PURPOSE: Thioredoxin is an endogenous antioxidant which directly scavenges reactive oxygen species(ROS) and regenerates oxidatively damaged protein by reducing potential at the redox active disulfide(-Cys-Gly-Pro-Cys-) site. Under oxidative stress, thiredoxin plays a protective and adaptative role by inducing expressions. The aim of this study was to clarify the role of thioredoxin on oxidative neuronal cell injury. We investigated the protective effects of E. coli thioredoxin, also acting as a substrate for mammalian thioredoxin reductase, against oxidative neuronal cell injury under oxidative stresses such as hydrogen peroxide and diamide. METHODS: PC 12 cells were cultured in RPMI 1640 media containing 10% fetal calf serum and subcultured in 96-well plates. Each well contained 30,000 cells. Cells were treated with hydrogen peroxide or diamide 30 minutes after thioredoxin treatment and then incubated for 24 hours. Cytotoxicity and cellular viability were assessed by measuring of lactate dehydrogenase(LDH) release and MTT reduction. RESULTS: Thioredoxin not only decreased the cytotoxicity of PC 12 cell treated with hydrogen peroxide by decreasing LDH release and preventing the decrease of MTT reduction but also thioredoxin showed greater protective effects when simultaneously treated with hydrogen peroxide. Also, thioredoxin decreased cytotoxicity by decreasing LDH release from PC 12 cells damaged by diamide. Thioredoxin did not prevent the decrease of MTT reduction on PC 12 cells damaged by diamide. CONCLUSION: Thioredoxin protected PC 12 cells under oxidative stresses by directly scavenging and inhibiting oxidants such as hydrogen peroxide and diamide.
Diamide ; Hydrogen Peroxide ; Lactic Acid ; Neurons* ; Oxidants ; Oxidation-Reduction ; Oxidative Stress ; Oxygen ; Thioredoxin-Disulfide Reductase ; Thioredoxins*

BACKGROUND AND OBJECTIVES: This study was designed to examine the effects of diamide and thioredoxin (TRX) on vascular endothelial cells in order to clarify the mechanism by which vascular damage is mediated by oxygen free radicals. MATERIALS AND METHODS: The pulmonary artery endothelial cell (PAEC) line derived from bovine serum was cultured for 8 hours in media supplemented with various concentrations of diamide and TRX. The XTT assay, MTS assay, SRB assay, LDH activity and lipid peroxidation tests were perfomed. RESULTS: In XTT and MTS assays, diamide significantly decreased the cell viability of cultured PAEC in a dose- and time-dependent manner. Diamide showed a decrease in the amount of total protein, although it showed an increase of lipid peroxidation and LDH activity in cultured PAEC. In regards to the protective effect of TRX on diamide-induced cytotoxicity, this showed an increase of total protein, however it showed a decrease of lipid peroxidation and LDH activity. CONCLUSION: Our results suggest that diamide has a vasculotoxic effect on cultured bovine PAEC and that TRX is very effective in the protection of diamide-induced cytotoxicity by duye to the increase of total protein and the decrease of lipid peroxidation and LDH activity in these cultures.
Cell Survival ; Diamide* ; Endothelial Cells* ; Endothelium, Vascular ; Free Radicals ; Lipid Peroxidation ; Oxygen ; Pulmonary Artery ; Thioredoxins*

PURPOSE: Thioredoxin is an endogenous antioxidant. It regulates the activities of transcriptional factors such as NF-kB(nuclear factor kappa B)and AP-1(activator protein-1) and it increases the synthesis of cytokines, preventing cellular proliferation and apoptosis. The aim of this study was to clarify the role of thioredoxin on apoptosis-inducing neuronal cell injury. We investigated the protective effects of thioredoxin against apoptosis-inducing neuronal cell injury through intracellular mechanism by 6-hydroxydopamine and serum deprivation. METHODS: PC 12 cells were cultured in RPMI 1640 media containing 10% fetal calf serum and subcultured in 96-well plates. Each well contained 30,000 cells. Cells were treated with hydrogen peroxide, diamide or 6-hydroxydopamine 30 minutes after thioredoxin treatment and then incubated for 24 hours. Cytotoxicity and cellular viability were assessed by measuring lactate dehydrogenase(LDH) release and MTT reduction. RESULTS: Thioredoxin increased cytotoxicity of PC cells treated with 6-hydroxydopamine by increasing LDH release and decreasing MTT reduction. In the serum deprivation condition, thioredoxin increased cytotoxicity of PC cells by increasing LDH release. CONCLUSION: Thioredoxin potentiates oxidative injury through intracellular mechanisms by 6-hydroxydopamine and serum deprivation instead of protecting. The cytotoxicity of thioredoxin may be mediated by decreasing the activity of NF-kB, which has been reported recently to protect against cellular apoptosis. Evidence suppors that the cytotoxic effect was not increased in the presence of serum in this study. Therefore, we found that the antioxidant effects of thioredoxin depended on mechanisms of injuries.
Antioxidants ; Apoptosis ; Cell Proliferation ; Cytokines ; Diamide ; Hydrogen Peroxide ; Lactic Acid ; Neurons* ; NF-kappa B ; Oxidopamine ; Thioredoxins*

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
Cells, Cultured ; Chemokine CCL3 ; Chemokine CCL4 ; Chemotaxis, Leukocyte ; physiology ; Diamide ; pharmacology ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Lipid Peroxidation ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Sulfhydryl Reagents ; pharmacology ; Umbilical Veins ; cytology

OBJECTIVETo study the effect of diamide on the expression of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in cultured human umbilical vein endothelial cells.

METHODSAfter exposure of the endothelial cells (ECs) to different concentrations of diamide for 4 hours, the MIP-1 alpha mRNA in the cells was detected by nuclease S1 protection assay and the MIP-1 alpha protein in those cells was determined by cell enzyme-linked immunosorbent assay. The chemotactic activity of MIP-1 alpha in the conditioned medium of ECs treated with diamide for peripheral blood monocytes was tested by microfilter method using modified Boyden chambers.

RESULTSIncubation of ECs with 5 micro mol/L diamide resulted in a 2.4-fold increase in the level of MIP-1 alpha mRNA expression as compared with the control group (t = 8.70, P < 0.05). Exposure of ECs to 1 micro mol/L, 5 micro mol/L and 10 micro mol/L diamide resulted in a 0.9-fold, 1.2-fold, and 0.7-fold increase in the level of MIP-1 alpha protein expression respectively, as compared with the control group (F = 35.65, P < 0.05). Chemotactic assay showed that the migration distance of monocytes towards the conditioned medium (CM) of ECs treated with 5 micromol/L diamide was 99.50 microm +/- 4.31 microm, which was significantly more than the 66.47 microm +/- 3.25 microm towards the conditioned medium of ECs in the non-diamide group, the chemokinetic group (67.03 microm +/- 6.83 microm) and the random migration group (65.40 microm +/- 3.36 microm) (F = 404.31, P < 0.05). The results revealed that there might be chemotactic substances in the conditioned medium of 5 micro mol/L diamide treated ECs. The migration distance of monocytes towards the conditioned medium of the ECs exposed to 5 micromol/L diamide was significantly reduced to 82.80 microm +/- 6.88 microm after the addition of goat anti-human MIP-1 alpha antibody (F = 192.25, P < 0.05), which indicates the chemotactic activity of MIP-1 alpha in the conditioned medium of the ECs in the diamide group.

CONCLUSIONSDiamide, a lipid peroxidation inducer, could stimulate ECs to produce high levels of MIP-1 alpha with chemotactic activity, and may play an important role in atherogenesis through attraction of peripheral blood monocytes into arterial intima.

Arteriosclerosis ; pathology ; Cells, Cultured ; Chemokine CCL4 ; Diamide ; pharmacology ; Endothelium, Vascular ; drug effects ; metabolism ; Gene Expression ; drug effects ; Humans ; Macrophage Inflammatory Proteins ; metabolism ; RNA, Messenger ; drug effects ; metabolism ; Radiation-Sensitizing Agents ; pharmacology

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
Cells, Cultured ; Chemotaxis, Leukocyte/physiology ; Diamide/*pharmacology ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Lipid Peroxidation ; Macrophage Inflammatory Protein-1/*biosynthesis ; Macrophage Inflammatory Protein-1/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Sulfhydryl Reagents/pharmacology ; Umbilical Veins/cytology

A nucleoside diphosphate-linked moiety X (Nudix) hydrolase-like gene, YSA1, has been identified as one of the gromwell plant extract-responsive genes in Cryptococcus neoformans. Ysa1 is known to control intracellular concentrations of ADP-ribose or O-acetyl-ADP-ribose, and has diverse biological functions, including the response to oxidative stress in the ascomycete yeast, Saccharomyces cerevisiae. In this study, we characterized the role of YSA1 in the stress response and adaptation of the basidiomycete yeast, C. neoformans. We constructed three independent deletion mutants for YSA1, and analyzed their mutant phenotypes. We found that ysa1 mutants did not show increased sensitivity to reactive oxygen species-producing oxidative damage agents, such as hydrogen peroxide and menadione, but exhibited increased sensitivity to diamide, which is a thiol-specific oxidant. Ysa1 was dispensable for the response to most environmental stresses, such as genotoxic, osmotic, and endoplasmic reticulum stress. In conclusion, modulation of YSA1 may regulate the cellular response and adaptation of C. neoformans to certain oxidative stresses and contribute to the evolution of antifungal drug resistance.
Adenosine Diphosphate Ribose ; Ascomycota ; Basidiomycota ; Cryptococcus neoformans* ; Cryptococcus* ; Diamide ; Drug Resistance, Fungal ; Endoplasmic Reticulum Stress ; Hydrogen Peroxide ; Lithospermum ; O-Acetyl-ADP-Ribose ; Oxidative Stress* ; Oxygen ; Phenotype ; Plants ; Saccharomyces cerevisiae ; Vitamin K 3 ; Yeasts

Reactive oxygen species (ROS) has been implicated as an inducer of NF-kappaB activity in numbers of cell types where exposure of cells to ROS such as H2O2 leads to NF-kappaB activation. In contrast, exposure to oxidative stress in certain cell types induced reduction of tumor necrosis factor (TNF)-induced NF-kappaB activation. And various thiol-modifying agents including gold compounds and cyclopentenone prostaglandins inhibit NF-kappaB activation by blocking IkappaB kinase (IKK). To understand such conflicting effect of oxidative stress on NF-kappaB activation, HeLa cells were incubated with H2O2 or diamide and TNF-induced expression of NF-kappaB reporter gene was measured. NF-kappaB activation was significantly blocked by these oxidizing agents, and the inhibition was accompanied with reduced nuclear NF-kappaB and inappropriate cytosolic IkappaB degradation. H2O2 and diamide also inhibited IKK activation in HeLa and RAW 264.7 cells stimulated with TNF and lipopolysaccharide, respectively, and directly blocked IKK activity in vitro. In cells treated with H2O2 alone, nuclear NF-kappaB was induced after 2 h without detectible degradation of cytosolic IkBa or activation of IKK. Our results suggest that ROS has a dual effect on NF-kappaB activation in the same HeLa cells: it inhibits acute IKK-mediated NF-kappaB activation induced by inflammatory signals, while longer-term exposure to ROS induces NF-kappaB activity through an IKK-independent pathway.
Cell Nucleus/drug effects/metabolism ; Cytosol/drug effects/metabolism ; Diamide/pharmacology ; Hela Cells/drug effects/metabolism ; Human ; Hydrogen Peroxide/pharmacology ; I-kappa B/drug effects/metabolism ; NF-kappa B/drug effects/genetics/*metabolism ; Oxidants/pharmacology ; *Oxidative Stress ; Protein-Serine-Threonine Kinases/metabolism ; Signal Transduction/drug effects ; Time Factors ; Transcription, Genetic ; Tumor Necrosis Factor/pharmacology

OBJECTIVETo investigate the effects of mitochondrial membrane permeability transition pore (MPT)-opened agent diamide and MPT-closed agent cyclosporin A on arsenic trioxide (As(2)O(3))-induced apoptosis in acute promyelocytic leukemia (APL) cell line NB4.

METHODSNB4 cells were treated with As(2)O(3) alone or in combination with diamide or cyclosporin A in different concentrations. Cell apoptosis was assessed by the morphological observation, Annexin-V assay, distribution of cellular DNA contents and genomic DNA electrophoresis. The mitochondrial transmembrane potentials (DeltaPsim) were detected by flow cytometry according to the intensity of rhodamine 123 uptake in cells.

RESULTSBoth diamide and cyclosporin A significantly enhanced As(2)O(3)-induced apoptosis in NB4 cells. The DeltaPsim collapse induced by As(2)O(3) was also enforced by combined treatment with diamide or cyclospo-rin A. 1 micromol/L As(2)O(3) alone treatment for 72 hours led to DeltaPsim disruption in 27.9% of cells, while combined treatment of As(2)O(3) and diamide or cyclosporin A increased DeltaPsim disruption cells to 59.7% and 42.2%, respectively.

CONCLUSIONSAs(2)O(3)-induced DeltaPsim disruption possibly involves with thiol oxidation or crosslink of important components especially ANT-related molecules.

Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; Arsenicals ; pharmacology ; therapeutic use ; Cyclosporine ; pharmacology ; Diamide ; pharmacology ; Drug Synergism ; Enzyme Inhibitors ; pharmacology ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; pathology ; Membrane Potentials ; drug effects ; physiology ; Mitochondria ; physiology ; Oxides ; pharmacology ; therapeutic use ; Sulfhydryl Reagents ; pharmacology ; Tumor Cells, Cultured