Food and Chemical Toxicology 72 (2014) 147–153
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Protective effect of polydatin, a natural precursor of resveratrol, against cisplatin-induced toxicity in rats Sinan Ince a,⇑, Damla Arslan Acaroz b, Ondrˇej Neuwirth c, Hasan Huseyin Demirel d, Baris Denk b, Ismail Kucukkurt b, Ruhi Turkmen a a
Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, 03030 Afyonkarahisar, Turkey Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Biochemistry, 03030 Afyonkarahisar, Turkey University of Veterinary and Pharmaceutical Sciences, Faculty of Pharmacy, Department of Natural Drugs, CZ 612 42 Brno, Czech Republic d Afyon Kocatepe University, Bayat Vocational School, Afyonkarahisar, Turkey b c
a r t i c l e
i n f o
Article history: Received 20 May 2014 Accepted 12 July 2014 Available online 19 July 2014 Keywords: Cisplatin Polydatin Oxidative stress DNA damage Toxicity Rat
a b s t r a c t The aim of the present study was to evaluate the possible protective effect of polydatin (PD) on cisplatin (Cis) induced oxidative stress in rats. Totally, thirty male Wistar albino rats were fed standard rodent diet and divided into 5 equal groups: the control group (vehicle treated) was treated with physiological saline for ten days both orally and intraperitoneally (i.p.), the second group was orally treated with physiological saline and 7 mg/kg single i.p. injection of Cis on the seventh day, and third, fourth, and fifth groups were treated orally PD at 25, 50, and 100 mg/kg/day, respectively for 10 days starting seven days before Cis injection and 7 mg/kg single i.p. Cis was injected on the seventh day. Cis resulted in significant increase malondialdehyde levels and decreased glutathione levels. In addition, Cis treatment decreased superoxide dismutase and catalase activities in erythrocyte and tissues. Also, Cis treatment caused to increase DNA damage and affected serum biochemical parameters whereas slightly decreased AchE activity. However, treatment of PD resulted in reversal of Cis-induced oxidative stress, lipid peroxidation, and activities of antioxidant enzymes. In conclusion, PD has protective effect in rats against Cis-induced oxidative stress, enhances antioxidant defence mechanism, and regenerates their tissues. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Cisplatin (Cis), cis-diaminedichloroplatinum(II), is a highly effective antineoplastic drug commonly used for treatment of wide variety malignant and solid tumors (Tayem et al., 2006). However, its toxicity especially nephrotoxicity, is the major adverse effect representing a dose-limiting factor in Cis therapy. There is substantial evidence supporting the role of reactive oxygen species and lipid peroxidation in damage that develops in response to Cis (Vickers et al., 2004). Also, several studies have demonstrated the protective effect of antioxidants in Cis-induced toxicity (Zeki et al., 2003; Atessahin et al., 2005). Polygonum cuspidatum root (RPC), is a widely used traditional medicine in Asia which has been shown to exhibit antibacterial (Shan et al., 2008), antioxidant (Pan et al., 2007) and antiangiogenesis (Wang et al., 2004) activities. There are numerous constituents existing in RPC, such as emodin, chrysophanol, rhein, resveratrol, polydatin (PD), catechin, quercetin, and so on (Shan ⇑ Corresponding author. Tel.: +90 2722281312/142; fax: +90 2722281349. E-mail address: [email protected] (S. Ince). http://dx.doi.org/10.1016/j.fct.2014.07.022 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
et al., 2008). PD, the principal constituent in RPC, is an important stilbenoid and a glycoside of resveratrol, whose content in RPC is six times higher than the resveratrol (Zhou et al., 2005). PD possesses several activities proved by previous studies: it reduces lipid profile in hyperlipidemic rabbits (Xing et al., 2009), exerts a neuroprotective effect on cerebral injury induced by ischemia/reperfusion (Cheng et al., 2006), promotes weight loss and enhances diet attitudes in low-income mothers of young children (Jordan et al., 2008), protects the primarily cultured rat hepatocytes against CCl4-induced injury (Huang et al., 1999), and reduces lipid oxidation (Pan et al., 2007). Nevertheless, our knowledge, there has been no report on the effect of polydatin on Cis-induced toxicity in rats, yet. The present study aimed to evaluate the protective effect of PD against Cis-induced acute toxicity in male Wistar albino rats. Biochemical parameters and 8-OHdG as a marker of DNA damage were measured in serum. Also, blood and tissue malondialdehyde (MDA) and reduced glutathione (GSH) levels, acetylcholinesterase (AchE), catalase (CAT) and superoxide dismutase (SOD) activities were determined. In addition, light microscopic examination of liver, kidney, and heart tissues were performed.
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2. Materials and methods 2.1. Materials Polydatin and Cis purchased from Sigma–Aldrich (Interlab A.S., Istanbul, Turkey), and Kocak Farma (Istanbul, Turkey), respectively. All the other chemicals and reagents were of analytical reagent grade purchased from commercial sources. 2.2. Experimental protocol Healthy male Wistar albino rats, 60 days of age and weighing 250–300 g, were purchased from The Animal Breeding Laboratories of the Experimental Animal Research and Application Center (Afyon, Turkey). The animals were kept at room temperature (25 °C) and relative humidity (50–55%) in a 12 h light/dark cycle with ad libitum access to standard rodent diet and water. Rats were allowed to acclimatise to the animal facility for at least seven days before experiment started. Prior to the experiments, rats were fed with standard rodent diet for one week in order to adapt to the laboratory conditions. In this study, totally 30 male rats were randomly allocated into 5 groups, 6 rats in each group. All animals were fasted overnight before the experiment. The animals in the control group (vehicle treated) was treated orally and also treated intraperitoneally (i.p.) physiological saline for ten days, the second group was treated orally, physiological saline and 7 mg/kg single i.p. injection of Cis (Fouad et al., 2008) on the seventh day, and third, fourth, and fifth groups were treated orally PD at 25, 50, and 100 mg/kg/day, respectively for 10 days starting seven days before Cis injection and 7 mg/kg single i.p. Cis was injected on the seventh day. The experimental protocols were also approved by the Animal Care and Use Committee at Afyon Kocatepe University and were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. 2.3. Blood collection Blood samples from each group were collected by cardiac puncture into heparinised and non-heparinised tubes under light ether anaesthesia at the end of experimental period. 2.4. Erythrocytes preparation Within 30 min of blood collection, the erythrocytes were precipitated by centrifugation at 3500 rpm for 15 min at 4 °C, afterwards plasma and serum were removed. The erythrocytes were washed three times with isotonic saline and the puffy coat was discarded. Then, same volume of isotonic saline and erythrocyte were added into vials and stored at 20 °C in the deep freeze. Erythrocyte suspension was destroyed by osmotic pressure, use five times of cold deionised water. The erythrocyte lysate was stored at 4 °C until measurements within 3 days (Winterbourn et al., 1975). 2.5. Preparation of homogenates Animals were sacrificed by cervical dislocation and their liver, kidney, and heart tissues were washed immediately with ice cold 0.9% NaCl. Each tissue was trimmed free of extraneous tissue, rinsed in chilled 0.15 M Tris–HCl buffer (pH 7.4). These tissues were blotted dry, and homogenized in 0.15 M Tris–HCl buffer (pH 7.4) to yield a 10% (w/v) homogenate. Then, they were centrifuged at 3500 rpm for 10 min at 4 °C. The pellets represented the nuclear fraction and the supernatants were subjected to centrifugation at 20,000 rpm for 20 min at 4 °C. The resultant pellets and the supernatants represented the mitochondrial fraction and the cytosolic (including microsomal fraction) fraction, respectively. Reactive oxygen species generation was observed in these fractions as well as whole homogenate (Kucukkurt et al., 2008). 2.6. Preparation of tissues for histopathological analysis At the end of experimental period, 30 male rats were sacrificed. Then, they were dissected and liver, kidney, and heart tissues from each animal were collected and fixed into 10% formalin solution for 48 h and then dehydrated through graded alcohol series (70–100%), cleared in xylene and embedded in paraffin. 5–6 lm thick paraffin sections were cut and stained with haematoxylin–eosin (H&E) and analyzed under a light microscope (Olympus Bx51 model, Tokyo, Japan) equipped with camera (Olympus DP20, Tokyo, Japan).
(TBA) with MDA and its absorbance was measured spectrophotometrically at 532 nm. The concentration of MDA was calculated by the absorbance coefficient of MDA–TBA complex and expressed in nmol/ml blood and nmol/g wet tissue.
2.8. Measurement of GSH in whole blood and tissue homogenates GSH concentration was measured using the method described by Beutler et al. (1993) in whole blood and tissue homogenates. Briefly, 0.2 ml sample was added to 1.8 ml distilled water. 3 ml of precipitating solution (1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl in 100 ml distilled water) was mixed with sample. The mixture was allowed to stand for approximately 5 min and then filtered (Whatman No. 42). 2 ml of filtrate was taken then it was added into another tube and 8 mL of the phosphate solution (0.3 M disodium hydrogen phosphate) and 1 ml 5,50 -Dithiobis(2-nitrobenzoic acid) (DTNB) were added. A blank was prepared with 8 ml of phosphate solution; 2 ml diluted precipitating solution and 1 mL DTNB reagent. A standard solution of the GSH was prepared (40 mg/100 ml). The optical density was measured at 412 nm on the spectrophotometer. Results were communicated as nmol/ml blood and nmol/g wet tissue.
2.9. Measurement of SOD activity in erythrocyte lysate and tissue homogenates The antioxidant enzyme activity of SOD in erythrocyte lysate and tissue homogenate was measured according to the method of Sun et al. (1988). The measurement of SOD is based on the principle in which xanthine reacts with xanthine oxidase as a source of substrate (superoxide) and reduces nitroblue tetrazolium (NBT) as an indicator of superoxide. In this method, xantine–xantine oxidase was utilized to generate a superoxide flux. The absorbance obtained from NBT reduction to blue formazon by superoxide was determined at 560 nm spectrophotometrically. SOD activity was expressed U/gHb in erythrocyte and U/lg protein in tissue.
2.10. Measurement of CAT activity in erythrocyte lysate and tissue homogenate CAT activities in erythrocyte lysate and tissue homogenate were determined according to the method of Luck (1955) and Aebi (1974), respectively. These methods are based on the decomposition of H2O2 by catalase. The reaction mixture was composed of 50 mM phosphate buffer of pH 7.0; 10 mM H2O2 and sample. The reduction rate of H2O2 was followed at 240 nm for 45 s. at room temperature. One unit of catalase is the amount of catalase decomposes 1.0 lmol of H2O2 per min at pH 4.5 at 25 °C, and the catalase activity (k; nmol/min) is expressed k/gHb in erythrocyte and k/lg protein in tissue.
2.11. Measurement of 8-OHdG level, AchE and biochemical parameters in serum The serum samples were examined for their concentration of 8-OHdG using a competitive enzyme immunoassay (EIA) kit (Cayman Chemical Company, Ann Arbor, MI, USA) (Michoulas et al., 2006; Devries et al., 2008) and intra-assay and inter-assay CV were determinate 5.4% and 8.5%, respectively. AchE activity was also measured quantitative sandwich EIA kit (Cusabio Chemical Company, Wuhan, Hubei Province, P.R. China) and intra-assay and inter-assay CV were determinate 6.1% and 8.3%, respectively. Blood urea nitrogen (BUN), serum levels of creatinine, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total cholesterol (TCh), glucose, low density lipoprotein-cholesterol (LDL-c), high density lipoprotein-cholesterol (HDL-c) and triglyceride (TG) were determined using COBAS test kits (Roche Diagnostics Systems, Istanbul, Turkey), according to the manufacturer’s instructions, in the Laboratory of Internal Medicine, Faculty of Veterinary Medicine, University of Afyon Kocatepe.
2.12. Measurement of hemoglobin (Hb) and protein concentrations The Hb was determined colorimetric cyanomethemoglobin method according to Drabkin and Austin, 1935, and tissue protein content was assayed according to the colorimetric method of Lowry et al. (1951).
2.13. Spectrophotometric measurements The spectrophotometric measurements were performed by using a Shimadzu 1601 UV–VIS spectrophotometer (Tokyo, Japan).
2.14. Statistical analyses 2.7. Measurement of LPO in whole blood and tissue homogenates Malondialdehyde (MDA), as a marker for LPO, was determined according to the methods of Draper and Hardley (1990) in whole blood and in tissue homogenates (Ohkawa et al., 1979). The principle of the methods is based on spectrophotometric measurement of the colour production during the reaction of thiobarbituric acid
Data obtained from experimental animals were expressed as means and standard deviation of means (±SD) and analysed using one-way analysis of variance (ANOVA), followed by Duncan post hoc tests on the SPSS (11.5) software computer program. A difference in the mean values of p < 0.05 was considered to be significant.
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3. Results 3.1. Effect on lipid peroxidation and reduced glutathione MDA level is widely used as a marker of free-radical mediated LPO. A highly significant elevation was observed in whole blood, kidney, heart (p < 0.001), and liver (p = 0.001) MDA levels of Cis administered rats compared with control rats. In contrast, supplementation of B in a dose-dependent manner showed significantly decreased in whole blood and all tissue than the Cis group (Table 1). GSH is a nonenzymatic antioxidant in the detoxification pathway that reduces the toxic metabolites of xenobiotics. GSH levels of whole blood (p < 0.05), kidney (p < 0.01), liver, and heart (p < 0.001) of Cis groups were found to be lower than the control group. In contrast, administration of PD in a dose-dependent manner showed higher GSH levels of blood, liver, kidney and heart than the Cis groups (Table 2). 3.2. Effect on antioxidant enzymes Antioxidant enzymes, SOD and CAT activities were determined in erythrocyte, liver, kidney, and heart tissue of rats and they are shown in Table 3 and Table 4, respectively. In the Cis group, SOD activities were found to be lower in erythrocyte (p = 0.001), liver, kidney (p < 0.001), and heart (p < 0.05) tissues compared to control group. Nonetheless, CAT activities were decreased in erythrocyte (p = 0.001), liver, kidney, and heart tissues (p < 0.001) compared to control group. In contrast, administration of PD in a dose dependent manner was reversed Cis-induced alteration of SOD and CAT activities. 3.3. Effect on biochemical parameters Cis administration significantly increased BUN (p < 0.05), creatinine (p = 0.001), ALT, ALP, and AST (p < 0.001) levels as compared to the control group. Administration of PD in a dose dependent manner resulted in a significant reduction in these parameters (Table 5). Also, glucose and TG levels were found to be low level in Cis group. The results revealed that PD treatment significantly increased glucose and TG levels elevated by Cis administration. HDL-c, LDL-c and TCh levels were also determined in rats and shown in Table 5. 3.4. Effect on 8-OHdG level and AchE activity 8-OHdG level and AchE activity of rats were shown in Fig. 1A. In the Cis group, 8-OHdG levels were found to be high level (13.95 ± 1.54 pg/L) compared with control group (3.68 ± 1.21 pg/ L) (p < 0.001). 8-OHdG were also found to be at 9.71 ± 1.95 (p < 0.001), 7.67 ± 1.13 (p < 0.01), and 6.59 ± 1.78 (p < 0.05) pg/L in PD25, PD50, and PD100 plus Cis, respectively. These results suggested that administration of PD could not alleviate enough the 8-OHdG compared to control but in a dose dependent manner it
prevented the Cis-induced alteration of 8-OHdG compared with Cis group. In the Cis group, AchE levels (Fig. 1B) were found to be low level (24.38 ± 4.95 pg/L) compared with control group (34.06 ± 5.41 pg/ L) (p > 0.05). AchE levels were also found to be at 29.01 ± 6.73, 31.67 ± 8.41, and 31.22 ± 4.91 pg/L in PD25, PD50, and PD100 plus Cis, respectively. These results suggested that administration of Cis could not significantly affect on the AchE activity of rats. 3.5. Histopathological examination Histopathological changes in organs of experimental group were largely described and shown in Fig. 2. In Cis group, necrosis and degeneration have been observed in vena centralise of liver (Fig. 2-B1). There was vacuole degeneration in epithelial cell of the kidney tubuli (Fig. 2-B2) and hyaline degeneration was found in heart (Fig. 2-B3) of rats. In PD groups, especially PD100, slight histopathological changes have been observed in liver, kidney, and heart tissues of rats (Fig. 2C, D, E-1, 2, and 3, respectively). In the control group, no significant histopathological changes were observed in liver, kidney, and heart tissues of rats (Fig. 2A-1, 2, and 3, respectively). 4. Discussion Increased oxidative stress represents an imbalance between intracellular product of free radicals and the cellular defence mechanisms; notably, MDA is one of the most important markers of oxidative stress (Ince et al., 2010). Many researches demonstrated that Cis is a chemotherapeutic agent causes oxidative stress in a dose- and time-dependent manner (Naziroglu et al., 2004; Hannemann and Baumann, 1988), and increases levels of MDA, depletes GSH, and decreases activities of antioxidant enzymes such as SOD and CAT (Husain et al., 1996; Pandir et al., 2013). These reports suggested that the generation of oxidative products are mainly related to the DNA damage caused by Cis. In addition, Prieto González et al., 2009 reported that Cis at a dose of 10 mg/ kg, i.p. significantly caused DNA damages in murine peripheral lymphocyte cells and increased MDA contents in mouse blood. In our study, the enhanced production of blood and tissue lipid peroxides observed in agreement with other studies. PD administration in a dose dependent manner was found to produce significantly less lipid peroxides than Cis-treated rats. Consistent with our results, PD acted as scavenger of superoxide, hydroxyl radical and singlet molecular oxygen (Lanzilli et al., 2012; Chen et al., 2013). Previous studies have shown that GSH plays a key role in the detoxification of the reactive toxic metabolites of Cis, and that cell death begins when GSH stores are markedly depleted (Atessahin et al., 2005). Also, Zhang et al. (2012) have demonstrated that PD supplementation induces reduced glutathione activity in mice. In this study, the observed increase in lipid peroxidation and concomitant depletion of GSH levels in Cis group suggests that the increased peroxidation could be a consequence of
Table 1 Effects of cisplatin (Cis) at dose 7 mg/kg and Cis + polydatin (PD) at doses 25 (PD25), 50 (PD50), and 100 (PD100) mg/kg on malondialdehyde levels in blood, liver, kidney, and heart of rats. Experimental design
Blood (nmol/ml)
Liver (nmol/g tissue)
Kidney (nmol/g tissue)
Heart (nmol/g tissue)
Control Cis PD25 + Cis PD50 + Cis PD100 + Cis P value
4.47 ± 0.63c 7.31 ± 0.39a 5.31 ± 0.24b 5.29 ± 0.13b 5.13 ± 0.43b 0.000
0.98 ± 0.15b 2.13 ± 0.62a 2.12 ± 0.83a 1.86 ± 0.43a 1.13 ± 0.18b 0.001
1.32 ± 0.49c 3.69 ± 0.75a 3.32 ± 0.47ab 2.81 ± 0.44b 1.91 ± 0.58c 0.000
0.03 ± 0.02d 0.18 ± 0.04a 0.16 ± 0.04ab 0.14 ± 0.03b 0.08 ± 0.04c 0.000
Values are mean ± Standard deviations; n = 6. In the same column values with different letters show statistically significant differences in blood (p < 0.001), and liver (p=0.001).
a,b,c,d
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Table 2 Effects of cisplatin (Cis) at dose 7 mg/kg and Cis + polydatin (PD) at doses 25 (PD25), 50 (PD50), and 100 (PD100) mg/kg on glutathione levels in blood, liver, kidney, and heart of rats. Experimental design
Blood (nmol/ml)
Liver (nmol/g tissue)
Kidney (nmol/g tissue)
Heart (nmol/g tissue)
Control Cis PD25 + Cis PD50 + Cis PD100 + Cis P value
47.60 ± 6.32a 37.11 ± 5.57b 41.69 ± 2.67ab 43.33 ± 3.93ab 45.24 ± 6.14a 0.019
11.59 ± 0.51a 8.13 ± 0.63d 9.15 ± 0.28c 9.81 ± 0.30b 10.17 ± 0.27b 0.000
9.24 ± 1.03a 6.86 ± 1.19b 6.94 ± 1.31ab 8.71 ± 0.79ab 9.07 ± 1.61ab 0.003
7.99 ± 0.53a 6.05 ± 0.73c 6.44 ± 0.75c 6.69 ± 0.93ab 7.35 ± 0.27ab 0.000
Values are mean ± Standard deviations; n = 6. In the same column values with different letters show statistically significant differences in blood (p < 0.05), kidney (p
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Protective effect of polydatin, a natural precursor of resveratrol, against cisplatin-induced toxicity in rats Sinan Ince a,⇑, Damla Arslan Acaroz b, Ondrˇej Neuwirth c, Hasan Huseyin Demirel d, Baris Denk b, Ismail Kucukkurt b, Ruhi Turkmen a a
Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, 03030 Afyonkarahisar, Turkey Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Biochemistry, 03030 Afyonkarahisar, Turkey University of Veterinary and Pharmaceutical Sciences, Faculty of Pharmacy, Department of Natural Drugs, CZ 612 42 Brno, Czech Republic d Afyon Kocatepe University, Bayat Vocational School, Afyonkarahisar, Turkey b c
a r t i c l e
i n f o
Article history: Received 20 May 2014 Accepted 12 July 2014 Available online 19 July 2014 Keywords: Cisplatin Polydatin Oxidative stress DNA damage Toxicity Rat
a b s t r a c t The aim of the present study was to evaluate the possible protective effect of polydatin (PD) on cisplatin (Cis) induced oxidative stress in rats. Totally, thirty male Wistar albino rats were fed standard rodent diet and divided into 5 equal groups: the control group (vehicle treated) was treated with physiological saline for ten days both orally and intraperitoneally (i.p.), the second group was orally treated with physiological saline and 7 mg/kg single i.p. injection of Cis on the seventh day, and third, fourth, and fifth groups were treated orally PD at 25, 50, and 100 mg/kg/day, respectively for 10 days starting seven days before Cis injection and 7 mg/kg single i.p. Cis was injected on the seventh day. Cis resulted in significant increase malondialdehyde levels and decreased glutathione levels. In addition, Cis treatment decreased superoxide dismutase and catalase activities in erythrocyte and tissues. Also, Cis treatment caused to increase DNA damage and affected serum biochemical parameters whereas slightly decreased AchE activity. However, treatment of PD resulted in reversal of Cis-induced oxidative stress, lipid peroxidation, and activities of antioxidant enzymes. In conclusion, PD has protective effect in rats against Cis-induced oxidative stress, enhances antioxidant defence mechanism, and regenerates their tissues. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Cisplatin (Cis), cis-diaminedichloroplatinum(II), is a highly effective antineoplastic drug commonly used for treatment of wide variety malignant and solid tumors (Tayem et al., 2006). However, its toxicity especially nephrotoxicity, is the major adverse effect representing a dose-limiting factor in Cis therapy. There is substantial evidence supporting the role of reactive oxygen species and lipid peroxidation in damage that develops in response to Cis (Vickers et al., 2004). Also, several studies have demonstrated the protective effect of antioxidants in Cis-induced toxicity (Zeki et al., 2003; Atessahin et al., 2005). Polygonum cuspidatum root (RPC), is a widely used traditional medicine in Asia which has been shown to exhibit antibacterial (Shan et al., 2008), antioxidant (Pan et al., 2007) and antiangiogenesis (Wang et al., 2004) activities. There are numerous constituents existing in RPC, such as emodin, chrysophanol, rhein, resveratrol, polydatin (PD), catechin, quercetin, and so on (Shan ⇑ Corresponding author. Tel.: +90 2722281312/142; fax: +90 2722281349. E-mail address: [email protected] (S. Ince). http://dx.doi.org/10.1016/j.fct.2014.07.022 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
et al., 2008). PD, the principal constituent in RPC, is an important stilbenoid and a glycoside of resveratrol, whose content in RPC is six times higher than the resveratrol (Zhou et al., 2005). PD possesses several activities proved by previous studies: it reduces lipid profile in hyperlipidemic rabbits (Xing et al., 2009), exerts a neuroprotective effect on cerebral injury induced by ischemia/reperfusion (Cheng et al., 2006), promotes weight loss and enhances diet attitudes in low-income mothers of young children (Jordan et al., 2008), protects the primarily cultured rat hepatocytes against CCl4-induced injury (Huang et al., 1999), and reduces lipid oxidation (Pan et al., 2007). Nevertheless, our knowledge, there has been no report on the effect of polydatin on Cis-induced toxicity in rats, yet. The present study aimed to evaluate the protective effect of PD against Cis-induced acute toxicity in male Wistar albino rats. Biochemical parameters and 8-OHdG as a marker of DNA damage were measured in serum. Also, blood and tissue malondialdehyde (MDA) and reduced glutathione (GSH) levels, acetylcholinesterase (AchE), catalase (CAT) and superoxide dismutase (SOD) activities were determined. In addition, light microscopic examination of liver, kidney, and heart tissues were performed.
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2. Materials and methods 2.1. Materials Polydatin and Cis purchased from Sigma–Aldrich (Interlab A.S., Istanbul, Turkey), and Kocak Farma (Istanbul, Turkey), respectively. All the other chemicals and reagents were of analytical reagent grade purchased from commercial sources. 2.2. Experimental protocol Healthy male Wistar albino rats, 60 days of age and weighing 250–300 g, were purchased from The Animal Breeding Laboratories of the Experimental Animal Research and Application Center (Afyon, Turkey). The animals were kept at room temperature (25 °C) and relative humidity (50–55%) in a 12 h light/dark cycle with ad libitum access to standard rodent diet and water. Rats were allowed to acclimatise to the animal facility for at least seven days before experiment started. Prior to the experiments, rats were fed with standard rodent diet for one week in order to adapt to the laboratory conditions. In this study, totally 30 male rats were randomly allocated into 5 groups, 6 rats in each group. All animals were fasted overnight before the experiment. The animals in the control group (vehicle treated) was treated orally and also treated intraperitoneally (i.p.) physiological saline for ten days, the second group was treated orally, physiological saline and 7 mg/kg single i.p. injection of Cis (Fouad et al., 2008) on the seventh day, and third, fourth, and fifth groups were treated orally PD at 25, 50, and 100 mg/kg/day, respectively for 10 days starting seven days before Cis injection and 7 mg/kg single i.p. Cis was injected on the seventh day. The experimental protocols were also approved by the Animal Care and Use Committee at Afyon Kocatepe University and were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. 2.3. Blood collection Blood samples from each group were collected by cardiac puncture into heparinised and non-heparinised tubes under light ether anaesthesia at the end of experimental period. 2.4. Erythrocytes preparation Within 30 min of blood collection, the erythrocytes were precipitated by centrifugation at 3500 rpm for 15 min at 4 °C, afterwards plasma and serum were removed. The erythrocytes were washed three times with isotonic saline and the puffy coat was discarded. Then, same volume of isotonic saline and erythrocyte were added into vials and stored at 20 °C in the deep freeze. Erythrocyte suspension was destroyed by osmotic pressure, use five times of cold deionised water. The erythrocyte lysate was stored at 4 °C until measurements within 3 days (Winterbourn et al., 1975). 2.5. Preparation of homogenates Animals were sacrificed by cervical dislocation and their liver, kidney, and heart tissues were washed immediately with ice cold 0.9% NaCl. Each tissue was trimmed free of extraneous tissue, rinsed in chilled 0.15 M Tris–HCl buffer (pH 7.4). These tissues were blotted dry, and homogenized in 0.15 M Tris–HCl buffer (pH 7.4) to yield a 10% (w/v) homogenate. Then, they were centrifuged at 3500 rpm for 10 min at 4 °C. The pellets represented the nuclear fraction and the supernatants were subjected to centrifugation at 20,000 rpm for 20 min at 4 °C. The resultant pellets and the supernatants represented the mitochondrial fraction and the cytosolic (including microsomal fraction) fraction, respectively. Reactive oxygen species generation was observed in these fractions as well as whole homogenate (Kucukkurt et al., 2008). 2.6. Preparation of tissues for histopathological analysis At the end of experimental period, 30 male rats were sacrificed. Then, they were dissected and liver, kidney, and heart tissues from each animal were collected and fixed into 10% formalin solution for 48 h and then dehydrated through graded alcohol series (70–100%), cleared in xylene and embedded in paraffin. 5–6 lm thick paraffin sections were cut and stained with haematoxylin–eosin (H&E) and analyzed under a light microscope (Olympus Bx51 model, Tokyo, Japan) equipped with camera (Olympus DP20, Tokyo, Japan).
(TBA) with MDA and its absorbance was measured spectrophotometrically at 532 nm. The concentration of MDA was calculated by the absorbance coefficient of MDA–TBA complex and expressed in nmol/ml blood and nmol/g wet tissue.
2.8. Measurement of GSH in whole blood and tissue homogenates GSH concentration was measured using the method described by Beutler et al. (1993) in whole blood and tissue homogenates. Briefly, 0.2 ml sample was added to 1.8 ml distilled water. 3 ml of precipitating solution (1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl in 100 ml distilled water) was mixed with sample. The mixture was allowed to stand for approximately 5 min and then filtered (Whatman No. 42). 2 ml of filtrate was taken then it was added into another tube and 8 mL of the phosphate solution (0.3 M disodium hydrogen phosphate) and 1 ml 5,50 -Dithiobis(2-nitrobenzoic acid) (DTNB) were added. A blank was prepared with 8 ml of phosphate solution; 2 ml diluted precipitating solution and 1 mL DTNB reagent. A standard solution of the GSH was prepared (40 mg/100 ml). The optical density was measured at 412 nm on the spectrophotometer. Results were communicated as nmol/ml blood and nmol/g wet tissue.
2.9. Measurement of SOD activity in erythrocyte lysate and tissue homogenates The antioxidant enzyme activity of SOD in erythrocyte lysate and tissue homogenate was measured according to the method of Sun et al. (1988). The measurement of SOD is based on the principle in which xanthine reacts with xanthine oxidase as a source of substrate (superoxide) and reduces nitroblue tetrazolium (NBT) as an indicator of superoxide. In this method, xantine–xantine oxidase was utilized to generate a superoxide flux. The absorbance obtained from NBT reduction to blue formazon by superoxide was determined at 560 nm spectrophotometrically. SOD activity was expressed U/gHb in erythrocyte and U/lg protein in tissue.
2.10. Measurement of CAT activity in erythrocyte lysate and tissue homogenate CAT activities in erythrocyte lysate and tissue homogenate were determined according to the method of Luck (1955) and Aebi (1974), respectively. These methods are based on the decomposition of H2O2 by catalase. The reaction mixture was composed of 50 mM phosphate buffer of pH 7.0; 10 mM H2O2 and sample. The reduction rate of H2O2 was followed at 240 nm for 45 s. at room temperature. One unit of catalase is the amount of catalase decomposes 1.0 lmol of H2O2 per min at pH 4.5 at 25 °C, and the catalase activity (k; nmol/min) is expressed k/gHb in erythrocyte and k/lg protein in tissue.
2.11. Measurement of 8-OHdG level, AchE and biochemical parameters in serum The serum samples were examined for their concentration of 8-OHdG using a competitive enzyme immunoassay (EIA) kit (Cayman Chemical Company, Ann Arbor, MI, USA) (Michoulas et al., 2006; Devries et al., 2008) and intra-assay and inter-assay CV were determinate 5.4% and 8.5%, respectively. AchE activity was also measured quantitative sandwich EIA kit (Cusabio Chemical Company, Wuhan, Hubei Province, P.R. China) and intra-assay and inter-assay CV were determinate 6.1% and 8.3%, respectively. Blood urea nitrogen (BUN), serum levels of creatinine, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total cholesterol (TCh), glucose, low density lipoprotein-cholesterol (LDL-c), high density lipoprotein-cholesterol (HDL-c) and triglyceride (TG) were determined using COBAS test kits (Roche Diagnostics Systems, Istanbul, Turkey), according to the manufacturer’s instructions, in the Laboratory of Internal Medicine, Faculty of Veterinary Medicine, University of Afyon Kocatepe.
2.12. Measurement of hemoglobin (Hb) and protein concentrations The Hb was determined colorimetric cyanomethemoglobin method according to Drabkin and Austin, 1935, and tissue protein content was assayed according to the colorimetric method of Lowry et al. (1951).
2.13. Spectrophotometric measurements The spectrophotometric measurements were performed by using a Shimadzu 1601 UV–VIS spectrophotometer (Tokyo, Japan).
2.14. Statistical analyses 2.7. Measurement of LPO in whole blood and tissue homogenates Malondialdehyde (MDA), as a marker for LPO, was determined according to the methods of Draper and Hardley (1990) in whole blood and in tissue homogenates (Ohkawa et al., 1979). The principle of the methods is based on spectrophotometric measurement of the colour production during the reaction of thiobarbituric acid
Data obtained from experimental animals were expressed as means and standard deviation of means (±SD) and analysed using one-way analysis of variance (ANOVA), followed by Duncan post hoc tests on the SPSS (11.5) software computer program. A difference in the mean values of p < 0.05 was considered to be significant.
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3. Results 3.1. Effect on lipid peroxidation and reduced glutathione MDA level is widely used as a marker of free-radical mediated LPO. A highly significant elevation was observed in whole blood, kidney, heart (p < 0.001), and liver (p = 0.001) MDA levels of Cis administered rats compared with control rats. In contrast, supplementation of B in a dose-dependent manner showed significantly decreased in whole blood and all tissue than the Cis group (Table 1). GSH is a nonenzymatic antioxidant in the detoxification pathway that reduces the toxic metabolites of xenobiotics. GSH levels of whole blood (p < 0.05), kidney (p < 0.01), liver, and heart (p < 0.001) of Cis groups were found to be lower than the control group. In contrast, administration of PD in a dose-dependent manner showed higher GSH levels of blood, liver, kidney and heart than the Cis groups (Table 2). 3.2. Effect on antioxidant enzymes Antioxidant enzymes, SOD and CAT activities were determined in erythrocyte, liver, kidney, and heart tissue of rats and they are shown in Table 3 and Table 4, respectively. In the Cis group, SOD activities were found to be lower in erythrocyte (p = 0.001), liver, kidney (p < 0.001), and heart (p < 0.05) tissues compared to control group. Nonetheless, CAT activities were decreased in erythrocyte (p = 0.001), liver, kidney, and heart tissues (p < 0.001) compared to control group. In contrast, administration of PD in a dose dependent manner was reversed Cis-induced alteration of SOD and CAT activities. 3.3. Effect on biochemical parameters Cis administration significantly increased BUN (p < 0.05), creatinine (p = 0.001), ALT, ALP, and AST (p < 0.001) levels as compared to the control group. Administration of PD in a dose dependent manner resulted in a significant reduction in these parameters (Table 5). Also, glucose and TG levels were found to be low level in Cis group. The results revealed that PD treatment significantly increased glucose and TG levels elevated by Cis administration. HDL-c, LDL-c and TCh levels were also determined in rats and shown in Table 5. 3.4. Effect on 8-OHdG level and AchE activity 8-OHdG level and AchE activity of rats were shown in Fig. 1A. In the Cis group, 8-OHdG levels were found to be high level (13.95 ± 1.54 pg/L) compared with control group (3.68 ± 1.21 pg/ L) (p < 0.001). 8-OHdG were also found to be at 9.71 ± 1.95 (p < 0.001), 7.67 ± 1.13 (p < 0.01), and 6.59 ± 1.78 (p < 0.05) pg/L in PD25, PD50, and PD100 plus Cis, respectively. These results suggested that administration of PD could not alleviate enough the 8-OHdG compared to control but in a dose dependent manner it
prevented the Cis-induced alteration of 8-OHdG compared with Cis group. In the Cis group, AchE levels (Fig. 1B) were found to be low level (24.38 ± 4.95 pg/L) compared with control group (34.06 ± 5.41 pg/ L) (p > 0.05). AchE levels were also found to be at 29.01 ± 6.73, 31.67 ± 8.41, and 31.22 ± 4.91 pg/L in PD25, PD50, and PD100 plus Cis, respectively. These results suggested that administration of Cis could not significantly affect on the AchE activity of rats. 3.5. Histopathological examination Histopathological changes in organs of experimental group were largely described and shown in Fig. 2. In Cis group, necrosis and degeneration have been observed in vena centralise of liver (Fig. 2-B1). There was vacuole degeneration in epithelial cell of the kidney tubuli (Fig. 2-B2) and hyaline degeneration was found in heart (Fig. 2-B3) of rats. In PD groups, especially PD100, slight histopathological changes have been observed in liver, kidney, and heart tissues of rats (Fig. 2C, D, E-1, 2, and 3, respectively). In the control group, no significant histopathological changes were observed in liver, kidney, and heart tissues of rats (Fig. 2A-1, 2, and 3, respectively). 4. Discussion Increased oxidative stress represents an imbalance between intracellular product of free radicals and the cellular defence mechanisms; notably, MDA is one of the most important markers of oxidative stress (Ince et al., 2010). Many researches demonstrated that Cis is a chemotherapeutic agent causes oxidative stress in a dose- and time-dependent manner (Naziroglu et al., 2004; Hannemann and Baumann, 1988), and increases levels of MDA, depletes GSH, and decreases activities of antioxidant enzymes such as SOD and CAT (Husain et al., 1996; Pandir et al., 2013). These reports suggested that the generation of oxidative products are mainly related to the DNA damage caused by Cis. In addition, Prieto González et al., 2009 reported that Cis at a dose of 10 mg/ kg, i.p. significantly caused DNA damages in murine peripheral lymphocyte cells and increased MDA contents in mouse blood. In our study, the enhanced production of blood and tissue lipid peroxides observed in agreement with other studies. PD administration in a dose dependent manner was found to produce significantly less lipid peroxides than Cis-treated rats. Consistent with our results, PD acted as scavenger of superoxide, hydroxyl radical and singlet molecular oxygen (Lanzilli et al., 2012; Chen et al., 2013). Previous studies have shown that GSH plays a key role in the detoxification of the reactive toxic metabolites of Cis, and that cell death begins when GSH stores are markedly depleted (Atessahin et al., 2005). Also, Zhang et al. (2012) have demonstrated that PD supplementation induces reduced glutathione activity in mice. In this study, the observed increase in lipid peroxidation and concomitant depletion of GSH levels in Cis group suggests that the increased peroxidation could be a consequence of
Table 1 Effects of cisplatin (Cis) at dose 7 mg/kg and Cis + polydatin (PD) at doses 25 (PD25), 50 (PD50), and 100 (PD100) mg/kg on malondialdehyde levels in blood, liver, kidney, and heart of rats. Experimental design
Blood (nmol/ml)
Liver (nmol/g tissue)
Kidney (nmol/g tissue)
Heart (nmol/g tissue)
Control Cis PD25 + Cis PD50 + Cis PD100 + Cis P value
4.47 ± 0.63c 7.31 ± 0.39a 5.31 ± 0.24b 5.29 ± 0.13b 5.13 ± 0.43b 0.000
0.98 ± 0.15b 2.13 ± 0.62a 2.12 ± 0.83a 1.86 ± 0.43a 1.13 ± 0.18b 0.001
1.32 ± 0.49c 3.69 ± 0.75a 3.32 ± 0.47ab 2.81 ± 0.44b 1.91 ± 0.58c 0.000
0.03 ± 0.02d 0.18 ± 0.04a 0.16 ± 0.04ab 0.14 ± 0.03b 0.08 ± 0.04c 0.000
Values are mean ± Standard deviations; n = 6. In the same column values with different letters show statistically significant differences in blood (p < 0.001), and liver (p=0.001).
a,b,c,d
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Table 2 Effects of cisplatin (Cis) at dose 7 mg/kg and Cis + polydatin (PD) at doses 25 (PD25), 50 (PD50), and 100 (PD100) mg/kg on glutathione levels in blood, liver, kidney, and heart of rats. Experimental design
Blood (nmol/ml)
Liver (nmol/g tissue)
Kidney (nmol/g tissue)
Heart (nmol/g tissue)
Control Cis PD25 + Cis PD50 + Cis PD100 + Cis P value
47.60 ± 6.32a 37.11 ± 5.57b 41.69 ± 2.67ab 43.33 ± 3.93ab 45.24 ± 6.14a 0.019
11.59 ± 0.51a 8.13 ± 0.63d 9.15 ± 0.28c 9.81 ± 0.30b 10.17 ± 0.27b 0.000
9.24 ± 1.03a 6.86 ± 1.19b 6.94 ± 1.31ab 8.71 ± 0.79ab 9.07 ± 1.61ab 0.003
7.99 ± 0.53a 6.05 ± 0.73c 6.44 ± 0.75c 6.69 ± 0.93ab 7.35 ± 0.27ab 0.000
Values are mean ± Standard deviations; n = 6. In the same column values with different letters show statistically significant differences in blood (p < 0.05), kidney (p
