diethyl-maleate and Urinary-Bladder-Neoplasms

This study was undertaken to investigate the intracellular induction of reactive oxygen species (ROS) by cis-dichlorodiammineplatinum (CDDP) and the augmentation of their cytotoxicity in bladder cancer cells (KU7) by enhancement of ROS generation by the glutathione (GSH) depletors buthionine sulphoximine (BSO) and diethylmaleate (DEM). CDDP-induced cytotoxicity in KU7 cells and its modulation by GSH depletors were determined using spectrophotometric measurement with crystal violet staining. The effects of GSH depletors on intracellular GSH levels were confirmed using the GSH reductase-DTNB recycling method. Intracellular ROS generation induced by CDDP with or without GSH depletors was estimated from the amount of intracellular dichlorofluorescein (DCF), an oxidized product of dichlorofluorescein (DCFH), which was measured with an anchored cell analysis and sorting system. The cytotoxic effects of CDDP (IC50 15.0 +/- 2.5 microM) were significantly enhanced by BSO (IC50 9.3 +/- 2.6 microM, P < 0.01) and DEM (IC50 10.3 +/- 0.3 microM, P <0.01). BSO and DEM produced a significant depletion in intracellular GSH levels (9.6 +/- 0.4 nmol 10(-6) cells, 17.9 +/- 1.0 nmol 10(-6) cells) compared with the controls (30.5 +/- 0.6 nmol 10(-6) cells). Intracellular DCF production in KU7 cells treated with CDDP (1.35 +/- 0.33 microM) was significantly enhanced by the addition of BSO (4.43 +/- 0.33 microM) or DEM (3.12 +/- 0.22 microM) at 150 min. These results suggest that ROS may play a substantial role in CDDP-induced cytotoxicity and that GSH depletors augment its cytotoxicity through an enhancement of ROS generation in bladder cancer cells.

Topics: Antimetabolites; Antineoplastic Agents; Buthionine Sulfoximine; Carcinoma, Transitional Cell; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Fluoresceins; Glutathione; Humans; Hydrogen Peroxide; Maleates; Reactive Oxygen Species; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The modifying potential of diethylmaleate (DEM) and NH4Cl on promotion by butylated hydroxyanisole (BHA) or NaHCO3 of urinary bladder carcinogenesis in rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was investigated. Six week old animals received 0.05% BBN for 4 weeks and then BHA (2%) + DEM (0.15%), BHA + NH4Cl (1%), NaHCO3 (3%) + DEM, NaHCO3 + NH4Cl, BHA, DEM, NH4Cl or no supplement, administered during experimental weeks 5-36. BHA and NaHCO3 clearly amplify the induction of papillary or nodular (PN) hyperplasias and papillomas in rats initiated with BBN. The promoting activity of BHA was not affected by simultaneous administration of DEM or NH4Cl. The enhancing effects of NaHCO3, in contrast, were clearly diminished by concurrent administration of either of these agents. DEM itself did not influence lesion development whereas NH4Cl reduced the incidence of papillomas. In a second experiment, rats exposed to the same protocol were killed at week 8, and assessed for levels of lipid peroxides in the bladder tissue. No remarkable alterations were observed in any group. Thus, the fact that DEM did exert inhibiting effects on tumor promotion by NaHCO3 without decreasing the urinary sodium ion concentration or pH and influence on lipidperoxide levels, suggests essential differences in the mechanisms of action of different types of bladder promoters.

Topics: Ammonium Chloride; Animals; Butylated Hydroxyanisole; Butylhydroxybutylnitrosamine; Lipid Peroxidation; Male; Maleates; Papilloma; Rats; Rats, Inbred F344; Sodium Bicarbonate; Urinary Bladder Neoplasms

The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S-transferases. Higher concentrations of monochlorobimane achieved more complete labelling of the human cellular GSH pool but gave increased background fluorescence due to non-GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with approximately 20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, o-phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labeling.

Topics: Animals; Breast Neoplasms; Bridged Bicyclo Compounds; Buthionine Sulfoximine; Carcinoma; Carcinosarcoma; CHO Cells; Colonic Neoplasms; Cricetinae; Ethylmaleimide; Eukaryotic Cells; Evaluation Studies as Topic; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Glutathione; Humans; Maleates; Mammary Neoplasms, Experimental; Methionine Sulfoximine; Mice; o-Phthalaldehyde; Phenylmercury Compounds; Pyrazoles; Species Specificity; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The effect of pretreating the C3H/He mouse MBT-2 tumor with diethyl maleate (DEM), buthionine-S R-sulfoximine (BSO), or misonidazole (MISO) before administration of cyclophosphamide (CTX) was studied with the use of tumor volume-doubling time delay as an endpoint. The kinetics of glutathione (GSH) depletion and regeneration in the tumor and in the host liver were determined after treatment with the thiol-depleting agents. CTX was administered at appropriate time points. MISO was the most effective chemosensitizer at a time point at which tumor GSH content was 80-85% of the control value. Both BSO and DEM were chemosensitizers in relation to the degree they had reduced tumor GSH levels. This chemosensitization was significant at 50% GSH reduction. By combining MISO and BSO at doses lower than previously used for each agent alone, highly effective sensitization of subsequent CTX was obtained.

Topics: Animals; Buthionine Sulfoximine; Carcinoma, Transitional Cell; Cyclophosphamide; Drug Combinations; Drug Resistance; Female; Glutathione; Liver; Maleates; Methionine Sulfoximine; Mice; Mice, Inbred C3H; Misonidazole; Neoplasm Transplantation; Urinary Bladder Neoplasms