|Year : 2014 | Volume
| Issue : 1 | Page : 105-108
Comparision of uroprotective activity of reduced glutathione with Mesna in Ifosfamide induced hemorrhagic cystitis in rats
Syed Amir Ali1, Sandeep Kumar Danda2, Syed Abdul Azeez Basha1, Asif Rasheed1, Osman Ahmed1, M Muqtedar Ahmed1
1 Deccan School of Pharmacy, Hyderabad, Andhra Pradesh, India
2 Bharat Institute of Technology, Hyderabad, Andhra Pradesh, India
|Date of Submission||01-Jul-2013|
|Date of Decision||12-Aug-2013|
|Date of Acceptance||01-Dec-2013|
|Date of Web Publication||16-Jan-2014|
Syed Amir Ali
Deccan School of Pharmacy, Hyderabad, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
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 6 th 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.
Keywords: Glutathione, hemorrhagic cystitis, ifosfamide, solid tumors
|How to cite this article:|
Ali SA, Danda SK, Basha SA, Rasheed A, Ahmed O, Ahmed M M. Comparision of uroprotective activity of reduced glutathione with Mesna in Ifosfamide induced hemorrhagic cystitis in rats. Indian J Pharmacol 2014;46:105-8
|How to cite this URL:|
Ali SA, Danda SK, Basha SA, Rasheed A, Ahmed O, Ahmed M M. Comparision of uroprotective activity of reduced glutathione with Mesna in Ifosfamide induced hemorrhagic cystitis in rats. Indian J Pharmacol [serial online] 2014 [cited 2021 Oct 22];46:105-8. Available from: https://www.ijp-online.com/text.asp?2014/46/1/105/125188
| » 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.  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.  Hemorrhagic cystitis occurs in up to 70% of patients exposed to high doses of cyclophosphamide or ifosphamide chemotherapy. ,, 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. ,
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. ,,,, 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.  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. ,
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).
Bladders were examined grossly for edema and hemorrhage according to Gray's criteria.  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.
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 [Figure 2], [Figure 3] and [Figure 4]
|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)|
Click here to view
|Figure 2: Normal bladder in rats in control group without ulcerations, edema, leukocyte infiltration, hemorrhage and fibrin deposition|
Click here to view
|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)|
Click here to view
|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)|
Click here to view
|Table 1: Compared scores for gross evaluation and histological grading of bladder changes in ifosfamide induced hemorrhagic cystitis in rats|
Click here to view
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,  which can damage DNA and contribute to toxicity through interference in the central dogma in the cell.  Hemorrhagic cystitis is a common complication associated with the use of ifosfamide and cyclophosphamide during cancer therapeutics.  Although, these alkylating agents are known to induce hemorrhagic cystitis, the incidence of this side effect is greater with ifosfamide treatment.  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.  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.  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.  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.  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.  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. ,,,, 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. 
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. 
| » 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.
| » References|| |
|1.||Wang D, Wang H. Oxazaphosphorine bioactivation and detoxification: The role of xenobiotic receptors. Acta Pharmaceutica Sinica B 2012;2:107-17. |
|2.||Zhang J, Tian Q, Yung Chan S, Chuen Li S, Zhou S, Duan W, et al. Metabolism and transport of oxazaphosphorines and the clinical implications. Drug Metab Rev 2005;37:611-703. |
|3.||Cannon J, Linke CA, Cos LR. Cyclophosphamide-associated carcinoma of urothelium: Modalities for prevention. Urology 1991;38:413-6. |
|4.||Efros M, Ahmed T, Choudhury M. Cyclophosphamide-induced hemorrhagic pyelitis and ureteritis associated with cystitis in marrow transplantation. J Urol 1990;144:1231-2. |
|5.||Fairchild WV, Spence CR, Solomon HD, Gangai MP. The incidence of bladder cancer after cyclophosphamide therapy. J Urol 1979;122:163-4. |
|6.||Batista CK, Mota JM, Souza ML, Leitão BT, Souza MH, Brito GA, et al. Amifostine and glutathione prevent ifosfamide-and acrolein-induced hemorrhagic cystitis. Cancer Chemother Pharmacol 2007;59:71-7. |
|7.||Srivastava A, Nair SC, Srivastava VM, Balamurugan AN, Jeyaseelan L, Chandy M, et al. Evaluation of uroprotective efficacy of amifostine against cyclophosphamide induced hemorrhagic cystitis. Bone Marrow Transplant 1999;23:463-7. |
|8.||Batista CK, Brito GA, Souza ML, Leitão BT, Cunha FQ, Ribeiro RA. A model of hemorrhagic cystitis induced with acrolein in mice. Braz Journal Med Biol Res 2006;39:1475-81. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17146560. |
|9.||Ehrlich RM, Freedman A, Goldsobel AB, Stiehm ER. The use of sodium 2-mercaptoethane sulfonate to prevent cyclophosphamide cystitis. J Urol 1984;131:960-2. |
|10.||Goren MP, McKenna LM, Goodman TL. Combined intravenous and oral mesna in outpatients treated with ifosfamide. Cancer Chemother Pharmacol 1997;40:371-5. |
|11.||Habs MR, Schmähl D. Prevention of urinary bladder tumors in cyclophosphamide-treated rats by additional medication with the uroprotectors sodium 2-mercaptoethane sulfonate (mesna) and disodium 2, 2′-dithio-bis-ethane sulfonate (dimesna). Cancer 1983;51:606-9. |
|12.||Kanat O, Kurt E, Yalcinkaya U, Evrensel T, Manavoglu O. Comparison of uroprotective efficacy of mesna and amifostine in cyclophosphamide-induced hemorrhagic cystitis in rats. Indian J Cancer 2006;43:12-5. |
|13.||Morais MM, Belarmino-Filho JN, Brito GA, Ribeiro RA. Pharmacological and histopathological study of cyclophosphamide-induced hemorrhagic cystitis - comparison of the effects of dexamethasone and Mesna. Braz J Med Biol Res 1999;32:1211-5. |
|14.||Katz A, Epelman S, Anelli A, Gorender EF, Cruz SM, Oliveira RM, et al. A prospective randomized evaluation of three schedules of mesna administration in patients receiving an ifosfamide-containing chemotherapy regimen: Sustained efficiency and simplified administration. J Cancer Res Clin Oncol 1995;121:128-31. |
|15.||Gray KJ, Engelmann UH, Johnson EH, Fishman IJ. Evaluation of misoprostol cytoprotection of the bladder with cyclophosphamide (Cytoxan) therapy. J Urol 1986;136:497-500. |
|16.||Ribeiro RA, Freitas HC, Campos MC, Santos CC, Figueiredo FC, Brito GA, et al. Tumor necrosis factor-alpha and interleukin-1beta mediate the production of nitric oxide involved in the pathogenesis of ifosfamide induced hemorrhagic cystitis in mice. J Urol 2002;167:2229-34. |
|17.||Duarte DB, Vasko MR. The role of DNA damage and repair in neurotoxicity caused by cancer therapies. In: Kelley MR, editor. DNA Repair in Cancer Therapy. Ch. 13. San Diego: Academic Press; 2012. p. 283-299. |
|18.||Kiuchi H, Takao T, Yamamoto K, Nakayama J, Miyagawa Y, Tsujimura A, et al. Sesquiterpene lactone parthenolide ameliorates bladder inflammation and bladder overactivity in cyclophosphamide induced rat cystitis model by inhibiting nuclear factor-kappa B phosphorylation. J Urol 2009;181:2339-48. |
|19.||Ratliff TR, Williams RD. Hemorrhagic cystitis, chemotherapy, and bladder toxicity. J Urol 1998;159:1044. |
|20.||Zaki EL, Springate JE, Taub M. Comparative toxicity of ifosfamide metabolites and protective effect of mesna and amifostine in cultured renal tubule cells. Toxicol In Vitro. 2003;17:397-402. |
|21.||Pastore A, Federici G, Bertini E, Piemonte F. Analysis of glutathione: Implication in redox and detoxification. Clin Chim Acta 2003;333:19-39. |
|22.||Chai YC, Ashraf SS, Rokutan K, Johnston RB Jr, Thomas JA. S-thiolation of individual human neutrophil proteins including actin by stimulation of the respiratory burst: Evidence against a role for glutathione disulfide. Arch Biochem Biophys 1994;310:273-81. |
|23.||Saydam N, Kirb A, Demir Ö, Hazan E, Oto O, Saydam O, et al. Determination of glutathione, glutathione reductase, glutathione peroxidase and glutathione S-transferase levels in human lung cancer tissues. Cancer Lett 1997;119:13-9. |
|24.||Arafa HM. Unprotective effects of curcumin in cyclophosphamide induced haemorrhagic cystitis paradigm. Basic Clin Pharmacol Toxicol 2009;104:393-9. |
|25.||Talesnik J, Tsoporis JN. Reduced glutathione modulates the arachidonic acid induced coronary reactions. Can J Physiol Pharmacol 1984;62:1261-7. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]