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Protective effects of Pycnogenol® on carbon tetrachloride-induced hepatotoxicity in Sprague–Dawley rats
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Food and Chemical Toxicology 46 (2008) 380–387 www.elsevier.com/locate/foodchemtox
Protective effects of Pycnogenol on carbon tetrachloride-induced hepatotoxicity in Sprague–Dawley rats Young-Su Yang a, Tai-Hwan Ahn a, Jong-Chan Lee a, Chang-Jong Moon a, Sung-Ho Kim a, Woojin Jun b, Seung-Chun Park c, Hyoung-Chin Kim d, Jong-Choon Kim a,* a
Department of Toxicology, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea b Department of Food and Nutrition, Chonnam National University, Gwangju 500-757, Republic of Korea c College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea d Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea Received 31 January 2007; accepted 10 August 2007
Abstract Oxidative damage is implicated in the pathogenesis of various liver injuries. In the present study the ability of Pycnogenol (PYC) as an antioxidant to protect against CCl4-induced oxidative stress and hepatotoxicity in rats was investigated. Four experimental groups of six rats each were constructed: a vehicle control group received the respective vehicles (distilled water and corn oil) only; a CCl4 group received a 14-day repeated intraperitoneal (i.p.) dose of distilled water and then a single oral dose of CCl4 at 1.25 ml/kg; and the CCl4&PYC 10 and CCl4&PYC 20 groups received a 14-day repeated i.p. dose of PYC 10 and 20 mg/kg, respectively, and then a single oral dose of CCl4 at 1.25 ml/kg. Hepatotoxicity was assessed 24 h after the CCl4 treatment by measurement of serum aminotransferase (AST) and alanine aminotransferase (ALT) activities, hepatic malondialdehyde (MDA) and glutathione (GSH) concentrations, and catalase, superoxide dismutase (SOD), and glutathione-S-transferase (GST) activities. The results were confirmed histopathologically. The single oral dose of CCl4 produced significantly elevated levels of serum AST and ALT activities. Histopathological examinations showed extensive liver injuries, characterized by extensive hepatocellular degeneration/necrosis, fatty changes, inflammatory cell infiltration, congestion, and sinusoidal dilatation. In addition, an increased MDA concentration and decreased GSH, catalase, SOD, and GST were observed in the hepatic tissues. On the contrary, PYC treatment prior to the administration of CCl4 significantly prevented the CCl4-induced hepatotoxicity, including the elevation of serum AST and ALT activities and histopathological hepatic lesions, in a dose-dependent manner. Moreover, MDA and GSH levels and catalase, SOD, and GST activities in hepatic tissues were not affected by administration of CCl4, indicating that the pretreatment of PYC efficiently protects against CCl4-induced oxidative damage in rats. The results indicate that PYC has a protective effect against acute hepatotoxicity induced by the administration of CCl4 in rats, and that the hepatoprotective effects of PYC may be due to both the inhibition of lipid peroxidation and the increase of antioxidant activity. 2007 Elsevier Ltd. All rights reserved. Keywords: Pycnogenol; Carbon tetrachloride; Hepatotoxicity; Oxidative stress; Protective effect
1. Introduction Liver disease is considered to be a serious health problem, as the liver is an important organ for the detoxification and deposition of endogenous and exogenous substances. *
Corresponding author. Tel.: +82 62 530 2827; fax: +82 62 530 2809. E-mail address: [email protected] (J.-C. Kim).
0278-6915/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.08.016
Steroids, vaccines, and antiviral drugs, which have been employed as therapies for liver diseases, have potential adverse effects, especially when administered for long terms. Therefore, herbal products and traditional medicines with improved effectiveness and safety profiles are needed as a substitute for chemical therapeutics. It is reported that a number of herbal products have been shown to protect against liver injury, and many possess
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one or a combination of antioxidant, antifibrotic, immune modulatory, or antiviral activities (Seeff et al., 2001; Lee and Jeong, 2002; Shin et al., 2006). In recent years, there has been a substantial increase in the use of so-called complementary and alternative therapies that utilize herbal medicines by patients with liver disease (Strader et al., 2002; Fogden and Neuberger, 2003; Hanje et al., 2006). Pycnogenol (PYC), a standardized extract from the bark of the French maritime pine (Pinus maritima), is used extensively in dietary supplements, multi-vitamins, and health products because of its direct, strong antioxidant activity (Rohdewald, 2002). Major constituents of PYC are polyphenols, specifically monomeric and oligomeric units of catechin, epicatechin, and taxifolin (Rohdewald, 2002; Grimm et al., 2006). It is well documented that polyphenols comprise a wide area of natural substances of plant origin, and almost all of them exhibit a marked antioxidant activity (Bors and Michel, 2002; Cai et al., 2002). Oxidative stress is defined in general as excess formation and/or insufficient removal of highly reactive molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Johansen et al., 2005; Valko et al., 2007). ROS include free radicals such as superoxide anion (O2), hydroxyl (OH), peroxyl (RO2), hydroperoxyl (HRO2) as well as nonradical species such as hydrogen peroxide (H2O2) and hydrochlorous acid (HOCl). RNS include free radicals like nitric oxide (NO) and nitrogen dioxide (NO2), as well as nonradicals such as peroxynitrite (ONOO), nitrous oxide (HNO2) and alkyl peroxynitrates (RONOO). The effects of antioxidants on oxidative stress are mainly measured through certain observable biomarkers, including the enzymatic activities of catalase, SOD, GSH-Px, and GSH-reductase, as well as thiobarbituric acid reactants (TBARS) levels, an indirect measurement of free-radical production (Maritim et al., 2003; Johansen et al., 2005; Valko et al., 2007). Previous studies showed that PYC is a very potent antioxidant to scavenge reactive oxygen and nitrogen species such as superoxide anion radical, hydroxyl radical, lipid peroxyl radical (LOO), peroxynitrite radical, and singlet oxygen (1O2). It has also been shown to bind to proteins, thereby affecting both structural and functional characteristics of key enzymes and other proteins involved in metabolism; to participate in the cellular antioxidant network, especially in prolonging the lifetime of ascorbyl radical, possibly through a one-electron reduction of dehydroascorbic acid; and to protect endogenous vitamin E and glutathione (Packer et al., 1999; Maritim et al., 2003). It has been claimed that PYC has diverse beneficial effects on a wide range of medical conditions, including inflammation, diabetes, asthma, hypertension, attention deficiency hyperactivity disorder, cancer, immune disease, and others (Rohdewald, 2002). Despite the favorable pharmacological properties of PYC, the protective capacity of PYC against hepatotoxicity has not previously been explored. PYC may have a protective effect on the deteriorated hepatic function that results from free radicals in the toxic
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chemical-induced hepatotoxicity. To test this hypothesis, the present investigation examined the ability of PYC to protect against carbon tetrachloride (CCl4)-induced oxidative stress and hepatotoxicity. We selected CCl4 as a model hepatotoxicant because this chemical is a potent hepatotoxin and a single administration can rapidly lead to both oxidative stress via the excessive production of free radicals and acute liver injuries such as centrilobular necrosis and steatosis in rats (Recknagel et al., 1989; Janakat and Al-Merie, 2002; Weber et al., 2003). Because CCl4 is a common contaminant found in both ambient outdoor and indoor air, the human health risk is very high. It is therefore important to investigate the antioxidant activity of PYC against CCl4-induced hepatic oxidative damages. 2. Materials and methods 2.1. Animal husbandry and maintenance Thirty male Sprague–Dawley rats aged 8 weeks were obtained from a specific pathogen-free colony at Bio Genomics Inc. (Seoul, Republic of Korea) and used after 1 week of quarantine and acclimatization. The animals were housed in a room maintained at a temperature of 23 ± 3 C and a relative humidity of 50 ± 10% with artificial lighting from 08:00 to 20:00 and 13 to 18 air changes per hour. The animals were housed two per cage in stainless steel wire mesh cages and were allowed sterilized tap water and commercial rodent chow (Samyang Feed Co., Wonju, Republic of Korea) ad libitum. Our Institutional Animal Care and Use Committee approved the protocols for the animal study, and the animals were cared for in accordance with the Guidelines for Animal Experiments of the Chonnam National University.
2.2. Materials and treatment PYC (US patent # 4,698,360), extracted exclusively from the bark of French marine pine, was donated from Horphag Research Ltd. (Route de Belis, 40420 Le Sen, France). CCl4 and corn oil were purchased from the Sigma–Aldrich Co. (St. Louis, MO, USA). All other chemicals were of the highest grade commercially available. PYC and CCl4 were dissolved in sterilized distilled water and corn oil, respectively, and were prepared immediately before treatment. PYC was administered intraperitoneally (i.p.) to rats once daily for 14 days at dose levels of 10 and 20 mg/kg. Three hours after the final PYC treatment, the rats were given a single oral dose of CC14 (1.25 ml/kg, 20% in corn oil) to induce liver injury. All of the animals were sacrificed 24 h after administration of CCl4.
2.3. Experimental groups and dose selection Prior to testing, rats were evaluated by clinical observations and body weight determinations during a 7-day quarantine period to assure freedom from potential confounding variables. Twenty-four healthy male rats were randomly assigned to four experimental groups of six rats each: a vehicle control group received the respective vehicles only; a CCl4 group received a 14-day repeated i.p. dose of distilled water and then a single oral dose of CCl4 at 1.25 ml/kg, which is well documented to induce acute hepatotoxicity in rats (Janakat and Al-Merie, 2002; Wong et al., 2003); and the CCl4&PYC 10 and CCl4&PYC 20 groups received a 14-day repeated i.p. dose of PYC 10 and 20 mg/kg, respectively, and then a single oral dose of CCl4 at 1.25 ml/kg. The effective doses of PYC were based on earlier reports and clinical dosages (Packer et al., 1999; Maritim et al., 2003). We used only male rats because of their constant metabolism compared to the variations in the female physiology.
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2.4. Autopsy and serum biochemistry All treated animals were anesthetized by ether inhalation for blood sample collection 24 h after administration of CCl4. Blood samples were drawn from the posterior vena cava using a syringe with 24-gauge needle under ether anesthesia. The samples were centrifuged at 1256g for 10 min within 1 h after collection. The sera were stored in the 80 C freezer before they were analyzed. Enzyme activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in blood serum were evaluated by an autoanalyzer (Shimadzu CL-7200, Shimadzu, Japan). After bleeding, complete gross postmortem examinations were performed on all terminated rats. The absolute and relative (organ-to-body weight ratio) weights of the liver were also measured for all rats when they were sacrificed.
2.5. Histopathological examinations A portion of the median lobe of the liver was dissected and fixed in 10% neutral buffered formalin solution for 24 h. The remaining livers were frozen quickly in dry ice and stored at 70 C for biochemical analysis. The fixed tissues were processed routinely, and were then embedded in paraffin, sectioned to 3–5 lm thickness, deparaffinized, and rehydrated using standard techniques. The extent of CC14-induced necrosis and steatosis was evaluated by assessing morphological changes in liver sections stained with hematoxylin and eosin (H&E), using standard techniques.
2.6. Preparation of hepatic homogenate The weighed frozen liver tissue was homogenized in a glass-Teflon homogenizer with 50 mM phosphate buffer (pH 7.4) to obtain 1:9 (w/v) whole homogenate. The homogenates were then centrifuged at 11,000g for 15 min at 4 C to discard any cell debris, and the supernatant was used for the measurement of malondialdehyde (MDA), reduced glutathione (GSH), catalase, superoxide dismutase (SOD), and glutathione-S-transferase (GST). Total protein contents were determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard.
2.7. Determination of lipid peroxidation and GSH levels Lipid peroxidation was measured by the thiobarbituric acid (TBA) reaction method (Berton et al., 1998). In brief, samples were mixed with TBA reagent consisting of 0.375% TBA and 15% trichloroacetic acid in 0.25-N hydrochloric acid. The reaction mixtures were placed in a boiling water bath for 30 min and centrifuged at 1811g for 5 min. The absorbance of the supernatant was measured at 535 nm. MDA, a measure of lipid peroxidation, was calculated using an extinction coefficient of 1.56 · 105/ M cm. The results were expressed as lM/mg protein. GSH content was measured by the method of Moron et al. (1979). In brief, samples were mixed with 0.1 M of sodium phosphate buffer (pH 7.5) containing 5 lM EDTA, 0.6 mM of 5,5-dithiol-bis (2-nitrobenzoic acid), 0.2 mM NADPH and glutathione reductase. The mixture was incubated for 2 min at room temperature. The absorbance of the product was measured at 412 nm. GSH content was determined using a standard curve generated from known concentrations of GSH. The results were expressed as lM/mg protein.
2.8. Determination of antioxidant enzymes Catalase activity was measured according to the method of Aebi (1984). One unit of catalase was defined as the amount of enzyme required to decompose 1 lM of H2O2 in 1 min. The reaction was initiated by the addition of 1.0 ml of freshly prepared 20 mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm for 1 min. The enzyme activity was expressed as U/mg protein. The activity of SOD was measured according to the method of McCord (1994). For the determination of SOD activity, xanthine and xanthine oxidase were used to generate superoxide radicals reacting with
2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyl tetrazolium chloride to form a red formazan dye. SOD activity was then measured at 505 nm. Hepatic GST activity was assayed according to the method of Habig et al. (1984) with some modifications. The reaction was carried out in 0.1 M potassium phosphate buffer (pH 6.5), 1 mM GSH, and 1 mM 1-chloro-2,4-dinitrobenzene in a 50 uL sample. An increase in absorbance was monitored with a wavelength of 340 nm at 25 C for a 4 min time period, and the activity of enzyme was expressed as lmol/min/mg protein.
2.9. Statistical analyses The results are expressed as mean ± SD, and all statistical comparisons were made by means of one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison test. The data were analyzed with GraphPad InStat v. 3.0 (GraphPad Software, Inc., CA, USA). The difference showing a P level of 0.05 or lower was considered to be statistically significant.
3. Results 3.1. Effects of PYC on CCl4-induced hepatotoxicity The single oral dose of CCl4 caused severe hepatotoxicity in rats, as evidenced by the significant elevation of serum AST and ALT activities and the increased incidence of histopathological hepatic injury after the administration of CCl4. In contrast, no treatment-related effects on the absolute and relative weights of the liver and necropsy findings were observed in the CCl4-treated rats (data not shown). The protective effects of pretreatment with PYC on the CCl4-induced elevation of serum AST and ALT activities are presented in Table 1. The activities of serum AST and ALT in the CCl4 group were much higher than those in the control group. However, pretreatment with PYC significantly prevented the elevation of serum AST and ALT activities induced by CCl4 treatment in a dosedependent manner. Pretreatment with PYC at a dose of 10 mg/kg partially prevented the elevation of AST and ALT levels, and 20 mg/kg almost completely prevented changes in the serum levels. The results of hepatic histopathological examination are shown in Table 2. When compared with the normal liver tissues of the vehicle controls, liver tissue in the rats treated with CCl4 revealed extensive liver injuries, characterized by moderate to severe hepatocellular degeneration and necrosis around the central vein, fatty changes, inflammatory cell
Table 1 Effects of PYC pretreatment on serum AST and ALT activities in CCl4induced hepatotoxicity in adult rats Group
Serum AST (IU/l)
Serum ALT (IU/l)
Control CCl4 CCl4&PYC 10 CCl4&PYC 20
131 ± 20.5 3238 ± 411.4** 883 ± 129.6**,## 591 ± 101.1**##
47 ± 3.9 743 ± 124.7** 246 ± 57.3**,## 173 ± 46.4*,##
Each value represents the mean ± SD of six rats. *,** Significant difference at P < 0.05 and P < 0.01 levels compared with the control group, respectively. ## Significant difference at P < 0.01 level compared with the CCl4 group.
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Table 2 Effects of PYC pretreatment on hepatic histopathology in CCl4-induced hepatotoxicity in adult rats Parameter
Hepatocyte degeneration/ necrosis Fatty change
Inflammatory cell infiltration Congestion
Sinusoidal dilatation
Grades Group
+ ++ +++ + ++ + ++ + ++ +++ + ++
Control CCl4 CCl4&PYC 10
CCl4&PYC 20
6 0 0 0 6 0 0 5 1 0 6 0 0 0 6 0 0
2 3 1 0 1 5 0 4 2 0 3 3 0 0 4 1 1
0 0 4 2 0 2 4 0 2 4 0 2 2 2 1 3 2
1 4 1 0 1 4 1 3 2 1 1 3 2 0 4 1 1
Grades are as follows: (normal), + (mild), ++ (moderate), and +++ (severe).
infiltration, congestion, and sinusoidal dilatation (Plate 1). However, the histopathological hepatic lesions induced by administration of CCl4 were remarkably ameliorated by pretreatment with PYC in a dose-dependent manner, and this was in good agreement with the results of serum aminotransferase activity and hepatic oxidative stress level. 3.2. Effects of PYC on lipid peroxidation and GSH levels As shown in Fig. 1, the concentration of MDA, an end product of lipid peroxidation, in the rats treated with CCl4 was increased 2.7-fold when compared with the vehicle control rats. Consistent with the serum AST and ALT activities, pretreatment with PYC resulted in a significant dose-dependent decrease in the concentration of hepatic MDA when compared with the CCl4 group. Conversely, as shown in Fig. 2, the concentration of hepatic GSH in the CCl4 group was decreased by 33% when compared with the vehicle control group. However, pretreatment with PYC also resulted in a statistically significant increase in hepatic GSH concentration in comparison with that of the CCl4 group. 3.3. Effects of PYC on antioxidant enzymes As presented in Fig. 3, the activity of catalase in the CCl4 group was decreased by more than 50% when compared with the vehicle control group. However, pretreatment with PYC resulted in a statistically significant, dosedependent increase in catalase activity when compared with the CCl4 group. As shown in Fig. 4, the hepatic SOD activity in the CCl4 group was also reduced by 38%
Plate 1. Representative photographs of liver sections stained with hematoxylin & eosin. (A) Hepatic tissue of a control rat, showing normal appearance. (B) Hepatic tissue of a CCl4-treated rat, showing moderate hepatocyte necrosis (*) around the central vein region, moderate fatty changes (.), and mild inflammatory cell infiltration (#). (C) Hepatic tissue of a PYC- (20 mg/kg) and CCl4-treated rat, showing mild hepatocyte necrosis (*) and fatty changes (.).
when compared with the vehicle control group. However, the SOD activity was significantly increased by PYC pretreatment in a dose-dependent manner when compared with the CCl4 group. As depicted in Fig. 5, the activity of GST in the CCl4 group was decreased by 35% as compared to normal control rats. However, pretreatment with PYC significantly protected against the depletion of GST activity induced by administration of CCl4 in a dose-dependent manner.
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Y.-S. Yang et al. / Food and Chemical Toxicology 46 (2008) 380–387 1.0
8
** 7
## ##
**
0.6
##
** ##
0.4
Catalase (unit/mg protein)
MDA (umol/mg protein)
0.8
6
5
4
** 3
2
0.2
1
0
0.0
Control
CCl4
CCl4 & PYC 10
Control
CCl4 & PYC 20
CCl4
CCl4 & PYC 20
Group
Group
Fig. 1. Effects of PYC on the hepatic MDA concentration in CCl4induced hepatotoxicity in adult rats. Each bar represents the mean ± SD of six rats. **Significant difference at P < 0.01 level compared with the control group. ##Significant difference at P < 0.01 level compared with the CCl4 group.
Fig. 3. Effects of PYC on the hepatic catalase activity in CCl4-induced hepatotoxicity in adult rats. Each bar represents the mean ± SD of six rats. **Significant difference at P < 0.01 level compared with the control group. ##Significant difference at P < 0.01 level compared with the CCl4 group.
3.0
2.5
**
**
2.5
2.0
##
2.0
**
1.5
1.0
SOD (unit/mg protein)
## GSH (umol/mg protein)
CCl4 & PYC 10
## ##
1.5
**
1.0
0.5 0.5
0.0
0.0
Control
CCl4
CCl4 & PYC 10
CCl4 & PYC 20
Group
Control
CCl4
CCl4 & PYC 10
CCl4 & PYC 20
Group
Fig. 2. Effects of PYC on the hepatic GSH concentration in CCl4-induced hepatotoxicity in adult rats. Each bar represents the mean ± SD of six rats. **Significant difference at P < 0.01 level compared with the control group. ##Significant difference at P < 0.01 level compared with the CCl4 group.
Fig. 4. Effects of PYC on the hepatic SOD activity in CCl4-induced hepatotoxicity in adult rats. Each bar represents the mean ± SD of six rats. **Significant difference at P < 0.01 level compared with the control group. ##Significant difference at P < 0.01 level compared with the CCl4 group.
4. Discussion
bility of PYC to protect against CCl4-induced hepatotoxicity and oxidative stress was investigated. In this study, we used an experimental model of CCl4-induced acute hepatotoxicity in rats, because this chemical is a potent hepatotoxin and a single exposure can rapidly lead to severe hepatic necrosis and steatosis (Recknagel et al., 1989; Janakat and Al-Merie, 2002). Previous studies reported that CCl4 is activated by cytochrome P-450 to form a trichloromethyl radical, CCl3
It has been reported that PYC shows a number of beneficial effects against various types of degenerative diseases in humans, largely because the major ingredients of PYC, polyphenols, have very potent antioxidant activity (Packer et al., 1999; Rohdewald, 2002). Therefore, we considered that PYC is useful in the prevention of various liver injuries induced by oxidative stress. In the present study, the capa-
Y.-S. Yang et al. / Food and Chemical Toxicology 46 (2008) 380–387 500
GST (umol/mg protein)
400
##
##
300
** 200
100
0
Control
CCl4
CCl4 & PYC 10
CCl4 & PYC 20
Group
Fig. 5. Effects of PYC on the hepatic GST activity in CCl4-induced hepatotoxicity in adult rats. Each bar represents the mean ± SD of six rats. **Significant difference at P < 0.01 level compared with the control group. ##Significant difference at P < 0.01 level compared with the CCl4 group.
(Weber et al., 2003). This radical can bind to cellular molecules (nucleic acid, protein, lipid), impairing crucial cellular processes such as lipid metabolism, with the potential outcome of fatty degeneration. The covalent binding of trichloromethyl radicals to cellular macromolecules is considered to function as the initiator of membrane lipid peroxidation and cell necrosis. This radical can also react with oxygen to form a trichloromethylperoxy radical, CCl3OO•, a highly reactive species. The trichloromethylperoxy radical initiates the chain reaction of lipid peroxidation, which attacks and destroys polyunsaturated fatty acids, particularly those associated with phospholipids. As expected, a single oral dose of CCl4 at 1.25 ml/kg showed significant hepatotoxicity, as evidenced by a dramatic elevation in the serum AST and ALT activities and an increased incidence and severity of histopathological hepatic lesions in rats. In addition, CCl4 treatment produced high levels of oxidative damage, as evidenced by a significant elevation in hepatic MDA level and a significant decrease in GSH concentration and catalase, SOD, and GST activities, which suggest a role of oxidative stress in CCl4 hepatotoxicity. However, pretreatment with PYC showed a significant protective effect against CCl4-induced acute hepatotoxicity and oxidative stress in rats. Serum aminotransferase activities have long been considered as sensitive indicators of hepatic injury (Molander et al., 1955). Injury to the hepatocytes alters their transport function and membrane permeability, leading to leakage of enzymes from the cells (Zimmerman and Seeff, 1970). Therefore, the marked release of AST and ALT into the circulation indicates severe damage to hepatic tissue membranes during CCl4 intoxication. In the present study, the single oral dose of CCl4 at 1.25 ml/kg caused a dramatic
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elevation in serum AST and ALT activities, indicating an acute hepatotoxicity induced by administration of CCl4 (Table 1). The acute hepatotoxic effects induced by CCl4 administration were confirmed histopathologically, revealing extensive hepatocellular degeneration and necrosis, fatty changes, inflammatory cell infiltration, congestion, and sinusoidal dilatation (Table 2 and Plate 1). The obtained results are in accordance with those of the previous reports (Recknagel et al., 1989; Lee and Jeong, 2002; Wong et al., 2003). Pretreatment with PYC efficiently prevented the CCl4-induced elevation of serum AST and ALT activities in a dose-dependent manner, indicating the hepatoprotective activity of PYC against the acute intoxication of CCl4. This phenomenon was also confirmed by the results of histopathological examination, as evidenced by a dose-related decrease in the incidence and severity of histopathological hepatic lesions. Lipid peroxidation, a reactive oxygen species-mediated mechanism, has been implicated in the pathogenesis of various liver injuries and subsequent liver fibrogenesis in experimental animals and humans (Niemela et al., 1994; Paradis et al., 1997; Liu et al., 2006). MDA is a major reactive aldehyde that appears during the peroxidation of biological membrane polyunsaturated fatty acid (Vaca et al., 1988). Therefore, the hepatic content of MDA is used as an indicator of liver tissue damage involving a series of chain reactions (Ohkawa et al., 1979). It has been accepted that lipid peroxidation of hepatocyte membranes is one of the principal causes of CCl4-induced hepatotoxicity, and is mediated by the production of free radical derivatives of CCl4 (Recknagel et al., 1989; Basu, 2003; Weber et al., 2003). In the present study, a single oral dose of CCl4 at 1.25 ml/kg resulted in a significant increase in the hepatic MDA concentration, indicating increased lipid peroxidation caused by administration of CCl4 (Fig. 1). The significant dose-dependent decrease in the hepatic MDA concentration confirms that pretreatment with PYC could effectively protect against the hepatic lipid peroxidation induced by CCl4. GSH (y-glutamylcysteinylglycine) acts as a non-enzymatic antioxidant both intracellularly and extracellularly in conjunction with various enzymatic processes that reduce hydrogen peroxide (H2O2) and hydroperoxides (Kadiska et al., 2000). It is involved in the maintenance of the normal structure and function of cells, probably by its redox and detoxification reactions (Gueeri, 1995). Reduced levels of GSH play a key role in the detoxification of the reactive toxic metabolites of CCl4; liver necrosis is initiated when reserves of GSH are markedly depleted (Recknagel et al., 1989; Williams and Burk, 1990). In the present study, the hepatic content of GSH was found to be decreased significantly in CCl4-intoxicated rats compared with control rats (Fig. 2). However, pretreatment with PYC significantly prevented the CCl4-induced depletion of hepatic GSH, indicating the antioxidant effect of PYC in CCl4-intoxicated rats. The antioxidant effects of PYC in the present study were determined at the doses
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of PYC 10 and 20 mg/kg to examine the dose–response relationship. Although the result of hepatic GSH concentration obtained in this study was not dose-dependent, most of the data showed a clear dose–response relationship. Therefore, the higher concentration of hepatic GSH observed in the CCl4&PYC 10 group than both the control and CCl4&PYC 20 groups is believed to be an accidental finding. Antioxidant enzymes such as catalase, SOD, and GST are easily inactivated by lipid peroxides or reactive oxygen species, which results in decreased activities of these enzymes in CCl4 toxicity. Of the antioxidant enzymes, catalase is a major antioxidant enzyme with hematin as the prosthetic group, and it is ubiquitously present in all aerobic cells containing a cytochrome system. It is most abundant in the liver and is responsible for the catalytic decomposition of H2O2 to oxygen and water (Baudrimont et al., 1997; Reiter et al., 2000). SOD is an extremely effective antioxidant enzyme, and is responsible for catalytic dismutation of highly reactive and potentially toxic superoxide radicals to H2O2 (Reiter et al., 2000). GST is a soluble protein that is located both in the cytosol and the endoplasmic reticulum of the liver, and catalyzes the conjugation of GSH with many xenobiotics and their reactive metabolites to form more water-soluble compounds (Boyer et al., 1984). Therefore, the GST enzyme also plays an important role in the detoxification of xenobiotics, catalyzing their conjugation with reduced GSH. The role of free radicals in CCl4 intoxication has been shown to be a major pathway of non-enzymatically induced lipid peroxidation, which subsequently affects various enzyme activities of the body and thereby may also be linked to enzymatically induced lipid peroxidation (Basu, 2003). Therefore, we theorize that the increased production of free radicals caused by administration of CCl4 is a major cause of the significantly decreased activities of catalase, SOD, and GST in rats treated with CCl4 compared with control rats (Figs. 3–5). In rats pretreated with PYC for 14 days, however, the activities of these antioxidant enzymes were significantly higher, and occurred in a dose-dependent manner, compared to the rats exposed to CCl4 alone, and were very similar to the values noted in normal control rats. Previous studies showed that intracellular antioxidant defense systems including vitamin C and E, SOD, catalase, and GSH are stabilized by PYC in the presence of oxidative stressors and, concomitantly, PYC decreases toxic byproducts of lipid peroxidation and reactive oxygen species such as H2O2, OH, nitric oxide (NO), O2, and ONOO (Kobuchi et al., 1999; Packer et al., 1999). The antioxidant activity of PYC has been demonstrated to protect against various degenerative conditions caused by free radicals, including diabetes-induced oxidative stress (Maritim et al., 2003), ethanol-induced neuronal cell death (Siler-Marsiglio et al., 2004), 2,4,6-trinitrobenzene sulfonic acid-induced inflammatory bowel disease (Mochizuki and Hasegawa, 2004), and ionizing radiation-induced intestinal damage (Ramos et al., 2006). The results of the above stud-
ies and our investigation strongly suggest that the hepatoprotective effect of PYC observed in this study largely resulted from antioxidant activity, including stabilization in the intracellular antioxidant defense systems and reductions in the lipid peroxidation products and the production of reactive oxygen species. As far as we know, this is the first report to demonstrate that pretreatment with PYC prevented CCl4-induced acute hepatotoxicity and oxidative stress in rats. On the whole, it can be concluded that PYC has a protective effect against acute hepatotoxicity induced by the administration of CCl4 in rats, and that the hepatoprotective effects of PYC may be due to both the inhibition of lipid peroxidation and the increase of antioxidant activity. Acknowledgements This work was supported by the Regional Research Centers Program (Bio-housing Research Institute), granted by the Korean Ministry of Education&Human Resources Development. References Aebi, H., 1984. Catalase in vitro. In: Packer, L. (Ed.), Methods in Enzymology, vol. 105. Academic Press, San Diego, pp. 121–126. Basu, S., 2003. Carbon tetrachloride-induced lipid peroxidation: eicosanoid formation and their regulation by antioxidant nutrients. Toxicology 189, 113–127. Baudrimont, I., Ahouandjivo, R., Creppy, E.E., 1997. Prevention of lipid peroxidation induced by ochratoxin A in Vero cells in culture by several agents. Chem. Biol. Interact. 104, 29–40. Berton, T.R., Conti, C.J., Mitchell, D.L., Aldaz, C.M., Lubet, R.A., Fischer, S.M., 1998. The effect of vitamin E acetate on ultravioletinduced mouse skin carcinogenesis. Mol. Carcinog. 23, 175–184. Bors, W., Michel, C., 2002. Chemistry of the antioxidant effect of polyphenols. Ann. NY Acad. Sci. 957, 57–69. Boyer, T.D., Vessey, D.A., Holcomb, C., Saley, N., 1984. Studies of the relationship between the catalytic activity and binding of nonsubstrate ligands by the glutathione S-transferases. Biochem. J. 217, 179–185. Cai, Y.J., Ma, L.P., Hou, L.F., Zhou, B., Yang, L., Liu, Z.L., 2002. Antioxidant effects of green tea polyphenols on free radical initiated peroxidation of rat liver microsomes. Chem. Phys. Lipids 120, 109– 117. Fogden, E., Neuberger, J., 2003. Alternative medicines and the liver. Liver Int. 23, 213–220. Grimm, T., Skrabala, R., Chovanova´, Z., Muchova´, J., Sumegova´, K., ˇ uracˇkova´, Z., Ho¨gger, P., 2006. Single and multiple Lipta´kova´, A., D dose pharmacokinetics of maritime pine bark extract (Pycnogenol) after oral administration to healthy volunteers. BMC Clin. Pharmacol. 6, 4. Gueeri, H., 1995. Influence on prolonged ethanol intake on the level and turnover of alcohol and aldehyde dehydrogenase and glutathione. Adv. Exp. Med. Biol. 23, 133–134. Habig, W.H., Jakoby, W.B., Guthenberg, C., Mannervik, B., Vander Jagt, D.L., 1984. 2-Propylthiouracil does not replace glutathione for the glutathione transferases. J. Biol. Chem. 259, 7409–7410. Hanje, B., Fortune, A.J., Song, M., Hill, D., McClain, C., 2006. The use of selected nutrition supplements and complementary and alternative medicine in liver disease. Nutr. Clin. Pract. 21, 255–272. Janakat, S., Al-Merie, H., 2002. Optimization of the dose and route of injection, and characterisation of the time course of carbon
Y.-S. Yang et al. / Food and Chemical Toxicology 46 (2008) 380–387 tetrachloride-induced hepatotoxicity in the rat. J. Pharmacol. Toxicol. Methods 48, 41–44. Johansen, J.S., Harris, A.K., Rychly, D.J., Ergul, A., 2005. Oxidative stress and the use of antioxidants in diabetes: linking basic science to clinical practice. Cardiovas. Diabetol. 4, 5. Kadiska, M.B., Gladen, B.C., Baird, D.D., Dikalov, A.E., Sohal, R.S., Hatch, G.B., Jones, D.P., Mason, R.P., Barret, J.C., 2000. Biomarkers of oxidative stress study: are plasma antioxidants markers of CCl4 poisoning? Free Rad. Biol. Med. 28, 838–845. Kobuchi, H., Virgili, F., Packer, L., 1999. Assay of inducible form of nitric oxide synthase activity: effect of flavonoids and plant extracts. Methods Enzymol. 301, 504–513. Lee, K.J., Jeong, H.G., 2002. Protective effect of Platycodi radix on carbon tetrachloride-induced hepatotoxicity. Food Chem. Toxicol. 40, 517– 525. Liu, J.Y., Chen, C.C., Wang, W.H., Hsu, J.D., Yang, M.Y., Wang, C.J., 2006. The protective effects of Hibiscus sabdariffa extract on CCl4induced liver fibrosis in rats. Food Chem. Toxicol. 44, 336–343. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265–275. Maritim, A., Dene, B.A., Sanders, R.A., Watkins, J.B., 2003. Effects of pycnogenol treatment on oxidative stress in streptozotocin-induced diabetic rats. J. Biochem. Mol. Toxicol. 17, 193–199. McCord, J.M., 1994. Mutant mice, Cu, Zn superoxide dismutase, and motor neuron degeneration. Science 266, 1586–1587. Mochizuki, M., Hasegawa, N., 2004. Therapeutic efficacy of pycnogenol in experimental inflammatory bowel diseases. Phytother. Res. 18, 1027–1028. Molander, D.W., Wroblewsk, F., La Due, J.S., 1955. Transaminase compared with cholinesterase and alkaline phosphatase an index of hepatocellular integrity. Clin. Res. Proc. 3, 20–24. Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathione reductase and glutathione-S-transferase activities in rat lung and liver. Biochem. Biophys. Acta 582, 67–78. Niemela, O., Parkkila, S., Yla-Herttuala, S., Halsted, C., Lanca, A., Israel, Y., 1994. Covalent protein adducts in the liver as a result of ethanol metabolism and lipid peroxidation. Lab. Invest. 70, 537–546. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay of lipid peroxides in animal tissue by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Packer, L., Rimbach, G., Virgili, F., 1999. Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus maritima) bark, pycnogenol. Free Rad. Biol. Med. 27, 704–724. Paradis, V., Kollinger, M., Fabre, M., Holstege, A., Poynard, T., Beddosa, P., 1997. In situ detection of lipid peroxidation by-products in chronic liver diseases. Hepatology 26, 135–142.
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Ramos, F.M.D.M., Schonlau, F., Novaes, P.D., Manzi, F.R., Boscolo, F.N., Almeida, S.M.D., 2006. Pycnogenol protects against ionizing radiation as shown in the intestinal mucosa of rats exposed to X-rays. Phytother. Res. 20, 676–679. Recknagel, R.O., Glende, E.A., Dolak, J.A., Waller, R.L., 1989. Mechanisms of carbon tetrachloride toxicity. Pharmacol. Therapeut. 43, 139–154. Reiter, R.J., Tan, D., Osuna, C., Gitto, E., 2000. Actions of melatonin in the reduction of oxidative stress. J. Biomed. Sci. 7, 444–458. Rohdewald, P., 2002. A review of the French maritime pine bark extract (Pycnogenol), a herbal medication with a diverse clinical pharmacology. Int. J. Clin. Pharmacol. Therapeut. 40, 158–168. Seeff, L.B., Lindsay, K.L., Bacon, B.R., Kresina, T.F., Hoofnaqle, J.H., 2001. Complementary and alternative medicine in chronic liver disease. Hepatology 34, 595–603. Shin, J.W., Son, J.Y., Oh, S.M., Han, S.H., Wang, J.H., Cho, J.H., Cho, C.K., Yoo, H.S., Lee, Y.W., Lee, M.M., Hu, X.P., Son, C.G., 2006. An herbal formula, CGX, exerts hepatotherapeutic effects on dimethylnitrosamine-induced chronic liver injury model in rats. World J. Gastroenterol. 12, 6142–6148. Siler-Marsiglio, K.I., Shaw, G., Heaton, M.B., 2004. Pycnogenol and vitamin E inhibit ethanol-induced apoptosis in rat cerebellar granule cells. J. Neurobiol. 59, 261–271. Strader, D.B., Bacon, B.R., Lindsay, K.L., La Breque, D.R., Morgan, T., Wright, E.C., Allen, J., Khokar, M.F., Hoofnagle, J.H., Seeff, L.B., 2002. Use of complementary and alternative medicine in patients with liver disease. Am. J. Gastroenterol. 97, 2391–2397. Vaca, C.E., Wilhelm, J., Harms-Rihsdahl, M., 1988. Interaction of lipid peroxidation product with DNA. A Rev. Mutat. Res. Rev. Genet. Toxicol. 195, 137–149. Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T.D., Mazur, M., Telser, J., 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39, 44–84. Weber, L.W.D., Bull, M., Stampfl, A., 2003. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit. Rev. Toxicol. 33, 105–136. Williams, A.T., Burk, R.F., 1990. Carbon tetrachloride hepatotoxicity: an example of free radical-mediated injury. Semin. Liver Dis. 10, 279–284. Wong, C.K., Ooi, V.E.C., Wong, C.K., 2003. Protective effects of N-acetylcysteine against carbon tetrachloride and trichloroethyleneinduced poisoning in rats. Environ. Toxicol. Pharmacol. 14, 109– 116. Zimmerman, H.J., Seeff, L.B., 1970. Enzymes in hepatic disease. In: Goodley, E.L. (Ed.), Diagnostic Enzymology. Lea and Febiger, Philadelphia, pp. 1–38.
Food and Chemical Toxicology 46 (2008) 380–387 www.elsevier.com/locate/foodchemtox
Protective effects of Pycnogenol on carbon tetrachloride-induced hepatotoxicity in Sprague–Dawley rats Young-Su Yang a, Tai-Hwan Ahn a, Jong-Chan Lee a, Chang-Jong Moon a, Sung-Ho Kim a, Woojin Jun b, Seung-Chun Park c, Hyoung-Chin Kim d, Jong-Choon Kim a,* a
Department of Toxicology, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea b Department of Food and Nutrition, Chonnam National University, Gwangju 500-757, Republic of Korea c College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea d Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea Received 31 January 2007; accepted 10 August 2007
Abstract Oxidative damage is implicated in the pathogenesis of various liver injuries. In the present study the ability of Pycnogenol (PYC) as an antioxidant to protect against CCl4-induced oxidative stress and hepatotoxicity in rats was investigated. Four experimental groups of six rats each were constructed: a vehicle control group received the respective vehicles (distilled water and corn oil) only; a CCl4 group received a 14-day repeated intraperitoneal (i.p.) dose of distilled water and then a single oral dose of CCl4 at 1.25 ml/kg; and the CCl4&PYC 10 and CCl4&PYC 20 groups received a 14-day repeated i.p. dose of PYC 10 and 20 mg/kg, respectively, and then a single oral dose of CCl4 at 1.25 ml/kg. Hepatotoxicity was assessed 24 h after the CCl4 treatment by measurement of serum aminotransferase (AST) and alanine aminotransferase (ALT) activities, hepatic malondialdehyde (MDA) and glutathione (GSH) concentrations, and catalase, superoxide dismutase (SOD), and glutathione-S-transferase (GST) activities. The results were confirmed histopathologically. The single oral dose of CCl4 produced significantly elevated levels of serum AST and ALT activities. Histopathological examinations showed extensive liver injuries, characterized by extensive hepatocellular degeneration/necrosis, fatty changes, inflammatory cell infiltration, congestion, and sinusoidal dilatation. In addition, an increased MDA concentration and decreased GSH, catalase, SOD, and GST were observed in the hepatic tissues. On the contrary, PYC treatment prior to the administration of CCl4 significantly prevented the CCl4-induced hepatotoxicity, including the elevation of serum AST and ALT activities and histopathological hepatic lesions, in a dose-dependent manner. Moreover, MDA and GSH levels and catalase, SOD, and GST activities in hepatic tissues were not affected by administration of CCl4, indicating that the pretreatment of PYC efficiently protects against CCl4-induced oxidative damage in rats. The results indicate that PYC has a protective effect against acute hepatotoxicity induced by the administration of CCl4 in rats, and that the hepatoprotective effects of PYC may be due to both the inhibition of lipid peroxidation and the increase of antioxidant activity. 2007 Elsevier Ltd. All rights reserved. Keywords: Pycnogenol; Carbon tetrachloride; Hepatotoxicity; Oxidative stress; Protective effect
1. Introduction Liver disease is considered to be a serious health problem, as the liver is an important organ for the detoxification and deposition of endogenous and exogenous substances. *
Corresponding author. Tel.: +82 62 530 2827; fax: +82 62 530 2809. E-mail address: [email protected] (J.-C. Kim).
0278-6915/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.08.016
Steroids, vaccines, and antiviral drugs, which have been employed as therapies for liver diseases, have potential adverse effects, especially when administered for long terms. Therefore, herbal products and traditional medicines with improved effectiveness and safety profiles are needed as a substitute for chemical therapeutics. It is reported that a number of herbal products have been shown to protect against liver injury, and many possess
Y.-S. Yang et al. / Food and Chemical Toxicology 46 (2008) 380–387
one or a combination of antioxidant, antifibrotic, immune modulatory, or antiviral activities (Seeff et al., 2001; Lee and Jeong, 2002; Shin et al., 2006). In recent years, there has been a substantial increase in the use of so-called complementary and alternative therapies that utilize herbal medicines by patients with liver disease (Strader et al., 2002; Fogden and Neuberger, 2003; Hanje et al., 2006). Pycnogenol (PYC), a standardized extract from the bark of the French maritime pine (Pinus maritima), is used extensively in dietary supplements, multi-vitamins, and health products because of its direct, strong antioxidant activity (Rohdewald, 2002). Major constituents of PYC are polyphenols, specifically monomeric and oligomeric units of catechin, epicatechin, and taxifolin (Rohdewald, 2002; Grimm et al., 2006). It is well documented that polyphenols comprise a wide area of natural substances of plant origin, and almost all of them exhibit a marked antioxidant activity (Bors and Michel, 2002; Cai et al., 2002). Oxidative stress is defined in general as excess formation and/or insufficient removal of highly reactive molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Johansen et al., 2005; Valko et al., 2007). ROS include free radicals such as superoxide anion (O2), hydroxyl (OH), peroxyl (RO2), hydroperoxyl (HRO2) as well as nonradical species such as hydrogen peroxide (H2O2) and hydrochlorous acid (HOCl). RNS include free radicals like nitric oxide (NO) and nitrogen dioxide (NO2), as well as nonradicals such as peroxynitrite (ONOO), nitrous oxide (HNO2) and alkyl peroxynitrates (RONOO). The effects of antioxidants on oxidative stress are mainly measured through certain observable biomarkers, including the enzymatic activities of catalase, SOD, GSH-Px, and GSH-reductase, as well as thiobarbituric acid reactants (TBARS) levels, an indirect measurement of free-radical production (Maritim et al., 2003; Johansen et al., 2005; Valko et al., 2007). Previous studies showed that PYC is a very potent antioxidant to scavenge reactive oxygen and nitrogen species such as superoxide anion radical, hydroxyl radical, lipid peroxyl radical (LOO), peroxynitrite radical, and singlet oxygen (1O2). It has also been shown to bind to proteins, thereby affecting both structural and functional characteristics of key enzymes and other proteins involved in metabolism; to participate in the cellular antioxidant network, especially in prolonging the lifetime of ascorbyl radical, possibly through a one-electron reduction of dehydroascorbic acid; and to protect endogenous vitamin E and glutathione (Packer et al., 1999; Maritim et al., 2003). It has been claimed that PYC has diverse beneficial effects on a wide range of medical conditions, including inflammation, diabetes, asthma, hypertension, attention deficiency hyperactivity disorder, cancer, immune disease, and others (Rohdewald, 2002). Despite the favorable pharmacological properties of PYC, the protective capacity of PYC against hepatotoxicity has not previously been explored. PYC may have a protective effect on the deteriorated hepatic function that results from free radicals in the toxic
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chemical-induced hepatotoxicity. To test this hypothesis, the present investigation examined the ability of PYC to protect against carbon tetrachloride (CCl4)-induced oxidative stress and hepatotoxicity. We selected CCl4 as a model hepatotoxicant because this chemical is a potent hepatotoxin and a single administration can rapidly lead to both oxidative stress via the excessive production of free radicals and acute liver injuries such as centrilobular necrosis and steatosis in rats (Recknagel et al., 1989; Janakat and Al-Merie, 2002; Weber et al., 2003). Because CCl4 is a common contaminant found in both ambient outdoor and indoor air, the human health risk is very high. It is therefore important to investigate the antioxidant activity of PYC against CCl4-induced hepatic oxidative damages. 2. Materials and methods 2.1. Animal husbandry and maintenance Thirty male Sprague–Dawley rats aged 8 weeks were obtained from a specific pathogen-free colony at Bio Genomics Inc. (Seoul, Republic of Korea) and used after 1 week of quarantine and acclimatization. The animals were housed in a room maintained at a temperature of 23 ± 3 C and a relative humidity of 50 ± 10% with artificial lighting from 08:00 to 20:00 and 13 to 18 air changes per hour. The animals were housed two per cage in stainless steel wire mesh cages and were allowed sterilized tap water and commercial rodent chow (Samyang Feed Co., Wonju, Republic of Korea) ad libitum. Our Institutional Animal Care and Use Committee approved the protocols for the animal study, and the animals were cared for in accordance with the Guidelines for Animal Experiments of the Chonnam National University.
2.2. Materials and treatment PYC (US patent # 4,698,360), extracted exclusively from the bark of French marine pine, was donated from Horphag Research Ltd. (Route de Belis, 40420 Le Sen, France). CCl4 and corn oil were purchased from the Sigma–Aldrich Co. (St. Louis, MO, USA). All other chemicals were of the highest grade commercially available. PYC and CCl4 were dissolved in sterilized distilled water and corn oil, respectively, and were prepared immediately before treatment. PYC was administered intraperitoneally (i.p.) to rats once daily for 14 days at dose levels of 10 and 20 mg/kg. Three hours after the final PYC treatment, the rats were given a single oral dose of CC14 (1.25 ml/kg, 20% in corn oil) to induce liver injury. All of the animals were sacrificed 24 h after administration of CCl4.
2.3. Experimental groups and dose selection Prior to testing, rats were evaluated by clinical observations and body weight determinations during a 7-day quarantine period to assure freedom from potential confounding variables. Twenty-four healthy male rats were randomly assigned to four experimental groups of six rats each: a vehicle control group received the respective vehicles only; a CCl4 group received a 14-day repeated i.p. dose of distilled water and then a single oral dose of CCl4 at 1.25 ml/kg, which is well documented to induce acute hepatotoxicity in rats (Janakat and Al-Merie, 2002; Wong et al., 2003); and the CCl4&PYC 10 and CCl4&PYC 20 groups received a 14-day repeated i.p. dose of PYC 10 and 20 mg/kg, respectively, and then a single oral dose of CCl4 at 1.25 ml/kg. The effective doses of PYC were based on earlier reports and clinical dosages (Packer et al., 1999; Maritim et al., 2003). We used only male rats because of their constant metabolism compared to the variations in the female physiology.
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2.4. Autopsy and serum biochemistry All treated animals were anesthetized by ether inhalation for blood sample collection 24 h after administration of CCl4. Blood samples were drawn from the posterior vena cava using a syringe with 24-gauge needle under ether anesthesia. The samples were centrifuged at 1256g for 10 min within 1 h after collection. The sera were stored in the 80 C freezer before they were analyzed. Enzyme activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in blood serum were evaluated by an autoanalyzer (Shimadzu CL-7200, Shimadzu, Japan). After bleeding, complete gross postmortem examinations were performed on all terminated rats. The absolute and relative (organ-to-body weight ratio) weights of the liver were also measured for all rats when they were sacrificed.
2.5. Histopathological examinations A portion of the median lobe of the liver was dissected and fixed in 10% neutral buffered formalin solution for 24 h. The remaining livers were frozen quickly in dry ice and stored at 70 C for biochemical analysis. The fixed tissues were processed routinely, and were then embedded in paraffin, sectioned to 3–5 lm thickness, deparaffinized, and rehydrated using standard techniques. The extent of CC14-induced necrosis and steatosis was evaluated by assessing morphological changes in liver sections stained with hematoxylin and eosin (H&E), using standard techniques.
2.6. Preparation of hepatic homogenate The weighed frozen liver tissue was homogenized in a glass-Teflon homogenizer with 50 mM phosphate buffer (pH 7.4) to obtain 1:9 (w/v) whole homogenate. The homogenates were then centrifuged at 11,000g for 15 min at 4 C to discard any cell debris, and the supernatant was used for the measurement of malondialdehyde (MDA), reduced glutathione (GSH), catalase, superoxide dismutase (SOD), and glutathione-S-transferase (GST). Total protein contents were determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard.
2.7. Determination of lipid peroxidation and GSH levels Lipid peroxidation was measured by the thiobarbituric acid (TBA) reaction method (Berton et al., 1998). In brief, samples were mixed with TBA reagent consisting of 0.375% TBA and 15% trichloroacetic acid in 0.25-N hydrochloric acid. The reaction mixtures were placed in a boiling water bath for 30 min and centrifuged at 1811g for 5 min. The absorbance of the supernatant was measured at 535 nm. MDA, a measure of lipid peroxidation, was calculated using an extinction coefficient of 1.56 · 105/ M cm. The results were expressed as lM/mg protein. GSH content was measured by the method of Moron et al. (1979). In brief, samples were mixed with 0.1 M of sodium phosphate buffer (pH 7.5) containing 5 lM EDTA, 0.6 mM of 5,5-dithiol-bis (2-nitrobenzoic acid), 0.2 mM NADPH and glutathione reductase. The mixture was incubated for 2 min at room temperature. The absorbance of the product was measured at 412 nm. GSH content was determined using a standard curve generated from known concentrations of GSH. The results were expressed as lM/mg protein.
2.8. Determination of antioxidant enzymes Catalase activity was measured according to the method of Aebi (1984). One unit of catalase was defined as the amount of enzyme required to decompose 1 lM of H2O2 in 1 min. The reaction was initiated by the addition of 1.0 ml of freshly prepared 20 mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm for 1 min. The enzyme activity was expressed as U/mg protein. The activity of SOD was measured according to the method of McCord (1994). For the determination of SOD activity, xanthine and xanthine oxidase were used to generate superoxide radicals reacting with
2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyl tetrazolium chloride to form a red formazan dye. SOD activity was then measured at 505 nm. Hepatic GST activity was assayed according to the method of Habig et al. (1984) with some modifications. The reaction was carried out in 0.1 M potassium phosphate buffer (pH 6.5), 1 mM GSH, and 1 mM 1-chloro-2,4-dinitrobenzene in a 50 uL sample. An increase in absorbance was monitored with a wavelength of 340 nm at 25 C for a 4 min time period, and the activity of enzyme was expressed as lmol/min/mg protein.
2.9. Statistical analyses The results are expressed as mean ± SD, and all statistical comparisons were made by means of one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison test. The data were analyzed with GraphPad InStat v. 3.0 (GraphPad Software, Inc., CA, USA). The difference showing a P level of 0.05 or lower was considered to be statistically significant.
3. Results 3.1. Effects of PYC on CCl4-induced hepatotoxicity The single oral dose of CCl4 caused severe hepatotoxicity in rats, as evidenced by the significant elevation of serum AST and ALT activities and the increased incidence of histopathological hepatic injury after the administration of CCl4. In contrast, no treatment-related effects on the absolute and relative weights of the liver and necropsy findings were observed in the CCl4-treated rats (data not shown). The protective effects of pretreatment with PYC on the CCl4-induced elevation of serum AST and ALT activities are presented in Table 1. The activities of serum AST and ALT in the CCl4 group were much higher than those in the control group. However, pretreatment with PYC significantly prevented the elevation of serum AST and ALT activities induced by CCl4 treatment in a dosedependent manner. Pretreatment with PYC at a dose of 10 mg/kg partially prevented the elevation of AST and ALT levels, and 20 mg/kg almost completely prevented changes in the serum levels. The results of hepatic histopathological examination are shown in Table 2. When compared with the normal liver tissues of the vehicle controls, liver tissue in the rats treated with CCl4 revealed extensive liver injuries, characterized by moderate to severe hepatocellular degeneration and necrosis around the central vein, fatty changes, inflammatory cell
Table 1 Effects of PYC pretreatment on serum AST and ALT activities in CCl4induced hepatotoxicity in adult rats Group
Serum AST (IU/l)
Serum ALT (IU/l)
Control CCl4 CCl4&PYC 10 CCl4&PYC 20
131 ± 20.5 3238 ± 411.4** 883 ± 129.6**,## 591 ± 101.1**##
47 ± 3.9 743 ± 124.7** 246 ± 57.3**,## 173 ± 46.4*,##
Each value represents the mean ± SD of six rats. *,** Significant difference at P < 0.05 and P < 0.01 levels compared with the control group, respectively. ## Significant difference at P < 0.01 level compared with the CCl4 group.
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Table 2 Effects of PYC pretreatment on hepatic histopathology in CCl4-induced hepatotoxicity in adult rats Parameter
Hepatocyte degeneration/ necrosis Fatty change
Inflammatory cell infiltration Congestion
Sinusoidal dilatation
Grades Group
+ ++ +++ + ++ + ++ + ++ +++ + ++
Control CCl4 CCl4&PYC 10
CCl4&PYC 20
6 0 0 0 6 0 0 5 1 0 6 0 0 0 6 0 0
2 3 1 0 1 5 0 4 2 0 3 3 0 0 4 1 1
0 0 4 2 0 2 4 0 2 4 0 2 2 2 1 3 2
1 4 1 0 1 4 1 3 2 1 1 3 2 0 4 1 1
Grades are as follows: (normal), + (mild), ++ (moderate), and +++ (severe).
infiltration, congestion, and sinusoidal dilatation (Plate 1). However, the histopathological hepatic lesions induced by administration of CCl4 were remarkably ameliorated by pretreatment with PYC in a dose-dependent manner, and this was in good agreement with the results of serum aminotransferase activity and hepatic oxidative stress level. 3.2. Effects of PYC on lipid peroxidation and GSH levels As shown in Fig. 1, the concentration of MDA, an end product of lipid peroxidation, in the rats treated with CCl4 was increased 2.7-fold when compared with the vehicle control rats. Consistent with the serum AST and ALT activities, pretreatment with PYC resulted in a significant dose-dependent decrease in the concentration of hepatic MDA when compared with the CCl4 group. Conversely, as shown in Fig. 2, the concentration of hepatic GSH in the CCl4 group was decreased by 33% when compared with the vehicle control group. However, pretreatment with PYC also resulted in a statistically significant increase in hepatic GSH concentration in comparison with that of the CCl4 group. 3.3. Effects of PYC on antioxidant enzymes As presented in Fig. 3, the activity of catalase in the CCl4 group was decreased by more than 50% when compared with the vehicle control group. However, pretreatment with PYC resulted in a statistically significant, dosedependent increase in catalase activity when compared with the CCl4 group. As shown in Fig. 4, the hepatic SOD activity in the CCl4 group was also reduced by 38%
Plate 1. Representative photographs of liver sections stained with hematoxylin & eosin. (A) Hepatic tissue of a control rat, showing normal appearance. (B) Hepatic tissue of a CCl4-treated rat, showing moderate hepatocyte necrosis (*) around the central vein region, moderate fatty changes (.), and mild inflammatory cell infiltration (#). (C) Hepatic tissue of a PYC- (20 mg/kg) and CCl4-treated rat, showing mild hepatocyte necrosis (*) and fatty changes (.).
when compared with the vehicle control group. However, the SOD activity was significantly increased by PYC pretreatment in a dose-dependent manner when compared with the CCl4 group. As depicted in Fig. 5, the activity of GST in the CCl4 group was decreased by 35% as compared to normal control rats. However, pretreatment with PYC significantly protected against the depletion of GST activity induced by administration of CCl4 in a dose-dependent manner.
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Y.-S. Yang et al. / Food and Chemical Toxicology 46 (2008) 380–387 1.0
8
** 7
## ##
**
0.6
##
** ##
0.4
Catalase (unit/mg protein)
MDA (umol/mg protein)
0.8
6
5
4
** 3
2
0.2
1
0
0.0
Control
CCl4
CCl4 & PYC 10
Control
CCl4 & PYC 20
CCl4
CCl4 & PYC 20
Group
Group
Fig. 1. Effects of PYC on the hepatic MDA concentration in CCl4induced hepatotoxicity in adult rats. Each bar represents the mean ± SD of six rats. **Significant difference at P < 0.01 level compared with the control group. ##Significant difference at P < 0.01 level compared with the CCl4 group.
Fig. 3. Effects of PYC on the hepatic catalase activity in CCl4-induced hepatotoxicity in adult rats. Each bar represents the mean ± SD of six rats. **Significant difference at P < 0.01 level compared with the control group. ##Significant difference at P < 0.01 level compared with the CCl4 group.
3.0
2.5
**
**
2.5
2.0
##
2.0
**
1.5
1.0
SOD (unit/mg protein)
## GSH (umol/mg protein)
CCl4 & PYC 10
## ##
1.5
**
1.0
0.5 0.5
0.0
0.0
Control
CCl4
CCl4 & PYC 10
CCl4 & PYC 20
Group
Control
CCl4
CCl4 & PYC 10
CCl4 & PYC 20
Group
Fig. 2. Effects of PYC on the hepatic GSH concentration in CCl4-induced hepatotoxicity in adult rats. Each bar represents the mean ± SD of six rats. **Significant difference at P < 0.01 level compared with the control group. ##Significant difference at P < 0.01 level compared with the CCl4 group.
Fig. 4. Effects of PYC on the hepatic SOD activity in CCl4-induced hepatotoxicity in adult rats. Each bar represents the mean ± SD of six rats. **Significant difference at P < 0.01 level compared with the control group. ##Significant difference at P < 0.01 level compared with the CCl4 group.
4. Discussion
bility of PYC to protect against CCl4-induced hepatotoxicity and oxidative stress was investigated. In this study, we used an experimental model of CCl4-induced acute hepatotoxicity in rats, because this chemical is a potent hepatotoxin and a single exposure can rapidly lead to severe hepatic necrosis and steatosis (Recknagel et al., 1989; Janakat and Al-Merie, 2002). Previous studies reported that CCl4 is activated by cytochrome P-450 to form a trichloromethyl radical, CCl3
It has been reported that PYC shows a number of beneficial effects against various types of degenerative diseases in humans, largely because the major ingredients of PYC, polyphenols, have very potent antioxidant activity (Packer et al., 1999; Rohdewald, 2002). Therefore, we considered that PYC is useful in the prevention of various liver injuries induced by oxidative stress. In the present study, the capa-
Y.-S. Yang et al. / Food and Chemical Toxicology 46 (2008) 380–387 500
GST (umol/mg protein)
400
##
##
300
** 200
100
0
Control
CCl4
CCl4 & PYC 10
CCl4 & PYC 20
Group
Fig. 5. Effects of PYC on the hepatic GST activity in CCl4-induced hepatotoxicity in adult rats. Each bar represents the mean ± SD of six rats. **Significant difference at P < 0.01 level compared with the control group. ##Significant difference at P < 0.01 level compared with the CCl4 group.
(Weber et al., 2003). This radical can bind to cellular molecules (nucleic acid, protein, lipid), impairing crucial cellular processes such as lipid metabolism, with the potential outcome of fatty degeneration. The covalent binding of trichloromethyl radicals to cellular macromolecules is considered to function as the initiator of membrane lipid peroxidation and cell necrosis. This radical can also react with oxygen to form a trichloromethylperoxy radical, CCl3OO•, a highly reactive species. The trichloromethylperoxy radical initiates the chain reaction of lipid peroxidation, which attacks and destroys polyunsaturated fatty acids, particularly those associated with phospholipids. As expected, a single oral dose of CCl4 at 1.25 ml/kg showed significant hepatotoxicity, as evidenced by a dramatic elevation in the serum AST and ALT activities and an increased incidence and severity of histopathological hepatic lesions in rats. In addition, CCl4 treatment produced high levels of oxidative damage, as evidenced by a significant elevation in hepatic MDA level and a significant decrease in GSH concentration and catalase, SOD, and GST activities, which suggest a role of oxidative stress in CCl4 hepatotoxicity. However, pretreatment with PYC showed a significant protective effect against CCl4-induced acute hepatotoxicity and oxidative stress in rats. Serum aminotransferase activities have long been considered as sensitive indicators of hepatic injury (Molander et al., 1955). Injury to the hepatocytes alters their transport function and membrane permeability, leading to leakage of enzymes from the cells (Zimmerman and Seeff, 1970). Therefore, the marked release of AST and ALT into the circulation indicates severe damage to hepatic tissue membranes during CCl4 intoxication. In the present study, the single oral dose of CCl4 at 1.25 ml/kg caused a dramatic
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elevation in serum AST and ALT activities, indicating an acute hepatotoxicity induced by administration of CCl4 (Table 1). The acute hepatotoxic effects induced by CCl4 administration were confirmed histopathologically, revealing extensive hepatocellular degeneration and necrosis, fatty changes, inflammatory cell infiltration, congestion, and sinusoidal dilatation (Table 2 and Plate 1). The obtained results are in accordance with those of the previous reports (Recknagel et al., 1989; Lee and Jeong, 2002; Wong et al., 2003). Pretreatment with PYC efficiently prevented the CCl4-induced elevation of serum AST and ALT activities in a dose-dependent manner, indicating the hepatoprotective activity of PYC against the acute intoxication of CCl4. This phenomenon was also confirmed by the results of histopathological examination, as evidenced by a dose-related decrease in the incidence and severity of histopathological hepatic lesions. Lipid peroxidation, a reactive oxygen species-mediated mechanism, has been implicated in the pathogenesis of various liver injuries and subsequent liver fibrogenesis in experimental animals and humans (Niemela et al., 1994; Paradis et al., 1997; Liu et al., 2006). MDA is a major reactive aldehyde that appears during the peroxidation of biological membrane polyunsaturated fatty acid (Vaca et al., 1988). Therefore, the hepatic content of MDA is used as an indicator of liver tissue damage involving a series of chain reactions (Ohkawa et al., 1979). It has been accepted that lipid peroxidation of hepatocyte membranes is one of the principal causes of CCl4-induced hepatotoxicity, and is mediated by the production of free radical derivatives of CCl4 (Recknagel et al., 1989; Basu, 2003; Weber et al., 2003). In the present study, a single oral dose of CCl4 at 1.25 ml/kg resulted in a significant increase in the hepatic MDA concentration, indicating increased lipid peroxidation caused by administration of CCl4 (Fig. 1). The significant dose-dependent decrease in the hepatic MDA concentration confirms that pretreatment with PYC could effectively protect against the hepatic lipid peroxidation induced by CCl4. GSH (y-glutamylcysteinylglycine) acts as a non-enzymatic antioxidant both intracellularly and extracellularly in conjunction with various enzymatic processes that reduce hydrogen peroxide (H2O2) and hydroperoxides (Kadiska et al., 2000). It is involved in the maintenance of the normal structure and function of cells, probably by its redox and detoxification reactions (Gueeri, 1995). Reduced levels of GSH play a key role in the detoxification of the reactive toxic metabolites of CCl4; liver necrosis is initiated when reserves of GSH are markedly depleted (Recknagel et al., 1989; Williams and Burk, 1990). In the present study, the hepatic content of GSH was found to be decreased significantly in CCl4-intoxicated rats compared with control rats (Fig. 2). However, pretreatment with PYC significantly prevented the CCl4-induced depletion of hepatic GSH, indicating the antioxidant effect of PYC in CCl4-intoxicated rats. The antioxidant effects of PYC in the present study were determined at the doses
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of PYC 10 and 20 mg/kg to examine the dose–response relationship. Although the result of hepatic GSH concentration obtained in this study was not dose-dependent, most of the data showed a clear dose–response relationship. Therefore, the higher concentration of hepatic GSH observed in the CCl4&PYC 10 group than both the control and CCl4&PYC 20 groups is believed to be an accidental finding. Antioxidant enzymes such as catalase, SOD, and GST are easily inactivated by lipid peroxides or reactive oxygen species, which results in decreased activities of these enzymes in CCl4 toxicity. Of the antioxidant enzymes, catalase is a major antioxidant enzyme with hematin as the prosthetic group, and it is ubiquitously present in all aerobic cells containing a cytochrome system. It is most abundant in the liver and is responsible for the catalytic decomposition of H2O2 to oxygen and water (Baudrimont et al., 1997; Reiter et al., 2000). SOD is an extremely effective antioxidant enzyme, and is responsible for catalytic dismutation of highly reactive and potentially toxic superoxide radicals to H2O2 (Reiter et al., 2000). GST is a soluble protein that is located both in the cytosol and the endoplasmic reticulum of the liver, and catalyzes the conjugation of GSH with many xenobiotics and their reactive metabolites to form more water-soluble compounds (Boyer et al., 1984). Therefore, the GST enzyme also plays an important role in the detoxification of xenobiotics, catalyzing their conjugation with reduced GSH. The role of free radicals in CCl4 intoxication has been shown to be a major pathway of non-enzymatically induced lipid peroxidation, which subsequently affects various enzyme activities of the body and thereby may also be linked to enzymatically induced lipid peroxidation (Basu, 2003). Therefore, we theorize that the increased production of free radicals caused by administration of CCl4 is a major cause of the significantly decreased activities of catalase, SOD, and GST in rats treated with CCl4 compared with control rats (Figs. 3–5). In rats pretreated with PYC for 14 days, however, the activities of these antioxidant enzymes were significantly higher, and occurred in a dose-dependent manner, compared to the rats exposed to CCl4 alone, and were very similar to the values noted in normal control rats. Previous studies showed that intracellular antioxidant defense systems including vitamin C and E, SOD, catalase, and GSH are stabilized by PYC in the presence of oxidative stressors and, concomitantly, PYC decreases toxic byproducts of lipid peroxidation and reactive oxygen species such as H2O2, OH, nitric oxide (NO), O2, and ONOO (Kobuchi et al., 1999; Packer et al., 1999). The antioxidant activity of PYC has been demonstrated to protect against various degenerative conditions caused by free radicals, including diabetes-induced oxidative stress (Maritim et al., 2003), ethanol-induced neuronal cell death (Siler-Marsiglio et al., 2004), 2,4,6-trinitrobenzene sulfonic acid-induced inflammatory bowel disease (Mochizuki and Hasegawa, 2004), and ionizing radiation-induced intestinal damage (Ramos et al., 2006). The results of the above stud-
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