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Research Article
Comparision of uroprotective activity of reduced glutathione with Mesna in Ifosfamide induced hemorrhagic cystitis in rats Syed Amir Ali, Sandeep Kumar Danda1, Syed Abdul Azeez Basha, Asif Rasheed, Osman Ahmed, M. Muqtedar Ahmed
ABSTRACT 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.
Deccan School of Pharmacy, Hyderabad, 1Bharat Institute of Technology, Hyderabad, Andhra Pradesh, India Received: 01-07-2013 Revised: 12-08-2013 Accepted: 01-12-2013 Correspondence to: Mr. Syed Aamir Ali, E-mail: [email protected]
KEY WORDS: Glutathione, hemorrhagic cystitis, ifosfamide, solid tumors
Introduction Ifosfamide (IFO) is among the most widely used DNAalkylating 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 Access this article online Website: www.ijp-online.com DOI: 10.4103/0253-7613.125188
Quick Response Code:
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. [3-5] 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 Indian Journal of Pharmacology | February 2014 | Vol 46 | Issue 1 105
[Downloaded free from http://www.ijp-online.com on Tuesday, January 21, 2014, IP: 202.164.45.14] || Click here to download free Android application for this journa Ali, et al.: Comparison of uroprotective activity of reduced glutathione with Mesna
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.[6,7] 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.[8-12] 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.[13,14] 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. 106 Indian Journal of Pharmacology | February 2014 | Vol 46 | Issue 1
The results were expressed as median (min-max) and 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 Table 1: Compared scores for gross evaluation and histological grading of bladder changes in ifosfamide induced hemorrhagic cystitis in rats Group
Treatment
Edema
Hemorrhage
Microscopic analysis
I II III
Control IFO IFO and Mesna IFO and glutathione
0 (0-0)* 2.5 (2-3)† 0 (0-1)*
0 (0-0)* 2 (2-3)† 0 (0-1)*
0 (0-0)* 2 (1-2)† 0 (0-0)*
0 (0-1)*
0 (0-1)*
0 (0-1)*
IV
Medians and Range (n = 6 per group), *P < 0.01, †P < 0.001 when compared with control group
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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 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)
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,
Figure 2: Normal bladder in rats in control group without ulcerations, edema, leukocyte infiltration, hemorrhage and fibrin deposition
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)
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.[9-13] 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] Indian Journal of Pharmacology | February 2014 | Vol 46 | Issue 1 107
[Downloaded free from http://www.ijp-online.com on Tuesday, January 21, 2014, IP: 202.164.45.14] || Click here to download free Android application for this journa Ali, et al.: Comparison of uroprotective activity of reduced glutathione with Mesna
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]
12. 13.
14.
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 cyclophosphamidetreated rats by additional medication with the uroprotectors sodium
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2-mercaptoethane sulfonate (mesna) and disodium 2, 2′-dithio-bis-ethane sulfonate (dimesna). Cancer 1983;51:606-9. 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. 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. 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. 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. 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. 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. 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. Ratliff TR, Williams RD. Hemorrhagic cystitis, chemotherapy, and bladder toxicity. J Urol 1998;159:1044. 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. Pastore A, Federici G, Bertini E, Piemonte F. Analysis of glutathione: Implication in redox and detoxification. Clin Chim Acta 2003;333:19-39. 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. 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. Arafa HM. Unprotective effects of curcumin in cyclophosphamide induced haemorrhagic cystitis paradigm. Basic Clin Pharmacol Toxicol 2009;104:393-9. Talesnik J, Tsoporis JN. Reduced glutathione modulates the arachidonic acid induced coronary reactions. Can J Physiol Pharmacol 1984;62:1261-7.
Cite this article as: Ali SA, Danda SK, Basha SA, Rasheed A, Ahmed O, Ahmed MM. Comparision of uroprotective activity of reduced glutathione with Mesna in Ifosfamide induced hemorrhagic cystitis in rats. Indian J Pharmacol 2014;46:105-8. Source of Support: Nil, Conflict of Interest: No.
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Research Article
Comparision of uroprotective activity of reduced glutathione with Mesna in Ifosfamide induced hemorrhagic cystitis in rats Syed Amir Ali, Sandeep Kumar Danda1, Syed Abdul Azeez Basha, Asif Rasheed, Osman Ahmed, M. Muqtedar Ahmed
ABSTRACT 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.
Deccan School of Pharmacy, Hyderabad, 1Bharat Institute of Technology, Hyderabad, Andhra Pradesh, India Received: 01-07-2013 Revised: 12-08-2013 Accepted: 01-12-2013 Correspondence to: Mr. Syed Aamir Ali, E-mail: [email protected]
KEY WORDS: Glutathione, hemorrhagic cystitis, ifosfamide, solid tumors
Introduction Ifosfamide (IFO) is among the most widely used DNAalkylating 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 Access this article online Website: www.ijp-online.com DOI: 10.4103/0253-7613.125188
Quick Response Code:
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. [3-5] 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 Indian Journal of Pharmacology | February 2014 | Vol 46 | Issue 1 105
[Downloaded free from http://www.ijp-online.com on Tuesday, January 21, 2014, IP: 202.164.45.14] || Click here to download free Android application for this journa Ali, et al.: Comparison of uroprotective activity of reduced glutathione with Mesna
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.[6,7] 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.[8-12] 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.[13,14] 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. 106 Indian Journal of Pharmacology | February 2014 | Vol 46 | Issue 1
The results were expressed as median (min-max) and 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 Table 1: Compared scores for gross evaluation and histological grading of bladder changes in ifosfamide induced hemorrhagic cystitis in rats Group
Treatment
Edema
Hemorrhage
Microscopic analysis
I II III
Control IFO IFO and Mesna IFO and glutathione
0 (0-0)* 2.5 (2-3)† 0 (0-1)*
0 (0-0)* 2 (2-3)† 0 (0-1)*
0 (0-0)* 2 (1-2)† 0 (0-0)*
0 (0-1)*
0 (0-1)*
0 (0-1)*
IV
Medians and Range (n = 6 per group), *P < 0.01, †P < 0.001 when compared with control group
[Downloaded free from http://www.ijp-online.com on Tuesday, January 21, 2014, IP: 202.164.45.14] || Click here to download free Android application for this journa Ali, et al.: Comparison of uroprotective activity of reduced glutathione with Mesna
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 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)
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,
Figure 2: Normal bladder in rats in control group without ulcerations, edema, leukocyte infiltration, hemorrhage and fibrin deposition
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)
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.[9-13] 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] Indian Journal of Pharmacology | February 2014 | Vol 46 | Issue 1 107
[Downloaded free from http://www.ijp-online.com on Tuesday, January 21, 2014, IP: 202.164.45.14] || Click here to download free Android application for this journa Ali, et al.: Comparison of uroprotective activity of reduced glutathione with Mesna
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]
12. 13.
14.
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 cyclophosphamidetreated rats by additional medication with the uroprotectors sodium
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Cite this article as: Ali SA, Danda SK, Basha SA, Rasheed A, Ahmed O, Ahmed MM. Comparision of uroprotective activity of reduced glutathione with Mesna in Ifosfamide induced hemorrhagic cystitis in rats. Indian J Pharmacol 2014;46:105-8. Source of Support: Nil, Conflict of Interest: No.
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