mesna and chloroacetaldehyde

Depletion of cellular glutathione (GSH) enhances the efficacy of many anticancer agents in preclinical systems. Limited published data showing depletion of GSH in vitro and in patients by ifosfamide and/or mesna provided the rationale for a Phase I trial. Ifosfamide and mesna were infused over 24 and 36 h, respectively, at equal daily doses; carboplatin was given after ifosfamide to a target plasma area under the curve of 4 mg x min x ml(-1). Plasma and peripheral WBC thiols were quantitated by high-performance liquid chromatography. The dose of ifosfamide was escalated from 2 to 8 g/m2; the maximum tolerated dose was 6 g/m2. Significant depletion in plasma cysteine and homocysteine, precursors for GSH synthesis, was observed (maximum, 95% to >99% at 8 g/m2). Plasma mesna and cysteine/ homocysteine levels were inversely correlated; nadir levels of cysteine/homocysteine were maintained for several hours after ifosfamide infusion had stopped and while mesna infusion was continuing. In vitro coincubation experiments confirmed that mesna reduces these thiols from disulfides to sulfhydryls, which are readily cleared, as evidenced by the significantly increased rate of excretion of cysteine in urine. In contrast, ifosfamide/mesna treatment caused a moderate depletion of plasma GSH in only 60% of the patients, with a nadir at 24 h and recovery immediately after the end of ifosfamide infusion. The GSH depletion in these patients was not dose related. The profile of GSH recovery in plasma after ifosfamide and the fact that mesna could not reduce GSH disulfides in vitro suggest that the observed GSH depletion in plasma in 60% of the patients may be related to direct reactions of GSH with ifosfamide metabolites and/or mesna. Our results indicate that mesna is a modulator of GSH precursors and that a prolonged infusion of mesna may be required to achieve GSH precursor starvation and the consequent GSH depletion in cells.

Topics: Acetaldehyde; Antineoplastic Agents, Alkylating; Cysteine; Dose-Response Relationship, Drug; Glutathione; Homocysteine; Humans; Ifosfamide; Infusions, Intravenous; Leukocytes; Mesna; Neoplasms; Oxidation-Reduction; Protective Agents; Sulfhydryl Compounds

Chloroacetaldehyde (CAA) is the putative metabolite responsible for ifosfamide-induced nephrotoxicity. Whereas evidence suggests that sodium 2-mercaptoethanesulfonate (mesna) and amifostine protect renal cells against CAA toxicity in vitro, their efficacy in clinical studies is controversial. To better understand the discrepancy between in vivo and in vitro results, we combined the in vivo intraperitoneal administration of either saline or mesna (100 mg/kg) or amifostine (200 mg/kg) in rats and the in vitro study of CAA toxicity to both proximal tubules and precision-cut renal cortical slices. The measured renal cortical concentrations of mesna and amifostine were 0.6+/-0.1 micromol/g and 1.2+/-0.2 micromol/g, respectively; these drugs did not cause renal toxicity. Despite this, none of the adverse effects of 0.5 mM CAA was prevented by the previous in vivo administration of mesna or amifostine. Toxicity of 0.5 mM CAA to rat proximal tubules was shown by the fall of cellular adenosine triphosphate (ATP), total glutathione and coenzyme A + acetyl-coenzyme A levels and by the altered metabolic viability of renal cells. Long-term exposure of cortical slices to CAA concentrations > or =30 microM caused severe cell toxicity (i.e. decrease in cellular ATP, total glutathione, and coenzyme A + acetyl-coenzyme A levels), which was not prevented by the in vivo administration of mesna or amifostine.

Topics: Acetaldehyde; Acetyl Coenzyme A; Adenosine Triphosphate; Amifostine; Animals; Disease Models, Animal; Drug Therapy, Combination; Glutathione; Glutathione Disulfide; Injections, Intraperitoneal; Kidney; Kidney Diseases; Kidney Tubules, Proximal; Male; Mesna; Organ Culture Techniques; Radiation-Protective Agents; Rats; Rats, Wistar

Renal injury is a common side effect of the chemotherapeutic agent ifosfamide. Current evidence suggests that ifosfamide metabolites, particularly chloroacetaldehyde, produced within the kidney contribute to nephrotoxicity. The present study examined the effects of ifosfamide and its metabolites, chloroacetaldehyde and acrolein, on rabbit proximal renal tubule cells in primary culture, using a transwell culture system that allows separate access to apical and basolateral cell surfaces. The ability of the uroprotectant medications sodium 2-mercaptoethanesulfonate (mesna) and amifostine to prevent chloroacetaldehyde-and acrolein-induced renal cell injury was also assessed. Ifosfamide (2,000-4,000 microM) did not affect transcellular inulin diffusion but caused a modest but significant impairment in organic ion transport; this impairment was greater when ifosfamide was added to the basolateral compartment of the transwell. Chloroacetaldehyde and acrolein (6.25-100 microM) produced dose-dependent impairments in transcellular inulin diffusion and organic ion transport. Chloroacetaldehyde was a more potent toxin than acrolein. Co-administration of mesna or amifostine prevented metabolite toxicity. Amifostine was only protective when added to the apical compartment of transwells. These results show that ifosfamide is taken up by renal tubule cells preferentially through their basolateral surfaces, and supports the hypothesis that chloroacetaldehyde is primarily responsible for ifosfamide-induced nephrotoxicity. The protective effect of mesna and amifostine in vitro contrasts with clinical experience showing that these medications do not eliminate ifosfamide nephrotoxicity in vivo.

Topics: Acetaldehyde; Acrolein; Amifostine; Animals; Cell Culture Techniques; Cells, Cultured; Dose-Response Relationship, Drug; Ifosfamide; Inulin; Ion Transport; Kidney Tubules, Proximal; Male; Mesna; p-Aminohippuric Acid; Protective Agents; Rabbits; Tetraethylammonium

Chloroacetaldehyde (CAA) is formed in the body from the chemotherapeutically used drug ifosfamide (IFO). CAA leads to cell death in proximal tubule cells mainly through the mechanism of necrosis rather than apoptosis. During chemotherapy, 2-mercaptosulfonic acid (mesna) is used with IFO to protect the urothel from cell damage. Little is known of the effect of mesna on renal proximal tubule cells, the primary site of damage after IFO treatment. Mesna contains a sulfhydryl (SH) group. To clarify whether SH-group-containing molecules can prevent CAA-induced cell death, we studied the effect of mesna and cysteine on necrosis, apoptosis, and protein content in a human proximal tubule-derived cell line (IHKE cells) treated with CAA. Both substances prevented CAA-induced necrotic cell death and protein loss and restored CAA-inhibited caspase-3 activity. CAA also prevented cisplatin-induced apoptosis. This inhibition was reversible in the presence of glutathione (GSH). We conclude that SH-containing molecules can protect proximal tubule cells from cell death because they interact with CAA before CAA can disturb other important cellular SH groups. A sufficient supply of intra- and extracellular SH groups during IFO chemotherapy may therefore have the ability to protect renal tubule cells from cell death.

Topics: Acetaldehyde; Apoptosis; Cell Line; Cysteine; Dose-Response Relationship, Drug; Drug Antagonism; Drug Therapy, Combination; Glutathione; Humans; Kidney Tubules, Proximal; Mesna; Necrosis; Protective Agents

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

Renal injury is a common side effect of the chemotherapeutic agent ifosfamide. Current evidence suggests that the ifosfamide metabolite chloroacetaldehyde contributes to this nephrotoxicity. The present study examined the effects of chloroacetaldehyde and acrolein, another ifosfamide metabolite, on rabbit proximal renal tubule cells in primary culture. The ability of the uroprotectant medications sodium 2-mercaptoethanesulfonate (mesna) and amifostine to prevent chloroacetaldehyde- and acrolein-induced renal cell injury was also assessed. Chloroacetaldehyde and acrolein (25-200 M) produced dose-dependent declines in neutral red dye uptake, glucose transport and glutathione content. Chloroacetaldehyde was a more potent toxin than acrolein. Pretreatment of cells with the glutathione-depleting agent buthionine sulfoximine enhanced the toxicity of both chloroacetaldehyde and acrolein while co-administration of mesna or amifostine prevented metabolite toxicity. These results support the hypothesis that chloroacetaldehyde is responsible for ifosfamide-induced nephrotoxicity. The protective effect of mesna and amifostine in vitro contrasts with clinical experience showing that these medications do not eliminate ifosfamide nephrotoxicity.

Topics: Acetaldehyde; Acrolein; Amifostine; Animals; Antineoplastic Agents; Cells, Cultured; Dose-Response Relationship, Drug; Glucose; Glutathione; Ifosfamide; Kidney Tubules; Mesna; Neutral Red; Rabbits

Renal injury is a common side effect of the chemotherapeutic agent ifosfamide. Current evidence suggests that the ifosfamide metabolite chloroacetaldehyde may contribute to this nephrotoxicity. The present study examined the effects of ifosfamide and chloroacetaldehyde on rabbit proximal renal tubule cells in primary culture. The ability of the uroprotectant medication sodium 2-mercaptoethanesulfonate (mesna) to prevent chloroacetaldehyde-induced renal cell injury was also assessed. Chloroacetaldehyde (12.5-150 microM) produced dose-dependent declines in neutral red dye uptake, ATP levels, glutathione content, and cell growth. Coadministration of mesna prevented chloroacetaldehyde toxicity while pretreatment of cells with the glutathione-depleting agent buthionine sulfoximine enhanced the toxicity of chloroacetaldehyde. Ifosfamide (1000-10,000 microM) toxicity was detected only at concentrations of 4000 microM or greater. Analysis of media collected from ifosfamide-treated cell cultures revealed the presence of several ifosfamide metabolites, demonstrating that renal proximal tubule cells are capable of biotransforming this chemotherapeutic agent. This primary renal cell culture system should prove useful in studying the cause and prevention of ifosfamide nephrotoxicity.

Topics: Acetaldehyde; Adenosine Triphosphate; Animals; Antineoplastic Agents, Alkylating; Cell Division; Cells, Cultured; Glucose; Glutathione; Ifosfamide; Kidney Tubules, Proximal; Male; Mesna; Rabbits

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

Based on clinical data, indicating that chloroacetaldehyde (CAA) is an important metabolite of oxazaphosphorine cytostatics, an experimental study was carried out in order to elucidate the role of CAA in the development of hemorrhagic cystitis. The data demonstrate that CAA after i.v. administration does not contribute to bladder damage. When instilled directly into the bladder, CAA exerts urotoxic effects, it is, however, susceptible to detoxification with mesna.

Topics: Acetaldehyde; Animals; Cyclophosphamide; Cystitis; Female; Hemorrhage; Ifosfamide; Male; Mesna; Rats; Rats, Inbred Strains