Comparision of uroprotective activity of reduced glutathione with mesna in ifosfamide induced hemorrhagic cystitis in rats.

BACKGROUND: Ifosfamide (IFO) is widely used DNA-alkylating agents in cancer chemotherapy for management of solid tumors and hematological malignancies. However, hemorrhagic cystitis limits the use of IFO.
OBJECTIVES: To compare the efficiency of reduced glutathione with 2-Mesna in reducing Ifosfamide (IFO) induced hemorrhagic cystitis (HC) in wistar rats.
MATERIALS AND METHODS: Ifosfamide and 2-Mesna were dissolved in sterile water for injection and administered to wistar rats of albino strains. The rats were randomly assigned to one of the four groups of 6 rats each: Group I: Vehicle control; Group II: 120 mg/kg of IFO alone by intraperitoneal injection (i.p); Group III: 40 mg/kg Mesna i.p., at the same time and at 4 and 8 h after IFO administration; Group IV: 500 mg/kg of glutathione i.p., 30 min prior to IFO as above. The animals were observed for 5 days. On 6th day, rats were sacrificed by dissecting the intrajugular vein. The bladders were macroscopically and histopathologically evaluated.
RESULTS: Control animals had normal bladders with assigned scores of '0' for the three parameters of edema, hemorrhage and histopathological changes. All the animals receiving IFO (group II) had evidence of HC as evidenced by alterations of edema and hemorrhages. These alterations were almost abolished (P < 0.001) by the glutathione (group III) or Mesna (group IV) in IFO-treated animals.
CONCLUSION: Glutathione could be as useful as Mesna in the preventive management of IFO-induced HC.

Introduction

Ifosfamide (IFO) is among the most widely used DNA-alkylating agents in cancer chemotherapy. Like other oxazaphosphorines, it is used as a single agent, but more frequently, in combination with other anticancer agents in management of a wide spectrum of solid tumors and hematological malignancies.[1] It is administered as a prodrug that requires activation by hepatic cytochrome P450. It yields cytotoxic nitrogen mustards capable of reacting with DNA molecules, leading to cell death and the metabolic product acrolein which induces urotoxicity.[2] Hemorrhagic cystitis occurs in up to 70% of patients exposed to high doses of cyclophosphamide or ifosphamide chemotherapy.[345] It has been proposed that urothelial damage occurs because of reduction of endogenous glutathione and inducing the generation of free radicals, such as superoxide anion and hydroxyl radical, which initiate lipid peroxidation and other cell damage. This damage is followed by bladder edema, ulceration, neovascularization, hemorrhage, and necrosis. Various anti-oxidants have been tried for prevention of this toxicity, which includes Mesna (2-mercaptoethanesulfonic acid), amifostine, pentosan polysulfate sodium, etc. Amifostine has been used with mixed results in preventing radiation damage, but its use is probably limited to clinical trials. Amifostine is prodrug that is dephosphorylated by alkaline phosphatase to a pharmacologically active free thiol metabolite, which in studies of head and neck cancer, is believed to be responsible for the reduced renal toxicity of cisplatin and reduced side effects of radiation on the normal oral tissues. The active thiol metabolite is thought to scavenge reactive oxygen species generated in irradiated cells. Amifostine is thought to protect the normal tissues, since they have higher pH, higher alkaline phosphatase activity (and produce more active metabolite), and better vascularity than the tumor tissue.[67]

Cyclophosphamide and ifosphamide toxicity can be minimized with the concurrent use of drugs such as N-acetylcysteine, S-2-(3-aminopropylamino) ethyl phosphorothioic acid (amifostine), pentosan polysulfate, and Mesna. These drugs bind to acrolein resulting in an inert thioether, which passes innocuously through urine and limits damage to the uroepithelium.[89101112] Adequate hydration and the concurrent administration of Mesna are the most widely employed methods for prevention of IFO induced HC in clinical practice. However, HC still occurs in 10-40% of Mesna-treated patients.[12] Therefore, there is a need for new effective agents for prevention. Here we compare the uroprotective activity of glutathione, an antioxidant, with Mesna in reducing the adverse effects of IFO that can lead to better tolerance of IFO and optimization of chemotherapy.

Materials and Methods

Ifosfamide (Ifex®) and 2-MESNA (Mesnex®) were obtained from Ranbaxy Labs and reduced glutathione (GSH) from Qualigens fine chemicals, India. Chemicals used were ofanalytical grade and were obtained locally. All compounds were dissolved in sterile water for injection prior to administration. Wistar rats of albino strains were procured locally. The rats were randomly assigned to one of the four groups of 6 rats each: Group I: Vehicle control; Group II: 120 mg/kg of IFO alone by intraperitoneal (i.p) injection; Group III: 40 mg/kg mesna i.p., at the same time and at 4 and 8 h after IFO administration; Group IV: 500 mg/kg of glutathione i.p., 30 min prior to IFO.[1314]

The animals were kept under physical observation for five days. On 6th day, rats were sacrificed by dissecting the intrajugular vein. All the procedures were carried out in accordance with the guidelines of CPCSEA and Institutional Animal Ethics committee (Registration number: 1656/B/12 CPCSEA).

Macroscopic Evaluation

Bladders were examined grossly for edema and hemorrhage according to Gray's criteria.[15] Edema was considered severe (3+) when fluid was seen externally and internally in the bladder walls; moderate (2+) when confined to the internal mucosa; mild (1+) between normal to moderate; and absent (0). Hemorrhage was scored as follows: (3+) intravesical clots; (2+) mucosal hematomas; (1+) telangiectasia or dilatation of the bladder vessels; and (0) normal.

Histopathological Evaluation

Bladders were fixed in formalin, embedded in paraffin, and processed for hematoxylin and eosin staining. Histopathological changes were scored as follows: (0), normal epithelium and absence of inflammatory cell infiltration and ulceration; (1+), mild changes involving reduction of epithelial cells, flattening with submucosal edema, mild hemorrhage, and few ulcerations; (2+), severe changes including mucosal erosion, inflammatory cell infiltration, fibrin deposition, hemorrhage, and multiple ulcerations. Blinding to histopathological analysis was done.

The results were expressed as median (min-max) and P < 0.05 was considered as statistically significant. The data was analyzed using Kruskal-Wallis test to find whether there is difference between the groups, followed by Wilcoxon sum rank test to analyze two groups, consecutively.

Results

Control animals had grossly normal bladders with assigned scores of ‘0’ for the three parameters of edema, hemorrhage and histopathological changes [Table 1]. Abnormalities observed in group II were bleeding from eyes and nose, hematuria, thrombocytopenia around femur bone due to rupture of blood vessels, penis was protruded out. Blood clot in the testis, Atrial clots, parts of duodenum and jejunum were abnormal [Figure 1]. All the animals receiving IFO (group II) had evidence of HC. It was characterized macroscopically by the presence of moderate to severe edema, receiving a score of 2.5 (2-3); and marked hemorrhage with mucosal hematomas and intravesical clots, receiving a score of 2 (2-3); that was significantly (P < 0.001) different from the control group which received a score of 0 (0-0) for edema and hemorrhage. Significant histological changes, including extensive mucosal erosion with multiple ulcerations, inflammatory cell infiltration, and fibrin deposition, receiving a score of 2 (1-2) were also present in this group compared with control group, which received a score of 0 (0-0) (P < 0.001). These alterations were almost abolished (P < 0.001) by the glutathione (group III) or mesna (group IV) in IFO-treated animals. No abnormal observations were recorded in Group I, III, IV [Figures 2, 3 and 4]

Table 1

Compared scores for gross evaluation and histological grading of bladder changes in ifosfamide induced hemorrhagic cystitis in rats

Figure 1

Cystitis in a rat treated with ifosfamide (120 mg/kg, i.p.) alone, showing severe ulcerations, edema, leukocyte infiltration, hemorrhage and fibrin deposition; score of 2 (Gray's criteria). (H&E, 200X)

Figure 2

Normal bladder in rats in control group without ulcerations, edema, leukocyte infiltration, hemorrhage and fibrin deposition

Figure 3

Bladder of a rat treated with three doses of Mesna (20% of ifosfamide dose, 30 mg/kg, and i.p.) showing edema and dilatation of bladder vessels (Score of 1) (H&E, 200X)

Figure 4

Bladder of a rat treated with ifosfamide and glutathione (500 mg/kg) showing urothelium preservation and edema (Score of 1) (H&E, 200X)

Dilatation of the bladder vessels was noted in four bladders. Only one animal had mild histopathological changes suggestive of HC.

In Group IV, mild mucosal edema was noted in four, and dilatation of bladder vessels was found in two bladders. When the scores (median and range) for bladder damage of group I, III and IV were compared; there was no significant difference among these groups (P > 0.05).

Discussion

Ifosfamide is an oxazaphosphorine alkylating agent with a broad spectrum of antineoplastic activity. It interacts with DNA to form cross-linking adducts. Recent evidence suggests that these alkylating agents increase production of reactive oxygen species (ROS) such as nitric oxide,[16] which can damage DNA and contribute to toxicity through interference in the central dogma in the cell.[17] Hemorrhagic cystitis is a common complication associated with the use of ifosfamide and cyclophosphamide during cancer therapeutics.[18] Although, these alkylating agents are known to induce hemorrhagic cystitis, the incidence of this side effect is greater with ifosfamide treatment.[19] In the absence of adequate uroprotection with MESNA (2-mercaptoethanesulfonic acid) but with standard prophylaxis, that is high fluid intake, diuretics, forced diuresis and urine alkalization, HC becomes dose limiting but not prevented.[16] Also with Mesna, approximately 40% of IFO-treated children develop a permanent subclinical renal tubulopathy and 5% have a persistent De Toni-Debre-Fanconi syndrome.[20] Alternatively, there is a need to search for newer agents which could provide better solutions to the problem.

Glutathione is a ubiquitous molecule that is produced in all organs and is present in all mammalian tissues. Intracellularly, it is found in millimolar concentrations; plasma and urine containing lower total GSH levels.[21] In normal conditions, the glutathione redox couple is present in mammalian cells in concentrations between 1 and 10 mM, with the reduced GSH predominating. In the resting cell, the ratio exceeds 100, whereas in various models of oxidative stress, this ratio was reported to decrease.[22] Recent reports have demonstrated that glutathione dependent enzymes activities are increased in several human tumors. Reduced glutathione levels in the normal tissues were found to be higher than the tumor tissues. This decrease in tumor tissue GSH levels may be a consequence of the increased detoxification activity in the tumor cells.[23] Many studies have been done on the uroprotective activities of MESNA, amifostine, pentosan polysulfate, dexamethasone, etc and they had a common finding that the level of anti-oxidants in the tumor cells have decreased due to the activity of alkylating agents.[910111213] In a similar study, bladder contents of adenosine triphosphate, reduced glutathione and glutathione-S-transferase activity were markedly reduced whereas malondialdehyde level, myeloperoxidase activity and urinary nitrite-nitrate levels, expressed as nitric oxide, were dramatically increased, demonstrating increased oxidant activity.[24]

The animals treated with ifosfamide had evidence of HC along with histological alteration [Figure 3]. When treated with IFO and GSH simultaneously, these alteration were completely abolished [Figure 4]. This could also provide useful information for predicting a possible mechanism of action of GSH with IFO in prevention of DNA damage. Animals treated with GSH had dilated vessels. This can be postulated by a marked development and enhancement of the vasodilator action of arachidonic acid modulated by GSH.[25]

Conclusion

We investigated the role of glutathione in the prevention of ifosfamide-induced HC and compared its efficacy with Mesna. We found that glutathione could be a useful agent in the preventive management of IFO-induced HC. We assume that this observation provides evidence that could modify the prophylactic approaches to IFO-induced bladder damage. Thus, glutathione should be investigated clinically as an alternative treatment to prevent HC observed in patients undergoing Ifosfamide treatment.