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 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

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

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