Free Glutathione Research Articles

Selenium- and Tellurium-Based Antioxidants for Modulating Inflammation and Effects on Osteoblastic Activity.

Increased oxidative stress plays a significant role in the etiology of bone diseases. Heightened levels of H2O2 disrupt bone homeostasis, leading to greater bone resorption than bone formation. Organochalcogen compounds could act as free radical trapping agents or glutathione peroxidase mimetics, reducing oxidative stress in inflammatory diseases. In this report, we synthesized and screened a library of organoselenium and organotellurium compounds for hydrogen peroxide scavenging activity, using macrophagic cell lines RAW264.7 and THP-1, as well as human mono- and poly-nuclear cells. These cells were stimulated to release H2O2, using phorbol 12-myristate 13-acetate, with and without organochalogens. Released H2O2 was then measured using a chemiluminescent assay over a period of 2 h. The screening identified an organoselenium compound which scavenged H2O2 more effectively than the vitamin E analog, Trolox. We also found that this organoselenium compound protected MC3T3 cells against H2O2-induced toxicity, whereas Trolox did not. The organoselenium compound exhibited no cytotoxicity to the cells and had no deleterious effects on cell proliferation, viability, or alkaline phosphatase activity. The rapidity of H2O2 scavenging and protection suggests that the mechanism of protection is due to the direct scavenging of extracellular H2O2. This compound is a promising modulators of inflammation and could potentially treat diseases involving high levels of oxidative stress.

АНТИОКСИДАНТНЫЙ СТАТУС ПРИ РАЗЛИЧНЫХ ПОДТИПАХ ИШЕМИЧЕСКОГО ИНСУЛЬТА В ОСТРЫЙ ПЕРИОД ЗАБОЛЕВАНИЯ

The aim of the study was to detect significant biochemical changes in antioxidant status during acute period of ischemic stroke in its various pathogenic subtypes in patients aged 45–74years. The most important results were blood levels of ascorbic acid, malondialdehyde and free/ oxidized glutathione ratio. Cardioembolic stroke was associated with: the reduced ascorbic acid level of 62–74% comparable to control group; 5-times increased malondialdehyde level at the end of the first week of the disease; and also 50% decreasing of cell antioxidant capacity comparable to control group and other stroke subtypes. In lacunary stroke minimal changes in antioxidant status were measured, possibly because of small size of brain damage. Positive clinical dynamics was followed by decreasing of free glutathione levels. It could be related with its previous significant expenditure in process of cell recovery in penumbra area. In case of favorable outcome all of the markers return to the levels which were comparable to control group. The differences in the dynam-ics of the cells antioxidant capacity and consumption of native antioxidants, the time from the onset of the disease, the severity of the clinical picture, depending on the stroke subtype, were revealed. Further research of this problem may help to create new methods of diagnostics and selective pharmacological correction of this pathology.

Read-across of 90-day rat oral repeated-dose toxicity: A case study for selected β-olefinic alcohols

There are no in vivo repeated-dose data for the vast majority of β-olefinic alcohols. However, there are robust and consistent ex vivo data suggesting many of these chemicals are metabolically transformed, especially in the liver, to reactive electrophilic toxicants which react in a mechanistically similar manner to acrolein, the reactive metabolite of 2-propen-1-ol. Hence, an evaluation was conducted to determine suitability of 2-propen-1-ol as a read-across analogue for other β-olefinic alcohols. The pivotal issue to applying read-across to the proposed category is the confirmation of the biotransformation to metabolites having the same mechanism of electrophilic reactivity, via the same metabolic pathway, with a rate of transformation sufficient to induce the same in vivo outcome. The applicability domain for this case study was limited to small (C3 to C6) primary and secondary β-olefinic alcohols. Mechanistically, these β-unsaturated alcohols are considered to be readily metabolised by alcohol dehydrogenase to polarised α,β-unsaturated aldehydes and ketones. These metabolites are able to react via the Michael addition reaction mechanism with thiol groups in proteins resulting in cellular apoptosis and/or necrosis. The addition of the non-animal in chemico reactivity data (50% depletion of free glutathione) reduced the uncertainty so the read-across prediction for the straight-chain olefinic β-unsaturated alcohols is deemed equivalent to a standard test. Specifically, the rat oral 90-day repeated-dose No Observed Adverse Effect Level (NOAEL) for 2-propen-1-ol of 6mg/kg body weight (bw)/d in males based on increase in relative weight of liver and 25mg/kg bw/d in females based on bile duct hyperplasia and periportal hepatocyte hypertrophy in the liver, is read across to fill data gaps for the straight-chained analogues.

Relationship between plasma glycation with membrane modification, oxidative stress and expression of glucose trasporter-1 in type 2 diabetes patients with vascular complications

Background of studyEnhanced protein glycation in diabetes causes irreversible cellular damage through membrane modifications. Erythrocytes are persistently exposed to plasma glycated proteins; however, little are known about its consequences on membrane. Aim of this study was to examine the relationship between plasma protein glycation with erythrocyte membrane modifications in type 2 diabetes patients with and without vascular complications. MethodWe recruited 60 healthy controls, 85 type 2 diabetic mellitus (DM) and 75 type 2 diabetic patients with complications (DMC). Levels of plasma glycation adduct with antioxidants (fructosamine, protein carbonyl, β-amyloids, thiol groups, total antioxidant status), erythrocyte membrane modifications (protein carbonyls, β-amyloids, free amino groups, erythrocyte fragility), antioxidant profile (GSH, catalase, lipid peroxidation) and Glut-1 expression were quantified. ResultCompared with controls, DM and DMC patients had significantly higher level of glycation adducts, erythrocyte fragility, lipid peroxidation and Glut-1 expression whereas declined levels of plasma and cellular antioxidants. Correlation studies revealed positive association of membrane modifications with erythrocyte sedimentation rate, fragility, peroxidation whereas negative association with free amino groups, glutathione and catalase. ConclusionOur data suggest that plasma glycation is associated with oxidative stress, Glut-1 expression and erythrocyte fragility in DM patients. This may further contribute to progression of vascular complications.

Glibenclamide impairs responses of neutrophils against Burkholderia pseudomallei by reduction of intracellular glutathione.

The major risk factor for melioidosis, an infectious disease caused by B. pseudomallei, is diabetes mellitus. More than half of diabetic melioidosis patients in Thailand were prescribed glibenclamide. Recent evidence demonstrates that glibenclamide reduces pro-inflammatory cytokine production by polymorphonuclear neutrophils (PMNs) of diabetic individuals in response to this bacterial infection. However, the mechanisms by which glibenclamide affects cytokine production are unknown. We found that PMNs from glibenclamide-treated diabetic individuals infected with live B. pseudomallei in vitro showed lower free glutathione (GSH) levels compared with those of healthy individuals. Glibenclamide decreased GSH levels and glutathione peroxidase (GPx) of PMNs after exposed to live B. pseudomallei. Moreover, glibenclamide reduced cytokine production and migration capacity of infected PMNs, whereas GSH could restore these functions. Taken together, our data show a link between the effect of glibenclamide on GSH and PMN functions in response to B. pseudomallei that may contribute to the susceptibility of diabetic individuals to B. pseudomallei infection.

Structure and Ligand-Binding Mechanism of a Cysteinyl Leukotriene-Binding Protein from a Blood-Feeding Disease Vector.

Blood-feeding disease vectors mitigate the negative effects of hemostasis and inflammation through the binding of small-molecule agonists of these processes by salivary proteins. In this study, a lipocalin protein family member (LTBP1) from the saliva of Rhodnius prolixus, a vector of the pathogen Trypanosoma cruzi, is shown to sequester cysteinyl leukotrienes during feeding to inhibit immediate inflammatory responses. Calorimetric binding experiments showed that LTBP1 binds leukotrienes C4 (LTC4), D4 (LTD4), and E4 (LTE4) but not biogenic amines, adenosine diphosphate, or other eicosanoid compounds. Crystal structures of ligand-free LTBP1 and its complexes with LTC4 and LTD4 reveal a conformational change during binding that brings Tyr114 into close contact with the ligand. LTC4 is cleaved in the complex, leaving free glutathione and a C20 fatty acid. Chromatographic analysis of bound ligands showed only intact LTC4, suggesting that cleavage could be radiation-mediated.

Covalent adducts of melphalan with free amino acids and a model peptide studied by liquid chromatography/tandem mass spectrometry.

Melphalan is a frequently used chemotherapeutical agent for the treatment of myeloma, breast cancer, ovarian cancer and sarcoma of soft tissue. A good knowledge of the reactivity of the drug toward the different amino acids, e.g. covalent adduct formation, is crucial for the understanding of its activity and side effects during cancer treatment. The reactivity of melphalan and sites of adduct formation were studied by in vitro incubation of melphalan with free amino acids and glutathione as a model peptide. The formed covalent adducts were investigated using ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) using a triple-quadrupole instrument. Accurate mass measurements for the confirmation of characteristic product ions were performed on a quadrupole time-of-flight (QTOF) mass spectrometer. The incubation of melphalan with different classes of amino acids resulted in the formation of adducts on the amino and carboxyl termini, as well as adduct formation in the reactive side chains of Cys, Met, Tyr, His, Lys, Asp and Glu. All these melphalan adducts could be identified by their characteristic collision-induced dissociation (CID) product ion patterns. The present study demonstrates the reactivity of melphalan towards the functional groups of amino acids. The different alkylation site products show distinctive fragmentation patterns, which enable a fast identification of the different melphalan adducts. This study is a first important step towards a better understanding of the adduct formation in more complex molecules, e.g. peptides and proteins.

The effects of buthionine sulfoximine treatment on diaphragm contractility and SERCA pump function in adult and middle aged rats.

This study examined the effects of 10 days of buthionine sulfoximine (BSO) treatment on in vitro contractility and sarcoplasmic reticulum calcium pump (SERCA) expression and function in adult (AD; 6–8 months old) and middle aged (MA; 14–17 months old) rat diaphragm in both the basal state and following fatiguing stimulation. BSO treatment reduced the cellular concentrations of free glutathione (GSH) by >95% and oxidized glutathione (GSSG) by >80% in both age cohorts. GSH content in AD Control diaphragm was 32% higher (P < 0.01) than in MA Control, with no differences in GSSG. The ratio of GSH:GSSG, an indicator of cellular oxidative state, was 34.6 ± 7.4 in MA Control, 52.5 ± 10.1 in AD Control, 10.6 ± 1.7 in MA BSO, and 9.5 ± 1.1 in AD BSO (BSO vs. Control, P < 0.05). Several findings suggest that the effects of BSO treatment are age dependent. AD BSO diaphragm had 26% higher twitch and 28% higher tetanic force (both P < 0.05) than AD Controls, whereas no significant difference existed between the two MA groups. In contrast to our previous work on BSO-treated AD rats, BSO treatment did not influence maximal SERCA ATPase activity in MA rat diaphragm, nor did SERCA2a expression increase in BSO-treated MA diaphragm. Biotinylated iodoacetamide binding to SERCA1a, a specific marker of free cysteine residues, was reduced by 35% (P < 0.05) in AD Control diaphragm following fatiguing stimulation, but was not reduced in any other group. Collectively, these results suggest an important role for redox regulation in both contractility and SERCA function which is influenced by aging.

Electrochemical determination of free and total glutathione in human saliva samples

Glutathione (GSH) is a small peptide of great relevance in many biological processes. Its determination is a valuable aid to understand the cellular health status. Important information is obtained not only from the quantification of its free reduced form (GSH) but also of its oxidized form (GSSG) and both the ratio and absolute amounts of these two species. We report an electro-analytical methodology for determining the reduced and total GSH using square wave voltammetry and a screen-printed electrode modified with carbon nanotubes (CNT–SPE). For the total glutathione determination, sodium borohydride was chosen as a reducing agent for its effectiveness and inert electrochemical activity. Human saliva samples were successfully analysed by the developed methodology as a proof-of-concept and validated independently with a commercially available enzyme assay kit.

Effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine primary hepatocytes.

To investigate the cytotoxic effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine hepatocytes. Commercially available cryopreserved canine primary hepatocytes. The study consisted of 2 trials. In trial 1, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 6 concentrations (0.468, 0.937, 1.875, 3.750, 7.500, or 15.000 μmol/L) for 24, 48, or 72 hours. At each time, cell viability and lactate dehydrogenase (LDH) activity were determined for each thiopurine-concentration combination, and alanine aminotransferase (ALT) activity was determined for cells incubated with each thiopurine at a concentration of 15 μmol/L. In trial 2, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 3 concentrations (18.75, 37.50, or 75.00 μmol/L) for 24 hours, after which the free glutathione concentration was determined for each thiopurine-concentration combination and compared with that for hepatocytes incubated without a thiopurine (control). Incubation of hepatocytes with each of the 3 thiopurines adversely affected cell viability in a time- and concentration-dependent manner; however, this decrease in cell viability was not accompanied by a concurrent increase in LDH or ALT activity. Likewise, free glutathione concentration for hepatocytes incubated for 24 hours with supratherapeutic thiopurine concentrations (> 18.75 μmol/L) did not differ significantly from that of control cells. Results indicated that thiopurines adversely affected the viability of canine hepatocytes in a time- and concentration-dependent manner but had a nonsignificant effect on the LDH and ALT activities and free glutathione depletion of those hepatocytes.

APR-246 overcomes resistance to cisplatin and doxorubicin in ovarian cancer cells.

Two main causes of platinum resistance are mutation in the tumor suppressor gene TP53 and drug-induced increase in intracellular glutathione concentration. Mutations in TP53 occur in about 50% of human tumors. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53 and induces apoptosis. APR-246 is a prodrug that is converted to the active compound methylene quinuclidinone (MQ), a Michael acceptor that binds to cysteine residues in mutant p53 and restores its wild-type conformation. Here, we show that MQ also binds to cysteine in glutathione, thus decreasing intracellular free glutathione concentration. We also show that treatment with APR-246 completely restores the cisplatin and doxorubicin sensitivity to p53-mutant drug-resistant ovarian cancer cells. We propose that this unique ability of APR-246/MQ to bind to cysteines in both mutant p53 and glutathione has a key role in the resensitization as well as in the outstanding synergistic effects observed with APR-246 in combination with platinum compounds in ovarian cancer cell lines and primary cancer cells. However, MQ binding to cysteines in other targets, for example, thioredoxin reductase, may contribute as well. Strong synergy was also observed with the DNA-damaging drugs doxorubicin and gemcitabine, while additive effects were found with the taxane docetaxel. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with platinum-based therapy in patients with p53-mutant recurrent high-grade serous (HGS) ovarian cancer. More than 96% of these patients carry TP53 mutations. Combined treatment with APR-246 and platinum or other DNA-damaging drugs could allow dramatically improved therapy of a wide range of therapy refractory p53 mutant tumors.

Detection of Labile Low-Molecular-Mass Transition Metal Complexes in Mitochondria.

Liquid chromatography was used with an online inductively coupled plasma mass spectrometer to detect low-molecular-mass (LMM) transition metal complexes in mitochondria isolated from fermenting yeast cells, human Jurkat cells, and mouse brain and liver. These complexes constituted 20-40% of total mitochondrial Mn, Fe, Zn, and Cu ions. The major LMM Mn complex in yeast mitochondria, called Mn1100, had a mass of ∼1100 Da and a concentration of ∼2 μM. Mammalian mitochondria contained a second Mn species with a mass of ∼2000 Da at a comparable concentration. The major Fe complex in mitochondria isolated from exponentially growing yeast cells had a mass of ∼580 Da; the concentration of Fe580 in mitochondria was ∼100 μM. When mitochondria were isolated from fermenting cells in postexponential phase, the mass of the dominant LMM Fe complex was ∼1100 Da. Upon incubation, the intensity of Fe1100 declined and that of Fe580 increased, suggesting that the two are interrelated. Mammalian mitochondria contained Fe580 and two other Fe species (Fe2000 and Fe1100) at concentrations of ∼50 μM each. The dominant LMM Zn species in mitochondria had a mass of ∼1200 Da and a concentration of ∼110 μM. Mammalian mitochondria contained a second major LMM Zn species at 1500 Da. The dominant LMM Cu species in yeast mitochondria had a mass of ∼5000 Da and a concentration in yeast mitochondria of ∼16 μM; Cu5000 was not observed in mammalian mitochondria. The dominant Co species in mitochondria, Co1200, had a concentration of 20 nM and was probably a cobalamin. Mammalian but not yeast mitochondria contained a LMM Mo species, Mo730, at a concentration of ∼1 μM. Increasing Mn, Fe, Cu, and Zn concentrations 10-fold in the medium increased the concentration of the same element in the corresponding isolated mitochondria. Treatment with metal chelators confirmed that these LMM species were labile. The dominant S species at 1100 Da was not free glutathione or glutathione disulfide.

Endogeous sulfur dioxide protects against oleic acid-induced acute lung injury in association with inhibition of oxidative stress in rats

The role of endogenous sulfur dioxide (SO2), an efficient gasotransmitter maintaining homeostasis, in the development of acute lung injury (ALI) remains unidentified. We aimed to investigate the role of endogenous SO2 in the pathogenesis of ALI. An oleic acid (OA)-induced ALI rat model was established. Endogenous SO2 levels, lung injury, oxidative stress markers and apoptosis were examined. OA-induced ALI rats showed a markedly downregulated endogenous SO2/aspartate aminotransferase 1 (AAT1)/AAT2 pathway and severe lung injury. Chemical colorimetry assays demonstrated upregulated reactive oxygen species generation and downregulated antioxidant capacity in OA-induced ALI rats. However, SO2 increased endogenous SO2 levels, protected against oxidative stress and alleviated ALI. Moreover, compared with OA-treated cells, in human alveolar epithelial cells SO2 downregulated O2− and OH− generation. In contrast, L-aspartic acid-β-hydroxamate (HDX, Sigma-Aldrich Corporation), an inhibitor of endogenous SO2 generating enzyme, promoted free radical generation, upregulated poly (ADP-ribose) polymerase expression, activated caspase-3, as well as promoted cell apoptosis. Importantly, apoptosis could be inhibited by the free radical scavengers glutathione (GSH) and N-acetyl-L-cysteine (NAC). The results suggest that SO2/AAT1/AAT2 pathway might protect against the development of OA-induced ALI by inhibiting oxidative stress.

Genetic variability in proso millet [Panicum miliaceum] germplasm of Central Himalayan Region based on morpho-physiological traits and molecular markers

Sixteen representative a ccessions of proso millet [Panicum miliaceum] having distinct traits of agronomic importance were collected from altitudinal range of 510–2,695 m asl in the Central Himalayan Region (CHR) of India. Considerable diversity was found in morpho-physiological traits viz., leaf length (16.80–32.00 cm), leaf width (1.7–2.1 cm), plant height (57.00–134.00 cm), days to 50 % flowering (34–54), days to 80 % maturity (111–144), and 1,000 seed weight (0.68–1.86 g). Collected accessions have been evaluated for a battery of biochemical parameters viz., chlorophyll, carotenoids, lipid peroxidation, cellular hydrogen peroxide, activity of nitrate reductase, lipoxygenase, catalase, peroxidase, superoxide dismutase along with super oxide free radical formation, glutathione (total, reduced, and oxidized), glutathione reductase, glutathione S-transferase, ascorbate (total, reduced, and oxidized), monodehydroascorbate reductase, and didehydroascorbate reductase. The sets of 12 genic-SSRs (simple sequence repeats), 54 ISSR (inter simple sequence repeats), and 40 SRAP (sequence related amplified polymorphism) markers were used to study the level of genetic diversity, and Nei’s gene diversity value of 0.20 was obtained with both ISSR and SRAP markers. SRAP markers showed higher average number of polymorphic bands, % polymorphism, polymorphic information content (PIC), and Shannon information index compared to ISSR markers; genic-SSRs showed no allelic variation. Cluster analysis shows close groupings of germplasm based on morpho-physiological traits as well as molecular markers. The diverse germplasm identified based on molecular markers with considerable diversity in morpho-physiological traits may be utilized for development of climate resilient cultivars.

Molecular speciated isotope dilution mass spectrometric methods for accurate, reproducible and direct quantification of reduced, oxidized and total glutathione in biological samples.

Novel protocols were developed to accurately quantify reduced (GSH), oxidized (GSSG) and total (tGSH) glutathione in biological samples using molecular speciated isotope dilution mass spectrometry (SIDMS). For GSH and GSSG measurement, the sample was spiked with isotopically enriched analogues of the analytes ((310)GSH and (616)GSSG), along with N-ethylmaleimide (NEM), and treated with acetonitrile to solubilize the endogenous analytes via protein precipitation and equilibrate them with the spikes. The supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the analytes were quantified with simultaneous tracking and correction for auto-oxidation of GSH to GSSG. For tGSH assay, a (310)GSH-spiked sample was treated with dithiothreitol (DTT) to convert disulfide-bonded glutathione to GSH. After removing the protein, the supernatant was analyzed by LC-MS/MS and the analyte was quantified by single-spiking isotope dilution mass spectrometry (IDMS). The mathematical relationships in IDMS and SIDMS quantifications are based on isotopic ratios and do not involve calibration curves. The protocols were validated using spike recovery tests and by analyzing synthetic standard solutions. Red blood cell (RBC) and saliva samples obtained from healthy subjects, and whole blood samples collected and shipped from a remote location were analyzed. The concentrations of tGSH in the RBC and whole blood samples were 2 orders of magnitude higher than those found in saliva. The fractions of GSSG were 0.2-2.2% (RBC and blood) and 15-47% (saliva) of the free glutathione (GSH + 2xGSSG) in the corresponding samples. Up to 3% GSH was auto-oxidized to GSSG during sample workup; the highest oxidations (>1%) were in the saliva samples.

An antinitrosative system as a factor in malignant tumor resistance to the cytotoxic effect of nitrogen monoxide

Inhibition of growth of a transplanted solid tumor in BDF1 mice bearing Lewis lung carcinoma cells on days 1–5 and 7–11 after tumor transplantation was observed at daily intraperitoneal administration of an aqueous solution of binuclear form of dinitrosyl iron complexes with glutathione at a dose of 200 μmol/kg (relative to one Fe(NO)2 group in the complexes). Inhibition of tumor growth during the administration of complexes (for 11 days) was 70 and 85% when an iron complex: free glutathione ratio in solution was equal to 1: 1 and 1: 10, respectively. Accelerated tumor growth that was faster than in the control began after cessation of dinitrosyl iron complex administration. The selective accumulation of dinitrosyl iron complexes in the tumor, as well as the accumulation of nitrosyl complexes of heme proteins was detected by the EPR method. The latter were observed in the tumor and control animals. It is hypothesized that delayed tumor development during administration of the binuclear form of dinitrosyl iron complexes to mice is caused by inactivation of heme-containing proteins under the action of NO released from these complexes, which provides antinitrosative defenses produced in malignant tumors.

Is development of high-grade gliomas sulfur-dependent?

We characterized γ-cystathionase, rhodanese and 3-mercaptopyruvate sulfurtransferase activities in various regions of human brain (the cortex, thalamus, hypothalamus, hippocampus, cerebellum and subcortical nuclei) and human gliomas with II to IV grade of malignancy (according to the WHO classification). The human brain regions, as compared to human liver, showed low γ-cystathionase activity. The activity of rhodanese was also much lower and it did not vary significantly between the investigated brain regions. The activity of 3-mercaptopyruvate sulfurtransferase was the highest in the thalamus, hypothalamus and subcortical nuclei and essentially the same level of sulfane sulfur was found in all the investigated brain regions. The investigations demonstrated that the level of sulfane sulfur in gliomas with the highest grades was high in comparison to various human brain regions, and was correlated with a decreased activity of γ-cystathionase, 3-mercaptopyruvate sulfurtransferase and rhodanese. This can suggest sulfane sulfur accumulation and points to its importance for malignant cell proliferation and tumor growth. In gliomas with the highest grades of malignancy, despite decreased levels of total free cysteine and total free glutathione, a high ratio of GSH/GSSG was maintained, which is important for the process of malignant cells proliferation. A high level of sulfane sulfur and high GSH/GSSG ratio could result in the elevated hydrogen sulfide levels. Because of the disappearance of γ-cystathionase activity in high-grade gliomas, it seems to be possible that 3-mercaptopyruvate sulfurtransferase could participate in hydrogen sulfide production. The results confirm sulfur dependence of malignant brain tumors.

Piperlongumine Blocks JAK2-STAT3 Pathway to Inhibit Collagen-Induced Platelet Reactivity Independent of Reactive Oxygen Species

Piperlongumine (PL) from piper longum induces cancerous, but not normal cells to undergo reactive oxygen species (ROS)-mediated apoptosis and blocks the activation of the transcription factor Stat3 in breast cancer cells. Paradoxically, this ROS-inducing agent also inhibits platelet activation and aggregation induced by collagen, which is reported to induce ROS production in platelets. Several types of ROS are known to activate platelets. We test the hypothesis that PL inhibits platelet reactivity to collagen by regulating a non-transcriptional activity of Stat3 that we have recently characterized. Consistent with previous reports, we found that PL dose-dependently blocked collagen-induced platelet activation, aggregation, and thrombus formation with a maximal inhibition at 100 mM. PL was equally effective in blocking platelet aggregation induced by a collagen-related peptide, suggesting that it primarily targets the collagen-receptor GP VI. Several lines of evidence suggest that this inhibitory effect of PL on collagen-induced platelet reactivity is mediated through blocking JAK2-Stat3 signaling. First, PL blocked the tyrosine phosphorylation of JAK2 and Stat3 in collagen-stimulated platelets. This inhibitory effect was significantly reduced in platelets that were pretreated with the Stat3 inhibitor STA21. Second, the JAK2 inhibitor AG490 blocked collagen-induced platelet aggregation and thrombus formation. Third, PL did not inhibit the tyrosine phosphorylation of Syk, the key kinase for collagen-GP VI signaling in platelets. However, it was effective in blocking PLCγ2 phosphorylation, a regulatory profile that is identical to that of Stat3 inhibitors to block collagen-induced platelet activation. Consistent with a report on cancer cells, PL at 100 mM induced platelets to release ROS detected by two specific probes and by a mass spectrometric measurement of platelet free glutathione (GSH). This effect was additive to that of collagen. Quenching oxidants by 20 µM of GSH (cell non-permeable) and 0.5 mM of L-Cysteine (cell permeable) did not block the inhibitory effects of PL. Neither was Apocynin, an inhibitor to reduced nicotinamide adenine dinucleotide phosphate, which blocks collagen-induced ROS production in platelets. The PL treated platelets were viable and responsive to other platelet agonists, suggesting that PL did not induce platelets to undergo apoptosis. Finally, two PL analogs that are active (MZ370) and inactive (MZ306) in inhibiting the growth of breast cancer cells were synthesized and found to be equally active in blocking collagen-induced platelet activation. In summary, we made several observations that define a mechanism for PL to inhibit collagen-induced platelet reactivity and provide new information regarding differential effects of ROS on platelet reactivity to collagen. First, we demonstrate that the inhibitory activity of PL on collagen-induced platelet reactivity was primarily mediated by blocking the activation of JAK2 and Stat3, but not a direct inhibition of collagen-GP IV signaling. Second, both PL and collagen induced ROS release in platelets, but PL’s inhibitory activity was not affected by ROS. The results suggest that PL and collagen induce the release of different oxidants that have differential effects on platelets. Studying these differential effects may uncover new mechanisms of regulating platelet functions by oxidants. DisclosuresNo relevant conflicts of interest to declare.

Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

Protein S-glutathionylation is a posttranslational modification that links oxidative stimuli to reversible changes in cellular function. Protein–glutathione mixed disulfide (PSSG) is commonly quantified by reduction of the disulfide and detection of the resultant glutathione species. This methodology is susceptible to contamination by free unreacted cellular glutathione (GSH) species, which are present in 1000-fold greater concentration. A liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based method was developed for quantification of glutathione and glutathione disulfide (GSSG), which was used for the determination of PSSG in biological samples. Analysis of rat liver samples demonstrated that GSH and GSSG coprecipitated with proteins similar to the range for PSSG in the sample. The use of [13C2,5N]GSH and [13C4,5N2]GSSG validated these results and demonstrated that the release of GSH from PSSG did not occur during sample preparation and analysis. These data demonstrate that GSH and GSSG contamination must be accounted for when determining PSSG content in cellular/tissue preparations. A protocol for rinsing samples to remove the adventitious glutathione species is demonstrated. The fragmentation patterns for glutathione were determined by high-resolution mass spectrometry, and candidate ions for detection of PSSG on protein and protein fragments were identified.

Redox activities of mono- and binuclear forms of low-molecular and protein-bound dinitrosyl iron complexes with thiol-containing ligands

EPR, optical, electrochemical and stopped-flow methods were used to demonstrate that Fe(NO)2 fragments in paramagnetic mononuclear and diamagnetic binuclear forms of dinitrosyl iron complexes with glutathione are reversibly reduced by a two-electron mechanism to be further transformed from the initial state with d7 configuration into states with the d8 and d9 electronic configurations of the iron atom. Under these conditions, both forms of DNIC display identical optical and EPR characteristics in state d9 suggesting that reduction of the binuclear form of DNIC initiates their reversible decomposition into two mononuclear dinitrosyl iron fragments, one of which is EPR-silent (d8) and the other one is EPR-active (d9). Both forms of DNIC produce EPR signals with the following values of the g-factor: g⊥=2.01, g||=1.97, gaver.=2.0. M-DNIC with glutathione manifest an ability to pass into state d9, however, only in solutions with a low content of free glutathione. Similar transitions were established for protein-bound М- and B-DNIC with thiol-containing ligands.