- Email: [email protected]
Protective Effects of Bilirubin Against Cyclophosphamide Induced Hemorrhagic Cystitis in Rats
Protective Effects of Bilirubin Against Cyclophosphamide Induced Hemorrhagic Cystitis in Rats Yoh Matsuoka,* Hitoshi Masuda, Minato Yokoyama and Kazunori Kihara From the Department of Urology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
Purpose: The end product of the heme oxygenase pathway, bilirubin, is the most abundant endogenous antioxidant in mammals. We report the heme oxygenase-1 mediated production of bilirubin and its cytoprotective roles in cyclophosphamide induced hemorrhagic cystitis in rats. Materials and Methods: Female Sprague-Dawley rats received intraperitoneal administration of cyclophosphamide. In the first experiment hemin (an inducer of heme oxygenase-1) with or without zinc protoporphyrin IX (an inhibitor of heme oxygenase activity) was given before cyclophosphamide injection. Endogenous bilirubin production was analyzed in bladder tissues immunohistochemically. In another experiment bilirubin solution was administered before the cyclophosphamide injection. Changes in bladder weight, microscopic feature and expression levels of inducible nitric oxide synthase, proinflammatory cytokines and heme oxygenase were evaluated using polymerase chain reaction and immunostaining. Results: Bilirubin was generated in bladders with cyclophosphamide induced cystitis, especially in the urothelium and suburothelium. Hemin pretreatment provided increased production of endogenous bilirubin, which was decreased by zinc protoporphyrin IX. In an evaluation of the roles of bilirubin exogenous bilirubin administration ameliorated cyclophosphamide induced inflammatory changes and reduced the increase in bladder weight. The elevated expression of inducible nitric oxide synthase and interleukin-1 in cyclophosphamide induced cystitis was significantly downregulated by exogenously applied bilirubin. The expression of heme oxygenase-1 and 2 was not modified by bilirubin administration. Conclusions: Cyclophosphamide induced hemorrhagic cystitis is accompanied by endogenous bilirubin production through heme oxygenase-1 induction in the bladder. Bilirubin has cytoprotective roles in association with the down-regulation of inducible nitric oxide synthase expression. Our results suggest that bilirubin may have therapeutic potential against bladder inflammatory insults such as cyclophosphamide induced cystitis. Key Words: bladder; rats, Sprague-Dawley; cystitis; cyclophosphamide; bilirubin
eme oxygenase is the rate limiting enzyme in the cleavage of the heme molecule to equimolar quantities of CO, free iron and biliverdin. Free iron is sequestered into ferritin and biliverdin is rapidly converted to bilirubin through the action of biliverdin reductase.1,2 The HO pathway was recently proposed to function as an effective system to counteract environmental stresses and maintain cellular homeostasis.3 To date 3 isoforms have been identified, including inducible HO-1, and constitutive HO-2 and 3. HO-1, which acts as a ubiquitous defensive system in various cell types, is induced by a wide range of stimuli, such as cytokines, bacterial endotoxin, reactive oxygen species, ischemia/reperfusion and hypoxia.1,2,4 Although the cytoprotective effects of HO-1 have been confirmed in various organ systems,2,4 the mechanism of HO-1 action has not fully been elucidated. In recent years attention has focused on the downstream products of HO activity, CO and bilirubin, which
H
Submitted for publication June 2, 2007. * Correspondence: Department of Urology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan (telephone: ⫹81 3 5803 5295; FAX: ⫹81 3 5803 5295; e-mail: [email protected] or [email protected]).
0022-5347/08/1793-1160/0 THE JOURNAL OF UROLOGY® Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION
have been suggested to be key mediators of the beneficial effects of HOs.5 Bilirubin has long been considered a potentially toxic waste product of heme metabolism that causes jaundice and deposits in the brain to elicit the neurotoxicity associated with kernicterus at high concentrations. However, this bile pigment has also been known to have antioxidant properties and function as an efficient peroxyl radical scavenger at physiological concentration.6 Increased serum levels of bilirubin have been associated with a decreased incidence of coronary artery diseases and cancer mortality.7,8 In a rat model ligation of the common bile duct caused cholestasis with an increase in the plasma bilirubin concentration and it conferred resistance to glycerol induced acute renal failure.9 Thus, cytoprotective effects of bilirubin have been demonstrated in animal and human studies. CYP is an alkylating agent extensively used in the treatment of various neoplastic diseases. It causes hemorrhagic cystitis as a complication. The initial urothelial damage occurs by direct contact with acrolein, a urotoxic metabolite of CYP, and it is followed by the involvement of NO produced by iNOS.10,11 Recently we reported the involvement of HO-1 and its anti-inflammatory effects in bladders with CYP in-
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Vol. 179, 1160-1166, March 2008 Printed in U.S.A. DOI:10.1016/j.juro.2007.10.031
BILIRUBIN AND CYCLOPHOSPHAMIDE INDUCED HEMORRHAGIC CYSTITIS duced hemorrhagic cystitis.12 However, little is known about the end product of the HO system, bilirubin, in urinary tract disease. We determined the expression profile of endogenous bilirubin through HO-1 induction and evaluated the roles of bilirubin in urological inflammatory states using the rat CYP induced cystitis model. MATERIALS AND METHODS Animals A total of 102 nonpregnant female Sprague-Dawley rats weighing 180 to 200 gm were randomly divided into groups. All efforts were made to use the minimum number of animals. This study complied with the animal welfare regulation of Tokyo Medical and Dental University. Protocol All chemicals were obtained from Sigma-Aldrich™ and administered intraperitoneally. Animals received an injection of cyclophosphamide monohydrate (200 mg/kg) for cystitis induction. Bilirubin was dissolved in 0.1 N sodium hydroxide and then diluted with sodium phosphate buffer. Care was taken to shield bilirubin from visible light to prevent its inactivation. In the first experiment 50 mg/kg hemin (a chemical inducer of HO-1) was given 24 hours before CYP injection with or without 30 mg/kg ZnPP-IX (an inhibitor of HO activity) 1 hour before CYP injection. The animals were sacrificed by exsanguination 24 hours after CYP injection. The bladders were harvested and used for analyses. In the second experiment the rats were treated with various doses of bilirubin (10, 30 and 100 mg/kg, respectively) 30 minutes before CYP injection and the bladders were harvested at various time points after CYP injection. Each bladder was weighed in the wet condition to evaluate the degree of inflammation.11,13 Bilirubin (30 mg/kg) pretreated rat bladders excised 24 hours after CYP injection were used for further evaluation, as described. Control animals were given vehicle only. RT-PCR Total RNA extracted was reverse transcribed to generate first-strand cDNA. Semiquantitative RT-PCR was performed for 30 cycles of amplification using a certain protocol, including HO-1 (denaturation at 94C for 1 minute, annealing at 55C for 2 minutes and extension at 72C for 3 minutes), HO-2 (94C for 1 minute, 55C for 1 minute and 72C for 2 minutes) and GAPDH (94C for 45 seconds, 55C for 45 seconds and 72C for 45 seconds). PCR products were electrophoresed on agarose gels, which were stained with ethidium bromide to visualize the bands. HO expression was quantified by densitometric scanning and normalized to the GAPDH level. Primer sequences were HO-1 forward 5=-ACTTTCAGAAGGGTCAGGTGTCC-3= and reverse 5=TTGAGCAGGAAGGCGGTCTTAG-3=, and HO-2 forward 5=-GAAGTGAGGGCAGCACAAAC-3= and reverse 5=-CTTCTTCAGCACCTGGCCT-3=. A primer pair for rat GAPDH (R & D Systems®) was used. Real-Time PCR Specific primer sets for rat iNOS, IL-1, TNF-␣ and GAPDH (Nihon Gene Research Laboratories, Sendai, Japan) were used. Real-time PCR was performed to quantify the products
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using a LightCycler® and a LightCycler-FastStart DNA master SYBR® Green I kit according to the manufacturer protocol. Data on iNOS, IL-1 and TNF-␣ were standardized to GAPDH expression in each sample. PCR experiments were repeated at least 2 times. Microscopic Evaluation Tissues were fixed in formol, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Histological damage was scored on a scale of 0 —normal epithelium, 1—mild changes involving a decrease in epithelial cells, flattening with submucosal edema, mild hemorrhage and few ulcerations, and 2—severe changes, including mucosal erosion, inflammatory cell infiltration, fibrin deposition, hemorrhage and multiple ulcerations.13 Two of us separately evaluated slides in a blind fashion. Immunohistochemical Analysis Bilirubin and iNOS immunostaining was performed in CYP treated rat bladders with hemin/ZnPP-IX pretreatments and with bilirubin pretreatment, respectively. Sections were pretreated with microwave irradiation for antigen retrieval. Endogenous peroxidase was blocked by incubation in hydrogen peroxide and nonspecific protein binding was minimized by treatment with normal goat serum. The preparations were incubated at 4C for 24 hours with mouse anti-bilirubin monoclonal antibody (24G7, Dojindo, Kumamoto, Japan) or mouse anti-iNOS monoclonal antibody (Wako, Osaka, Japan) and then treated with secondary antibody conjugated with peroxidase (Nichirei, Tokyo, Japan) for 30 minutes at room temperature. Negative control sections incubated in the absence of the primary antibody were also processed and evaluated for specificity or background staining levels. In bilirubin immunostaining each step was performed in a dark room to prevent isomerization and photo-oxidation of bilirubin. Evaluation of Immunoreactivity Slides were independently reviewed by 2 of us, who were blinded to treatment data on each sample. A combined intensity and percent positive scoring method was used to semiquantify immunoreactivity in the suburothelium.12,14 Staining intensity was scored as 3—strong, 2—moderate, 1—weak and 0 –negative. The percent of cells with each intensity score was estimated. A combined weighted score was defined as the sum of the percent of cells staining at each intensity level multiplied by the intensity score, eg a case with 20% strong staining, 50% moderate staining and 30% weak staining would be scored as 190 (20 ⫻ 3 ⫹ 50 ⫻ 2 ⫹ 30 ⫻ 1). Statistical Analysis Results are expressed as means ⫾ SEM. Statistical comparisons were performed by 1-way ANOVA, followed by Bonferroni’s test. For histopathological and immunohistochemical data median values were used and the difference was assessed using the nonparametric Kruskal-Wallis test, followed by the Mann-Whitney U test with p ⬍0.05 considered statistically significant.
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FIG. 1. Representative immunohistochemical staining for bilirubin in bladder. A, in controls no apparent immunostaining for bilirubin was. B, bilirubin immunoreactivity in urothelial cells and suburothelial stroma cells in rats treated with CYP. C, bilirubin immunoreactivity in urothelial cells and suburothelial stroma cells in rats treated with CYP plus hemin. D, bilirubin immunoreactivity in urothelial cells and suburothelial stroma cells in rats treated with CYP plus hemin and ZnPP-IX pretreatments. Scale bars indicate 100 m.
RESULTS HO-1 Mediated Bilirubin Production in CYP Induced Cystitis We analyzed the accumulation and localization of endogenous bilirubin in bladder tissues to determine HO-1 mediated heme degradation in situ. Bilirubin immunoreactivity was absent or weak in control rat bladders. In the bladders of CYP treated rats positive immunostaining for bilirubin was detected in the urothelium and suburothelium. Hemin further increased the number and distribution of bilirubin immunoreactive cells. Especially in the suburothelium intense immunostaining for cytoplasmic bilirubin were identified. This increased immunoreactivity was decreased by ZnPP-IX (figs. 1 and 2, A). No immunostaining was observed in negative control sections (data not shown). Semiquantitative RT-PCR analysis showed that CYP injection increased the expression level of HO-1 mRNA in bladders, which was further enhanced by hemin (fig. 2, B). These results indicated that endogenous bilirubin production in bladder tissue was on a parallel with the HO-1 expression level. Effects of Exogenously Applied Bilirubin on CYP Induced Bladder Tissue Injury CYP injection induced a time dependent increase in bladder weight, which was significantly inhibited by 30 mg/kg bilirubin pretreatment at each time point (fig. 3, A). In the
analysis of dose-response relationships 24 hours after CYP injection the bladder weight increase was significantly decreased by pretreatment with 30 and 100 mg/kg bilirubin. Differences were not significant between the 30 and 100 mg/kg groups (fig. 3, B). We then examined the histological appearance of bladders in each condition (fig. 4). CYP treated rat bladders showed extensive cystitis, characterized by acute inflammation with vascular congestion, edema, hemorrhage, neutrophil infiltration and epithelial denudation. Pretreatment with 30 mg/kg bilirubin ameliorated CYP induced inflammatory changes. Median morphological scores were 0 (range 0), 2 (range 2, vs controls p ⬍0.05) and 1 (range 1 to 2, vs CYP p ⬍0.05) for control, CYP only and CYP plus bilirubin treated rats, respectively.
Effects of Bilirubin Pretreatment on HO Expression in CYP Induced Cystitis We performed semiquantitative RT-PCR to evaluate the effects of exogenous bilirubin administration on HO-1 and HO-2 mRNA expression in bladders. Although CYP injection led to a prominent induction of HO-1 expression, exogenously applied bilirubin did not modify its expression in our experimental conditions (fig. 5, A and B). On the other hand, HO-2 was constitutively expressed and not influenced by CYP treatment or by CYP plus bilirubin (fig. 5, A and C).
BILIRUBIN AND CYCLOPHOSPHAMIDE INDUCED HEMORRHAGIC CYSTITIS
FIG. 2. A, semiquantitative results of immunohistochemistry of bilirubin in bladders in 6 to 8 rats per group. Boxes represent 25th to 75th percentiles. Central lines indicate median. Whiskers indicate 5th and 95th percentiles. Circles represent outliers. Asterisk indicates p ⬍0.05. B, densitometric analysis of HO-1 mRNA in bladder of CYP treated rats. Data are shown as mean ⫾ SE HO-1-toGAPDH ratio compiled from duplicated determinations, considering 1 as control value, in 5 or 6 bladders per group. Asterisk indicates p ⬍0.01.
Bilirubin Down-Regulated Inflammation Related Gene Expression in CYP Induced Cystitis To discern how bilirubin protects against CYP mediated urotoxicity we measured the expression levels of iNOS, IL-1 and TNF-␣ mRNA in the bladders of each group by real-time PCR. Although these 3 mRNAs were expressed in control rat bladders at a low level, CYP injection mark-
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FIG. 3. Effect of CYP administration on increase in bladder wet weight was decreased by bilirubin pretreatment (BR). A, time course of bladder wet weight after CYP injection with or without 30 mg/kg bilirubin pretreatment. Single asterisk indicates p ⬍0.05. Double asterisks indicate p ⬍0.01. B, bilirubin dose-response relationship 24 hours after CYP injection in 6 to 8 bladders per group. Histograms represent mean ⫾ SE.
edly induced their expression. Up-regulated expression of iNOS and IL-1 was significantly decreased by 30 mg/kg bilirubin (fig. 6, A and B). The decrease in TNF-␣ expression by bilirubin was not significant (vs CYP p ⫽ 0.10, fig. 6, C). Immunohistochemical analyses confirmed that exogenously applied bilirubin down-regulated the increased expression of iNOS protein in bladders with CYP induced cystitis (figs. 7 and 8). Although CYP administration increased the number and distribution of iNOS immunoreactive cells in all bladder regions, especially the suburothelium, this immunoreactivity was significantly
FIG. 4. Representative bladder staining. A, in controls intact urothelium projecting to lumen and blood vessels with thin walls were observed. B, sections from rats treated with CYP showed extensive urothelial damage, marked edema in suburothelium, vascular congestion and marked inflammatory infiltrates with lymphocytes and leukocytes. C, in bladders of rats treated with CYP plus bilirubin urothelium was preserved. H & E. Scale bars indicate 200 m.
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BILIRUBIN AND CYCLOPHOSPHAMIDE INDUCED HEMORRHAGIC CYSTITIS oxidant actions of bilirubin reflect an amplification cycle, whereby bilirubin is oxidized to biliverdin, which is recycled back to bilirubin by biliverdin reductase, resulting in a 10,000-fold amplification of bilirubin available to sequester reactive oxygen species.16 In a CYP induced cystitis model we previously noted that the level of HO-1 expression was increased in the urothelium and suburothelium by CYP injection, and enhanced further by hemin pretreatment.12 The current study demonstrated that endogenous bilirubin as a consequence of HO-1 induction accumulated in bladder tissue in a fashion similar to that of the increase in the HO-1 expression. The co-
FIG. 5. RT-PCR analysis of HO-1 and HO-2 mRNA expression. A, effects of exogenous bilirubin administration on HO expression in bladder of CYP treated rats. GAPDH bands are shown as internal control. B, densitometric analysis of HO-1. C, densitometric analysis of HO-2-to-GAPDH ratio. Data are shown as mean ⫾ SE of duplicated determinations in 6 bladders per group with 1 considered control value. Asterisk indicates p ⬍0.01 vs control.
inhibited by bilirubin pretreatment. No immunostaining was observed in negative control sections (data not shown). DISCUSSION There is increasing evidence to support the suggestion that bilirubin acts as a powerful antioxidant with a wide spectrum. Several experimental models have shown that endogenously produced bilirubin manifests cytoprotective properties.15 It is considered that the physiological anti-
FIG. 6. Effects of exogenously applied bilirubin on mRNA expression in bladders of 8 CYP treated rats per group per experiment. A, iNOS. B, IL-1. C, TNF-␣. Data are shown as means ⫾ SE of duplicated determinations from 2 independent experiments. Asterisk indicates p ⬍0.05 vs CYP treated group.
BILIRUBIN AND CYCLOPHOSPHAMIDE INDUCED HEMORRHAGIC CYSTITIS
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FIG. 7. Representative immunohistochemical staining for iNOS. A, control rat bladders show weak iNOS expression in urothelium. B, CYP treated rat bladder with intense iNOS staining in all bladder regions. C, CYP treated rat bladder with bilirubin pretreatment demonstrated decreased iNOS expression. Scale bars indicate 100 m.
localized expression of HO-1 and bilirubin proves that heme degrading products through HO-1 reaction are actually generated in bladder tissue and it suggests the possibility that bilirubin is involved in the HO-1 dependent anti-inflammatory system. Meanwhile, we evaluated the roles of bilirubin in the bladder by exogenous supplementation of this bile pigment and noted its anti-inflammatory effects. Our experiments demonstrated that exogenously applied bilirubin decreased IL-1 expression as an indicator of the severity of inflammation and it ameliorated the histological damage, including vascular damage, caused by CYP. Exogenous bilirubin has previously been shown to ameliorate oxidative injury in experimental models of ischemia/ reperfusion and organ transplantation.5,17,18 Wang et al reported that the total bilirubin concentration in the rat serum peaked at levels 4-fold above baseline by 30 to 60 minutes after a single intraperitoneal administration of bilirubin in the same dose as that used in our experiment.19 Extracellular bilirubin is then rapidly taken up into cells, mainly by spontaneous transmembrane diffusion. It has been reported that increasing NO produced by iNOS is responsible for the detrimental effects of CYP on the bladder10 and cytoprotective functions of HO-1 in the bladder are associated with down-regulation of NO pro-
FIG. 8. Semiquantitative results of immunohistochemistry of iNOS in bladder of 8 rats per group. Box represents 25th to 75th percentiles. Central line indicates median. Whiskers indicate 5th and 95th percentiles. Circles represent outliers. Asterisk indicates p ⬍0.05.
duction and iNOS expression.12 In the current study exogenously applied bilirubin significantly decreased the up-regulated expression of iNOS in the bladder, suggesting that the cytoprotective roles of bilirubin are mediated at least in part through the inhibition of iNOS expression. Since bilirubin is the end product of the HO pathway, our findings that endogenous generation of bilirubin is associated with HO-1 up-regulation in inflamed bladder tissue imply the possibility that the production of CO, another catabolite of heme degradation, is also enhanced. Although CO has an anti-inflammatory property, which depends on modulation of the p38 mitogen-activated protein kinase signaling pathway,1 toxic potential of this molecule complicates the clinical application of exogenous CO supplementation. In our CYP induced cystitis model exogenously applied bilirubin did not affect the level of HO-1 expression in the bladder, suggesting that the protective roles of bilirubin are not mediated through the function of HO-1 itself or the other byproducts of HO-1, such as CO and iron. Indeed, bilirubin works as a key mediator of HO-1 and it provides cytoprotection in bladder tissue. Manipulation of the HO-1 pathway to increase intracellular bilirubin generation may represent a feasible strategy to counteract oxidative stress15 and ultimately it may have potential clinical implications in the prevention of inflammatory diseases in the urinary tract. Although pharmacological or genetic approaches targeting HO-1 are the proposed therapeutic strategies in several clinical settings2 and hemin has already been used clinically in humans to treat acute porphyries, recent studies demonstrated that HO induction also has pro-oxidant properties, primarily through the accumulation of iron.1 For this reason exogenous bilirubin supplementation may be a safer and more practical means to substitute for HO-1 induction in clinical models.5,18 Furthermore, we suggest that bilirubin supplementation may offer a simple and inexpensive method that can be used in conjunction with CYP therapy to decrease its side effects. Murat et al reported the relaxant effects of bilirubin on ureteral and bladder muscles in vitro, and suggested the possibility that hyperbilirubinemia may contribute to the spontaneous recovery of prenatal hydronephrosis.20 Further functional studies may provide important insights and relevant information on the roles of HO and its downstream products in urinary tract diseases.
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CONCLUSIONS In rat bladders with CYP induced hemorrhagic cystitis intracellular bilirubin generation increased in proportion to HO-1 expression. Bilirubin production represents an adaptive response to CYP induced bladder insults. Supplementation of this bile pigment provides cytoprotective effects, which are mediated in part via the suppression of iNOS induction.
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Abbreviations and Acronyms CYP ⫽ cyclophosphamide GAPDH ⫽ glyceraldehyde-3-phosphate dehydrogenase HO ⫽ heme oxygenase IL-1 ⫽ interleukin-1 iNOS ⫽ inducible NO synthase NO ⫽ nitric oxide RT-PCR ⫽ reverse-transcriptase polymerase chain reaction TNF-␣ ⫽ tumor necrosis factor-␣ ZnPP-IX ⫽ zinc protoporphyrin IX REFERENCES 1.
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Ryter SW, Otterbein LE, Morse D and Choi AM: Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance. Mol Cell Biochem 2002; 234 – 235: 249. Sikorski EM, Hock T, Hill-Kapturczak N and Agarwal A: The story so far: molecular regulation of the heme oxygenase-1 gene in renal injury. Am J Physiol Renal Physiol 2004; 286: F425. Foresti R and Motterlini R: The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis. Free Radic Res 1999; 31: 459. Taille C, Foresti R, Lanone S, Zedda C, Green C, Aubier M et al: Protective role of heme oxygenases against endotoxininduced diaphragmatic dysfunction in rats. Am J Respir Crit Care Med 2001; 163: 753. Kirkby KA and Adin CA: Products of heme oxygenase and their potential therapeutic applications. Am J Physiol Renal Physiol 2006; 290: F563. Dore S, Takahashi M, Ferris CD, Zakhary R, Hester LD, Guastella D et al: Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury. Proc Natl Acad Sci U S A 1999; 96: 2445. Hopkins PN, Wu LL, Hunt SC, James BC, Vincent GM and Williams RR: Higher serum bilirubin is associated with
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decreased risk for early familial coronary artery disease. Arterioscler Thromb Vasc Biol 1996; 16: 250. Temme EH, Zhang J, Schouten EG and Kesteloot H: Serum bilirubin and 10-year mortality risk in a Belgian population. Cancer Causes Control 2001; 12: 887. Leung N, Croatt AJ, Haggard JJ, Grande JP and Nath KA: Acute cholestatic liver disease protects against glycerolinduced acute renal failure in the rat. Kidney Int 2001; 60: 1047. Souza-Fiho MV, Lima MV, Pompeu MM, Ballejo G, Cunha FQ and Ribeiro Rde A: Involvement of nitric oxide in the pathogenesis of cyclophosphamide-induced hemorrhagic cystitis. Am J Pathol 1997; 150: 247. Masuda H, Chancellor MB, Kihara K and Yoshimura N: 15deoxy-Delta12,14-prostaglandin J2 attenuates development of cyclophosphamide-induced cystitis in rats. Urology 2006; 67: 435. Matsuoka Y, Masuda H, Yokoyama M and Kihara K: Protective effects of heme oxygenase-1 against cyclophosphamideinduced haemorrhagic cystitis in rats. BJU Int 2007; 100: 1402. Gray KJ, Engelmann UH, Johnson EH and Fishman IJ: Evaluation of misoprostol cytoprotection of the bladder with cyclophosphamide (Cytoxan) therapy. J Urol 1986; 136: 497. Luo J, Zha S, Gage WR, Dunn TA, Hicks JL, Bennett CJ et al: Alpha-methylacyl-CoA racemase: a new molecular marker for prostate cancer. Cancer Res 2002; 62: 2220. Clark JE, Foresti R, Sarathchandra P, Kaur H, Green CJ and Motterlini R: Heme oxygenase-1-derived bilirubin ameliorates postischemic myocardial dysfunction. Am J Physiol Heart Circ Physiol 2000; 278: H643. Baranano DE, Rao M, Ferris CD and Snyder SH: Biliverdin reductase: a major physiologic cytoprotectant. Proc Natl Acad Sci U S A 2002; 99: 16093. Kato Y, Shimazu M, Kondo M, Uchida K, Kumamoto Y, Wakabayashi G et al: Bilirubin rinse: a simple protectant against the rat liver graft injury mimicking heme oxygenase-1 preconditioning. Hepatology 2003; 38: 364. Adin CA, Croker BP and Agarwal A: Protective effects of exogenous bilirubin on ischemia-reperfusion injury in the isolated, perfused rat kidney. Am J Physiol Renal Physiol 2005; 288: F778. Wang WW, Smith DL and Zucker SD: Bilirubin inhibits iNOS expression and NO production in response to endotoxin in rats. Hepatology 2004; 40: 424. Murat N, Kasap B, Kavukcu S, Soylu A, Türkmen M and Gidener S: In vitro analysis of the effect of hyperbilirubinemia on rabbit ureter and bladder. Pediatr Nephrol 2006; 21: 328.
Purpose: The end product of the heme oxygenase pathway, bilirubin, is the most abundant endogenous antioxidant in mammals. We report the heme oxygenase-1 mediated production of bilirubin and its cytoprotective roles in cyclophosphamide induced hemorrhagic cystitis in rats. Materials and Methods: Female Sprague-Dawley rats received intraperitoneal administration of cyclophosphamide. In the first experiment hemin (an inducer of heme oxygenase-1) with or without zinc protoporphyrin IX (an inhibitor of heme oxygenase activity) was given before cyclophosphamide injection. Endogenous bilirubin production was analyzed in bladder tissues immunohistochemically. In another experiment bilirubin solution was administered before the cyclophosphamide injection. Changes in bladder weight, microscopic feature and expression levels of inducible nitric oxide synthase, proinflammatory cytokines and heme oxygenase were evaluated using polymerase chain reaction and immunostaining. Results: Bilirubin was generated in bladders with cyclophosphamide induced cystitis, especially in the urothelium and suburothelium. Hemin pretreatment provided increased production of endogenous bilirubin, which was decreased by zinc protoporphyrin IX. In an evaluation of the roles of bilirubin exogenous bilirubin administration ameliorated cyclophosphamide induced inflammatory changes and reduced the increase in bladder weight. The elevated expression of inducible nitric oxide synthase and interleukin-1 in cyclophosphamide induced cystitis was significantly downregulated by exogenously applied bilirubin. The expression of heme oxygenase-1 and 2 was not modified by bilirubin administration. Conclusions: Cyclophosphamide induced hemorrhagic cystitis is accompanied by endogenous bilirubin production through heme oxygenase-1 induction in the bladder. Bilirubin has cytoprotective roles in association with the down-regulation of inducible nitric oxide synthase expression. Our results suggest that bilirubin may have therapeutic potential against bladder inflammatory insults such as cyclophosphamide induced cystitis. Key Words: bladder; rats, Sprague-Dawley; cystitis; cyclophosphamide; bilirubin
eme oxygenase is the rate limiting enzyme in the cleavage of the heme molecule to equimolar quantities of CO, free iron and biliverdin. Free iron is sequestered into ferritin and biliverdin is rapidly converted to bilirubin through the action of biliverdin reductase.1,2 The HO pathway was recently proposed to function as an effective system to counteract environmental stresses and maintain cellular homeostasis.3 To date 3 isoforms have been identified, including inducible HO-1, and constitutive HO-2 and 3. HO-1, which acts as a ubiquitous defensive system in various cell types, is induced by a wide range of stimuli, such as cytokines, bacterial endotoxin, reactive oxygen species, ischemia/reperfusion and hypoxia.1,2,4 Although the cytoprotective effects of HO-1 have been confirmed in various organ systems,2,4 the mechanism of HO-1 action has not fully been elucidated. In recent years attention has focused on the downstream products of HO activity, CO and bilirubin, which
H
Submitted for publication June 2, 2007. * Correspondence: Department of Urology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan (telephone: ⫹81 3 5803 5295; FAX: ⫹81 3 5803 5295; e-mail: [email protected] or [email protected]).
0022-5347/08/1793-1160/0 THE JOURNAL OF UROLOGY® Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION
have been suggested to be key mediators of the beneficial effects of HOs.5 Bilirubin has long been considered a potentially toxic waste product of heme metabolism that causes jaundice and deposits in the brain to elicit the neurotoxicity associated with kernicterus at high concentrations. However, this bile pigment has also been known to have antioxidant properties and function as an efficient peroxyl radical scavenger at physiological concentration.6 Increased serum levels of bilirubin have been associated with a decreased incidence of coronary artery diseases and cancer mortality.7,8 In a rat model ligation of the common bile duct caused cholestasis with an increase in the plasma bilirubin concentration and it conferred resistance to glycerol induced acute renal failure.9 Thus, cytoprotective effects of bilirubin have been demonstrated in animal and human studies. CYP is an alkylating agent extensively used in the treatment of various neoplastic diseases. It causes hemorrhagic cystitis as a complication. The initial urothelial damage occurs by direct contact with acrolein, a urotoxic metabolite of CYP, and it is followed by the involvement of NO produced by iNOS.10,11 Recently we reported the involvement of HO-1 and its anti-inflammatory effects in bladders with CYP in-
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Vol. 179, 1160-1166, March 2008 Printed in U.S.A. DOI:10.1016/j.juro.2007.10.031
BILIRUBIN AND CYCLOPHOSPHAMIDE INDUCED HEMORRHAGIC CYSTITIS duced hemorrhagic cystitis.12 However, little is known about the end product of the HO system, bilirubin, in urinary tract disease. We determined the expression profile of endogenous bilirubin through HO-1 induction and evaluated the roles of bilirubin in urological inflammatory states using the rat CYP induced cystitis model. MATERIALS AND METHODS Animals A total of 102 nonpregnant female Sprague-Dawley rats weighing 180 to 200 gm were randomly divided into groups. All efforts were made to use the minimum number of animals. This study complied with the animal welfare regulation of Tokyo Medical and Dental University. Protocol All chemicals were obtained from Sigma-Aldrich™ and administered intraperitoneally. Animals received an injection of cyclophosphamide monohydrate (200 mg/kg) for cystitis induction. Bilirubin was dissolved in 0.1 N sodium hydroxide and then diluted with sodium phosphate buffer. Care was taken to shield bilirubin from visible light to prevent its inactivation. In the first experiment 50 mg/kg hemin (a chemical inducer of HO-1) was given 24 hours before CYP injection with or without 30 mg/kg ZnPP-IX (an inhibitor of HO activity) 1 hour before CYP injection. The animals were sacrificed by exsanguination 24 hours after CYP injection. The bladders were harvested and used for analyses. In the second experiment the rats were treated with various doses of bilirubin (10, 30 and 100 mg/kg, respectively) 30 minutes before CYP injection and the bladders were harvested at various time points after CYP injection. Each bladder was weighed in the wet condition to evaluate the degree of inflammation.11,13 Bilirubin (30 mg/kg) pretreated rat bladders excised 24 hours after CYP injection were used for further evaluation, as described. Control animals were given vehicle only. RT-PCR Total RNA extracted was reverse transcribed to generate first-strand cDNA. Semiquantitative RT-PCR was performed for 30 cycles of amplification using a certain protocol, including HO-1 (denaturation at 94C for 1 minute, annealing at 55C for 2 minutes and extension at 72C for 3 minutes), HO-2 (94C for 1 minute, 55C for 1 minute and 72C for 2 minutes) and GAPDH (94C for 45 seconds, 55C for 45 seconds and 72C for 45 seconds). PCR products were electrophoresed on agarose gels, which were stained with ethidium bromide to visualize the bands. HO expression was quantified by densitometric scanning and normalized to the GAPDH level. Primer sequences were HO-1 forward 5=-ACTTTCAGAAGGGTCAGGTGTCC-3= and reverse 5=TTGAGCAGGAAGGCGGTCTTAG-3=, and HO-2 forward 5=-GAAGTGAGGGCAGCACAAAC-3= and reverse 5=-CTTCTTCAGCACCTGGCCT-3=. A primer pair for rat GAPDH (R & D Systems®) was used. Real-Time PCR Specific primer sets for rat iNOS, IL-1, TNF-␣ and GAPDH (Nihon Gene Research Laboratories, Sendai, Japan) were used. Real-time PCR was performed to quantify the products
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using a LightCycler® and a LightCycler-FastStart DNA master SYBR® Green I kit according to the manufacturer protocol. Data on iNOS, IL-1 and TNF-␣ were standardized to GAPDH expression in each sample. PCR experiments were repeated at least 2 times. Microscopic Evaluation Tissues were fixed in formol, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Histological damage was scored on a scale of 0 —normal epithelium, 1—mild changes involving a decrease in epithelial cells, flattening with submucosal edema, mild hemorrhage and few ulcerations, and 2—severe changes, including mucosal erosion, inflammatory cell infiltration, fibrin deposition, hemorrhage and multiple ulcerations.13 Two of us separately evaluated slides in a blind fashion. Immunohistochemical Analysis Bilirubin and iNOS immunostaining was performed in CYP treated rat bladders with hemin/ZnPP-IX pretreatments and with bilirubin pretreatment, respectively. Sections were pretreated with microwave irradiation for antigen retrieval. Endogenous peroxidase was blocked by incubation in hydrogen peroxide and nonspecific protein binding was minimized by treatment with normal goat serum. The preparations were incubated at 4C for 24 hours with mouse anti-bilirubin monoclonal antibody (24G7, Dojindo, Kumamoto, Japan) or mouse anti-iNOS monoclonal antibody (Wako, Osaka, Japan) and then treated with secondary antibody conjugated with peroxidase (Nichirei, Tokyo, Japan) for 30 minutes at room temperature. Negative control sections incubated in the absence of the primary antibody were also processed and evaluated for specificity or background staining levels. In bilirubin immunostaining each step was performed in a dark room to prevent isomerization and photo-oxidation of bilirubin. Evaluation of Immunoreactivity Slides were independently reviewed by 2 of us, who were blinded to treatment data on each sample. A combined intensity and percent positive scoring method was used to semiquantify immunoreactivity in the suburothelium.12,14 Staining intensity was scored as 3—strong, 2—moderate, 1—weak and 0 –negative. The percent of cells with each intensity score was estimated. A combined weighted score was defined as the sum of the percent of cells staining at each intensity level multiplied by the intensity score, eg a case with 20% strong staining, 50% moderate staining and 30% weak staining would be scored as 190 (20 ⫻ 3 ⫹ 50 ⫻ 2 ⫹ 30 ⫻ 1). Statistical Analysis Results are expressed as means ⫾ SEM. Statistical comparisons were performed by 1-way ANOVA, followed by Bonferroni’s test. For histopathological and immunohistochemical data median values were used and the difference was assessed using the nonparametric Kruskal-Wallis test, followed by the Mann-Whitney U test with p ⬍0.05 considered statistically significant.
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FIG. 1. Representative immunohistochemical staining for bilirubin in bladder. A, in controls no apparent immunostaining for bilirubin was. B, bilirubin immunoreactivity in urothelial cells and suburothelial stroma cells in rats treated with CYP. C, bilirubin immunoreactivity in urothelial cells and suburothelial stroma cells in rats treated with CYP plus hemin. D, bilirubin immunoreactivity in urothelial cells and suburothelial stroma cells in rats treated with CYP plus hemin and ZnPP-IX pretreatments. Scale bars indicate 100 m.
RESULTS HO-1 Mediated Bilirubin Production in CYP Induced Cystitis We analyzed the accumulation and localization of endogenous bilirubin in bladder tissues to determine HO-1 mediated heme degradation in situ. Bilirubin immunoreactivity was absent or weak in control rat bladders. In the bladders of CYP treated rats positive immunostaining for bilirubin was detected in the urothelium and suburothelium. Hemin further increased the number and distribution of bilirubin immunoreactive cells. Especially in the suburothelium intense immunostaining for cytoplasmic bilirubin were identified. This increased immunoreactivity was decreased by ZnPP-IX (figs. 1 and 2, A). No immunostaining was observed in negative control sections (data not shown). Semiquantitative RT-PCR analysis showed that CYP injection increased the expression level of HO-1 mRNA in bladders, which was further enhanced by hemin (fig. 2, B). These results indicated that endogenous bilirubin production in bladder tissue was on a parallel with the HO-1 expression level. Effects of Exogenously Applied Bilirubin on CYP Induced Bladder Tissue Injury CYP injection induced a time dependent increase in bladder weight, which was significantly inhibited by 30 mg/kg bilirubin pretreatment at each time point (fig. 3, A). In the
analysis of dose-response relationships 24 hours after CYP injection the bladder weight increase was significantly decreased by pretreatment with 30 and 100 mg/kg bilirubin. Differences were not significant between the 30 and 100 mg/kg groups (fig. 3, B). We then examined the histological appearance of bladders in each condition (fig. 4). CYP treated rat bladders showed extensive cystitis, characterized by acute inflammation with vascular congestion, edema, hemorrhage, neutrophil infiltration and epithelial denudation. Pretreatment with 30 mg/kg bilirubin ameliorated CYP induced inflammatory changes. Median morphological scores were 0 (range 0), 2 (range 2, vs controls p ⬍0.05) and 1 (range 1 to 2, vs CYP p ⬍0.05) for control, CYP only and CYP plus bilirubin treated rats, respectively.
Effects of Bilirubin Pretreatment on HO Expression in CYP Induced Cystitis We performed semiquantitative RT-PCR to evaluate the effects of exogenous bilirubin administration on HO-1 and HO-2 mRNA expression in bladders. Although CYP injection led to a prominent induction of HO-1 expression, exogenously applied bilirubin did not modify its expression in our experimental conditions (fig. 5, A and B). On the other hand, HO-2 was constitutively expressed and not influenced by CYP treatment or by CYP plus bilirubin (fig. 5, A and C).
BILIRUBIN AND CYCLOPHOSPHAMIDE INDUCED HEMORRHAGIC CYSTITIS
FIG. 2. A, semiquantitative results of immunohistochemistry of bilirubin in bladders in 6 to 8 rats per group. Boxes represent 25th to 75th percentiles. Central lines indicate median. Whiskers indicate 5th and 95th percentiles. Circles represent outliers. Asterisk indicates p ⬍0.05. B, densitometric analysis of HO-1 mRNA in bladder of CYP treated rats. Data are shown as mean ⫾ SE HO-1-toGAPDH ratio compiled from duplicated determinations, considering 1 as control value, in 5 or 6 bladders per group. Asterisk indicates p ⬍0.01.
Bilirubin Down-Regulated Inflammation Related Gene Expression in CYP Induced Cystitis To discern how bilirubin protects against CYP mediated urotoxicity we measured the expression levels of iNOS, IL-1 and TNF-␣ mRNA in the bladders of each group by real-time PCR. Although these 3 mRNAs were expressed in control rat bladders at a low level, CYP injection mark-
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FIG. 3. Effect of CYP administration on increase in bladder wet weight was decreased by bilirubin pretreatment (BR). A, time course of bladder wet weight after CYP injection with or without 30 mg/kg bilirubin pretreatment. Single asterisk indicates p ⬍0.05. Double asterisks indicate p ⬍0.01. B, bilirubin dose-response relationship 24 hours after CYP injection in 6 to 8 bladders per group. Histograms represent mean ⫾ SE.
edly induced their expression. Up-regulated expression of iNOS and IL-1 was significantly decreased by 30 mg/kg bilirubin (fig. 6, A and B). The decrease in TNF-␣ expression by bilirubin was not significant (vs CYP p ⫽ 0.10, fig. 6, C). Immunohistochemical analyses confirmed that exogenously applied bilirubin down-regulated the increased expression of iNOS protein in bladders with CYP induced cystitis (figs. 7 and 8). Although CYP administration increased the number and distribution of iNOS immunoreactive cells in all bladder regions, especially the suburothelium, this immunoreactivity was significantly
FIG. 4. Representative bladder staining. A, in controls intact urothelium projecting to lumen and blood vessels with thin walls were observed. B, sections from rats treated with CYP showed extensive urothelial damage, marked edema in suburothelium, vascular congestion and marked inflammatory infiltrates with lymphocytes and leukocytes. C, in bladders of rats treated with CYP plus bilirubin urothelium was preserved. H & E. Scale bars indicate 200 m.
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BILIRUBIN AND CYCLOPHOSPHAMIDE INDUCED HEMORRHAGIC CYSTITIS oxidant actions of bilirubin reflect an amplification cycle, whereby bilirubin is oxidized to biliverdin, which is recycled back to bilirubin by biliverdin reductase, resulting in a 10,000-fold amplification of bilirubin available to sequester reactive oxygen species.16 In a CYP induced cystitis model we previously noted that the level of HO-1 expression was increased in the urothelium and suburothelium by CYP injection, and enhanced further by hemin pretreatment.12 The current study demonstrated that endogenous bilirubin as a consequence of HO-1 induction accumulated in bladder tissue in a fashion similar to that of the increase in the HO-1 expression. The co-
FIG. 5. RT-PCR analysis of HO-1 and HO-2 mRNA expression. A, effects of exogenous bilirubin administration on HO expression in bladder of CYP treated rats. GAPDH bands are shown as internal control. B, densitometric analysis of HO-1. C, densitometric analysis of HO-2-to-GAPDH ratio. Data are shown as mean ⫾ SE of duplicated determinations in 6 bladders per group with 1 considered control value. Asterisk indicates p ⬍0.01 vs control.
inhibited by bilirubin pretreatment. No immunostaining was observed in negative control sections (data not shown). DISCUSSION There is increasing evidence to support the suggestion that bilirubin acts as a powerful antioxidant with a wide spectrum. Several experimental models have shown that endogenously produced bilirubin manifests cytoprotective properties.15 It is considered that the physiological anti-
FIG. 6. Effects of exogenously applied bilirubin on mRNA expression in bladders of 8 CYP treated rats per group per experiment. A, iNOS. B, IL-1. C, TNF-␣. Data are shown as means ⫾ SE of duplicated determinations from 2 independent experiments. Asterisk indicates p ⬍0.05 vs CYP treated group.
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FIG. 7. Representative immunohistochemical staining for iNOS. A, control rat bladders show weak iNOS expression in urothelium. B, CYP treated rat bladder with intense iNOS staining in all bladder regions. C, CYP treated rat bladder with bilirubin pretreatment demonstrated decreased iNOS expression. Scale bars indicate 100 m.
localized expression of HO-1 and bilirubin proves that heme degrading products through HO-1 reaction are actually generated in bladder tissue and it suggests the possibility that bilirubin is involved in the HO-1 dependent anti-inflammatory system. Meanwhile, we evaluated the roles of bilirubin in the bladder by exogenous supplementation of this bile pigment and noted its anti-inflammatory effects. Our experiments demonstrated that exogenously applied bilirubin decreased IL-1 expression as an indicator of the severity of inflammation and it ameliorated the histological damage, including vascular damage, caused by CYP. Exogenous bilirubin has previously been shown to ameliorate oxidative injury in experimental models of ischemia/ reperfusion and organ transplantation.5,17,18 Wang et al reported that the total bilirubin concentration in the rat serum peaked at levels 4-fold above baseline by 30 to 60 minutes after a single intraperitoneal administration of bilirubin in the same dose as that used in our experiment.19 Extracellular bilirubin is then rapidly taken up into cells, mainly by spontaneous transmembrane diffusion. It has been reported that increasing NO produced by iNOS is responsible for the detrimental effects of CYP on the bladder10 and cytoprotective functions of HO-1 in the bladder are associated with down-regulation of NO pro-
FIG. 8. Semiquantitative results of immunohistochemistry of iNOS in bladder of 8 rats per group. Box represents 25th to 75th percentiles. Central line indicates median. Whiskers indicate 5th and 95th percentiles. Circles represent outliers. Asterisk indicates p ⬍0.05.
duction and iNOS expression.12 In the current study exogenously applied bilirubin significantly decreased the up-regulated expression of iNOS in the bladder, suggesting that the cytoprotective roles of bilirubin are mediated at least in part through the inhibition of iNOS expression. Since bilirubin is the end product of the HO pathway, our findings that endogenous generation of bilirubin is associated with HO-1 up-regulation in inflamed bladder tissue imply the possibility that the production of CO, another catabolite of heme degradation, is also enhanced. Although CO has an anti-inflammatory property, which depends on modulation of the p38 mitogen-activated protein kinase signaling pathway,1 toxic potential of this molecule complicates the clinical application of exogenous CO supplementation. In our CYP induced cystitis model exogenously applied bilirubin did not affect the level of HO-1 expression in the bladder, suggesting that the protective roles of bilirubin are not mediated through the function of HO-1 itself or the other byproducts of HO-1, such as CO and iron. Indeed, bilirubin works as a key mediator of HO-1 and it provides cytoprotection in bladder tissue. Manipulation of the HO-1 pathway to increase intracellular bilirubin generation may represent a feasible strategy to counteract oxidative stress15 and ultimately it may have potential clinical implications in the prevention of inflammatory diseases in the urinary tract. Although pharmacological or genetic approaches targeting HO-1 are the proposed therapeutic strategies in several clinical settings2 and hemin has already been used clinically in humans to treat acute porphyries, recent studies demonstrated that HO induction also has pro-oxidant properties, primarily through the accumulation of iron.1 For this reason exogenous bilirubin supplementation may be a safer and more practical means to substitute for HO-1 induction in clinical models.5,18 Furthermore, we suggest that bilirubin supplementation may offer a simple and inexpensive method that can be used in conjunction with CYP therapy to decrease its side effects. Murat et al reported the relaxant effects of bilirubin on ureteral and bladder muscles in vitro, and suggested the possibility that hyperbilirubinemia may contribute to the spontaneous recovery of prenatal hydronephrosis.20 Further functional studies may provide important insights and relevant information on the roles of HO and its downstream products in urinary tract diseases.
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CONCLUSIONS In rat bladders with CYP induced hemorrhagic cystitis intracellular bilirubin generation increased in proportion to HO-1 expression. Bilirubin production represents an adaptive response to CYP induced bladder insults. Supplementation of this bile pigment provides cytoprotective effects, which are mediated in part via the suppression of iNOS induction.
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Abbreviations and Acronyms CYP ⫽ cyclophosphamide GAPDH ⫽ glyceraldehyde-3-phosphate dehydrogenase HO ⫽ heme oxygenase IL-1 ⫽ interleukin-1 iNOS ⫽ inducible NO synthase NO ⫽ nitric oxide RT-PCR ⫽ reverse-transcriptase polymerase chain reaction TNF-␣ ⫽ tumor necrosis factor-␣ ZnPP-IX ⫽ zinc protoporphyrin IX REFERENCES 1.
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