent for the induction of ssb and dsb and about 50 per cent for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen.     

Glutathione depletion by DL-buthionine-SR-sulfoximine (BSO) potentiates X-ray-induced chromosome lesions after liquid holding recovery

The impact of intracellular glutathione depletion on chromosome damage induced by X irradiation under aerobic conditions was investigated in two different cell lines, Ehrlich ascites tumor cells (EATC) and Chinese hamster ovary cells (CHO-K1). Thiol-depleted cell cultures in plateau phase were obtained by prolonged incubation in growth medium containing DL-buthionine-SR-sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase. Cells were then assayed using the procedures of G. L. Ellmann (Arch. Biochem. Biophys. 82, 70-77 (1959)), F. Tietze (Anal. Biochem. 27, 502-522 (1969)), and J. Sedlack and R.H. Lindsay (Anal. Biochem. 25, 192-205 (1968)) for non-protein bound SH (NPSH), glutathione (GSH), and total SH (TSH). In both cell lines GSH was reduced to less than 10% of controls at higher BSO concentrations around 1 mM, whereas TSH and NPSH were affected to only 40-60%. In EATC pretreated with up to 1 mM BSO for 72 h, increased levels of spontaneously occurring micronuclei were found. At BSO concentrations above 200 microM, both cell lines showed a potentiation of chromosome lesions scored as micronuclei and induced under aerobic X irradiation when liquid holding recovery in the original nutrient-depleted medium was performed; the extent of chromosome damage eventually reached that which could be obtained by application of beta-arabinofuranosyladenine (beta-araA), known to inhibit DNA repair processes by blocking DNA polymerases. It is therefore suggested that GSH depletion causes impairment of repair of lesions leading to chromosome deletions and subsequently to micronuclei. In contrast to CHO cell cultures, EATC showed a reversion of the potentiation effect as indicated by a decrease in the micronucleus content during prolonged incubation in the presence of BSO in the millimolar range. This effect could not be correlated to the remaining GSH content of less than 10% but may be due to some accumulation of unknown NPSH components. Since addition of L-cysteine to EATC cultures pretreated with BSO decreased the micronucleus content, cysteine/cystine or a related thiol within the NPSH fraction may be involved in the reestablishment of repair. Thus at least in one cell line, a rather complex response to BSO administration indicated that not only GSH but also other thiols may determine the level of chromosome damage after liquid holding recovery.     

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.     

Radiosensitizing and cytotoxic properties of misonidazole on glutathione synthetase deficient human fibroblasts

The cytotoxic and radiosensitizing effects of misonidazole have been studied on glutathione synthetase deficient fibroblasts and on their controls. At any concentration from 0.1 to 4 mM, deficient cells are more sensitive to the cytotoxic effect of misonidazole than the control cells. The differential effect between the two cell strain concerns both the shoulder and the slope of the survival curve, thus suggesting that NPSH play a role in the determination of misonidazole cytotoxicity. Like oxygen, misonidazole clearly sensitizes deficient cells to a lesser extent than control cells. For both cell strains, the maximum sensitizing effect of misonidazole is very close to that of oxygen (1.5 and 1.5 for deficient cells, 2.8 and 2.9 for control cells, respectively). The sensitizing effect of misonidazole appears in the same concentration range for both cell strains, with a maximal effect at lower concentrations for deficient cells.     

Reduced PLD repair ability in glutathione synthetase deficient human fibroblasts after UV irradiation

Using a human cell strain deficient in glutathione synthetase and a related control, the role of glutathione in repair mechanisms has been investigated. UV light has been used in order to avoid the interaction between thiols and free radicals. When potentially lethal damage repair is completed, deficient cells in plateau phase exhibit smaller surviving fractions than do control cells. The ratio of surviving fractions in control/deficient cells is about 2 for the same radiation dose. These results indicate that thiols and especially GSH are involved in repair mechanisms.     

Influence of buthionine sulfoximine and misonidazole on glutathione level and radiosensitivity of human tumor xenografts

We have studied the effect of buthionine sulfoximine (BSO; a gamma-glutamylcysteine synthetase inhibitor) administration, either alone or combined with misonidazole (MISO), on five human tumor xenografts (three melanomas: Bell, Mall, and Nall; and two rectocolic adenocarcinomas: HT29 and HRT18) transplanted into mice. Two criteria were used, the nonprotein bound sulfhydryl (NPSH) level (glutathione (GSH) and cysteine (CYS] and the fraction of surviving tumor cells after gamma irradiation. GSH and CYS were estimated by HPLC and cell survival by in vivo-in vitro clonogenic assay. Administration of BSO alone (three injections of 10 mumol/g) prior to irradiation always produced a significant reduction in the GSH level while MISO administration (1 mg/g) did not consistently influence the NPSH level. While BSO had little or no radiosensitizing effect, MISO always induced radiosensitization (enhancement ratio between 1.6 and 1.8). This effect did not depend on the fraction of surviving hypoxic cells. An increase in MISO-induced radiosensitization produced by BSO was cell-line dependent. Results do not seem to support the hypothesis of a relationship between the GSH level at the time of irradiation and the radiosensitization induced by BSO or BSO + MISO. However, BSO treatment may not have been able to reduce endogenous thiols to a low enough level to test the hypothesis.     

The role of thiols in cellular response to radiation and drugs

Cellular nonprotein thiols (NPSH) consist of glutathione (GSH) and other low molecular weight species such as cysteine, cysteamine, and coenzyme A. GSH is usually less than the total cellular NPSH, and with thiol reactive agents, such as diethyl maleate (DEM), its rate of depletion is in part dependent upon the cellular capacity for its resynthesis. If resynthesis is blocked by buthionine-S,R-sulfoximine(BSO), the NPSH, including GSH, is depleted more rapidly, Cellular thiol depletion by diamide, N-ethylmaleimide, and BSO may render oxygenated cells more sensitive to radiation. These cells may or may not show a reduction in the oxygen enhancement ratio (OER). Human A549 lung carcinoma cells depleted of their NPSH either by prolonged culture or by BSO treatment do not show a reduced OER but do show increased aerobic responses to radiation. Some nitroheterocyclic radiosensitizing drugs also deplete cellular thiols under aerobic conditions. Such reactivity may be the reason that they show anomalous radiation sensitization (i.e., better than predicted on the basis of electron affinity). Other nitrocompounds, such as misonidazole, are activated under hypoxic conditions to radical intermediates. When cellular thiols are depleted peroxide is formed. Under hypoxic conditions thiols are depleted because metabolically reduced intermediates react with GSH instead of oxygen. Thiol depletion, under hypoxic conditions, may be the reason that misonidazole and other nitrocompounds show an extra enhancement ratio with hypoxic cells. Thiol depletion by DEM or BSO alters the radiation response of hypoxic cells to misonidazole. In conclusion, we propose an altered thiol model which includes a mechanism for thiol involvement in the aerobic radiation response of cells. This mechanism involves both thiol-linked hydrogen donation to oxygen radical adducts to produce hydroperoxides followed by a GSH peroxidase-catalyzed reduction of the hydroperoxides to intermediates entering into metabolic pathways to produce the original molecule.     

Novel kinetics of mammalian glutathione synthetase: characterization of gamma-glutamyl substrate cooperative binding

Glutathione (GSH) synthetase [L-gamma-glutamyl-L-cysteinyl:glycine ligase (ADP-forming), EC 6.3.2.3] catalyzes the final step in GSH biosynthesis. Mammalian glutathione synthetase is a homodimer with each subunit containing an active site. We report the detailed kinetic data for purified recombinant rat glutathione synthetase. It has the highest specific activity (11 micromol/min/mg) reported for any mammalian glutathione synthetase. The apparent K(m) values for ATP and glycine are 37 and 913 microM, respectively. The Lineweaver-Burk double reciprocal plot for gamma-glutamyl substrate binding revealed a departure from linearity indicating cooperative binding. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0. 576, which shows the negative cooperativity. Neither ATP, the other substrate involved in forming the enzyme-bound gamma-glutamyl phosphate intermediate, nor glycine, which attacks this intermediate to form GSH, exhibit any cooperativity. The cooperative binding of gamma-glutamyl substrate is not affected by ATP concentration. Thus, mammalian glutathione synthetase is an allosteric enzyme.     

Glutathione synthesis in Streptococcus agalactiae. One protein accounts for gamma-glutamylcysteine synthetase and glutathione synthetase activities

Gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS), distinct enzymes that together account for glutathione (GSH) synthesis, have been isolated and characterized from several Gram-negative prokaryotes and from numerous eukaryotes including mammals, amphibians, plants, yeast, and protozoa. Glutathione synthesis is relatively uncommon among the Gram-positive bacteria, and, to date, neither the genes nor the proteins involved have been identified. In the present report, we show that crude extracts of Streptococcus agalactiae catalyze the gamma-GCS and GS reactions and can synthesize GSH from its constituent amino acids. The putative gene for S. agalactiae gamma-GCS was identified and cloned, and the corresponding protein was expressed and purified. Surprisingly, it was found that the isolated enzyme catalyzes both the ATP-dependent synthesis of L-gamma-glutamyl-L-cysteine from L-glutamate and L-cysteine and the ATP-dependent synthesis of GSH from L-gamma-glutamyl-L-cysteine and glycine. This novel bifunctional enzyme, referred to as gamma-GCS-GS, has been characterized in terms of catalytic activity, substrate specificity, and inhibition by GSH, cystamine, and transition state analog sulfoximines. The N-terminal 518 amino acids of gamma-GCS-GS (total M(r) 85,000) show 32% identity and 43% similarity with E. coli gamma-GCS (M(r) 58,000), but the C-terminal putative GS domain (remaining 202 amino acids) of gamma-GCS-GS shows no significant homology with known GS sequences. The C terminus (360 amino acids) is, however, homologous to D-Ala, D-Ala ligase (24% identity; 38% similarity), an enzyme having the same protein fold as known GS proteins. These results are discussed in terms of the evolution of GSH synthesis and the possible occurrence of a similar bifunctional GSH synthesis enzyme in other bacterial species.     

Endogenous thiol levels in heterogeneous murine tumor cells as a function of the physiological state and the response to X-irradiation

The endogenous thiols (PSH, protein sulfhydryls; NPSH, nonprotein sulfhydryls; and GSH, glutathione) were measured in the 66 and 67 murine carcinoma cells growing under different physiological conditions in vitro (e.g., proliferation, P; nutrient-deprived quiescence QI; and QI cells stimulated by refeeding the monolayer in situ and assayed 4 (St4) and 14 (St14) h later). The aerobic radiation response was also studied as a function of the physiological state and thiol concentration. The changes in PSH levels suggest that the proportion of thiol-containing proteins changed whenever the cells were in transition between different physiological states (e.g., when QI cells were stimulated by refeeding, the proportion of PSH was elevated dramatically over either QI or P cells). The NPSH and GSH levels were both down significantly in the QI vs. P cells as was the total thiol level (PSH plus NPSH). Fourteen h but not 4 h after stimulation, the NPSH and GSH levels had returned to or exceeded the P-cell levels. Also, the proportion of GSH in the NPSH fraction varied as a function of the physiological state. The 66 and 67 QI cells were both more radiosensitive than the respective P cells. Also, the 66 cell radiation-induced cytotoxicity had returned to the P response by about 4 h after refeeding but the stimulated 67 cells had not. However, no overall correlation was apparent between the various aerobic radiation responses and the pool sizes of either the total thiols or of the various subsets of thiols. The depressed total thiol level and the increased radiosensitivity of the QI cells could represent a cause-and-effect relationship or these parameters could be independent phenomena only related indirectly through the reduced metabolic activity of the quiescent cells.     

Changes in organ nonprotein sulfhydryl and glutathione concentrations during acute and chronic administration of inorganic lead to chicks

The effects of dietary and injected lead (Pb) on organ nonprotein sulfhydryl (NPSH) and glutathione (GSH) concentrations in the chick were studied. Lead acetate·3H₂O was administered either in the diet for 3 wk at 2000 ppm Pb or by intraperitoneal (ip) injection of 3-wkold chicks with 52 mg Pb/100 g body wt. In Exp. 1, NPSH concentrations in liver and kidney were increased by both dietary and injected Pb in comparison to chicks not receiving Pb. Thigh muscle NPSH was decreased by injected Pb, whereas dietary Pb had no effect. In Expt. 2, whole blood and plasma NPSH were measured at 0, 0.5, 1.0, 2.0, and 4.0 h following ip Pb injection. Both whole blood and plasma NPSH were increased by 30 min. Whole blood NPSH concentrations plateaued at 30 min, and plasma NPSH continued to rise for 2 h. In Expt. 3, injected Pb increased hepatic NPSH, but not GSH concentrations. The ratio of GSH/NPSH was therefore lowered. The incorporation of [1-¹⁴C]glycine into hepatic GSH was stimulated by injected Pb. Buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, reduced hepatic NPSH and [¹⁴C]glycine incorporation in Pb-treated chicks to below control (non-Pb injected) values. In Expt. 4, dietary Pb fed for 3 wk increased the hepatic concentrations of both NPSH and GSH such that the ratio of GSH/NPSH was unchanged in comparison to chicks not fed Pb. The data suggest that the initial response to acute Pb intoxication may involve a mobilization of nonprotein thiols via the interorgan translocation system for GSH. Such a response would help to maintain adequate levels of GSH in organs crucial to detoxification.     

Oxidative DNA damage in cultured fibroblasts from patients with hereditary glutathione synthetase deficiency

The SH compound glutathione (GSH) is involved in several fundamental functions in the cell, including protection against reactive oxygen species (ROS). Here, we studied the effect on oxidative DNA damage in cultured skin fibroblasts from patients with hereditary GSH synthetase deficiency. Our hypothesis was that GSH-deficient cells are more prone to DNA damage than control cells. Single cell gel electrophoresis (the comet assay) in combination with the formamidopyrimidine DNA glycosylase enzyme, which recognizes oxidative base modifications, was used on cultured fibroblasts from 11 patients with GSH synthetase deficiency and five control subjects. Contrary to this hypothesis, we found no significant difference in background levels of DNA damage between cells from patients and control subjects. To study the induction of oxidative DNA damage without simultaneous DNA repair, the cells were γ-irradiated on ice and DNA single-strand breaks measured. The patient and control cells were equally sensitive to induction of single strand breaks by γ-irradiation. Therefore, factors other than GSH protect DNA from oxidative damage. However, cells with a high background level of oxidative DNA damage were found to be more sensitive to ionizing radiation. This suggests that differences in background levels of oxidative DNA damage may depend on the cells' intrinsic protection against induction of oxidative damage.     

Nutritional biochemistry of cellular glutathione

Glutathione (GSH) has emerged to be one of the most fascinating endogenous molecules virtually present in all animal cells often in quite high (mM) concentrations. In addition to the detoxicant, antioxidant, and cysteine-reservoir functions of cellular glutathione, the potential of this ubiquitous thiol to modulate cellular signal transduction processes has been recently evident. Lowered tissue GSH levels have been observed in several disease conditions. Restoration of cell GSH levels in a number of these conditions have proven to be beneficial. Thus, strategies to boost cell glutathione level are of marked therapeutic significance. Availability of cysteine, a precursor for glutathione synthesis, inside the cell is a critical determinant of cellular glutathione level. N-acetylcysteine and -lipoic acid are two pro-glutathione agents that have remarkable clinical potential. The ability of these two clinical drugs to enhance cellular glutathione level, coupled with their favorable effect on the molecular biology of HIV infection may make them useful tools for AIDS treatment.     

Nuclear glutathione

Glutathione (GSH) is a linchpin of cellular defences in plants and animals with physiologically-important roles in the protection of cells from biotic and abiotic stresses. Moreover, glutathione participates in numerous metabolic and cell signalling processes including protein synthesis and amino acid transport, DNA repair and the control of cell division and cell suicide programmes. While it is has long been appreciated that cellular glutathione homeostasis is regulated by factors such as synthesis, degradation, transport, and redox turnover, relatively little attention has been paid to the influence of the intracellular partitioning on glutathione and its implications for the regulation of cell functions and signalling. We focus here on the functions of glutathione in the nucleus, particularly in relation to physiological processes such as the cell cycle and cell death. The sequestration of GSH in the nucleus of proliferating animal and plant cells suggests that common redox mechanisms exist for DNA regulation in G1 and mitosis in all eukaryotes. We propose that glutathione acts as “redox sensor” at the onset of DNA synthesis with roles in maintaining the nuclear architecture by providing the appropriate redox environment for the DNA replication and safeguarding DNA integrity. In addition, nuclear GSH may be involved in epigenetic phenomena and in the control of nuclear protein degradation by nuclear proteasome. Moreover, by increasing the nuclear GSH pool and reducing disulfide bonds on nuclear proteins at the onset of cell proliferation, an appropriate redox environment is generated for the stimulation of chromatin decompaction. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Effect of glutathione depletion on the cytotoxicity of xenobiotics and induction of single-strand DNA breaks by ionizing radiation in isolated hamster round spermatids

The role of glutathione (GSH) in cellular protection mechanisms in round spermatids from hamsters was studied. Isolated spermatids were largely depleted of GSH by treating the cells for 2 h with the GSH conjugating agent diethyl maleate (DEM). This treatment resulted in a 90% decrease of the cellular GSH content, but did not affect the ATP content. Exposure of isolated spermatids to cumene hydroperoxide (CHP), a compound which is detoxicated by the GSH redox cycle, showed that the cytotoxicity of the peroxide was markedly potentiated by GSH depletion of the cells. The cytotoxicity was reflected by the cellular ATP content. A decrease of the ATP content of the GSH-depleted spermatids was observed at 5-6-fold lower CHP concentrations, as compared to control cells. An increased cytotoxicity in GSH-depleted cells was also observed using 1-chloro-2,4-dinitrobenzene (CDNB), which is a reactive compound that is detoxicated by glutathione conjugation. The induction of single-strand DNA breaks by gamma radiation was 3-5-fold higher in GSH-depleted spermatids as compared to control cells. This radiation-induced damage was estimated under hypoxic conditions (500 p.p.m. O2 in N2). GSH depletion did not affect the repair of single-strand DNA breaks following the irradiation. The present results indicate that cellular GSH has an important function in the defence mechanisms of round spermatids against peroxides, electrophilic xenobiotics and radiation-induced DNA damage.     

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.     

Transcriptional regulation of glutathione synthetase in the fission yeast Schizosaccharomyces pombe

Glutathione (GSH), an important antioxidant involved in the stress response, is synthesized in two sequential reactions involving glutamylcysteine synthetase (GCS), followed by glutathione synthetase (GS). Expression of the unique GS gene in the fission yeast Schizosaccharomyces pombe was previously found to be regulated by nitric oxide and by L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of GCS. In this work, expression of S. pombe GS gene is shown to be induced by menadione (MD), which generates superoxide. The responsible DNA sequence between -365 and -234 bp from the translation start site, was convinced using five GS-lacZ fusion plasmids. Expression of GS gene is also induced by low glucose, fructose and disaccharides, apparently dependent on Pap1 protein; GS mRNA increases in low concentrations of glucose in wild type S. pombe but not in Pap1-negative cells. Although nonfermentable carbon sources such as acetate and ethanol stimulate expression of GS gene, they also arrest the growth of the yeast cells. These results indicate that the biosynthesis of glutathione is regulated by superoxide radicals and carbon source limitation.