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Puerarin protects the rat liver against oxidative stress-mediated DNA damage and apoptosis induced by lead
Experimental and Toxicologic Pathology 64 (2012) 575–582
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Puerarin protects the rat liver against oxidative stress-mediated DNA damage and apoptosis induced by lead Chan-Min Liu a,∗ , Jie-Qiong Ma b , Yun-Zhi Sun a a b
School of Life Science, Xuzhou Normal University, No. 101, Shanghai Road, Tangshan New Area, Xuzhou City 221116, Jiangsu Province, PR China School of Chemical Engineering, China University of Mining and Technology, Xuzhou City 221008, Jiangsu Province, PR China
a r t i c l e
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Article history: Received 30 April 2010 Accepted 17 November 2010 Keywords: Puerarin Lead Oxidative stress DNA damage Apoptosis Rat liver
a b s t r a c t Puerarin (PU), a natural flavonoid, has been reported to have many benefits and medicinal properties. In this study, we valuated the protective effect of puerarin against lead-induced oxidative DNA damage and apoptosis in rat liver. A total of forty male Wistar rats (8-week-old) was divided into 4 groups: control group; lead-treated group (500 mg Pb/l as the only drinking fluid); lead + puerarin treated group (500 mg Pb/l as the only drinking fluid plus 400 mg PU/kg bwt intra-gastrically once daily); and puerarintreated group (400 mg PU/kg bwt intra-gastrically once daily). The experimental period was lasted for 75 successive days. Our data showed that puerarin significantly effectively improved the lead-induced histology changes in rat liver and decreased the serum ALT and AST activities in lead-treated rats. Puerarin markedly restored Cu/Zn-SOD, CAT and GPx activities and the GSH/GSSG ratio in the liver of lead-treated rat. Furthermore, the increase of 8-hydroxydeoxyguanosine induced by lead was effectively suppressed by puerarin. The enhanced caspase-3 activity in the rat liver induced by lead was also inhibited by puerarin. TUNEL assay showed that lead-induced apoptosis in rat liver was significantly inhibited by puerarin, which might be attributed to its antioxidant property. In conclusion, these results suggested that puerarin could protect the rat liver against lead-induced injury by reducing ROS production, renewing the activities of antioxidant enzymes and decreasing DNA oxidative damage. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved.
1. Introduction Puerarin, a major isoflavone compound isolated from Pueraria lobata, has a variety of biological actions in cardiovascular diseases, gynecology disease, osteoporosis, cognitive capability, diabetic nephropathy (Zhang et al., 2006; Yeung et al., 2006; Sun et al., 2007; Li et al., 2009). Many reports have demonstrated that puerarin possesses a lot of activities including anti-oxidative activity (Guerra et al., 2000; Yan et al., 2006; Xiong et al., 2006; Han et al., 2007; Wu et al., 2007; Chung et al., 2008; Chang et al., 2009; Zhao et al., 2010), anti-inflammation (Yang et al., 2010; Kim et al., 2010; Zheng et al., 2008, 2009; Singh et al., 2007a,b) and anti-apoptosis (Xiong et al., 2006; Cheng et al., 2009; Bo et al., 2005; Zheng and Xu, 2007; Mercer et al., 2005). Increasing evidence shows that puerarin can protect liver from injury induced by hepatoxins (Guerra et al., 2000; Chung et al., 2008; Wu et al., 2007; Zheng et al., 2008, 2009;
Abbreviations: PU, puerarin; PbAc, lead-acetate; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; 8-OHdG, 8-hydroxy-2deoxyguanosine; TUNEL, deoxyribonucleotidyl transferase (TdT)-mediated dUTPfluorescein isothiocyanate (FITC) nick-end labeling. ∗ Corresponding author. Tel.: +86 516 83500170; fax: +86 516 83500171. E-mail addresses: [email protected], [email protected] (C.-M. Liu).
Zhao et al., 2010; Hwang et al., 2007; Yang et al., 2009; Benlhabib et al., 2004; Zhang et al., 2006). Lead (Pb) is one of the most widely used metals in industries and in many countries exposure to lead continues to be a widespread problem. Because it cannot be rendered harmless by chemical or biological remediation processes, lead is particularly worrisome among the environmental toxins (Xiang et al., 2010). Many investigations have indicated that lead exposure could induce a wide range of biochemical and physiological dysfunctions in humans and laboratory animals (Courtois et al., 2003). Mechanisms of leadinduced liver injury were to increase production of reactive oxygen species (ROS), and to induce oxidative stress, excitotoxicity, DNA damage and apoptosis (Sivaprasad et al., 2004; Jurczuk et al., 2007; Sieg and Billings, 1997; Xu et al., 2006, 2008; Franco et al., 2009; Pulido and Parrish, 2003). Reports from our laboratory and others have demonstrated that lead can alter Bcl-2/Bax ratio in liver, and these effects were shown to be associated with ROS formation resulting in caspase-3 dependent apoptosis (Liu et al., 2010; Pulido and Parrish, 2003; Gargioni et al., 2006; Xu et al., 2008; Franco et al., 2009). However, the molecular mechanisms of lead-induced liver injury and hepatoprotective effects of puerarin are not yet completely understood. The aim of this study was to investigate the possible hepatoprotective mechanisms of puerarin against leadinduced oxidative DNA damage and apoptosis in rat liver.
0940-2993/$ – see front matter. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2010.11.016
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2. Materials and methods
2.3. Assay of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity
2.1. Animals and treatment Adult male Wistar rats (8-week-old weighing approximately 170 g) were purchased from the Branch of National Breeder Center of Rodents (Shanghai). The rats were maintained under constant conditions (23 ± 1 ◦ C and 60% humidity) and had free access to rodent food and tap water under 12 h light/dark schedule (lights on from 08:30 to 20:30 h) (Liu et al., 2010). After acclimatization to the laboratory conditions, the animals were randomly divided into four groups (ten rats in each). (1) Control group, the rats received lead-free redistilled water and daily given physiological saline (0.9% NaCl) by oral gavage during the whole course of the experiment; (2) lead-treated group, animals received an aqueous solution of lead acetate (Pb(CH3 COO)2 ) (Sigma–Aldrich, MO, USA) at a concentration of 500 mg Pb/l as the only drinking fluid (Jurczuk et al., 2007; Liu et al., 2010; Moniuszko-Jakoniuk et al., 2003); (3) lead + puerarin treated group, animals received an aqueous solution of lead acetate (Pb(CH3 COO)2 ) (Sigma–Aldrich, MO, USA) (500 mg Pb/l in the drinking water) and received a daily oral gavage administration of puerarin (Sigma–Aldrich, MO, USA) at dose of 400 mg/((kg day) body weight) dissolved in distilled water (Zhang et al., 2006); (4) puerarin treated group, animals received a daily oral gavage administration of puerarin (Sigma–Aldrich, MO, USA) at dose of 400 mg/((kg day) body weight) dissolved in distilled water. Puerarin (400 mg/kg day) was administrated orally during the lead exposure. The experiment lasted for 75 days. After the experiment termination, seven rats in each group were used for the biochemical analysis; the others were used for histological evaluations. Rats were sacrificed and 10 ml of blood was drawn from heart. The serum was collected after centrifugation at 5000 rpm for 10 min and stored at −70 ◦ C freezer for further analysis. The liver was removed quickly and placed in ice-cold 0.9% NaCl solution, perfused with the physiological saline solution to remove blood cells, blotted on filter paper. And then the removed liver was immediately collected, respectively for experiments or stored at −70 ◦ C for later use. The dose of puerarin was selected on the basis of the previous studies (Zhang et al., 2006) and our preliminary experiment. The dose that rats consumed a solution of Pb (500 mg/l) as only drinking water exposed to lead in drinking water was selected on the basis of previous studies on the effect of lead exposure on hepatic peroxidative damage to simulate drinking water lead exposure (Jurczuk et al., 2007; Moniuszko-Jakoniuk et al., 2003; Liu et al., 2010). It had been reported that lead concentrations in the blood of rats continuously intoxicated with 500 mg Pb/l are within the range of values noted in lead workers (Jurczuk et al., 2007). The present research reported in this paper was conducted in accordance with the Chinese legislation on the use and care of laboratory animals and were approved by the respective university committees for animal experiments.
The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed by the method of Reitman and Frankel (1957). 2.4. Assay of ROS level ROS was measured as described previously, based on the oxidation of 2 7 -dichlorodihydrofluorescein diacetate to 2 7 dichloro-fluorescein (Shinomol and Muralidhara, 2007). Briefly, the homogenate was diluted 1:20 times with ice-cold Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3 , 2.0 mM CaCl2 , 10 mM d-glucose, and 5 mM HEPES, pH 7.4) to obtain a concentration of 5 mg tissue/ml. The reaction mixture (1 ml) containing Locke’s buffer (pH 7.4), 0.2 ml homogenate and 10 ml of DCFH-DA (5 mM) was incubated for 15 min at room temperature to allow the DCFHDA to be incorporated into any membrane-bound vesicles and the diacetate group cleaved by esterases. After 30 min of further incubation, the conversion of DCFH-DA to the fluorescent product DCF was measured using a spectrofluorimeter with excitation at 484 nm and emission at 530 nm. Background fluorescence (conversion of DCFH-DA in the absence of homogenate) was corrected by the inclusion of parallel blanks. ROS formation was quantified from a DCF-standard curve and data are expressed as pmol DCF formed/min/mg protein. 2.5. Assay of lipid peroxidation level Chemicals, including n-butanol, thiobarbutiric acid, 1,1,3,3tetramethoxypropane and all other reagents, were purchased from Sigma Chemical Company (St. Louis, MO, USA). The level of malondialdehyde (MDA) concentrations (as a marker of lipid peroxidation) in liver tissue homogenates was determined using the method of Uchiyama and Mihara (1978). MDA levels were expressed as nmol/mg protein. 2.6. Assay of liver reduced glutathione (GSH) level and oxidized glutathione (GSSG) level Liver GSH and GSSG levels were measured using the commercial kits (Jiancheng Institute of Biotechnology, Nanjing, China) (Qu et al., 2009). GSH and GSSG content was expressed as nmol/mg protein. 2.7. Assay of Cu/Zn-SOD activity Superoxide dismutase (SOD) kits were purchased from Jiancheng Institute of Biotechnology (Nanjing, China) (Qu et al., 2009). Data were expressed as units of SOD activity per milligram of protein. 2.8. Assay of CAT activity
2.2. Histological evaluations The rats were perfused transcardially with 100 ml of normal saline (0.9%). The liver tissues were fixed in a fresh solution of 4% paraformaldehyde (pH 7.4) at 4 ◦ C for 24 h, incubated overnight at 4 ◦ C in 100 mM sodium phosphate buffer (pH 7.4) containing 30% sucrose; and embedded in optimal cutting temperature (OCT) compound (Leica, CA, Germany). Cryosections were collected on 3aminopropyl-trimethoxysilane-coated slides (Sigma–Aldrich). The liver slices were stained with hematoxylin and eosin, and examined by an expert in liver pathology (S.M.) blinded to the type of treatment received by the animals.
Catalase (CAT) activity was assayed by the method of Aebi (1984). CAT activity was calculated as nM H2 O2 consumed/min/mg of tissue protein. 2.9. Assay of GPx activity The glutathione peroxidases (GPx) activity assay was based on the method of Paglia and Valentine (1967). Tertbutylhydroperoxide was used as substrate. GPx activity was computed using the molar extinction coefficient of 6.22 mM−1 cm−1 . One unit of GPx was defined as the amount
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of enzyme that catalyzed the oxidation of 1.0 mol of NADPH to NADP+ per minute at 25 ◦ C. 2.10. Caspase-3 enzymatic activity assay The caspase-3 enzymatic activity in the nuclear fraction was carried out by caspase-3 colorimetric kit (R&D Systems, USA). The analysis was done according to the manufacturer’s instructions. The results were expressed as ng/mg protein (Kuhad and Chopra, 2009). 2.11. Deoxyribonucleotidyl transferase (TdT)-mediated dUTP-fluorescein isothiocyanate (FITC) nick-end labeling (TUNEL) assay For the TUNEL staining, the standard protocol for frozen sections was followed (BD ApoAlertTM DNA Fragmentation assay kit, BD Biosciences Clontech, Palo Alto, CA, USA). The sections were immersed in a Coplin jar (VWR International, Aurora, CO, USA) containing fresh 4% formaldehyde/PBS, and incubated at room temperature for 5 min. The sections were washed twice with PBS for 5 min. The liquid was allowed to drain thoroughly, and the slides were placed on a flat surface. Each section was covered with 100 ml of 20 mg/ml proteinase K solution (section III) and incubated at 37 ◦ C for 5 min. After two washes of 5 min each with PBS, the sections were transferred into a Coplin jar containing 4% formaldehyde/PBS and then washed in PBS again. The cells were covered in equilibration buffer (from the kit) and equilibrated at room temperature for 5 min. The equilibration buffer was drained, and TdT incubation buffer was added to the tissue sections. To perform the tailing reaction, the slice was placed in a dark and humidified 37 ◦ C incubator for 1 h. The tailing reaction was terminated by immersing the samples in 2× saline-sodium citrate (SSC) at room temperature for 15 min. Samples were washed three times with PBS for 5 min to remove unincorporated fluorescein-dUTP. Finally, strong, nuclear green fluorescence in apoptotic cells were observed on a fluorescent microscopy equipped with a standard fluorescein filter (520 nm ± 20 nm). All cells stained with propidium iodide exhibit strong red cytoplasmic fluorescence when viewed at 620 nm. Specimens were analyzed with a Zeiss Axioskop 40TM microscope equipped for light microscopy (Carl Zeiss, Oberkochen, Germany). The images were taken with a CCD camera (CoolSNAPTM Color, Photometrics, Roper Scientific Inc., Trenton, NJ, USA) and processed with Image-Pro® Plus 6.0 software (Media Cybernetics Inc., Newburyport, MA, USA). For analysis; plaque areas were excluded, and the number of stained cells in 0.01 mm2 was estimated by blind manual counting of seven regions located at a consistent position per section. 2.12. Statistic analysis All statistical analyses were performed using the SPSS software, version 11.5. A one-way analysis of variance (ANOVA; P < 0.05) was used to determine significant differences between groups and the individual comparisons were obtained by Turkey’s HSD post hoc test. Statistical significance was set at P ≤ 0.05. 3. Results 3.1. Effects of puerarin on activities of the aspartate and alanine aminotransferase (AST and ALT) in lead-treated rat liver In order to determine whether puerarin can attenuate the liver damage in the lead-treated rats, we measured the activities of ALT and AST (Fig. 1). In lead-treated rats, the activities of ALT and AST significantly increased by 281% and 120% as compared with vehicle controls, respectively (P < 0.01). However, the ALT and AST activities
Fig. 1. Effect of puerarin on lead-induced changes in hepatic functional markers in rats: (A) serum ALT and (B) serum AST. All values are expressed as mean ± S.E.M. (n = 7). **P < 0.01, compared with the control group; ## P < 0.01, vs. lead-treated group.
in the puerarin + lead group decreased by 48% and 30% as compared with lead-treated group, respectively (P < 0.01). There were no significant differences in the level of aminotransferase activity among the puerarin treated rats, the vehicle controls and puerarin + lead rats. 3.2. Effects of puerarin on histology changes in lead-treated rat liver As shown in Fig. 2, the results of histopathological evaluation showed that puerarin exhibited hepatoprotective effect against lead-induced liver injury. Lead treatment caused visible histology changes including structure damage hepatocellular necrosis, leukocyte infiltration in rat liver. In addition, the sinusoids between the plates of hepatocytes were markedly enlarged in the liver of lead-treated rats (Fig. 2B), and these histology changes were invisible in the liver of normal rats and the puerarin + lead treated rats (Fig. 2A and C). Whereas, puerarin significantly alleviated the liver damage in lead-treated rats, no appearance difference could be observed in the liver between the puerarin + lead group and the vehicle control rats (Fig. 2A and C). Compared with normal rats, there were no visible histopathological changes induced by puerarin in lead-treated rat liver and the puerarin treated rat liver (Fig. 2C and D). 3.3. Effects of puerarin on levels of ROS, MDA and ratio of GSH/GSSG in lead-treated rat liver The results showed that puerarin could decrease lead-induced ROS and MDA levels (Fig. 3). Hepatic levels of ROS were enhanced in the lead-treated rats by 77% and 25% as compared with vehicle controls, respectively (P < 0.01). Whereas, the hepatic ROS and MDA levels of the puerarin + lead treated rats were significantly reduced by 32% and 14% as compared with lead-treated rats (P < 0.01). There were no significant differences in the levels of ROS and MDA among the lead + puerarin treated rats, puerarin treated rats and the vehicle controls (Fig. 3A). Many studies suggested that the GSH/GSSG ratio might be an indicator of oxidative stress. As shown in Fig. 3C, the GSH/GSSG
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Fig. 2. Hematoxylin and eosin-stained liver slices from the treated rat. (A) The vehicle control rat; (B) rats treated with lead (500 mg Pb/l in the drinking water); (C) rats treated with lead (500 mg Pb/l in the drinking water) and fed with puerarin (400 mg PU/kg bwt intra-gastrically once daily); (D) rat fed with puerarin (400 mg PU/kg bwt intra-gastrically once daily). The black arrow indicates infiltrating leukocytes. The green arrow indicates hepatic cell necrosis. The yellow arrow indicates the enlarged sinusoids between the plates of hepatocytes. Original magnification, 10 × 10. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
ratio in lead-treated rats was markedly decreased as compared with that of the vehicle controls (P < 0.01). There were no significant differences in the GSH/GSSG ratio among the lead + puerarin treated rats, puerarin treated rats and the vehicle controls (Fig. 3C). 3.4. Effects of puerarin on activities of the antioxidant enzymes in lead-treated rat liver In order to determine whether puerarin can attenuate the oxidative damage in the liver of lead-treated rats, we measured the activities of major antioxidant enzymes, including Cu/Zn-SOD, CAT and GPx in rat liver. The results showed that puerarin could renew the activities of these antioxidant enzymes in the liver of leadtreated rats (Fig. 4). In lead-treated rats, hepatic Cu/Zn-SOD, CAT and GPx activities were significantly decreased by 31%, 46% and 42% as compared with vehicle controls, respectively (P < 0.01). However, the hepatic Cu/Zn-SOD, CAT and GPx activities in the puerarin + lead treated rats was markedly increased by 34%, 85% and 57% as compared with lead-treated rats, respectively (P < 0.01) (Fig. 4). Interestingly, there were no significant differences in hepatic activities of Cu/Zn-SOD, CAT and GPx among the lead + puerarin treated rats, puerarin treated rats and the vehicle controls (Fig. 4).
can attenuate the liver DNA damage in the lead-treated rats, we measured the level of 8-OHdG. As shown in Fig. 5A, the level of 8-OHdG in lead-treated rats was significantly increased by 41% as compared with vehicle controls (P < 0.01). Interestingly, puerarin could decrease the level of 8-OHdG in the liver by 18% (P < 0.01) as compared to the lead-treated rats. There were no significant differences in level of 8-OHdG among the control group, the puerarin group and the puerarin + lead group (Fig. 5A). 3.6. Effect of puerarin on the level of caspase-3 in rat liver Caspase-3 executes the final apoptotic processes in the nucleus that result in DNA and cell fragmentation. Caspase-3 activation is one of the biomarkers indicating mitochondrial dysfunction and oxidative stress-mediated cell apoptosis (Herrera et al., 2001). In order to determine whether puerarin can attenuate the oxidative DNA damage and apoptosis in the liver of lead-treated rats, the activity of caspase-3 was also examined. As shown in Fig. 5B, hepatic caspase-3 activity in the lead-treated rats was significantly elevated by 134% as compared with vehicle controls. In the present study, we found that puerarin could markedly decrease the activity of caspase-3 (30%) in lead-treated rat liver (P < 0.01). There were no significant differences in hepatic activities of caspase-3 among the control group, the puerarin group and the puerarin + lead group.
3.5. Effects of puerarin on level of 8-OHdG in the liver of lead-treated rats
3.7. Effect of puerarin on lead-induced apoptosis in rat liver
8-OHdG is one of such important oxidative DNA lesions formed by the oxidation of the C-8 position of 2 -deoxyguanosine, which has commonly been used as a biomarker of oxidative DNA damage (Toyokuni et al., 1995; Singh et al., 2007a,b; Pilger and Rudiger, 2006; Chen et al., 2010). In order to determine whether puerarin
We used the TUNEL assay to investigate the effect of puerarin on lead-induced apoptosis (Fig. 6). The number of TUNEL-positive cells in the liver of lead-treated rats was significantly increased (P < 0.01). There were significantly fewer TUNEL-positive cells in the liver of rats co-treated with lead and puerarin as compared with the
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Fig. 3. Effect of puerarin on the levels of ROS and MDA and ratio of GSH/GSSG in lead-treated rat liver. (A) Level of ROS; (B) level of MDA; (C) ratio of GSH/GSSG. Each value is expressed as mean ± S.E.M. (n = 7). **P < 0.01, compared with the control group; ## P < 0.01, vs. lead-treated group.
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Fig. 4. Effect of puerarin on the activities of antioxidant enzymes in lead-treated rat liver. (A) Cu/Zn-SOD activity; (B) CAT activity; (C) GPx activity. All values are expressed as mean ± S.E.M. (n = 7). **P < 0.01, compared with the control group; ## P < 0.01, vs. lead-treated group.
rats treated with lead (P < 0.01). However, there were no significant differences in the number of TUNEL-positive cells in the liver among the control group, the puerarin group and the puerarin + lead group (Fig. 6). 4. Discussion Liver is the primary organ site for xenobiotic metabolism. In most cases, the metabolic process is accomplished without injury to the liver itself, whereas many compounds are toxic themselves or produce metabolites that can cause liver injury. Accumulating evidence have demonstrated that low, medium or high dose of lead can cause liver injury. It was also shown that lead causes oxidative stress by inducing the generation of reactive oxygen species (ROS), including hydroperoxides, singlet oxygen, hydrogen peroxide and superoxide (Sivaprasad et al., 2004; Jurczuk et al., 2007; Liu et al., 2010; Franco et al., 2009; Pulido and Parrish, 2003). In the present study, we demonstrated overproduction of ROS by lead. ROS could damage DNA, proteins and lipids within cells, which led to liver injury (Franco et al., 2009; Liu et al., 2010). In lead-treated animals, the histological changes of liver, such as structure damage, hepatocellular necrosis, leukocyte infiltration and massive hemorrhage, had been observed (Campana et al., 2003; Liu et al., 2010; Narayana and Al-Bader, 2010; El-Neweshy and El-Sayed, 2010). Puerarin has been used for various medicinal purposes in traditional oriental medicine for thousands of years. Even with high doses, puerarin had safety and tolerability profiles to animals (Zhang et al., 2006; Wang et al., 2008). In this study, puerarin by oral gavage administration markedly decreased activities of serum ALT and AST in lead-treated
Fig. 5. Effect of puerarin on the level of 8-OHdG and the activity of caspase-3 in lead-treated rat liver. (A) Level of 8-OHdG; (B) activity of caspase-3. Each value is expressed as mean ± S.E.M. (n = 7). **P < 0.01, compared with the control group; # P < 0.05 and ## P < 0.01, vs. lead-treated group.
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Fig. 6. (A) In situ detection of fragmented DNA [deoxyribonucleotidyl transferase-mediated dUTP-FITC nick-endlabeling (TUNEL) assay] in the liver of rat. The liver tissues were processed for TUNEL and photographed using a fluorescence microscope with either propidium iodide (PI) filter alone (left) or an FITC filter alone (middle). The merged images show that apoptotic cells appear yellow and non-apoptotic cells appear red (right). Scale bars = 100 mm. (B) The histogram showed the relative proportion of TUNEL-positive cells in the liver of rat. All values are expressed as mean ± S.E.M. **P < 0.01, compared with the control group; ## P < 0.01, vs. Pb-treated group.
rats (Fig. 1) and improved lead-induced histopathologic changes in rat liver (Fig. 2). These results suggest that puerarin could protect rat liver against lead-induced dysfunction and histopathologic damage. Recent studies indicated that elevation of severe oxidative stress biomarkers could be observed in the liver of lead-treated rodents (Sivaprasad et al., 2004; Zhang et al., 2006; Jurczuk et al., 2007; Liu et al., 2010). ROS and MDA are considered major oxidative stress markers. It have been reported that lead exposure could induce changes in the fatty acid composition of membrane (Knowles and Donaldson, 1990) and increase of MDA level in liver (Jurczuk et al., 2007; Xu et al., 2008; Liu et al., 2010). Lead induced arachidonic acid elongation might be due to the enhanced lipid peroxidation in the membrane because fatty acid chain length and unsaturation are associated with membrane susceptibility to peroxidation (Lawton
and Donaldson, 1991). In the present study, levels of MDA and ROS were markedly increased in lead-treated rat liver as compared with the controls, indicating that lead exposure induced oxidative stress. However, puerarin markedly decreased levels of MDA and ROS in lead-treated rat liver (Fig. 3A and B). This may be due to the powerful antioxidant and free radical scavenging activities (Xu, 2003; Hwang et al., 2007; Yang et al., 2009; Zhao et al., 2010). Our finding suggests that puerarin could at least partly attenuate oxidative stress by decreasing levels of ROS and lipid peroxide in lead-treated rat liver. Antioxidant enzymes can protect cellular compounds against damage induced by free radicals. Catalase (CAT), superoxide dismutases (SOD) and glutathione peroxidases (GPx) are important antioxidant enzymes (Boots et al., 2008). The SOD decomposes • superoxide radicals (O2 − ) and produce H2 O2 . H2 O2 is subsequently
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removed to water by CAT in peroxisomes, or by GPx oxidizing GSH in the cytosol (Dröge, 2002; Lee and Choi, 2003). Therefore, activities of these enzymes have been used to assess oxidative stress in cells. Whereas GPx, CAT and SOD depend on various essential trace elements for proper molecular structure and activity, these antioxidant enzymes are potential targets for lead toxicity (Gelman et al., 1978; Hsu and Guo, 2002). Many studies have indicated that lead can also alter antioxidant activities by inhibiting functional SH groups in several enzymes such as SOD, CAT and GPx, because of its high affinity for sulfhydryl (SH) groups in these enzymes (Hsu, 1981; McGowan and Donaldson, 1986; Chiba et al., 1996; Hsu and Guo, 2002). Moreover, if the balance between ROS production and antioxidant defenses is broken, the enzyme may be exhausted and its concentration therefore depleted. In the present study, activities of antioxidant enzymes in rat liver, including SOD, CAT and GPx, were dramatically decreased by the treatment with lead. It was demonstrated that lead exposure induced oxidative stress due to the inhibition of antioxidant enzymes activities. Interestingly, puerarin could markedly restore activities of those antioxidant enzymes in the liver of lead-treated rats (Fig. 4). GSH can act as a nonenzymatic antioxidant by direct interaction of SH group with ROS, or it can be involved in the enzymatic detoxification reaction for ROS, as a cofactor or a coenzyme, because it is a tripeptide containing cysteine that has a reactive SH group with reductive potency (Sivaprasad et al., 2004; Hsu and Guo, 2002). In this study, we observed significant decreases in GSH/GSSG ratio in the liver of lead-treated rats which is in agreement with several previous studies (Aykin-Burns et al., 2003; Sivaprasad et al., 2004; Flora et al., 2007; Liu et al., 2010). It may be due to the high ability of lead to bind with the sulfhydryl (SH) group of GSH and lead-induced oxidative stress. Interestingly, we found that puerarin markedly increased GSH level and GSH/GSSG ratio in lead-treated rat liver (Fig. 3B). It suggested that puerarin could at least partly attenuate oxidative stress by renewing the activities of antioxidant enzymes and increasing GSH/GSSG ratio in lead-treated rat liver. ROS are known to attack other cellular components such as lipids, leaving behind reactive species that in turn can couple to DNA bases (Marnett, 2000). The most extensively studied oxidative DNA lesion is 8-OHdG, which have been widely used as a sensitive ´ biomarker of DNA oxidation [Kinoshita et al., 2007; GałazynSidorczuk et al., 2009]. It was reported that copper, inorganic arsenic, methylmercury and iron overload can induce increase of 8-OHdG level in the liver (Toyokuni and Sagripanti, 1994; Kinoshita et al., 2007; Jin et al., 2008; Asare et al., 2008, 2009). In this study, the level of 8-OHdG was markedly increased in the liver of leadtreated rats, which correlated with the level of ROS, suggesting that DNA is a common target of ROS induced by lead in liver (Fig. 5A). Interestingly, the hepatic 8-OHdG level in the puerarin + lead group was markedly decreased as compared with lead-treated group. Our findings indicate that puerarin may play an effective role in protecting liver function from lead-induced oxidative DNA damage by decreasing 8-OHdG level in the liver of lead-treated rats. High ROS concentrations may contribute to the apoptotic cell death whenever they are generated in the context of apoptotic process (Liu et al., 2010; Franco et al., 2009; Pulido and Parrish, 2003; Gargioni et al., 2006; Xu et al., 2008). Based on labeling of DNA strand breaks in the liver of rats, the TUNEL method was used to analyze apoptotic cells in situ (Fig. 6). Recent studies from our laboratory have shown that pro-apoptotic BAK protein and cleave caspase-3 expression were increased after lead expose in rat liver (Liu et al., 2010). The present study also showed that lead increased the number of TUNEL-positive cells in the liver of rats (Fig. 6). Moreover, caspase-3 activity was significantly elevated in the lead-treated rat liver, suggesting that apoptosis induced by lead is occurred due to activation of caspase-3 (Pulido and Parrish, 2003; Gargioni et al., 2006; Xu et al., 2006; Franco et al., 2009; Kiran Kumar
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et al., 2009) (Fig. 5B). Our finding that puerarin could markedly decrease the number of TUNEL-positive cells and reduce caspase-3 activity in rats treated with lead suggests that puerarin has a potential hepatoprotective effect against lead-induced DNA damage and apoptosis due to its antioxidant activity. In conclusion, our results indicated that puerarin administration attenuated lead-induced hepatic dysfunction and histopathologic changes in the rat liver. Puerarin restored antioxidant enzymes activities, down-regulated levels of ROS, MDA and 8-OHdG and inhibited apoptosis due to suppression of caspase-3 activity. Furthermore, it consequently improved lead-induced oxidative DNA damage and apoptosis in the liver of rats. Recent studies indicated isoflavone compounds like genistein and daizein at high doses have been shown to possess estrogenic activity in animals and to promote reproductive cancer (Martfnez-Montemayor et al., 2010; Messina and Wood, 2008). However, there are fewer reports on the impact of puerarin administration on cancer risk. The question warrants further investigation. Acknowledgments This work is supported by the Fundamental Research Program of Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 07KJD180210), and Grants from Natural Science Foundation by Xuzhou Normal University (Nos. 08XLR08 and 09XLY06). References Aebi H. Catalase in vitro. Meth Enzymol 1984;105(1):121–6. Asare GA, Bronz M, Naidoo V, Kew MC. Synergistic interaction between excess hepatic iron and alcohol ingestion in hepatic mutagenesis. Toxicology 2008;254(1–2):11–8. Asare GA, Kew MC, Mossanda KS, Paterson AC, Siziba K, Kahler-Venter CP. Effects of exogenous antioxidants on dietary iron overload. J Clin Biochem Nutr 2009;44(1):85–94. Aykin-Burns N, Laegeler A, Kellogg G, Ercal N. Oxidative effects of lead in young and adult Fisher 344 rats. Arch Environ Contam Toxicol 2003;44(3):417–20. Benlhabib E, Baker JI, Keyler DE, Singh AK. Effects of purified puerarin on voluntary alcohol intake and alcohol withdrawal symptoms in P rats receiving free access to water and alcohol. J Med Food 2004;7(2):180–6. Bo J, Ming BY, Gang LZ, Lei C, Jia AL. Protection by puerarin against MPP+-induced neurotoxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction and caspase-3-like activation. Neurosci Res 2005;53(2):183–8. Boots AW, Haenen GR, Bast A. Health effects of quercetin: from antioxidant to nutraceutical. Eur J Pharmacol 2008;585(2–3):325–37. Campana O, Sarasquete C, Blasco J. Effect of lead on ALAD activity, metallothionein levels, and lipid peroxidation in blood, kidney, and liver of the toadfish Halobatrachus didactylus. Ecotoxicol Environ Saf 2003;55(1):116–25. Chang Y, Hsieh CY, Peng ZA, Yen TL, Hsiao G, Chou DS, et al. Neuroprotective mechanisms of puerarin in middle cerebral artery occlusion-induced brain infarction in rats. J Biomed Sci 2009;16(1):1–13. Chen H, Chu G, Cao Z, Liang J, Lu J, Zhou G. Magnetic bead-based approach to monitoring of cigarette smoke-induced DNA oxidation damage and screening of natural protective compounds. Talanta 2010;80(3):1216–21. Cheng YF, Zhu GQ, Wang M, Cheng H, Zhou A, Wang N, et al. Involvement of ubiquitin proteasome system in protective mechanisms of puerarin to MPP+-elicited apoptosis. Neurosci Res 2009;63(1):52–8. Chiba M, Shinohara A, Matsushita K, Watanabe H, Inaba Y. Indices of lead-exposure in blood and urine of lead-exposed workers and concentrations of major and trace elements and activities of SOD, GSH-Px and catalase in their blood. Tohoku J Exp Med 1996;178(1):49–62. Chung MJ, Sung NJ, Park CS, Kweon DK, Mantovani A, Moon TW, et al. Antioxidative and hypocholesterolemic activities of water-soluble puerarin glycosides in HepG2 cells and in C57BL/6J mice. Eur J Pharmacol 2008;578(2–3):159–70. Courtois E, Marques M, Barrientos A. Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxygenase-2. J Am Soc Nephrol 2003;14(6):1464–70. Dröge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82(1):47–95. El-Neweshy MS, El-Sayed YS. Influence of vitamin C supplementation on leadinduced histopathological alterations in male rats. Exp Toxicol Pathol 2010. Flora SJ, Saxena G, Mehta A. Reversal of lead-induced neuronal apoptosis by chelation treatment in rats: role of reactive oxygen species and intracellular Ca(2+). J Pharmacol Exp Ther 2007;322(1):108–16. Franco R, Sánchez-Olea R, Reyes-Reyes EM, Panayiotidis MI. Environmental toxicity, oxidative stress and apoptosis: ménage à trois. Mutat Res 2009;674(1–2):3–22.
582
C.-M. Liu et al. / Experimental and Toxicologic Pathology 64 (2012) 575–582
´ M, Brzóska MM, Jurczuk M, Moniuszko-Jakoniuk J. Oxidative Gałazyn-Sidorczuk damage to proteins and DNA in rats exposed to cadmium and/or ethanol. Chem Biol Interact 2009;180(1):31–8. Gargioni R, Filipak Neto F, Buchi DF, Randi MAF, Franco CRC, Paludo KS, et al. Cell death and DNA damage in peritoneal macrophages of mice (Mus musculus) exposed to inorganic lead. Cell Biol Int 2006;30(7):615–23. Gelman BB, Michaelson IA, Bus JS. The effect of lead on oxidative hemolysis and erythrocyte defense mechanisms in the rat. Toxicol Appl Pharmacol 1978;45(1):119–29. Guerra MC, Speroni E, Broccoli M, Cangini M, Pasini P, Minghetti A, et al. Comparison between Chinese medical herb Pueraria lobata crude extract and its main isoflavone puerarin antioxidant properties and effects on rat liver CYP-catalysed drug metabolism. Life Sci 2000;67(24):2997–3006. Han RM, Tian YX, Becker EM, Andersen ML, Zhang JP, Skibsted LH. Puerarin and conjugate bases as radical scavengers and antioxidants: molecular mechanism and synergism with -carotene. J Agric Food Chem 2007;55(6):2384–91. Herrera B, Alvarez AM, Sanchez A, Fernandez M, Roncero C, Benito M, et al. Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor {beta} in fetal hepatocytes. FASEB J 2001;15(3):741–51. Hsu JM. Lead toxicity related to glutathione metabolism. J Nutr 1981;111(1):26–33. Hsu PC, Guo YL. Antioxidant nutrients and lead toxicity. Toxicology 2002;180(1):33–44. Hwang YP, Choi CY, Chung YC, Jeon SS, Jeong HG. Protective effects of puerarin on carbon tetrachloride-induced hepatotoxicity. Arch Pharm Res 2007;30(10):1309–17. Jin X, Chan HM, Lok E, Kapal K, Taylor M, Kubow S, et al. Dietary fats modulate methylmercury-mediated systemic oxidative stress and oxidative DNA damage in rats. Food Chem Toxicol 2008;46(5):1706–20. Jurczuk M, Moniuszko-Jakoniuk J, Brzóska MM. Hepatic and renal concentrations of vitamins E and C in lead- and ethanol-exposed rats. An assessment of their involvement in the mechanisms of peroxidative damage. Food Chem Toxicol 2007;45(8):1478–86. Kim KM, Jung DH, Jang DS, Kim YS, Kim JM, Kim HN, et al. Puerarin suppresses AGEs-induced inflammation in mouse mesangial cells: a possible pathway through the induction of heme oxygenase-1 expression. Toxicol Appl Pharmacol 2010;244(2):106–13. Kinoshita A, Wanibuchi H, Wei M, Yunoki T, Fukushima S. Elevation of 8hydroxydeoxyguanosine and cell proliferation via generation of oxidative stress by organic arsenicals contributes to their carcinogenicity in the rat liver and bladder. Toxicol Appl Pharmacol 2007;221(3):295–305. Kiran Kumar B, Prabhakara Rao Y, Noble T, Weddington K, McDowell VP, Sharada R, et al. Lead-induced alteration of apoptotic proteins in different regions of adult rat brain. Toxicol Lett 2009;184(1):56–60. Knowles SO, Donaldson WE. Dietary modification of lead toxicity: effects on fatty acid and eicosanoid metabolism in chicks. Comp Biochem Physiol 1990;95(1):99–104. Kuhad A, Chopra K. Attenuation of diabetic nephropathy by tocotrienol: involvement of NFkB signaling pathway. Life Sci 2009;84(9–10):296–301. Lawton L, Donaldson WE. Lead-induced tissue fatty acid alterations and lipid peroxidation. Biol Trace Elem Res 1991;28(2):83–97. Lee PJ, Choi AMK. Pathways of cell signaling in hyperoxia. Free Radic Biol Med 2003;35(4):341–50. Li Q, Xiao Y, Gong H, Shen D, Zhu F, Wu Q, et al. Effect of puerarin on the expression of extracellular matrix in rats with streptozotocin-induced diabetic nephropathy. Natl Med J India 2009;22(1):9–12. Liu CM, Zheng YL, Lu J, Zhang ZF, Fan SH, Wu DM, et al. Quercetin protects rat liver against lead-induced oxidative stress and apoptosis. Environ Toxicol Pharmacol 2010;29(2):158–66. Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis 2000;21(3):361–70. Martfnez-Montemayor MM, Otero-Franqui E, Martinez J, De La Mota-Peynado A, Cubano LA, Dharmawardhane S. Individual and combinedsoy isoflavones exert differential effects on metastatic cancer progression. Clin Exp Metastasis 2010;27(7):465–80. McGowan C, Donaldson WE. Changes in organ nonprotein sulfhydryl and glutathione concentrations during acute and chronic administration of inorganic lead to chicks. Biol Trace Elem Res 1986;10(1):37–46. Mercer LD, Kelly BL, Horne MK, Beart PM. Dietary polyphenols protect dopamine neurons from oxidative insults and apoptosis: investigations in primary rat mesencephalic cultures. Biochem Pharmacol 2005;69(2):339–45. Messina MJ, Wood CE. Soy isoflavones, estrogen therapy, and breast cancer risk: analysis and commentary. Nutr J 2008;7(17):1–12. ´ Moniuszko-Jakoniuk J, Jurczuk M, Gałazyn-Sidorczuk M, Brzóska MM. Lead turnover and changes in the body status of chosen micro- and macroelements in rats exposed to lead and ethanol. Pol J Environ Stud 2003;12(1):335–44. Narayana K, Al-Bader M. Ultrastructural and DNA damaging effects of lead nitrate in the liver. Exp Toxicol Pathol 2010. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70(1):158– 69.
Pilger A, Rudiger HW. 8-hydroxydeoxyguanosine as a marker of oxidative damage related to occupational and environmental exposure. Int Arch Occup Environ Health 2006;80(1):1–15. Pulido MD, Parrish AR. Metal-induced apoptosis: mechanisms. Mutat Res 2003;533(1–2):227–41. Qu XJ, Xia X, Wang YS, Song MJ, Liu LL, Xie YY, et al. Protective effects of salvia plebeia compound homoplantaginin on hepatocyte injury. Food Chem Toxicol 2009;47(7):1710–5. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957;28(1):56–63. Shinomol GK, Muralidhara. Differential induction of oxidative impairments in brain regions of male mice following subchronic consumption of Khesari dhal (Lathyrus sativus) and detoxified Khesari dhal. Neurotoxicology 2007;28:798–806. Sieg DJ, Billings RE. Lead/cytokine-mediated oxidative DNA damage in cultured mouse hepatocytes. Toxicol Appl Pharmacol 1997;142(1):106–15. Singh AK, Jiang Y, Benlhabib E, Gupta S. Herbal mixtures consisting of puerarin and either polyenylphosphatidylcholine or curcumin provide comprehensive protection against alcohol-related disorders in P rats receiving free choice water and 15% ethanol in pure water. J Med Food 2007a;10(3):526–42. Singh R, Sram RJ, Binkova B, Kalina I, Popov TA, Georgieva T, et al. The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans. Mutat Res 2007b;620(1–2):83–92. Sivaprasad R, Nagaraj M, Varalakshmi P. Combined efficacies of lipoic acid and 2,3dimercaptosuccinic acid against lead-induced lipid peroxidation in rat liver. J Nutr Biochem 2004;15(1):18–23. Sun XH, Ding JP, Li H, Pan N, Gan L, Yang XL, et al. Activation of large-conductance calcium-activated potassium channels by puerarin: the underlying mechanism of puerarin-mediated vasodilation. J Pharmacol Exp Ther 2007;323(1):391–7. Toyokuni S, Okamoto K, Yodoi J, Hiai H. Persistent oxidative stress in cancer. FEBS Lett 1995;358(1):1–3. Toyokuni S, Sagripanti JL. Increased 8-hydroxydeoxyguanosine in kidney and liver of rats continuously exposed to copper. Toxicol Appl Pharmacol 1994;126(1):91–7. Uchiyama M, Mihara M. Determination of malondialdehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978;86(1):271–8. Wang Y, Yin L, Li Y, Liu P, Cui Q. Preventive Effects of Puerarin on Alcohol-induced Osteonecrosis. Clin Orthop Relat Res 2008;466(5):1059–67. Wu L, Qiao H, Li Y, Li L. Protective roles of puerarin and Danshensu on acute ischemic myocardial injury in rats. Phytomedicine 2007;14(10):652–8. Xiang JJ, Zhai YF, Tang Y, Wang H, Liu B, Guo CW. A competitive indirect enzymelinked immunoassay for lead ion measurement using mAbs against the leadDTPA complex. Environ Pollut 2010;158(5):1376–80. Xiong FL, Sun XH, Gan L, Yang XL, Xu HB. Puerarin protects rat pancreatic islets from damage by hydrogen peroxide. Eur J Pharmacol 2006;529(1–3):1–7. Xu J, Ji LD, Xu LH. Lead-induced apoptosis in PC12 cells: involvement of p53, Bcl-2 family and caspase-3. Toxicol Lett 2006;166(2):160–7. Xu J, Lian LJ, Wu C, Wang XF, Fu WY, Xu LH. Lead induces oxidative stress, DNA damage and alteration of p53, Bax and Bcl-2 expressions in mice. Food Chem Toxicol 2008;46(5):1488–94. Xu XH. Effects of puerarin on fatty superoxide in aged mice induced by d-galactose. Zhongguo Zhong Yao Za Zhi 2003;28(1):66–9. Yan LP, Chan SW, Chan AS, Chen SL, Ma XJ, Xu HX. Puerarin decreases serum total cholesterol and enhances thoracic aorta endothelial nitric oxide synthase expression in diet-induced hypercholesterolemic rats. Life Sci 2006;79(4):324–30. Yang S, Lou JL, Wang Q. Effect of puerarin on liver injury in KKAy mice with type 2 diabetes mellitus. Zhongguo Zhong Xi Yi Jie He Za Zhi 2009;29(8):707–10. Yang X, Hu W, Zhang Q, Wang Y, Sun L. Puerarin inhibits C-reactive protein expression via suppression of nuclear factor kappaB activation in lipopolysaccharide-induced peripheral blood mononuclear cells of patients with stable angina pectoris. Basic Clin Pharmacol Toxicol 2010. Yeung DK, Leung SW, Xu YC, Vanhoutte PM, Man RY. Puerarin, an isoflavonoid derived from Radix puerariae, potentiates endothelium-independent relaxation via the cyclic AMP pathway in porcine coronary artery. Eur J Pharmacol 2006;552(1–3):105–11. Zhang SH, Ji G, Liu JW. Reversal of chemical-induced liver fibrosis in Wistar rats by puerarin. J Nutr Biochem 2006;17(7):485–91. Zhao M, Du YQ, Yuan L, Wang NN. Protective effect of puerarin on acute alcoholic liver injury. Am J Chin Med 2010;38(2):241–9. Zheng P, Ji G, Ma Z, Liu T, Xin L, Wu H, et al. Therapeutic effect of puerarin on non-alcoholic rat fatty liver by improving leptin signal transduction through JAK2/STAT3 pathways. Am J Chin Med 2009;37(1):69–83. Zheng PY, Ma ZS, Hua YQ, Liu T, Xing LJ, Ji G. Effects of puerarin on expressions of leptin receptor mRNA, phosphorylated Janus kinase 2/phosphorylated signal transducers and activators of transcription 3 proteins in the liver of rats with non-alcoholic fatty liver. Zhong Xi Yi Jie He Xue Bao 2008;6(9):921–7. Zheng X, Xu X. Potential involvement of calcium and nitric oxidein protective effects of puerarin on oxygen–glucose deprivation in cultured hippocampal neurons. J Ethnopharmacol 2007;113(3):421–6.
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Puerarin protects the rat liver against oxidative stress-mediated DNA damage and apoptosis induced by lead Chan-Min Liu a,∗ , Jie-Qiong Ma b , Yun-Zhi Sun a a b
School of Life Science, Xuzhou Normal University, No. 101, Shanghai Road, Tangshan New Area, Xuzhou City 221116, Jiangsu Province, PR China School of Chemical Engineering, China University of Mining and Technology, Xuzhou City 221008, Jiangsu Province, PR China
a r t i c l e
i n f o
Article history: Received 30 April 2010 Accepted 17 November 2010 Keywords: Puerarin Lead Oxidative stress DNA damage Apoptosis Rat liver
a b s t r a c t Puerarin (PU), a natural flavonoid, has been reported to have many benefits and medicinal properties. In this study, we valuated the protective effect of puerarin against lead-induced oxidative DNA damage and apoptosis in rat liver. A total of forty male Wistar rats (8-week-old) was divided into 4 groups: control group; lead-treated group (500 mg Pb/l as the only drinking fluid); lead + puerarin treated group (500 mg Pb/l as the only drinking fluid plus 400 mg PU/kg bwt intra-gastrically once daily); and puerarintreated group (400 mg PU/kg bwt intra-gastrically once daily). The experimental period was lasted for 75 successive days. Our data showed that puerarin significantly effectively improved the lead-induced histology changes in rat liver and decreased the serum ALT and AST activities in lead-treated rats. Puerarin markedly restored Cu/Zn-SOD, CAT and GPx activities and the GSH/GSSG ratio in the liver of lead-treated rat. Furthermore, the increase of 8-hydroxydeoxyguanosine induced by lead was effectively suppressed by puerarin. The enhanced caspase-3 activity in the rat liver induced by lead was also inhibited by puerarin. TUNEL assay showed that lead-induced apoptosis in rat liver was significantly inhibited by puerarin, which might be attributed to its antioxidant property. In conclusion, these results suggested that puerarin could protect the rat liver against lead-induced injury by reducing ROS production, renewing the activities of antioxidant enzymes and decreasing DNA oxidative damage. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved.
1. Introduction Puerarin, a major isoflavone compound isolated from Pueraria lobata, has a variety of biological actions in cardiovascular diseases, gynecology disease, osteoporosis, cognitive capability, diabetic nephropathy (Zhang et al., 2006; Yeung et al., 2006; Sun et al., 2007; Li et al., 2009). Many reports have demonstrated that puerarin possesses a lot of activities including anti-oxidative activity (Guerra et al., 2000; Yan et al., 2006; Xiong et al., 2006; Han et al., 2007; Wu et al., 2007; Chung et al., 2008; Chang et al., 2009; Zhao et al., 2010), anti-inflammation (Yang et al., 2010; Kim et al., 2010; Zheng et al., 2008, 2009; Singh et al., 2007a,b) and anti-apoptosis (Xiong et al., 2006; Cheng et al., 2009; Bo et al., 2005; Zheng and Xu, 2007; Mercer et al., 2005). Increasing evidence shows that puerarin can protect liver from injury induced by hepatoxins (Guerra et al., 2000; Chung et al., 2008; Wu et al., 2007; Zheng et al., 2008, 2009;
Abbreviations: PU, puerarin; PbAc, lead-acetate; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; 8-OHdG, 8-hydroxy-2deoxyguanosine; TUNEL, deoxyribonucleotidyl transferase (TdT)-mediated dUTPfluorescein isothiocyanate (FITC) nick-end labeling. ∗ Corresponding author. Tel.: +86 516 83500170; fax: +86 516 83500171. E-mail addresses: [email protected], [email protected] (C.-M. Liu).
Zhao et al., 2010; Hwang et al., 2007; Yang et al., 2009; Benlhabib et al., 2004; Zhang et al., 2006). Lead (Pb) is one of the most widely used metals in industries and in many countries exposure to lead continues to be a widespread problem. Because it cannot be rendered harmless by chemical or biological remediation processes, lead is particularly worrisome among the environmental toxins (Xiang et al., 2010). Many investigations have indicated that lead exposure could induce a wide range of biochemical and physiological dysfunctions in humans and laboratory animals (Courtois et al., 2003). Mechanisms of leadinduced liver injury were to increase production of reactive oxygen species (ROS), and to induce oxidative stress, excitotoxicity, DNA damage and apoptosis (Sivaprasad et al., 2004; Jurczuk et al., 2007; Sieg and Billings, 1997; Xu et al., 2006, 2008; Franco et al., 2009; Pulido and Parrish, 2003). Reports from our laboratory and others have demonstrated that lead can alter Bcl-2/Bax ratio in liver, and these effects were shown to be associated with ROS formation resulting in caspase-3 dependent apoptosis (Liu et al., 2010; Pulido and Parrish, 2003; Gargioni et al., 2006; Xu et al., 2008; Franco et al., 2009). However, the molecular mechanisms of lead-induced liver injury and hepatoprotective effects of puerarin are not yet completely understood. The aim of this study was to investigate the possible hepatoprotective mechanisms of puerarin against leadinduced oxidative DNA damage and apoptosis in rat liver.
0940-2993/$ – see front matter. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2010.11.016
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2. Materials and methods
2.3. Assay of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity
2.1. Animals and treatment Adult male Wistar rats (8-week-old weighing approximately 170 g) were purchased from the Branch of National Breeder Center of Rodents (Shanghai). The rats were maintained under constant conditions (23 ± 1 ◦ C and 60% humidity) and had free access to rodent food and tap water under 12 h light/dark schedule (lights on from 08:30 to 20:30 h) (Liu et al., 2010). After acclimatization to the laboratory conditions, the animals were randomly divided into four groups (ten rats in each). (1) Control group, the rats received lead-free redistilled water and daily given physiological saline (0.9% NaCl) by oral gavage during the whole course of the experiment; (2) lead-treated group, animals received an aqueous solution of lead acetate (Pb(CH3 COO)2 ) (Sigma–Aldrich, MO, USA) at a concentration of 500 mg Pb/l as the only drinking fluid (Jurczuk et al., 2007; Liu et al., 2010; Moniuszko-Jakoniuk et al., 2003); (3) lead + puerarin treated group, animals received an aqueous solution of lead acetate (Pb(CH3 COO)2 ) (Sigma–Aldrich, MO, USA) (500 mg Pb/l in the drinking water) and received a daily oral gavage administration of puerarin (Sigma–Aldrich, MO, USA) at dose of 400 mg/((kg day) body weight) dissolved in distilled water (Zhang et al., 2006); (4) puerarin treated group, animals received a daily oral gavage administration of puerarin (Sigma–Aldrich, MO, USA) at dose of 400 mg/((kg day) body weight) dissolved in distilled water. Puerarin (400 mg/kg day) was administrated orally during the lead exposure. The experiment lasted for 75 days. After the experiment termination, seven rats in each group were used for the biochemical analysis; the others were used for histological evaluations. Rats were sacrificed and 10 ml of blood was drawn from heart. The serum was collected after centrifugation at 5000 rpm for 10 min and stored at −70 ◦ C freezer for further analysis. The liver was removed quickly and placed in ice-cold 0.9% NaCl solution, perfused with the physiological saline solution to remove blood cells, blotted on filter paper. And then the removed liver was immediately collected, respectively for experiments or stored at −70 ◦ C for later use. The dose of puerarin was selected on the basis of the previous studies (Zhang et al., 2006) and our preliminary experiment. The dose that rats consumed a solution of Pb (500 mg/l) as only drinking water exposed to lead in drinking water was selected on the basis of previous studies on the effect of lead exposure on hepatic peroxidative damage to simulate drinking water lead exposure (Jurczuk et al., 2007; Moniuszko-Jakoniuk et al., 2003; Liu et al., 2010). It had been reported that lead concentrations in the blood of rats continuously intoxicated with 500 mg Pb/l are within the range of values noted in lead workers (Jurczuk et al., 2007). The present research reported in this paper was conducted in accordance with the Chinese legislation on the use and care of laboratory animals and were approved by the respective university committees for animal experiments.
The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed by the method of Reitman and Frankel (1957). 2.4. Assay of ROS level ROS was measured as described previously, based on the oxidation of 2 7 -dichlorodihydrofluorescein diacetate to 2 7 dichloro-fluorescein (Shinomol and Muralidhara, 2007). Briefly, the homogenate was diluted 1:20 times with ice-cold Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3 , 2.0 mM CaCl2 , 10 mM d-glucose, and 5 mM HEPES, pH 7.4) to obtain a concentration of 5 mg tissue/ml. The reaction mixture (1 ml) containing Locke’s buffer (pH 7.4), 0.2 ml homogenate and 10 ml of DCFH-DA (5 mM) was incubated for 15 min at room temperature to allow the DCFHDA to be incorporated into any membrane-bound vesicles and the diacetate group cleaved by esterases. After 30 min of further incubation, the conversion of DCFH-DA to the fluorescent product DCF was measured using a spectrofluorimeter with excitation at 484 nm and emission at 530 nm. Background fluorescence (conversion of DCFH-DA in the absence of homogenate) was corrected by the inclusion of parallel blanks. ROS formation was quantified from a DCF-standard curve and data are expressed as pmol DCF formed/min/mg protein. 2.5. Assay of lipid peroxidation level Chemicals, including n-butanol, thiobarbutiric acid, 1,1,3,3tetramethoxypropane and all other reagents, were purchased from Sigma Chemical Company (St. Louis, MO, USA). The level of malondialdehyde (MDA) concentrations (as a marker of lipid peroxidation) in liver tissue homogenates was determined using the method of Uchiyama and Mihara (1978). MDA levels were expressed as nmol/mg protein. 2.6. Assay of liver reduced glutathione (GSH) level and oxidized glutathione (GSSG) level Liver GSH and GSSG levels were measured using the commercial kits (Jiancheng Institute of Biotechnology, Nanjing, China) (Qu et al., 2009). GSH and GSSG content was expressed as nmol/mg protein. 2.7. Assay of Cu/Zn-SOD activity Superoxide dismutase (SOD) kits were purchased from Jiancheng Institute of Biotechnology (Nanjing, China) (Qu et al., 2009). Data were expressed as units of SOD activity per milligram of protein. 2.8. Assay of CAT activity
2.2. Histological evaluations The rats were perfused transcardially with 100 ml of normal saline (0.9%). The liver tissues were fixed in a fresh solution of 4% paraformaldehyde (pH 7.4) at 4 ◦ C for 24 h, incubated overnight at 4 ◦ C in 100 mM sodium phosphate buffer (pH 7.4) containing 30% sucrose; and embedded in optimal cutting temperature (OCT) compound (Leica, CA, Germany). Cryosections were collected on 3aminopropyl-trimethoxysilane-coated slides (Sigma–Aldrich). The liver slices were stained with hematoxylin and eosin, and examined by an expert in liver pathology (S.M.) blinded to the type of treatment received by the animals.
Catalase (CAT) activity was assayed by the method of Aebi (1984). CAT activity was calculated as nM H2 O2 consumed/min/mg of tissue protein. 2.9. Assay of GPx activity The glutathione peroxidases (GPx) activity assay was based on the method of Paglia and Valentine (1967). Tertbutylhydroperoxide was used as substrate. GPx activity was computed using the molar extinction coefficient of 6.22 mM−1 cm−1 . One unit of GPx was defined as the amount
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of enzyme that catalyzed the oxidation of 1.0 mol of NADPH to NADP+ per minute at 25 ◦ C. 2.10. Caspase-3 enzymatic activity assay The caspase-3 enzymatic activity in the nuclear fraction was carried out by caspase-3 colorimetric kit (R&D Systems, USA). The analysis was done according to the manufacturer’s instructions. The results were expressed as ng/mg protein (Kuhad and Chopra, 2009). 2.11. Deoxyribonucleotidyl transferase (TdT)-mediated dUTP-fluorescein isothiocyanate (FITC) nick-end labeling (TUNEL) assay For the TUNEL staining, the standard protocol for frozen sections was followed (BD ApoAlertTM DNA Fragmentation assay kit, BD Biosciences Clontech, Palo Alto, CA, USA). The sections were immersed in a Coplin jar (VWR International, Aurora, CO, USA) containing fresh 4% formaldehyde/PBS, and incubated at room temperature for 5 min. The sections were washed twice with PBS for 5 min. The liquid was allowed to drain thoroughly, and the slides were placed on a flat surface. Each section was covered with 100 ml of 20 mg/ml proteinase K solution (section III) and incubated at 37 ◦ C for 5 min. After two washes of 5 min each with PBS, the sections were transferred into a Coplin jar containing 4% formaldehyde/PBS and then washed in PBS again. The cells were covered in equilibration buffer (from the kit) and equilibrated at room temperature for 5 min. The equilibration buffer was drained, and TdT incubation buffer was added to the tissue sections. To perform the tailing reaction, the slice was placed in a dark and humidified 37 ◦ C incubator for 1 h. The tailing reaction was terminated by immersing the samples in 2× saline-sodium citrate (SSC) at room temperature for 15 min. Samples were washed three times with PBS for 5 min to remove unincorporated fluorescein-dUTP. Finally, strong, nuclear green fluorescence in apoptotic cells were observed on a fluorescent microscopy equipped with a standard fluorescein filter (520 nm ± 20 nm). All cells stained with propidium iodide exhibit strong red cytoplasmic fluorescence when viewed at 620 nm. Specimens were analyzed with a Zeiss Axioskop 40TM microscope equipped for light microscopy (Carl Zeiss, Oberkochen, Germany). The images were taken with a CCD camera (CoolSNAPTM Color, Photometrics, Roper Scientific Inc., Trenton, NJ, USA) and processed with Image-Pro® Plus 6.0 software (Media Cybernetics Inc., Newburyport, MA, USA). For analysis; plaque areas were excluded, and the number of stained cells in 0.01 mm2 was estimated by blind manual counting of seven regions located at a consistent position per section. 2.12. Statistic analysis All statistical analyses were performed using the SPSS software, version 11.5. A one-way analysis of variance (ANOVA; P < 0.05) was used to determine significant differences between groups and the individual comparisons were obtained by Turkey’s HSD post hoc test. Statistical significance was set at P ≤ 0.05. 3. Results 3.1. Effects of puerarin on activities of the aspartate and alanine aminotransferase (AST and ALT) in lead-treated rat liver In order to determine whether puerarin can attenuate the liver damage in the lead-treated rats, we measured the activities of ALT and AST (Fig. 1). In lead-treated rats, the activities of ALT and AST significantly increased by 281% and 120% as compared with vehicle controls, respectively (P < 0.01). However, the ALT and AST activities
Fig. 1. Effect of puerarin on lead-induced changes in hepatic functional markers in rats: (A) serum ALT and (B) serum AST. All values are expressed as mean ± S.E.M. (n = 7). **P < 0.01, compared with the control group; ## P < 0.01, vs. lead-treated group.
in the puerarin + lead group decreased by 48% and 30% as compared with lead-treated group, respectively (P < 0.01). There were no significant differences in the level of aminotransferase activity among the puerarin treated rats, the vehicle controls and puerarin + lead rats. 3.2. Effects of puerarin on histology changes in lead-treated rat liver As shown in Fig. 2, the results of histopathological evaluation showed that puerarin exhibited hepatoprotective effect against lead-induced liver injury. Lead treatment caused visible histology changes including structure damage hepatocellular necrosis, leukocyte infiltration in rat liver. In addition, the sinusoids between the plates of hepatocytes were markedly enlarged in the liver of lead-treated rats (Fig. 2B), and these histology changes were invisible in the liver of normal rats and the puerarin + lead treated rats (Fig. 2A and C). Whereas, puerarin significantly alleviated the liver damage in lead-treated rats, no appearance difference could be observed in the liver between the puerarin + lead group and the vehicle control rats (Fig. 2A and C). Compared with normal rats, there were no visible histopathological changes induced by puerarin in lead-treated rat liver and the puerarin treated rat liver (Fig. 2C and D). 3.3. Effects of puerarin on levels of ROS, MDA and ratio of GSH/GSSG in lead-treated rat liver The results showed that puerarin could decrease lead-induced ROS and MDA levels (Fig. 3). Hepatic levels of ROS were enhanced in the lead-treated rats by 77% and 25% as compared with vehicle controls, respectively (P < 0.01). Whereas, the hepatic ROS and MDA levels of the puerarin + lead treated rats were significantly reduced by 32% and 14% as compared with lead-treated rats (P < 0.01). There were no significant differences in the levels of ROS and MDA among the lead + puerarin treated rats, puerarin treated rats and the vehicle controls (Fig. 3A). Many studies suggested that the GSH/GSSG ratio might be an indicator of oxidative stress. As shown in Fig. 3C, the GSH/GSSG
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Fig. 2. Hematoxylin and eosin-stained liver slices from the treated rat. (A) The vehicle control rat; (B) rats treated with lead (500 mg Pb/l in the drinking water); (C) rats treated with lead (500 mg Pb/l in the drinking water) and fed with puerarin (400 mg PU/kg bwt intra-gastrically once daily); (D) rat fed with puerarin (400 mg PU/kg bwt intra-gastrically once daily). The black arrow indicates infiltrating leukocytes. The green arrow indicates hepatic cell necrosis. The yellow arrow indicates the enlarged sinusoids between the plates of hepatocytes. Original magnification, 10 × 10. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
ratio in lead-treated rats was markedly decreased as compared with that of the vehicle controls (P < 0.01). There were no significant differences in the GSH/GSSG ratio among the lead + puerarin treated rats, puerarin treated rats and the vehicle controls (Fig. 3C). 3.4. Effects of puerarin on activities of the antioxidant enzymes in lead-treated rat liver In order to determine whether puerarin can attenuate the oxidative damage in the liver of lead-treated rats, we measured the activities of major antioxidant enzymes, including Cu/Zn-SOD, CAT and GPx in rat liver. The results showed that puerarin could renew the activities of these antioxidant enzymes in the liver of leadtreated rats (Fig. 4). In lead-treated rats, hepatic Cu/Zn-SOD, CAT and GPx activities were significantly decreased by 31%, 46% and 42% as compared with vehicle controls, respectively (P < 0.01). However, the hepatic Cu/Zn-SOD, CAT and GPx activities in the puerarin + lead treated rats was markedly increased by 34%, 85% and 57% as compared with lead-treated rats, respectively (P < 0.01) (Fig. 4). Interestingly, there were no significant differences in hepatic activities of Cu/Zn-SOD, CAT and GPx among the lead + puerarin treated rats, puerarin treated rats and the vehicle controls (Fig. 4).
can attenuate the liver DNA damage in the lead-treated rats, we measured the level of 8-OHdG. As shown in Fig. 5A, the level of 8-OHdG in lead-treated rats was significantly increased by 41% as compared with vehicle controls (P < 0.01). Interestingly, puerarin could decrease the level of 8-OHdG in the liver by 18% (P < 0.01) as compared to the lead-treated rats. There were no significant differences in level of 8-OHdG among the control group, the puerarin group and the puerarin + lead group (Fig. 5A). 3.6. Effect of puerarin on the level of caspase-3 in rat liver Caspase-3 executes the final apoptotic processes in the nucleus that result in DNA and cell fragmentation. Caspase-3 activation is one of the biomarkers indicating mitochondrial dysfunction and oxidative stress-mediated cell apoptosis (Herrera et al., 2001). In order to determine whether puerarin can attenuate the oxidative DNA damage and apoptosis in the liver of lead-treated rats, the activity of caspase-3 was also examined. As shown in Fig. 5B, hepatic caspase-3 activity in the lead-treated rats was significantly elevated by 134% as compared with vehicle controls. In the present study, we found that puerarin could markedly decrease the activity of caspase-3 (30%) in lead-treated rat liver (P < 0.01). There were no significant differences in hepatic activities of caspase-3 among the control group, the puerarin group and the puerarin + lead group.
3.5. Effects of puerarin on level of 8-OHdG in the liver of lead-treated rats
3.7. Effect of puerarin on lead-induced apoptosis in rat liver
8-OHdG is one of such important oxidative DNA lesions formed by the oxidation of the C-8 position of 2 -deoxyguanosine, which has commonly been used as a biomarker of oxidative DNA damage (Toyokuni et al., 1995; Singh et al., 2007a,b; Pilger and Rudiger, 2006; Chen et al., 2010). In order to determine whether puerarin
We used the TUNEL assay to investigate the effect of puerarin on lead-induced apoptosis (Fig. 6). The number of TUNEL-positive cells in the liver of lead-treated rats was significantly increased (P < 0.01). There were significantly fewer TUNEL-positive cells in the liver of rats co-treated with lead and puerarin as compared with the
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Fig. 3. Effect of puerarin on the levels of ROS and MDA and ratio of GSH/GSSG in lead-treated rat liver. (A) Level of ROS; (B) level of MDA; (C) ratio of GSH/GSSG. Each value is expressed as mean ± S.E.M. (n = 7). **P < 0.01, compared with the control group; ## P < 0.01, vs. lead-treated group.
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Fig. 4. Effect of puerarin on the activities of antioxidant enzymes in lead-treated rat liver. (A) Cu/Zn-SOD activity; (B) CAT activity; (C) GPx activity. All values are expressed as mean ± S.E.M. (n = 7). **P < 0.01, compared with the control group; ## P < 0.01, vs. lead-treated group.
rats treated with lead (P < 0.01). However, there were no significant differences in the number of TUNEL-positive cells in the liver among the control group, the puerarin group and the puerarin + lead group (Fig. 6). 4. Discussion Liver is the primary organ site for xenobiotic metabolism. In most cases, the metabolic process is accomplished without injury to the liver itself, whereas many compounds are toxic themselves or produce metabolites that can cause liver injury. Accumulating evidence have demonstrated that low, medium or high dose of lead can cause liver injury. It was also shown that lead causes oxidative stress by inducing the generation of reactive oxygen species (ROS), including hydroperoxides, singlet oxygen, hydrogen peroxide and superoxide (Sivaprasad et al., 2004; Jurczuk et al., 2007; Liu et al., 2010; Franco et al., 2009; Pulido and Parrish, 2003). In the present study, we demonstrated overproduction of ROS by lead. ROS could damage DNA, proteins and lipids within cells, which led to liver injury (Franco et al., 2009; Liu et al., 2010). In lead-treated animals, the histological changes of liver, such as structure damage, hepatocellular necrosis, leukocyte infiltration and massive hemorrhage, had been observed (Campana et al., 2003; Liu et al., 2010; Narayana and Al-Bader, 2010; El-Neweshy and El-Sayed, 2010). Puerarin has been used for various medicinal purposes in traditional oriental medicine for thousands of years. Even with high doses, puerarin had safety and tolerability profiles to animals (Zhang et al., 2006; Wang et al., 2008). In this study, puerarin by oral gavage administration markedly decreased activities of serum ALT and AST in lead-treated
Fig. 5. Effect of puerarin on the level of 8-OHdG and the activity of caspase-3 in lead-treated rat liver. (A) Level of 8-OHdG; (B) activity of caspase-3. Each value is expressed as mean ± S.E.M. (n = 7). **P < 0.01, compared with the control group; # P < 0.05 and ## P < 0.01, vs. lead-treated group.
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Fig. 6. (A) In situ detection of fragmented DNA [deoxyribonucleotidyl transferase-mediated dUTP-FITC nick-endlabeling (TUNEL) assay] in the liver of rat. The liver tissues were processed for TUNEL and photographed using a fluorescence microscope with either propidium iodide (PI) filter alone (left) or an FITC filter alone (middle). The merged images show that apoptotic cells appear yellow and non-apoptotic cells appear red (right). Scale bars = 100 mm. (B) The histogram showed the relative proportion of TUNEL-positive cells in the liver of rat. All values are expressed as mean ± S.E.M. **P < 0.01, compared with the control group; ## P < 0.01, vs. Pb-treated group.
rats (Fig. 1) and improved lead-induced histopathologic changes in rat liver (Fig. 2). These results suggest that puerarin could protect rat liver against lead-induced dysfunction and histopathologic damage. Recent studies indicated that elevation of severe oxidative stress biomarkers could be observed in the liver of lead-treated rodents (Sivaprasad et al., 2004; Zhang et al., 2006; Jurczuk et al., 2007; Liu et al., 2010). ROS and MDA are considered major oxidative stress markers. It have been reported that lead exposure could induce changes in the fatty acid composition of membrane (Knowles and Donaldson, 1990) and increase of MDA level in liver (Jurczuk et al., 2007; Xu et al., 2008; Liu et al., 2010). Lead induced arachidonic acid elongation might be due to the enhanced lipid peroxidation in the membrane because fatty acid chain length and unsaturation are associated with membrane susceptibility to peroxidation (Lawton
and Donaldson, 1991). In the present study, levels of MDA and ROS were markedly increased in lead-treated rat liver as compared with the controls, indicating that lead exposure induced oxidative stress. However, puerarin markedly decreased levels of MDA and ROS in lead-treated rat liver (Fig. 3A and B). This may be due to the powerful antioxidant and free radical scavenging activities (Xu, 2003; Hwang et al., 2007; Yang et al., 2009; Zhao et al., 2010). Our finding suggests that puerarin could at least partly attenuate oxidative stress by decreasing levels of ROS and lipid peroxide in lead-treated rat liver. Antioxidant enzymes can protect cellular compounds against damage induced by free radicals. Catalase (CAT), superoxide dismutases (SOD) and glutathione peroxidases (GPx) are important antioxidant enzymes (Boots et al., 2008). The SOD decomposes • superoxide radicals (O2 − ) and produce H2 O2 . H2 O2 is subsequently
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removed to water by CAT in peroxisomes, or by GPx oxidizing GSH in the cytosol (Dröge, 2002; Lee and Choi, 2003). Therefore, activities of these enzymes have been used to assess oxidative stress in cells. Whereas GPx, CAT and SOD depend on various essential trace elements for proper molecular structure and activity, these antioxidant enzymes are potential targets for lead toxicity (Gelman et al., 1978; Hsu and Guo, 2002). Many studies have indicated that lead can also alter antioxidant activities by inhibiting functional SH groups in several enzymes such as SOD, CAT and GPx, because of its high affinity for sulfhydryl (SH) groups in these enzymes (Hsu, 1981; McGowan and Donaldson, 1986; Chiba et al., 1996; Hsu and Guo, 2002). Moreover, if the balance between ROS production and antioxidant defenses is broken, the enzyme may be exhausted and its concentration therefore depleted. In the present study, activities of antioxidant enzymes in rat liver, including SOD, CAT and GPx, were dramatically decreased by the treatment with lead. It was demonstrated that lead exposure induced oxidative stress due to the inhibition of antioxidant enzymes activities. Interestingly, puerarin could markedly restore activities of those antioxidant enzymes in the liver of lead-treated rats (Fig. 4). GSH can act as a nonenzymatic antioxidant by direct interaction of SH group with ROS, or it can be involved in the enzymatic detoxification reaction for ROS, as a cofactor or a coenzyme, because it is a tripeptide containing cysteine that has a reactive SH group with reductive potency (Sivaprasad et al., 2004; Hsu and Guo, 2002). In this study, we observed significant decreases in GSH/GSSG ratio in the liver of lead-treated rats which is in agreement with several previous studies (Aykin-Burns et al., 2003; Sivaprasad et al., 2004; Flora et al., 2007; Liu et al., 2010). It may be due to the high ability of lead to bind with the sulfhydryl (SH) group of GSH and lead-induced oxidative stress. Interestingly, we found that puerarin markedly increased GSH level and GSH/GSSG ratio in lead-treated rat liver (Fig. 3B). It suggested that puerarin could at least partly attenuate oxidative stress by renewing the activities of antioxidant enzymes and increasing GSH/GSSG ratio in lead-treated rat liver. ROS are known to attack other cellular components such as lipids, leaving behind reactive species that in turn can couple to DNA bases (Marnett, 2000). The most extensively studied oxidative DNA lesion is 8-OHdG, which have been widely used as a sensitive ´ biomarker of DNA oxidation [Kinoshita et al., 2007; GałazynSidorczuk et al., 2009]. It was reported that copper, inorganic arsenic, methylmercury and iron overload can induce increase of 8-OHdG level in the liver (Toyokuni and Sagripanti, 1994; Kinoshita et al., 2007; Jin et al., 2008; Asare et al., 2008, 2009). In this study, the level of 8-OHdG was markedly increased in the liver of leadtreated rats, which correlated with the level of ROS, suggesting that DNA is a common target of ROS induced by lead in liver (Fig. 5A). Interestingly, the hepatic 8-OHdG level in the puerarin + lead group was markedly decreased as compared with lead-treated group. Our findings indicate that puerarin may play an effective role in protecting liver function from lead-induced oxidative DNA damage by decreasing 8-OHdG level in the liver of lead-treated rats. High ROS concentrations may contribute to the apoptotic cell death whenever they are generated in the context of apoptotic process (Liu et al., 2010; Franco et al., 2009; Pulido and Parrish, 2003; Gargioni et al., 2006; Xu et al., 2008). Based on labeling of DNA strand breaks in the liver of rats, the TUNEL method was used to analyze apoptotic cells in situ (Fig. 6). Recent studies from our laboratory have shown that pro-apoptotic BAK protein and cleave caspase-3 expression were increased after lead expose in rat liver (Liu et al., 2010). The present study also showed that lead increased the number of TUNEL-positive cells in the liver of rats (Fig. 6). Moreover, caspase-3 activity was significantly elevated in the lead-treated rat liver, suggesting that apoptosis induced by lead is occurred due to activation of caspase-3 (Pulido and Parrish, 2003; Gargioni et al., 2006; Xu et al., 2006; Franco et al., 2009; Kiran Kumar
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et al., 2009) (Fig. 5B). Our finding that puerarin could markedly decrease the number of TUNEL-positive cells and reduce caspase-3 activity in rats treated with lead suggests that puerarin has a potential hepatoprotective effect against lead-induced DNA damage and apoptosis due to its antioxidant activity. In conclusion, our results indicated that puerarin administration attenuated lead-induced hepatic dysfunction and histopathologic changes in the rat liver. Puerarin restored antioxidant enzymes activities, down-regulated levels of ROS, MDA and 8-OHdG and inhibited apoptosis due to suppression of caspase-3 activity. Furthermore, it consequently improved lead-induced oxidative DNA damage and apoptosis in the liver of rats. Recent studies indicated isoflavone compounds like genistein and daizein at high doses have been shown to possess estrogenic activity in animals and to promote reproductive cancer (Martfnez-Montemayor et al., 2010; Messina and Wood, 2008). However, there are fewer reports on the impact of puerarin administration on cancer risk. The question warrants further investigation. Acknowledgments This work is supported by the Fundamental Research Program of Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 07KJD180210), and Grants from Natural Science Foundation by Xuzhou Normal University (Nos. 08XLR08 and 09XLY06). References Aebi H. Catalase in vitro. Meth Enzymol 1984;105(1):121–6. Asare GA, Bronz M, Naidoo V, Kew MC. Synergistic interaction between excess hepatic iron and alcohol ingestion in hepatic mutagenesis. Toxicology 2008;254(1–2):11–8. Asare GA, Kew MC, Mossanda KS, Paterson AC, Siziba K, Kahler-Venter CP. Effects of exogenous antioxidants on dietary iron overload. J Clin Biochem Nutr 2009;44(1):85–94. Aykin-Burns N, Laegeler A, Kellogg G, Ercal N. Oxidative effects of lead in young and adult Fisher 344 rats. Arch Environ Contam Toxicol 2003;44(3):417–20. Benlhabib E, Baker JI, Keyler DE, Singh AK. Effects of purified puerarin on voluntary alcohol intake and alcohol withdrawal symptoms in P rats receiving free access to water and alcohol. J Med Food 2004;7(2):180–6. Bo J, Ming BY, Gang LZ, Lei C, Jia AL. Protection by puerarin against MPP+-induced neurotoxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction and caspase-3-like activation. Neurosci Res 2005;53(2):183–8. Boots AW, Haenen GR, Bast A. Health effects of quercetin: from antioxidant to nutraceutical. Eur J Pharmacol 2008;585(2–3):325–37. Campana O, Sarasquete C, Blasco J. Effect of lead on ALAD activity, metallothionein levels, and lipid peroxidation in blood, kidney, and liver of the toadfish Halobatrachus didactylus. Ecotoxicol Environ Saf 2003;55(1):116–25. Chang Y, Hsieh CY, Peng ZA, Yen TL, Hsiao G, Chou DS, et al. Neuroprotective mechanisms of puerarin in middle cerebral artery occlusion-induced brain infarction in rats. J Biomed Sci 2009;16(1):1–13. Chen H, Chu G, Cao Z, Liang J, Lu J, Zhou G. Magnetic bead-based approach to monitoring of cigarette smoke-induced DNA oxidation damage and screening of natural protective compounds. Talanta 2010;80(3):1216–21. Cheng YF, Zhu GQ, Wang M, Cheng H, Zhou A, Wang N, et al. Involvement of ubiquitin proteasome system in protective mechanisms of puerarin to MPP+-elicited apoptosis. Neurosci Res 2009;63(1):52–8. Chiba M, Shinohara A, Matsushita K, Watanabe H, Inaba Y. Indices of lead-exposure in blood and urine of lead-exposed workers and concentrations of major and trace elements and activities of SOD, GSH-Px and catalase in their blood. Tohoku J Exp Med 1996;178(1):49–62. Chung MJ, Sung NJ, Park CS, Kweon DK, Mantovani A, Moon TW, et al. Antioxidative and hypocholesterolemic activities of water-soluble puerarin glycosides in HepG2 cells and in C57BL/6J mice. Eur J Pharmacol 2008;578(2–3):159–70. Courtois E, Marques M, Barrientos A. Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxygenase-2. J Am Soc Nephrol 2003;14(6):1464–70. Dröge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82(1):47–95. El-Neweshy MS, El-Sayed YS. Influence of vitamin C supplementation on leadinduced histopathological alterations in male rats. Exp Toxicol Pathol 2010. Flora SJ, Saxena G, Mehta A. Reversal of lead-induced neuronal apoptosis by chelation treatment in rats: role of reactive oxygen species and intracellular Ca(2+). J Pharmacol Exp Ther 2007;322(1):108–16. Franco R, Sánchez-Olea R, Reyes-Reyes EM, Panayiotidis MI. Environmental toxicity, oxidative stress and apoptosis: ménage à trois. Mutat Res 2009;674(1–2):3–22.
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C.-M. Liu et al. / Experimental and Toxicologic Pathology 64 (2012) 575–582
´ M, Brzóska MM, Jurczuk M, Moniuszko-Jakoniuk J. Oxidative Gałazyn-Sidorczuk damage to proteins and DNA in rats exposed to cadmium and/or ethanol. Chem Biol Interact 2009;180(1):31–8. Gargioni R, Filipak Neto F, Buchi DF, Randi MAF, Franco CRC, Paludo KS, et al. Cell death and DNA damage in peritoneal macrophages of mice (Mus musculus) exposed to inorganic lead. Cell Biol Int 2006;30(7):615–23. Gelman BB, Michaelson IA, Bus JS. The effect of lead on oxidative hemolysis and erythrocyte defense mechanisms in the rat. Toxicol Appl Pharmacol 1978;45(1):119–29. Guerra MC, Speroni E, Broccoli M, Cangini M, Pasini P, Minghetti A, et al. Comparison between Chinese medical herb Pueraria lobata crude extract and its main isoflavone puerarin antioxidant properties and effects on rat liver CYP-catalysed drug metabolism. Life Sci 2000;67(24):2997–3006. Han RM, Tian YX, Becker EM, Andersen ML, Zhang JP, Skibsted LH. Puerarin and conjugate bases as radical scavengers and antioxidants: molecular mechanism and synergism with -carotene. J Agric Food Chem 2007;55(6):2384–91. Herrera B, Alvarez AM, Sanchez A, Fernandez M, Roncero C, Benito M, et al. Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor {beta} in fetal hepatocytes. FASEB J 2001;15(3):741–51. Hsu JM. Lead toxicity related to glutathione metabolism. J Nutr 1981;111(1):26–33. Hsu PC, Guo YL. Antioxidant nutrients and lead toxicity. Toxicology 2002;180(1):33–44. Hwang YP, Choi CY, Chung YC, Jeon SS, Jeong HG. Protective effects of puerarin on carbon tetrachloride-induced hepatotoxicity. Arch Pharm Res 2007;30(10):1309–17. Jin X, Chan HM, Lok E, Kapal K, Taylor M, Kubow S, et al. Dietary fats modulate methylmercury-mediated systemic oxidative stress and oxidative DNA damage in rats. Food Chem Toxicol 2008;46(5):1706–20. Jurczuk M, Moniuszko-Jakoniuk J, Brzóska MM. Hepatic and renal concentrations of vitamins E and C in lead- and ethanol-exposed rats. An assessment of their involvement in the mechanisms of peroxidative damage. Food Chem Toxicol 2007;45(8):1478–86. Kim KM, Jung DH, Jang DS, Kim YS, Kim JM, Kim HN, et al. Puerarin suppresses AGEs-induced inflammation in mouse mesangial cells: a possible pathway through the induction of heme oxygenase-1 expression. Toxicol Appl Pharmacol 2010;244(2):106–13. Kinoshita A, Wanibuchi H, Wei M, Yunoki T, Fukushima S. Elevation of 8hydroxydeoxyguanosine and cell proliferation via generation of oxidative stress by organic arsenicals contributes to their carcinogenicity in the rat liver and bladder. Toxicol Appl Pharmacol 2007;221(3):295–305. Kiran Kumar B, Prabhakara Rao Y, Noble T, Weddington K, McDowell VP, Sharada R, et al. Lead-induced alteration of apoptotic proteins in different regions of adult rat brain. Toxicol Lett 2009;184(1):56–60. Knowles SO, Donaldson WE. Dietary modification of lead toxicity: effects on fatty acid and eicosanoid metabolism in chicks. Comp Biochem Physiol 1990;95(1):99–104. Kuhad A, Chopra K. Attenuation of diabetic nephropathy by tocotrienol: involvement of NFkB signaling pathway. Life Sci 2009;84(9–10):296–301. Lawton L, Donaldson WE. Lead-induced tissue fatty acid alterations and lipid peroxidation. Biol Trace Elem Res 1991;28(2):83–97. Lee PJ, Choi AMK. Pathways of cell signaling in hyperoxia. Free Radic Biol Med 2003;35(4):341–50. Li Q, Xiao Y, Gong H, Shen D, Zhu F, Wu Q, et al. Effect of puerarin on the expression of extracellular matrix in rats with streptozotocin-induced diabetic nephropathy. Natl Med J India 2009;22(1):9–12. Liu CM, Zheng YL, Lu J, Zhang ZF, Fan SH, Wu DM, et al. Quercetin protects rat liver against lead-induced oxidative stress and apoptosis. Environ Toxicol Pharmacol 2010;29(2):158–66. Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis 2000;21(3):361–70. Martfnez-Montemayor MM, Otero-Franqui E, Martinez J, De La Mota-Peynado A, Cubano LA, Dharmawardhane S. Individual and combinedsoy isoflavones exert differential effects on metastatic cancer progression. Clin Exp Metastasis 2010;27(7):465–80. McGowan C, Donaldson WE. Changes in organ nonprotein sulfhydryl and glutathione concentrations during acute and chronic administration of inorganic lead to chicks. Biol Trace Elem Res 1986;10(1):37–46. Mercer LD, Kelly BL, Horne MK, Beart PM. Dietary polyphenols protect dopamine neurons from oxidative insults and apoptosis: investigations in primary rat mesencephalic cultures. Biochem Pharmacol 2005;69(2):339–45. Messina MJ, Wood CE. Soy isoflavones, estrogen therapy, and breast cancer risk: analysis and commentary. Nutr J 2008;7(17):1–12. ´ Moniuszko-Jakoniuk J, Jurczuk M, Gałazyn-Sidorczuk M, Brzóska MM. Lead turnover and changes in the body status of chosen micro- and macroelements in rats exposed to lead and ethanol. Pol J Environ Stud 2003;12(1):335–44. Narayana K, Al-Bader M. Ultrastructural and DNA damaging effects of lead nitrate in the liver. Exp Toxicol Pathol 2010. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70(1):158– 69.
Pilger A, Rudiger HW. 8-hydroxydeoxyguanosine as a marker of oxidative damage related to occupational and environmental exposure. Int Arch Occup Environ Health 2006;80(1):1–15. Pulido MD, Parrish AR. Metal-induced apoptosis: mechanisms. Mutat Res 2003;533(1–2):227–41. Qu XJ, Xia X, Wang YS, Song MJ, Liu LL, Xie YY, et al. Protective effects of salvia plebeia compound homoplantaginin on hepatocyte injury. Food Chem Toxicol 2009;47(7):1710–5. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957;28(1):56–63. Shinomol GK, Muralidhara. Differential induction of oxidative impairments in brain regions of male mice following subchronic consumption of Khesari dhal (Lathyrus sativus) and detoxified Khesari dhal. Neurotoxicology 2007;28:798–806. Sieg DJ, Billings RE. Lead/cytokine-mediated oxidative DNA damage in cultured mouse hepatocytes. Toxicol Appl Pharmacol 1997;142(1):106–15. Singh AK, Jiang Y, Benlhabib E, Gupta S. Herbal mixtures consisting of puerarin and either polyenylphosphatidylcholine or curcumin provide comprehensive protection against alcohol-related disorders in P rats receiving free choice water and 15% ethanol in pure water. J Med Food 2007a;10(3):526–42. Singh R, Sram RJ, Binkova B, Kalina I, Popov TA, Georgieva T, et al. The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans. Mutat Res 2007b;620(1–2):83–92. Sivaprasad R, Nagaraj M, Varalakshmi P. Combined efficacies of lipoic acid and 2,3dimercaptosuccinic acid against lead-induced lipid peroxidation in rat liver. J Nutr Biochem 2004;15(1):18–23. Sun XH, Ding JP, Li H, Pan N, Gan L, Yang XL, et al. Activation of large-conductance calcium-activated potassium channels by puerarin: the underlying mechanism of puerarin-mediated vasodilation. J Pharmacol Exp Ther 2007;323(1):391–7. Toyokuni S, Okamoto K, Yodoi J, Hiai H. Persistent oxidative stress in cancer. FEBS Lett 1995;358(1):1–3. Toyokuni S, Sagripanti JL. Increased 8-hydroxydeoxyguanosine in kidney and liver of rats continuously exposed to copper. Toxicol Appl Pharmacol 1994;126(1):91–7. Uchiyama M, Mihara M. Determination of malondialdehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978;86(1):271–8. Wang Y, Yin L, Li Y, Liu P, Cui Q. Preventive Effects of Puerarin on Alcohol-induced Osteonecrosis. Clin Orthop Relat Res 2008;466(5):1059–67. Wu L, Qiao H, Li Y, Li L. Protective roles of puerarin and Danshensu on acute ischemic myocardial injury in rats. Phytomedicine 2007;14(10):652–8. Xiang JJ, Zhai YF, Tang Y, Wang H, Liu B, Guo CW. A competitive indirect enzymelinked immunoassay for lead ion measurement using mAbs against the leadDTPA complex. Environ Pollut 2010;158(5):1376–80. Xiong FL, Sun XH, Gan L, Yang XL, Xu HB. Puerarin protects rat pancreatic islets from damage by hydrogen peroxide. Eur J Pharmacol 2006;529(1–3):1–7. Xu J, Ji LD, Xu LH. Lead-induced apoptosis in PC12 cells: involvement of p53, Bcl-2 family and caspase-3. Toxicol Lett 2006;166(2):160–7. Xu J, Lian LJ, Wu C, Wang XF, Fu WY, Xu LH. Lead induces oxidative stress, DNA damage and alteration of p53, Bax and Bcl-2 expressions in mice. Food Chem Toxicol 2008;46(5):1488–94. Xu XH. Effects of puerarin on fatty superoxide in aged mice induced by d-galactose. Zhongguo Zhong Yao Za Zhi 2003;28(1):66–9. Yan LP, Chan SW, Chan AS, Chen SL, Ma XJ, Xu HX. Puerarin decreases serum total cholesterol and enhances thoracic aorta endothelial nitric oxide synthase expression in diet-induced hypercholesterolemic rats. Life Sci 2006;79(4):324–30. Yang S, Lou JL, Wang Q. Effect of puerarin on liver injury in KKAy mice with type 2 diabetes mellitus. Zhongguo Zhong Xi Yi Jie He Za Zhi 2009;29(8):707–10. Yang X, Hu W, Zhang Q, Wang Y, Sun L. Puerarin inhibits C-reactive protein expression via suppression of nuclear factor kappaB activation in lipopolysaccharide-induced peripheral blood mononuclear cells of patients with stable angina pectoris. Basic Clin Pharmacol Toxicol 2010. Yeung DK, Leung SW, Xu YC, Vanhoutte PM, Man RY. Puerarin, an isoflavonoid derived from Radix puerariae, potentiates endothelium-independent relaxation via the cyclic AMP pathway in porcine coronary artery. Eur J Pharmacol 2006;552(1–3):105–11. Zhang SH, Ji G, Liu JW. Reversal of chemical-induced liver fibrosis in Wistar rats by puerarin. J Nutr Biochem 2006;17(7):485–91. Zhao M, Du YQ, Yuan L, Wang NN. Protective effect of puerarin on acute alcoholic liver injury. Am J Chin Med 2010;38(2):241–9. Zheng P, Ji G, Ma Z, Liu T, Xin L, Wu H, et al. Therapeutic effect of puerarin on non-alcoholic rat fatty liver by improving leptin signal transduction through JAK2/STAT3 pathways. Am J Chin Med 2009;37(1):69–83. Zheng PY, Ma ZS, Hua YQ, Liu T, Xing LJ, Ji G. Effects of puerarin on expressions of leptin receptor mRNA, phosphorylated Janus kinase 2/phosphorylated signal transducers and activators of transcription 3 proteins in the liver of rats with non-alcoholic fatty liver. Zhong Xi Yi Jie He Xue Bao 2008;6(9):921–7. Zheng X, Xu X. Potential involvement of calcium and nitric oxidein protective effects of puerarin on oxygen–glucose deprivation in cultured hippocampal neurons. J Ethnopharmacol 2007;113(3):421–6.