Elsevier

Food and Chemical Toxicology

Volume 46, Issue 11, November 2008, Pages 3368-3374
Food and Chemical Toxicology

Protective effect of S-allylcysteine against cyclophosphamide-induced bladder hemorrhagic cystitis in mice

https://doi.org/10.1016/j.fct.2008.08.011Get rights and content

Abstract

S-Allylcysteine (SAC), an organosulfur compound of aged garlic extract (AGE) regulates the thiol status of the cell and scavenges free radicals. Depletion of thiols along with free radical generation has been implicated in cyclophosphamide (CP)-induced urotoxicity. We studied modulatory effect of SAC on CP-induced urotoxicity in mice focusing on hemorrhagic cystitis (HC). SAC (150 and 300 mg kg−1) was administered in CP treated animals (200 mg kg−1) and bladder was observed for histological and biochemical changes. CP treatment caused a marked increase in the lumen exudates, edema, vasodilation and HC in lamina propia in the bladder. These changes were accompanied by increase in lipid peroxidation (LPO), and decrease in reduced glutathione (GSH) and activities of antioxidant enzymes. SAC not only showed protection in tissue histology but also improved the decreased activities of antioxidant enzymes. SAC treatment also reduced LPO and increased GSH levels. Although SAC treatment did not ensure full recovery, the marked improvement in histology and antioxidants of bladder suggests that it has a significant modulatory effect on CP-induced urotoxicity. Since decrease in antioxidant level is the major cause of CP urotoxicity, the protective effect of SAC deserves its further exploration involving laboratory and clinical investigations.

Introduction

Garlic (Allium sativum L.) is a known source of herbal remedies (Imai et al., 1994). Fresh garlic extract contains lipid soluble volatile compound allicin, which is an unstable and transient compound with oxidant activity (Freeman and Kodera, 1995). However, aged garlic extract (AGE) has antioxidant properties and contains more stable and bioavailable water-soluble organosulfur compounds (Kodera et al., 2002). The AGE is reported for scavenging the reactive oxygen species (ROS), enhancing the cellular antioxidant enzymes and increasing the reduced glutathione (GSH) in the cells (Numagami and Ohnishi, 2001). Some major identified organosulfur compounds of aged garlic extract are S-allylcysteine (SAC), S-allylmercaptocysteine (SAMC), allicin, alliin and agoene (Ayaz and Alpsoy, 2007). Compared to other compounds, SAC and SAMC are in high quantities in the AGE because they are produced during the process of aging (Imai et al., 1994).

SAC has been reported for antioxidant, anticancer, antihepatotoxic and neurotrophic activities (Nakagawa et al., 1989, Welch et al., 1992, Moriguchi et al., 1997, Numagami and Ohnishi, 2001). SAC content in the fresh intact garlic is low (<30 μg/g-fresh weight). However, it is produced in a soaking preparation through hydrolysis of γ-glutamyl-S-allylcysteine, which exists in raw garlic as a precursor to SAC (Kodera et al., 2002). Yeh and Lijuan (2001) reported on cholesterol lowering effects of SAC. The lipid lowering effect of SAC on isoproterenol-induced cardiotoxicity in rats has been reported by Padmanabhan and Prince (2006). SAC also showed in vivo and in vitro anti-genotoxic activities (Velmurugan et al., 2004, Belloir et al., 2006) and it protected vascular endothelial cells from hydrogen peroxide-induced injury (Yamasaki et al., 1993) and hepatocytes from carbon tetrachloride toxicity (Nakagawa et al., 1989). It has also shown ameliorative effect on doxorubicin-induced toxicity in the heart and liver of mice (Mostafa et al., 2000). Antioxidant property of SAC is one of the main mechanisms by which exerts its protective effects. Therefore, it may be a good candidate for preventing or mitigating the toxicity of cytotoxic anticancer drugs such as cyclophosphamide (CP).

CP is one of the most effective antineoplastic drugs in the treatment of solid tumors as well as B cell malignant diseases (Baumann and Preiss, 2001). However, its use also entails toxic manifestations such as immunotoxicity, teratogenicity, genotoxicity and urotoxicity (Bin-Hafeez et al., 2001, Sharma et al., 2001, Bailey et al., 2005, Franke et al., 2005). The deleterious effects of CP in the urinary bladder include mucosal edema, hemorrhage, ulceration, subendothelial telangiectasia and fibrosis (Levine and Richie, 1989). In most of the toxic manifestations of CP including urotoxicity, reactive oxygen species (ROS) have been implicated to play a major role (Korkmaz et al., 2007). In the absence of adequate uroprotection, the incidence of urotoxic side effect of CP varies from 2 to 40% in patients taking low doses of drug on a long-term basis. Mortality with massive bladder hemorrhage in up to 2–4% patients is reported in under high dose intravenous CP treatment regimen (Topal et al., 2005, Korkmaz et al., 2007).

In CP-induced urotoxicity hemorrhagic cystitis (HC) poses a major risk in cancer patients (Korkmaz et al., 2007). The incidence of HC is related to the CP dose and can be as high as 75% (Coggins et al., 1960, Levine and Richie, 1989). CP-induced urotoxicity in which its reactive metabolite acrolein plays a major role can be counteracted by the endogenous or exogenous GSH (Roberts et al., 1994). However, detoxification of acrolein does not completely prevent HC as free radicals such as NO are also involved in the damage to the bladder (Souza-Filho et al., 1997, Korkmaz et al., 2007). Besides GSH, some thiol compounds such as N-acetyl cysteine, L-cysteine, diallyl sulphide, mesna and a number of herbal extracts with antioxidant potential have shown to reduce CP toxicity including urotoxicity (Cavalletti et al., 1986, Roberts et al., 1994, Bhatia et al., 2006a, Bhatia et al., 2006b). Recently, bilirubin has shown protective effect against CP-induced HC mainly by downregulating the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in rats (Matsuoka et al., 2008). Previously, we showed that herbal extracts with antioxidant constituents would elevate the GSH levels in bladder and reduce the CP urotoxicity in animals (Bhatia et al., 2006a, Bhatia et al., 2006b). One such herbal extract, walnut (Juglans regia L.) extract contains a number of antioxidants such as polyphenols, ellagitannins and flavanoids (Haque et al., 2003, Bhatia et al., 2006b). The present study was designed to know the protective effect of SAC, a compound that is of natural origin found in foods, on CP-induced urotoxicity. We report here on the protective effect of SAC (a product of aged garlic) treatment against CP-induced hemorrhagic cystitis in mouse model.

Section snippets

Chemicals

Bovine serum albumin (BSA), butylated hydroxy toluene (BHT), ethylenediamine tetra acetic acid (EDTA) disodium salt, Folin’s reagent, nitroblue tetrazolium (NBT) and sulfosalicylic acid were procured from SRL (Mumbai, India). CP, dithio-bis-2-nitrobenzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), GSH and nicotinamide adenine dinucleotide phosphate reduced (NADPH) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Ortho phosphoric acid (OPA) was procured from CDH Chemicals

Mortality and other toxicity signs and symptoms

No treatment related mortality was observed in any of the groups of animals and there were no toxicity signs and symptoms, which could be attributed to treatments (data not shown).

Histological Investigations

Urinary bladder from control animals (Group I) showed keratinized squamous epithelial lining of the urinary bladder and narrow lamina propria and normal bladder wall structure with epithelial lining in folds (Fig. 1A and B, Table 2). In CP (200 mg kg−1) treated animals (Group II), widened lamina propria with severe

Discussion

The urotoxicity of CP is due to the cumulative effect of the two mechanisms: firstly the decrease in the levels of the predominant nucleophiles in the cells such as GSH on interaction with acrolein and secondly the formation of peroxynitrite by coupling of O2- and NO (Kehrer and Biswal, 2000, Korkmaz et al., 2007). Since bladder is the site for storage of urine and acrolein is excreted via urine, the overall exposure to bladder is increased resulting in decrease in GSH in the cells as acrolein

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

Kanchan Bhatia is a recipient of a Senior Research Fellowship (SRF) from the Council of Scientific and Industrial Research (CSIR), Government of India. We thank Dr. Abel Santamaria for providing us SAC used in this work.

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