Cancer Therapy Vol 6, 563-570, 2008

Alterations in antioxidant enzyme activities and increased oxidative stress in cyclophosphamide- induced hemorrhagic cystitis in the rat

Research Article

Premila Abraham^1,*, Indirani Kanakasabapathy², Preethi kulothungan¹

¹Department of Biochemistry

²Departments of Anatomy, Christian Medical College, Bagayam, Vellore 632002, Tamil Nadu, India

__________________________________________________________________________________

*Correspondence: Dr Premila Abraham, Associate Professor, Department of Biochemistry; Christian Medical College, Bagayam, Vellore 632002, Tamil Nadu, India Tel: +91-416-2284267, +91-416-2284458; Fax: +91-416-2262788; E-mail: premilaabraham@yahoo.com, premilaabraham@cmcvellore.ac.in

Key words: cyclophosphamide; hemorrhagic cystitis; Oxidative stress; Myeloperoxidase; Antioxidant enzymes

Abbreviations: 1-chloro-2, 4-dinitrobenzene, (CDNB); cyclophosphamide, (CP); glutathione peroxidase, (GPO); Glutathione S transferase, (GSTase); haematoxylin and eosin, (H & E); hemorrhagic cystitis, (HC); malondialdehye, (MDA); Protein carbonyl, (Pco); Reactive Oxygen species, (ROS); Superoxide dismutase, (SOD)

Received: 16 July 2008; Revised: 4 August 2008

Accepted: 7 August 2008; electronically published: September 2008

Summary

The mechanism of cyclophosphamide (CP) induced hemorrhagic cystitis (HC) is not clear although the production of acrolein is suggested to be involved. It has been demonstrated that detoxifying acrolein does not prevent HC symptoms completely. This suggests that acrolein production may not be the sole cause of HC; other mechanisms may be involved. It is important to verify the mechanism of cyclophosphamide induced bladder damage in order to perform cancer chemotherapy effectively by minimising the side effect. Our aim is to verify whether there is any alteration in the activities of the antioxidant enzymes and oxidative stress in cyclophosphamide induced HC, using a rat model. Adult male rats were administered a single injection of CP at the dose of 150/ kg body wt intraperitoneally and sacrificed 6 hours or 16 hours after the dose of CP. Vehicle treated rats served as control. The bladder was used for light microscopic studies and biochemical studies. Myeloperoxidase activity, a marker of neutrophil infiltration was measured in bladder homogenates. The markers of oxidative damage including protein carbonyl content, protein thiol, malondialdehyde and conjugated dienes were assayed in the homogenates. The activities of the antioxidant enzymes, superoxide dismutase, glutathione peroxidase, catalase, and glutathione reductase and glutathione S transferase were assayed in the bladder . Six-hour after treatment with CP the mucosa became edematous and the cells of the urothelium were not compact. Cellular exudates were observed in the lumen. The condition became worse in sixteen hours, wherein edema of lamina propria with epithelial and sub-epithelial hemorrhage was seen. Myeloperoxidase activity was increased significantly 6 hours after treatment with CP and increased further at 16 hours. All the parameters of oxidative stress that were studied were significantly elevated. The activities of the antioxidant enzymes were significantly lowered. The results of the present study suggest that alteration in the activities of antioxidant enzymes and oxidative stress is responsible, at least in part, for CP induced bladder damage. It is suggested that CP treatment of rats induces oxidative stress in the urinary bladder, depletion of antioxidant enzymes, and the oxidative stress contributes to neutrophil infiltration into bladder and hence inflammation.

I. Introduction

Cyclophosphamide is widely used in the treatment of solid tumors and B cell malignant disease, such as lymphoma, myeloma, chronic lymphocytic leukemia and Waldenstrom’s macroglobulinemia. Hemorrhagic cystitis (HC) is a major dose-limiting side effect of cyclophosphamide (CP) and ifosfamide (Levine and Richie, 1989). The incidence of this side effect is related to the dosage and can be as high as 75% in patients receiving a high intravenous dose. The urological side effects vary from transient irritative voiding symptoms to life-threatening HC. The urotoxicity of these nitrogen mustard cytostatics is believed to be based on the formation of 4-hydroxy metabolites, in particular, renal excretion of acrolein, which is formed from hepatic microsomal enzymatic hydroxylation. Mesna, an acrolein binding and detoxifying compound within the urinary collecting system, has been widely used as an effective agent against CP induced cystitis, but significant HC is still being encountered clinically (Brock et al, 1981; West, 1997). Since detoxifying acrolein does not remove symptoms of HC completely, it is proposed that mechanisms other than direct contact of acrolein with bladder mucosa may also be involved in CP induced HC. It is important to elucidate the mechanism of CP induced HC in order minimize the toxic and dose limiting side effect of CP. Elimination of the side effects of CP can lead to better tolerance of the drug and a more efficient and comfortable therapy can be achieved for patients in need of CP treatment. Reactive oxygen species (ROS) production in inflammatory states is considered to play an important role in the initiation and progression of tissue injury. Inflammatory cells such as macrophages, neutrophils and monocytes produce ROS which react with macromolecules such as lipids, proteins and nucleic acids because of their high reactivity, causing tissue injury. The cellular antioxidant systems helps to minimize ROS induced tissue injury. These include enzymes such as superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase and glutathione S transferase as well as non-enzymatic anti-oxidants as glutathione, protein thiol, ascorbic acid and tocopherol (Babiak et al, 1998).

The present study focused on oxidative stress and alteration in antioxidant enzymes and neutrophil infiltration in order to elucidate the mechanism of CP induced cystitis. To this end, a time course study was carried out in order to study the effect of CP on the histology of the urinary bladder and markers of oxidative stress, antioxidant enzyme activities and neutrophil infiltration. The results of the present study demonstrate that CP induced hemorrhagic cystitis is associated with depletion of antioxidant enzymes, increased oxidative stress, and neutrophil infiltration.

II. Materials and methods

A. Animals

Adult male Wistar rats (200-250g) were used for the experiments. The study was approved by animal ethics Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA), Government of India. The guidelines were followed. Dosage and route of administration of cyclophosphamide (CP) were determined from that described in literature (Ahluwalia et al, 1994).

B. Animal treatment

The rats were divided into three groups and were treated as follows.

The rats in group I (n = 8) received a single intraperitoneal injection of CP in saline at the dose of 150 mg/kg body weight .The rats in group II (n=8) received a single intraperitoneal injection of CP in saline at the dose of 150 mg/kg body weight. The rats (n=6) in group III received saline alone as a vehicle control.

The rats in group I were killed 6 hours after the dose of CP and the rats in group II were killed 16 hour after the dose of CP.

C. Tissue procurement

Rats were killed by exsanguination. The urinary bladder was removed and blotted dry before weighing. A part of the bladder was used for biochemical assays and another part for histological assessment.

D. Histology

The tissues were fixed overnight in 10 % buffered neutral formalin, processed to paraffin wax, sectioned at 5 mm, and stained with haematoxylin and eosin (H & E) for examination by light microscopy.

E. Biochemical assays

Bladder tissue was weighed and homogenized in appropriate buffers and used for the following assays.

1. Myeloperoxidase (Wallace et al, 1989)

Myeloperoxidase activity was measured with O- dianisidine- H O assay. The rate of decomposition of H2 O2by myeloperoxidase was determined by measuring the rate of color development at 460 nm. To 10ml of sample, 11ml of H O, 17ml of O- dianisidine and 962 ml of phosphate buffer were added and the color read at 460nm at an interval of 30 seconds for 4 minutes and the rate of change/minute was determined. Extinction coefficient of 1.13 X 104 cm -1 was used for the calculation. One unit is the amount of enzyme decomposing 1mmole of peroxide per minute.

2. Malondialdehyde

Malonaldehyde content was measured as described by Ohkawa and colleagues in 1979.The mixture consisted of 0.8 ml of sample (1mg), 0.2 ml of 8.1 % SDS, 1.5 ml of 20 % glacial acetic acid adjusted to pH 3.5, and 1.5 ml of 0.8 % aqueous solution of TBA. The mixture was made up to 4ml with distilled water and heated at 95 C for 60 min using a glass ball as condenser. After cooling with tap water, 1ml distilled water and 5ml n-butanol and pyridine mixture (15:1) were added and the solution was shaken vigorously. After centrifugation at 2000g for 10 minutes the absorbance of the organic layer was measured at 532nm. Amount of thiobarbituric reacting substances formed is calculated from standard curve prepared using 1, 1’, 3, 3’ tetramethoxy propane and the values expressed as nmoles per mg protein.

3. Conjugated diene

Total lipids were extracted as described by Chan and Levett, in 1977 and evaporated to dryness under nitrogen. This was dissolved in 1 ml heptane and the absorbance was measured at 233nm. The amount of conjugated diene formed in the sample is calculated using a molar absorption co-efficient of 2.52 x10⁴.

4. Protein carbonyl content

Protein carbonyl content was measured using DNPH as described by Sohal and colleagues in 1993.To 0.5 ml of sample (1-2mg), an equal volume of 10 mM DNPH in 2 N HCl was added and incubated for 1 hr shaking intermittently at room temperature. Corresponding blank was carried out by adding only 2N HCL to the sample. After incubation, the mixture was precipitated with 10 % TCA (final concentration) and centrifuged. The precipitate was washed twice with ethanol:ethylacetate (1:1) and finally dissolved in 1 ml of 6 M guanidine HCl, centrifuged at low speed and the supernatant was read at 366nm. The difference in absorbance between the DNPH treated and HCl treated sample is determined and expressed as nmoles of carbonyl groups per mg of protein, using extinction co-efficient of 22 mM^-1cm¹.

5. Protein thiol groups

Thiol groups were measured as described by Sedlak and Lindsay in 1968. To 1 ml of the sample suspension (1 mg protein/ml), 1 ml of 10 % TCA containing 1 mM EDTA was added. The protein precipitate was separated by high speed centrifugation for 10 min. For total thiol estimation the sample was taken directly with out precipitation. To this, 1 ml of solution I and 0.5 % SDS were added followed by 2 ml of solution II and 30 ml of DTNB. The tubes were mixed well and kept in the dark for 15 min at room temperature. The intense yellow colour of the nitromercapto benzoate anion formed from the DTNB reaction with the thiol was read at 412 nm which -1 -1has a molar absorption of 13,600 mM^-1cm¹.

6. Assay of anti-oxidant enzyme activities

i. Superoxide dismutase

Superoxide dismutase was measured as described by Ohkuma and colleagues in 1982. The assay mixture consisted of 100ml ofphosphate buffer, 10ml of BSC, 50mlof Triton X-100, 5ml of EDTA, 5ml of xanthine oxidase, 50ml ofxanthine is added. To this finally 150 ml MTT and sample (50-150 mg protein) were added and, the volume is made up to 1 ml with water. The mixture was incubated for 5 minutes at room temperature (30°C) and the reaction was terminated with the addition of 1ml of stop buffer. This was read at 540nm. Amount of superoxide formed is calculated using the molar extinction coefficient of MTT formazan -of 17,000 M^-1cm^-1 at pH 7.4 to 10.5. The percentage of inhibition by the presence of SOD is calculated 540from the reduction of the MTT colour formation as compared to the MTT formazan formed in the absence of SOD, which is taken as 100 %. One unit of SOD is defined as the amount of protein required to inhibit MTT reduction by 50%.

ii. Catalase

Catalase activity is estimated by measuring the change in absorption at 240 nm using H₂O₂ as substrate (Aebi, 1984). To 1 ml of 30 mM buffered H₂O₂, the enzyme (sample) was added to start the reaction. The final volume was made up to 2 ml with 0.05 M phosphate buffer pH 7.0. Change in OD was observed for 2 min at 240 nm. One unit is the activity that disproportionates H₂O₂at the rate of 10-3 absorbance/sec.

iii. Glutathione-S-transferase (GSTase)

The activity of GSTase is measured spectrophotometrically using the substrate 1-chloro-2, 4-dinitrobenzene (CDNB) (Awasthi et al, 1980). To 0.1 ml of 1M potassium phosphate buffer pH 6.5, following reagents were added: 0.1 ml of 10 mM GSH, 0.05 ml 20 mM CDNB and water and made up the volume to 1 ml. The reaction was started by adding the enzyme and change in OD at 340 nm is measured for 1-2 min. One unit of enzyme is the amount required to conjugate 1 mmole of substrate with glutathione in one minute.

iv. Glutathione reductase

In the presence of enzyme, hydrogen is transferred from NADPH to GSSG and the reaction can be measured at 340 nm (Racker, 1955). To the reaction mixture containing 0.05 ml of 1 M phosphate buffer pH 7.6, 0.15 ml of 10 mM EDTA, 0.1 ml of 1 mM NADPH, and 0.1 ml 10 mM GSSG, the enzyme was added. The volume was made up to 1 ml and the decrease in OD at 340 nm was measured for 2-3 min. One unit is the amount of enzyme needed to oxidise 1 mmole of NADPH/min.

v. Glutathione peroxidase

Total peroxidase is determined by following the oxidation of NADPH at 340 nm using hydrogen peroxide (Nakamura and Hosada, 1974). To 0.25 ml of 0.4 M phosphate buffer, 0.2 ml of 4 mM EDTA, 0.2 ml of 10 mM GSH, 0.2 ml of NaN₃, 0.2 ml of 1.6 mM NADPH, 0.03 ml glutathione reductase (one unit) and the enzyme (sample) was added. Total volume was made up to 2 ml with water. Reaction was started by adding 0.2 ml of H₂O₂and change in OD at 340 nm was followed. Extinction coefficient of 6.1 mm^-1 was used for the calculation. One unit is the amount needed to oxidize 1 nmole of NADPH/min.

F. Statistical Analysis

The results are expressed as mean ± S.D. Comparison between groups was done using ANOVA. P value of < 0.05 was considered as statistically significant.

III. Results

A. Histology (Figure 1)

In control animal, the urinary bladder had the urothelium formed by tightly packed cells with little intercellular space. The basement membrane that separates the epithelium from the underlying lamina propria was intact. There was no breach in it. The connective tissue that constituted the lamina propria was dense with normal vascular supply. The mucosa was thrown into folds and there were sub-epithelial crypts. The smooth muscle coat underlying the lamina propria displayed circular and longitudinal muscle coat with minimal connective tissue packing. There were no exudates in the lumen (Figure 1A).

Contrary to the normal picture presented above, the bladder wall in treated animals showed damages, which became severe with increased time after treatment with the drug. In six-hour case the mucosa became edematous and the cells of the urothelium were not compact. There seemed to be cellular exudates in the lumen. Mucosal content formed follicular cystitis (Figure 1B). However hemorrhage was not seen in this group. The condition became worse in sixteen hours, where edema of lamina propria with epithelial and sub-epithelial hemorrhage was seen (arrows in Figure 1C).

B. Biochemical findings

The biochemical results obtained for the bladder are shown in figure 2. Myeloperoxidase activity, a marker of neutrophil infiltration was increased by 72 % and by 122% in the urinary bladder, six hour and 16 hour respectively after treatment with CP as compared with control (Figure 2A). Protein carbonyl (Pco) content, an early and sensitive marker of oxidative damage to proteins was increased by 113% at 6 hour and by 50 % sixteen hour after treatment with CP (Figure 2B). Protein thiol, one of the important antioxidants and indicators of oxidative damage to proteins was decreased by 62 % and by 42 % 6 hours and 16 hours after treatment with CP respectively (Figure 2C). The markers of lipid peroxidation, namely malondialdehye (MDA) and conjugated diene were increased significantly in bladders of CP treated rats 16 hours after treatment as compared with control (Figure 2D,E). Conjugated diene was elevated by 367% and MDA by 131 %.

With regard to the antioxidant enzymes, a significant decrease in glutathione peroxidase (GPO) activity (50 %) was observed 16 hours after treatment with CP (Figure 2F). With respect to Superoxide dismutase (SOD) activity, a 54 % decrease was observed 16 hour after treatment with CP (Figure 2G). Theactivity of the drug detoxifying enzyme, Glutathione S transferase (GSTase) was decreased by 52 % and by 66 % 6 hour and 16 hour after treatment with CP respectively as compared with control (Figure 2H). Glutathione reductase activity was increased by 53 % and 25 % six hour and 16 hour after treatment with CP respectively as compared with control as shown in Figure 2I. No significant alteration in the activity of catalase was observed in the bladders of CP treated rats (Figure 2J).

IV. Discussion

Urotoxicity is one of the major dose limiting side effects of CP. The urotoxicity of CP is thought to be due to the formation of acrolein that damages the urothelium. Therefore, Mesna, an acrolein binding and detoxifying compound within the urinary collecting system, has been widely used as an agent against CP induced cystitis, but significant HC is still being encountered clinically (Brock et al, 1981). Since detoxifying acrolein does not remove symptoms of HC completely, it is speculated that mechanisms other than direct contact of acrolein with bladder mucosa may be involved in CP induced HC. Oxidative stress is known to be involved in several diseases and to be the cause of toxicity of many drugs. Therefore, in the present study the parameters of oxidative stress and the activities of antioxidant enzymes were studied and compared with histological findings.

The parameters of oxidative stress i.e. conjugated dienes, malondialdehyde and protein carbonyl content were found to be markedly increased in the bladders of CP treated rats suggesting that CP treatment caused oxidative damage to the lipids and proteins of the bladder. Protein carbonyl content was increased significantly 6 hour after treatment with CP and, MDA and Conjugated diene were elevated significantly 16 hours after treatment with CP. In parallel with their production, protein thiol content, an important antioxidant in the bladder was lowered. Recently, Topal and colleagues in 2005 and Sadir colleagues in 2007 have demonstrated that MDA levels increase in the bladder of rats after the administration of CP.

Antioxidant enzyme activities were measured to evaluate the antioxidant capacity. Antioxidant enzyme activities are reported to be altered in drug induced inflammatory conditions. In NSAIDs induced intestinal toxicity, SOD and GPO activities were shown to be decreased in parallel with the severity of damage (de la Lastra et al, 2000; Villegas et al, 2001). In the present study a decrease in the activities of the important antioxidant enzymes namely SOD, GPO, GSTase was observed in the bladders of CP treated rats. Since oxidative stress preceded the decrease in the activities of antioxidant enzymes it is suggested that the decrease in the activities of antioxidant enzymes is a consequence of increased oxidative stress in the bladder. It is proposed that the decrease in the activities of the antioxidant enzymes contribute to the urotoxicity of CP. An increase in the activity of glutathione reductase is considered to be protective response of the body to the oxidative stress induced by CP.

It has been demonstrated that activated neutrophils secrete enzymes such as myeloperoxidase, elastase and other proteases. MPO plays a fundamental role in the oxidant production by the neutrophils and has been used as an effective index of inflammation due to the correlation between MPO activity and the histological analysis of neutrophil infiltration (Sekizuka and Grisham, 1988). In the present study, MPO levels were elevated indicating that neutrophil infiltration contributes to CP induced cystitis.

ROS production in inflammatory diseases is considered to play an important role in the initiation and progression of tissue injury. Inflammatory cells such as macrophages, neutrophils and monocytes produce ROS which can react with biological macromolecules such as lipids, proteins and nucleic acids because of their high reactivity, causing tissue injury. On the other hand, the ROS produced are thought to contribute to the subsequent neutrophil infiltration to tissue (Petrone et al, 1980; Hotter et al, 1997). It is known that ROS and cellular redox status regulate expressions of proinflammatory cytokines in inflammatory or non-inflammatory cells at gene level (DeForge et al, 1993; Shi et al, 1999; Haddad 2000; Haddad et al, 2001). Increased ROS production has been shown to initiate and facilitate neutrophil infiltration leading to the inflammation (Bradley et al, 1982). In the present study, the early and sensitive indicator of oxidative stress namely Pco was significantly increased 6 hr after treatment with CP and began to decrease thereafter, but still remained elevated as compared with control. Besides, protein thiol content in the bladder was reduced 6 hour after treatment with CP. Increase in MPO activity was observed 6 hour after treatment with CP. The MPO activity increased further at 16 hours. Therefore it is suggested that oxidative stress initiates and facilitates neutrophil infiltration. The activated neutrophils generate reactive oxygen species leading to inflammation.

ROS not only regulate cytokine expression through nuclear transcriptional factors but also affect adhesion molecules (Suzuki et al, 1997; Droge 2002). ROS contribute towards increased transendothelial and transepithelial permeability (Rao et al, 2000; Meyer et al, 2001). The increase of transepithelial permeability allows toxins to permeate through the barrier, which leads to inflammation. ROS produced by the CP treatment may possibly cause the above events, leading to the CP-induced hemorrhagic cystitis.

Comparing the histological findings with that of the biochemical findings, it was observed that oxidative stress and neutrophil infiltration was evident 6 hours after treatment with CP, when the mucosa became edematous and cellular exudates were seen in the lumen. Sixteen hours after treatment with CP the activity of MPO increased further, the parameters of oxidative stress were elevated and histologically a more severe damage was observed. Increase in oxidant stress and decrease in the antioxidant status was accompanied by neutrophil infiltration and bladder damage. This study clearly shows that increased oxidative stress, depletion of antioxidant enzymes, and neutrophil infiltration contribute to CP induced hemorrhagic cystitis.

In the light of the current data, is suggested that oxidative stress contributes at least in part, to CP induced bladder damage. CP treatment of rats induces oxidative stress, depletes cellular antioxidants in the urinary bladder which contribute to neutrophil infiltration into bladder. In the future, investigation of the effect of administration of MPO inhibitors and natural antioxidants such as vitamin C in the prevention of hemorrhagic cystitis may be planned. It is believed that the proposed study will increase the clinical utility of the drug by overcoming cystitis, the most limiting side effect of cyclophosphamide.

Acknowledgements

The study was supported by Department of Science and Technology (DST) New Delhi.

References

Aebi IH (1984) Catalase in vitro. Methods Enzymol 105, 121-6.

Ahluwalia A, Maggi CA, Santicioli P, Lecci A, Giuliani S (1994) Characterization of the capsaicin-sensitive component of cyclophosphamide-induced inflammation in the rat urinary bladder. Br J Pharmacol 111, 1017-22.

Awasthi YC, Dao DD, Saneto RP (1980) Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver. Biochem J 191, 1-10.

Babiak RMV, Campello AP, Carnieri EGS (1998) Methotrexate: pentose cycle and oxidative stress. Cell Biochem Funct 16, 283-293.

Bradley PP, Priebat DA, Christensen RD, Rothstein G (1982) Measurement of cutaneous Inflammation: estimation of neutrophil content with an enzyme marker. J Inves Dermatol 78, 206-209.

Brock N, Pohl J, Stekar J (1981) Studies on the urotoxicity of oxazaphosphorine cytostatics and its prevention. 1. Experimental studies on the urotoxicity of alkylating compounds. Eur J Cancer 17, 595-601.

Chan HW, Levett G (1977) Autoxidation of methyl linoleate. Separation and analysis of isomeric mixtures of methyl linoleate hydroperoxides and methyl hydroxylinoleates. Lipids 12, 99-104.

de la Lastra CA, Nieto A, Motilva V, Martin MJ, Herrerias JM, Cabre F (2000) Intestinal toxicity of ketoprofen-trometamol vs its enantiomers in rat. Role of oxidative stress. Inflamm Res 49, 627-632.

DeForge LE, Preston AM, Takeuchi E, Kenney J, Boxer LA, Remick DG (1993) Regulation of interleukin 8 gene expression by oxidant stress. J Biol Chem 268, 25568-25576.

Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82, 47-95

Haddad JJ (2000) Glutathione depletion is associated with augmenting a proinflammatory signal: evidence for an antioxidant/prooxidant mechanism regulating cytokines in the alveolar epithelium. Cytokines Cell Mol Ther 6, 177-187.

Haddad JJ, Safieh-Garabedian B, Saade NE, Land SC (2001) Thiol regulation of pro-inflammatory cytokines reveals a novel immunopharmacological potential of glutathione in the alveolar epithelium. J Pharmacol Exp Ther 296, 996-1005.

Hotter G, Closa D, Prats N, Pi F, Gelpi E, Rosello-Catafau J (1997) Free radical enhancement promotes leucocyte recruitment through a PAF and LTB4 dependent mechanism. Free Radic Biol Med 22, 947-954.

Levine AL, Richie PJ (1989) Urological complications of cyclophosphamide. J Urol 141, 1063-1069.

Meyer TN, Schwesinger C, Ye J, Denker BM, Nigam SK (2001) Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity. J Biol Chem 276, 22048-22055.

Nakamura W, Hosada S (1974) Purification and properties of rat liver glutathione peroxidase. Biochim Biophys Acta 358, 251-261.

Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95, 351-8.

Ohkuma N, Matsuo S, Tutsui M, Ohkawara A. [Superoxide dismutase in the epidermis (author's transl)] (1982) Nippon Hifuka Gakkai Zasshi 92, 583-50.

Petrone WF, English DK, Wong K, McCord JM (1980) Free radicals and inflammation: superoxide-dependent activation of a neutrophil chemotactic factor in plasma. Proc Natl Acad Sci USA 77, 1159-1163.

Racker E (1955) Glutathione reductase from bakers' yeast and beef liver. J Biol Chem 217, 855-865.

Rao RK, Li L, Baker RD, Baker SS, Gupta A (2000) Glutathione oxidation and ATPase inhibition by hydrogen peroxide in Caco-2 cell monolayer. Am J Physiol Gastrointest Liver Physiol 279, G332-340.

Sair S, Deveci S, Korkmaz A (2007) Alpha tocopherol, beta carotene, and melatonin administration protects cyclophosphamide induced oxidative damage to bladder tissue in rats. Cell Biochem Funct 25, 521-52

Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 25, 192-205.

Sekizuka E, Grisham MB, Li M (1988) Inflammation induced intestinal hyperemia in the rat: role of neutrophils. Gastroenterology 95, 1528-1534.

Shi MM, Chong I, Godleski JJ, Paulauskis JD (1999) Regulation of macrophage inflammatory protein-2 gene expression by oxidative stress in rat alveolar macrophages. Immunology 97, 309-315.

Sohal RS, Agarwal S, Dubey A , Orr WC (1993) Protein oxidative damage is associated with life expectancy of houseflies. Proc Natl Acad Sci U S A 90, 7255-7259.

Suzuki YJ, Forman HJ, Sevanian A (1997) Oxidants as stimulators of signal transduction. Free Radic Biol Med 22, 269-285.

Topal P, Oztas Y, Korkmaz A (2005) Melatonin ameliorates bladder damage induced by cyclophosphamide in rats. J Pineal Research 38, 272-277.

Villegas I, Alarcon de la Lastra C, La Casa C, Motilva V, Martin MJ (2001)Effects of food intake and oxidative stress on intestinal lesions caused by meloxicam and piroxicam in rats. Eur J Pharmacol 414, 79-86.

Wallace JL, MacNaughton W K, Morris GP, Beck PL (1989) Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology 96, 29-36.

West NJ (1997) Prevention and treatment of hemorrhagic cystitis. Pharmacotherapy 4, 696-706.