mesna and hydroperoxyisophosphamide

Renal Fanconi syndrome occurs in about 1-5% of all children treated with Ifosfamide (Ifo) and impairment of renal phosphate reabsorption in about 20-30% of them. Pathophysiological mechanisms of Ifo-induced nephropathy are ill defined. The aim has been to investigate whether Ifo metabolites affect the type IIa sodium-dependent phosphate transporter (NaPi-IIa) in viable opossum kidney cells. Ifo did not influence viability of cells or NaPi-IIa-mediated transport up to 1 mM/24 h. Incubation of confluent cells with chloroacetaldehyde (CAA) and 4-hydroperoxyIfosfamide (4-OH-Ifo) led to cell death by necrosis in a concentration-dependent manner. At low concentrations (50-100 microM/24 h), cell viability was normal but apical phosphate transport, NaPi-IIa protein, and -mRNA expression were significantly reduced. Coincubation with sodium-2-mercaptoethanesulfonate (MESNA) prevented the inhibitory action of CAA but not of 4-OH-Ifo; DiMESNA had no effect. Incubation with Ifosfamide-mustard (Ifo-mustard) did alter cell viability at concentrations above 500 microM/24 h. At lower concentrations (50-100 microM/24 h), it led to significant reduction in phosphate transport, NaPi-IIa protein, and mRNA expression. MESNA did not block these effects. The effect of Ifo-mustard was due to internalization of NaPi-IIa. Cyclophosphamide-mustard (CyP-mustard) did not have any influence on cell survival up to 1000 microM, but the inhibitory effect on phosphate transport and on NaPi-IIa protein was the same as found after Ifo-mustard. In conclusion, CAA, 4-OH-Ifo, and Ifo- and CyP-mustard are able to inhibit sodium-dependent phosphate cotransport in viable opossum kidney cells. The Ifo-mustard effect took place via internalization and reduction of de novo synthesis of NaPi-IIa. Therefore, it is possible that Ifo-mustard plays an important role in pathogenesis of Ifo-induced nephropathy.

Topics: Acetaldehyde; Animals; Antineoplastic Agents, Alkylating; Biological Transport; Cell Death; Cell Line; Dose-Response Relationship, Drug; Gene Expression Regulation; Ifosfamide; Kidney; Mesna; Opossums; Phosphates; Phosphoramide Mustards; RNA, Messenger; Sodium-Phosphate Cotransporter Proteins, Type IIa

We studied the effects of ifosfamide and major metabolites on intracellular glutathione (GSH) levels in human peripheral blood lymphocytes (PBL). In vitro exposure of PBL to 4-hydroperoxyifosfamide (4-OOH-IF), acrolein or chloroacetaldehyde at 37 degrees C for 60 min led to a concentration dependent depletion of intracellular GSH. The concentration of the three metabolites to cause a 50% depletion of GSH in PBL was in the micromolar range (acrolein: 16 +/- 4 microM; 4-OOH-IF: 22 +/- 9 microM; chloroacetaldehyde: 30 +/- 7 microM). Exposure to ifosfamide, the non-activated drug, had no effects on the intracellular GSH levels. Pretreatment with 4-OOH-IF suppressed dose-dependently the interleukin-2-induced proliferation of PBL. Incubation of PBL together with 2-mercaptoethanesulfonate (mesna) and 4-OOH-IF, acrolein or chloroacetaldehyde prevented the GSH depletion. The protecting effect of mesna in combination with 4-OOH-IF was independent of GSH biosynthesis, because addition of buthionine sulfoximine had no significant influence on this effect. These findings indicate a novel protective mechanism of mesna against intracellular GSH depletion of PBL during exposure to metabolites of ifosfamide.

Topics: Acetaldehyde; Acrolein; Animals; CHO Cells; Cricetinae; Glutathione; Humans; Ifosfamide; Intracellular Fluid; Lymphocytes; Mesna

Ifosfamide, an isomer of cyclophosphamide, has been shown to be one of the most effective antineoplastic agents for the treatment of human malignancies. There is considerable evidence that the intracellular status of glutathione (GSH) plays a major role in modifying the cytotoxicity of ifosfamide in cells and tissues. We have studied the effects of 4-hydroperoxy-ifosfamide (4-OOH-IF) upon the proliferation of human peripheral blood lymphocytes (PBL) and the intracellular GSH content. The major finding was that occurrence of significant inhibition of [3H]-thymidine incorporation in interleukin-2 (IL-2) expanded PBL after exposure with 4-OOH-IF was accompanied by substantial depletion of intracellular GSH content in these cells. PBL seemed to be more sensitive to this drug induced effect comparing our results obtained in other cells (e.g. Ewing sarcoma, Chinese hamster ovary). In PBL 4-OOH-IF also induced rapid phosphorylation of the small heat shock protein (HSP27) signaling a similar type of stress response as reported for several other agents (e.g. arsenite, phorbol ester, tumor necrosis factor). Reconstitution of the depleted GSH content in PBL after treatment with 4-OOH-IF could be achieved by GSH-monoethylester and mesna within 24 hours of postincubation time. From these results we conclude that human lymphocytes are sensitive targets for ifosfamide induced metabolic stress during treatment. This might have further importance in regard to the immunological function of these cells.

Topics: Blotting, Western; Cells, Cultured; DNA; Glutathione; Heat-Shock Proteins; Humans; Ifosfamide; Interleukin-2; Isoelectric Focusing; Kinetics; Lymphocyte Activation; Lymphocytes; Mesna; Recombinant Proteins; Stress, Physiological; Thymidine

Drug interference of ifosfamide and sodium 2-mercaptoethanesulphonate (MESNA) was studied in three malignant glioma cell cultures (HTZ-17, HTZ-209B, and HTZ-243) by a recently developed in vitro method for evaluation of multimodal treatment interactions. Glioma cell cultures were treated in monolayer 96-well tissue-culture plates for 2 h each, with 4-hydroperoxyifosfamide and MESNA combined in both sequences, or alone. Concentrations ranged from 0.01 microM to 50 microM in single-modality exposures, and from 0.01 microM to 10 microM in combination exposures. After five population doubling times, DNA synthesis was determined by a standard [3H]Tdr-incorporation liquid-scintillation-counting protocol. Data points were evaluated for mono- and combined treatment dose effects (adapted with a probit function), and a model-free three-dimensional response surface was created that was compared to the theoretical additive, anticipated response surface. Local additivity was analysed for any ratio of combined treatment. No tumour effects were seen with MESNA in single-drug exposure, whereas ifosfamide resulted in more than 90% inhibition of tumour DNA synthesis. In combination experiments, MESNA could be confirmed to be inert: the anticipated theoretical combination response surfaces formed a three-dimensional extension of the single-drug ifosfamide dose/response curves--the experimental combination response surfaces displayed an identical appearance (P < or = 0.05). In conclusion, these results indicate no drug interference of MESNA and ifosfamide in malignant glioma cells.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Combined Modality Therapy; Drug Interactions; Drug Screening Assays, Antitumor; Glioma; Humans; Ifosfamide; Mesna; Models, Biological; Tumor Cells, Cultured