Inhibitory Effect Of Glutathione Research Articles

Manganese coordination polymer nanoparticles with excellent oxidase-like activity for the rapidly and selectively colorimetric detection of glutathione

Novel kinds of manganese coordination polymer nanoparticles (Mn-IMZs CPNs) were fabricated through self-assemble of imidazole with Mn2+ ions in aqueous solution. The prepared Mn-IMZs CPNs, with remarkable stability, exhibit excellent oxidase-like activity that can effectively catalyze the conversion of the dissolved O2 to O2•−, turning the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB (ox-TMB). Based on the inhibitory effect of glutathione (GSH) on the TMB oxidation, a rapid and highly sensitive colorimetric method for the detection of GSH was developed, and the linear ranges are 1.25–50 μM and 50–112.5 μM. Besides, the discriminative detection of GSH from cysteine was achieved by using the time lag between N-ethylmaleimide on the destruction of thiol compounds. Finally, the developed method was successfully applied in the quantification of GSH in tablets and rabbit plasma with high accuracy and good recovery.

2DG Affects Liver Cancer Cell Ferroptosis By Affecting

Cancer cells are characterized by high proliferation but at the expense of enhanced metabolic rates. If the activeoxygen content in the cell exceeds the average level, cancer cells activate antioxidant defenses against this increase.Ferroptosis occurs when glutathione levels are reduced due to a lack of cysteine in the cell. Ferroptosis is a new type ofasymptomatic apoptosis. We chose to investigate the sensitivity of cancer cells to the effect of 2DG on ferroptosis. Theseinclude some effects of 2DG on intracellular NADPH solubility and NADPH’s role in converting cystine to cysteine incancer cells. The effect of cysteine on glutathione, the inhibitory effect of glutathione on ROS in cancer cells, and theaccumulation of ROS can lead to ferroptosis in cancer cells.

Inhibitory effect of glutathione S-transferase A3 in the progression of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most common cutaneous malignancy with an incidence rate increasing each year. Glutathione S-transferase A3 (GSTA3), a member of the glutathione S-transferase family, is considered an antioxidative protease, but its role in cSCC remains unclear. The present study was designed to explore the effect of GSTA3 on cSCC. Through previous systematic studies, we screened GSTA3 to be a key gene with lower expression in cSCC. In the present study, we selected cSCC tissues and para-carcinoma tissue specimens from 20 patients in plastic surgery department. A431 cells were treated with GSTA3 transfection. The cell proliferation, apoptosis, colony formation, and cell migration as well as invasion were examined, respectively. And the expressions of GSTA3, TGF-β/Smad, and HIF-1α signalings were measured by Western blot and qRT-PCR. GSTA3 was downregulated in both cSCC tissues and A431 cells. Additionally, overexpression of GSTA3 induced a phenotype with a lower degree of malignancy, while GSTA3 silencing induced more malignant phenotypes, including cell proliferation, colony formation, apoptosis, migration, and invasion. Moreover, we found that the TGF-β/Smad2/3 and HIF-1α signalings were activated in cSCC under hypoxic conditions. GSTA3 could inhibit cSCC progression through suppression of the TGF-β/Smad and HIF-1α signalings. Therefore, GSTA3 may prove to be a prospective therapeutic target for cSCC.

Timely Addition of Glutathione for Its Interaction with Deoxypentosone To Inhibit the Aqueous Maillard Reaction and Browning of Glycylglycine-Arabinose System.

The inhibitory effects of glutathione (GSH) and oxiglutathione (GSSG) on Maillard browning were compared, and it was clarified that free sulfhydryl was the key substance for the inhibition. The Amadori rearrangement product (ARP) derived from glycylglycine (Gly-Gly) and arabinose (Ara) was prepared by aqueous Maillard reaction, and LC-MS/MS was used to investigate the reaction products of GSH and purified ARP. Reaction between GSH and deoxypentosone (DP) was found to alter the pathway of aqueous Maillard reaction, which reduced the production of glyoxal, methylglyoxal, and furfural and thereby inhibited the formation of melanoidins. To determine the optimal conditions for browning inhibition, a stepwise increase of temperature was used to prepare Maillard reaction products (MRPs). The results showed that the optimum browning inhibitory effect was obtained by adding GSH after Gly-Gly and Ara heating at 80 °C for 60 min.

Inhibitory effects of glutathione 2 on excessive migration of human lens epithelial cells induced by hyperoxia

Objective To observe the influences of hyperoxia on the migration of human lens epithelial cells (HLECs) and the inhibitory effects of glutathione 2 (Grx2) on excessive migration of HLECs induced by hyperoxia. Methods The HLECs were divided into the hyperoxic model group and normoxic control group.HLECs were cultured in 80% oxygen mixture for 2 days to establish the oxidative damage model of HLECs induced by hyperoxia in vitro (hyperoxia model group), and cultured in normoxia mixture as the normoxic control group.Cell migration was observed by scratch test, cytoskeleton morphology was observed by phalloidin staining, and Grx2 mRNA level was detected by real-time quantitative PCR.HLECs in the hyperoxic model group and the normoxic control group were subdivided into empty plasmid transfection group and Grx2 plasmid transfection group, respectively.The migration behavior and cytoskeleton changes of hyperoxic model cells in different transfection groups were observed.MitoSOX staining was used to detect reactive oxygen species (ROS) content of mitochondria under flow cytometry, and JC-1 staining was used to observe the changes of mitochondrial membrane potential under laser-scanning confocal microscopy. Results In the process of cell culture, the cells grew well.Twenty-four hours and 48 hours after scratching, the scratch distance of the hyperoxic model group was smaller than that of the normoxic control group, with significant differences between them (both at P<0.05). The cell circumference of the hyperoxic model group was increased compared with that of the normoxic group, with a significant difference between them (t= -2.254, P<0.05). After 2 days culture, the relative expression level of Grx2 mRNA in the hyperoxic model group was 0.54±0.11, which was significantly lower than that in the normoxic control group (t=7.128, P<0.01). The transfection efficiency of Grx2 was higher than 90%.At 24 hours and 48 hours after scratching, the scratch distance in the Grx2 transfected hyperoxic model group was wider than that in the empty plasmid transfected hyperoxic model group, with significant differences between them (both at P<0.05). The relative expression level of Grx2 mRNA in Grx2 transfected cells was up-regulated compared with that in empty plasmid transfected cells, with a significant difference between them (P<0.01). Compared with the normoxic control group, the cell circumference of the hyperoxic model group was decreased, with a significant difference between them (P<0.05). The relative mean fluorescence intensity of mitochondrial ROS in the Grx2 transfected hyperoxic model group was lower than that in the empty plasmid transfected hyperoxic model group, with a significant difference between them (P<0.05). Under the hyperoxic condition, the red/green fluorescence intensity of Grx2 transfected group was higher than that of the empty plasmid transfected group, with a significant difference between them (P<0.01). Conclusions Grx2 may inhibit the excessive migration of HLECs under hyperoxia by regulating oxidative stress in mitochondria. Key words: Lens epithelial cells; Hyperoxia; Glutathione 2; Cell migration; Cell culture

Assessment of genotoxicity of catecholics using impedimetric DNA-biosensor

The potential toxicity of catecholics is a big concern, because the catechol-derived semiquinone radical after the oxidation of catechol (CA) can donate an H-atom to generate quinone, and during this process a superoxide anion radical may be produced. Considering the fact that catecholics are highly consumed in our daily life and some drugs also contain one or more CA moieties, we speculate that CA's toxicity might not be insurmountable. Therefore, finding approaches to investigate catecholics potential toxicity is of great significance. Here in, an electrochemical protocol for direct monitoring of genotoxicity of catecholics is described. CA encapsulated on MWCNTs (CA@MWCNT) through continuous cyclic voltammetric on the surface of pencil graphite electrode (PGE). Subsequently, a DNA functionalized biosensor (DNA/CA@MWCNT/PGE) was prepared and characterized for the detection and the investigation of DNA damage induced by radicals generated from catecholics. The change in the charge transfer resistance (Rct) after the incubation of the DNA biosensor in the damaging solution for a certain time was used as an indicator for DNA damage. Incubation of DNA-modified electrode with CA solution containing Cu(II), Cr(VI) and Fe(III) has been shown to result in oxidative damage to the DNA and change in the electrochemical properties. It was found that the presence of Cu(II), Cr(VI) and Fe(III) in solution caused damage to DNA. The inhibitory effect of glutathione and plumbagin on the CA-mediated DNA damage has also been investigated using the biosensor. The minimum concentration of the metal ions for CA induced DNA damage was investigated. Recognition of suitable matrixes for CA-mediated DNA damage can be assessed using proposed DNA biosensor. Such direct monitoring of the DNA damage holds great promise for designing new biosensors with modification of the biosensor with different damaging agents.

Inhibitory Effect of Glutathione on Oxidative Liver Injury Induced by Dengue Virus Serotype 2 Infections in Mice

The pathogenesis of dengue virus (DV) infection has not been completely defined and change of redox status mediated by depletion of glutathione (GSH) in host cell is a common result of viral infection. Our previous study has demonstrated that DV serotype 2 (DV2) infection alters host intracellular GSH levels, and exogenous GSH inhibits viral production by modulating the activity of NF-κB in HepG2 cells. GSH is the most powerful intracellular antioxidant and involved in viral infections. Thus, this study was to investigate whether DV2 infection can induce alteration in redox balance and effect of GSH on the disease in HepG2 xenografts SCID mice. Our results revealed that mice infected with DV2 showed alterations in oxidative stress by increasing the level of malondialdehyde (MDA), an end product of lipid peroxidation, and GSSG/GSH ratio. DV2-infected mice also showed a decrease in the activity of catalase (CAT) and total superoxide dismutase (T-SOD) in the serum and/or observed organs, especially the liver. Moreover, DV2 infection resulted in elevated serum levels of the cytokines tumor necrosis factor-α and interlukin-6 and obvious histopathological changes in the liver. The administration of exogenous GSH significantly reversed all of the aforementioned pathological changes and prevented significant liver damage. Furthermore, in vitro treatment of HepG2 cells with antioxidants such as GSH inhibited viral entry as well as the production of reactive oxygen species in HepG2 cells. These results suggest that GSH prevents DV2-induced oxidative stress and liver injury in mice by inhibiting proinflammatory cytokine production, and GSH and may be a promising therapeutic agent for prevention of oxidative liver damage during DV infection.

Partial characterization of polyphenoloxidase from a hybridized wheat (Triticum aestivum L.)

Polyphenol oxidase was extracted and partially purified from wheat leaves by a procedure that included ammonium sulfate fractionation followed by dialysis and gel filtration chromatography. These procedures led to 35.21-fold purification with 17.65% recovery. Optimum pH, temperature, and ionic strength were determined with six substrates. Some kinetic properties of the enzyme such as Vmax,KM, and kcat were calculated for the substrates. The kcat/KM values of the PPO for catechol, catechin, pyrogallol, l-dopa, dopamine, and 4-methyl catechol were 31408, 31167, 28404, 15378, 4865, and 4967 mM/min, respectively. The best substrate of wheat PPO was found to be catechol. The native molecular weight of the PPO was estimated to be 243 kDa based on its mobility in gel filtration column. The inhibitory effects of glutathione, sodium azide, ascorbic acid, oxalic acid, l-cysteine, and thiourea on the reaction catalyzed by the enzyme were tested, and I50 values were estimated to be 8.0 mM, 10.12 mM, 11.18 mM, 77.33 mM, 183 mM, and 413 mM, and Ki constants were also calculated as 0.416 ± 0.244 mM, 0.317 ± 0.208 mM, 0.820 ± 0.111 mM, 13.80 ± 1.179 mM, 14.10 ± 5.069 mM, and 130 ± 62.45 mM, respectively, by means of Lineweaver–Burk graphs. The most effective inhibitor was glutathione. Glutathione, sodium azide, oxalic acid, and thiourea were competitive inhibitors, whereas ascorbic acid and l-cysteine were also noncompetitive inhibitors.

Inhibitory effects of glutathione on dengue virus production

Reduced glutathione (GSH) is the most powerful intracellular antioxidant and also involved in viral infections. The pathogenesis of dengue virus (DV) infection has not been completely clarified. This study investigated the relationship between DV serotype 2 (DV2) infections and host intracellular GSH content. Results showed infection with DV2 resulted in a decrease in intracellular GSH, which caused NF-κB activation and increased DV2 production. Supplemental GSH significantly inhibited activation of NF-κB, resulting in a decreased production of DV2 in HepG2 cells. Furthermore, high activity of NF-κB and increased production of DV2 was observed in HepG2 cells treated with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. In conclusion, DV2 infection could reduce host intracellular GSH concentration and benefited from this process. Supplemental GSH could inhibit viral production, indicating GSH might be valuable in the prevention and treatment of DV2 infection.

The inhibitory effect of glutathione on buccal enzymatic degradation of therapeutic peptides (leu-enkephalin, luteinizing hormone-releasing hormone and pituitary adenylate cyclase activating peptide)

The purpose of this study was to evaluate the potential of glutathione (GSH) as an auxiliary agent for buccal drug delivery systems to protect therapeutic peptides from enzymatic degradation. Stability of the model peptide drugs leu-enkephalin (Leu-Enke), luteinizing hormone-releasing hormone (LHRH) and pituitary adenylate cyclase activating peptide (PACAP) in the absence and presence of GSH on degradation by buccal enzymes was investigated. Inhibition studies were performed with isolated aminopeptidase N representing the most abundant peptidase on the buccal mucosa and on excised intact buccal mucosa from pigs. Enzymatic degradation of peptides was quantified by HPLC. The absorption of GSH across freshly excised porcine buccal mucosa was studied using Ussing type diffusion chambers. The proteolytic activity of isolated aminopeptidase N was significantly inhibited by the addition of 2% (m/v) GSH. Without the addition of enzyme inhibitors Leu-Enke was completely degraded by aminopeptidase N, whereas 34.8 ± 27.2% of LHRH and 74.2 ± 12.0% of PACAP remained intact. In contrast, by the addition of 2% (m/v) GSH 33.2 ± 2.2% of Leu-Enke, 88.6 ± 12.8% of LHRH and 97.0 ± 6.2% of PACAP remained stable after 5 h of incubation. Additionally, it was shown that only 0.2 ± 0.0% of GSH was able to permeate the buccal mucosa within 5 h, demonstrating that GSH remains concentrated at the site of drug absorption. After incubation for 5 h on the buccal mucosa 0.8 ± 0.2% of Leu-Enke, 73.2 ± 6.6% of LHRH and 82.9 ± 8.8% of PACAP remained stable. In contrast, in the presence of 2% (m/v) GSH 103.1 ± 12.7% of Leu-Enke, 98.5% ± 5.4% of LHRH and 97.2 ± 4.2% of PACAP were stable. According to these results GSH seems to represent a promising inhibitor for the buccal administration of peptide drugs.

Absorption Enhancement of a Protein Drug by Nitric Oxide Donor: Effect on Nasal Absorption of Human Granulocyte Colony-Stimulating Factor

The objective of this study was to evaluate the effect of a nitric oxide (NO) donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the nasal absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rabbits and to evaluate the irritation (cytotoxicity) potential of the NO donor on the mucosal membrane using a cultured cell system (strain KB, human epidermoid carcinoma of the floor of the mouth). Significantly higher serum G-CSF concentration and increased total leukocyte count in the peripheral blood were observed after coadministration of rhG-CSF (100 μg/kg) with SNAP at various doses (0.3-3.3 mg/kg). The serum G-CSF concentration and the increased total leukocyte count were markedly decreased by the presence of the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazole-l-oxyl 3-oxide sodium salt (carboxy-PTIO), in combination with rhG-CSF and SNAP. However, no significant inhibitory effect of glutathione (peroxynitrite scavenger) on the absorption-enhancing effect of SNAP was observed. These results suggest that carboxy-PTIO inhibits the absorption-enhancing effect of NO released from SNAP. We found that SNAP has a very low potential for cytotoxicity, as evaluated by the cell detachment assay, release of lactate dehydrogenase (LDH) from cultured cells and morphological observations of nasal tissue of rabbits. It is concluded that a NO donor such as SNAP is a promising absorption enhancer for nasal protein-drug delivery.

Differential non-redox inhibitory effects of glutathione against protein kinase C isozyme family members.

Glutathione (GSH) and the GSH metabolic precursor N-acetylcysteine (NAC) are potent antioxidants that have clear potential either as cancer chemopreventive agents or as lead compounds for new cancer chemopreventive agents. The potential efficacy of GSH and NAC in clinical cancer chemoprevention is suggested by their antagonism of tumor promotion in animal models. Protein kinase C (PKC) is an isozyme family that plays a critical role in phorbol ester-mediated tumor promotion. We recently found that GSH and NAC exert direct inhibitory effects against a purified PKC isozyme mixture by a mechanism that did not involve their antioxidant properties. In this report, we characterize non-redox inhibitory effects of glutathiones on PKC isozymes that have been shown to produce partially or fully transformed phenotypes in mammalian cells. We show that GSH, NAC, and oxidized GSH analogs exert potent inhibition of the isozyme cPKC-ç and are somewhat less effective against cPKC- 1. In contrast, the oncogenic isozyme nPKC-â was unaffected by NAC, and it was inhibited by GSH and oxidized GSH analogs very modestly. Our results suggest that the potential impact of non-redox GSH/NAC-mediated PKC inhibition on cellular responses to tumor promoters and indeed, on cell growth regulation in general, may depend upon the pattern of PKC isozyme expression in the cells.

Inhibitory effect of glutathione on the generation of hydroxyl radicals in the reaction system of glutathione-alloxan.

Deoxyribose (DOR) degradation, an indicator of hydroxyl radical (OH·) generation, was observed in the reaction system of glutathione (GSH)-alloxan in the presence of Fe3+-ethylenediaminetetraacetic acid (EDTA). GSH had a biphasic action on the DOR degradation, i.e. GSH at low concentrations caused marked activation and at high concentrations had an inhibitory effect. GSH at high concentrations had an ability to scavenge OH· in the hypoxanthin-xanthine oxidase in the presence of Fe3+-EDTA and to inhibit the oxygen consumption and the generation of the alloxan radical (AH·) and superoxide anion radical in the GSH-alloxan system. At GSH : alloxan ratio of 10 : 1 led to an immediate formation of dialuric acid. The autoxidation of dialuric acid was suppressed by GSH in a concentration-dependent fashion. These results indicate that autoxidation of dialuric acid is strongly suppressed by excess GSH in the alloxan-dialuric acid redox cycle, resulting in the decrease of AH·generation and oxygen consumption.On the other hand, the reduction of Fe3+-EDTA to Fe2+-EDTA by dialuric acid and AH· was scarcely inhibited by any concentrations of GSH. These results indicate that adding an excess of GSH suppresses the generation of OH· in the GSH-alloxan system through the inhibition of the redox cycling reaction between alloxan and dialuric acid independently on the reduction of Fe3+-EDTA to Fe2+-EDTA.

Metabolic activation of benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide to protein-binding products and the inhibitory effect of glutathione and cysteine.

Trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) and the anti-isomer of trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) were found to be activated by microsomes isolated from 3-methylcholanthrene (MC)-treated rats to reactive intermediates that bound covalently to microsomal proteins. The extent of binding was markedly reduced by the presence of reduced glutathione (GSH) or cysteine. Fluorescence spectroscopic studies on the products derived from BP-7,8-diol and BPDE after microsomal activation in presence of GSH or cysteine revealed the formation of a common reactive intermediate with unique fluorescence properties. The involvement of cytochrome P-448 in the activation of BP-7,8-diol and BPDE to protein-binding products was inferred by the requirement for NADPH and almost complete inhibition by alpha-naphthoflavone. Furthermore, microsomes from MC-treated rats could be replaced by a reconstituted system containing purified cytochrome P-448, NADPH-cytochrome reductase and co-factors. The conjugation of the reactive intermediates from BP-7,8-diol and BPDE with GSH or cysteine did not require the presence of either microsomes or cytosol, thus indicating a non-catalytic reaction. These results emphasize the importance of cellular nucleophiles such as GSH and cysteine in the deactivation of reactive benzo[a]pyrene (BP) intermediates and also provides evidence for the further activation of the ultimate carcinogen BPDE to more reactive electrophiles and may thus have relevance concerning the regulation of BP-induced carcinogenesis.

The inhibitory effect of glutathione on some processes of animalization

The inhibitory effect of glutathione on some processes of animalization