A nested case-control study of untargeted albumin adductomics and acute myeloid leukemia.

Environmental exposures often produce reactive electrophiles in vivo, leading to oxidative stress, which plays a major role in carcinogenesis. These electrophiles frequently form adducts with human albumin, which can be measured to assess in vivo oxidative stress. Here, we aimed to examine the associations between circulatory albumin adducts and acute myeloid leukemia (AML), the most common adult myeloid leukemia that showed consistent associations with environmental exposures. We conducted a nested case-control study of 52 incident AML cases and 103 controls matched on age, sex and race within two prospective cohorts: the CLUE and PLCO studies. We measured 42 untargeted albumin adducts in prediagnostic samples using liquid chromatography-high-resolution mass spectrometry. Circulatory albumin adducts were associated with AML in conditional logistic regression models. For instance, higher levels of Cys34 disulfide adduct of the S-γ-glutamylcysteine, a precursor of the essential antioxidant, glutathione were associated with a lower risk of AML (odds ratios [95% confidence intervals]) for the 1st, 2nd and 3rd tertiles were 1.0, 0.65 (0.31-1.36) and 0.31 (0.12-0.80), respectively (P-trend = .01). These associations were largely driven by effects present among cases diagnosed at or above the median follow-up time of 5.5 years. In conclusion, applying a novel approach to characterize exposures in the prediagnostic samples, we found evidence supporting the notion that oxidative stress may play a role in the pathogenesis of AML. Our findings offer insight into AML etiology and may be relevant in identifying novel therapeutic targets.

γ-Glutamylcysteine rescues mice from TNBS-driven inflammatory bowel disease through regulating macrophages polarization.

OBJECTIVE: To explore the molecular mechanism of γ-glutamylcysteine (γ-GC) in response to inflammation in vivo and in vitro on regulating the polarization of macrophages. METHODS: The expressions of gene or protein were assessed by qPCR and Western blot assays, respectively. Cell viability was investigated by CCK-8 assay. Eight-week-old male BALB/c mice were established to examine the therapeutic effects of γ-GC in vivo. The release of TNF-α and IL-4 was determined by ELISA assay. Macrophages polarization was identified by flow cytometry assay. RESULTS: Our data showed that γ-GC treatment significantly improved the survival, weight loss, and colon tissue damage of IBD mice. Furthermore, we established M1- and M2-polarized macrophages, respectively, and our findings provided evidence that γ-GC switched M1/M2-polarized macrophages through activating AMPK/SIRT1 axis and inhibiting inflammation-related signaling pathway. CONCLUSION: Collectively, both in vivo and in vitro experiments suggested that γ-GC has the potential to become a promising novel therapeutic dipeptide for the treatment of IBD, which provide new ideas for the treatment of inflammatory diseases in the future.

Characterization of GshAB of Tetragenococcus halophilus: a two-domain glutathione synthetase.

The γ-glutamyl tripeptide glutathione (γ-Glu-Cys-Gly) is a low molecular thiol that acts as antioxidant in response to oxidative stress in eukaryotes and prokaryotes. γ-Glutamyl dipeptides including γ-Glu-Cys, γ-Glu-Glu, and γ-Glu-Gly also have kokumi activity. Glutathione is synthesized by first ligating Glu with Cys by γ-glutamylcysteine ligase (Gcl/GshA), and then the resulting dipeptide γ-glutamylcysteine is ligated with Gly by glutathione synthetase (Gs/GshB). GshAB/GshF enzymes that contain both Gcl and Gs domains are capable of catalyzing both reactions. The current study aimed to characterize GshAB from Tetragenococcus halophilus after heterologous expression in Escherichia coli. The optimal conditions for GshAB from T. halophilus were pH 8.0 and 25 °C. The substrate specificity of the Gcl reaction of GshAB was also determined. GshAB has a high affinity to Cys. γ-Glu-Cys was the only dipeptide generated when Glu, Cys, Gly, and other amino acids were present in the reaction system. This specificity differentiates GshAB from T. halophilus from Gcl of heterofermentative lactobacilli and GshAB of Streptococcus agalactiae, which also use amino acids other than Cys as glutamyl-acceptor. Quantification of gshAB in cDNA libraries from T. halophilus revealed that gshAB was overexpressed in response to oxidative stress but not in response to acid, osmotic, or cold stress. In conclusion, GshAB in T. halophilus served as part of the oxidative stress response but this study did not provide any evidence for a contribution to the resistance to other stressors.Key points Glutathione synthesis in Tetragenococcus halophilus is carried out by the two-domain enzyme GshAB. GshAB is inhibited by glutathione and is highly specific for Cys as acceptor. T. halophilus synthesizes glutathione in response to oxidative stress.

Assay of the enzymes of glutathione biosynthesis.

This article is dedicated to the late long-time Editor-in-Chief of Analytical Biochemistry, William Jakoby. As a graduate student, I remember reading many articles in Analytical Biochemistry and Methods in Enzymology, both volumes that Bill edited. I first met him as a graduate student presenting at the American Society of Biochemistry (and Molecular Biology) meetings. My Ph.D. advisor, Alton Meister, would bring over well-known biochemists and introduce me as Dr. Anderson, leaving me a bit tongue-tied being that I was still actually a humble graduate student! I next met Bill at my first Analytical Biochemistry Executive Editors meeting in San Diego when he was Editor-in-Chief Emeritus; I felt honored to be on the same board with him and serving the journal to which he had brought to prominence. His eyes were piercing and he was so sharp; his knowledge was both broad and deep. Since much of the large body of Bill's research was on glutathione S-transferases, my article focuses on the assay of the enzymes that synthesize glutathione, a substrate for glutathione S-transferases.

Protective effect of γ-glutamylcysteine against UVB radiation in NIH-3T3 cells.

BACKGROUND: Ultraviolet (UV) radiation-induced oxidative stress is the main cause of photodamage to the skin. Glutathione (GSH) serves important physiological functions, including scavenging oxygen-free radicals and maintaining intracellular redox balance. γ-glutamylcysteine (γ-GC), as an immediate precursor of GSH and harboring antioxidant and anti-inflammatory properties, represents an unexplored option for skin photodamage treatment. PURPOSE: The purpose of this study was to investigate whether γ-GC can reduce UVB-induced NIH-3T3 cell damage. METHODS: The experimental groups were as follows: control, UVB radiation, UVB radiation after pretreatment with γ-GC. Cell counting kit-8 (CCK-8) assays were used to measure cell proliferation, flow cytometry, and immunoblotting to detect the apoptosis rate and apoptosis-associated proteins. The levels of Reactive Oxygen Species (ROS), Superoxide Dismutase (SOD), and GSH/GSSG (oxidized GSH) were measured to assess oxidative stress. Immunoblotting and immunofluorescence were used to detect DNA damage. The members of the MAPK signaling pathways were detected by immunoblotting. RESULTS: UVB irradiation significantly reduced cell viability and destroyed the oxidative defense system. Pretreatment with γ-GC reduced UVB-induced cytotoxicity, restored the oxidation defense system, and inhibited activation of the MAPK pathway. It also reduced the apoptosis rate, downregulated the levels of cleaved caspase 3 and cleaved PARP. Furthermore, pretreatment with γ-GC reduced the accumulation of γH2AX after UVB radiation exposure, indicating that γ-GC could protect cells from DNA damage. CONCLUSION: γ-GC protected NIH-3T3 from damage caused by UVB irradiation. The photoprotective effect of γ-GC is mediated via strengthening the endogenous antioxidant defense system, which prevents DNA damage and inhibits the activation of the MAPK pathway.

The Isothiocyanate Sulforaphane Depends on the Nrf2/γ-GCL/GSH Axis to Prevent Mitochondrial Dysfunction in Cells Exposed to Methylglyoxal.

Methylglyoxal (MG) is a reactive dicarbonyl presenting both endogenous (e.g. glycolysis) and exogenous (e.g. food cooking) sources. MG induces neurotoxicity, at least in part, by affecting mitochondrial function, including a decline in the oxidative phosphorylation (OXPHOS) system activity, bioenergetics failure, and redox disturbances. Sulforaphane (SFN) is an isothiocyanate found mainly in cruciferous vegetables and exerts antioxidant and anti-inflammatory effects in mammalian cells. SFN also decreases mitochondrial vulnerability to several chemical stressors. SFN is a potent activator of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which is a master regulator of the mammalian redox biology. Here, we have investigated whether and how SFN would be able to prevent the MG-induced mitochondrial collapse in the human neuroblastoma SH-SY5Y cells. The cells were exposed to SFN at 5 µM for 24 h prior to the administration of MG at 500 µM for additional 24 h. We found that SFN prevented the MG-induced OXPHOS dysfunction and mitochondrial redox impairment. SFN stimulated the activity of the enzyme γ-glutamylcysteine ligase (γ-GCL), leading to increased synthesis of glutathione (GSH). Inhibition of γ-GCL with buthionine sulfoximine (BSO) or silencing of Nrf2 using small interfering RNA (siRNA) against this transcription factor reduced the levels of GSH and abolished the mitochondrial protection promoted by SFN in the MG-treated cells. Thus, SFN protected mitochondria of the MG-challenged cells by a mechanism involving the Nrf2/γ-GCL/GSH axis.

Effects of trypanocidal drugs on DNA synthesis: new insights into melarsoprol growth inhibition.

Trypanothione is the primary thiol redox carrier in Trypanosomatids whose biosynthesis and utilization pathways contain unique enzymes that include suitable drug targets against the human parasites in this family. Overexpression of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GSH1), can increase the intracellular concentration of trypanothione. Melarsoprol directly inhibits trypanothione and has predicted the effects on downstream redox biology, including ROS management and dNTP synthesis that require further investigation. Thus, we hypothesized that melarsoprol treatment would inhibit DNA synthesis, which was tested using BrdU incorporation assays and cell cycle analyses. In addition, we analysed the effects of eflornithine, which interfaces with the trypanothione pathway, fexinidazole, because of the predicted effects on DNA synthesis, and pentamidine as an experimental control. We found that melarsoprol treatment resulted in a cell cycle stall and a complete inhibition of DNA synthesis within 24 h, which were alleviated by GSH1 overexpression. In contrast, the other drugs analysed had more subtle effects on DNA synthesis that were not significantly altered by GSH1 expression. Together these findings implicate DNA synthesis as a therapeutic target that warrants further investigation in the development of antitrypanosomal drugs.

Optimization of Reaction Conditions for γ-Glutamylcysteine Production from Glutathione Using a Phytochelatin Synthase-Like Enzyme from Nostoc sp. Pasteur Culture Collection 7120.

γ-Glutamylcysteine (γ-EC) has antioxidant properties similar to those of glutathione (GSH) and acts as its precursor in mammals. There are a few procedures for the production of γ-EC, such as chemical synthesis or enzymatic synthesis from glutamate and cysteine; however, they are very costly and not suitable for industrial production. A phytochelatin synthase-like enzyme derived from Nostoc sp. Pasteur Culture Collection 7120 (NsPCS) catalyzes the hydrolysis of GSH to γ-EC and glycine in the absence of ATP or other additives. Our research aims to establish an alternative γ-EC production procedure with low cost and high productivity. To this end, we optimized the reaction conditions of NsPCS and characterized its properties in this study. We found that 200 mM potassium phosphate buffer, pH 8.0, at 37 °C, had the highest NsPCS activity among the conditions we tested. Under these conditions, NsPCS had a Km of 385 µM and a Vmax of 26 mol/min/mg-protein. In addition, NsPCS converted 100 mM GSH into γ-EC with high yields. These results suggest that the NsPCS reaction has great potential for the low-cost, industrial-scale production of γ-EC.

γ-Glutamylcysteine synthetase and γ-glutamyl transferase as differential enzymatic sources of γ-glutamylpeptides in mice.

Some γ-glutamylpeptides in blood plasma are putative biomarkers for pathological conditions of the liver. γ-Glutamyltransferase (GGT) and γ-glutamylcysteine synthetase (γ-GCS) are two such potential enzymes that are responsible for the production of γ-glutamylpeptides. GGT produces γ-glutamylpeptides by transferring the γ-glutamyl moiety from glutathione to an amino acid or a peptide. γ-GCS normally catalyzes the production of γ-glutamylcysteine from glutamate and cysteine in the glutathione-synthesizing reaction, but other amino acids can also serve as an acceptor of a γ-glutamyl group, thus resulting in the formation of a variety of γ-glutamylpeptides. Based on liquid chromatography-mass spectrometry analyses, we observed differences in the distribution of γ-glutamylpeptides between the liver and kidney and were able to measure the activities of γ-GCS as well as the GGT reactions by quantifying the resulting γ-glutamylpeptides. The enzymatic characterization of γ-GCS in liver homogenates indicated that several γ-glutamylpeptides including γ-glutamyltaurine are actually produced. Cys showed the lowest Km value (0.06 mM) while other amino acids had much higher Km values (ranging from 21 to 1800 mM). The moderate Km values for these amino acids suggest that they were not the preferred amino acids in this conversion but were utilized as acceptor substrates for the production of the corresponding γ-glutamylpeptides by the γ-GCS reaction under Cys-deficient conditions. Thus, the production of these γ-glutamylpeptides by γ-GCS is directly correlated with a low Cys content, suggesting that their measurement in blood plasma could be useful for predicting the presymptomatic disease state of the liver with a defect in GSH redox balance.

Development of a highly efficient and specific L-theanine synthase.

γ-Glutamylcysteine synthetase (γ-GCS) from Escherichia coli, which catalyzes the formation of L-glutamylcysteine from L-glutamic acid and L-cysteine, was engineered into an L-theanine synthase using L-glutamic acid and ethylamine as substrates. A high-throughput screening method using a 96-well plate was developed to evaluate the L-theanine synthesis reaction. Both site-saturation mutagenesis and random mutagenesis were applied. After three rounds of directed evolution, 13B6, the best-performing mutant enzyme, exhibited 14.6- and 17.0-fold improvements in L-theanine production and catalytic efficiency for ethylamine, respectively, compared with the wild-type enzyme. In addition, the specific activity of 13B6 for the original substrate, L-cysteine, decreased to approximately 14.6% of that of the wild-type enzyme. Thus, the γ-GCS enzyme was successfully switched to a specific L-theanine synthase by directed evolution. Furthermore, an ATP-regeneration system was introduced based on polyphosphate kinases catalyzing the transfer of phosphates from polyphosphate to ADP, thus lowering the level of ATP consumption and the cost of L-theanine synthesis. The final L-theanine production by mutant 13B6 reached 30.4 ± 0.3 g/L in 2 h, with a conversion rate of 87.1%, which has great potential for industrial applications.

Inhibition of the glutathione biosynthetic pathway increases phytochemical toxicity to Spodoptera litura and Nilaparvata lugens.

Phytochemicals are toxic to insects, but their insecticidal efficiencies are usually low compared to synthetic insecticides. Understanding the mechanism of insect adaptation to phytochemicals will provide guidance for increasing their efficacy. Reduced glutathione (GSH) is a scavenger of reactive oxygen species (ROS) induced by phytochemicals. However, in insects, the pathway of GSH biosynthesis in response to phytochemicals is unclear. We found that exposure to 0.5% indole-3-methanol (I3C), xanthotoxin, and rotenone (ROT) significantly retarded the growth of Spodoptera litura larvae. The oxidative stress in S. litura larvae exposed to phytochemicals was increased. The up-regulation of glutamate cysteine ligase but not glutathione reductase revealed that the de novo synthesis pathway is responsible for GSH synthesis in phytochemical-treated larvae. Treatment with the inhibitor (BSO) of γ-glutamylcysteine synthetase (gclc), a subunit of glutamate cysteine ligase, resulted in decreases of GSH levels and GST activities, increases of ROS levels in I3C-treated larvae, which finally caused midgut necrosis and larval death. Treatment with BSO or I3C alone did not cause larval death. The addition of GSH could partly reduce the influence of I3C and BSO on S. litura growth. Nilaparvata lugens gclc RNAi confirmed the result of BSO treatment in S. litura. N. lugens gclc RNAi significantly increased the mortality of ROT-sprayed N. lugens, in which ROS levels were significantly increased. All data indicate that gclc is involved in insect response to phytochemical treatment. Treatment with dsgclc will increase the insecticidal efficacy of plant-derived compounds.

The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins.

In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes.

Cadmium induced glutathione bioaccumulation mediated by γ-glutamylcysteine synthetase in ectomycorrhizal fungus Hebeloma cylindrosporum.

Ectomycorrhizal fungi hold a potential role in bioremediation of heavy metal polluted areas because of its metal accumulation and detoxification property. We investigated the cadmium (Cd) induced bioaccumulation of glutathione (GSH) mediated by γ-glutamylcysteine synthetase (γ-GCS) in the ectomycorrhizal fungus Hebeloma cylindrosporum. In H. cylindrosporum, a demand driven synthesis of GSH has been observed in response to Cd. The expression and enzyme activity of H. cylindrosporum γ-GCS (Hcγ-GCS) increased as a function of external Cd stress resulting in increased GSH production. The function of Hcγ-GCS in providing heavy metal tolerance to H. cylindrosporum was justified by complementing the gene in gsh1Δ mutant of Saccharomyces cerevisiae. The metal sensitive mutant gsh1Δ successfully restored its metal tolerance ability when transformed with Hcγ-GCS gene. Sequence analysis of Hcγ-GCS showed homology with most of the reported γ-GCS proteins from basidiomycetes family. The active site of the Hcγ-GCS protein is composed of amino acids that were found to be conserved not only in fungi, but also in plants and mammals. From these results, it was concluded that Hcγ-GCS plays an important role in bioaccumulation of GSH, which is a core component in the mycorrhizal defense system under Cd stress for Cd homeostasis and detoxification.

Shenduning Granule Attenuates Renal Injury from Oxidative Stress through the Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway.

BACKGROUND: Systemic oxidative stress has been reported to play a central role in the pathogenesis of kidney function decline. The nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is one of the important endogenous antioxidant stress pathways in cells. This study aims to investigate whether shenduning granule can ameliorate oxidative stress in kidney tissues by activating the Nrf2/ARE pathway, and explores the detailed underlying mechanism. METHODS: A total of 120 male Sprague-Dawley rats were randomly assigned to the sham-operated and operation groups. Rats in the operation group underwent 5/6 nephrectomy. Two weeks later, rats in the operation group were further randomly divided into 5 groups: model group, low-dose, medium-dose and high-dose shenduning granule groups, and losartan group. Rats in each group were given the same volume of corresponding liquid orally. Serum creatinine (SCr), blood urea nitrogen (BUN), 24-h urinary protein, malondialdehyde (MDA) and superoxide dismutase (SOD), Nrf2, heme oxygenase-1 (HO-1), and γ-glutamyl-cysteine synthetase (γ-GCS) were determined. RESULTS: Shenduning granule could markedly elevate HO-1, NRF2, γ-GCS and SOD (p < 0.05), and significantly decreased MDA, 24-h urinary protein, SCr and BUN in rats (p < 0.05). CONCLUSION: Shenduning granule can improve renal antioxidative stress activity in rats, exhibiting a renoprotective effect. The potential mechanism is likely exerted by the activation of the Nrf2/ARE pathway.

HNF1ß drives glutathione (GSH) synthesis underlying intrinsic carboplatin resistance of ovarian clear cell carcinoma (OCCC).

Chemoresistance to platinum-based antineoplastic agents is a consistent feature among ovarian carcinomas; however, whereas high-grade serous carcinoma (OSC) acquires resistance during chemotherapy, ovarian clear cell carcinoma (OCCC) is intrinsically resistant. The main objective of this study was to explore, in vitro and in vivo, if hepatocyte nuclear factor 1ß (HNF1ß) and glutaminolysis contribute for the resistance of OCCC to carboplatin through the intrinsically increased GSH bioavailability. To disclose the role of HNF1ß, experiments were also performed in an OSC cell line, which does not express HNF1ß. Metabolic profiles, GSH quantification, HNF1ß, and γ-glutamylcysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) expression, cell cycle, and death were assessed in ES2 cell line (OCCC) and OVCAR3 cell line (OSC); HNF1ß knockdown was performed in ES2 and murine model of subcutaneous and peritoneal OCCC tumors was established to test buthionine sulphoxamine (BSO), as a sensitizer to carboplatin. Glutaminolysis is activated in ES2 and OVCAR3, though ES2 exclusively synthesizes amino acids and GSH. ES2 cells are more resistant to carboplatin than OVCAR3 and the abrogation of GSH production by BSO sensitizes ES2 to carboplatin. HNF1ß regulates the expression of GCLC, but not GCLM, and consequently GSH production in ES2. In vivo, BSO prior to carboplatin reduces dramatically subcutaneous tumor size and GSH levels, as well as peritoneal dissemination. Our study discloses HNF1ß as the mediator of intrinsic OCCC chemoresistance and sheds a light to re-explore a cancer adjuvant therapeutic approach using BSO to overcome the lack of efficient therapy in OCCC.

Glutamate cysteine ligase and the age-related decline in cellular glutathione: The therapeutic potential of γ-glutamylcysteine.

A consistent underlying index of aging is a decline in the cellular levels of the tripeptide glutathione (GSH). GSH is an essential thiol antioxidant produced in the cytosol of all cells and plays a key role in protecting against oxidative stress by neutralising free radicals and reactive oxygen species (ROS). The decline in GSH has been associated with changes in the expression and activity of the rate-limiting enzyme glutamate cysteine ligase (GCL), which produces the intermediate dipeptide γ-glutamylcysteine (γ-GC). The molecular mechanisms that affect these age-related changes remain unclear due to the complexity of GCL regulation. Impairment of the transcriptional activity of Nrf2 has been demonstrated to contribute to GCL dysregulation in aged rats. However, considering the complex nature of GCL regulation, relatively little research has been conducted to investigate the age-associated post-transcriptional controls of the enzyme. Defining these unknown mechanisms may inform our understanding of the aetiology of many age-related diseases and assist in formulating appropriate therapeutic strategies. This review focuses on the suitability of treatment with exogenous γ-GC to raise GSH levels by circumventing the age-related dysregulation of the rate-limiting step of GSH, providing promise for future research for the treatment of chronic oxidative stress-related diseases.

Glutathione - From antioxidant to post-translational modifier.

Helmut Sies is one of the leading investigators in the multiple roles of glutathione (GSH) in biology. He has pioneered work on the role of GSH in preventing oxidative stress, in transport of GSSG, in protection of protein thiols from irreversible oxidation through mixed disulfide formation and demonstrated a role of protein glutathionylation in response to hormonal stimulation well before redox signaling became a major subject of investigation. Here I will describe the roles of GSH in several aspects of biology, the work of my laboratory in those findings, and how Helmut Sies work influenced our studies.

Dichlorodiphenyldichloroethylene exposure reduces r-GCS via suppressed Nrf2 in HepG2 cells.

p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), the major isomer of persistent 1,1-Bis(4-chlorophenyl)-2,2,2-trichloroethane metabolite, is highly associated with the risk of liver cancer. γ-glutamyl-cysteine synthetase (γ-GCS), which is the rate-limiting enzyme of glutathione (GSH) biosynthesis and an important scavenger of reactive oxygen species (ROS), is considered as a potential therapeutic target for many cancers. However, the association between the body burden of p,p'-DDE and γ-GCS has not been fully established. Here, we indicated that low doses of p,p'-DDE exposure promoted the proliferation and decreased γ-GCS activity of HepG2 cells in a dose- and time-dependent manner. In addition, p,p'-DDE elevated ROS content and attenuated glutathione peroxidase activity. This was accompanied with inhibitions of NF-E2-related factor 2 (Nrf2) at the mRNA and protein levels. ROS inhibitor supplement could significantly reverse these effects. Moreover, the addition of the proteasome inhibitor, MG132, strongly reversed the p,p'-DDE-reduced Nrf2 expression and γ-GCS activity. Consistently, GSH content was in line with the alteration of γ-GCS. Collectively, the results indicate that p,p'-DDE treatment downregulates γ-GCS activity in HepG2 cells by inducing ROS-mediated Nrf2 loss.

Glutathione is involved in physiological response of Candida utilis to acid stress.

Candida utilis often encounters an acid stress environment when hexose and pentose are metabolized to produce acidic bio-based materials. In order to reveal the physiological role of glutathione (GSH) in the response of cells of this industrial yeast to acid stress, an efficient GSH-producing strain of C. utilis CCTCC M 209298 and its mutants deficient in GSH biosynthesis, C. utilis Δgsh1 and Δgsh2, were used in this study. A long-term mild acid challenge (pH 3.5 for 6 h) and a short-term severe acid challenge (pH 1.5 for 2 h) were conducted at 18 h during batch culture of the yeast to generate acid stress conditions. Differences in the physiological performances among the three strains under acid stress were analyzed in terms of GSH biosynthesis and distribution; intracellular pH; activities of γ-glutamylcysteine synthetase, catalase, and superoxide dismutase; intracellular ATP level; and ATP/ADP ratio. The intracellular GSH content of the yeast was found to be correlated with changes in physiological data, and a higher intracellular GSH content led to greater relief of cells to the acid stress, suggesting that GSH may be involved in protecting C. utilis against acid stress. Results presented in this manuscript not only increase our understanding of the impact of GSH on the physiology of C. utilis but also help us to comprehend the mechanism underlying the response to acid stress of eukaryotic microorganisms.

α-Lipoic acid protects against the oxidative stress and cytotoxicity induced by cadmium in HepG2 cells through regenerating glutathione regulated by glutamate-cysteine ligase.

Alpha-lipoic acid (α-LA) is an important antioxidant that is capable of regenerating other antioxidants, such as glutathione (GSH). In the present study, we examined the protective effects of α-LA against the oxidative stress and cytotoxicity induced by cadmium in human hepatoma cell lines (HepG2) and investigated if the process was mediated through regenerating GSH. Our results showed that after exposure to 25 µM cadmium for 16 h, there was a significant decrease in the cell viability and glutathione levels and a significant increase in lipid peroxidation (p<0.01) compared with untreated cells. The presence of α-LA significantly attenuated cadmium-induced cytotoxicity and lipid peroxidation, and reversed cellular GSH levels compared with cadmium-treated cells (p<0.05). Compared with the cells treated with cadmium, co-treatment with α-LA and cadmium significantly increased the activities of γ-glutamylcysteine ligase (γ-GCL), the rate limiting enzyme in GSH biosynthesis and the mRNA and the protein levels of γ-GCL catalytic subunit (GCLC) and a modifier subunit (GCLM). In conclusion, our results indicated that α-LA is an effective agent to reduce the oxidative stress and cytotoxicity induced by cadmium by regenerating GSH levels through increasing the activities and the expressions of γ-GCL.