Cytoprotective and antioxidant role of diallyl tetrasulfide on cadmium induced renal injury: An in vivo and in vitro study

Life Sciences 80 (2007) 650 – 658 www.elsevier.com/locate/lifescie

Cytoprotective and antioxidant role of diallyl tetrasulfide on cadmium induced renal injury: An in vivo and in vitro study L. Pari a,⁎, P. Murugavel a , S.L. Sitasawad b , K. Sandeep Kumar b a

Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar — 608002, Tamilnadu, India b National Centre for Cell Science, NCCS Complex, Ganeshkhind Road, Pune — 411007, India Received 19 February 2006; accepted 18 October 2006

Abstract Cadmium (Cd) is an environmental and industrial pollutant that affects various organs in humans and animals. A body of evidence has accumulated implicating the free radical generation with subsequent oxidative stress in the biochemical and molecular mechanisms of Cd toxicity. Since kidney is the critical target of Cd toxicity, we carried out this study to investigate the effects of diallyl tetrasulfide (DTS), an organosulfur compound derived from garlic on Cd induced toxicity in the kidney of rats and also in the kidney cell line (vero cells). In experimental rats, subcutaneous administration of Cd (3 mg/kg bw/day) for 3 weeks induced renal damage, which was evident from significantly increased levels of serum urea and creatinine with significant decrease in creatinine clearance. A markedly increased levels of lipid peroxidation markers (thiobarbituric acid reactive substances and lipid hydroperoxides) and protein carbonyl contents with significant decrease in nonenzymic antioxidants (total sulphydryl groups, reduced glutathione, vitamin C and vitamin E) and enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase) as well as glutathione metabolizing enzymes (glutathione reductase, and glucose-6phosphate dehydrogenase) were also observed in Cd intoxicated rats. Coadministration of DTS (40 mg/kg bw/day) and Cd resulted in the reversal of the kidney function accompanied by a significant decrease in lipid peroxidation and increase in the antioxidant defense system. In vitro studies with vero cells showed that incubation of DTS (5–50 μg/ml) with Cd (10 μM) significantly reduced the cell death induced by Cd. DTS at 40 μg/ ml effectively blocked the cell death and lipid peroxidation induced by Cd (10 μM) indicating its cytoprotective property. Further, the flow cytometric assessment on the level of intracellular reactive oxygen species using a fluorescent probe 2V, 7V-dichlorofluorescein diacetate (DCF-DA) confirmed the Cd induced intracellular oxidative stress in vero cells, which was significantly suppressed by DTS (40 μg/ml). The histopathological studies in the kidney of rats also showed that DTS (40 mg/kg bw/day) markedly reduced the toxicity of Cd and preserved the architecture of renal tissue. The present study suggests that the cytoprotective potential of DTS in Cd toxicity might be due to its antioxidant and metal chelating properties, which could be useful for achieving optimum effects in Cd induced renal damage. © 2006 Elsevier Inc. All rights reserved. Keywords: Cadmium; Diallyl tetrasulfide; Kidney; Oxidative stress; Vero cells; Rats

Introduction Cadmium (Cd) is an inorganic toxicant of great environmental and occupational concern, which was classified as a group I human carcinogen (Waalkes, 2000). Sources of Cd are discharges mainly from industries such as electroplating, plastic production, pigments, battery manufactures, pesticides, etc. Products of vegetable origins are the main carriers of Cd compounds in food (Lyn Patrick, 2003; Noel et al., 2004). Cd ⁎ Corresponding author. Tel.: +91 04144 238343; fax: +91 04144 238145. E-mail address: [email protected] (L. Pari). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.10.013

largely accumulates in the liver and the kidney, which makes up the bulk of total body burden (Klaasen et al., 1999). The kidney is the critical target organ for Cd and is documented by a number of studies in humans and animals. Cd can produce a variety of renal effects involving the proximal tubules and the glomerulus, which is believed to be irreversible at advanced stages (WHO, 1992; Ahn et al., 1999). The kidney stones and glomerular damage have been seen in those with occupational exposure to Cd (Hu, 2000). Several lines of evidence indicate that oxidative stress and reactive oxygen species (ROS) formed in the presence of Cd could be responsible for its toxic effects in many organs or cells (Wang et al., 2004; Watjen

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and Beyermann, 2004). Cd-induced cytotoxicity is known to be intimately associated with the induction of oxidative stress (Hart et al., 1999; Pathak and Khandelwal, 2006). These evidences indicate that apoptosis probably plays an important role in acute and chronic intoxication with Cd (Li et al., 2000). Although several chelating agents and antagonists are established to reduce the Cd toxicity, some of them are burdened with undesirable side effects. Due to the intrinsic limitations and variability of efficacy of heavy metal chelating agents, Cd intoxication therapy is looking for the development of new therapeutic agents with different mode of actions especially from the medicinal plants. Garlic is a widely consumed spice in foodstuffs and medicines. Despite the extensive medicinal use of garlic and its organosulfur compounds (OSCs), limited knowledge is available regarding their role in heavy metal toxicity. Research has shown that concomitant use of garlic extract had considerably reduced the metal accumulation in the tissues indicating the potential therapeutic value of garlic against various pollutants and heavy metals (Cha, 1987; Lee et al., 1999; Senapati et al., 2001). The garlic and related OSCs are known to exert antioxidant, antimutagenic, detoxifying and other properties (Wang et al., 1996; Guyonnet et al., 2001). Diallyl tetrasulfide (DTS) is one of the major sulfide break down products from garlic. The sulfoxides in garlic are degraded enzymatically to thiosulfonates, which on decomposition predominantly forms tetrasulfide with mono, di and trisulfides (Block, 1992). Metabolic studies indicate that such sulfides are also formed through in vivo metabolism of thiosulfonates after ingestion of garlic (Egen-Schwind et al., 1992; Saurez et al., 1999). The antioxidant activity of DTS was more potent than crude aged garlic extract and other diallyl sulfides (mono, di and trisulfides) (Horie et al., 1992). Vero cells are cell line of renal origin, which has manifested a response to heavy metals similar to that of other renal cells, therefore considered as a model in this study. It has long been established that chelating agents possessing sulphydryl groups are more effective against heavy metal poisoning (Williams and Halstead, 1983). Our previous study has shown that DTS has the ability to ameliorate the Cd induced biochemical changes in rats (Pari and Murugavel, 2005). In this view, the present study was carried out to evaluate the antioxidant and cytoprotective potential of DTS against Cd induced oxidative damage especially in renal tissue.

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creatinine were from Sigma Diagnostics (I) Pvt Ltd (Baroda, India). Cadmium chloride and other fine chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). In vivo studies Animals Male inbred Wistar rats of initial body weight 170–200 g were used in this study. The rats were maintained under standard laboratory conditions (temperature 24 ± 2 °C; natural light– dark cycle). The rats had free access to drinking water and commercial standard pellet diet (Lipton India Ltd, Mumbai, India). The laboratory animal protocol used for this study was approved (Approval No: 157, 2003) by the committee for the purpose of control and supervision on experimental animals (CPCSEA) at Annamalai University, Annamalainagar, India. Experimental design The rats were randomly divided into four groups of six animals in each. Group I:

Control rats daily treated subcutaneously with isotonic saline and intragastrically with corn oil (2 ml/kg bw/day) for 3 weeks Group II: Rats daily received oral administration of DTS (40 mg/kg bw/day) dissolved in corn oil for 3 weeks using intragastric tube Group III: Rats subcutaneously received Cd as cadmium chloride (3 mg/kg bw/day) in isotonic saline daily for 3 weeks Group IV: Rats daily received a subcutaneous injection of Cd (3 mg/kg bw/day) followed by an oral administration of DTS (40 mg/kg bw/day) in corn oil for 3 weeks

At the end of the experimental period, the animals in different groups were sacrificed by cervical decapitation. Blood was collected and centrifuged for the separation of serum. The kidney was dissected out, weighed and washed using chilled saline solution. The tissue was minced and homogenized (10% w/v) in an appropriate buffer (pH 7.4) and centrifuged. The resulting supernatant was used for enzyme assays. Biochemical assays Estimation of urea, creatinine and creatinine clearance The levels of creatinine and urea in serum were estimated spectrophotometrically using commercial diagnostic kits (Sigma Diagnostics (I) Pvt Ltd, Baroda, India). Creatinine clearance as an index of glomerular filtration rate was calculated from serum creatinine and a 24 h urine sample creatinine levels.

Materials and methods Chemicals Diallyl tetrasulfide was a gift sample provided by Oxford Chemicals Ltd, Hartlepool, UK as diallyl polysulfide. The number of sulfur atoms was confirmed by using mass spectral study, which confirmed that the compound is DTS. Dulbecco's modified eagles medium (DMEM) and fetal calf serum (FCS) were obtained from GIBCO, Invitrogen Corporation, Carlsbad, California. The trace pure nitric acid (Merck, Germany) was used for metal analysis. Commercial kits to estimate urea and

Determination of cadmium concentration One gram of liver sample was weighed and transferred to a high-pressure quartz vessel. 5 ml of concentrated nitric acid was added to the sample and was allowed to stand for 1 h. After the initial acid attack, the quartz vessels were capped and heated for 5 min at 1400 W in a microwave oven. Then the digests were continuously preconcentrated and finally diluted with ultrapure water to 25 ml. The Cd level was determined at 228 nm by flame atomic absorption spectrometer (Perkin Elmer 5000 model) furnished with a Cd hollowcathode lamp.

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Determination of lipid peroxidation and protein carbonyl contents Lipid peroxidation in the renal tissue was estimated colorimetrically by measuring thiobarbituric acid reactive substances (TBARS) and hydroperoxides as described by Niehiaus and Samuelsson (1968), Jiang et al. (1992) respectively. As a hallmark of protein oxidation, total protein carbonyl was determined in the kidney by a spectrophotometric method described by Levine et al. (1990) and expressed as nmol/mg protein. Measurement of nonenzymic antioxidants Reduced glutathione (GSH) was determined by the method of Moron et al. (1979) based on the reaction with Ellman's reagent (19.8 mg dithionitrobis benzoic acid in 100 ml of 0.1% sodium citrate). Total sulphydryl groups (TSH) in the kidney homogenate was measured after the reaction with dithionitrobis benzoic acid using the method of Ellman (1959). Ascorbic acid (vitamin C) and vitamin E concentrations were measured by the methods of Omaye et al. (1979), Desai (1984) respectively. Assay of antioxidant and glutathione metabolizing enzymes Superoxide dismutase (SOD) activity was determined by the method of Kakkar et al. (1984) in which the inhibition of formation of NADH–Phenazinemethosulphate–nitroblue tetrazolium formazon was measured spectrophotometrically at 560 nm. Catalase (CAT) activity was assayed colorimetrically as described by Sinha (1972) using dichromate–acetic acid reagent. Glutathione peroxidase (GPx) activity was assayed by the method based on the reaction between glutathione remaining after the action of GPx and 5,5V-dithio bis-(2nitro benzoic acid) to form a complex that absorbs maximally at 412 nm (Rotruck et al., 1973). Glutathione-S-transferase (GST) activity was determined spectrophotometrically by using 1-chloro 2,4-dinitro benzene as the substrate (Habig et al., 1974). Glutathione reductase (GR) that utilizes NADPH to convert oxidized glutathione (GSSG) to the reduced form was assayed by the method of Horn and Burns (1978). The estimation of glucose-6-phosphate dehydrogenase (G6PD) was carried out by the method of Beutler (1983), where an increase in the absorbance was measured when the reaction was started by the addition of glucose-6-phosphate. Protein level was determined by using bovine serum albumin (BSA) as the standard at 660 nm (Lowry et al., 1951). Histopathological studies in the kidney For qualitative analysis of kidney histology, the tissue samples were fixed for 48 h in 10% formalin-saline and dehydrated by passing successfully in different mixtures of ethyl alcohol–water, cleaned in xylene and embedded in paraffin. Sections of the tissue (5–6 μm thick) were prepared by using a rotary microtome and stained with haematoxylin and eosin (H&E) dye, which was mounted in a neutral deparaffinated xylene medium for microscopic observations.

In vitro studies The vero (a normal African green monkey kidney cell line) cells were obtained from the cell bank of National Center for Cell Science (NCCS), Pune, India. The vero cells were maintained in DMEM supplemented with 10% FCS, 100IU penicillin/ml and 100 μg streptomycin/ml and incubated at 37 °C under a humidified 5% CO2 atmosphere. The cells were grown in 25 cm2 tissue culture flasks until confluent and subcultured for experimentation. The Cd was dissolved in DMEM without FCS and DTS was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was not more than 0.2%. Analysis of cell viability The viability of vero cells after treatment with DTS was assayed by the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyl-tetrazoliumbromide (MTT) to formazan as described previously (Plumb et al., 1989). Briefly, cells were seeded in 96well microtiter plates (1 × 104 cells per well), and left to adhere to the plastic plates overnight before being exposed to different concentrations of Cd and DTS. In each experiment, different concentrations of Cd (1, 2, 5, 10, 20 and 40 μM) and DTS (5, 10, 20, 30, 40 and 50 μg/ml) were tested in three separate wells and the cytotoxicity curve was constructed from three different experiments. After exposure to Cd and DTS, 50 μl of 5 mM MTT solution was added to each well, and the cells were incubated in the dark at 37 °C for an additional 4 h. Thereafter, the medium was removed, the formazan crystals were dissolved in 200 μl of DMSO and the absorbance was measured at 570 nm in a microplate reader (Molecular Devices, Spectra MAX 250). The data of the survival curves were expressed as the percentage of untreated controls. Lipid peroxidation in vero cells Lipid peroxidation was measured in terms of malondialdehyde (MDA) by the method of Konings and Drijver (1979). Vero cells were cultured in 25 cm2 tissue culture flasks and grown in DMEM supplemented with 10% FCS and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin) in a humidified atmosphere of 5% CO2 at 37 °C. On attaining 80–90% confluency, the cells were washed with DMEM medium and treated for 1 h at 37 °C with Cd (10 μM) and DTS (10, 20, 30 and 40 μg/ml). The cells were washed with PBS (pH 7.4) and the levels of MDAwere estimated. Vero cells were treated with 1 ml of 0.5 M KCl in 10 mM Tris– HCl buffer, 0.5 ml of 30% TCA and 0.5 ml of 52 mM TBA and heated in a water bath at 90 °C for 30 min. The mixture was then cooled in ice for 2 min and centrifuged at 800×g for 10 min. The absorbance of the supernatant was measured at 532 nm using a dual beam spectrophotometer (U-3210, Hitachi). The levels were expressed as nmol MDA/ml cell supernatant. Detection of intracellular ROS Intracellular reactive oxygen intermediate (ROI) production was monitored by flow cytometry using the fluorescent probe 2V,

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Fig. 2. Concentration of Cd in kidney of diallyl tetrasulfide (DTS) and Cd treated rats. Con, Control. Values are mean ± SD (n = 6). Values not sharing a common letter differ significantly at p b 0.05 (DMRT).

experiments performed in duplicate unless stated otherwise. The results were presented as mean ± SD. One-way ANOVA with a Student–Newman–Keuls post hoc comparison was used for statistical significance. Values of p b 0.001 were considered to be statistically significant. Results Fig. 1. Effect of diallyl tetrasulfide (DTS) on Cd induced changes in renal functional markers (A) serum urea and (B) serum creatinine and creatinine clearance in male albino Wistar rats. Con, Control. Values are mean ± SD (n = 6). Values not sharing a common letter differ significantly at p b 0.05 (DMRT).

7V-dichlorofluorescein diacetate (DCF-DA) (Kuo and Tang, 1998). After diffusing into the cells, the DCF-DA is hydrolyzed to polar nonfluorescent dichloroflurescein (DCFH) and trapped within cells. DCFH is further oxidized by ROI to a fluorescent — dichlorofluorencein (DCF). Briefly, confluent vero cells were harvested by trypsinisation and an aliquot of 3 × 105 cells were co-incubated with DCF-DA (5 μM) in the absence or presence of Cd (10 μM) and DTS (10, 20, 30 and 40 μg/ml) for 1 h at 37 °C in the dark. After treatment, the cells were immediately washed and resuspended in 1× PBS, filtered through 100 mm nylon mesh and analyzed by flow cytometry. The fluorescence emitted at 525 nm was measured with a FACS Calibur flow cytometer (Beckton Dickinson, San Jose, CA) equipped with an argon laser (488 nm emission, 15 mW output) and analyzed using the CELL Quest™ software. Ten thousand cells were examined for each sample with a flow rate of 300–600 cells/s. The fluorescence intensity was expressed as percent fluorescence intensity. Statistical analysis The data from in vivo experiments were expressed as mean ± SD (n = 6). The statistical significance was evaluated by oneway analysis of variance (ANOVA) using SPSS version 9.0 (SPSS, Cary, NC, USA) and the individual comparisons were obtained by Duncan's Multiple Range Test (DMRT). A p value b 0.05 was considered to be statistically significant. The data of in vitro studies represent a minimum of three independent

Effects of DTS on Cd concentration and renal functional markers A significantly ( p b 0.05) increased level of urea and creatinine in serum with significantly ( p b 0.05) decreased level of creatinine clearance was observed in Cd treated rats (Fig. 1). Administration of DTS at 40 mg/kg significantly ( p b 0.05) decreased urea and creatinine levels in serum and restored the level of creatinine clearance. The increased accumulation of Cd and its content observed in the kidney of Table 1 Effect of diallyl tetrasulfide (DTS) and cadmium (CdCl2) on the levels of lipid peroxidation, protein carbonyl contents and nonenzymic antioxidant status in kidney of control and experimental rats (values are mean ± SD for 6 rats in each group) Normal + DTS Normal + CdCl2 CdCl2 (3 mg/ kg) + DTS (3 mg/kg) (40 mg/kg) (40 mg/kg)

Parameters

Control

TBARS (mg/g tissue) Hydroperoxides (mmol/g tissue) Protein carbonyls (nmol/mg protein) Vitamin C (μmol/mg tissue) Vitamin E (μmol/mg tissue) GSH (μg/mg protein) TSH (μg/mg protein)

2.70 ± 0.17a 2.56 ± 0.14a 4.59 ± 0.35b

3.04 ± 0.20c

0.64 ± 0.05a 0.60 ± 0.05a 0.94 ± 0.07b

0.74 ± 0.06c

1.88 ± 0.16a 1.79 ± 0.13a 4.56 ± 0.35b

2.43 ± 0.20c

0.97 ± 0.05a 1.06 ± 0.06a 0.60 ± 0.03b

0.81 ± 0.05c

0.50 ± 0.04a 0.55 ± 0.04a 0.28 ± 0.02b

0.37 ± 0.03c

2.76 ± 0.19a 3.02 ± 0.29b 1.62 ± 0.18c

2.18 ± 0.22d

9.45 ± 0.92a 10.67 ± 0.85b 6.84 ± 0.49c

8.14 ± 0.63d

TBARS, thiobarbituric acid reactive substances; GSH, reduced glutathione; TSH, total sulphydryl groups. a,b,c,dWithin rows, means with different superscript letter differ significantly at p b 0.05 (DMRT).

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Table 2 Effect of diallyl tetrasulfide (DTS) and cadmium (CdCl2) on the activities of antioxidant and glutathione metabolizing enzymes in kidney of control and experimental rats (values are mean ± SD for 6 rats in each group) Parameters Control SOD CAT GPx GST GR G6PD

Normal + DTS Normal + CdCl2 CdCl2 (3 mg/kg) + (40 mg/kg) DTS (40 mg/kg) (3 mg/kg)

11.41 ± 0.85a 12.91 ± 0.79b 47.45 ± 3.65a 50.24 ± 2.34a 5.11 ± 0.33a 5.65 ± 0.45a 5.59 ± 0.31a 6.04 ± 0.39b 0.33 ± 0.03a 0.37 ± 0.03b 1.60 ± 0.09a 1.71 ± 0.11a

6.59 ± 0.47c 30.25 ± 1.62b 2.82 ± 0.16b 3.80 ± 0.21c 0.23 ± 0.02c 1.13 ± 0.06b

8.84 ± 0.67d 39.48 ± 2.88c 4.41 ± 0.28c 4.56 ± 0.27d 0.29 ± 0.01d 1.44 ± 0.08c

SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione-S-transferase; GR, glutathione reductase; G6PD, glucose-6-phosphate dehydrogenase. The units of enzyme activities are expressed as follows: SOD, one unit of activity was taken as the enzyme reaction, which gave 50% inhibition of NBT reduction in one minute/mg protein, CAT, μmol of hydrogen peroxide consumed/min/mg protein, GPx, μg of glutathione consumed/min/mg protein, GST, μmol of CDNB–GSH conjugate formed/min/mg protein, GR, nmol of NADPH oxidized/min/mg protein, G6PD, nmol of NADPH formed/min/mg protein. a,d,c,dWithin rows, means with different superscript letter differ significantly at p b 0.05 (DMRT).

Fig. 4. Effect of Cd and diallyl tetrasulfide (DTS) on cell viability of vero cells. The cells were treated with different concentrations of DTS (5, 10, 20, 30, 40 and 50 μg/ ml) with Cd (10 μM) for 18 h. The columns represent the % viability of vero cells. Data are mean ± SD. Con, Control. ⁎Significantly different from control ( p b 0.001); ♣significantly different from Cd treated cells ( p b 0.001); NS — non-significant, DTS with Cd treated group compared with Cd (10 μM) alone treated group; ⁎⁎no significant difference from control group; #no significant difference from Cd with DTS (40 μg/ml) treated group.

Cd intoxicated rats were significantly ( p b 0.05) reduced upon administration of DTS (40 mg/kg) (Fig. 2).

were observed. In contrast, Cd alone treated rats showed significantly ( p b 0.05) decreased levels of nonenzymic antioxidants (Table 1) and the activities of antioxidant and glutathione metabolizing enzymes (Table 2).

Effects of DTS on Cd induced lipid peroxidation and antioxidant status in the kidney

Cytotoxicity of Cd on vero cells

Exposure of Cd to rats showed a significantly ( p b 0.05) increased levels of TBARS, hydroperoxides and protein carbonyl contents in the kidney (Table 1). Treatment with DTS significantly ( p b 0.05) decreased the levels of lipid peroxidation products and protein carbonyl contents in the kidney of Cd treated rats. In rats administrated with DTS and Cd treated rats, a significant ( p b 0.05) increase in the level of nonenzymic antioxidants (TSH, GSH, vitamin C and vitamin E) and increased activities of enzymic antioxidants (SOD, CAT, GPx and GST) and glutathione metabolizing enzymes (GR and G6PD)

The viability of vero cells on Cd exposure was determined in a time dependent (2 to 24 h) and concentration (1, 2, 5, 10, 20 and 40 μM) dependent manner by MTT assay. The cell damage was concentration dependent and increased progressively throughout the time course (data not shown). The concentration dependent decrease in cell viability was observed in the presence of higher concentrations of Cd (Fig. 3). Based on these findings, the effects of DTS on Cd cytotoxicity were studied in vero cells exposed to 10 μM CdCl2 for 18 h, where 60% suppression of cell viability was observed.

Fig. 3. Effect of Cd on cell viability of vero cells. The cells were treated with different concentrations of Cd (1, 5, 10, 20, 30 and 40 μM) for 18h treatment. The columns represent the % viability of vero cells. Data are mean ± SD. Con, Control. ⁎Significantly different from control ( p b 0.001); NS — nonsignificant, Cd treated group compared with control; #no significant difference from Cd (20 μM) treated group.

Fig. 5. Effect of diallyl tetrasulfide (DTS) on Cd induced lipid peroxidation in vero cells. Vero cells were treated with (10, 20, 30 and 40 μg/ml) of DTS prior to Cd treatment. Lipid peroxidation was estimated and expressed in terms of nmol MDA/ml cell supernatant. Data are means ± SD (n = 5). Con, Control. ⁎Significantly different from control ( p b 0.001); ♣significantly different from Cd treated cells ( p b 0.001); NS — non-significant, DTS with Cd treated group compared with Cd (10 μM) alone treated group; #no significant difference from control group.

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Effect of DTS on Cd induced lipid peroxidation Treatment of vero cells with Cd for 1 h at 37 °C lead to a significantly ( p b 0.001) increased level of lipid peroxidation as compared to that of control (Fig. 5). However, cells concomitantly treated with Cd (10 μM) and different concentrations of DTS (5, 10, 20 and 40 μg/ml) showed a significant ( p b 0.001) decrease in lipid peroxidation as compared to Cd alone treated cells. DTS at 40 μg/ml effectively decreased the Cd induced lipid peroxidation. Fig. 6. Effect of diallyl tetrasulfide (DTS) on Cd induced ROS generation in vero cells. Vero cells were co-incubated with (10, 20, 30 and 40 μg/ml) of DTS with Cd (10 μM) and DCF-DA (5 μM). DCF fluorescence was measured with a flow cytometer. DCF fluorescence was excited at 488 nm. Emitted fluorescence was measured at 525 nm. Data are mean ± SD (n = 3). Con, Control. ⁎Significantly different from control ( p b 0.001); ♣significantly different from Cd treated cells ( p b 0.001).

Effect of DTS on Cd cytotoxicity The effects of various doses of DTS (5 to 50 μg/ml) on Cd cytotoxicity are shown in Fig. 4. The cells incubated with DTS and Cd for 18 h showed that DTS remarkably suppressed the Cd induced decrease in cell viability. DTS at a concentration of 40 μg/ml more effectively ( p b 0.001) decreased the Cd induced cytotoxicity. There was no significant difference in cell viability between cells incubated with DTS (50 μg/ml) for 18 h and control cells, which indicates that DTS has no toxic effect up to 50 μg/ml.

Effect of DTS on Cd-induced intracellular ROS production DCF-DA is a sensitive fluorescent probe, which is helpful to detect ROS generation in cells. To confirm the ability of DTS in the reduction of Cd-induced oxidative stress in cells, the intracellular ROS production was assessed. A rapid increase in the intracellular ROS levels were noted in the cells treated with 10 μM of Cd, as assessed by an increase in DCF fluorescence (17.2%), but the oxidant burden after Cd exposure was decreased in the presence of DTS in a dose-dependent manner (14% to 7%) (Fig. 6). These results demonstrate that DTS exhibit a significant antioxidant activity. Histopathology of the kidney Histopathological studies showed that Cd induced multiple foci of hemorrhage necrosis and cloudy swelling of tubules in the kidney (Fig. 7C). According to microscopic examinations,

Fig. 7. A — Normal rat kidney (H&E 20X). Normal glomeruli and tubules. B — Normal + DTS (40 mg/kg) treated rat kidney (H&E 20X). Normal appearance of glomeruli. C — Normal + Cd (3 mg/kg) treated rat kidney (H&E 20X). Multiple foci of hemorrhage, necrosis and cloudy swelling of tubules. D — Cd (3 mg/kg) + DTS (40 mg/kg) treated rat kidney (H&E 20X). Almost normal appearance of kidney of glomeruli and tubules.

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pathological lesions induced by Cd were remarkably reduced by the administration of DTS (Fig. 7D), which were in agreement with the results of renal functional markers and the kidney lipid peroxidation status. There was no change in the kidney histology of DTS (40 mg/kg) alone treated rats (Fig. 7B) when compared to control (Fig. 7A). Discussion Exposure of cells to Cd evokes a number of cellular responses to protect the cell from the metal-induced cytotoxicity (Beyersmann and Hechtenberg, 1997). Cd interferes with antioxidant defense mechanisms together with the production of ROS, which may act as a signaling molecule in the induction of cell death (Waisberg et al., 2003). Several studies have demonstrated that Cd induced cytotoxicity is associated with apoptotic cell death in various organs and cell systems (Tanimoto et al., 1993; Hamada et al., 1996; Hart et al., 1999; Li et al., 2000; Gennari et al., 2003). Garlic, garlic oil and aged garlic extract are well documented for the attenuation of oxidant mediated renal damage induced by various agents (Iqbal and Athar, 1998; Maldonadoa et al., 2003; Wongmekiat and Thamprasert, 2005; Kabasakal et al., 2005). The diallyl sulfur compounds in garlic have also been reported for its capability to decrease oxidative stress and to preserve the renal tissue from nephrotoxicants (Dwivedi et al., 1996; Pedraza-Chaverri et al., 2003). In our earlier study, we found the ability of DTS (40 mg/ kg) to ameliorate Cd induced changes in biochemical markers, which reflects the organ function (Pari and Murugavel, 2005). In this context, the present study also confirmed that administration of DTS at 40 mg/kg significantly restored the kidney function against the toxic effects of Cd. Further, histopathological studies showed that 40 mg/kg of DTS administration almost reduced the pathological changes such as hemorrhage, necrosis and swelling of tubules caused by Cd. Cd causes superficial irregularities on plasma membrane by binding to anionic sites of phospholipids in the membrane (Sorensen et al., 1984; Sorensen, 1988), and also form coordinate complexes with SH groups thereby alter the cellular homeostasis and membrane fluidity, which results in cell death and organ dysfunction (Rong et al., 1996). Cd causes specific alterations in mitochondria including a decrease in mitochondrial membrane potential, uncoupling of oxidative phosphorylation, disruption of electron transport, thereby enhances the generation of ROS and ultimately leads to cytotoxicity (Koizumi et al., 1994; Tang and Shaikh, 2001; Wang et al., 2004). Concomitant administration of DTS with Cd significantly improved the function of kidney, which might be due to the interference of DTS with the effects of Cd and a reduction in the accumulation of Cd by DTS. These findings are in agreement with the in vitro results, which display the preservation of cell viability by DTS against Cd, thus strengthening the protective efficacy of DTS on Cd toxicity. Redox disturbances are known to negatively impact the body system through ROS generation, which destroy proteins, lipids and DNA by oxidation (Halliwell and Gutteridge, 1990). The increased lipid peroxidation and protein carbonyl contents

observed after Cd exposure, implicates oxidative stress in Cd induced cytolethality (Stohs and Bagchi, 1995). Although Cd itself does not generate free radicals directly, it indirectly generates various radicals like superoxide, hydroxyl and nitric oxide thus causing damage consistent with oxidative stress (Hassoun and Stohs, 1996; Stohs et al., 2000). In this context, our in vivo and in vitro studies also showed an elevation in the levels of lipid peroxidation upon Cd exposure. Moreover, the generation of ROS and oxidative stress induced by Cd in cells were observed by flow cytometric analysis with DCF-DA, an indicator of oxidative stress in the cells. The increase of ROS in the presence of Cd suggests a higher degree of oxidative stress and extent of cellular damage in the tissues. The free radical scavenging nature of DTS (Imai et al., 1994) might lead to the direct reactions of DTS with various ROS, especially the free radicals initiating Fenton type reactions, which could be responsible for the decreased levels of lipid peroxidation in Cd intoxicated rats. Moreover, the OSCs has been reported for their nonenzymic antioxidant actions, which is mainly from reducing power and metal ion chelating effects as well as interaction with other antioxidants (Yinn et al., 2002). Our in vitro study with vero cells also support the ability of DTS to reduce ROS generation during Cd toxicity. The diallyl sulfides related to DTS have also been reported for their renoprotective effect, which could be associated with their antioxidant properties (Dwivedi et al., 1996; Pedraza-Chaverri et al., 2003). GSH and other thiol containing proteins play a key role in cellular defense against Cd toxicity. It is well established that the nonenzymic antioxidants such as vitamin C and vitamin E concomitantly decreased with GSH in Cd toxicity (Sunitha et al., 2001; Pari and Murugavel, 2005). It has been suggested that the depletion of intracellular sulphydryl groups (SH groups) by Cd is the prerequisite for ROS generation as well as disruption of intracellular organs (Valko et al., 2005). In agreement with this, a significant decrease in the levels of these nonenzymic antioxidants in Cd toxicity could lead to increased susceptibility of the tissue to free radical damage. The chelating property and the ability of DTS to inhibit the radical generation reduce the oxidative threat caused by Cd, which could mitigate the consumption of endogenous nonenzymic antioxidants and increase their levels in the kidney tissue. Suppression of free radical scavenging function and the enhancement of ROS contribute to Cd induced oxidative stress and its associated toxic effects (Szuster-Clesielska et al., 2000; Waisberg et al., 2003). Most of the antioxidant enzymes become inactive by Cd exposure due to the direct binding of Cd to enzyme active sites, if it contains –SH groups (Quig, 1998) or displacement of metal cofactors from active sites (Casalino et al., 2000). Further, the oxidative modification of enzymes was indicated by increased level of protein carbonyl contents and decreased level of protein thiols in Cd toxicity, a reasonable marker for free radical induced protein oxidation (Butterfield et al., 1998; Moskovitz et al., 2002). In consistent with these reports, the decreased activities of antioxidant enzymes (SOD, CAT, GPx and GST) were observed in the renal tissue, which indicate the failure of antioxidant defense system to overcome the influx of ROS on Cd exposure. Thus, the inhibition of

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enzymes involved in free radical removal leads to the accumulation of H2O2, which promotes lipid peroxidation and modulation of DNA, altered gene expression, and cell death (Stohs et al., 2000; Waisberg et al., 2003). GR is the enzyme responsible for the reduction of oxidized glutathione (GSSG) to GSH. G6PD supplies the cells with most of the NADPH, which keeps GSH at a constant level by providing NADPH for GR. G6PD is known to contain many – SH groups, which plays a critical role in maintaining its tertiary structure. The formation of Cd–sulphydryl complex with –SH groups of enzymes might lead to a decrease in the activities of GR and G6PD, and depletion of GSH level (Gerson and Shaikh, 1984; Yoshida and Huang, 1986). Our study also showed decreased activities of GR and G6PD along with a fall in the level of GSH in Cd treated rats. The chelating property of DTS enhances the elimination of Cd from the renal tissue, which might reduce the Cd burden with displacement of metal cofactors and/or Cd binding with enzymes. In addition, the capability of DTS to react with free radicals or with highly reactive byproducts of lipid peroxidation as well as enhancement of tissue thiol pools may be responsible for the reduction of oxidative modification of enzymes and a reversal of the activities of antioxidant and glutathione metabolizing enzymes. In conclusion, our study suggests that DTS has a protective effect against Cd induced oxidative damage in the rat kidney cells. The mechanisms contributing to its effectiveness involves the quenching of free radicals, antioxidant, metal chelating property, and cytoprotective action. Recently, much attention has been focused on the protective biochemical functions of naturally occurring antioxidants in biological systems against toxic heavy metals. This study therefore provides biological evidence supporting the usefulness of DTS, a compound from garlic to protect Cd induced toxic effects in renal cells. However, further studies are needed in order to explore the exact cellular mechanisms underlying the cytoprotective effects of DTS. Acknowledgement The financial assistance from the Indian Council of Medical Research, New Delhi, India in the form of SRF to P. Murugavel is gratefully acknowledged. The authors would like to thank Oxford Chemicals Ltd, UK for the gift sample of diallyl tetrasulfide. References Ahn, D.W., Kim, M.Y., Kim, K.R., Park, Y.S., 1999. Cadmium binding and sodium dependent solute transport in renal brush border membrane vesicles. Toxicology and Applied Pharmacology 154, 212–218. Beutler, E., 1983. Active transport of glutathione disulfide from erythrocytes. In: Larson, A., Orrenius, S., Holmgren, A., Mannerwik, B. (Eds.), Functions of Glutathione—Biochemical, Physiological, Toxicological and Clinical Aspects. Raven Press, New York, USA, p. 65. Block, A., 1992. The organosulfur chemistry of the genus allium: implications for the organic chemistry of sulfur. Angewandte Chemie. International Edition 31, 1135–1178. Butterfield, D.A., Koppal, T., Howard, B., Subramaniam, R., Hall, N., Hensley, K., Yatin, S., Allen, K., Aksenova, M., Carney, J., 1998. Structural and

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