Golgi SNAP receptor complex 1 (GS28) has been implicated in vesicular transport between intra-Golgi networks and between endoplasmic reticulum (ER) and Golgi. Additional role(s) of GS28 within cells have not been well characterized. We observed decreased expression of GS28 in rat ischemic hippocampus. In this study, we examined the role of GS28 and its molecular mechanisms in neuronal (SK-N-SH) cell death induced by hydrogen peroxide (H2O2). GS28 siRNA-transfected cells treated with H2O2 showed a significant increase in cytotoxicity under glutathione (GSH)-depleted conditions after pretreatment with buthionine sulfoximine, which corresponded to an increase of intracellular reactive oxygen species (ROS) in the cells. Pretreatment of GS28 siRNA-transfected cells with p38 chemical inhibitor significantly inhibited cytotoxicity; we also observed that p38 was activated in the cells by immunoblot analysis. We confirmed the role of p38 MAPK in cotransfected cells with GS28 siRNA and p38 siRNA in the cell viability assay, flow cytometry, and immunoblot. Involvement of apoptotic or autophagic processes in the cells was not shown in the cell viability, flow cytometry, and immunoblot analyses. However, pretreatment of the cells with necrostatin-1 completely inhibited H2O2-induced cytotoxicity, ROS generation, and p38 activation, indicating that the cell death is necroptotic. Collectively these data imply that H2O2 induces necroptotic cell death in the GS28 siRNA-transfected cells and that the necroptotic signals are mediated by sequential activations in RIP1/p38/ROS. Taken together, these results indicate that GS28 has a protective role in H2O2-induced necroptosis via inhibition of p38 MAPK in GSH-depleted neuronal cells.
Animals ; Buthionine Sulfoximine ; Cell Death ; Cell Survival ; Endoplasmic Reticulum ; Flow Cytometry ; Glutathione ; Hippocampus ; Hydrogen ; Hydrogen Peroxide ; Imidazoles ; Indoles ; Methionine ; Neurons ; p38 Mitogen-Activated Protein Kinases ; Rats ; Reactive Oxygen Species ; RNA, Small Interfering ; SNARE Proteins

Neural stem cells (NSCs) have mainly been applied to neurodegeneration in some medically intractable neurologic diseases. In this study, we established a novel NSC line and investigated the cytotoxic responses of NSCs to exogenous neurotoxicants, glutamates and reactive oxygen species (ROS). A multipotent NSC line, B2A1 cells, was established from long-term primary cultures of oligodendrocyte-enriched cells from an adult BALB/c mouse brain. B2A1 cells could be differentiated into neuronal, astrocytic and oligodendroglial lineages. The cells also expressed genotypic mRNA messages for both neural progenitor cells and differentiated neuronoglial cells. B2A1 cells treated with hydrogen peroxide and L-buthionine-(S,R)-sulfoximine underwent 30-40% cell death, while B2A1 cells treated with glutamate and kainate showed 25-35% cell death. Cytopathologic changes consisting of swollen cell bodies, loss of cytoplasmic processes, and nuclear chromatin disintegration, developed after exposure to both ROS and excitotoxic chemicals. These results suggest that B2A1 cells may be useful in the study of NSC biology and may constitute an effective neurotoxicity screening system for ROS and excitotoxic chemicals.
Animals ; Brain/*cytology ; Buthionine Sulfoximine/pharmacology ; Cell Differentiation ; Cell Line ; Cell Lineage ; Cytokines/pharmacology ; Enzyme Inhibitors/pharmacology ; Excitatory Amino Acid Agonists/pharmacology ; Glutamic Acid/pharmacology ; Humans ; Hydrogen Peroxide/pharmacology ; Intercellular Signaling Peptides and Proteins/pharmacology ; Kainic Acid/pharmacology ; Mice ; Mice, Inbred BALB C ; Multipotent Stem Cells/cytology/*drug effects/physiology ; Neuroglia/cytology/drug effects/physiology ; Neurons/cytology/*drug effects/physiology ; Neurotoxins/*pharmacology ; Oxidants/pharmacology ; Phenotype ; Reactive Oxygen Species/metabolism

OBJECTIVETo investigate the apoptosis-inhancing effect of the combination of arsenic trioxide (As2O3 ) and buthionine sulfoximine (BSO) on multidrug-resistant human leukemic K562/ADM cells, to compare the effect of As2O3 alone and As2O3 combined with BSO and As2O3 alone, and to determine the effect of intracellular GSH content on this treatment.

METHODSAs2O3 was used in a dose of 0.5 micromol/L, 2.0 micromol/L and 5.0 micromol/L, respectively, and BSO was used in a dose of 100 micromol/L in the culture of multidrug-resistant human leukenic K562/ADM cells. The cell proliferation activity was assessed with MTT assay. The cell apoptosis was detected by flow cytometry using Annexin-V and propidium iodide (PI) staining. Intracellular GSH content was measured using glutathione assay kit by spectrophotometry.

RESULTSAfter the GSH contents were reduced by the combination of arsenic in clinic dose (0.5, 2 micromol/L) and BSO (100 micromol/L), respectively, the K562/ADM cell proliferation activity was obviously inhibited and the cell apoptosis-inducing effect was advanced in 24 hours. In 48 and 72 hours, the effect of the combination group (clinic dose arsenic group) was significantly stronger than that of clinic dose arsenic alone group and the high dose arsenic alone group.

CONCLUSIONThe cell apoptosis-inducing effect of arsenic in combination of BSO on multidrug resistant human leukemia K562/ADM cells is significantly enhanced in comparison with that of arsenic alone. The reduction of intracellular glutathione content is closely correlated with this apoptosis-enhancing effect.

Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Buthionine Sulfoximine ; pharmacology ; Cell Proliferation ; drug effects ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; Drug Synergism ; Glutathione ; metabolism ; Humans ; K562 Cells ; Oxides ; pharmacology

OBJECTIVETo investigate the apoptosis-induction, P-glycoprotein (P-gp) and mdr1 mRNA inhibition effects of arsenic trioxide (As2O3) and buthionine sulfoximine (BSO) on multidrug-resistant cell line K562/ADM cells, and to determine the relationship between intracellular GSH content and arsenic effect.

METHODSK562/ADM cells were treated with arsenic (0.5, 2.0, 5.0 micromol/L) alone or combined with BSO (100 micromol/L). The cell proliferating capacity was assessed with MTT assay, and cell apoptosis by Annexin V and propidium iodide (PI) staining. Intracellular GSH contents were measured using a glutathione assay kit by spectrophotometry. P-gp expression was determined by flow cytometry, and mdr1 mRNA expression by semi-quantitative RT-PCR.

RESULTSThe GSH contents in K562/ADM cell was (81.13 +/- 3.91) mg/g protein. After the GSH contents were degraded by BSO, the K562/ADM cell proliferating capacity was obviously inhibited and the cells were induced apoptosis in 24 hours by the combination of clinic dose arsenic group (0.5, 2.0 micromol/L) and BSO (100 micromol/L). The cell apoptosis rates at 48 hours in arsenic alone group and combination group were (59.29 +/- 6.01)% and (65.06 +/- 8.29)%, and at 72 hours were (82.15 +/- 9.28)% and (92.72 +/- 9.41)% retrospectively. At 48 hours, the mdr1 mRNA inhibition effect of the combination group was obviously stronger than that of high dose arsenic alone group. At 72 hours, the P-gp inhibition effect of the combination group (clinic dose arsenic group, 0.5, 2.0 micromol/L) was obviously stronger than that of high dose arsenic alone group (5.0 micromol/L).

CONCLUSIONThe intracellular GSH contents are closely correlated with the arsenic effect. The combination of conventional dose arsenic and BSO significantly induces K562/ADM cell apoptosis and inhibits P-gp and mdr1 mRNA expression in the cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Buthionine Sulfoximine ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Genes, MDR ; drug effects ; Glutathione ; metabolism ; Humans ; K562 Cells ; Oxides ; pharmacology

OBJECTIVETo determine the effects of buthionine sulfoximine (BSO) and free radical scavenger, dimethyl sulfoxide (DMSO), on mutation frequency and the formation of 8-hydroxydeoxyganosine (8-OHdG) induced by crocidolite fibers in human-hamster hybrid (A(L)) cells.

METHODSThe cytotoxicity and mutagenicity were determined by the formation of colonies. 8-OHdG was examined by immunoperoxidase staining. Non-protein sulfhydryl (NPSH) compound was assayed by modified Tietze's method.

RESULTSThe level of NPSH in A(L) cell pretreated with 25 micro mol/L of BSO was decreased to 2 nmol/10(7) cells, only 5% of the control after 24 h. The mutation frequency of CD59 gene of A(L) cell in crocidolite alone treated group was 208 +/- 18 while that in BSO pretreated group (397 +/- 55) was about twice the former (P < 0.05). The mutation frequency of CD59 gene in the group treated with crocidolite and in the presence of DMSO (57 +/- 8) was 72.6% less than that in crocidolite alone treated group. Crocidolite fibers induced a dose-effect relationship in the formation of 8-OHdG in A(L) cells (y = 150 + 20x, r = 0.9621). The level of 8-OHdG in cells was 289 +/- 6 at the dose of 6 micro g/cm(2) crocidolite, which was about twice the control group (137 +/- 9). In the presence of DMSO, 8-OHdG level decreased to 170 +/- 3 at the same dose of crocidolite.

CONCLUSIONFree radicals are the important inducer of mutagenesis and DNA damage in A(L) cells caused by crocidolite, which has dose-effect relationship.

Animals ; Asbestos, Crocidolite ; pharmacology ; Buthionine Sulfoximine ; pharmacology ; CD59 Antigens ; genetics ; Cricetinae ; DNA ; drug effects ; genetics ; DNA Damage ; Deoxyguanosine ; analogs & derivatives ; metabolism ; Dimethyl Sulfoxide ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Free Radical Scavengers ; pharmacology ; Humans ; Hybrid Cells ; Immunoenzyme Techniques ; Mutation

OBJECTIVE: The purpose of this study is to determine the effects of glutathione on cisplatin-induced cytotoxicity of human cervical carcinoma cell lines (SiHa: squamous cell carcinoma cell, CaSki: epidermoid metastatic carcinoma cell). METHODS: Human cervical carcinoma cells (SiHa, CaSki) were incubated with culture media (RPMI1640) in the presence of cisplatin and/or buthionine sulfoximine (BSO), as a inhibitor of gamma-glutamyl- cysteine synthetase, and/or glutathione (GSH) and/or 2-oxo 4-thiazolidine carboxylic acid (OTC). The viable cells were examined by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and was determined by spectrophotometer at 570 nm. RESULTS: The incubation of cervical cells with cisplatin resulted in an decreasing cells viability by dose response. The MTT reduction rate were not different by BSO (5 mM) treatment in cervical cell lines. The viable cells were increased significantly by glutathione (5 mM) or OTC (5 mM) in cisplatin-treated cell lines. CONCLUSION: gamma-glutamylcysteine synthetase inhibitor had no effect on cisplatin toxicity. GSH and OTC had effect on cisplatin cytotoxicity. So, These result suggested that cervical cancer line cells were more susceptive to protective effects of glutathione and OTC than BSO on cisplatin induced-toxicity.
Buthionine Sulfoximine ; Carcinoma, Squamous Cell ; Cell Line* ; Cisplatin ; Culture Media ; Cysteine ; Glutathione* ; Humans* ; Ligases ; Uterine Cervical Neoplasms*

OBJECTIVES: To evaluate the effect of glutathione(GSH) on lead induced modulation of nitric oxide(NO) synthesis, and to examine how lead modulates NO production in macrophages. METHODS: This study was observed in a culture of RAW 264.7 cells, which originated from a tumor in a Balb/c mouse that was induced by the Abelson murine leukemia virus. The compounds investigated were lead chloride, N-acetyl-cystein(NAC), and Buthionine Sulfoximine(BSO). RESUJLTS: ATP synthesis in RAW 264.7 cells was unchanged by each lead concentration exposure in a dose dependent manner. The NO synthesis was decreased when exposed to lead(PbCl2) concentration 0.5 micro M. The presence of 300 micro M NAC, used as a pretreatment in the culture medium, caused the recovery of the lead induced decrease in NO synthesis, but in the presence of 300 micro M BSO as a pretreatment, there was no recoverey. Pretreatment with NAC and BSO had no affect on ATP synthesis at any of the lead concentrations used. CONCLUSIONS: These results indicated that GSH has a protective effect toward lead toxicity, and suggested that the inhibition of NO production in macrophage due to lead toxicity may be related to cofactors of iNOS (inducible nitric oxide synthase)
Abelson murine leukemia virus ; Acetylcysteine ; Adenosine Triphosphate ; Animals ; Buthionine Sulfoximine ; Glutathione* ; Macrophages ; Mice ; Nitric Oxide

OBJECTIVES: To evaluate the effect of glutathione(GSH) on lead induced modulation of nitric oxide(NO) synthesis, and to examine how lead modulates NO production in macrophages. METHODS: This study was observed in a culture of RAW 264.7 cells, which originated from a tumor in a Balb/c mouse that was induced by the Abelson murine leukemia virus. The compounds investigated were lead chloride, N-acetyl-cystein(NAC), and Buthionine Sulfoximine(BSO). RESUJLTS: ATP synthesis in RAW 264.7 cells was unchanged by each lead concentration exposure in a dose dependent manner. The NO synthesis was decreased when exposed to lead(PbCl2) concentration 0.5 micro M. The presence of 300 micro M NAC, used as a pretreatment in the culture medium, caused the recovery of the lead induced decrease in NO synthesis, but in the presence of 300 micro M BSO as a pretreatment, there was no recoverey. Pretreatment with NAC and BSO had no affect on ATP synthesis at any of the lead concentrations used. CONCLUSIONS: These results indicated that GSH has a protective effect toward lead toxicity, and suggested that the inhibition of NO production in macrophage due to lead toxicity may be related to cofactors of iNOS (inducible nitric oxide synthase)
Abelson murine leukemia virus ; Acetylcysteine ; Adenosine Triphosphate ; Animals ; Buthionine Sulfoximine ; Glutathione* ; Macrophages ; Mice ; Nitric Oxide

PURPOSE: Cycloporine A (CsA) is a immunosuppressive agent most widely used in organ transplanted patients for preventing immunorejection, but it has some side effects, including hypertension, nephrotoxicity, hepatotoxicity and diabetes. The mechanism of toxicity of CsA was not completely understood, but the reactive oxygen species has been proposed to be involved in the reaction of toxicity of CsA. The purpose of this study is to determine the effects of glutathione, as a physiological antioxidant on CsA induced cytotoxicity in rat insulinoma (RINm5F) cells. METHODS: RINm5F cells were incubated in the presence of CsA (105~108 M) and buthionine sulfoximine (BSO), as a inhibitor of r-glutamyl cysteine synthetase, was added to cultured media (RPMI1640). Twenty four hours of incubation with CsA and BSO, viable cells were determined by MTT method. RESULTS: CsA decreased cell viability in dose response in cultured RINm5F cells and significantly decreased according to redusing glutathione. CONCLUSION: These results suggested that CsA may induce the diabetes and glutathione have some roles in the pathogenesis of CsA-induced diabetes.
Animals ; Buthionine Sulfoximine ; Cell Survival ; Cyclosporine* ; Cysteine ; Glutathione* ; Humans ; Hypertension ; Insulinoma* ; Ligases ; Rats ; Reactive Oxygen Species

We examined the neurotoxic effects of 3 glutathione (GSH) depletors, buthionine sulfoximine (BSO), diethyl maleate (DEM) and phorone, under the presence of trolox, cycloheximide (CHX), pyrrolidine dithiocarbamate (PDTC) or MK-801 in primary mouse cortical cell cultures. All three depletors induced neuronal death in dose and exposure time dependent manner, and decreased total cellular GSH contents. The patterns of the neuronal death and the GSH decrements were dependent on the individual agents. DEM (200 micrometer) induced rapid and irreversible decrement of the GSH. BSO (1 mM) also decreased the GSH irreversibly but the rate of decrement was more progressive than that of DEM. Phorone (1 mM) reduced the GSH content to 40% by 4 hr exposure, that is comparable to the decrement of BSO, but the GSH recovered and reached over the control value by 36 hr exposure. BSO showed a minimal neurotoxicity (0-10%) at the end of 24 hr exposure, but marked neuronal cell death at the end of 48 hr exposure. The BSO (1 mM)-induced neurotoxicity was markedly inhibited by trolox or CHX and partially attenuated by MK-801. DEM induced dose-dependent cytotoxicity at the end of 24 hr exposure. Over the doses of 400 micrometer, glial toxicity also appeared. DEM (200 micrometer)-induced neurotoxicity was markedly inhibited by trolox or PDTC. Phorone (1 mM) induced moderate neurotoxicity (40%) at the end of 48 hr exposure. Only CHX showed significant inhibitory effect on the phorone-induced neurotoxicity. These results suggest that the GSH depletors induce neuronal injury via different mechanisms and that GSH depletors should be carefully employed in the researches of neuronal oxidative injuries.
Animals ; Buthionine Sulfoximine ; Cell Culture Techniques* ; Cell Death ; Cycloheximide ; Dizocilpine Maleate ; Glutathione* ; Mice* ; Neurons