|Year : 2013 | Volume
| Issue : 2 | Page : 126-129
Oral administration of vitamin C and histidine attenuate cyclophosphamide-induced hemorrhagic cystitis in rats
Amir Abbas Farshid1, Esmaeal Tamaddonfard2, Sepideh Ranjbar1
1 Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
2 Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
|Date of Submission||12-Mar-2012|
|Date of Decision||02-Jun-2012|
|Date of Acceptance||30-Dec-2012|
|Date of Web Publication||11-Mar-2013|
Amir Abbas Farshid
Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia
Source of Support: Faculty of Veterinary Medicine of Urmia University, Conflict of Interest: None
Objectives: Cyclophosphamide (CP), a widely used antineoplastic drug causes hemorrhagic cystitis (HC) mainly via induction of oxidative stress. Both vitamin C and histidine have antioxidant properties. The present study aimed to investigate the effects of oral (p.o.) administration of vitamin C and histidine on the CP-induced HC in rats.
Materials and Methods: The animals were divided into two major groups I and II with four subgroups (a, b, c, and d) in each. Groups I and II were treated with intraperitoneal (i.p.) injections of normal saline and CP (200 mg/kg), respectively, thereafter, normal saline, vitamin C (200 mg/kg), histidine (200 mg/kg) and vitamin C plus histidine were p.o. administered in subgroups a, b, c, and d, respectively, three times (2, 6, and 24 h) after i.p. injections of normal saline and CP. Blood samples were assayed for total antioxidant capacity (TAC) and malondialdehyde (MDA) levels. Histopathological changes of bladder wall were investigated.
Results: The decreased TAC and increased MDA levels of plasma and the severity of hemorrhages, congestion, edema, and leukocyte infiltration of bladder induced by CP were recovered with vitamin C and histidine treatments. Combined treatment with vitamin C and histidine showed a potentiation effect.
Conclusion: The results indicated that vitamin C and histidine attenuated the CP-induced HC by reducing of free radical-induced toxic effects.
Keywords: Cyclophosphamide, histidine, hemorrhagic cystitis, rats, vitamin C
|How to cite this article:|
Farshid AA, Tamaddonfard E, Ranjbar S. Oral administration of vitamin C and histidine attenuate cyclophosphamide-induced hemorrhagic cystitis in rats. Indian J Pharmacol 2013;45:126-9
|How to cite this URL:|
Farshid AA, Tamaddonfard E, Ranjbar S. Oral administration of vitamin C and histidine attenuate cyclophosphamide-induced hemorrhagic cystitis in rats. Indian J Pharmacol [serial online] 2013 [cited 2021 Feb 25];45:126-9. Available from: https://www.ijp-online.com/text.asp?2013/45/2/126/108283
| » Introduction|| |
Cyclophosphamide (CP) is widely used in the treatment of tumors and B cells malignant disease, such as lymphoma and myeloma and in non-malignant disease such as rheumatoid arthritis. Hemorrhagic cystitis (HC) is a common dose-limiting side effect of CP with a reported incidence of 40- 75% and mortality rate 4%.  It has been shown that reactive oxygen species (ROS) play an important role in CP-induced HC.  Systemic or i.p. injection of CP induces a reproducible dose- dependent chemical cystitis in both mice and rats, and therefore has been used as an experimental model of cystitis. 
Vitamin C (ascorbic acid) is a water-soluble vitamin and has a potent free radical scavenger activity in the plasma, protecting cells against oxidative damage caused by ROS.  Vitamin C has also anti-atherosclerotic, anti-rheumatoid, anti-cancer, and anti-cataract properties.  Some researchers have reported suppressive effects of vitamin C on the CP-induced oxidative stress changes. , Recently, Gurbuz et al.  reported suppressive effects of vitamin C on the histopathologic and genotoxic changes induced by CP in the bladder of mice.
Histidine is one of the most common natural amino acids, and has many biological functions. In the carrageenan-induced inflammation model, histidine reduced paw edema as well as neutrophil infiltration in paw tissues.  Histidine also attenuated the histopathological changes in the colon of acetic acid-induced colitis in rats.  Several studies have reported that histidine possessed marked antioxidant activities such as scavenging free radicals, binding divalent metal ions and anti-glycating action. ,
In the present study, we investigated the effects of oral administrations of vitamin C and histidine in separate and combined treatments on the plasma TAC, MDA levels, and histopathological changes of bladder induced by i.p. injection of CP in rats.
| » Materials and Methods|| |
Healthy adult male Wistar rats, weighing 250-270 g were used in this study. Rats were maintained in groups of 6 per cage in a light-dark cycle (light on at 07:00 h) at a controlled ambient temperature (22 ± 0.5°C) with ad libitum food and water. Six rats were used for each experiment. All research and animal care procedures were approved by the Veterinary Ethics Committee of the Faculty of Veterinary Medicine of Urmia University and were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Drugs and chemicals
Vitamin C (ascorbic acid) and histidine dihydrochloride powders purchased from Sigma Chemical (St. Louis, MO, USA). Cyclophosphamide (Endoxan Baxter ® ) powder, sodium dodecyl sulfate, acetic acid, thiobarbituric acid, n-butanol, pyridine, 2, 4, 6-tripyridyl-S-triazine (TPTZ), and FeCl 3 .6H 2 O purchased from Merck Chemical Co. (Darmstadt, Germany).
In this study, 48 rats were divided into two major groups I and II each containing 24 rats. Each major group were divided into four subgroups a, b, c, and d each containing six rats. Group Ia received i.p. and p.o. of normal saline, group Ib received i.p. of normal saline and p.o. of vitamin C (200 mg/ kg), group Ic received i.p. of normal saline and p.o. of histidine (200 mg/kg), group Id received i.p. of normal saline and p.o. of vitamin C (200 mg/kg) plus histidine (200 mg/kg), group IIa received CP (200 mg/kg, i.p.) followed by p.o. of normal saline, group IIb received CP (200 mg/kg, i.p.) followed by vitamin C (200 mg/ kg, p.o.), group IIc received CP (200 mg/ kg, i.p.) followed by histidine (200 mg/kg, p.o.) and group IId received CP (200 mg/ kg, i.p.) followed by vitamin C (200 mg/kg, p.o.) plus histidine (200 mg/kg, p.o.). Groups Ia, Ib, Ic, and Id served as control groups. The p.o. administrations of normal saline, vitamin C, and histidine were performed 2, 6, and 24 h after i.p. injections of normal saline and CP. The protocol for this study, including doses of treatment for CP, vitamin C and histidine were all designed according to previous studies. ,,
Forty-eight hours after CP injection, the animals were euthanized using diethyl ether and decapitated. Blood samples were collected in vials containing heparin. The plasma was separated and kept at -80°C until analysis of TAC and MDA. Necropsy was conducted and the bladder tissue was fixed in 10% buffer formal saline.
Plasma TAC was determined by measuring the ability to reduce Fe 3+ to Fe 2+ as named ferric-reducing antioxidant power (FRAP).  The reagent included TPTZ, FeCl 3 , and acetate buffer. Twenty microliter of water-diluted plasma was added to 600 microliter of freshly prepared reagent warmed at 37°C. The complex between Fe 2+ and TPTZ gives a blue color with absorbance at 593 nm. Plasma TAC levels were expressed as mmol/ml.
Malondialdehyde, an index of free radical generation/lipid peroxidation, was determined as described by Ohkawa et al.  Briefly, the reaction mixture consisted of 0.2 ml of 8.1% sodium dudecyl, 1.5 ml of 20% acetic acid (pH 3.5), 1.5 ml of 0.8% aqueous solution of thiobarbituric acid and 0.2 ml of blood plasma. The mixture was made up to 4 ml with distilled water and heated at 95°C for 60 min. After the cooling the contents under running tap water, 5 ml of n-butanol and pyridine (15:1 v/v) and 1 ml of distilled water was added. The contents were centrifuged at about 16000 × g for 3 min. The organic layer was separated out and its absorbance was measured at 532 nm using double beam UV-Visible spectrophotometer (JASCO, UV-975, Tokyo, Japan). Plasma MDA concentrations were expressed as nmol/ml.
The 10% buffer formal saline fixed bladders routinely processed for paraffin embedding. Thin sections (4-5 μm) were cut using a microtome and stained with hematoxylin and eosin (H&E) and then examined using a light microscope. The evaluation of the sections was based on pathological changes including edema, congestion, hemorrhages, and inflammatory cell infiltration on a scale from normal (0) sever changes (3).
The values are expressed as mean ± SEM of six animals. Differences between groups were assessed by one-way analysis of variance (ANOVA) followed by Duncan's test. Significance at P < 0.05 has been given receptive in the tables.
| » Results|| |
[Figure 1] shows the effects of vitamin C and histidine on CP- induced plasma changes of TAC [Figure 1]a and MDA [Figure 1]b levels. No significant changes on plasma levels of TAC were observed among normal saline, vitamin C, histidine, and vitamin C plus histidine-treated groups (511.7 ± 38.3, 520.6 ± 36.7, 493.8 ± 20.1, 502.2 ± 34.8 mmol/ ml, respectively) (F(3,20) = 0.124, P > 0.05, one-way ANOVA) [Figure 1]a. The i.p. injection of CP significantly decreased plasma TAC levels. Separate and combined treatments with vitamin C and histidine reversed the effect of CP on plasma TAC levels. The reversing effect of vitamin C plus histidine on plasma TAC levels was significantly more than those obtained from vitamin C and histidine when used alone (F(4,25) = 11.565, P < 0.05, one-way ANOVA) [Figure 1]a. Plasma levels of MDA was not significantly changed by treatments with normal saline, vitamin C, histidine, and vitamin C plus histidine (0.71 ± 0.06, 0.66 ± 0.05, 0.71 ± 0.05, 0.64 ± 0.08 nmol/ml, respectively, F(3,20) = 0.352, P > 0.05, one-way ANOVA) [Figure 1]b. Plasma MDA levels was significantly increased by i.p. injection of CP. Separate and combined treatments with vitamin C and histidine reversed the effect of CP on plasma MDA levels. The reversing effect of vitamin C plus histidine on plasma MDA levels was significantly more than those obtained from vitamin C and histidine when used alone (F(4,25) = 8.697, P < 0.05, one-way ANOVA) [Figure 1]b.
|Figure 1: Effects of vitamin C and histidine on plasma changes of total antioxidant capacity (TAC) (a) and malondialdehyde (MDA) (b) induced by cyclophosphamide (CP) in rats. Each column represents mean ± SEM (n = 6). *P < 0.05 vs. Ns + Ns group. †P < 0.05 vs. CP + Ns group. ×P < 0.05 vs. CP + Vit C and CP + His groups (one-way ANOVA followed by Duncan's test). Ns: Normal saline, CP: Cyclophosphamide, Vit C: Vitamin C, His: Histidine, i.p.: Intraperitoneal, p.o.: Oral administration|
Click here to view
[Figure 2] and [Figure 3] show the effects of vitamin C and histidine on CP-induced histopathological changes in the bladder. No histopathological changes were observed among Ia (Ns + Ns), Ib (Ns + Vit C), Ic (Ns + His), Id (Ns + Vit C + His) groups. The data of groups Ib, Ic, and Id were not shown in [Figure 2] and [Figure 3]. In Ia group, no histopathological changes were observed [Figure 2] and [Figure 3]a. In IIa group, i.p. injection of CP produced severe histopathological changes in the bladder, including congestion, hemorrhages, edema and leukocyte infiltration [Figure 2] and [Figure 3]b. Oral administrations of vitamin C (IIb group) produced no effect on congestion and hemorrhages, but significantly (P < 0.05) prevented edema and leukocyte infiltration induced by CP [Figure 2] and [Figure 3]c. Oral administration of histidine (IIc group) significantly (P < 0.05) attenuated all the histopathological changes induced by CP. [Figure 2] and [Figure 3]d. The suppressive effects of vitamin C plus histidine (IId group) on histopathological changes were more documented than those obtained from vitamin C (IIb group) and histidine (IIc group) when used alone [Figure 2] and [Figure 3]e.
|Figure 2: Effect of vitamin C and histidine on histopathological changes in the urinary bladder wall induced by cyclophosphamide in rats. Each column represents mean ± SEM (n = 6). *P < 0.05 vs. Ns + Ns group. †P < 0.05 vs. CP + Ns group. ×P < 0.05 vs. CP + Vit C and CP + His groups (one-way ANOVA followed by Duncan's test). Ns: Normal saline, CP: Cyclophosphamide, Vit C: Vitamin C, His: Histidine, i.p.: Intraperitoneal, p.o.: Oral administration|
Click here to view
|Figure 3: Histopathology of bladder wall in experimental groups (H&E, ×100). (a) normal architecture in control group. (b) CP-administered group showing congested blood vessels (c), edema (e), hemorrhages (h), and leukocyte infiltration (li), (c) CP-administered group showing extensive edema (e), (d, e, and f) CP plus vitamin C, CP plus histidine, and CP plus vitamin C plus histidine groups, showing nearly normal bladder wall|
Click here to view
| » Discussion|| |
In the present study, a single dose of CP suppressed the TAC, increased MDA and produced histological changes including congestion, edema, hemorrhages, and leukocyte infiltration in the wall of the urinary bladder. Ifosfamide, a synthetic analog of CP and CP are widely used in treatment of neoplastic and non-neoplastic disease.  In most of the toxic manifestations of CP including urotoxicity, ROS have been implicated to play a major role.  Living organisms have developed complex antioxidant systems to counteract reactive species and to reduce their damage. These antioxidant systems include enzymes such as superoxide dismutase, macromolecules such as ceruloplasmin, and an array of small molecules including ascorbic acid, β-carotene, uric acid and bilirubin.  However, the decreased and increased levels of plasma TAC and MDA, respectively, and induction of histopathological changes in the bladder wall induced by CP, observed in the present study, are consistent with the previous reports. ,
Vitamin C prevented the suppressive effect of CP on TAC and attenuated the histopathological changes induced by CP on the bladder wall. Vitamin C is a potent antioxidant, and antioxidant property of vitamin C attributes to its ability to reduce potentially damaging ROS, forming, instead, resonance stabilized and relatively stable ascorbate free radicals.  Some researchers have reported beneficial effects of vitamin C in CP-induced adverse changes in biological systems. Vitamin C reversed CP-induced testicular gametogenic and androgenic disorders in male rats.  Moreover, vitamin C restored the increased activities of acid phosphatase (ACP), glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in liver, kidney, and plasma of female rats treated with CP.  In addition, vitamin C showed an inhibitory effect on histopathological changes induced by CP, including inflammatory infiltration and hemorrhage in mucosa propria, in the bladder in mice. 
Histidine recovered the plasma TAC suppression, MDA elevation and attenuated the severity of congestion, edema, hemorrhage, and leukocyte infiltration induced by i.p. injection of CP. In thioacetamide-induced liver fibrosis in rats, i.p. injection of histidine reversed the decreased TAC.  Oral supplementation of L-histidinol, a structural analog of the essential amino acid L-histidine, strongly ameliorated the severity of renal disfunction and reduced the MDA accumulation induced by ifosfamide in rats.  Histidine significantly decreased MDA formation, increased GSH content and enhanced CAT and GPx activities when used after chronic alcoholic liver injury induced by ethanol in rats.  Moreover, i.p. injection of histidine attenuated the histopathological changes including congestion, hemorrhage, edema, leukocyte infiltration, and necrosis in the rat model of acetic acid-induced colitis.  Recently, Farshid et al.  reported that histidine suppressed both paw edema and neutrophil infiltration in paw tissues induced by intraplantar injection of carrageenan in rats.
The suppressive effects induced by a combined treatment of vitamin C and histidine on biochemical and histological changes induced by CP were more documented than those obtained from vitamin C and histidine when used alone. This indicates a potentiation effect that may exist between vitamin C and histidine in producing suppressive effects on CP-induced oxidative and histological changes. Vitamin C and some amino acids affect each other functions during oxidative stress status. In the caerulein-induced pancreatitis and associated liver injury in rats, coadministration of ascorbic acid with n-acetylcysteine decreased MDA levels in pancreas and liver and increased hepatic CAT and GPx activities.  In one study, vitamin C and histidine decreased the intracellular accumulation of adriamycin induced by ultrasound. 
In conclusion, the present study has shown that both vitamin C and histidine not only ameliorated the CP-induced TAC and MDA alternations, but also attenuated the histopathological changes of the wall of the urinary bladder induced by CP. Moreover, a potentiation effect was observed between the suppressive effects of vitamin C and histidine on CP-induced oxidative and histopathological changes.
| » Acknowledgment|| |
This work was supported by the Faculty of Veterinary Medicine of Urmia University. The authors declare that there is no conflict of interest.
| » References|| |
|1.||Levine AL, Richie PJ. Urological complications of cyclophosphamide. J Urol 1989;141:1063-9. |
|2.||Sadir S, Deveci S, Korkmaz A, Oter S. Alpha tocopherol, beta carotene, and melatonin administration protects cyclophosphamide-induced oxidative damage to bladder tissue in rats. Cell Biochem Funct 2007;25:521-6. |
|3.||Eichel E, Scheidweiler K, Kost J, Shojaie J, Schwarz E, Messing E, et al. Assessment of murine bladder permeability with fluorescein: Validation with cyclophosphamide and protamine. Urology 2001;58:113-8. |
|4.||Li Y, Schellhorn HE. New developments and novel therapeutic perspectives for vitamin C. J Nutr 2007;137:2171-84. |
|5.||Mandl J, Szarka A, Banhegyi G. Vitamin C: Update on physiology and pharmacology. Br J Pharmacol 2009;157:1097-110. |
|6.||Ghosh S, Ghosh D, Chattopadhyay S, Debnath J. Effect of ascorbic acid supplementation on liver and kidneys toxicity in cyclophosphamide-treated female albino rats. J Toxicol Sci 1999;24:141-4. |
|7.||Ray S, Sengupta C, Roy K. Evaluation of ascorbic acid as suppressor of cyclophosphamide induced lipid peroxidation using common laboratory markers. Acta Pol Pharm 2005;62:145-52. |
|8.||Gurbuz N, Ozkul A, Burgaz S. Effects of vitamin C and N-acetylcysteine against cyclophosphamide-induced genotoxicity in exfoliated bladder cells of mice in vivo. J BUON 2009;14:647-52. |
|9.||Farshid AA, Tamaddonfard E, Yahyaee F. Effects of histidine and N-acetylcysteine on diclofenac-induced anti-inflammatory response in acute inflammation in rats. Indian J Exp Biol 2010;48:1136-42. |
|10.||Farshid AA, Tamaddonfard E, Belasius MS, Hamzeh-Gooshchi N. Histopathological comparison of the effects of histidine and ketotifen in a rat model of colitis. Bull Vet Inst Pulawy 2009;53:795-800. |
|11.||Wade AM, Tucker HN. Antioxidant characteristics of L-histidine. J Nutr Biochem 1998;9:308-15. |
|12.||Lee YT, Hsu CC, Lin MH, Liu KS, Yin MC. Histidine and carnosine delay diabetic deterioration in mice and protect human low density lipoprotein against oxidation and glycation. Eur J Pharmacol 2005;513:145-50. |
|13.||Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay. Anal Biochem 1996;239:70-6. |
|14.||Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by tiobarbituric acid reaction. Anal Biochem 1979;95:351-8. |
|15.||Korkmaz A, Topal T, Oter S. Pathophysiological aspects of cyclophosphamide and ifosfamaide induced hemorrhagic cystitis: Implication of reactive oxygen and nitrogen species as well as PARP activation. Cell Biol Toxicol 2007;23:303-12. |
|16.||Yu BP. Cellular defense against damage from reactive oxygen species. Physiol Rev 1994;74:139-62. |
|17.||Rezvanfar MA, Farshid AA, Sadrkhanlou RA, Ahmadi A, Rezvanfar MA, Salehnia A, et al. Benefit of Satureja khuzestanica in subchronically rat model of cyclophosphamide-induced hemorrhagic cystitis. Exp Toxicol Pathol 2010;62:323-30. |
|18.||Motawi TM, Sadik NA, Refaat A. Cytoprotective effects of DL-alpha lipoic acid or squalene on cyclophosphamide-induced oxidative injury: An experimental study on rat myocardium, testicles and urinary bladder. Food Chem Toxicol 2010;48:2326-36. |
|19.||Das UB, Mallick M, Debnath JM, Ghosh D. Protective effects of ascorbic acid on cyclophosphamide-induced testicular gametogenic and androgenic disorders in male rats. Asian J Androl 2002;4:201-7. |
|20.||El-Batch M, Ibrahim W, Said S. Effect of histidine on autotoxin activity in experimentally induced liver fibrosis. J Biochem Mol Toxicol 2011;25:143-50. |
|21.||Badary OA. L-histidinol, attenuates Fanconi syndrome induced by ifosfamide in rats. Exp Nephrol 1999;7:323-7. |
|22.||Liu WH, Liu TC, Yin MC. Beneficial effects of histidine and carnosine on ethanol-induced chronic liver injury. Food Chem Toxicol 2008;46:1503-9. |
|23.||Esrefoglu M, Gul M, Ates B, Batcioglu K, Selimoglu MA. Antioxidative effect of melatonin, ascorbic acid and N-acetylcysteine on caerolein-induced pancreatitis and associated liver injury in rats. World J Gastroenterol 2006;14:259-64. |
|24.||Yu T, Bai J, Hu K, Wang Z. The effects of free radical scavenger and antioxidant on the increase in intracellular adriamycin accumulation induced by ultrasound. Ultrason Sonochem 2003;10:33-5. |
[Figure 1], [Figure 2], [Figure 3]
|This article has been cited by|
||Effects of histidine andn-acetylcysteine on experimental lesions induced by doxorubicin in sciatic nerve of rats
| ||Amir Abbas Farshid,Esmaeal Tamaddonfard,Sima Najafi |
| ||Drug and Chemical Toxicology. 2014; : 1 |
|[Pubmed] | [DOI]|
||Effects of Histidine and N-Acetylcysteine on Doxorubicin-Induced Cardiomyopathy in Rats
| ||Amir Abbas Farshid,Esmaeal Tamaddonfard,Naeime Simaee,Sanam Mansouri,Sima Najafi,Siamak Asri-Rezaee,Hossein Alavi |
| ||Cardiovascular Toxicology. 2013; |
|[Pubmed] | [DOI]|