Rhus verniciflua Stokes glycoprotein (36 kDa) has protective activity on carbon tetrachloride-induced liver injury in mice

Environmental Toxicology and Pharmacology 22 (2006) 8–14

Rhus verniciflua Stokes glycoprotein (36 kDa) has protective activity on carbon tetrachloride-induced liver injury in mice Jeong-Hyeon Ko, Sei-Jung Lee, Kye-Taek Lim ∗ # 521, Molecular Biochemistry Laboratory, Institute of Biotechnology, Chonnam National University, 300 Yongbong-Dong, Kwangju 500-757, South Korea Received 8 August 2005; accepted 31 October 2005 Available online 11 January 2006

Abstract This study was carried out to investigate the hepatoprotective effect of the glycoprotein isolated from Rhus verniciflua Stokes (RVS), which has traditionally been used for healing of inflammatory diseases. We evaluated the activities of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), thiobarbituric acid-reactive substances (TBARS), and antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)] activities in treatment with carbon tetrachloride (CCl4 ) in vivo. When mice were treated with CCl4 in the absence of RVS glycoprotein, the activities of ALT, LDH, and TBARS were increased, while the antioxidant enzymes activities were decreased. However, when the mice were treated with CCl4 in the presence of RVS glycoprotein, the activities of ALT, LDH, and TBARS were significantly reduced and SOD, CAT, and GPx activities were remarkably increased. In addition, RVS glycoprotein increased the nitric oxide (NO) production and decreased the nuclear factor-kappa B (NF-␬B) and activator protein-1 (AP-1) activation in CCl4 -treated mice. Collectively, these results pointed out that RVS glycoprotein can inhibit lipid peroxidation, enhance the activities of antioxidant enzymes, increase the NO production, and decrease the NF-␬B and AP-1 activations. Therefore, we speculate that RVS glycoprotein protects from liver damage through its radical scavenging ability. © 2005 Elsevier B.V. All rights reserved. Keywords: RVS glycoprotein; Carbon tetrachloride; Antioxidant enzymes; Nitric oxide; Nuclear factor-kappa B; Activator protein-1

1. Introduction Reactive oxygen species (ROS) including oxygen free radicals are causative factors in the etiology of degenerative diseases, including some hepatopathies (Poli, 1993). The enhanced production of free radicals and oxidative stress can be induced by a variety of factors such as ionizing radiation or exposure to drug and xenobiotics (e.g., carbon tetrachloride). Carbon tetrachloride (CCl4 ) has been used extensively one of chemical compounds in animal model system, with which it is studied liver injury induced by free radicals in mice. Liver damage caused by CCl4 is regarded to inflammation in early stage as the analogue of hepatotoxins in humans. In the principle of liver damage, CCl4 is reductively bioactivated by cytochrome P450 2E1 into a trichloromethyl radical (• CCl3 ), which is subsequently converted into a peroxyl radical (• OOCCl3 ) in the ∗

Corresponding author. Tel.: +82 62 530 2115; fax: +82 62 530 2129. E-mail addresses: [email protected], [email protected] (K.-T. Lim). 1382-6689/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2005.10.005

presence of oxygen. These reactive free radical metabolites can covalently bind to macromolecules and also initiate lipid peroxidation (Recknagel, 1983; Recknagel et al., 1989; Goeptar et al., 1995). Antioxidative action plays an important role in protection against CCl4 -induced liver injury. Protective effects of various natural products in CCl4 hepatotoxicity have been reported (Jeong et al., 1996; Halim et al., 1997; Hsiao et al., 2003). Rhus verniciflua Stokes (RVS) has traditionally been used for both the preservation of antique furniture and the healing of inflammatory diseases in Korea (Kim, 1996). In a previous study, we demonstrated that ethanol crude extracts of RVS contain cellular active agents that can scavenge ROS and prevent oxidationmediated apoptotic thymocytes (Lee et al., 2002). They also have an inhibitory activity of human cancer cell proliferation and an enhancing activity of detoxifying enzymes in hepatocytes (Lim and Shim, 1997; Lim et al., 2000). More recently, we isolated glycoprotein (36 kDa) from RVS fruit and investigated its scavenging activities against different oxygen radicals in the cell free system. In addition, we reported that RVS glycoprotein has a

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strong antioxidative activity and an antiapoptotic effect through the modulation activities of NF-␬B and AP-1 in NIH/3T3 cells (Ko et al., 2005). Based on the results from experiments, we speculated that RVS glycoprotein might be effective against diseases in which ROS play a role as potent causative factors. Therefore, we examined the antioxidative and hepatoprotective effects of RVS glycoprotein on CCl4 -induced mouse liver injury and elucidated the possible mechanisms of these protective effects. 2. Materials and methods 2.1. Chemicals 5-Bromo-4-chloro-3-indolylphosphate/nitrobluetetrazolium (BCIP/NBT) mixture solution, glutathione peroxidase (GPx), ␤-nicotinamide adenine dinucleotide (␤-NADH), olive oil, superoxide dismutase (SOD), 1,1,1,3tetraethoxypropane, and thiobarbituric acid (TBA) were obtained from Sigma (St. Louis, USA). Carbon tetrachloride (CCl4 ) was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). l-Alanine and l-lactate dehydrogenase (LDH) were obtained from Fluka (Buchs, Switzerland). Specific antibodies of NF-␬B (p50), c-fos, c-jun, ␣-tubulin, and alkaline phosphatase-conjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other chemicals and reagents were of the highest quality available.

2.2. Preparation of RVS glycoprotein Samples of RVS fruits were collected from Naju in the Chonnam province of South Korea, and glycoprotein was isolated from it as described previously (Ko et al., 2005). In this study, we used RVS glycoprotein with a molecular weight of 36 kDa.

2.3. Animals and treatments Male mice (ICR), aged 5 weeks, were purchased from Daehan Lab. (Animal Research Center Co. Ltd., Dae Jeon, Korea) and housed according to the animal care guidelines approved by the Animal-Care Committee of the American Society of Mammalogists (American Society of Mammalogists Animal Care and Use Committee, 1998) at the Experimental Animal Room of Veterinary College of Chonnam National University. All mice were fed a commercial diet and water ad libitum, and kept for at least 1 week prior to the experiments. Mice were divided into the following six study groups: (1) (2) (3) (4) (5) (6)

control (n = 6); control + RVS glycoprotein (80 mg/kg) (n = 6); CCl4 (n = 6); CCl4 + RVS glycoprotein (20 mg/kg) (n = 6); CCl4 + RVS glycoprotein (40 mg/kg) (n = 6); CCl4 + RVS glycoprotein (80 mg/kg) (n = 6).

RVS glycoprotein was dissolved in PBS and then administered orally with 20, 40, and 80 mg/kg body weight RVS glycoprotein into mice for 3 days. One hour after the final treatment, mice were treated with CCl4 at a dose of 0.5 ml/kg body weight intraperitoneally dissolved in olive oil (1:1, v/v). At 24 h after CCl4 administration, the mice of each group were anesthetized with ether and the blood drawn by cardiac puncture. The samples were centrifuged, isolated plasma, and stored at −70 ◦ C for use future experiments. Mice were weighed before and after the defined treatments for each group. Livers were also extracted after dissection and then were weighed.

2.4. Plasma alanine transaminase (ALT) and lactate dehydrogenase (LDH) activities The activity of plasma ALT and LDH were measured as a marker of liver injury by the method of Bergmeyer and Bernt (1974a,b). For the analysis of ALT,

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plasma was made to react with 80 mM phosphate buffer (pH 7.4), 800 mM lalanine, 0.18 mM NADH, and 3.7 U/ml LDH and allow to stand for 3 min. This was followed by addition of 18 mM 2-oxoglutarate. For LDH, plasma sample was mixed with reaction mixture containing 0.6 mM pyruvate in 48 mM potassium phosphate buffer (pH 7.5). The reaction was initiated by the addition of 0.18 mM ␤-NADH. The ALT and LDH activities were measured as the rate of loss of ␤-NADH absorption at 340 nm for 2 min.

2.5. Determination of hepatic lipid peroxidation The malondialdehyde (MDA) content, a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid-reactive substances (TBARS) by method of Uchiyama and Mihara (1978). Briefly, 1 g of liver was homogenized in 10 ml of KCl 1.15% (w/v) and the homogenate was filtered through fourfolded gauze. A volume of 0.5 ml of liver homogenate was mixed with 3 ml of H3 PO4 1% (v/v) and 1 ml of TBA 0.6% (w/v), and heated at 100 ◦ C for 45 min. The samples were allowed to reach room temperature and 3 ml of n-butanol was added. After a vigorous shaking, the butanolic phase was obtained by centrifugation at 4000 × g for 10 min to determinate the absorbance at 535 nm. 1,1,1,3-Tetraethoxypropane was used as the standard.

2.6. Antioxidant enzyme activities The livers from each mouse were dissected and homogenized in a 250 mM sucrose buffer (pH 7.5) containing 10 mM EDTA. The homogenates were then centrifuged at 600 × g for 10 min at 4 ◦ C to remove nuclear fractions, and the remaining separated supernatant was re-centrifuged at 10,000 × g for 20 min at 4 ◦ C to collect the mitochondrial fraction (pellet) for a catalase (CAT) assay. The supernatant was ultra-centrifuged at 100,000 × g for 1 h at 4 ◦ C to isolate the cytosolic fraction for a superoxide dismutase (SOD) and glutathione peroxidase (GPx) assays. The supernatant was used for activities of antioxidant enzymes as described below and the amount of protein was measured using the method of Lowry et al. (1951). SOD activity was measured according to the method of Beauchamp and Fridovich (1971). An adequate amount of the liver supernatant was mixed with the reaction mixtures, which contained 0.1 mM EDTA, 25 mM NBT, 0.1 mM xanthine, 50 mM sodium carbonate buffer (pH 10.2), and the final volume of the reaction mixture was brought up to 3 ml with distilled water. The reaction was initiated by the addition of 2 mU/ml xanthine oxidase and maintained under two 40 W lamps at 25 ◦ C. After 15 min, the inhibition rate of NBT reduction was spectrophotometrically determined at 560 nm. One unit of SOD is defined as the amount of enzyme required to reduce the NBT by 50%. The specific activity of SOD was expressed as U/mg protein in each supernatant. CAT activity was measured according to the method of Thomson et al. (1978). The liver supernatant was mixed with 2.8 ml of 50 mM phosphate buffer (pH 7.4). After equilibration at 30 ◦ C for 5 min, the reaction was started by the addition of 200 ml of 100 mM sodium perborate (pH 7.4). The CAT activity, which reduces the sodium perborate as a substrate, was assessed spectrophotometrically following the consumption of H2 O2 at 220 nm for 2 min. One unit of CAT was defined as the amount of enzyme required reducing 1 ␮M of H2 O2 /min. The results were expressed as U/mg protein in each supernatant. GPx activity was measured according to the method of Paglia and Valentine (1967). The liver supernatant was added to the reaction mixtures consisting of 1 mM EDTA, 1 unit of glutathione reductase, 1 mM glutathione, 0.25 mM H2 O2 and 1 mM sodium azide in 50 mM phosphate buffer (pH 7.0). The reaction was initiated by the addition of 0.2 mM NADPH and GPx activity was measured as the rate of NADPH oxidation at 340 nm. One unit of GPx was defined, as the amount required oxidizing 1 ␮M of NADPH/min. Results of GPx activity were expressed as U/mg protein in each supernatant.

2.7. Determination of nitric oxide (NO) The production of NO was assessed indirectly by measuring the nitrite levels in plasma determined by a calorimetric method based on the Griess reaction (Green et al., 1982). Plasma samples were diluted four times with distilled water and deproteinized by adding 1/20 volume of zinc sulfate (300 g/l) to a final concentration of 15 g/l. After centrifugation at 10,000 × g for 5 min at room tempera-

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ture, 100 ␮l supernatant was applied to a microtiter plate well, followed by 100 ␮l of Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 2.5% polyphosphoric acid). After 10 min of color development at room temperature, the absorbance was measured at 540 nm with a MicroReader (Hyperion, Inc., FL, USA). Nitrite was quantified by using sodium nitrate as a standard curve.

2.8. Western blot analysis Liver tissues were homogenized in lysis buffer (0.6% NP-40, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, and 0.5 mM PMSF) at 4 ◦ C. Nuclear extracts were prepared by modification of the method of Deryckere and Gannon (1994). Protein concentrations were measured by the method of Lowry et al. (1951). Fifty micrograms of protein was fractionated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). Membranes were incubated with primary antibodies overnight at 4 ◦ C using 1:3000 dilution of goat polyclonal anti-rabbit NF-␬B (p50), c-fos, c-jun, and ␣-tubulin antibodies. After washing, the membranes were incubated with 1:10,000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG as the second antibody for 1 h. Antibody binding was visualized by incubation with BCIP/NBT mixture solution. NF-␬B and AP-1 activities in the results of Western blot analysis were quantified by measuring the relative intensity compared to the control using Scion Imaging Software (Scion Image Beta 4.02, MD, USA) and represented in the relative intensities.

2.9. Statistical analysis All experiments were done three times in triplicates, and the results were represented as mean ± S.D. The Duncan test and a one-way analysis of variance (ANOVA) were used for multiple comparisons between treatments (SPSS program, Version 10.0).

3. Results 3.1. Effect of RVS glycoprotein on hepatotoxicity in CCl4 -treated mouse CCl4 significantly decreased body and liver weights, compared to control. However, RVS glycoprotein reversed the body weight loss and liver swelling induced by CCl4 to the levels of control group (Table 1). The levels of ALT and LDH were measured in the plasma to evaluate hepatic tissue damage (Table 2). A significant increase the levels of ALT and LDH were observed in the CCl4 -treated group. Administrations of RVS glycoprotein significantly reduced the increased

Table 2 Effects of RVS glycoprotein on the plasma ALT and LDH levels in CCl4 -treated mice Groups

ALT (U/l)

Control RVS glycoprotein (80 mg/kg) CCl4 (0.5 ml/kg) RVS glycoprotein (20 mg/kg) + CCl4 RVS glycoprotein (40 mg/kg) + CCl4 RVS glycoprotein (80 mg/kg) + CCl4

9.5 12.6 42.8 35.2 32.4 25.4

± ± ± ± ± ±

1.2 2.9 2.2*** 2.0 1.3* 0.9**

LDH (U/l) 126.6 134.8 305.6 285.3 226.9 183.6

± ± ± ± ± ±

25.4 23.1 51.9 21.0 11.5* 8.8**

ALT, alanine aminotransferase; LDH, lactate dehydrogenase. The values are mean ± S.D. from six mice done in triplicates. * p < 0.05. ** p < 0.01 as compared to CCl -treated group. 4 *** p < 0.01 as compared to control group.

plasma ALT and LDH levels. For instance, the level of ALT was 9.5 ± 1.2 and 12.6 ± 2.9 U/l, and LDH level was 126.6 ± 25.4 and 134.8 ± 23.1 U/l in the control and RVS glycoprotein administration alone group. The levels of ALT and LDH were increased to 42.8 ± 2.2 and 305.6 ± 51.9 U/l after CCl4 treatment. However, RVS glycoprotein pretreatment decreased the levels of ALT and LDH to 35.2 ± 2.0 and 285.3 ± 21.0 U/l at 20 mg/kg, 32.4 ± 1.3 and 226.9 ± 11.5 U/l at 40 mg/kg, 25.4 ± 0.9 and 183.6 ± 8.8 U/l at 80 mg/kg, respectively. 3.2. Effect of RVS glycoprotein on lipid peroxidation in CCl4 -treated mouse liver As shown in Fig. 1, hepatic levels of TBARS were assessed as an indicator of tissue lipid peroxides. CCl4 treatment significantly increased the level of TBARS in the liver. However, pretreatment of RVS glycoprotein significantly decreased the increased level of TBARS. For example, the TBARS level was 0.5 and 0.4 ␮M/g liver in the control and RVS glycoprotein treatment alone. The level was significantly increased to 1.1 ␮M/g liver in CCl4 -treated group, compared to the control group. However, the TBARS level was diminished approximately by 0.1, 0.3, and 0.4 ␮M/g liver at pretreatment with 20, 40, and 80 mg/kg of RVS glycoprotein in the

Table 1 Effects of RVS glycoprotein on body and liver weights in CCl4 -treated mice Groups

Relative weight (%/100 g body weight) Body

Control RVS glycoprotein (80 mg/kg) CCl4 (0.5 ml/kg) RVS glycoprotein (20 mg/kg) + CCl4 RVS glycoprotein (40 mg/kg) + CCl4 RVS glycoprotein (80 mg/kg) + CCl4

99.18 98.40 91.44 93.16 95.83 97.65

Liver ± ± ± ± ± ±

0.85 1.08 1.52** 0.09 1.13* 0.29*

The values are mean ± S.D. from six mice done in triplicates. * p < 0.05 as compared to CCl -treated group. 4 ** p < 0.05 as compared to control group.

4.02 3.84 5.61 5.42 5.27 5.08

± ± ± ± ± ±

0.21 0.16 0.49 0.04 0.05* 0.08* Fig. 1. Effect of RVS glycoprotein on TBARS formation in CCl4 -treated mice. The values are mean ± S.D. from six mice done in triplicates. # p < 0.01 as compared to control group. * p < 0.05, ** p < 0.01 as compared to CCl4 -treated group.

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Fig. 3. Effect of RVS glycoprotein on NO production in CCl4 -treated mice. The values are mean ± S.D. from six mice done in triplicates. # p < 0.05 as compared to control group. * p < 0.05, ** p < 0.01 as compared to CCl4 -treated group.

and 10.2 U/mg protein at 40 mg/kg, 30.2, 17.5, and 11.3 U/mg protein at 80 mg/kg, individually. 3.4. Effect of RVS glycoprotein on NO production in CCl4 -treated mouse plasma As shown in Fig. 3, the production of NO in mouse plasma was significantly reduced in CCl4 -treated mice, compared to the control group. However, pretreatment of RVS glycoprotein increased the NO production in CCl4 -treated mice. For example, NO production in control and RVS glycoprotein treatment group was 17.0 and 17.8 ␮M, while it was 12.3 ␮M at the treatment of CCl4 . However, the NO production in the CCl4 -treated mice was significantly augmented by 2.3, 4.5 and 7.2 ␮M at 20, 40, and 80 mg/kg RVS glycoprotein pretreatment, respectively. Fig. 2. Effect of RVS glycoprotein on antioxidant enzymes activities in CCl4 treated mice. SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase. The values are mean ± S.D. from six mice done in triplicates. # p < 0.05 as compared to control group. * p < 0.05, ** p < 0.01 as compared to CCl4 -treated group.

CCl4 -treated mice, respectively, as compare to the CCl4 -treated group. 3.3. Effect of RVS glycoprotein on antioxidant enzymes activities in CCl4 -treated mouse liver The hepatic antioxidant enzyme activities (SOD, CAT, and GPx) are shown in Fig. 2. The SOD, CAT, and GPx activities in CCl4 -treated mice were decreased, compared to the control. Interestingly, the pretreatment with RVS glycoprotein significantly increased the decreased SOD, CAT, and GPx activities in CCl4 -treated group. For instance, SOD activity was 25.1 and 27.0 U/mg protein, CAT activity was 15.7 and 16.2 U/mg protein, and GPx activity was 10.3 and 10.8 U/mg protein in the control and RVS glycoprotein administration alone group. The activities of SOD, CAT, and GPx were decreased to 16.3, 10.9, and 7.2 U/mg protein after CCl4 treatment. However, RVS glycoprotein pretreatment increased the activities of SOD, CAT, and GPx to 19.8, 12.8, and 8.5 U/mg protein at 20 mg/kg, 25.0, 15.7,

3.5. Effect of RVS glycoprotein on activities of NF-κB and AP-1 in CCl4 -treated mouse liver We investigated the changes of the activation of NF-␬B or AP-1 by RVS glycoprotein in CCl4 -treated mice (Fig. 4). The relative intensities of bands obtained from Western blot were calculated with the use of the Scion Imaging Software. The results showed that CCl4 treatment stimulates to increase activation of NF-␬B. For example, in CCl4 treatment group, the relative intensity of NF-␬B band was increased by 3.5, compared to the control. However, the treatment of RVS glycoprotein decreased the NF-␬B activation in CCl4 -induced mice. Namely, the relative intensities of bands about NF-␬B activation were reduced by 1.8 and 1.9 at 40 and 80 mg/kg of RVS glycoprotein, respectively, compared to CCl4 treatment alone. In activation of AP-1 (c-jun and c-fos) (Fig. 4), the activations of c-jun and c-fos proteins were increased at the CCl4 treatment alone, compared to control. However, when the mice were pretreated with RVS glycoprotein (40 and 80 mg/kg), c-jun and c-fos activities were reduced in the CCl4 -treated mice. In facts, at CCl4 treatment alone, the relative intensities of bands about c-jun and c-fos proteins activation were increased by 2.4 and 4.5 compared to the control. However, the pretreatment of RVS glycoprotein reduced the relative intensities of c-jun bands by 0.4 and 1.3 at 40 and 80 mg/kg in the CCl4 -treated mice, respectively, compared to CCl4 treatment

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Fig. 4. Effect of RVS glycoprotein on CCl4 -induced the activation of NF-␬B and AP-1 in mice. The relative intensities were evaluated with the use of the Scion Imaging Software. ␣-Tubulin was used as internal control for equal loading of proteins. The values are mean ± S.D. from six mice done in triplicates. # p < 0.01 as compared to control group. * p < 0.05, ** p < 0.01 as compared to CCl4 -treated group.

alone. RVS glycoprotein also decreased the relative intensities of c-fos bands by 0.5 and 1.6 at 40 and 80 mg/kg, respectively, compared to CCl4 treatment alone. 4. Discussion It has reported that liver injuries induced by CCl4 are the best characterized system of xenobiotic-induced hepatotoxicity and commonly used models for the screening of anti-hepatotoxic and/or hepatoprotective activities of drugs (Recknagel et al., 1989, 1991). The principle causes of CCl4 -induced hepatic damage is lipid peroxidation and decreased activities of antioxidant enzymes and generation of free radicals (Castro et al., 1974; Poli, 1993). For the therapeutic strategies of liver injury and disease, we postulate that it is important to find antioxidant compounds which are able to block liver injuries through trichloromethyl free radical generated by CCl4 . Therefore, we strongly speculated that RVS glycoprotein can protect against diseases which are caused by ROS, because it has a radical scavenging activity (Ko et al., 2005). The results of the present study demonstrate that the oral administration of RVS glycoprotein effectively protected mice against CCl4 -induced acute liver damage. There are two possible explanations about hepatoprotective effect of orally administered RVS glycoprotein. One of them is that the carbohydrate part of RVS glycoprotein cannot be digested in the mammalian small intestine and forms a viscous solution, which is also thought to delay the absorption of various chemicals including CCl4 , suggesting that the viscous RVS glycoprotein can interfere with CCl4 absorption in the small intestine, especially the ileum (Wolever et al., 1979; Moundras et al., 1994; Stedronsky, 1994).

The other explanation is that RVS glycoprotein has an ability to modulate the signal transduction to both liver parenchymal cells (hepatocytes) and nonparenchymal cells (Kupffer, endothelial, and stellate cells) through binding to the several receptors of intestinal epithelial cells on CCl4 -induced liver injury. From these speculations, we sophisticate that pretreatment of RVS glycoprotein has indirectly protective effects on CCl4 -induced liver injury. The general indicator of CCl4 -induced hepatotoxicity such as histopathological lesions was not observed, but biological and hepatic damage by CCl4 administration was clearly evident from the severe body weight loss and liver swelling (Table 1). CCl4 is known to cause hepatic damage with a marked elevation in serum levels of LDH and aminotransferases enzymes (AST and ALT), because these enzymes are cytoplasmic in location and are released into the blood after cellular damage (Recknagel et al., 1989, 1991). In agreement with those investigations, our results showed that a significant increase in the activities of ALT and LDH in CCl4 -treated mice (Table 2). The increase in the activities of these enzymes in plasma suggests enhanced hepatocellular damage by CCl4 . The activities of these enzymes were found to decrease after pretreatment with RVS glycoprotein (Table 2). Additionally, like lactate dehydrogenase (LDH), aspartate aminotransferase (AST) is found in every tissue of the body, and the amount of AST is particularly high in the cardiac muscle. In contrast, ALT is present in moderately high concentration in liver and low in cardiac, skeletal muscle and other tissues. That is why we evaluated only both LDH values instead of AST and ALT. RVS glycoprotein also showed the ability to prevent a CCl4 induced increment of hepatic TBARS level, suggesting that RVS glycoprotein inhibits lipid peroxidation and its propagation in

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the liver (Fig. 1). Moreover, antioxidant enzymes (SOD, CAT, and GPx) activities were increased by RVS glycoprotein treatment (Fig. 2). One of possible mechanisms for lowering of SOD, CAT, and GPx activities is that the treatment of mouse liver with CCl4 concurrently induces both processes in acute injury and regeneration; injury events are dominantly expressed in the early stage but this regeneration process is latent (Taniguchi et al., 2004). Therefore, the SOD, CAT, and GPx were degraded or saturated to block the CCl4 -induced massive free radical production at in the early stage. It has reported that antioxidant enzyme expression can be altered by hydrogen peroxide (Rohrdanz and Kahl, 1998). By contrast, a possible reason why SOD, CAT and GPx activities after RVS glycoprotein treatment were augmented in both the absence of CCl4 and in the presence of CCl4 is that RVS glycoprotein possesses not only the capacity to scavenge the small number of ROS which are inevitably generated due to the incomplete reduction of O2 in electron transfer reactions in normal aerobic metabolisms, but also the capacity to block the CCl4 -induced massive ROS production. The excessive activities of SOD, CAT, and GPx, and antioxidative activity of RVS glycoprotein can together contribute to synergic/additive scavenging activity to radicals, suggesting that the antioxidative potential of RVS glycoprotein seems to stimulate the activities of antioxidant enzymes, compared to control. Therefore, we speculate that RVS glycoprotein particularly plays on the early stage in CCl4 -induced liver injury. These findings indicated that administration of RVS glycoprotein decreased lipid peroxidation, improved antioxidant status and thereby prevented the damage to the liver and leakage of enzymes ALT and LDH. These results clearly show that antioxidant action of RVS glycoprotein significantly reduces the damage of CCl4 -induced liver injury and activates the biological defense system of the liver. Fig. 3 shows that the level of NO in plasma decreased significantly with administration of CCl4 . RVS glycoprotein blocked the reduction of plasma NO level in CCl4 -treated mice. There are two possible explanations for the observed decrease in NO levels after CCl4 treatment in our case. First, one or more of the substrates or any of the cofactors of nitric oxide synthase (NOS) was depleted or damaged, decreasing the NO production (Billiar and Harbrecht, 1997). Second, NO was used up continuously after the injury. It is possible that another mechanism of protective action of RVS glycoprotein against CCl4 -induced hepatotoxicity, due to the increased NO production. Several studies have found that NO protected against CCl4 -induced liver injury using a NOS knockout mice or a NOS inhibitor (Zhu and Fung, 2000; Morio et al., 2001). The mechanism underlying the protective effects of NO in CCl4 -induced hepatotoxicity has not been elucidated and may be related to its antioxidant properties (Wink et al., 1996; O’Donnell et al., 1997). NO has also been shown to interfere directly with the progression of lipid peroxidation (O’Donnell et al., 1997), which may contribute to its protective actions in this model (Muriel, 1998). CCl4 is also one of the well-known promoters of nuclear factor-kappa B (NF-␬B) and activator protein-1 (AP-1) in apoptotic response of hepatocytes, inflammatory response of Kupffer cells, and fibrogenetic response of stellate cells on liver injury (Zawaski et al., 1993; Camandola et al., 1997; Wu and Zern,

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1999; Cesaratto et al., 2004). In the present study, CCl4 treatment caused to increase the activations of NF-␬B and AP-1 (c-jun and c-fos), while the CCl4 -induced activations of these transcriptional signals markedly inhibited by treatment with RVS glycoprotein in CCl4 -induced system (Fig. 4). It has reported that NF-␬B activity is increased by metabolites of CCl4 , the agent that enhances hepatic cell injury and necrosis. The main action of NF-␬B in liver injury is to mediate the release of cytotoxic cytokines and inflammatory cytotoxins, such as TNF-␣, IL-1␤, and IFN-␥ (Siebenlist et al., 1994). Therefore, reduction of NF-␬B activity may decrease cell necrosis, perhaps through affecting cytokine expression (Liu et al., 1995). AP-1 participates in cell proliferation and differentiation by positively or negatively regulating the transcription of genes that contains AP1 binding sites including c-jun and c-fos expression (Brenner et al., 1989; Hanazawa et al., 1993). AP-1 activity has been demonstrated to be sensitive to ROS (Meyer et al., 1994) as well as to lipid peroxidation end-products (Camandola et al., 1997). Recently, it has been reported that c-jun and c-fos expression are elevated in livers of rodents following treatment with necrogenic doses of CCl4 (Herbst et al., 1991; Schmiedeberg et al., 1993). The results in our experiments indicated that CCl4 -induced acute liver injury, which is mediated by the activation of NF-␬B and AP-1 as a result of increased oxidative stress caused by CCl4 . Thus, the antioxidative properties of RVS glycoprotein may be of importance for their protective effect. In conclusion, the results of this study demonstrate that RVS glycoprotein has property to inhibit lipid peroxidation, to stimulate increasing activities of antioxidant enzymes, to increase in NO production, and to inhibit activities of NF-␬B and AP-1. Therefore, RVS glycoprotein was found to be effective against CCl4 -induced liver damage in mice. However, further research must be carried out to elucidate the mechanisms of hepatoprotective effect by RVS glycoprotein at the molecular biological level. and . Acknowledgments This study was financially supported by a research fund of Chonnam National University in 2005. This study was carried out for master degree at Chonnam National University in 2006. References American Society of Mammalogists Animal Care and Use Committee, 1998. Guidelines for the capture, handling, and care of mammals as approved by the American Society of Mammalogists. J. Mammal. 79, 1416–1431. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Bergmeyer, H.U., Bernt, E., 1974a. Lactate dehydrogenase: UV-assay with pyruvate and NADH. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, vol. 2. Academic Press, New York, NY, pp. 574–578. Bergmeyer, H.U., Bernt, E., 1974b. Glutamate-pyruvate transaminase: UVassay manual method. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, vol. 2. Academic Press, New York, NY, pp. 752–758. Billiar, T.R., Harbrecht, B.G., 1997. Resolving the nitric oxide paradox in acute tissue damage. Gastroenterology 113, 1405–1407.

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Brenner, D.A., O’Hara, M., Angel, P., Chojkier, M., Karin, M., 1989. Prolonged activation of jun and collagenase genes by tumour necrosis factoralpha. Nature 337, 661–663. Camandola, S., Scavazza, A., Leonarduzzi, G., Biasi, F., Chiarpotto, E., Azzi, A., Poli, G., 1997. Biogenic 4-hydroxy-2-nonenal activates transcription factor AP-1 but not NF-kappa B in cells of the macrophage lineage. Biofactors 6, 173–179. Castro, J.A., De Ferreyra, E.C., De Castro, C.R., De Fenos, O.M., Sasame, H., Gillette, J.R., 1974. Prevention of carbon tetrachloride-induced necrosis by inhibitors of drug metabolism—further studies on their mechanism of action. Biochem. Pharmacol. 23, 295–302. Cesaratto, L., Vascotto, C., Calligaris, S., Tell, G., 2004. The importance of redox state in liver damage. Ann. Hepatol. 3, 86–92. Deryckere, F., Gannon, F., 1994. A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques 16, 405. Goeptar, A.R., Scheerens, H., Vermeulen, N.P., 1995. Oxygen and xenobiotic reductase activities of cytochrome P450. Crit. Rev. Toxicol. 25, 25–65. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15 N]nitrate in biological fluids. Anal. Biochem. 126, 131–138. Halim, A.B., el-Ahmady, O., Hassab-Allah, S., Abdel-Galil, F., Hafez, Y., Darwish, A., 1997. Biochemical effect of antioxidants on lipids and liver function in experimentally-induced liver damage. Ann. Clin. Biochem. 34, 656–663. Hanazawa, S., Takeshita, A., Amano, S., Semba, T., Nirazuka, T., Katoh, H., Kitano, S., 1993. Tumor necrosis factor-alpha induces expression of monocyte chemoattractant JE via fos and jun genes in clonal osteoblastic MC3T3-E1 cells. J. Biol. Chem. 268, 9526–9532. Herbst, H., Milani, S., Schuppan, D., Stein, H., 1991. Temporal and spatial patterns of proto-oncogene expression at early stages of toxic liver injury in the rat. Lab. Invest. 65, 324–333. Hsiao, G., Shen, M.Y., Lin, K.H., Lan, M.H., Wu, L.Y., Chou, D.S., Lin, C.H., Su, C.H., Sheu, J.R., 2003. Antioxidative and hepatoprotective effects of Antrodia camphorata extract. J. Agric. Food Chem. 51, 3302–3308. Jeong, T.C., Kim, H.J., Park, J., Ha, C.S., Park, J.D., Kim, S., Roh, J.K., 1996. Protective effects of red ginseng saponins against carbon tetrachlorideinduced hepatotoxicity in sprague dawley rats. Planta Med. 63, 136–140. Kim, T.J., 1996. Korea Resource Plant II. Seoul University Press, Seoul, pp. 292–297. Ko, J.H., Lee, S.J., Lim, K.T., 2005. 36 kDa glycoprotein isolated from Rhus verniciflua Stokes fruit has a protective activity to glucose/glucose oxidase-induced apoptosis in NIH/3T3 cells. Toxicol. In Vitro 19, 353–363. Lee, J.C., Lim, K.T., Jang, Y.S., 2002. Identification of Rhus verniciflua Stokes compounds that exhibit free radical scavenging and anti-apoptotic properties. Biochim. Biophys. Acta 1570, 181–191. Lim, K.T., Lee, J.C., Jung, H.Y., Jo, S.K., 2000. Effects of Rhus verniciflua Stokes (RVS) on cell-associated detoxificant enzymes and glucose oxidase-mediated toxicity in cultured mouse hepatocytes. J. Toxicol. Public Health 16, 125–131. Lim, K.T., Shim, J.H., 1997. Antioxidative effects of ethanol extracts from Rhus verniciflua Stokes (RVS) on mouse whole brain cells. Food Sci. Biotechnol. 29, 1248–1254. Liu, S.L., Degli Esposti, S., Yao, T., Diehl, A.M., Zern, M.A., 1995. Vitamin E therapy of acute CCl4-induced hepatic injury in mice is associated with inhibition of nuclear factor kappa B binding. Hepatology 22, 1474–1481. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Meyer, M., Pahl, H.L., Baeuerle, P.A., 1994. Regulation of the transcription factors NF-kappa B and AP-1 by redox changes. Chem. Biol. Interact. 91, 91–100.

Morio, L.A., Chiu, H., Sprowles, K.A., Zhou, P., Heck, D.E., Gordon, M.K., Laskin, D.L., 2001. Distinct roles of tumor necrosis factor-alpha and nitric oxide in acute liver injury induced by carbon tetrachloride in mice. Toxicol. Appl. Pharmacol. 172, 44–51. Moundras, C., Behr, S.R., Demigne, C., Mazur, A., Remesy, C., 1994. Fermentable polysaccharides that enhance fecal bile acid excretion lower plasma cholesterol and apolipoprotein E-rich HDL in rats. J. Nutr. 124, 2179–2188. Muriel, P., 1998. Nitric oxide protection of rat liver from lipid peroxidation, collagen accumulation, and liver damage induced by carbon tetrachloride. Biochem. Pharmacol. 56, 773–779. O’Donnell, V.B., Chumley, P.H., Hogg, N., Bloodsworth, A., Darley-Usmar, V.M., Freeman, B.A., 1997. Nitric oxide inhibition of lipid peroxidation: kinetics of reaction with lipid peroxyl radicals and comparison with alphatocopherol. Biochemistry 36, 15216–15223. Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158–169. Poli, G., 1993. Liver damage due to free radicals. Br. Med. Bull. 49, 604–620. Recknagel, R.O., 1983. A new direction in the study of carbon tetrachloride hepatotoxicity. Life Sci. 33, 401–408. Recknagel, R.O., Glende E.A.Jr., Britton, R.S., 1991. Free radical damage and lipid peroxidation. In: Meeks, R.G. (Ed.), Hepatotoxicology. CRC Press, Boca Raton, FL, pp. 401–436. Recknagel, R.O., Glende E.A.Jr., Dolak, J.A., Waller, R.L., 1989. Mechanisms of carbon tetrachloride toxicity. Pharmacol. Ther. 43, 139–154. Rohrdanz, E., Kahl, R., 1998. Alterations of antioxidant enzyme expression in response to hydrogen peroxide. Free Radic. Biol. Med. 24, 27–38. Schmiedeberg, P., Biempica, L., Czaja, M.J., 1993. Timing of protooncogene expression varies in toxin-induced liver regeneration. J. Cell Physiol. 154, 294–300. Siebenlist, U., Franzoso, G., Brown, K., 1994. Structure, regulation and function of NF-kappa B. Annu. Rev. Cell Biol. 10, 405–455. Stedronsky, E.R., 1994. Interaction of bile acids and cholesterol with nonsystemic agents having hypocholesterolemic properties. Biochim. Biophys. Acta 1210, 255–287. Taniguchi, M., Takeuchi, T., Nakatsuka, R., Watanabe, T., Sato, K., 2004. Molecular process in acute liver injury and regeneration induced by carbon tetrachloride. Life Sci. 75, 1539–1549. Thomson, J.F., Nance, S.L., Tollaksen, S.L., 1978. Spectrophotometric assay of catalase with perborate as substrate. Proc. Soc. Exp. Biol. Med. 157, 33–35. Uchiyama, M., Mihara, M., 1978. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal. Biochem. 86, 271–278. Wink, D.A., Cook, J.A., Pacelli, R., DeGraff, W., Gamson, J., Liebmann, J., Krishna, M.C., Mitchell, J.B., 1996. The effect of various nitric oxidedonor agents on hydrogen peroxide-mediated toxicity: a direct correlation between nitric oxide formation and protection. Arch. Biochem. Biophys. 331, 241–248. Wolever, T.M., Jenkins, D.J., Nineham, R., Alberti, K.G., 1979. Guar gum and reduction of post-prandial glycaemia: effect of incorporation into solid food, liquid food, and both. Br. J. Nutr. 41, 505–510. Wu, J., Zern, M.A., 1999. NF-kappa B, liposomes and pathogenesis of hepatic injury and fibrosis. Front. Biosci. 4, D520–D527. Zawaski, K., Gruebele, A., Kaplan, D., Reddy, S., Mortensen, A., Novak, R.F., 1993. Evidence for enhanced expression of c-fos, c-jun, and the Ca(2+)-activated neutral protease in rat liver following carbon tetrachloride administration. Biochem. Biophys. Res. Commun. 197, 585–590. Zhu, W., Fung, P.C., 2000. The roles played by crucial free radicals like lipid free radicals, nitric oxide, and enzymes NOS and NADPH in CCl(4)induced acute liver injury of mice. Free. Radic. Biol. Med. 29, 870– 880.