Characterisation of naturally occurring isothiocyanates as glutathione reductase inhibitors.

Glutathione reductase (GR), an essential antioxidant enzyme against oxidative stress, has become an attractive drug target for the development of anticancer and antimalarial drugs. In this regard, we evaluated the naturally occurring isothiocyanates as promising GR inhibitors and elucidated the mechanism of action. It was found that benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) inhibited yeast GR (yGR) and human GR (hGR) in a time- and concentration-dependent manner. The Ki and kinact of BITC against yGR were determined to be 259.87 µM and 0.0266 min-1, respectively. The GR inhibition occurred only in the presence of NADPH and persisted after extensive dialysis. The tandem mass spectrometric analysis revealed that Cys61 rather than Cys66 at the active site of yGR was mono-benzyl thiocarbamoylated by BITC. Inhibition of intracellular GR by BITC and PEITC in cultured cancer cells was also observed. BITC and PEITC were evaluated as competitive and irreversible inhibitors of GR.

Effect of a Prolonged Dietary Iron Intake on the Gene Expression and Activity of the Testicular Antioxidant Defense System in Rats.

Despite the fact that iron represents a crucial element for the catalysis of many metabolic reactions, its accumulation in the cell leads to the production of reactive oxygen species (ROS), provoking pathological conditions such as cancer, cardiovascular diseases, diabetes, neurodegenerative diseases, and fertility. Thus, ROS are neutralized by the enzymatic antioxidant system for the purpose of protecting cells against any damage. Iron is a potential risk factor for male fertility. However, the mechanism of action of iron on the testicular antioxidant system at the gene and protein levels is not fully understood. Thus, the purpose of the current research was to ensure a better understanding of how the long-term iron treatment influences both gene expression and enzyme activities of the testicular antioxidant system in rat testis. The data of our study showed that a significant dose-dependent increase occurred in the iron level in rat testis. A reduction occurred in reduced glutathione (GSH) levels, which represent a marker of oxidative stress, along with long-term iron overload. The expression and activity of glucose 6-phosphate dehydrogenase (G6pd), glutathione reductase (Gr), glutathione peroxidase (Gpx), and glutathione S-transferases (Gst) were significantly affected by the presence of iron. The findings of the current research demonstrate that the long-term toxic dietary iron overload influences the gene expression and enzyme activity of the testicular antioxidant defense system, but the actual effect occurs at the protein level. This may modify the sperm function and dysfunction of the male reproductive system.

In vivo biochemical evaluations of some ß-lactam group antibiotics on glutathione reductase and glutathione S- transferase enzyme activities.

OBJECTIVES: The aim of this study was to investigate whether some of the cephalosporin group antibiotics have inhibition effects on GR and GST enzymes with important functions in the metabolic pathway. METHODS: In this study, some selected cephalosporin group antibiotics on GST and GR enzyme was carried out using 96 rats. 16 groups (16 × 6) were created from these rats, divided to another 4 groups (4 × 24). The resulting groups were named as sham groups, cefazolin groups, cefuroxime groups and cefoperazone groups, respectively. The antibiotics used were injected to cefazolin, cefuroxime and cefoperazone groups. The inhibition effects of the antibiotics were measured in the different time intervals (1st, 3th, 5th, 7th). The statistical investigation of the results was performed using the SPSS software program. RESULTS: Results revealed the complex effects of the tested substances on GR and GST activity at different time intervals and in different tissues (p < 0.05). This indicated that the tested substances could be exposed to different interactions in vivo. CONCLUSION: The tested antibiotics showed some significant inhibition effects on the GST and GR enzyme activity in some tissues of brain, eye and muscle. The interaction of enzyme - the drug is a key factor to highlight the toxicological mechanism. For this reason, the results obtained from in vivo experiments are crucial to explane the physiological properties of the enzymes.

Reactive Oxygen Species-Mediated Apoptosis and Cytotoxicity of Newly Synthesized Pyridazin-3-Ones In P815 (Murin Mastocytoma) Cell Line.

BACKGROUND: In cancer cells, the intracellular antioxidant capacity and the redox homeostasis are mainly maintained by the glutathione- and thioredoxin-dependent systems which are considered as promising targets for anticancer drugs. Pyridazinones constitute an interesting source of heterocyclic compounds for drug discovery. The present investigation focused on studying the in-vitro antitumor activity of newly synthesized Pyridazin-3(2h)-ones derivatives against P815 (Murin mastocytoma) cell line. METHODS: The in-vitro cytotoxic activities were investigated toward the P815 cell line using tetrazolium-based MTT assay. Lipid peroxidation and the specific activities of antioxidant enzymes were also determined. RESULTS: The newly compounds had a selective dose-dependent cytotoxic effect without affecting normal cells (PBMCs). Apoptosis was further confirmed through the characteristic apoptotic morphological changes and DNA fragmentation. Two compounds (6F: and 7H: ) were highly cytotoxic and were submitted to extend biological testing to determine the likely mechanisms of their cytotoxicity. Results showed that these molecules may induce cytotoxicity via disturbing the redox homeostasis. Importantly, the anticancer activity of 6F: and 7H: could be due to the intracellular reactive oxygen species hypergeneration through significant loss of glutathione reductase and thioredoxin reductase activities. This eventually leads to oxidative stress-mediated P815 cell apoptosis. Furthermore, the co-administration of 6F: or 7H: with Methotrexate exhibited a synergistic cytotoxic effect. CONCLUSIONS: considering their significant anticancer activity and chemosensitivity, 6F: and 7H: may improve the therapeutic efficacy of the current treatment for cancer.

The Relevance of Glutathione Reductase Inhibition by Fluoxetine to Human Health and Disease: Insights Derived from a Combined Kinetic and Docking Study.

Glutathione reductase (GR) is a homodimeric enzyme playing an important role in the regeneration of the central antioxidant molecule reduced glutathione (GSH) from oxidized glutathione (GSSG) at the expense of a molecule of NADPH. GSH scavenges and eliminates superoxide and hydroxyl radicals non-enzymatically or serves as an electron donor for several enzymes. Fluoxetine (FLU), a selective serotonin reuptake inhibitor, is widely prescribed in the treatment of major depressive disorder. Here, using enzyme kinetic studies and molecular docking simulations, we aimed at disclosing the mechanistic and structural aspects of the interaction between GR and FLU. Affecting enzyme activity in a dose-dependent manner, FLU was shown to be a moderately potent (IC50 = 0.88 mM) inhibitor of GR. When the variable substrate was GSSG, the type of inhibition was linear mixed-type competitive (Ki = 279 ± 32 µM; α = 5.48 ± 1.29). When the variable substrate was NADPH, however, the type of inhibition was non-competitive (Ki = 879 ± 82 µM). The observed difference in inhibition types was attributed to the binding of FLU in the large intermonomer cavity of GR, where it hampered catalysis and interfered with substrate binding. Overall, although it is anticipated that long-term use of FLU leads to acquired GR deficiency, the inhibitory action of FLU on GR may be therapeutically exploited in anti-cancer research.

Antitumor Action of Amygdalin on Human Breast Cancer Cells by Selective Sensitization to Oxidative Stress.

The treatment of MCF-7 and T47D human breast cancer cell lines with amygdalin was able to reduce the growth of both cells, in concentration and time-dependent manners. The potency of this inhibition against MCF-7 and T47D cells produced IC50 values of 39 and 45 mM, respectively. To investigate the correlation of this inhibition with oxidative stress, an amygdalin treatment of both cell lines was capable of inducing the generation of malondialdehyde (MDA) and oxidized glutathione levels. Also, this treatment caused the decrease of total glutathione and glutathione reductase activity. The proportional survival of tumor cells from this inhibition was positively correlated with the total glutathione, but it was inversely correlated with amygdalin or MDA levels (P < 0.001). In MCF-7 cells, the production of total glutathione was six times higher in the untreated than in amygdalin-treated cells, whereas this difference was reduced to 2.1 times in the T47D cells. Similarly, the production of MDA in MCF-7 cells was 2.4 times higher in the amygdalin-treated than in the untreated cultures, which were lowered to 1.3 times in the T47D cells. These data support a mechanism of amygdalin antitumor action against breast cancer cells based on the induction of oxidative stress.

Glutathione reductase-mediated thiol oxidative stress suppresses metastasis of murine melanoma cells.

Malignant melanoma is a highly metastatic and life-threatening cancer. Reactive oxygen species (ROS) play important roles in cancer initiation and progression including metastasis. It has been reported that the oxidative stress spontaneously generated in circulating melanoma cells was able to suppress distant metastasis in vivo. However, little is known regarding the effects and mechanism of glutathione reductase (GR) inhibition-induced oxidative stress in regulation of melanoma metastasis. Here, we demonstrate that GR inhibition generates oxidative stress and suppresses lung metastasis and subcutaneous growth of melanoma in vivo. In addition, inhibitory effects by GR activity reduction were observed on cell proliferation, colony formation, cell adhesion, migration and invasion in melanoma cells in vitro. GR inhibition-induced oxidative stress was also found to block epithelial-to-mesenchymal transition (EMT) by decreasing the expression of Vimentin, ERK1/2, transcription factor Snail and increasing the expression of E-cadherin. In addition, actin rearrangement, a key element involved in cell motility, was also affected by GR-mediated oxidative stress possibly through protein S-glutathionylation on actin. In conclusion, this study identifies GR as an effective regulator of oxidative stress that affects the multistep processes of metastasis in melanoma cells, and it becomes a potential target for melanoma therapy.

Aqueous and Ethanol Extracts of Daylily Flower (Hemerocallis fulva L.) Protect HUVE Cells Against High Glucose.

The content of several phenolic acids and flavonoids in aqueous extract (AE) and ethanol extract (EE) of daylily flower (Hemerocallis fulva L.) was analyzed. The effects of AE or EE at 0.5%, 1%, or 2% in HUVE cells against high glucose-induced cell death, oxidative, and inflammatory damage were examined. Results showed that seven phenolic acids and seven flavonoids could be detected in AE or EE, in the range of 29 to 205 and 41 to 273 mg/100 g, respectively. Compared with the control groups, high glucose raised the activity of caspase-3 and caspase-8; suppressed Bcl-2 mRNA expression and increased Bax mRNA expression; and induced HUVE cells apoptosis. The pretreatments from AE or EE at 1% or 2% reduced caspase-3 activity and Bax mRNA expression, and enhanced cell viability. High glucose decreased glutathione content; stimulated the production of reactive oxygen species, interleukin-6, tumor necrosis factor-alpha, and prostaglandin E2 ; raised the activity of cyclooxygenase-2 and nuclear factor kappa B p50/65 binding; and reduced the activity of glutathione peroxidase, glutathione reductase, and catalase in HUVE cells. AE pretreatments at 1% and 2% reversed these changes. These novel findings suggested that daylily flower was rich in phytochemicals, and could be viewed as a potent functional food against diabetes.

Mechanistic and structural insights into the in vitro inhibitory action of hypericin on glutathione reductase purified from baker's yeast.

This work aims at studying the interaction between glutathione reductase (GR) and hypericin. The type of inhibition was determined by measuring changes in GR activity at increasing concentrations of hypericin as well as at varying concentrations of glutathione disulfide (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH), and the binding pose of hypericin was predicted by molecular docking. Accordingly, hypericin emerges as an effective inhibitor of GR. When the variable substrate is GSSG, the type of inhibition is competitive. When the variable substrate is NADPH, however, the type of inhibition appears to be linear mixed-type competitive. Our computational analyses suggest that hypericin binds in the large intermonomer cavity of GR, and that it may interfere with the normal positioning/functioning of the redox-active disulfide center at the enzyme's active site. Overall, besides its contributory role in promoting oxidative stress via the formation of reactive oxygen species in photodynamic therapy, hypericin can also weaken cancer cells through inhibiting GR.

Cinobufagin Induces Apoptosis in Osteosarcoma Cells Via the Mitochondria-Mediated Apoptotic Pathway.

BACKGROUND/AIMS: Osteosarcoma is a common primary malignant bone tumor that mainly occurs in childhood and adolescence. Despite developments in the diagnosis and treatment of osteosarcoma, the prognosis is still very poor. Cinobufagin is an active component in the anti-tumor Chinese medicine called "Chan Su", and we previously revealed that cinobufagin induced apoptosis and reduced the viability of osteosarcoma cells; however, the underlying mechanism remains to be elucidated. Herein, the present study was undertaken to illuminate the molecular mechanism of cinobufagin-induced apoptosis of osteosarcoma cell. METHODS: U2OS and 143B cells were treated with different concentrations of cinobufagin. Cell viability, colony formation ability and morphological changes were assessed by a CCK-8 assay, a clonogenic assay and light microscopy, respectively. Cell apoptosis was detected by Hoechst 33258 and Annexin V-FITC/PI staining. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were determined by flow cytometry. Glutathione (GSH) levels were detected by a GSH and GSSG assay kit. The levels of apoptosis-related proteins were determined by western blotting, and 143B cells were introduced to establish a xenograft tumor model. The effect of cinobufagin on osteosarcoma was further investigated in vivo. RESULTS: Our results showed that cinobufagin significantly reduced the viability of U2OS and 143B cells in vitro in a dose-and time-dependent manner. In addition, cinobufagin-induced apoptosis in U2OS and 143B cells was concentration-dependent. Moreover, we found that cinobufagin treatment increased the level of intracellular ROS, decreased ΔΨm, reduced GSH and inhibited GSH reductase (GR). The effects of cinobufagin on cell proliferation, apoptosis, ROS generation and ΔΨm loss were dramatically reversed when the cells were pretreated with the thiol-antioxidants NAC or GSH. Moreover, cinobufagin treatment increased the expression of the pro-apoptotic protein Bax and decreased the expression of the anti-apoptitic protein Bcl-2, thus altering the ratio of Bax to Bcl-2. Furthermore, Cinobufagin treatment caused cytochrome c release from the mitochondria to cytoplasm, thus increasing the protein levels of cleaved-caspase family members to induce apoptosis. Ac-DEVD-CHO or Z-LEHD-FMK significantly reduced cinobufagin-induced apoptosis. Finally, a subcutaneous xenograft animal study verified that cinobufagin also significantly suppressed osteosarcoma growth in vivo. CONCLUSIONS: Our present data demonstrated that cinobufagin triggered cell apoptosis in osteosarcoma cells via the intrinsic mitochondria-dependent apoptosis pathway by the accumulation of ROS and the loss of ΔΨm. In an in vivo subcutaneous xenograft model, cinobufagin exhibited excellent tumor inhibitory effects. These results suggest that cinobufagin might potentially be further developed as an anti-tumor candidate for treating osteosarcoma patients in the clinic.

Glutathione reductase mediates drug resistance in glioblastoma cells by regulating redox homeostasis.

Glutathione (GSH) and GSH-related enzymes constitute the most important defense system that protects cells from free radical, radiotherapy, and chemotherapy attacks. In this study, we aim to explore the potential role and regulatory mechanism of the GSH redox cycle in drug resistance in glioblastoma multiforme (GBM) cells. We found that temozolomide (TMZ)-resistant glioma cells displayed lower levels of endogenous reactive oxygen species and higher levels of total antioxidant capacity and GSH than sensitive cells. Moreover, the expression of glutathione reductase (GSR), the key enzyme of the GSH redox cycle, was higher in TMZ-resistant cells than in sensitive cells. Furthermore, silencing GSR in drug-resistant cells improved the sensitivity of cells to TMZ or cisplatin. Conversely, the over-expression of GSR in sensitive cells resulted in resistance to chemotherapy. In addition, the GSR enzyme partially prevented the oxidative stress caused by pro-oxidant L-buthionine -sulfoximine. The modulation of redox state by GSH or L-buthionine -sulfoximine regulated GSR-mediated drug resistance, suggesting that the action of GSR in drug resistance is associated with the modulation of redox homeostasis. Intriguingly, a trend toward shorter progress-free survival was observed among GBM patients with high GSR expression. These results indicated that GSR is involved in mediating drug resistance and is a potential target for improving GBM treatment.

Alleviation of cadmium-induced oxidative stress by trehalose via inhibiting the Nrf2-Keap1 signaling pathway in primary rat proximal tubular cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates a cluster of oxidative stress-inducible genes in cells. Here, we aimed to investigate whether trehalose (Tre) protects primary rat proximal tubular (rPT) cells against cadmium (Cd)-induced oxidative stress via Nrf2 antioxidant pathway. Data showed that Tre treatment inhibited Nrf2 nuclear translocation and restored the decline in Kelch-like ECH-associated protein 1 (Keap1) protein level in Cd-exposed rPT cells. Moreover, Cd-activated Nrf2 target genes, including phase II detoxifying enzymes, that is, NAD(P)H quinone oxidoreductase 1 and heme oxygenase-1, direct antioxidant proteins, that is, glutathione peroxidase, superoxide dismutase, catalase, and glutathione biosynthesis-related proteins, that is, glutamatecysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione reductase, were all downregulated by co-treatment with Tre. Collectively, these findings demonstrate that Tre treatment alleviates Cd-induced oxidative stress in rPT cells by inhibiting the Nrf2-Keap1 signaling pathway.

Characterization of Hydrophilic Gold(I) N-Heterocyclic Carbene (NHC) Complexes as Potent TrxR Inhibitors Using Biochemical and Mass Spectrometric Approaches.

We report here on the synthesis of a series of mono- and dinuclear gold(I) complexes exhibiting sulfonated bis(NHC) ligands and novel hydroxylated mono(NHC) Au(I) compounds, which were also examined for their biological activities. Initial cell viability assays show strong antiproliferative activities of the hydroxylated mono(NHC) gold compounds (8 > 9 > 10) against 2008 human ovarian cancer cells even after 1 h incubation. In order to gain insight into the mechanism of biological action of the gold compounds, their effect on the pivotal cellular target seleno-enzyme thioredoxin reductase (TrxR), involved in the maintenance of intracellular redox balance, was investigated in depth. The compounds' inhibitory effects on TrxR and glutathione reductase (GR) were studied comparatively, using either the pure proteins or cancer cell extracts. The results show a strong and selective inhibitory effect of TrxR, specifically for the hydroxyl-functionalized NHC gold(I) complexes (8-10). Valuable information on the gold compounds' molecular reactivity with TrxR was gained using the BIAM (biotin-conjugated iodoacetamide) assay and performing competition experiments by mass spectrometry (MS). In good agreement, both techniques suggest the binding affinity of the mono(NHC) Au(I) complexes toward selenols and thiols. Notably, for the first time, bis-carbene formation from mono-carbenes in buffered solution could be observed by MS, which may provide new insights into the speciation mechanisms of bioactive Au(I) NHC complexes. Furthermore, the compounds' interactions with another relevant in cellulo target, namely telomeric G-quadruplex DNA-a higher-order DNA structure playing key roles in telomere function-was investigated by means of FRET melting assays. The lack of interactions with this type of nucleic acid secondary structure support the idea of selective targeting of the hydrophilic Au(I) NHC compounds toward proteins such as TrxR.

Inhibition of reductase systems by 2-AAPA modulates peroxiredoxin oxidation and mitochondrial function in A172 glioblastoma cells.

Thiol homeostasis has a critical role in the maintenance of proper cellular functions and survival, being coordinated by the action of several reductive enzymes, including glutathione (GSH)/glutathione reductase (GR) and thioredoxin (Trx)/thioredoxin reductase (TrxR) systems. Here, we investigated the effects of the GR inhibitor 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) on the activity of thiol reductases (GR and TrxR), redox balance and mitochondrial function of A172 glioblastoma cells. 2-AAPA inhibited cell GR (IC50=6.7µM) and TrxR (IC50=8.7µM). A significant decrease in the cellular ability to decompose cumene hydroperoxide was observed and associated to a greater susceptibility to this peroxide. The redox state of peroxiredoxins (Prx1, Prx2 and Prx3) was markedly shifted to dimer 30min after treatment with 100µM 2-AAPA, an event preceding 2-AAPA-induced decrease in cell viability. Furthermore, mitochondrial function was also severely impaired, leading to a decrease in the respiratory control ratio, reserve capacity, and ATP synthesis-coupled respiration, as well as an increase in mitochondrial membrane potential. Our results indicate that inhibition of GR and TrxR activities, disruption of the ability to detoxify peroxides, increased oxidation of Prxs, as well as compromised mitochondrial function represent early events mediating 2-AAPA toxicity to A172 glioblastoma cells.

Fluorimetric evaluation of glutathione reductase activity and its inhibitors using carbon quantum dots.

A highly sensitive fluorimetric assay has been developed for the evaluation of glutathione reductase (GR) activity and the screening of its inhibitors by using carbon quantum dots (CQDs) as the signal reporter. The detection mechanism is based on the following facts: (1) the fluorescence of CQDs can be quenched by Hg(II) through the strong CQDs-Hg(II) coordination; (2) GR can catalyze the reduction of oxidized glutathione (GSSG) into reduced glutathione (GSH), so that the fluorescence recovery of CQDs would take place resulting from the strong GSH-Hg(II) interaction; (3) GR can lose its catalytic reduction of GSSG in the presence of its inhibitors, which will inhibit the recovery of the quenched fluorescence of CQDs. The developed CQDs-based fluorimetric method can facilitate the sensitive evaluation of GR activity in the range of 0.10-2.0mUmL-1 with a detection limit of 0.050 mUmL-1. In addition, other kinds of enzymes like myoglobin, thrombin, alcohol dehyrogenase, amylase, pepsin, and trypsin could show no significant effects on the evaluation of GR activity. This work may expand the biological applications of CQDs as the fluorescent probes with low cost, easy preparation, and high photostability.

MitoNEET Protects HL-1 Cardiomyocytes from Oxidative Stress Mediated Apoptosis in an In Vitro Model of Hypoxia and Reoxygenation.

The iron-sulfur cluster containing protein mitoNEET is known to modulate the oxidative capacity of cardiac mitochondria but its function during myocardial reperfusion injury after transient ischemia is unknown. The purpose of this study was to analyze the impact of mitoNEET on oxidative stress induced cell death and its relation to the glutathione-redox system in cardiomyocytes in an in vitro model of hypoxia and reoxygenation (H/R). Our results show that siRNA knockdown (KD) of mitoNEET caused an 1.9-fold increase in H/R induced apoptosis compared to H/R control while overexpression of mitoNEET caused a 53% decrease in apoptosis. Necrosis was not affected. Apoptosis of both, mitoNEET-KD and control cells was diminished to comparable levels by using the antioxidants Tiron and glutathione compound glutathione reduced ethyl ester (GSH-MEE), indicating that mitoNEET-dependent apoptosis is mediated by oxidative stress. The interplay between mitoNEET and glutathione redox system was assessed by treating cardiomyocytes with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthio-carbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), known to effectively inhibit glutathione reductase (GSR) and to decrease the GSH/GSSG ratio. Surprisingly, inhibition of GSR-activity to 20% by 2-AAPA decreased apoptosis of control and mitoNEET-KD cells to 23% and 25% respectively, while at the same time mitoNEET-protein was increased 4-fold. This effect on mitoNEET-protein was not accessible by mitoNEET-KD but was reversed by GSH-MEE. In conclusion we show that mitoNEET protects cardiomyocytes from oxidative stress-induced apoptosis during H/R. Inhibition of GSH-recycling, GSR-activity by 2-AAPA increased mitoNEET-protein, accompanied by reduced apoptosis. Addition of GSH reversed these effects suggesting that mitoNEET can in part compensate for imbalances in the antioxidative glutathione-system and therefore could serve as a potential therapeutic approach for the oxidatively stressed myocardium.

Unexpected Thiols Triggering Photoluminescent Enhancement of Cytidine Stabilized Au Nanoclusters for Sensitive Assays of Glutathione Reductase and Its Inhibitors Screening.

The photoluminescence (PL) of nonthiolate ligand capped Au nanoclusters (NCs) is usually quenched by thiols due to the tight adsorption of thiols to the Au surface and formation of larger non-PL species. However, we here report an unexpected PL enhancement of cytidine stabilized Au (AuCyt) NCs triggered by thiols, such as reduced glutathione (GSH) at sub-µM level, while such phenomena have not been observed for Au NCs capped with similar adenosine/cytidine nucleotides. The mass spectroscopic results indicate that this enhancement may be caused by the formation of smaller, but highly fluorescent, Au species etched by thiols. This enables the sensitive detection of GSH from 20 nM to 3 µM, with an ultralow detection limit of 2.0 nM. Moreover, the glutathione reductase (GR) activity can be determined by the initial rate of GSH production, i.e., the maximum PL increasing rate, with a linear range of 0.34-17.0 U/L (1 U means reduction of 1.0 µmol of oxidized glutathione per min at pH 7.6 at 25 °C) and a limit of detection of 0.34 U/L. This method allows the accurate assays of GR in clinical serum samples as well as the rapid screening of GR inhibitors, indicating its promising biomedical applications.

Development of a functional assay to detect inhibitors of Plasmodium falciparum glutathione reductase utilizing liquid chromatography-mass spectrometry.

Plasmodium falciparum (Pf) like most other organisms, has a sophisticated antioxidant system, part of which includes glutathione reductase (GR). GR works by recycling toxic glutathione disulfide to glutathione, thereby reducing reactive oxygen species and making a form of glutathione (GSH) the parasite can use. Inhibition of this enzyme in Pf impedes parasite growth. In addition, it has been confirmed that PfGR is not identical to human GR. Thus, PfGR is an excellent target for antimalarial drug development. A functional assay utilizing liquid chromatography-mass spectrometry was developed to specifically identify and evaluate inhibitors of PfGR. Using recombinant PfGR enzyme and 1,4-naphthoquinone (1) as a reference compound and 4-nitrobenzothiadiazole (2) and methylene blue (3) as additional compounds, we quantified the concentration of GSH produced compared with a control to determine the inhibitory effect of these compounds. Our results coincide with that presented in literature: compounds 1-3 inhibit PfGR with IC50 values of 2.71, 8.38, and 19.23 µm, respectively. Good precision for this assay was exhibited by low values of intraday and interday coefficient of variation (3.1 and 2.4%, respectively). Thus, this assay can be used to screen for other potential inhibitors of PfGR quickly and accurately.

A direct comparison of methods used to measure oxidized glutathione in biological samples: 2-vinylpyridine and N-ethylmaleimide.

The ratio of glutathione disulfide (GSSG) to reduced glutathione (GSH) in biological samples is a frequently used parameter of oxidative stress. As a result, many methods are developed to measure GSSG. The most popular and convenient of these relies on enzymatic cycling following the chemical masking of GSH in the sample using 2-vinylpyridine (2VP). However, 2VP is a slow reactant and its use may result in artificially high GSSG values due to oxidation of the sample over time. Fast-reacting reagents such as N-ethylmaleimide (NEM) may provide more accurate results. We performed a direct comparison of methods using 2VP and NEM. With 2VP, the percentage of total glutathione (GSH+GSSG) in the oxidized form was significantly higher in all tested tissues (kidney, lung and liver) compared to the same procedure performed using NEM. We conclude that NEM, when coupled with a simple solid-phase extraction procedure, is more accurate for the determination of GSSG. We also tested the effects of various handling and storage conditions on GSSG. A detailed description and a discussion of other methods are also included.

LPO-Antioxidant Defense System in Newborns and Women Drinking Small Amounts of Alcohol During Pregnancy.

We studied the effects of consumption of low-alcohol drinks (no more than 2 doses) during pregnancy on the state of LPO-antioxidant defense system and glutathione level in the blood serum and erythrocytes of pregnant women and newborns. It was found that even single alcohol intake was followed by the development of oxidative stress in women and newborns. In pregnant women, the decrease in reduced glutathione content and glutathione reductase activity associated with increased level of oxidized glutathione and glutathione transferase activity were typical changes. In newborns, the decrease in reduced glutathione concentration was accompanied by a decrease in glutathione reductase, glutathione transferase, and glutathione peroxidase activities. It should be emphasized that even small alcohol doses produce delayed negative effects on metabolism of the mother and newborns.