Food and Chemical Toxicology 63 (2014) 174–185

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The involvement of Nrf2 in the protective effects of diallyl disulfide on carbon tetrachloride-induced hepatic oxidative damage and inflammatory response in rats In-Chul Lee a, Sung-Hwan Kim a, Hyung-Seon Baek a, Changjong Moon a, Seong-Soo Kang a, Sung-Ho Kim a, Yun-Bae Kim b, In-Sik Shin c,⇑,1, Jong-Choon Kim a,⇑,1 a

College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea c Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk 363-883, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 11 October 2013 Accepted 6 November 2013 Available online 15 November 2013 Keywords: Carbon tetrachloride Hepatotoxicity Diallyl disulfide Nuclear factor E2-related factor 2 Nuclear factor kappaB

a b s t r a c t This study investigated the potential effect of diallyl disulfide (DADS) against carbon tetrachloride (CCl4)induced oxidative hepatic damage and inflammatory response in rat liver. DADS at doses of 50 and 100 mg/kg/day was administered orally once daily for 5 days, prior to CCl4 administration. Pretreatment with DADS attenuated CCl4-induced elevated serum transaminase activities and histopathological alterations in liver. It prevented the hepatocellular apoptotic changes with induction of Bcl-2-associated X (Bax), cytochrome c, and caspase-3 caused by CCl4. An increase in the nuclear translocation of nuclear factor-kappaB (NF-jB) and phosphorylation of I kappaB alpha (IjBa) was observed in the livers of CCl4-treated rats that coincided with induction of inflammatory mediators or cytokines. In contrast, DADS inhibited NF-jB translocation and IjBa phosphorylation, and that subsequently decreased inflammatory mediators. Furthermore, DADS prevented CCl4-induced depletion of cytosolic nuclear factor E2-related factor 2 (Nrf2) and suppression of nuclear translocation of Nrf2, which, in turn, up-regulated phase II/ antioxidant enzyme activities. Taken together, these results demonstrate that DADS increases the expression of phase II/antioxidant enzymes and simultaneously decreases the expression of inflammatory mediators in CCl4-induced liver injury. These findings indicate that DADS induces antioxidant defense mechanism by activating Nrf2 pathway and reduces inflammatory response by inhibiting NF-jB activation. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Abbreviations: ALT, alanine aminotransferase; ANOVA, analysis of variance; ARE, antioxidant response element; AST, aspartate aminotransferase; Bax, Bcl-2-associated X; CCl4, carbon tetrachloride; Cox-2, cyclooxygenase-2; CYPs, cytochrome P450 isoenzymes; DADS, diallyl disulfide; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSTa, glutathione S-transferase alpha; H&E, hematoxylin and eosin; HO-1, heme oxygenase-1; IjBa, I kappaB alpha; IKK, IjB kinase; IL-1b, interleukin-1b; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; Keap1, Kelch like-ECH-associated protein 1; MDA, malondialdehyde; NF-jB, nuclear factor-kappaB; NQO1, NAD(P)H quinine oxidoreductase; Nrf2, nuclear factor E2related factor 2; SOD, superoxide dismutase; RT-PCR, real-time polymerase chain reaction; p-IjBa, phosphor-I kappa B alpha; ROS, reactive oxygen species; TNF-a, tumor necrosis factor-alpha; TUNEL, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. ⇑ Corresponding authors. Tel.: +82 43 240 6135; fax: +82 43 240 6129 (I.-S. Shin), tel.: +82 62 530 2827; fax: +82 62 530 2809 (J.-C. Kim). E-mail addresses: [email protected] (I.-S. Shin), [email protected] (J.-C. Kim). 1 These authors contributed equally to this work. 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.11.006

Carbon tetrachloride (CCl4) is widely used in animal models to induce acute liver injury and to evaluate the protective effects of drugs against such injury because CCl4-induced liver damage is regarded as the analog of liver damage caused by a variety of hepatotoxins (Reyes-Gordillo et al., 2007; Rudnicki et al., 2007). Hepatotoxicity caused by CCl4 is thought to be mediated by at least two sequential processes. The initial phase involves reductive dehalogenation by the cytochrome P450 isoenzymes (CYPs) system in the microsomal compartment of the liver, into the highly reactive trichloromethyl free radical, which initiates lipid peroxidation and leads to hepatocellular membrane damage (Lee and Jeong, 2002; Recknagel et al., 1989; Wong et al., 1998). The second phase implicates the release of inflammatory mediators from activated hepatic macrophages, which are thought to potentiate CCl4-induced hepatic injury (Badger et al., 1996; el Sisi et al., 1993). During the deteriorating phase, induction of inflammatory mediators including tumor

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necrosis factor-alpha (TNF-a), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (Cox-2) through nuclear factor-kappaB (NF-jB) activation occurs in the liver of rats after CCl4 treatment. Ongoing production of inflammatory mediators regulated by NF-jB is believed to aggravate CCl4-induced liver injury (Son et al., 2007). According to some reports, several herbal medicines and dietary compounds that possess antioxidant activities and inhibit activation of NF-jB could protect the liver against oxidative damage and inflammation caused by CCl4 (Domitrovic et al., 2012; Jeong, 1999; Rahman et al., 2006; Surh et al., 2001). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a member of the cap ‘‘n’’ collar basic region-leucine zipper transcription factor that protects a variety of tissues and cells against reactive oxygen species (ROS) through antioxidant response element (ARE)-mediated induction of diverse antioxidant and phase II detoxifying enzymes, including heme oxygenase-1 (HO-1), NAD(P)H quinine oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and glutamate-cysteine ligase (Lee and Johnson, 2004; Nguyen et al., 2003). Under basal condition, Nrf2-dependent transcription is suppressed by the negative regulator Kelch like-ECH-associated protein 1 (Keap1). Upon stimulation, Nrf2 escapes Keap1-mediated repression and is translocated from the cytosol to the nucleus, subsequently binds to ARE, resulting in up-regulation of antioxidant or phase II enzymes that confer cellular protection against oxidative stress damage and inflammation (Chen et al., 2006; Dhakshinamoorthy and Jaiswal, 2001; Kim et al., 2010). Garlic (Allium sativum L.) is a widely used flavoring agent and is a traditional medicine to control various diseases such as hyperlipidemia, microbial infection, and heart disease (Chen et al., 1998). It also possesses diverse biological activities, including anticarcinogenesis, antithrombosis, antiatherosclerotic, antidiabetic, antioxidant, and anti-inflammatory effects (Agarwal, 1996). Garlic oil contains more than 20 organosulfur compounds, which are believed to play a major role in the reported biological activities. Diallyl disulfide (DADS), a secondary component derived from garlic, has a potent antioxidant property (Sheen et al., 2001; Singh et al., 1998; Wu et al., 2002) and anti-inflammatory activity (Chang and Chen, 2005; Dirsch et al., 1998). This component also down-regulates the expression of numerous genes involved in hepatic oxidative stress and pro-inflammatory response (Chiang et al., 2006; Guyonnet et al., 1999; Keiss et al., 2003). A few reports have suggested the ameliorating effect of DADS against CCl4-induced hepatotoxicity and lipopolysaccharide-stimulated inflammatory response in vitro model (Fukao et al., 2004; Liu et al., 2006). However, the mechanism by which DADS elicits hepatoprotective and antioxidant effects in association with Nrf2 is unclear. Therefore, the aim of the present study was to evaluate the protective effects of DADS on CCl4-induced oxidative hepatic injury and inflammatory responses and to elucidate the mechanisms underlying these protective effects in rats. 2. Materials and methods 2.1. Animals and environmental conditions Thirty male Sprague–Dawley rats aged 6 weeks were obtained from a specific pathogen-free colony at Samtako Co. (Osan, Republic of Korea) and used after 1 week of quarantine and acclimation. Two animals per cage 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 with 13–18 air changes per hour. Commercial rodent chow (Samyang Feed, Wonju, Republic of Korea) sterilized by radiation and sterilized tap water were available ad libitum. The Institutional Animal Care and Use Committee of Chonnam National University approved the protocols for the animal study, and the animals were cared for in accordance with the Guidelines for Animal Experiments of Chonnam National University. 2.2. Test chemicals and treatment CCl4 (CAS No. 56-23-5) was purchased from Sigma Aldrich Co. (St. Louis, MO, USA). DADS was purchased from Tokyo Kasei Chemical Co. (Tokyo, Japan). All other chemicals were of the highest grade commercially available. Test chemicals were

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dissolved in corn oil, and were prepared immediately before treatment. The daily application volumes of CCl4 (10 ml/kg body weight) and DADS (2 ml/kg body weight) were calculated based on the most recently recorded body weight of the individual animal. DADS was gavaged to rats once daily for 5 days at dose levels of 50 and 100 mg/kg/day, respectively. Three hours after the final DADS treatment, the rats were given a single oral dose of CCl4 (2 ml/kg, 20% in corn oil) to induce liver injury (Lee et al., 2003). All animals were sacrificed 24 h after administration of CCl4. 2.3. Experimental groups and dose selection Thirty healthy male rats were randomly assigned to five experimental groups: (1) vehicle control, (2) DADS, (3) CCl4, (4) CCl4 + DADS 50, and (5) CCl4 + DADS 100 (n = 6 per group). The effective doses of DADS were based on previous studies (Guyonnet et al., 1999; Kalayarasan et al., 2009; Wu et al., 2002). 2.4. Necropsy and serum biochemical analysis All treated animals were euthanized by carbon dioxide inhalation for blood sample collection 24 h after administration of CCl4 on the scheduled termination day (test day 6). Blood samples were drawn from the posterior vena cava and serum samples were collected by centrifugation at 800g for 10 min within 1 h after collection and stored at 80 °C before analysis. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured with an autoanalyzer (Dri-chem 4000i, Fujifilm Co., Tokyo, Japan). 2.5. Histopathological examination A portion of liver was dissected and fixed in 10% neutral buffered formalin solution for 2 weeks. The remaining livers were frozen quickly in dry ice and stored at 80 °C for biochemical analysis. The fixed tissues were processed routinely, and were embedded in paraffin, sectioned to 4 lm thickness, deparaffinized, and rehydrated using standard techniques. The extent of CCl4-induced liver injury and the protective effects of DADS were evaluated by assessing morphological changes in liver sections stained with hematoxylin and eosin (H&E). All observations were made manually with a light microscope with 5, 10, 20, and 40 objective lenses and a 100 oil immersion lens in a totally blinded manner. The following variables were used for assessment of histological changes of the liver: (1) hepatocyte degeneration/necrosis; (2) fatty changes; (3) inflammatory cell infiltration; and (4) congestion. 2.6. Determination of lipid peroxidation and, reduced glutathione (GSH), and antioxidant enzymes A portion of frozen liver 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 to measure malondialdehyde (MDA) and GSH concentrations. Concentration of MDA was assayed by monitoring thiobarbituric acid reactive substance formation using the method of Berton et al. (1998). GSH content was measured by the method of Moron et al. (1979). Antioxidant enzyme activities, including catalase, superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx), and GST were also determined using commercial assay kits (Cayman Chemical, Ann Arbor, MI, USA). Total protein content was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard. 2.7. Hepatic cytoplasmic and nuclear protein isolation A frozen liver sample was cut into small pieces and washed in ice-cold (10 mM Tris–HCl, pH 7.4). Samples were homogenized in a glass–Teflon homogenizer with a suitable hypotonic lysis buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 Mm EGTA) containing protease inhibitor cocktail and dithiothretiol as a reducing agent for lysing the cell membranes. The lysate was incubated on ice for 15 min and added NP-40 to a final concentration of 0.5%, and then centrifuged at 250g for 15 min. The supernatant (cytosol fraction) was removed and stored at 80 °C for subsequent analysis. The pellet containing the nuclear fraction was resuspended in extraction buffer (20 mM HEPES, pH 7.9, 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and protease inhibitor cocktail) and vigorously vortexed for 15 min on ice. The nuclear suspension was centrifuged at 16,000g for 30 min. The supernatant (nuclear fraction) was stored at 80 °C for Western blot analysis. 2.8. Western blotting analysis Equal amounts of proteins (50 lg/well) from each sample were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Whatman, Maidenstone, UK), and blocked in blocking buffer (150 mM NaCl in 10 mM Tris, pH 7.5 containing 5% non-fat dry milk) for 1 h at room temperature. The membranes were incubated with primary rabbit

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Table 1 Primer sequences for quantitative real-time polymerase chain reaction. Target genes

Forward (50 ? 30 )

Reverse (50 ? 30 )

IL-1b IL-6 iNOS Cox-2 TNF-a NQO1 HO-1 GSTa GAPDH

CCC TGC AGC TGG AGA GTG TGG CGA GCC CAC CAG GAA CGA AAG TC GAT TCA GTG GTC CAA CCT GCA CCA GAG CAG AGA GAT GAA ATA CCA GAA GCC CCT CCC AGT TCT AGT TC GTG AGA AGA GCC CTG ATT GT TGC TCG CAT GAA CAC TCT GGA GAT GCC TTC TAC CCG AAG ACA CCT T AAC GGC ACA GTC AAG GCT GA

TGT GCT CTG CTT GAG AGG TGC T CTG GCT GGA AGT CTC TTG CGG AG CGA CCT GAT GTT GCC ACT GTT GCA GGG CGG GAT ACA GTT C CAC TCC CCA TCC TCC CTG GTC CCT GTG ATG TCG TTT CTG GA ATG GCA TAA ATT CCC ACT GCC ACG GTC AGC CTG TTC CCT ACA ACG CCA GTA GAC TCC ACG ACA T

IL-1b, interleukin-1b; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase, Cox-2, cyclooxygenase-2; TNF-a, tumor necrosis factor-alpha; NQO1, NAD(P)H quinine oxidoreductase 1; GSTa, glutathione S-transferase alpha; and glyceraldehydes-3-phosphate dehydrogenase, GAPDH. antibodies against rat Nrf2, phosphor-I kappaB alpha (p-IjBa), IjBa, a-tubulin, Lamin B1 (1:1000; Santa Cruz Biotechnology, CA, USA), Bax, cytochrome c, cleaved caspase-3, iNOS, Cox-2, NF-jB p65, TNF-a, and b-actin (1:1000; Cell Signaling Technology, Beverly, MA, USA) for 18 h at 4 °C. After incubation, the membranes were washed three times (20 mM Tris–HCl, pH 7.5, 137 mM NaCl, and 0.1% Tween 20), incubated with HRP-conjugated secondary antibodies (1:2000) for 1 h at room temperature, washed three times, and then detected by enhanced chemiluminescence. The protein concentration was determined with a BCA Protein Assay kit (Pierce, Rockford, IL, USA). Protein expression was quantified based on band density using TINA 20 Image software (Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany). Relative intensity was calculated by dividing the densities of respective loading control density value. Values are presented as means ± SD.

2.10. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay The level of DNA fragmentation was detected using a TUNEL assay, which was performed according to the manufacturer’s instructions (ApopTag Peroxidase In Situ Apoptosis Detection Kit; Chemicon, Billerica, MA, USA). The number of TUNEL-positive cells was counted in 10 arbitrary selected fields around centrilobular areas (200) of similar size per slide in a double-blinded manner and calculated as the total number of TUNEL-positive cells/10 fields. The results are presented as means ± SD.

2.11. Immunohistochemical analysis 2.9. Quantitative real-time polymerase chain reaction (RT-PCR) Total RNA was extracted from the liver using TRI reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s protocol. RNA concentration was quantified using a NanoDrop ND-1000 (Thermo Scientific, Waltham, MA, USA). Reverse transcription of an equal amount of target RNA was performed using A QuantiTect Reverse Transcription kit (Qiagen, Valencia, CA, USA). The primers for each gene are shown in Table 1. Quantitative RT-PCR was performed with iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) using a MyiQ2 thermocycler and the SYBR Green Detection System (Bio-Rad) with 0.5 lg of cDNA. Manufacturer-supplied (SuperArray, Bioscience Corp., Beltsville, MD, USA) primer pairs were used to measure TNF-a, iNOS, Cox-2, interleukin-1b (IL-1b), IL-6, NQO1, HO-1, and GSTa mRNA expression. Samples were run in triplicate to ensure amplification integrity. The standard PCR conditions were 95 °C for 15 min, 40 cycles at 95 °C, 30 cycles at 60 °C for 30 s, and 72 °C for 30 s, as recommended by the primer manufacturer. The threshold cycle (Ct; the cycle number at which the amount of amplified gene of interest reached a fixed threshold) was determined subsequently. Relative quantitation of each mRNA expression was calculated by the comparative Ct method. The relative quantitation values of targets was normalized to the endogenous glyceraldehydes-3-phosphate dehydrogenase (GAPDH) control gene and were expressed as 2DDCt (fold), where DCt = Ct of target gene – Ct of endogenous control gene and DDCt = DCt of samples for target gene – DCt of the calibrator for the target gene.

The fixed liver tissues were processed routinely, embedded in paraffin, sectioned to 4 lm thickness, deparaffinized, and rehydrated using standard techniques. After incubation with a protein block (Anti Rabbit-Specific HRP/DAB IHC Kit; Abcam, Cambridge, MA, USA), the sections were incubated overnight with anticaspase-3 antibody (1:200; Cell Signaling Technology) at 4 °C. Caspase-3 expression was visualized using an IHC kit (Abcam) according to the manufacturer’s protocol. The second antibody biotinylated goat anti-rabbit IgG was applied followed by streptavidin peroxidase and then DAB chromogen with its substrate buffer. The sections were counterstained with Harris’s hematoxylin before being mounted. Finally, caspase-3-positive cells and the total number of cells were quantified by counting 10 arbitrarily selected fields around centrilobular areas (200) in a double-blinded manner and calculated as the total number of caspase-3-positive cells/10 fields. The results are presented as means ± SD.

2.12. Statistical analysis Data are expressed as means ± SD, and all statistical comparisons were made by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Statistical analysis was performed comparing the treatment groups to the control group using GraphPad InStat v. 3.0 (GraphPad Software, Inc., La Jolla, CA, USA). Values of P < 0.05 were considered to be statistically significant.

Fig. 1. Effects of DADS on serum biochemical markers in CCl4-induced hepatotoxicity. DADS (50 and 100 mg/kg/day) was administered intragastrically for 5 days prior to CCl4 (2 ml/kg) treatment. Values are presented as means ± SD (n = 6). ⁄⁄ p < 0.01 compared with the control group;    p < 0.01 compared with the CCl4 group.

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Fig. 2. Effects of DADS on CCl4-induced histopathological alterations. Livers from (A) vehicle control and (B) DADS treated rats showing normal appearance. (C), (D) Liver from CCl4 treated rats showing moderate or severe degeneration/necrosis of hepatocytes around the central vein region (open arrow), fatty changes (closed arrow), inflammatory cell infiltration (reverse triangle), and congestion (asterisk). Livers from (E) CCl4 + DADS 50 and (F) CCl4 + DADS 100 treated rats showing mild degeneration of hepatocyte and fatty changes. DADS (50 and 100 mg/kg/day) was administered intragastrically for 5 days prior to CCl4 (2 ml/kg) treatment. H&E stain. Bar = 50 lm (400).

Table 2 Lipid peroxidation, reduced glutathione levels, and antioxidant enzyme activities in the liver of male rats treated with CCl4 and/or DADS. Items

No. of rats MDA (nmol/mg protein) GSH (pmol/mg protein) Catalase (units/mg protein) SOD (units/mg protein) GR (units/mg protein) GPx (units/mg protein) GST (units/mg protein)

Group Control

DADS

CCl4

CCl4 + DADS 50

CCl4 + DADS 100

6 2.05 ± 0.066a 0.72 ± 0.071 12.7 ± 1.75 0.93 ± 0.149 4.87 ± 0.615 1.49 ± 0.251 76.2 ± 3.43

6 2.15 ± 0.079 1.04 ± 0.063 11.6 ± 3.59 0.96±.0138 6.35 ± 0.856 1.59 ± 0.206 86.8 ± 4.78

6 2.78 ± 0.064** 0.62 ± 0.038** 8.1 ± 1.59* 0.44 ± 0.153* 3.57 ± 0.855* 1.13 ± 0.175* 64.6 ± 5.04**

6 2.51 ± 0.154  1.12 ± 0.040   12.1 ± 2.75  1.27 ± 0.249  5.21 ± 0.723   1.49 ± 0.139   74.3 ± 7.04

6 2.47 ± 0.073   1.47 ± 0.075   20.3 ± 3.10   1.63 ± 0.101   6.01 ± 0.829   1.83 ± 0.130   83.2 ± 7.45  

MDA, malondialdehyde; GSH, reduced glutathione; SOD, superoxide dismutase; GR, glutathione reductase; GPx, glutathione peroxidase; and GST, glutathione S-transferase. a Values are presented as means ± SD. * p < 0.05 compared with the control group. ** p < 0.01 compared with the control group.   p < 0.05, compared with the CCl4 group.    p < 0.01 compared with the CCl4 group.

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Fig. 3. Effects of DADS on CCl4-induced hepatic cellular apoptosis. Representative photographs of TUNEL assay performed on liver sections of (A) vehicle control and rats treated with (B) DADS, (C) CCl4, (D) CCl4 + DADS 50, and (E) CCl4 + DADS 100. The black arrows point to the apoptotic cells (TUNEL-positive cells). Bar = 50 lm. (F) The bar graphs show the total number of TUNEL-positive cells/10 fields in liver sections of vehicle control, DADS, CCl4, and CCl4 + DADS-treated rats and results are presented as means ± SD (n = 6 per group). DADS (50 and 100 mg/kg/day) was administered intragastrically for 5 days prior to CCl4 (2 ml/kg) treatment. ⁄⁄ p < 0.01 compared with the control group;    p < 0.01 compared with the CCl4 group.

3. Results

3.3. Effects of DADS on CCl4-induced hepatic oxidative stress

3.1. Effects of DADS on acute hepatic damage

As presented in Table 2, the concentration of MDA, an end product of lipid peroxidation, increased significantly, whereas GSH content decreased significantly in the CCl4 group compared to those in the control group. In addition, catalase, SOD, GR, GPx, and GST activities in the liver of CCl4-treated rats decreased significantly compared to those in the control group. However, MDA concentration decreased significantly and GSH content in the CCl4 + DADS groups increased significantly compared to those in the CCl4 group. Moreover, catalase, SOD, GR, GPx, and GST activities in the CCl4 + DADS groups were significantly higher than those in the CCl4 group in a dose-dependent manner.

As shown in Fig. 1, serum AST and ALT activities in the CCl4 group increased significantly when compared to those in the control group. In contrast, serum AST and ALT activities in the CCl4 + DADS groups decreased significantly when compared to those in the CCl4 group.

3.2. Effects of DADS on CCl4-induced histopathological alterations The control and DADS groups presented livers with normal architecture (Fig. 2A and B). However, liver tissues from all rats treated with CCl4 showed extensive histopathological changes, characterized by moderate or severe hepatocytes degeneration/ necrosis (n = 6), fatty changes (n = 6), inflammatory cell infiltration (n = 6), and congestion (n = 4) (Fig. 2C and D). Although these findings were also observed in the CCl4 + DADS groups (Fig. 2E and F), the incidence and severity of histopathological lesions were less than those in the CCl4 group.

3.4. Effects of DADS on CCl4-induced hepatic cell apoptosis The control and DADS groups showed few apoptotic hepatic cells (Fig. 3A and B). An increased number of hepatic apoptotic cells was observed in the CCl4 group (Fig. 3C), and the number of TUNEL-positive cell increased significantly compared to that in the control group (Fig. 3F). In contrast, the number of TUNEL-positive cells in the CCl4 + DADS groups decreased significantly compared to that in the CCl4 group in a dose-dependent manner (Fig. 3D–F).

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Fig. 4. Effects of DADS on CCl4-induced mitochondrial apoptosis pathway. (A) Western blot analysis of hepatic cytosolic Bax, cytochrome c, and caspase-3 expression in male rats treated with CCl4 and/or DADS (loading control: b-actin). The bar graphs show relative (B) Bax, (C) cytochrome c, and (D) caspase-3 protein levels in hepatic tissues for vehicle, DADS, CCl4, and CCl4 + DADS-treated rats. DADS (50 and 100 mg/kg/day) was administered intragastrically for 5 days prior to CCl4 (2 ml/kg) treatment. Values are presented as means ± SD (n = 6). ⁄⁄ p < 0.01 compared with the control group;   p < 0.05,    p < 0.01 compared with the CCl4 group.

3.5. Effects of DADS on CCl4-induced mitochondrial apoptosis pathway Mitochondrial-initiated apoptosis triggered by ROS plays an important role in CCl4-induced hepatotoxicity (Cai et al., 2005). To determine whether DADS exerts effects on CCl4-induced mitochondrial apoptotic pathway, we investigated cytosolic Bax, cytochrome c, and caspase-3 protein levels by Western blot analysis. As shown in Fig. 4, Bax, cytochrome c, and caspase-3 protein levels increased significantly in the CCl4 group compared to those in the control group. However, cytosolic protein levels of Bax, cytochrome c, and caspase-3 dose-dependently decreased in the CCl4 + DADS groups compared to those in the CCl4 group. 3.6. Effects of DADS on caspase-3 immunopositivity To further confirmed anti-apoptotic effects of DADS, we conducted an immunohistochemical analysis for caspase-3. Caspase3-positive cells were seldom seen in the control and DADS groups (Fig. 5A and B). However, the caspase-3-positive cell count in the CCl4 group increased significantly compared to that in the control group (Fig. 5C and F). In contrast, the number of caspase-3-positive cells in the CCl4 + DADS groups decreased significantly compared to that in the CCl4 group in a dose-dependent manner (Fig. 5D–F). 3.7. Effects of DADS on CCl4-induced hepatic NF-jB activation and IjBa phosphorylation The transcription factor NF-jB is a key regulator of the cellular inflammatory response. The inducible phosphorylation of IjBa is

mediated by IjB kinase (IKK) complex, and this degradation of IjB proteins from NF-jB is an essential step to activate NF-jB nuclear translocation. We examined nuclear/cytoplasmic NF-jB and p-IjBa/IjBa protein expression by Western blot analysis to investigate whether DADS exerts its effects by regulating NF-jB activation and IjBa phosphorylation. CCl4-treated rats showed decreased hepatic cytoplasmic NF-jB expression and increased nuclear NF-jB expression compared to those in the control group (Fig. 6A). The relative nuclear/cytoplasmic NF-jB ratio in CCl4 group increased significantly compared to that in the control group (Fig. 6B). In addition, p-IjBa protein level and relative p-IjBa/IjBa ratio increased significantly in the CCl4 group compared to that in the control group (Fig. 6C and D). However, hepatic cytoplasmic NF-jB protein levels were increased, whereas nuclear NF-jB expression levels were decreased in CCl4 + DADS groups compared to those in the CCl4 group. The relative nuclear/cytoplasmic NF-jB ratios in the CCl4 + DADS groups decreased significantly compared to that in the CCl4 group in a dose-dependent manner. Moreover, the relative ratios of p-IjBa/IjBa in the CCl4 + DADS groups also decreased significantly compared to the CCl4 group in a dosedependent manner. 3.8. Effects of DADS on CCl4-induced inflammatory mediators To determine whether DADS elicits its effects on NF-jB signaling-related inflammatory mediators, we confirmed the mRNA and protein levels of hepatic TNF-a, iNOS, and Cox-2. As shown in Fig. 7. TNF-a, iNOS and Cox-2 mRNA levels in the CCl4 group increased significantly compared to those in the control group

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Fig. 5. Effects of DADS on CCl4-induced caspase-3 activation. Representative photographs of immunohistochemical analysis of caspase-3 in liver sections of (A) vehicle controls and rats treated with (B) DADS, (C) CCl4, (D) CCl4 + DADS 50, and (E) CCl4 + DADS 100. The black arrows point to caspase-3-positive cells. Bar = 50 lm. (F) The bar graphs show the total number of caspase-3-positive cells/10 fields in liver sections of vehicle control, DADS, CCl4, and CCl4 + DADS-treated rats and results are presented as means ± SD (n = 6 per group). DADS (50 and 100 mg/kg/day) was administered intragastrically for 5 days prior to CCl4 (2 ml/kg) treatment. ⁄⁄ p < 0.01 compared with the control group;    p < 0.01 compared with the CCl4 group.

(Fig. 7E–G). Similarly, hepatic TNF-a, iNOS and Cox-2 protein levels also elevated in the CCl4 group compared to those in the control group (Fig. 7B–D). In contrast, hepatic TNF-a, iNOS and Cox-2 mRNA and protein levels decreased significantly in the CCl4 + DADS groups compared to those in the CCl4 group in a dose-dependent manner. 3.9. Effects of DADS on CCl4-induced inflammatory cytokines Hepatic IL-1b and IL-6 mRNA levels are presented in Fig. 8. IL-1b and IL-6 mRNA levels in the CCl4 group increased significantly compared to those in the control group. In contrast, hepatic IL-1b and IL-6 mRNA levels in the CCl4 + DADS groups decreased significantly compared to those in the CCl4 group. 3.10. Effects of DADS on NQO1, HO-1, and cytosolic and nuclear Nrf2 expression Nrf2 is sequestered in the cytoplasm by Keap1 under normal conditions and its translocation into the nucleus is essential for transcription of various phase II and/or antioxidant enzyme genes (Chen et al., 2006; Dhakshinamoorthy and Jaiswal, 2001). To determine whether DADS elicits its effects by regulating Nrf2, we inves-

tigated cytosolic and nuclear Nrf2 protein levels by Western blot analysis. As presented in Fig. 9, CCl4-treated rat liver showed a depletion of cytoplasmic Nrf2 and a significant decrease in nuclear Nrf2 compared to the control group. In contrast, cytoplasmic Nrf2 levels in the CCl4 + DADS groups were significantly higher than that in the CCl4 group and nuclear translocation of Nrf2 in the CCl4 + DADS groups increased significantly compared to that in the CCl4 group in a dose-dependent manner. To confirm these results, we examined HO-1 and NQO1 protein levels. No significant differences were observed in HO-1 protein levels between the control and the CCl4 groups. Unlike HO-1 expression, NQO1 protein level in the CCl4 group was significantly less than that in the control group. However, the protein level of HO-1 in the CCl4 + DADS 100 group and increased significantly compared to that in the CCl4 group. In addition, NQO1 protein levels in the CCl4 + DADS groups were significantly higher than that in the CCl4 group in a dosedependent manner. 3.11. Effects of DADS on Nrf2/ARE-related gene expression To further confirm whether Nrf2-related genes are induced by DADS, we examined the mRNA levels of HO-1, NQO1, and GSTa by quantitative RT-PCR. As presented in Fig. 10, CCl4-treated rat

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Fig. 6. Effects of DADS on CCl4-induced NF-jB activation and IjBa phosphorylation. Western blot analysis of (A) hepatic nuclear/cytosolic NF-jB and (C) IjBa/p-IjBa expression in the male rats treated with CCl4 and/or DADS (loading control: b-actin; cytosolic control: a-tubulin; nuclear control: Lamin B1). The bar graphs show relative (B) nuclear/cytoplasmic NF-jB and (D) p-IjBa/IjBa ratios in hepatic tissues for vehicle, DADS, CCl4, and CCl4 + DADS-treated rats, respectively. DADS (50 and 100 mg/kg/day) was administered intragastrically for 5 days prior to CCl4 (2 ml/kg) treatment. Values are presented as means ± SD (n = 6). ⁄⁄ p < 0.01 compared with the control group;    p < 0.01 compared with the CCl4 group.

liver showed a significant decrease in the mRNA levels of HO-1, NQO1, and GSTa, Nrf2/ARE pathway-related genes, compared to those in the control group. In contrast, the HO-1, NQO1, and GSTa mRNA levels in the CCl4 + DADS groups increased significantly compared to those in the CCl4 group in a dose-dependent manner.

4. Discussion CCl4 is one of the most intensively studied hepatotoxicants to date and is commonly used as a model for screening the anti-hepatotoxic and/or hepatoprotective properties of drugs (Brattin et al., 1985; Recknagel et al., 1989, 1991; Williams and Burk, 1990). Increased serum AST and ALT levels have been attributed to the damaged structural integrity of hepatocytes. This is because the altered permeability of the membrane causes enzymes from the cells to be released into the circulation after cellular damage (Recknagel et al., 1989, 1991). In this study, the single oral dose of CCl4 caused a significant elevation in serum AST and ALT levels, indicating the acute hepatotoxicity induced by CCl4. The hepatotoxic effects observed in the CCl4 treated rats were confirmed histopathologically, characterized by massive hepatocellular degeneration/necrosis, fatty changes, and inflammatory cell infiltration. However, pretreatment with DADS effectively improved the CCl4-induced elevation in serum AST and ALT levels, indicating the hepatoprotective effect of DADS against the acute intoxication of CCl4. This phenomenon was also confirmed by histopathological examination.

The hepatotoxicity of CCl4 is result of reductive dehalogenation, which is catalyzed by CYPs, to form the highly reactive trichloromethyl and trichloromethyl peroxyl radicals (Brattin et al., 1985; Recknagel et al., 1991; Williams and Burk, 1990). Covalent binding of these radicals to cellular macromolecules is assumed to initiate free radical-mediated lipid peroxidation, which is one of the principal causes of CCl4-induced liver injury (Recknagel et al., 1991; Williams and Burk, 1990). GSH conjugation in the liver plays a key role in detoxifying the trichloromethyl radical, reactive toxic metabolites of CCl4, and its depletion causes liver necrosis (Recknagel et al., 1991). Therefore, the antioxidant activity and/or inhibition of free radical generation are important in terms of protecting the liver from CCl4-induced damage (Jeong, 1999; Koop, 1992). In this study, CCl4 treatment caused high levels of oxidative damage, as evidenced by a significant elevation in hepatic MDA concentration and a significant decrease in GSH content and antioxidant enzyme activities. However, DADS attenuated the CCl4-induced elevation in hepatic MDA concentration and dosedependently improved CCl4-induced suppression of GSH content and antioxidant enzyme activities in hepatic tissue. It has been demonstrated that many of the beneficial effects of DADS result from modulation of phase I and II metabolizing enzymes (Jeong and Lee, 1998; Reicks and Crankshaw, 1996). In particular, this component not only inhibits CYP2E1, but also has potent antioxidant properties and elevates hepatic GSH levels (Dwivedi et al., 1998; Fanelli et al., 1998). In our study, this apparent ameliorative effect may be due to the ability of DADS to inhibit lipid peroxidation

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Fig. 7. Effects of DADS on CCl4-induced hepatic pro-inflammatory mediators. (A) Western blot analysis of hepatic iNOS, Cox-2, and TNF-a expression in male rats treated with CCl4 and/or DADS (loading control: b-actin). The bar graphs show relative expression of hepatic iNOS, Cox-2, and TNF-a at protein (B-D) and mRNA (E-G) levels in hepatic tissues for vehicle, DADS, CCl4, and CCl4 + DADS-treated rats (loading control: GAPDH). DADS (50 and 100 mg/kg/day) was administered intragastrically for 5 days prior to CCl4 (2 ml/kg) treatment. Values are presented as means ± SD (n = 6). ⁄⁄ p < 0.01 compared with the control group;   p < 0.05,    p < 0.01 compared with the CCl4 group.

and to enhance antioxidant enzyme activities. The protective effects of DADS on CCl4-induced liver damage may be attributed to inhibition of CYP2E1 by DADS and there is a need for further study for its effect on CYP2E1. Several studies have shown that liver damage induced by CCl4 is partly involved in the apoptosis pathway in vivo and in vitro (Cai et al., 2005; Sun et al., 2001). CCl4 damages liver mitochondria by inhibiting cytochrome oxidase and by enhancing oxidative stress (Ikeda et al., 1998). Mitochondrial initiated apoptosis triggered by ROS plays an important role in CCl4-induced hepatotoxicity, and antioxidants effectively reversed liver damage caused by CCl4 (Sun et al., 2001). In the present study, CCl4 caused a marked increase in the numbers of TUNEL-positive and caspase-3-positive cells in hepatic tissue. However, pretreatment with DADS effectively decreased the numbers of TUNEL-positive and caspase-3-positive cells induced by CCl4 in a dose-dependent manner. In addition, we investigated the expression of Bax, cytochrome c, and caspase-3, which have been implicated as stimulators of apop-

tosis, to further confirm the role of DADS in ROS-mediated hepatic cell apoptosis by CCl4. As a result, CCl4 intoxication caused a marked increase in Bax, cytochrome c, and caspase-3 protein levels in hepatic tissue. In contrast, DADS pretreatment dosedependently attenuates mitochondrial pathway-related protein expression. These observations indicate that DADS effectively attenuates ROS-mediated hepatic cell apoptosis induced by CCl4. Nrf2 has emerged as an indispensable regulator of both constitutive and inducible expression of detoxifying phase II and antioxidant enzyme genes in various tissues and cell types (Elbling et al., 2005; Enomoto et al., 2001). The induction of cytoprotective enzymes is an important event in the cellular stress response during which a variety of oxidative toxicants can be eliminated or inactivated before they damage critical cellular macromolecules (Copple et al., 2007; Rushmore and Kong, 2002). Nrf2-null mice are particularly susceptible to oxidative stress, contributing to increased hepatotoxicity by ethanol (Lamle et al., 2008) and acetaminophen (Reisman et al., 2009). In this study, rats treated with CCl4 showed

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Fig. 8. Effects of DADS on CCl4-induced hepatic inflammatory cytokines. The bar graphs show relative mRNA levels of hepatic (A) IL-1b and (B) IL-6 in male rats treated with treated with CCl4 and/or DADS (loading control: GAPDH). DADS (50 and 100 mg/kg/day) was administered intragastrically for 5 days prior to CCl4 (2 ml/kg) treatment. Values are presented as means ± SD (n = 6). ⁄⁄ p < 0.01 compared with the control group;    p < 0.01 compared with the CCl4 group.

Fig. 9. Effects of DADS on Nrf2 and phase II enzyme expression. (A) Western blot analysis of HO-1, NQO1, and cytoplasmic/nuclear Nrf2 expression in male rats treated with CCl4 and/or DADS (cytoplasmic control: a-tubulin; nuclear control: Lamin B1). The bar graphs show relative (B) cytoplasmic and (C) nuclear Nrf2, (D) HO-1, and (E) NQO1 protein levels for vehicle, DADS, CCl4, and CCl4 + DADS-treated rats. DADS (50 and 100 mg/kg/day) was administered intragastrically for 5 days prior to CCl4 (2 ml/kg) treatment. Values are presented as means ± SD (n = 6). ⁄ p < 0.05, ⁄⁄ p < 0.01 compared with the control group;    p < 0.01 compared with the CCl4 group.

depletion of cytoplasmic Nrf2 and suppression of Nrf2 nuclear translocation. Consistent with this observation, a dramatic downregulation of Nrf2 target genes, NQO1, HO-1, and GSTa levels was observed in the CCl4-treated rat liver. These findings were in accordance with those of previous studies (Domitrovic et al., 2012; Wu et al., 2013) and well correlated with the decreased antioxidant enzyme activities caused by CCl4. However, DADS pretreatment reversed cytoplasmic Nrf2 depletion and enhanced nuclear translocation of Nrf2. These results were also consistent with upregulation of Nrf2 target genes and the enhancement of antioxidant enzyme activities. Similar to Nrf2 target gene levels, CCl4 caused a decrease in NQO1 protein expression, but not HO-1, whereas DADS pretreatment effectively reversed NQO1 and HO-1 expression levels in rat liver. Several studies reported that activating Nrf2 pathway offers cytoprotective effects against various pathological conditions caused by oxidative stress (Kalayarasan et al., 2009; Wu et al., 2013). Our results indicate that DADS showed dose-dependent effects on stabilization of Nrf2 in cyto-

plasm and enhancement of Nrf2 nuclear translocation that promotes the transcription of detoxifying or antioxidant enzymes. Thus, activation of Nrf2 plays an important role in the hepatoprotective effects of DADS via induction of detoxifying phase II or antioxidant enzymes against CCl4-induced hepatotoxicity. NF-jB activation and its subsequent nuclear translocation induced by ROS, are responsible for modulation of liver injury by affecting cytokine production, such as TNF-a, and the induction of other pro-inflammatory mediators, such as iNOS and Cox-2 (Luedde and Schwabe, 2011; Reyes-Gordillo et al., 2007). In the resting cells, NF-jB is sequestered in the cytoplasm by the inhibitory IjBa subunit. Upon stimulation, IjBa is phosphorylated at serine residues by IKKs, ultimately leading to induction of inflammatory mediators. In the present study, CCl4 caused an increase in the phosphorylation of IjBa and nuclear translocation of NF-jB concurrent with induction of TNF-a, iNOS, and Cox-2 at the protein and mRNA levels. These observations well correlated with increased IL-1b and IL-6 mRNA levels. However, DADS

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Conflict of Interest The authors declare that there are no conflicts of interest.

Acknowledgements This study was financially supported by Chonnam National University, 2012. The animal experiment in this study was supported by the Animal Medical Institute of Chonnam National University.

References

Fig. 10. Effects of DADS on Nrf2/ARE pathway-related gene expression. The bar graphs show relative mRNA expression of hepatic HO-1, NQO1, and GSTa in male rats treated with treated with CCl4 and/or DADS (loading control: GAPDH). DADS (50 and 100 mg/kg/day) was administered intragastrically for 5 days prior to CCl4 (2 ml/kg) treatment. Values are presented as means ± SD (n = 6). ⁄ p < 0.05, ⁄⁄ p < 0.01 compared with the control group;    p < 0.01 compared with the CCl4 group.

pretreatment effectively prevented IjBa phosphorylation and suppressed activation of NF-jB, and that reduced the induction of inflammatory mediators or cytokines at the protein and mRNA levels. Induction of phase II detoxifying or antioxidant enzymes via Nrf2/ARE signaling provides an effective means for cellular protection as first line defense against a variety of electrophilic, reactive toxicants and pro-inflammatory stimuli (Chen et al., 2006; Khor et al., 2006; Li and Nel, 2006). Recent studies have suggested interplay between Nrf2 and NF-jB for the inflammatory response. Nrf2deficient mice display more NF-jB activation in response to lipopolysaccharides, whereas activation of Nrf2-antioxidant signaling attenuates the NF-jB-mediated inflammatory response (Zakkar et al., 2009). Several reports suggested that an increase in ROS level activates NF-jB signaling pathway, Nrf2 limits ROS level leading to inactivation of redox sensitive pro-inflammatory NF-jB pathway (Jin et al., 2008). Moreover, garlic organosulfur compounds including DADS induced detoxifying enzymes by activating transcription factor Nrf2 (Chen et al., 2004). We investigated the anti-inflammatory effects of DADS in CCl4-induced liver injury and found that DADS increases the phase II detoxifying and/or antioxidant enzyme expression by activating Nrf2 and simultaneously decreases inflammatory mediators by inhibiting NF-jB activation. Therefore, these findings indicate that the ability of DADS to suppress production of inflammatory mediators by the inhibiting NF-jB activation and the ability of DADS to mediate NF- jB inhibition may be achieved by activating Nrf2/ARE signaling, as well as by its direct anti-inflammatory effects. In conclusion, the results of this study indicate that DADS effectively ameliorates CCl4-induced oxidative hepatic injury and inflammatory responses in rats, and that the hepatoprotective effects of DADS may be due to its ability to induce antioxidant or detoxifying enzyme activities through activation of Nrf2 and suppressing production of inflammatory mediators by inhibiting NFjB activation. These findings suggest that DADS may be a useful protective agent against various hepatic injuries caused by oxidative stress and inflammatory response.

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