Food and Chemical Toxicology 64 (2014) 110–118

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Mechanism for the protective effect of diallyl disulfide against cyclophosphamide acute urotoxicity in rats Sung-Hwan Kim a, In-Chul Lee a, Hyung-Seon Baek a, In-Sik Shin a,b, Changjong Moon a, Chun-Sik Bae a, Sung-Ho Kim a, Jong-Choon Kim a,⇑, Hyoung-Chin Kim c,⇑ a b c

College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk 363-883, Republic of Korea Biomedical Mouse Resource Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk 363-883, Republic of Korea

a r t i c l e

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Article history: Received 16 September 2013 Accepted 20 November 2013 Available online 27 November 2013 Keywords: Cyclophosphamide Diallyl disulfide Urotoxicity Cytochrome P450 Oxidative stress Nrf-2

a b s t r a c t This study investigated the protective effects of diallyl disulfide (DADS) against cyclophosphamide (CP)induced acute urotoxicity in rats. CP caused severe hemorrhagic cystitis as shown by significant increases in bladder weight, edema, and hemorrhage as well as increased urinary bladder epithelial cell apoptosis, protein expression of nuclear factor erythroid 2-related factor-2 (Nrf-2) and phase II enzymes (i.e., NAD(P)H: quinone oxidoreductase-1 (NQO-1) and heme oxygenase-1 (HO-1)), immunostaining intensity of acrolein–protein adducts, and histopathological changes. The significant decreases in glutathione content and catalase, glutathione-S-transferase, and glutathione reductase activities and a significant increase in malondialdehyde content indicated that CP-induced bladder injury was mediated through oxidative stress. In contrast, pretreatment with DADS significantly attenuated the CP-induced urotoxic effects, including oxidative damage, histopathological lesions, apoptotic changes, and accumulation of acrolein–protein adducts in the bladder. DADS also significantly increased expression of CYP2B1/2, CYP3A1, Nrf-2, NQO-1, and HO-1 and significantly decreased expression of CYP2C11. These results indicate that DADS prevented CP-induced bladder toxicity, in part, by detoxifying acrolein. The protective effects of DADS may be due to its ability to decrease metabolic activation of CP by inhibiting CYP2C11 and inducing CYP3A1, and its potent antioxidant activity and antiapoptotic effects occurred via the Nrf-2-antioxidant response element pathway. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Cyclophosphamide (CP) is an alkylating agent that is used to treat solid tumors, Hodgkin’s disease, and nonneoplastic conditions, such as rheumatic arthritis. It is also a transplant-rejection combatant drug. However, the urological side effects of CP are a major factor limiting its use (West, 1997). The incidence of these side effects is related to dosage and varies from 2% to 40%. These effects include transient irritative voiding symptoms, such as dysuria, hemorrhagic cystitis, urgency, bladder fibrosis, necrosis, contracture, and strangury with microhematuria (Gray et al., 1986; Levine and Richie, 1989). Therefore, a potential therapeutic approach aimed at protecting against or reversing the CP-induced urotoxicity would have very important clinical consequences. Most xenobiotic compounds require metabolic activation by cytochrome P450 (CYP) to form ultimate carcinogens or toxicants (Nebert and Dalton, 2006). It has been demonstrated that CP is a ⇑ Corresponding authors. Tel.: +82 62 530 2827; fax: +82 62 530 2809 (J.-C. Kim). Tel.: +82 43 240 6565; fax: +82 43 240 6569 (H.-C. Kim). E-mail addresses: [email protected] (J.-C. Kim), [email protected] (H.-C. Kim). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.11.023

prodrug that requires hepatic biotransformation to exert its urotoxic effect (Fleming, 1997). An alternative, but minor, pathway is the inactivation of CP, and both pathways are dependent on CYP enzymes (Chang et al., 1993; Weber and Waxman, 1993). In male rats, CP is oxidatively activated by CYP2C11 and CYP2B. Alternatively, CP-catalyzed N-dechloroethylation, which produces chloroacetaldehyde without anticancer activity, is catalyzed by CYP3A in rats (McClure and Stupans, 1992). Thus, the alteration of hepatic CYP may change the rate and pattern of CP metabolism. The concomitant use of CP with other drugs that inhibit or induce the CYP2B, CYP2C, or CYP3A enzymes can lead to drug–drug interactions (Chang et al., 1997; Rae et al., 2002; Yu et al., 1999). The urotoxicity of CP is not based on its direct alkylating activity; rather, it stems from the formation of renally excreted 4-hydroxy metabolites. In particular, acrolein is formed from hepatic microsomal enzyme hydroxylation (Kurowski and Wagner, 1997). Oxidative stress induced by acrolein plays a major role in the pathogenesis of CP-induced acute urotoxicity (Bhatia et al., 2006; Ozcan et al., 2005; Tripathi and Jena, 2010). To avoid these toxic side effects, CP is typically used in combination with various detoxifying and protective agents, to reduce or eliminate its

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adverse toxic effects. Antioxidant agents, such as Trigonella foenum-graecum L. (Bhatia et al., 2006), flavonoids (Ozcan et al., 2005), melatonin (Tripathi and Jena, 2010), and taurine (Abd-Allah et al., 2005), have protective actions against CP-induced acute urotoxicity. Thus, the combination of CP with a potent antioxidant may be appropriate to reduce the urotoxic effects of the drug. Garlic oil contains more than 20 organosulfur compounds and has been considered a dietary anticancer component. Experimental animal studies have shown inhibition of chemically induced carcinogenesis in different organs by certain sulfur-containing compounds (Sparnins et al., 1988; Wattenberg et al., 1989). Among these compounds, diallyl disulfide (DADS), which is a major component of the secondary metabolites derived from garlic, has been well documented as a potent compound that prevents cancer, genotoxicity, nephrotoxicity, and hepatotoxicity (Fukao et al., 2004; Guyonnet et al., 2002; Nakagawa et al., 2001; Pedraza-Chaverrí et al., 2003). Previous studies have shown that DADS is effective in modulating not only phase I enzymes, such as hepatic CYP, and phase II enzymes, such as glutathione S-transferase (GST), nuclear factor erythroid 2-related factor-2 (Nrf-2), NAD(P)H: quinone oxidoreductase-1 (NQO-1), and heme oxygenase-1 (HO-1) (Fukao et al., 2004; Guyonnet et al., 2002; Pan et al., 1993; Singh et al., 1998), but also antioxidant-system capacity (Pedraza-Chaverrí et al., 2003; Wu et al., 2001). Although DADS has been confirmed to have a protective effect against CP-induced acute urotoxicity (Manesh and Kuttan, 2002), its effects on antioxidant enzymes and the molecular mechanisms underlying its protective action have not been fully elucidated. The present study investigated the protective effects of DADS against CP-induced acute urotoxicity in male rats. To study the protective mechanism of DADS, its potential effects of DADS on the expression of CYP, accumulation of acrolein–protein adducts, reactive oxygen species (ROS)-mediated apoptotic changes, and protein expression of Nrf-2 and the associated phase II enzymes HO-1 and NQO-1 were also assessed. 2. Materials and methods 2.1. Animals and environmental conditions Male Sprague–Dawley rats aged 12 weeks were obtained from a specific pathogen-free colony at Samtako Co. (Osan, Republic of Korea) and used after 1 week of quarantine and acclimation. Two animals per cage were housed in a room maintained at a temperature of 23 ± 3 °C and a relative humidity of 50 ± 10% with artificial lighting from 08:00 to 20:00 and with 13 to 18 air changes per hour. Commercial rodent chow (Samyang Feed, Wonju, Republic of Korea) sterilized by radiation and sterilized tap water were available ad libitum. The Institutional Animal Care and Use Committee of Chonnam National University approved the protocols for the animal study, and the animals were cared for in accordance with the Guidelines for Animal Experiments of Chonnam National University. 2.2. Test chemicals and treatment CP was purchased from Sigma Aldrich Co. (St. Louis, MO, USA). DADS was purchased from Tokyo Kasei Chemical Co. (Tokyo, Japan). All other chemicals were of the highest grade commercially available. CP and DADS were dissolved in saline and corn oil, respectively, and were prepared immediately before treatment. The daily application volumes of CP (2 ml/kg body weight) and DADS (5 ml/kg body weight) were calculated in advance based on the most recently recorded body weight of the individual animal. DADS was gavaged to rats once daily for a period of 5 days at 100 mg/kg/day. One hour after the final DADS treatment, the rats were given a single intraperitoneal dose of CP (100 mg/kg/day). All animals were sacrificed 12 h after CP administration.

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2.4. Tissue preparation After 12 h of acute hemorrhagic cystitis induction, all male rats were euthanized by carbon dioxide and exsanguination from the aorta. The absolute and relative (organ-to-body weight ratio) weights of the bladder were measured in all rats, and the bladders were assessed for severity of cystitis. Then, the bladders were cut into two equal pieces from dome to bottom. Half of the bladder was fixed for 24 h in 10% buffered formaldehyde for histopathological evaluation and immunohistochemical analysis and the remainder was stored at 80 °C to measure bladder oxidative damage and to conduct a Western blot analysis. 2.5. Bladder gross examination The gross examination included bladder edema and hemorrhage. The changes in bladder edema and hemorrhage were evaluated by Gray’s criteria (Gray et al., 1986). Edema was considered severe (score 3) when fluid was seen externally and internally in the bladder walls, moderate (score 2) – fluid confined to the internal mucosa, mild (score 1) – between normal and moderate, and none (score 0) – normal. Hemorrhage was scored as follows: severe (score 3) – intravesical clots, moderate (score 2) – mucosal hematoma, mild (score 1) – telangiectasis or dilatation of bladder vessels, and none (score 0) – normal. 2.6. Histopathological examination The bladders were routinely processed, embedded in paraffin, and sectioned at 4-lm thickness, deparaffinized, and rehydrated using standard techniques. The sections were stained with hematoxylin-eosin stain for microscopic examination. All sections were examined with a light microscope by a pathologist blinded to the sample treatments. A pathologist, blinded to the study groups, rated for mean histological damage, including edema, hemorrhage, and inflammation on a scale of 0 (normal) to 4 (severe changes). Mucosal ulceration was scored as 0 (normal), 1 (epithelial denuding), 2 (focal ulceration), 3 (widespread epithelial ulceration), and 4 (submucosal ulceration). 2.7. Immunohistochemistry (IHC) The paraffin embedded sections were deparaffinized and rehydrated. After an incubation with a protein block (Mouse and Rabbit Specific HRP/DAB Detection IHC Kit; Abcam, Cambridge, MA, USA), the sections were incubated overnight with anti-caspase-3 rabbit polyclonal antibody (1:200; #9664S; Cell Signaling Technology, Beverly, MA, USA) and anti-acrolein mouse monoclonal antibody (1:5000; ab48501; Abcam) at 4 °C. Caspase-3 and acrolein expression was visualized using the Mouse and Rabbit Specific HRP/DAB Detection IHC Kit (Abcam) according to the manufacturer’s protocol. The sections were counterstained with Harris’s hematoxylin before being mounted. The number of caspase-3 and acrolein positive cells was counted in 10 different fields in each section under 200 magnification. 2.8. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay The level of DNA fragmentation was detected by the TUNEL assay, according to the manufacturer’s instructions (ApopTagÒ In Situ Apoptosis Detection Kit; Chemicon, Billerica, MA, USA). Apoptotic cells were identified by their brownish staining. The number of TUNEL-positive cells was counted in 10 different fields in each section under 200 magnification. 2.9. Determination of lipid peroxidation (LPO), glutathione (GSH), and antioxidant enzymes The weighed frozen bladders were homogenized in a glass-Teflon homogenizer with 50 mM phosphate buffer (pH 7.4) to obtain 1:9 (w/v) whole homogenate. The homogenates were then centrifuged at 11,000g for 15 min at 4 °C to discard any cell debris, and the supernatant was used for the measurement of malondialdehyde (MDA), reduced GSH concentrations and activities of glutathione reductase (GR), catalase (CAT), and GST. The MDA concentration was assayed by monitoring thiobarbituric acid reactive substance formation by the method of Berton et al. (1998). GSH content was measured by the method of Moron et al. (1979). The activities of antioxidant enzymes including CAT (Aebi, 1984), GST (Habig et al., 1984), and GR (Carlberg and Mannervik, 1986) were also determined according to previously documented procedures. Total protein concentrations were determined using Bradford’s method (1976), using bovine serum albumin as the standard.

2.3. Experimental groups and dose selection 2.10. Preparation of hepatic microsomes A total of 24 healthy male rats were randomly assigned to four experimental groups as follows: (1) vehicle control, (2) CP, (3) DADS&CP, and (4) DADS (n = 6 per group). The CP dose was selected according to previous studies that demonstrated to induce acute hemorrhagic cystitis in rats (Ozcan et al., 2005). The effective DADS dose was based on earlier studies (Guyonnet et al., 1999; Wu et al., 2002).

The liver microsomes were prepared as described previously (Jeong and Yun, 1995). The frozen liver samples were cut into small pieces and homogenized in 2–5 volumes of 100 mM Tris–HCl (pH 7.4, 4 °C) containing 2 mM phenylmethyl sulfonyl fluoride and pepstatin (12.5 lg/ml), protease inhibitors, using a glass-Teflon

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homogenizer. The homogenate was centrifuged at 10,000g for 30 min (4 °C), and the supernatant was centrifuged at 200,000g for 60 min (4 °C) to sediment the microsomes. The microsomal pellets were suspended in 100 mM Tris–HCl and used for Western blot analysis of CYP2B1/2, CYP2C11, and CYP3A1.

2.11. Western blot analysis The frozen bladder tissues were lysed with a RIPA lysis buffer (Cell Signaling Technology, Lexington, KY, USA), and were centrifuged at 12,000g at 4 °C for 10 min to obtain the cellular proteins in the supernatant. The bladder tissues supernatant and the liver microsomal proteins (20 lg) were separated by SDS–PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), and blocked in blocking buffer (150 mM NaCl in 10 mM Tris, pH 7.5 containing 5% non-fat dry milk) for 1 h at room temperature. The membranes were incubated with primary antibodies for 18 h at 4 °C, washed three times (20 mM Tris–HCl, pH 7.5, 137 mM NaCl, and 0.1% Tween 20), incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, Thermo Scientific, Rockford, IL, USA) for 1 h at room temperature, washed three times, and then detected with enhanced chemiluminescence method (Supersignal West Pico, Pierce, IL, USA). Antibodies against b-actin (1:1000; ab8227; rabbit polyclonal), Nrf-2 (1:1000; ab137550; rabbit polyclonal), HO-1 (1:2000; ab13243; rabbit polyclonal), NQO-1 (1:1000; ab34173; rabbit polyclonal), CYP2B1/2 (1:1000 ab22719; mouse monoclonal), CYP2C11 (1:1000; ab3571; rabbit polyclonal), and CYP3A1 (1:1000; ab22733; rabbit polyclonal) were purchased from Abcam. The protein concentration was determined by BCA Protein Assay Kit (Pierce). The size was determined by using protein molecular weight marker (Thermo Scientific).

3.2. Effects of DADS on bladder gross pathology The results of the gross evaluation of the urinary bladder are presented in Table 1. Edema and hemorrhage in the CP group were significantly increased in comparison with those in the control group. In contrast, edema and hemorrhage in the DADS&CP group were significantly lower than those in the CP group. 3.3. Effects of DADS on bladder histopathology The results of the histopathological examination are shown in Fig. 2. The control and DADS groups presented bladders with normal architecture (Fig. 2A, B, G, and H). In the CP group, however, bladder tissues showed extensive histopathological changes, characterized by severe epithelial ulceration, hemorrhage, submucosal edema, desquamated granular uroepithelium cells, and inflammatory cells infiltration (Fig. 2C and D). All parameters in the CP group increased significantly compared with those in the control group. Although these findings were also observed in the DADS&CP group (Fig. 2E and F), the incidence and severity of the histopathological lesions were significantly decreased compared with those in the CP group (Fig. 2I–L). 3.4. Effects of DADS on oxidative damage

2.12. Statistical analyses The results are expressed as mean ± SD, and all statistical comparisons were made by means of one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Differences with a P-value of 0.05 or lower were considered to be statistically significant.

3. Results 3.1. Effects of DADS on bladder weight Fig. 1 shows the effects of DADS on CP-induced changes in urinary bladder weight. The CP treatment caused a significant increase in the relative weight of the urinary bladder when compared with that in the control group. Although no significant differences were observed between the groups, the relative weight of the urinary bladder in the DADS&CP group decreased compared to that in the CP group.

The MDA and GSH concentrations and the antioxidant enzymes activities in bladder tissues are presented in Table 2. The concentration of MDA, an end product of LPO, was significantly increased and GSH content and GR, GST, and CAT activities were significantly decreased in the CP group when compared with those in the control group. In contrast, the concentration of MDA in the DADS&CP group was significantly decreased compared with that in the CP group, whereas the GSH content and GR and GST activities were significantly increased. The CAT activity in the DADS&CP group was also slightly higher than that in the CP group but the difference was not significant between the CP and DADS&CP groups. 3.5. Effects of DADS on apoptosis IHC for active caspase-3 (Fig. 3A) and TUNEL (Fig. 3B) were used to identify urinary bladder apoptosis in rats. The control and DADS groups showed little urinary bladder apoptosis. However, rats treated with CP manifested many apoptotic cells, which were mainly distributed in urinary bladder epithelial cells. In contrast, apoptotic cells in the DADS&CP group were found only occasionally. Accordingly, the number of TUNEL-positive cells in the CP group was significantly higher than that in the control group. Although the number of TUNEL-positive cells in the DADS&CP group also increased when compared with that in the control group, the DADS treatment resulted in a decreased the number of TUNEL-positive cells when compared with that in the CP group. Similar to the TUNEL results, caspase-3-positive cells were seldom seen in the control

Table 1 Comparison of the scores on gross evaluation of urinary bladder for edema and hemorrhage of male rats treated CP and/or DADS. Items

No. of rats Edema Hemorrhage Fig. 1. Effects of DADS on CP-induced changes in the relative weight of the urinary bladder. Statistical analysis was performed using one-way ANOVA followed by the Tukey’s multiple comparison test. Data are expressed as means ± SD. P < 0.01 compared with the control group.

a

Group Control

CP

DADS&CP

DADS

6 0a 0

6 2.5 ± 0.84** 2.3 ± 0.82**

6 0.7 ± 0.52   1.3 ± 0.82**, 

6 0 0

Values are presented as means ± SD. Significant difference at P < 0.01 level compared with the control group.   Significant difference at P < 0.05 level compared with the CP group.    Significant difference at P < 0.01 level compared with the CP group. **

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Fig. 2. Representative photographs of bladder sections treated CP and/or DADS. Bladder from vehicle control (A and B) and DADS treated (G and H) rats showing normal appearance. However, bladder from a CP treated rat (C and D) showing severe hemorrhage (black arrow), submucosal edema (white arrow), inflammatory cells infiltration (black arrow head), epithelial ulceration (white arrow), and desquamated granular uroepithelium cells (black asterisk). Bladder from a DADS&CP treated rat (E and F) showing mild focal ulceration, hemorrhage, edema, and leucocytic infiltration. H&E stain. Bar = 20 lm. Comparison of the scores on histological damage of urinary bladder of male rats treated CP and/or DADS (I–L). Results are presented as means ± SD (n = 6). P < 0.01 compared with the control group;  P < 0.05 compared with the CP group;   P < 0.01 compared with the CP group.

Table 2 Antioxidant enzymes, glutathione, and lipid peroxidation in urinary bladder of male rats treated CP and/or DADS. Items

No. of rats Glutathione reductase (units/mg protein) Glutathione-S-transferase (units/mg protein) Catalase (units/mg protein) Glutathione (nmol/mg protein) Malondialdehyde (lmol/mg protein) a

Group Control

CP

DADS&CP

DADS

6 1.013 ± 0.29a 24.3 ± 4.21 10.2 ± 1.51 762 ± 29.2 1050 ± 209.9

6 0.521 ± 0.12** 12.5 ± 6.82** 6.8 ± 1.92* 652 ± 48.0* 1374 ± 131.5**

6 1.212 ± 0.08   20.8 ± 3.94  7.4 ± 0.88 922.3 ± 116.6   1102 ± 150.8 

6 1.067 ± 0.07 26.1 ± 4.18 9.85 ± 3.22 858.1 ± 23.82 989 ± 117.9

Values are presented as means ± SD. Significant difference at P < 0.05 level compared with the control group. ** Significant difference at P < 0.01 level compared with the control group.   Significant difference at P < 0.05 level compared with the CP group.    Significant difference at P < 0.01 level compared with the CP group. *

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Fig. 3. Representative photographs of TUNEL assay (B) and immunohistochemical analysis of caspase-3 (A) and acrolein–protein adducts (C) in bladder sections treated CP and/or DADS. Arrows indicate TUNEL-positive cells (brownish-stained cells). Bar = 20 lm. The number of TUNEL (B), caspase-3 (A), and acrolein positive cells (C) was counted in ten different fields in each section under 200 magnification. Results are presented as means ± SD (n = 6). P < 0.05 compared with the control group; P < 0.01 compared with the control group;   P < 0.01 compared with the CP group.

and DADS groups. In contrast, caspase-3-positive cells increased significantly in the CP group, whereas this effect was significantly attenuated in rats treated with DADS. On the other hand, although apoptosis was observed in the detrusor muscle, there were no obvious differences among the groups (data not shown).

3.6. Effects of DADS on acrolein–protein adducts accumulation To assess a possible contribution of the metabolite acrolein in the urinary bladder, IHC for acrolein–protein adducts was conducted. Acrolein–protein adducts were seldom seen in the control

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and DADS groups (Fig. 3C). The number of acrolein-positive cells was significantly increased in the CP group compared with that in the control group. In contrast, the number of acrolein-positive cells was attenuated in the DADS&CP group compared with that in the CP group. On the other hand, although acrolein–protein adducts were observed in the detrusor muscle, there were no obvious differences among the groups (data not shown). 3.7. Effects of DADS on Nrf-2, NQO-1, and HO-1 protein expression The protein expression of Nrf-2, NQO-1, and HO-1 is shown in Fig. 4. In the DADS group, the expression levels of Nrf-2 and the associated protective phase II enzymes HO-1 and NQO-1were significantly increased compared with those in the control group. The expression levels of Nrf-2, HO-1, and NQO-1 were also significantly increased in the CP group compared with those in the control group. Interestingly, the expression levels of Nrf-2, NQO-1, and HO-1 in the DADS&CP group were also significantly increased compared with those in the CP group. 3.8. Effects of DADS on hepatic microsomal CYP expression The protein expression of hepatic microsomal CYP is shown in Fig. 5. The expression levels of CYP2B1/2 and CYP3A1 were significantly increased in the DADS group compared with those in the control group, whereas the expression level of CYP2C11, a major CYP in the liver of adult male rats, was significantly decreased. The expression level of CYP2B1/2 was also significantly increased in the CP group compared with that in the control group, whereas the expression level of CYP2C11 was significantly decreased. In contrast, the expression levels of CYP2B1/2 and CYP3A1 were significantly increased in the DADS&CP group compared with those in the CP group, whereas the expression level of CYP2C11 was significantly decreased.

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4. Discussion CP is one of the cytotoxic alkylating agents used most widely to treat various human cancers; however, its clinical utility is often limited by the urotoxicity of this drug (West, 1997). Thus, a strategy aimed at diminishing the urotoxic side effect of CP, while preserving its chemotherapeutic efficacy, is needed. A combination of several agents that scavenge or interfere with ROS production effectively ameliorates the CP-induced urotoxic side effects (Sadir et al., 2007; Tripathi and Jena, 2010; Xu and Malavé, 2001). In particular, the protective effect of S-allylcysteine (aged garlic extract) and of garlic extract on CP toxicity was associated with the prevention of an increase in LPO and the preservation of antioxidant enzyme activities (Bhatia et al., 2008; Unnikrishnan et al., 1990). The present study was undertaken to investigate the potential uroprotective effect of DADS, which is a garlic-derived compound with antioxidant properties, and to determine the molecular mechanisms underlying the protection offered by DADS in rats. Based on previous studies of the actions of DADS and related garlic components, several mechanisms may have contributed to the uroprotective effects of DADS observed in our study. It is well-known that LPO is one of the principal causes of CP-induced toxicity and is mediated by the production of acrolein, a metabolite that is responsible for much of its toxicity (Ilbey et al., 2009; Motawi et al., 2010). Acrolein interferes with the tissue antioxidant defense system, produces highly reactive oxygen free radicals, and interacts with protein amino acids, thus causing structural and functional changes in enzymes (Haenen et al., 1988). Recent investigations have confirmed that acrolein binds covalently with proteins in vivo to form acrolein–protein adducts, and that these are considered to be putative markers of oxidative stress (Uchida et al., 1998). As expected, CP treatment resulted in an increase in MDA concentration and a decrease in GSH levels and in GR, GST, and CAT activities. However, DADS prevented the CP-induced depletion of GSH, suppression of GR, GST, and CAT activities, and increase in

Fig. 4. (A) Western blot analysis of Nrf-2, HO-1, and NQO-1 expression in the urinary bladder of male rats treated with CP and/or DADS. Detection of b-actin expression was used as a loading control. The bar graphs show quantitative relative level of Nrf-2 (B), HO-1 (C), and NQO-1 (D) protein expressions for vehicle, CP, DADS&CP, and DADStreated rats. Values are presented as means ± SD (n = 3). P < 0.01 compared with the control group;  P < 0.05 compared with the CP group;   P < 0.01 compared with the CP group.

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Fig. 5. (A) Western blot analysis of hepatic microsomal CYP2B1/2, CYP2C11, and CYP3A1 expressions in male rats treated with CP and/or DADS. Detection of b-actin expression was used a loading control. The bar graphs show quantitative relative levels of CYP2B1/2 (B), CYP2C11 (C), and CYP3A1 (D) protein expressions for vehicle, CP, DADS&CP, and DADS-treated rats. Values are presented as means ± SD (n = 3). P < 0.05 compared with the control group; **P < 0.01 compared with the control group;   P < 0.05 compared with the CP group;   P < 0.01 compared with the CP group.

MDA concentration in bladder tissues. These changes were accompanied by a reduction in the accumulation of protein–acrolein adducts in the bladder. These results indicate clearly that pretreatment with DADS protected effectively against LPO and oxidative damage induced by acrolein. Previous studies have also demonstrated that DADS treatment results in increased GSH content, induction of quinone oxidoreductase, and modulation of enzymic and non-enzymic antioxidant activities such as scavenging of superoxide ions (Wu et al., 2001; Yin et al., 2002). Among the known antioxidant pathways, the Nrf-2-antioxidant response element (ARE) pathway has been characterized as an important endogenous mechanism. Nrf-2 is a redox-sensitive transcription factor that is a pleiotropic regulator of cell-survival mechanisms (Owuor and Kong, 2002). Under basal conditions, Nrf-2 is sequestered in the cytoplasm by the cytosolic regulatory protein Keap1. However, it is translocated from the cytoplasm to the nucleus under conditions of oxidative damage, and induces the expression and upregulation of downstream cytoprotective enzymes, including HO-1 and NQO-1, which, in turn, attenuate tissue injury (Jain et al., 2005; Ma, 2013). The induction of the protein expression of Nrf-2 and the associated phase II enzymes HO-1 and NQO-1 observed in the CP group was consistent with the results of previous studies (Matsuoka et al., 2007; Tripathi and Jena, 2010), which showed that urotoxicity itself induces Nrf-2 and the associated phase II enzymes expression as a cytoprotective mechanism. A previous study also demonstrated that acrolein increases the level of Nrf-2 and the subsequent expression of the phase-II enzymes HO-1 and NQO-1 (Tirumalai et al., 2002). In the present study, DADS treatment led to a significant increase in the protein expression of Nrf-2 and the associated phase II enzymes HO-1 and NQO1. This inductive effect appeared to be mediated by the binding of the Nrf-2 nuclear protein to the resident ARE enhancer sequence present in the promoters of the NQO-1 and HO-1 genes. Chen et al. (2004) reported that treatment of human hepatoma HepG2 cells with DADS resulted in the activation of Nrf-2 leading to the induction of HO-1 and NQO-1. Tripathi and Jena (2010) reported that melatonin exerts its antioxidant defense activity against CPinduced bladder injury in rats by inducing the Nrf-2-ARE pathway

and phase II enzymes, such as NQO-1 and HO-1. Therefore, our results suggest that the DADS-mediated improvement in acute urotoxicity might be mediated, at least in part, by its ability to induce the Nrf-2-ARE pathway in the urinary bladder. These results are in accordance with the decreased oxidative damage observed in the urinary bladder. To the best of our knowledge, the present study was the first to demonstrate the in vivo induction of the Nrf-2-ARE pathway by DADS. Apoptosis (programmed cell death) is a process of normal cell death that maintains tissue homeostasis and is triggered by various physiological and pathological stimuli, including oxidative damage (Simon et al., 2000). This supports the argument that ROS and resulting oxidative stress play a pivotal role in apoptosis (Kannan and Jain, 2000). Excessive apoptosis or its dysregulation can lead to various pathological processes, such as nephrotoxicity and urotoxicity (Sinanoglu et al., 2012). It was reported previously that CP can cause DNA damage and induce apoptosis in the bladder tissue of rats (Tripathi and Jena, 2010). In the present study, CP treatment caused apoptosis in urinary bladder epithelial cells, as shown by the significant elevation in the number of TUNEL positive cells and caspase-3 positive cells. However, DADS pretreatment decreased significantly the number of apoptosis-positive cells induced by CP treatment. The apoptotic results obtained in rats treated with DADS were well correlated with decreased urotoxicity and reduced oxidative stress. Koh et al. (2005) demonstrated the role of DADS as an inhibitor of apoptosis in PC12 cells. These findings indicate that the protective effects of DADS may reflect its role as an antioxidant and antiapoptotic agent. Another contributing mechanism to the DADS-mediated reduction of oxidative stress and acute bladder injury observed in our study may be its ability to inhibit CYP2C11 and induce CYP3A1. The conversion of CP into its active metabolites acrolein and phosphoramide mustard is catalyzed mainly by the CYP2B and 2C subfamily enzymes (Chen et al., 1996; Marinello et al., 1984; Yu and Waxman, 1996), whereas CYP3A metabolizes CP to dechloroethyl-CP and chloroacetaldehyde (Yu and Waxman, 1996) and, thus, seems to play a key role in CP detoxification. In this study, we observed that the potent downregulation of CYP2C11 produced by

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Fig. 6. Proposed pathway depicting mechanism of DADS in preventing CP-induced oxidative stress, apoptosis, and urotoxicity in male rats.

DADS was related to a strong inhibition of acrolein urotoxicity. Although pretreatment with DADS also significantly increased the expression of CYP2B1/2, CYP2C11 is the predominant isoform in the male rat liver, constituting up to 50% of the total CYP content (Morgan et al., 1985). Furthermore, we demonstrated that DADS increased the expression of CYP3A1, which is the main CYP3A form in the rat liver and participates in CP detoxification. According to a previous study, DADS lowers the mutagenicity of aflatoxin B1 (AFB1) by inducing phase I enzymes, including CYP2B1/2 and CYP3A1/2, as well as phase II enzymes, including GST A5 and AFB1 aldehyde reductase 1 (Guyonnet et al., 2002). The results of that study and of our investigation suggest that the inhibition of CYP2C11 and the induction of CYP3A1 by DADS contribute to its protective effects against CP-induced acute urotoxicity. These results are in accordance with the decreased accumulation of acrolein–protein adducts observed in the urinary bladder. In conclusion, DADS had protective effects against CP-induced acute urotoxicity, apoptosis, and oxidative damage in rats. Moreover, the protective effects of DADS may be due to a reduction in oxidative stress via, at least in part, the upregulation of the Nrf2-ARE pathway and reduction of the accumulation of protein–acrolein adducts (Fig. 6). This study also demonstrated that the protection against CP-induced acute urotoxicity conferred by DADS was related to the modulation of enzymes involved in CP metabolism (Fig. 6). These findings suggest that DADS may be a useful protective agent against various adverse effects induced by chemotherapeutic agents with mechanisms of action similar to those of CP, although our results, which were obtained in rats, may be difficult to translate into a human context. Finally, DADS may be effective in preventing chronic CP urotoxicity.

Conflict of Interest The authors declare that there are no conflicts of interest.

Acknowledgments This work was supported by a grant from the KRIBB Research Initiative Program. The animal experiment in this study was sup-

ported by the Animal Medical Institute of Chonnam National University.

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