The degree of pigmentation modulates the radiosensitivity of human melanoma cells

The relationship between cell pigmentation and radiosensitivity was investigated in two selected human melanoma cell lines with different melanin content (mixed type: eumelanin and pheomelanin, and pheomelanotic phenotypes). The same study was also done after stimulation of melanogenesis (1) by addition of the melanin precursor l-tyrosine to each of the cell lines separately and (2) by irradiation alone with doses ranging from 0 to 10 Gy. We found that a decrease in cell radiosensitivity was correlated with the type of melanin, with a clear involvement of eumelanin rather than pheomelanin. Increasing the intracellular content of both melanins promoted the growth of irradiated cells. Moreover, at a dose of 10 Gy, both tyrosinase activity and melanin cell content were significantly increased in the absence of any other melanogenesis promoter. Our data suggest that the amount of intracellular melanin is inversely related to the radiosensitivity of melanoma cells and may explain at least in part the controversial responses to ionizing radiations reported for melanoma.     

Up-regulation of P-glycoprotein expression by glutathione depletion-induced oxidative stress in rat brain microvessel endothelial cells

Glutathione (GSH) depletion has been implicated in the pathogenesis of neurological diseases. During GSH depletion, cells of the blood-brain barrier (BBB) are subjected to chronic oxidative stress. In this study, we investigated the effect of such stress, produced with the GSH synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO), on expression of P-glycoprotein (Pgp) in primary cultured rat brain microvessel endothelial cells that comprise the blood-brain barrier (BBB). Application of BSO to cell monolayers at concentrations up to 800 microm caused increases in expression of Pgp. Concentrations >or= 400 microm BSO decreased cell viability. Application of 200 microm BSO caused a significant increase in Pgp function activity, as assessed by rhodamine 123 (Rh123) accumulation experiments. At this concentration, BSO produced time-dependent decreases in levels of intracellular GSH and increases in levels of intracellular reactive oxygen species (iROS). The increases were also observed within 48 h following BSO treatment in mdr1a and mdr1b mRNA. Exposure of cells to BSO for 24 h produced maximal effects in the accumulation of iROS, and in expression and function of Pgp. The ROS scavenger N-acetylcysteine prevented ROS generation and attenuated the changes of both expression and activity of Pgp induced by BSO. Therefore, the transport of Pgp substrates may be affected by changing Pgp expression under conditions of chronic oxidative stress induced by GSH depletion.     

Differential effects of glutathione depletion on T cell subsets

Glutathione (GSH) is known to play an important role in various lymphocyte functions. We now report that different T cell subsets express different requirements for intracellular GSH. Depletion of intracellular GSH by buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis, decreases the proportion of CD8+ cells (i.e., increases the CD4+/CD8+ ratio), and inhibits particularly the generation of large blast-like CD8+ cells and cytotoxic T lymphocyte (CTL) activity. CTL activity is restored by administration of exogenous GSH. Differential effects of GSH depletion were also seen at the level of individual T cell clones. The CD4+ helper T cell clone D10.G4.1.HD was found to express a high rate of interleukin 2 (IL-2) dependent DNA synthesis even after severe depletion of intracellular GSH, whereas other T cell clones including the clone 29 were severely inhibited by BSO. The results of these studies suggest that the decreased intracellular GSH levels of HIV-1 seropositive persons are probably not (directly) responsible for the selective depletion of the CD4+ T cell subset but may be responsible for a cellular dysfunction of the CD8+ subset and for the ultimate failure of the CTL to control the viral infection in these patients.     

Buthionine sulfoximine induced growth inhibition in human lung carcinoma cells does not correlate with glutathione depletion

Treatment of A549 human lung carcinoma cells with L-buthionine-[S,R]-sulfoximine (BSO) results concomitantly in cellular glutathione (GSH) depletion and growth inhibition. The nature of BSO effects on cell growth and the relationships between BSO inhibition of cell growth and BSO effects on cellular GSH levels were determined in this study. A dose dependent effect of BSO on cell growth was observed, but this effect was found not to correlate with BSO effects on cellular GSH levels. Treatment with BSO for 60 h at concentrations of 5 and 10 mM was found to deplete cellular GSH at similar rates and to an undetectable level (below 0.5 nmol/mg protein). However, cessation of growth occured in 10 mM BSO whereas growth continued at better than one half the control rate in 5 mM BSO. The results suggest there may be a distinct threshold level of intracellular G GSH (on the order of or less than 0.5 nmol/mg protein) required for cell growth and for cells to protect themselves from the antiproliferative effects of BSO. At a concentration of 10 mM, BSO inhibited both DNA and protein synthesis and arrested growth of A549 cells throughout rather than at a specific phase of the cell cycle. BSO inhibition of growth was not, as indicated by colony-forming efficiency (CFE) and electron microscopy studies, accompanied by indications of cytotoxic effects. A stimulatory effect of 0.1 mM BSO on the growth of A549 cells was found also.Abbreviations BSO L-buthionine-[S,R]-sulfoximine - GSH Glutathione (reduced form) - GSSG Glutathione disulfide - DTNB 5,5-dithiobis (2-nitrobenzoate) - PBS Phosphate buffered saline - BSA Bovine serum albumin - PI Propidium iodide - CFE Colony-forming efficiency - EM Electron microscopy     

Co-regulation of melanin precursors and tyrosinase in human pigment cells: roles of cysteine and glutathione.

Glutathione (GSH) and cysteine (CysH) have both been implicated in the biogenesis of the pheomelanin precursor 5-S-cysteinyldopa (5-S-CD). However, recent studies have shown that only CysH is transported across the membrane of isolated melanosomes, and that the positive regulation of CysH in pigment cells leads to an increased production of 5-S-CD. In the present study, the question was examined as to whether melanin precursors and tyrosinase could be coregulated by cellular thiols. To address this issue, the levels of CysH and GSH were varied in normal melanocytes and melanoma cells using buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis. Treatment with 50-100 microM BSO decreased GSH levels to less than 10% of control, and increased CysH levels between two- and five-fold in both cell types. Concomitant with this, an increase in the ratio of 5-S-CD to DOPA and a decrease in the pigment content of the cells were observed. The decrease in cell pigmentation was associated with strong decreases in tyrosine hydroxylase activity and 14C-melanin production. Only melanoma cells showed a modified tyrosinase isozyme pattern on Western immunoblots in response to BSO, while the mRNA expression of tyrosinase and TRP-1 were unchanged in both cell types. These results suggest that the balance between CysH and GSH, which is partly determined by the rate of utilization of CysH for GSH biosynthesis, regulates not only the levels of 5-S-CD and DOPA but also the melanogenic activity of pigment cells. Since DOPA functions as a cofactor in the monophenolase reaction of tyrosinase, it is proposed that the ratio of 5-S-CD to DOPA may be an important factor in the regulation of tyrosinase activity in situ.     

Buthionine sulfoximine diverts the melanogenesis pathway toward the production of more soluble and degradable pigments

Buthionine sulfoximine (BSO) is a specific inhibitor of γ-glutamylcysteine synthetase, thus blocking the synthesis of glutathione (GSH). It is known that this makes that BSO affects melanin synthesis because of the role of thiols in melanogenesis. However, BSO may also react with the intermediate oxidation products of melanogenesis, a possibility that has not been investigated from the initial steps of the pathway. We created in vitro conditions simulating eumelanogenesis (oxidation of l-DOPA in the absence of GSH) and pheomelanogenesis (oxidation of l-DOPA in the presence of GSH) under presence or absence of BSO. BSO made that eumelanogenesis results in pigments more soluble and less resistant to degradation by hydrogen peroxide than pigments obtained without BSO. A similar but less marked effect was observed for pheomelanogenesis only at subsaturating concentrations of GSH. These results suggest that BSO diverts the melanogenesis pathway toward the production of more soluble and degradable pigments.     

Depletion of glutathione after gamma irradiation modifies survival

The relationship between the intracellular glutathione (GSH) concentration and the aerobic radiation response was studied in Chinese hamster ovary cells. Various degrees of GSH depletion were produced by exposure to buthionine sulfoximine (BSO) and/or diethyl maleate (DEM). Diethyl maleate did not act as a classical radiosensitizer under the experimental conditions employed, nor did exposure to DEM/BSO nonspecifically affect protein thiols as measured by thiol blotting. Dose-response curves were obtained using cells irradiated in the absence or presence of DEM/BSO, which decreased GSH levels by 90-95%. Exposure to DEM/BSO did not affect the formation of DNA single-strand breaks or DNA-protein crosslinks measured immediately after irradiation performed at ice temperatures. Analysis of survival curves indicated that the Dq was decreased by 18% when GSH depletion occurred prior to, during, and after irradiation. The DEM/BSO exposure did not affect D0. To study postirradiation conditions, cells were exposed to 10 microM DEM prior to and during irradiation, which was performed at ice temperatures. Levels of GSH were depleted by 75% by this protocol. Immediately after irradiation, the cells were rapidly warmed by the addition of 37 degrees C growth medium containing either 10 or 90 microM DEM. Addition of 10 microM DEM after irradiation did not affect the degree of depletion, which remained constant at 75%. In contrast, GSH depletion was increased to 90% 10 min after addition of the 90 microM DEM. Addition of 90 microM DEM after irradiation produced a statistically significant difference in survival compared to addition of 10 microM DEM. In a second depletion protocol, cells were exposed to 100 microM DEM at room temperature for 5 min, irradiated, incubated at 37 degrees C for 1 h, washed, and then incubated in 50 microM BSO for 24 h. This depletion protocol reduced survival by a factor of 2.6 compared to cells not exposed to the combination of DEM/BSO. Survival was not affected if the cells were exposed to the DEM or BSO alone. This was interpreted to indicate that survival was not affected by GSH depletion occurring after irradiation unless depletion was rapid and sustained. The rate of repair of sublethal and potentially lethal damage was measured and found to be independent of the DEM/BSO exposure. These experimental results in addition to previous ones (Freeman and Meredith, Int. J. Radiat. Oncol. Biol. Phys. 13, 1371-1375, 1987) were interpreted to indicate that under aerobic conditions GSH depletion may alter the expression of radiation damage by affecting metabolic fixation.     

Intracellular glutathione is a cofactor in methylseleninic acid-induced apoptotic cell death of human hepatoma HEPG(2) cells

Selenium is a widely studied dietary anticancer agent. Among various selenium compounds, the methylated forms appear to be particularly effective in cancer prevention. Intracellular glutathione (GSH) is known to be involved in the metabolism of many methylated forms of selenium. In this study, we investigated the role of intracellular GSH in methylseleninic acid (MSeA)-induced apoptosis in human hepatoma (HepG(2)) cells. MSeA was shown to deplete intracellular GSH rapidly, preceding the typical apoptotic changes such as DNA fragmentation as measured by the TUNEL assay. When the intracellular GSH concentration was enhanced using N-acetylcysteiene (NAC) (a GSH synthesis precursor) and decreased using buthionine sufoxamine (BSO) (a GSH synthesis inhibitor), NAC markedly augmented MSeA-induced apoptosis, while BSO significantly inhibited MSeA-induced apoptosis. Different from the effect of sodium selenite, there was no measurable superoxide radical level in MSeA-treated cells. These observations suggest that intracellular GSH mainly acts as a cofactor to facilitate MSeA-induced apoptosis, while its antioxidant function becomes largely irrelevant. It is thus postulated that some cancer cells, such as liver cancer cells with higher level of intracellular GSH, would be more susceptible to MSeA cytotoxicity.     

Possible involvement of oxidative stress in trichloroethylene-induced genotoxicity in human HepG2 cells

Trichloroethylene (TCE) is an environmental and industrial pollutant whose hepatotoxicity has been demonstrated in experimental animals. However, the mechanisms of the effects, in particular those related to its genotoxicity in humans, are not well understood. The aim of this study was to assess the genotoxic effects of TCE and to identify and clarify the mechanisms, using human hepatoma HepG2 cells. Exposure of the cells to TCE caused significant increase of DNA migration in comet assay and of micronuclei (MN) frequencies at all tested concentrations (0.5-4mM), respectively, which suggests that TCE caused DNA strand breaks and chromosome damage. The involvement of lipid peroxidation in the genotoxic properties of TCE was confirmed by using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). To elucidate the role of glutathione (GSH) in these effects, the intracellular GSH level was modulated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and by co-treatment with N-acetylcysteine (NAC), a GSH precursor. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to TCE-induced cytotoxicity and DNA damage, while when the intracellular GSH content was elevated by NAC, the DNA damage induced by TCE was almost completely prevented. These results indicate that TCE exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress; GSH, as a main intracellular antioxidant, is responsible for cellular defense against TCE-induced DNA damage.     

Ebselen induces apoptosis in HepG(2) cells through rapid depletion of intracellular thiols

Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, is a synthetic seleno-organic compound with antioxidant capability. In the present study, we systematically examined the ability of ebselen to induce apoptosis in a human hepatoma cell line, HepG(2). Ebselen-induced apoptosis was evaluated by (i) TdT-mediated dUTP nick end labeling assay; (ii) analysis of sub-G1 cells; (iii) cell morphology, including cell size and granularity examination; and (iv) DNA gel electrophoresis. The results showed that ebselen was able to induce typical apoptosis in HepG(2) cells in a dose- and time-dependent manner. In order to explore the possible mechanisms involved in ebselen-induced apoptosis, the effect of ebselen on intracellular thiol concentrations including reduced glutathione (GSH) and protein thiols and the effect of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) pretreatment on ebselen-induced apoptosis were investigated. It was found that (i) ebselen rapidly depleted intracellular GSH and protein thiols, moreover, the depletion preceded the occurrence of apoptosis; (ii) NAC, a precursor of intracellular GSH synthesis, significantly alleviated ebselen-induced apoptosis; and (iii) BSO, a specific inhibitor of intracellular GSH synthesis, augmented ebselen-induced apoptosis significantly. Taken together, the present study demonstrates that ebselen is able to induce apoptosis in HepG(2) cells, most probably through rapid depletion of intracellular thiols.     

<Emphasis Type="Italic">ENPP1</Emphasis> 121Q functional variant enhances susceptibility to coronary artery disease in South Indian patients with type 2 diabetes mellitus

Resveratrol is a dietary polyphenol that displays neuroprotective properties in several in vivo and in vitro experimental models, by modulating oxidative and inflammatory responses. Glutathione (GSH) is a key antioxidant in the central nervous system (CNS) that modulates several cellular processes, and its depletion is associated with oxidative stress and inflammation. Therefore, this study sought to investigate the protective effects of resveratrol against GSH depletion pharmacologically induced by buthionine sulfoximine (BSO) in C6 astroglial cells, as well as its underlying cellular mechanisms. BSO exposure resulted in several detrimental effects, decreasing glutamate-cysteine ligase (GCL) activity, cystine uptake, GSH intracellular content and the activities of the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR). Moreover, BSO increased reactive oxygen/nitrogen species (ROS/RNS) levels and pro-inflammatory cytokine release. Resveratrol prevented these effects by protecting astroglial cells against BSO-induced cytotoxicity, by modulating oxidative and inflammatory responses. Additionally, we observed that pharmacological inhibition of heme oxygenase 1 (HO-1), an essential cellular defense against oxidative and inflammatory injuries, abolished all the protective effects of resveratrol. These observations suggest HO-1 pathway as a cellular effector in the mechanism by which resveratrol protects astroglial cells against GSH depletion, a condition that may be associated to neurodegenerative diseases.     

Metabolic requirements associated with GSH synthesis during in vitro maturation of cattle oocytes

Glutathione (GSH) concentration increases in bovine oocytes during in vitro maturation (IVM). The constitutive amino acids involved in GSH synthesis are glycine (Gly), glutamate (Glu) and cysteine (Cys). The present study was conducted to investigate the effect of the availability of glucose, Cys, Gly and Glu on GSH synthesis during IVM. The effect of the amino acid serine (Ser) on intracellular reduced/oxidized glutathione (GSH/GSSG) content in both oocytes and cumulus cells was also studied. Cumulus-oocyte complexes (COC) of cattle obtained from ovaries collected from an abattoir were matured in synthetic oviduct fluid (SOF) medium containing 8 mg/ml bovine serum albumin-fatty acid-free (BSA-FAF), 10 microg/ml LH, 1 microg/ml porcine FSH (pFSH) and 1 microg/ml 17 beta-estradiol (17beta-E2). GSH/GSSG content was measured using a double-beam spectrophotometer. The COC were cultured in SOF supplemented with 1.5mM or 5.6mM glucose (Exp. 1); with or without Cys+Glu+Gly (Exp. 2); with the omission of one constitutive GSH amino acid (Exp. 3); with 0.6mM Cys or Cys+Ser (Exp. 4). The developmental capacity of oocytes matured in IVM medium supplemented with Cys and the cell number per blastocyst were determined (Exp. 5). The results reported here indicate (1) no differences in the intracellular GSH/GSSG content at any glucose concentrations. Also, cumulus cell number per COC did not differ either before or after IVM (Exp. 1). (2) Glutathione content in oocytes matured in SOF alone were significantly different from oocytes incubated with SOF supplemented with Cys+Glu+Gly (Exp. 2). (3) Addition of Cys to maturation medium, either with or without Gly and Glu supplementation resulted in an increase of GSH/GSSG content. However, when Cys was omitted from the IVM medium intracellular GSH in oocytes or cumulus cells was less but not significantly altered compared to SOF alone (Exp. 3). (4) Glutathione content in both oocytes and cumulus cells was significantly reduced by incubation with 5mM Ser (Exp.4). (5) There was a significant increase in cleavage and blastocyst rates when Cys was added to maturation medium. In contrast, the cleavage, morula and blastocyst rates were significantly different when 5mM Ser was added to maturation media. There was also a significant difference in mean cell number per blastocyst, obtained from oocytes matured with 5mM Ser (Exp. 5). This study provides evidence that optimal embryo development in vitro is partially dependent on the presence of precursor amino acids for intracellular GSH production. Moreover, the availability of Cys might be a critical factor for GSH synthesis during IVM in cattle oocytes. Greater Ser concentration in IVM medium altered "normal" intracellular GSH in both oocytes and cumulus cells with negative consequences for subsequent developmental capacity.     

Cell cycle progression of glutathione-depleted human peripheral blood mononuclear cells is inhibited at S phase

Glutathione (GSH) the most abundant nonprotein thiol, is involved in the maintenance of the cellular redox state. In this capacity it may influence lymphocyte responsiveness to various stimuli. We have investigated the requirement of GSH during the activation and proliferation of PBMC. The intracellular GSH content of PBMC was altered by continuous culture or pretreatment with buthionine-S,R-sulfoximine (BSO), a specific and irreversible inhibitor of GSH synthesis. Initial experiments demonstrated that the addition of BSO at the initiation of culture, or shortly thereafter (6 hr), inhibited DNA synthesis and produced a simultaneous decrease in intracellular GSH. It was necessary that the BSO be present in the culture for at least 24 hr prior to the initiation of DNA synthesis for maximal inhibition. Cell cycle analysis revealed that BSO did not affect the entry and progression of PBMC through G1 of the cell cycle, however, entry into S-phase was inhibited in a dose-dependent fashion. These results were further substantiated by the inability of BSO to inhibit IL-2 production and expression of the IL-2R. In addition the timely expression of the transferrin receptor by BSO-treated cells indicated that the block occurred at the G1/S transition. The influence of GSH on early activation events was determined by BSO pretreatments. Lowering the intracellular GSH level of PBMC to less than 10% of the initial content prior to mitogenic stimulation did not impair the ability of these cells to produce IL-2 and express IL-2R, indicating that GSH may not be involved in the generation and response to early activation signals. Furthermore, the removal of BSO from these cultures rapidly reversed its inhibitory effects on DNA and GSH synthesis. In the course of these studies we also observed a modest (17%) albeit consistent increase during activation in the total thiol levels of GSH-depleted PBMC. These thiols may have a key role in the activation process. These data support our hypothesis that GSH is required for lymphocyte proliferation and that additional thiols are involved during the activation process.     

Glutathione modulates Ca(2+) influx and oxidative toxicity through TRPM2 channel in rat dorsal root ganglion neurons

Glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells and maintains thiol redox in the cells. GSH depletion has been implicated in the neurobiology of sensory neurons. Because the mechanisms that lead to melastatin-like transient receptor potential 2 (TRPM2) channel activation/inhibition in response to glutathione depletion and 2-aminoethyldiphenyl borinate (2-APB) administration are not understood, we tested the effects of 2-APB and GSH on oxidative stress and buthionine sulfoximine (BSO)-induced TRPM2 cation channel currents in dorsal root ganglion (DRG) neurons of rats. DRG neurons were freshly isolated from rats and the neurons were incubated for 24 h with BSO. In whole-cell patch clamp experiments, TRPM2 currents in the rat were consistently induced by H₂O₂ or BSO. TRPM2 channels current densities and cytosolic free Ca²⁺ content of the neurons were higher in BSO and H₂O₂ groups than in control. However, the current densities and cytosolic Ca²⁺ release were also higher in the BSO + H₂O₂ group than in the H₂O₂ alone. When intracellular GSH is introduced by pipette TRPM2 channel currents were not activated by BSO, H₂O₂ or rotenone. BSO and H₂O₂-induced Ca²⁺ gates were blocked by the 2-APB. Glutathione peroxidase activity, lipid peroxidation and GSH levels in the DRG neurons were also modulated by GSH and 2-APB inhibition. In conclusion, we observed the protective role of 2-APB and GSH on Ca²⁺ influx through a TRPM2 channel in intracellular GSH depleted DRG neurons. Since cytosolic glutathione depletion is a common feature of neuropathic pain and diseases of sensory neuron, our findings are relevant to the etiology of neuropathology in DRG neurons.     

Melanosomal pH controls rate of melanogenesis, eumelanin/phaeomelanin ratio and melanosome maturation in melanocytes and melanoma cells.

The skin pigment melanin is produced in melanocytes in highly specialized organelles known as melanosomes. Melanosomes are related to the organelles of the endosomal/lysosomal pathway and can have a low internal pH. In the present study we have shown that melanin synthesis in human pigment cell lysates is maximal at pH 6.8. We therefore investigated the role of intramelanosomal pH as a possible control mechanism for melanogenesis. To do this we examined the effect of neutralizing melanosomal pH on tyrosinase activity and melanogenesis in 11 human melanocyte cultures and in 3 melanoma lines. All melanocyte cultures (9 of 9) from Caucasian skin as well as two melanoma cell lines with comparable melanogenic activity showed rapid (within 24 h) increases in melanogenesis in response to neutralization of melanosomal pH. Chemical analysis of total melanin indicated a preferential increase in eumelanin production. Electron microscopy revealed an accumulation of melanin and increased maturation of melanosomes in response to pH neutralization. In summary, our findings show that: (i) near neutral melanosomal pH is optimal for human tyrosinase activity and melanogenesis; (ii) melanin production in Caucasian melanocytes is suppressed by low melanosomal pH; (iii) the ratio of eumelanin/phaeomelanin production and maturation rate of melanosomes can be regulated by melanosomal pH. We conclude that melanosomal pH is an essential factor which regulates multiple stages of melanin production. Furthermore, since we have recently identified that pink locus product (P protein) mediates neutralization of melanosomal pH, we propose that P protein is a key control point for skin pigmentation. We would further propose that the wide variations in both constitutive and facultative skin pigmentation seen in the human population could be associated with the high degree of P-locus polymorphism.     

Toxic effects of acute glutathione depletion by buthionine sulfoximine and dimethylfumarate on murine mammary carcinoma cells

Glutathione (GSH) depletion to approximately equal to 5% of control for 48 h or longer by 0.05 mM L-buthionine sulfoximine (BSO) led to appreciable toxicity for the 66 murine mammary carcinoma cells growing in vitro [L.A. Dethlefsen et al., Int. J. Radiat. Oncol. Biol. Phys. 12, 1157-1160 (1986)]. Such toxicity in normal, proliferating cells in vivo would be undesirable. Thus the toxic effects after acute GSH depletion to approximately equal to 5% of control by BSO plus dimethylfumarate (DMF) were evaluated in these same 66 cells to determine if this anti-proliferative effect could be minimized. Two hours of 0.025 mM DMF reduced GSH to 45% of control, while 6 h of 0.05 mM BSO reduced it to 16%. However, BSO (6 h) plus DMF (2 h) and BSO (24 h) plus DMF (2 h) reduced GSH to 4 and 2%, respectively. The incorporation (15-min pulses) of radioactive precursors into protein and RNA were unaffected by these treatment protocols. In contrast, cell growth was only modestly affected, but the incorporation of [3H]thymidine into DNA was reduced to 64% of control by the BSO (24 h) plus DMF (2 h) protocol even though it was unaffected by the BSO (6 h) plus DMF (2 h) treatment. The cellular plating efficiencies from both protocols were reduced to approximately equal to 75% of control cells. However, the aerobic radiation response, as measured by cell survival, was not modified at doses of either 4.0 or 8.0 Gy. The growth rates of treated cultures, after drug removal, quickly returned to control rates and the resynthesis of GSH in cells from both protocols was also rapid. The GSH levels after either protocol were slightly above control by 12 h after drug removal, dramatically over control (approximately equal to 200%) by 24 h, and back to normal by 48 h. Thus even a relatively short treatment with BSO and DMF resulting in a GSH depletion to 2-5% of control had a marked effect on DNA synthesis and plating efficiency and a modest effect on cellular growth. One cannot rule out a direct effect of the drugs, but presumably the antiproliferative effects are due to a depletion of nuclear GSH with the subsequent inhibition of the GSH/glutaredoxin-mediated conversion of ribonucleotides to deoxyribonucleotides. However, even after extended treatment, upon drug removal, GSH was rapidly resynthesized and cellular DNA synthesis and growth quickly resumed.     

The role of thiol reduction in hydroquinone-induced apoptosis in HEK293 cells

Hydroquinone (HQ) is a chemical used as a reducing agent, antioxidant, polymerization inhibitor, and chemical intermediate. It has a minor use as a bleaching agent in dermatologic preparations. HQ also occurs as a main metabolite of benzene. In the present study, HQ-induced apoptosis was evaluated by cell morphology changes, determination of phosphatidylserine (PS) externalization and analysis of sub-G1 cells. The effect of HQ on intracellular thiol concentration, including glutathione and protein thiol, and the effect of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) pretreatment on HQ-induced apoptosis were investigated. The results showed that HQ was able to induce typical apoptosis in HEK293 cells (human embryonic kidney cells) in a dose-dependent manner. Intracellular thiol, including glutathione and protein thiol, was decreased following treatment with HQ. NAC, a precursor of intracellular GSH synthesis, significantly inhibited HQ-induced apoptosis. However, BSO, a specific inhibitor of intracellular GSH synthesis, enhanced HQ-induced apoptosis significantly. Taken together, the present study demonstrates that HQ is able to induce apoptosis in HEK293 cells, most probably through depletion of intracellular thiol. The results also suggest that, at least in HEK293 cells, the control of intracellular redox homeostasis has a central role in the regulation of cell death induced by HQ.     

Glutathione modulates lipid composition of human colon derived HT-29 cells

Glutathione (GSH) is important in maintaining intracellular thiol status. The present study looked at the effect of GSH depletion on lipid composition of colon-derived HT-29 cells. GSH was depleted in HT-29 cells by incubation either with buthionine-S, R-sulfoximine (BSO) or diethylmaleate (DEM). GSH was restored during early periods of cell growth by supplementation of growth medium with either GSH ester or N-acetyl cysteine (NAC). Lipids were analysed following GSH depletion and supplementation. Among the neutral lipids, an increase in free cholesterol and diacylglycerol and decrease in cholesteryl ester and triacylglycerol were seen in GSH-depleted cells as compared to control cells. There were no detectable free fatty acids either in control or GSH-depleted cells. Among the phospholipids, a decrease in phosphatidylcholine and phosphatidylinositol and an increase in phosphatidylethanolamine were observed. These changes were almost completely reversed by supplementation of BSO-treated cells with GSH ester and partially reversed by N-acetyl cysteine. These results suggest that the GSH status of the cell plays an important role in the lipid composition of the cells.     

Altered differentiation in rat and rabbit limb bud micromass cultures by glutathione modulating agents

Glutathione (GSH) is the primary source of reducing equivalents in most cells, contributes significantly to the cellular redox potential and can control differentiation, proliferation, and apoptosis. Using limb bud micromass cultures from Sprague Dawley rats and New Zealand White rabbits, GSH modulating agents, L-buthionine-S,R-sulfoximine (BSO) and diethyl maleate (DEM) altered the formation of Alcian blue positive chondrogenic foci. Limb bud micromass cultures were treated for 5 d with BSO (50 or 100 μM) or DEM (5–25 μM). GSH content was determined by HPLC analysis. In rat cultures, BSO treatment did not affect differentiation but did show GSH depletion. In rabbit cultures, BSO completely inhibited differentiation and significantly depleted GSH. Treatment of rat cultures with DEM resulted in the dose-dependent decrease of chondrogenic foci, which correlated with a dose-dependent depletion of GSH. DEM completely inhibited rabbit limb bud cell differentiation and depleted GSH by 44%. Inhibition of differentiation was confirmed in rabbit cultures by the reduction in BMP-4 content. Addition of N-acetylcysteine to rabbit micromass cultures restored chondrogenic foci differentiation seen following treatment with both DEM and BSO. These results show species differences in GSH depletion in rat vs. rabbit limb bud cells and implicate GSH and cysteine in affecting pathways involved in chondrocyte differentiation.     

Glutathione content and growth in A549 human lung carcinoma cells

The relationship between glutathione content and cell growth was investigated in A549 human lung carcinoma cells. A decreased cellular glutathione content was achieved by exposing the cells to L-buthionine-SR-sulfoximine (BSO). It also occurred in these cells as they approached their plateau phase of growth. During exponential growth, a lower initial glutathione content correlated with a longer lag phase in subcultured cells. Further, depletion of cellular glutathione by BSO inhibited cell growth. This inhibition became apparent 36 h after the addition of BSO. These observations raise the possibility that a critical concentration of GSH may be required for optimal growth of A549 human lung carcinoma cells.