Monochlorobimane Research Articles

Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay.

Thiol compounds including predominantly glutathione (GSH) are key components of redox homeostasis, which are involved in the protection and regulation of mammalian cells. The assessment of cell redox status by means of in situ analysis of GSH in living cells is often preferable over established assays in cell lysates due to fluctuations of the GSH pool. For this purpose, we propose a microplate assay with monochlorobimane (MCB) as an available fluorescent probe for GSH, although poorly detected in the microplate format. In addition to the new procedure for improved MCB-assisted GSH detection in plate-grown cells and its verification with GSH modulators, this study provides a useful methodology for the evaluation of cell redox status probed through relative GSH content and responsiveness to both supplemented thiols and variation in oxygen pressure. The roles of extracellular interactions of thiols and natural variability of cellular glutathione on the assay performance were emphasized and discussed. The results are of broad interest in cell biology research and should be particularly useful for the characterization of pathological cells with decreased GSH status and increased oxidative status as well as redox-modulating factors.

Role of Natural Antioxidant Products in Colorectal Cancer Disease: A Focus on a Natural Compound Derived from Prunus spinosa, Trigno Ecotype.

Colorectal cancer (CRC) is on the rise in industrialized countries, which is why it is important to find new compounds that are effective, with little or no adverse health effects. CRC arises from some cells of the epithelium which, following a series of genetic or epigenetic mutations, obtain a selective advantage. This work consists of a review on endogenous and exogenous antioxidant products that may have an efficacy in the treatment of CRC and an experimental study, in which the treatment was carried out with a natural compound with antitumor and antiproliferative activity, Prunus spinosa Trigno ecotype, patented by us, on HCT116 colorectal carcinoma cell line. The superoxide content was quantified after the treatments at different concentrations (2, 5, or 10 mg/mL) by means of the DHR123 probe; loss of the mitochondrial membrane potential with the tetramethylrodamine methyl ester (TMRM) cationic probe and reduced glutathione content (GSH) from monochlorobimane (MCB). This study revealed the importance of a careful choice of the concentration of the natural compound to be used in the CRC, due to the presence of a paradoxical effect, both antioxidant and pro-oxidant, depending on the different physiological conditions of the cell.

Automated quantification of live articular chondrocyte fluorescent staining using a custom image analysis framework.

The goal of this study was to develop, validate, and implement an image analysis framework to automatically analyze chondrocytes in 3D image stacks of cartilage acquired using a fluorescent confocal microscope. Source specimens consist of viable osteochondral tissue co-stained with multiple live-cell dyes. Our framework utilizes a seeded watershed-based algorithm to automatically segment individual chondrocytes in each 2D slice of the confocal image stack. The resulting cell segmentations are colocalized in 3D to eliminate duplicate segmentation of the same cell resulting from the visibility of fluorescence signal in multiple imaging planes, and the 3D cell distribution is used to automatically define the cartilage tissue volume. The algorithm then provides chondrocyte density data, and the associated segmentation can be used as a mask to extract and quantify per cell intensity of a secondary, functional dye co-staining the chondrocytes. The accuracy of the automated chondrocyte segmentation was validated against manual segmentations (average IOU = 0.79). When applied to a cartilage surrogate, this analysis framework estimated chondrocyte density within 10% of the true density and demonstrated a good agreement between framework's counts and manual counts (R2 = 0.99). In a real application, the framework was able to detect the increased dye signal of monochlorobimane (MCB) in chondrocytes treated with N-acetylcysteine (NAC) after mechanical injury, quantifying intracellular biochemical changes in living cells. This new framework allows for fast and accurate quantification of intracellular activities of chondrocytes, and it can be adapted for broader application in many imaging and treatment modalities, including therapeutic OA research.

Comparative analyses of glutathione system of vacuoles and leucoplasts isolated from the storage parenchyma cells of dormant red beetroots (Beta vulgaris L.)

The role of glutathione in the plant vacuole is still being debated. In the present paper, the redox state of glutathione and the activity of glutathione S-transferase (GST, E 2.5.1.18) in the vacuole compared to those in leucoplast have been studied. Organelles were isolated from dormant red beet (Beta vulgaris L.) taproots. Two generally used approaches have been applied to quantitatively assess the content of glutathione. Initially, levels of glutathione were measured in isolated organelles after labeling with monochlorobimane (MCB) and imaging with the use of confocal laser scanning microscopy. However, there are factors limiting the specificity of this method, because of which the resulting concentrations of vacuolar GSH have been underestimated. Another approach used was HPLC, which allows to simultaneously quantify the reduced glutathione (GSH) and glutathione disulfide (GSSG). The concentration of the total glutathione (GSHt) and GSSG in vacuoles determined with the aid of HPLC-UV was higher in comparison to that in the leucoplasts. The reduction potential (Eh) for the glutathione couple in the vacuoles was more positive (−163 mV), than that in plastids (−282 mV). The relatively rapid increase in fluorescence in the isolated vacuoles and plastids during MCB-labeling has indicated to the contribution of GSTs, since the conjugation of GSH to bimane is catalysed by these enzymes. The GST activity in the vacuoles has been assessed to be quite high compared to that of leucoplasts. The number of isoforms of GSTs also differed markedly in vacuoles and plastids. Collectively, our findings suggest the idea that the glutathione accumulated by central vacuole seems to contribute to the redox processes and to the detoxification, which can take place in this compartment.

Mapping glutathione utilization in the developing zebrafish (Danio rerio) embryo

Glutathione (GSH), the most abundant vertebrate endogenous redox buffer, plays key roles in organogenesis and embryonic development, however, organ-specific GSH utilization during development remains understudied. Monochlorobimane (MCB), a dye conjugated with GSH by glutathione-s-transferase (GST) to form a fluorescent adduct, was used to visualize organ-specific GSH utilization in live developing zebrafish (Danio rerio) embryos. Embryos were incubated in 20 μM MCB for 1 h and imaged on an epifluorescence microscope. GSH conjugation with MCB was high during early organogenesis, decreasing as embryos aged. The heart had fluorescence 21-fold above autofluorescence at 24 hpf, dropping to 8.5-fold by 48 hpf; this increased again by 72 hpf to 23.5-fold, and stayed high till 96 hpf (18-fold). The brain had lower fluorescence (10-fold) at 24 and 48 hpf, steadily increasing to 30-fold by 96 hpf. The sensitivity and specificity of MCB staining was then tested with known GSH modulators. A 10-min treatment at 48 hpf with 750 μM tert-butylhydroperoxide, caused organ-specific reductions in staining, with the heart losing 30% fluorescence, and, the brain ventricle losing 47% fluorescence. A 24 h treatment from 24-48 hpf with 100 μM of N-Acetylcysteine (NAC) resulted in significantly increased fluorescence, with the brain ventricle and heart showing 312% and 240% increases respectively, these were abolished upon co-treatment with 5 μM BSO, an inhibitor of the enzyme that utilizes NAC to synthesize GSH. A 60 min 100 μM treatment with ethacrynic acid, a specific GST inhibitor, caused 30% reduction in fluorescence across all measured structures. MCB staining was then applied to test for GSH disruptions caused by the toxicants perfluorooctanesulfonic acid and mono-(2-ethyl-hexyl)phthalate; MCB fluorescence responded in a dose, structure and age-dependent manner. MCB staining is a robust, sensitive method to detect spatiotemporal changes in GSH utilization, and, can be applied to identify sensitive target tissues of toxicants.

Molecular characterization of zebrafish Gstr1, the only member of teleost-specific glutathione S- transferase class

Glutathione S-transferases (GSTs) are multifunctional phase II detoxification enzymes with primary function of glutathione conjugation of various endogenous and exogenous compounds. Teleost-specific Gstr1 in zebrafish (Danio rerio) was previously shown to have high expression in toxicologically relevant tissues and high activity towards model substrates. The aim of this study was a detailed functional characterization of zebrafish Gstr1. Molecular docking analyses were used to get novel insight into structural characteristics of Gstr1 and elucidation of the mechanistic interactions with both GSH and various Gstr1 substrates or inhibitors. An initial screening inhibition assay performed using model fluorescence substrate monochlorobimane (MCB) revealed interactions of different endogenous compounds and environmentally relevant xenobiotics with zebrafish Gstr1. All interacting compounds were further analyzed to determine their inhibition type and Ki values. Our data revealed that pregnenolone, progesterone, testosterone, DHEAS and corticosterone competitively inhibited transformation of MCB by Gstr1 with the calculated Ki values in the range 14–26 μM, implying that these hormones are physiological substrates of zebrafish Gstr1. Estrogens had no effect on Gstr1 activity. Taurochenodeoxycholate (TCDC) expressed lower inhibition potency toward Gstr1 with the Ki value of 33 μM. Among tested xenobiotics tributyltin chloride and rifampicin non-enzymatically bound Gstr1 enzyme (the calculated Ki values are 0.26 μM and 65 μM, respectively) and inhibited its activity, showing that these compounds are reversible noncompetitive inhibitors of zebrafish Gstr1. Insecticide diazinon competitively inhibited Gstr1 activity with calculated Ki value of 27 μM, while other Gstr1-interacting insecticides, chlorpyrifos-methyl (CPF-methyl) and malathion, showed allosteric activation-like effect. Among tested pharmaceuticals, tetracycline, erythromycin and methotrexate demonstrated competitive type of inhibition with the calculated Ki values of 17.5, 36.5 and 29 μM, respectively. In summary, we suggest that zebrafish Gstr1 has an important role in steroidogenesis, metabolism and/or physiological actions of androgens, but not estrogens in fish. Finally, our results imply the role of Gstr1 in metabolism of xenobiotics and protection of fish against deleterious environmental contaminants such as organophosphate insecticides and pharmaceuticals.

283 - Mapping glutathione utilization in the developing zebrafish embryo

Glutathione (GSH), the most abundant endogenous redox buffer in vertebrates, plays a key role in organogenesis and embryonic development. Toxic insults during development are commonly neutralized by glutathionylation and excretion. Despite its importance, scant information exists regarding organ-specific GSH utilization during development. Monochlorobimane (MCB) is a dye that is conjugated with GSH by glutathione-s-transferase (GST) to form a fluorescent adduct. Here, we use MCB staining to visualize organ-specific location of GSH utilization in live developing zebrafish embryos at developmental timepoints commonly used in the OECD fish embryo acute toxicity test. At 24 hours post-fertilization (hpf), GSH conjugation with MCB was highest in the heart, brain and yolk, increasing in the gut and otolith at 48 hpf. By 72 hpf, MCB staining was higher in the endoderm-derived lineage, and at 96hpf it was brightest in the liver, gut, and gill arches. We then tested the sensitivity and specificity of MCB staining with known GSH modulators . A 10-minute treatment at 48 hpf with the prooxidant tert-butyl hydroperoxide, caused organ-specific reductions in MCB staining, with the heart and brain losing 30% fluorescence, and, the gut and myotome losing 50%. A 24 hour treatment from 24-48 hpf with the GSH precursor N-Acetylcysteine resulted in significantly increased fluorescence, with the brain showing a 640% increase and the heart showing a 350% increase. The gut and myotome showed a 100% increase. Ethacrynic acid, a highly specific GST inhibitor caused decreased fluorescence; the heart, brain and myotome lost 30% fluorescence and the gut lost 50%. 2-AAPA, an inhibitor of Glutathione Reductase, caused similar organ specific losses in fluorescence with the gut being more susceptible than the heart and brain. Our data indicate highly specific spatio-temporal GSH utilization, with different organs having differing resilience against GSH system disruptions during embryonic development.

Glutathione in Intact Vacuoles: Comparison of Glutathione Pools in Isolated Vacuoles, Plastids, and Mitochondria from Roots of Red Beet

Proportions between oxidized and reduced glutathione forms were determined in vacuoles isolated from red beet (Beta vulgaris L.) taproots. The pool of vacuolar glutathione was compared with glutathione pools in isolated plastids and mitochondria. The ratio of glutathione forms was assessed by approved methods, such as fluorescence microscopy with the fluorescent probe monochlorobimane (MCB), high-performance liquid chromatography (HPLC), and spectrophotometry with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). The fluorescence microscopy revealed comparatively low concentrations of reduced glutathione (GSH) in vacuoles. The GSH content was 104 μM on average, which was lower than the GSH levels in mitochondria (448 μM) and plastids (379 μM). The content of reduced (GSH) and oxidized (GSSG) glutathione forms was quantified by means of HPLC and spectrophotometric assays with DTNB. The glutathione concentrations determined by HPLC in the vacuoles were 182 nmol GSH and 25 nmol GSSG per milligram protein. The respective concentrations of GSH and GSSG in the plastids were 112 and 6 nmol/mg protein and they were 228 and 10 nmol/mg protein in the mitochondria. The levels of GSH determined with DTNB were 1.5 times lower, whereas the amounts of GSSG were, by contrast, 1.5–2 times higher than in the HPLC assays. Although the glutathione redox ratios depended to some extent on the method used, the GSH/GSSG ratios were always lower for vacuoles than for plastids and mitochondria. In vacuoles, the pool of oxidized glutathione was higher than in other organelles.

Melatonin Modifies Histone Acetylation During In Vitro Maturation of Mouse Oocytes.

Objective We evaluated the effect of melatonin, as a potent antioxidant agent, on glutathione (GSH) and reactive oxygen species (ROS) levels, as well as histone H3 lysine 9 (H3K9), and H4 lysine 12 (H4K12) acetylation when added to oocytes culture medium. Materials and Methods In this experimental study, two in vitro and in vivo groups were used. In the in vitro group, cumulus oocyte complexes (COCs) from the ovaries of B6D2F1 mice were cultured in maturation medium containing two doses of melatonin (10-9 and 10-6 M) and without melatonin [control group treated with dimethyl sulfoxide (DMSO)] for 22-24 hour. The cumulus expansion and nuclear status were monitored by an inverted microscope. Next, COCs were isolated from the oviducts of superovulated mice and studied as the in vivo group. In in vitro and in vivo matured oocytes, GSH and ROS levels were assessed by monochlorobimane (MCB) and 2-7-dichlorodihydrofluorescein diacetate (H2DCFDA) staining, respectively. Changes in histone acetylation were examined by immunofluorescent staining with specific antibodies against acetylated H3K9 and H4K12. Results The H4K12 acetylation and ROS levels were significantly higher in the oocytes matured in the in vitro group compared to the in vivo group (P<0.05). Furthermore, glutathione levels in the in vitro group were considerably lower than that of the in vivo group (P<0.05). Melatonin at the concentration of 10-6 M had the most substantial effect on nuclear maturation and histone acetylation as well as glutathione and ROS levels in the in vitro group (P<0.05). ConclusionExogenous melatonin improves the competence of mouse oocytes during in vitro maturation (IVM).

Selective fluorescence detection method for selenide and selenol using monochlorobimane

The low redox potential of selenide and selenol is physiologically important, as it confers efficient catalytic abilities to selenoproteins. Quantitative determination of selenol and selenide provide important clues for understanding the metabolism and physiological function of selenium. However, selective detection of selenol and selenide is extremely difficult because of their chemical similarity to thiol and sulfide. In this study, we established a highly sensitive, selective, quantitative, and simple method for detection of selenol and selenide, using a reaction with monochlorobimane (MCB), followed by ethyl acetate extraction of the product syn-(methyl,methyl)bimane. We analyzed selenide production from selenite, catalyzed by human glutathione reductase, and also determined selenide and selenol concentrations in Hepa1-6 cells using the MCB method, to demonstrate its practical applications. This study provides a new tool for selenium detection in biology.

Involvement of glutathione in β-cyclodextrin-hemin complex-induced lateral root formation in tomato seedlings

β-cyclodextrin-hemin complex (β-CDH) was shown to induce lateral root (LR) formation in tomato. However, the molecular mechanism is still elusive. In this report, the role of reduced glutathione (GSH) in the induction of lateral root triggered by β-CDH was investigated. Similar to the responses of β-CDH, exogenously applied with 0.1 mο GSH not only increased endogenous GSH content determined by spectrophotography and the monochlorobimane (MCB)-dependent fluorescent analysis, but also induced, thereafter, LR formation. Meanwhile, both β-CDH- and GSH-induced lateral root primordia (LRP) exhibited a similar accelerated anatomic structure. Above inducible responses were blocked significantly when the L-buthionine-(S,R)-sulfoximine (BSO), a potent and specific inhibitor of the enzyme catalyzing the first step of GSH biosynthesis, was separately applied. Upon β-CDH treatment, the changes of endogenous GSH content determined by spectrophotography and fluorescent analysis were consistent with the transcripts of two GSH synthetic genes, GSH1 and GSH2 encoding γ-glutamyl cysteine synthetase and glutathione synthetase, respectively. Exogenously applied with β-CDH could rescue N-1-naphthylphthalamic acid (NPA; IAA depletion)-triggered inhibition of LR formation. Further molecular evidence revealed that both β-CDH and GSH modulated gene expression of cell cycle regulatory genes (CYCA2;1, CYCA3;1, CYCD3;1, and CDKA1) and auxin signaling genes (ARF7 and RSI-1), six marker genes responsible for LR formation. By contrast, above changes were sensitive to the co-treatment with BSO. All together, these results suggest a role for GSH in the regulation of tomato LR development triggered by β-CDH.

Evaluation of Mouse Oocyte In Vitro Maturation Developmental Competency in Dynamic Culture Systems by Design and Construction of A Lab on A Chip Device and Its Comparison with Conventional Culture System.

In conventional assisted reproductive technology (ART), oocytes are cultured in static microdrops within Petri dishes that contain vast amounts of media. However, the in vivo environment is dynamic. This study assesses in vitro oocyte maturation through the use of a new microfluidic device. We evaluate oocyte fertilization to the blastocyct stage and their glutathione (GSH) contents in each experimental group. In this experimental study, we established a dynamic culture condition. Immature oocytes were harvested from ovaries of Naval Medical Research Institute (NMRI) mice. Oocytes were randomly placed in static (passive) and dynamic (active) in vitro maturation (IVM) culture medium for 24 hours. In vitro matured oocytes underwent fertilization, after which we placed the pronucleus (PN) stage embryos in microdrops and followed their developmental stages to blastocyst formation after 3 days. GSH content of the in vitro matured oocytes was assessed by monochlorobimane (MCB) staining. We observed significantly higher percentages of mature metaphase II oocytes (MII) in the passive and active dynamic culture systems (DCS) compared to the static group (P<0.01). There were significantly less mean numbers of germinal vesicle (GV) and degenerated oocytes in the passive and active dynamic groups compared to the static group (P<0.01). Fertilization and blastocyst formation rate in the dynamic systems were statistically significant compared to the static cultures (P<0.01). There was significantly higher GSH content in dynamically matured oocytes compared to statically matured oocytes (P<0.01). Dynamic culture for in vitro oocyte maturation improves their developmental competency in comparison with static culture conditions.

Cellular glutathione content in the organ of Corti and its role during ototoxicity.

Glutathione (GSH) is the major scavenger of reactive oxygen species (ROS) inside cells. We used live confocal imaging in order to clarify the role of GSH in the biology of the organ of Corti, the sensory epithelium of the cochlea, before, during and after the onset of hearing and in ~1 year old mice. GSH content was measured using monochlorobimane (MCB), a non-fluorescent cell permeant bimane that reacts with GSH, forming a fluorescent adduct through a reaction catalyzed by glutathione-S-transferase. GSH content increased significantly in inner hair cells during maturation in young adult animals, whereas there was no significant change in the outer hair cells. However, the GSH content in inner hair cells was significantly reduced in ~1 year old mice. The GSH content of supporting cells was comparatively stable over these ages. To test whether the GSH content played a significant protective role during ototoxicity, GSH synthesis was inhibited by buthionine sulfoximine (BSO) in organotypic cochlear explant cultures from immature mice. BSO treatment alone, which reduced GSH by 65 and 85% in inner hair cells and outer hair cells respectively, did not cause any significant cell death. Surprisingly, GSH depletion had no significant effect on hair cell survival even during exposure to the ototoxic aminoglycoside neomycin. These data suggest that the involvement of ROS during aminoglycoside-induced hair cell death is less clear than previously thought and requires further investigation.

Enhanced Growth-inhibitory Effect of Microemulsified Curcumin Formulation in Human Prostate Cancer LNCaP Cells

Aim: To assess the effect of curcumin microemulsified with non-ionic surfactant surfynol 465 W or dispersed using edible oils on prostate LNCaP cancer cell viability and glutathione status.Methodology: LNCaP cells were treated for 72-144 hr with curcumin dissolved with fish or corn oil and microemulsified using non-ionic surfactant surfynol 465 W; alternatively LNCaP cells were treated with curcumin directly dispersed in fish or corn oil (0-50 μM) for 24 -72-144 hr. Cell viability was determined using resazurin (Vision blueTM) fluorescence assay. Glutathione status was determined by monochlorobimane (MCB) assay.Results: Treatment with 0-34 μM of microemulsified curcumin produced moderate cytotoxic effect on LNCaP cells, no 50% reduction of cell viability was observed graphically. However, when LNCaP cells were treated with curcumin dispersed with corn oil the concentration or 50% reduction of cell viability (IC50) was 12-45 μM. Similarly for cells treated with curcumin dispersed with fish oil, the IC50 was between 20-40 μM. Cytotoxic doses of curcumin dispersed with corn or fish oil increased GST status in cells by 272-656% (p =<0.01).Conclusion: Microemulsified curcumin formulation prepared using fish or corn oil and surfynol 465 W surfactant had an inhibitory effect on viability of LNCaP cells as did direct dispersion of curcumin in fish or corn oil coupled with the ability for inducing intracellular GST status in LNCaP cells. Keywords : Curcumin; phase II enzymes; prostate cancer cells (LNCaP); glutathione (GSH); glutathione-S-transferase (GST); monochlorobimane (MCB); resazurin; fish oil; corn oil.- See more at: http://www.sciencedomain.org/abstract.php?iid=881&id=14&aid=7465#sthash.GPXT4ChK.dpuf

Characterization of glutathione-S-transferases in zebrafish (Danio rerio)

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28mM (Gstp2) to 3.69mM (Gstm3), while affinity toward MCB was in the range of 5μM (Gstt1a) to 250μM (Gstp1). Affinity toward GSH varied from 0.27mM (Gstz1) to 4.45mM (Gstt1a). Turnover number for CDNB varied from 5.25s−1 (Gstt1a) to 112s−1 (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the first comprehensive analysis of GST superfamily in zebrafish, presents new insight into distinct functions of individual Gsts, and offers methodological protocols that can be used for further verification of interaction of environmental contaminants with fish Gsts.

Curcumin restores glutathione-S-transferase activity for LNCaP prostate cancer cells

Prostate cancer is a leading cause of death in males aged fifty and over. Glutathione transferase (GST) activity is depressed in prostate cancer cells. The aim of this study was to assess GST reactivation in LNCaP prostate cancer cells treated with curcumin or 5-azacitidine (5-Aza) which is a known hypomethylation agent. GST activity was determined using monochlorobimane (MCB). Cell viability was assessed with resazurin (Vision blue TM) or 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium- bromide (MTT). From the results, treatment with >5 μM of curcumin or 5-Aza for 3 or 6 days depressed LNCaP cell viability. The concentrations of curcumin leading to 50% reduction of LNCaP cell viability (IC50) was 10-25 μM or 2-3 μM for 3 days or 6 days of treatment, respectively. The IC50 with 5-Aza was 17-23 μM (3 days) or 50-52 μM (6 days).Combination treatment using curcumin and 5-Aza showed complimentary interactions affecting cell viability. Low levels of curcumin or 5-Aza had no effect on GST activity. By contrast, cytotoxic doses of curcumin or 5-Aza increased GST activity by 450-750 % (3days) or 161-2800 % (6days). In conclusion, GST reactivation was feasible but only when LNCaP prostate cancer cells were treated with cytotoxic doses of curcumin or 5-azacytidne.

Coronary Artery Spasm Related to Thiol Oxidation and Senescence Marker Protein-30 in Aging

Senescence marker protein-30 (SMP30) decreases with aging, and SMP30 knockout (KO) mice show a short life with increased oxidant stress. We assessed the effect of oxidant stress with SMP30 deficiency in coronary artery spasm and clarify its underlying mechanisms. We measured vascular responses to acetylcholine (ACh) and sodium nitroprusside (SNP) of isolated coronary arteries from SMP30 KO and wild-type (WT) mice. In SMP30 KO mice, ACh-induced vasoconstriction occurred, which was changed to vasodilation by dithiothreitol (DTT), a thiol-reducing agent. However, Nω-nitro-L-arginine-methyl ester, nitric oxide (NO) synthase inhibitor, or tetrahydrobiopterin did not change the ACh response. In isolated coronary arteries of WT mice, ACh-induced vasodilation occurred. Inhibition of glutathione reductase by 1, 3-bis(2-chloroethyl)-1-nitrosourea decreased ACh-induced vasodilation (n=10, p<0.01), which was restored by DTT. To evaluate the thiol oxidation, we measured the fluorescence of monochlorobimane (MCB) in coronary arteries, which covalently labels the total. The fluorescence level to MCB decreased in SMP30 KO mice, but with DTT treatment restored to a level comparable to that of WT mice. The reduced glutathione and total thiol levels were also low in the aorta of SMP30 KO mice compared with those of WT mice. Administration of ACh into the aortic sinus in vivo of SMP30 KO mice induced coronary artery spasm. The thiol redox state is a key regulator of endothelial NO synthase activity, and thiol oxidation was associated with endothelial dysfunction in the SMP30 deficiency model. These results suggest that chronic thiol oxidation by oxidant stress is a trigger of coronary artery spasm, resulting in impaired endothelium-dependent vasodilation.

Astrocytes Prevent Ethanol Induced Apoptosis of Nrf2 Depleted Neurons by Maintaining GSH Homeostasis

Glutathione (GSH), a major cellular antioxidant protects cells against oxidative stress injury. Nuclear factor erythroid 2-related factor 2 (NFE2L2/Nrf2) is a redox sensitive master regulator of battery of antioxidant enzymes including those involved in GSH antioxidant machinery. Earlier we reported that ethanol (ETOH) elicits apoptotic death of primary cortical neurons (PCNs) which in partly due to depletion of intracellular GSH levels. Further a recent report from our laboratory illustrated that ETOH exacerbated the dysregulation of GSH and caspase mediated cell death of cortical neurons that are compromised in Nrf2 machinery (Narasimhan et al., 2011). In various experimental models of neurodegeneration, neuronal antioxidant defenses mainly GSH has been shown to be supported by astrocytes. We therefore sought to determine whether astrocytes can render protection to neurons against ETOH toxicity, particularly when the function of Nrf2 is compromised in neurons. The experimental model consisted of co-culturing primary cortical astrocytes (PCA) with Nrf2 downregulated PCNs that were exposed with 4 mg/mL ETOH for 24 h. Monochlorobimane (MCB) staining followed by FACS analysis showed that astrocytes blocked ETOH induced GSH decrement in Nrf2-silenced neurons as opposed to exaggerated GSH depletion in Nrf2 downregulated PCNs alone. Similarly, the heightened activation of caspase 3/7 observed in Nrf2-compromised neurons was attenuated when co-cultured with astrocytes as measured by luminescence based caspase Glo assay. Furthermore, annexin-V-FITC staining followed by FACS analysis revealed that Nrf2 depleted neurons showed resistance to ETOH induced neuronal apoptosis when co-cultured with astrocytes. Thus, the current study identifies ETOH induced dysregulation of GSH and associated apoptotic events observed in Nrf2-depleted neurons can be blocked by astrocytes. Further our results suggest that this neuroprotective effect of astrocyte despite dysfunctional Nrf2 system in neurons could be compensated by astrocytic GSH supply.

Comparative kinetic model of fluorescence enhancement in selective binding of monochlorobimane to glutathione

The highly specific binding of fluorescent label monochlorobimane (MCB) to thiols has been investigated using a tripeptide thiol, glutathione (GSH), a biomolecule protecting cells against oxidative stress and damaging radicals. Due to the reported uneven distribution of MCB in living cells and compartmentalization of GSH in cells, we have explored binding of MCB to GSH in a higher concentration range than normally applied in fluorimetric analysis. Since at higher concentrations, a concentration quenching may interfere with the analysis and because MCB binding to GSH is controlled by kinetics, a special comparative kinetic model of the binding reaction has been developed and utilized in fluorimetric determinations of GSH and in an independent non-optical isothermal reaction-heat measurement. The rate constants: k 1 = 0.274 ± 0.012 and 0.315 ± 0.004 s −1 M −1, have been obtained from fluorescence and microcalorimetric methods, respectively. The molar enthalpy of binding, Δ H = −48.46 ± 0.61 kJ/mol, has been determined. The low value of k 1 obtained from fluorimetric measurements precludes concentration quenching for MCB concentrations up to 330 μM and GSH concentrations up to 5 mM. The DFT quantum mechanical calculations have been performed to elucidate changes in vibrational characteristics of bimane fused rings upon GSH binding. The predicted relaxing of rings vibrational modes has been confirmed in Raman scattering spectra and is consistent with the increased fluorescence emission of the adduct. Plausible elucidation of the mechanism of this process is discussed.

Degradation of Glutathione S-Conjugates in Physcomitrella patens is Initiated by Cleavage of Glycine

Glutathione-dependent detoxification is a key pathway that allows plants to efficiently remove toxic compounds like heavy metals or electrophilic xenobiotics. Under persistent exposure to toxins plants need to respond to continuous demand with efficient synthesis of glutathione (GSH) and ideally fast and efficient removal of potentially toxic glutathione S-conjugates. With the aim of studying the respective degradation pathway in Physcomitrella patens we initially characterized fluorescence labeling of protonema cultures with GSH-specific xenobiotic monochlorobimane (MCB). Incubation of protonema with 200 μM MCB for 24 h resulted in a steady increase of total bimane label, which was not confined to glutathione S-bimane (GS-B), but predominantly present in γ-glutamylcysteine S-bimane (γ-EC-B) and cysteine S-bimane (Cys-B). Pulse-chase experiments identified γ-EC-B and Cys-B as degradation products of GS-B, suggesting initial cleavage of the C-terminal glycine, followed by cleavage of the γ-glutamyl bond. The amount of GS-B formed, increased linearly at 90 nmol GSH g fw⁻¹ h⁻¹ for 24 h and after ∼1.5 h already surpassed the amount of GSH present in control protonema. This demand-driven biosynthesis of GSH depends on sufficient supply of sulfate in the incubation medium.