Accepted Manuscript Protective effect and mechanism of action of diallyl disulfide against acetaminopheninduced acute hepatotoxicity Je-Won Ko, Sung-Hyeuk Park, Na-Rae Shin, Jin-Young Shin, Jeong-Won Kim, In-Sik Shin, Changjong Moon, Jeong-Doo Heo, Jong-Choon Kim, In-Chul Lee PII:

S0278-6915(17)30480-5

DOI:

10.1016/j.fct.2017.08.029

Reference:

FCT 9246

To appear in:

Food and Chemical Toxicology

Received Date: 1 June 2017 Revised Date:

30 July 2017

Accepted Date: 22 August 2017

Please cite this article as: Ko, J.-W., Park, S.-H., Shin, N.-R., Shin, J.-Y., Kim, J.-W., Shin, I.-S., Moon, C., Heo, J.-D., Kim, J.-C., Lee, I.-C., Protective effect and mechanism of action of diallyl disulfide against acetaminophen-induced acute hepatotoxicity, Food and Chemical Toxicology (2017), doi: 10.1016/ j.fct.2017.08.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Protective effect and mechanism of action of diallyl disulfide against

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acetaminophen-induced acute hepatotoxicity

Je-Won Ko a, Sung-Hyeuk Park a, Na-Rae Shin a, Jin-Young Shin b, Jeong-Won Kim c, In-

a

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Sik Shin a, Changjong Moon a, Jeong-Doo Heo d, Jong-Choon Kim a,*, In-Chul Lee e, **

College of Veterinary Medicine BK21 Plus Project Team, Chonnam National University,

b

c

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Gwangju 61186, Republic of Korea

Ministry of Food and Drug Safety, Cheongju 28159, Republic of Korea Department of Food and Nutrition, Duksung Women's University, Seoul 01369, Republic of

Korea d

Gyeongnam Department of Environment & Toxicology, Korea Institute of Toxicology,

e

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Gyeongnam 52834, Republic of Korea

Natural Product Research Center, Korea Research Institute of Bioscience and Biotechnology,

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Jeongeup 56212, Republic of Korea

* Corresponding author.

** Corresponding author. E-mail addresses: [email protected] (J.-C. Kim), [email protected] (I.-C. Lee).

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1. Introduction

Acetaminophen (AAP) is a widely used, effective analgesic and antipyretic drug, which is

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safe when prescribed at the therapeutic level (Bessems and Vermeulen, 2001). At therapeutic doses, approximately 80% of AAP is conjugated to glucuronic acid or sulfate in hepatocytes and excreted through the bile or urine (Mitchell and Jollows, 1975). The remaining 5–10% of AAP is

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metabolized by cytochrome P450 (CYP), a superfamily of hemecontaining monoxygenases that metabolize a large number of compounds (Nelson, 1990). Among the CYPs, there are 57 genes

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that have been found to be functionally significant, of which CYP2E1 is known to play an important and predominant roles in metabolism and subsequent toxicity of AAP (Gonzalez, 2005, Zanger and Schwab, 2013). By CYP2E1, AAP metabolized to a highly reactive intermediate Nacetyl-p-benzoquinone imine (NAPQI) (Nelson, 1990). NAPQI is rapidly detoxified via conjugation with reduced glutathione (GSH). However, AAP overdose can cause excessive

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production of NAPQI, which in turn depletes of GSH levels in hepatocytes and leads to subsequent hepatocellular death (Mitchell et al., 1973). Recent studies have demonstrated that production of reactive oxygen species induced by AAP causes mitochondrial damage and early

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activation of mitogen-activated protein kinases, especially c-Jun-N-terminal protein kinase (JNK) (Latchoumycandane et al., 2007; Hanawa et al., 2008).

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Oxidative stress activates signal transduction pathways involving transcription factors, such as nuclear factor kappa B (NF-κB) (Dambach et al., 2006). NF-κB is known to regulate the gene expression controlling inflammatory mediators, including inducible nitric oxide (iNOS), cyclooxygenase-2 (Cox-2), and tumor necrosis factor-α (TNF-α). It is an important regulator of numerous inflammatory mediators, several of which have been implicated in AAP-induced hepatotoxicity (Laskin and Gardner, 2003). Many studies have shown that antioxidant and antiinflammatory agents effectively protect against acute hepatotoxicity induced by AAP overdose (Girish et al., 2009; Nagi et al., 2010; Oz and Chen, 2008). In this regard, inhibition of AAP

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metabolic activation achieved by blocking CYP2E1, and/or inhibition of NAPQI-mediated oxidative stress achieved by increasing GSH levels and activating the antioxidant defense system, can confer protection from AAP-induced hepatotoxicity (Jaeschke et al., 2011).

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Garlic (Allium sativum L.) possesses diverse biological activities, including anticarcinogenic, antidiabetic, antioxidant, and anti-inflammatory properties (Agarwal, 1996; Wang et al., 1996). Garlic oil contains more than 20 organosulfur compounds (OSCs) that are believed to play a major role in the reported biological activities of garlic (Wu et al., 2002). Among various OSCs,

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diallyl disulfide (DADS) is beneficial to human health, particularly because of its protective

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effects against carcinogenesis and chemically induced toxicity (Siess et al., 1997; Sheen et al., 2001; Shin et al., 2016). Many of the pharmacological effects of DADS may result from modulation of phase I and II metabolizing enzymes (Reicks and Crankshaw, 1996; Guyonett et al., 1999). DADS not only inhibits CYP2E1-related metabolism but also enhances antioxidant properties (Dwivedi et al., 1998; Ko et al., 2017). However, the mechanism by which DADS

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elicits hepatoprotective and antioxidant effects against AAP-induced hepatic damage is still unclear. Therefore, the aim of the present study was to evaluate the protective effects of DADS against AAP-induced oxidative hepatic injury and to elucidate the mechanisms underlying these

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protective effects in rats.

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2. Materials and methods

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2.1. Animals and environmental conditions

Twenty-four male Sprague–Dawley rats aged 9 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

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temperature of 23 ± 3 °C and a relative humidity of 50 ± 10% with 12 h light/dark cycles and 12–

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18 air changes/h. Commercial rodent chow (Samyang Feed, Wonju, Republic of Korea) sterilized by radiation and sterilized tap water were provided 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

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of Chonnam National University.

2.2. Test chemicals and treatment

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AAP (CAS No. 103-90-2) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). DADS was purchased from Tokyo Kasei Chemical Co. (Tokyo, Japan). All other chemicals

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were of the highest grade commercially available. DADS was dissolved in corn oil, and AAP was dissolved in a saline solution that was kept in a boiling water bath. The volumes of AAP (20 mL/kg body weight) and DADS (10 mL/kg body weight) administered daily were calculated based on the most recently recorded body weight of the individual animal. DADS was administered to rats by oral gavage once daily for 5 days at 100 mg/kg/day. The rats were given a single oral dose of AAP (4 g/kg) to induce liver injury 3 h after the final DADS treatment (Sharifudin et al., 2013; Verma et al., 2013). All animals were sacrificed 24 h after AAP administration.

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2.3. Experimental groups and dose selection

Twenty four healthy male rats were randomly assigned to four experimental groups: 1)

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vehicle control, 2) DADS, 3) AAP, and 4) DADS + AAP (n = 6 per group). The effective dose of DADS was based on an earlier study and our previous study (Wu et al., 2002; Lee et al., 2014).

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2.4. Necropsy and serum biochemical analysis

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All treated animals were anesthetized via carbon dioxide inhalation for blood sample collection 24 h after administration of AAP. Blood samples were drawn from the posterior vena cava and all rats were euthanized via exsanguination after bleeding. The serum samples were collected via centrifugation at 800 g for 10 min within 1 h after collection and stored in the – 80 °C freezer before analysis. Aspartate aminotransferase (AST) and alanine aminotransferase

Tokyo, Japan).

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(ALT) activities were determined using the Fuji Dri-chem 4000i automatic analyzer (Fujifilm Co.,

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2.5. Histopathological examination

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After bleeding, a portion of the livers was dissected and fixed in 10% neutral buffered formalin solution for 2 weeks. The remaining livers were snap-frozen in dry ice and stored at – 80 °C for subsequent biochemical analyses. The fixed tissues were processed routinely, and were embedded in paraffin, sectioned to 4-µm thickness, deparaffinized, and rehydrated using standard techniques. The sections were stained with Harris’ hematoxylin and eosin stain for microscopic examination (Leica DM LB2; Leica, Wetzlar, Germany). All observations were performed manually with a light microscope with ×10 and ×20 objective lenses and a ×100 oil immersion lens in a totally blinded manner.

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2.6. Determination of lipid peroxidation and GSH levels, and antioxidant enzymes activities

Oxidative stress in the livers was assessed by evaluating the levels of malondialdehyde

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(MDA, a marker of lipid peroxidation) and GSH, as well as glutathione reductase (GR), glutathione S-transferase (GST), superoxide dismutase (SOD), and catalase activities. The concentration of MDA was assayed by monitoring thiobarbituric acid reactive substance formation using the method described by Berton et al. (1998). GSH content was measured using

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the method described by Moron et al. (1979). The activities of antioxidant enzymes including GR,

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GST, SOD, and catalase were determined using commercial assay kits (Cayman Chemical, Ann Arbor, MI, USA). Total protein contents were determined using the method by Lowry et al. (1951), using bovine serum albumin as a standard.

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2.7. Preparation of hepatic cytosolic/nuclear fraction

A frozen liver samples were cut into small pieces and washed with ice-cold (10 mM TrisHCl, pH 7.4). Samples were homogenized in a glass-Teflon homogenizer with a suitable

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hypotonic lysis buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, and 0.1 mM EGTA) containing protease inhibitor cocktail and dithiothreitol (DTT) as a reducing agent to lyse the cell

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membranes. The lysate was incubated on ice for 15 min and NP-40 was added to a final concentration of 0.5%, and then centrifuged at 250 g for 15 min. The supernatant (cytosolic fraction) was collected and stored at –80 °C for subsequent analyses. 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 16,000 g for 30 min. The supernatant (nuclear fraction) was stored at –80 °C for western blot analysis.

2.8. Preparation of hepatic microsomes

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Hepatic microsomes were prepared via differential centrifugation, as described previously (Jeong and Yun, 1995). A frozen liver samples were cut into small pieces and homogenized

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using a glass-Teflon homogenizer in 2–5 volumes of 100 mM Tris-HCl (pH 7.4, 4 °C) containing 2 mM phenylmethyl sulfonyl fluoride, pepstatin (12.5 µg/mL), and protease inhibitors. The homogenate was centrifuged at 10,000 g for 30 min (4 °C), and the supernatant was centrifuged at 200,000 g for 60 min (4 °C) to sediment the microsomes. The microsomal pellets were

2.9. Western blot analysis

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suspended in 100 mM Tris-HCl and used for western blot analysis of hepatic CYP2E1.

Equal amounts of proteins (40 µg/well) from each sample were resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene

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difluoride membranes (Whatman, Maidenstone, UK). The membranes were blocked with blocking buffer (5% skim milk) at room temperature for 1 h and incubated with primary antibodies for 18 h at 4 °C. The membranes were washed and incubated with horseradish

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peroxidase-conjugated secondary antibody (1:5,000; Sigma-Aldrich) at room temperature for 1 h. The blots were washed three times and then detected via enhanced chemiluminescence

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(Thermo Scientific, Waltham, MA, USA). The following antibodies and dilutions were used: CYP2E1, iNOS, Cox-2, and TNF-α (1:1,000; Abcam, Cambridge, MA, USA); JNK, phosphorJNK (p-JNK), NF-κB, and β-actin (1:1,000; Cell Signaling Technology, Beverly, MA, USA); α-tubulin and lamin B1 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA). To determine protein expressions, the density of each band was quantified using the TINA 20 Image software (Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany).

2.10. TUNEL and immunohistochemical analysis for evaluation of caspase-3 expression

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The fixed liver tissues were processed routinely, embedded in paraffin, sectioned to 4-µm thickness, deparaffinized, and rehydrated using standard techniques. After incubation with a protein block (Anti-rabbit Specific HRP/DAB IHC Kit; Abcam), the sections were incubated

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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 secondary antibody (biotinylated goat anti-rabbit IgG) was applied followed by streptavidin peroxidase and 3,3-diaminobenzidine (DAB) chromogen with its substrate buffer. Apoptotic

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changes in the livers were detected via TUNEL assay according to the manufacturer’s

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instructions (ApopTag Peroxidase In Situ Apoptosis Detection Kit; Chemicon, Billerica, MA, USA). The slides were visualized with DAB chromogen and counterstained with Harris’ hematoxylin before being mounted. Each slide was examined manually with a light microscope (Leica) with ×10 and ×20 objective lenses and a ×100 oil immersion lens in a double-blinded

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manner.

2.11. Statistical analyses

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The data are expressed as means ± standard deviations (SD) and all statistical comparisons were performed via one-way analyses of variance followed by Tukey’s multiple comparison tests.

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Statistical analyses comparing the treatment groups to the vehicle control group were performed using GraphPad InStat v. 3.0 (GraphPad Software, Inc., La Jolla, CA, USA). Values of p < 0.05 were considered statistically significant.

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3. Results

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3.1. Effects of DADS on AAP-induced acute hepatic injury and histopathological alterations

There were no significant changes in body weights and organ weights in animals treated with AAP and/or DADS during the study period (data not shown). The levels of serum AST

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(360.0 ± 52.33 vs. 72.3 ± 19.89, p < 0.01) and ALT (97.0 ± 13.79 vs. 54.4 ± 14.68, p < 0.01) in the AAP group significantly increased compared to those in the vehicle control group (Fig. 1A

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and 1B). In contrast, serum AST (118.0 ± 33.82, p < 0.01) and ALT (60.7 ± 7.63, p < 0.01) activities in the AAP + DADS group significantly decreased compared to those in the AAP group. On histological examination, the vehicle control and DADS groups showed livers with normal architecture (Fig. 1C and 1D). However, the liver tissues from rats treated with AAP revealed extensive histopathological changes, characterized by mild to moderate hepatocyte

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degeneration/necrosis, vacuolation, sinusoidal dilation, and inflammatory cell infiltration (Fig. 1E). In contrast, the AAP + DADS group showed only sinusoidal dilation, and the incidence and

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severity of histopathological changes decreased compared to those of the AAP group (Fig. 1F).

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3.2. Effects of DADS on AAP-induced hepatic oxidative stress

As presented in Fig. 2, the levels of MDA, an end product of lipid peroxidation,

significantly increased (0.62 ± 0.033 vs. 0.45 ± 0.034, p < 0.01) in the AAP group compared to those in the vehicle control group, whereas GSH content in the livers significantly decreased (3.91 ± 0.36 vs. 5.36 ± 0.34, p < 0.01). In addition, the activities of GR (3.65 ± 0.18 vs. 4.22 ± 0.34, p < 0.01), GST (46.02 ± 5.09 vs. 68.74 ± 4.60, p < 0.01), SOD (14.47 ± 3.39 vs. 29.35 ± 2.96, p < 0.01), and catalase (13.03 ± 2.29 vs. 19.24 ± 1.64, p < 0.01) in the livers from AAPtreated rats were significantly lower than those in the vehicle control group. In the AAP + DADS

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group, however, MDA levels significantly decreased (0.53 ± 0.059, p < 0.01), and GSH content significantly increased (4.94 ± 0.52, p < 0.01) compared to those of the AAP group. Moreover, the activities of GR (4.09 ± 0.27, p < 0.05), GST (69.83 ± 9.08, p < 0.01), SOD (23.01 ± 3.55, p

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< 0.01), and catalase (17.12 ± 1.48, p < 0.01) were significantly higher than those of the AAP group (Fig. 2C-2F).

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3.3. Effects of DADS on AAP-induced activation of JNK and hepatocellular apoptosis

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To determine whether DADS elicits its effects on the JNK-mediated cell death pathway activated by AAP, we evaluated the protein expression of p-JNK/JNK by western blot analysis, hepatocellular apoptosis by TUNEL, and caspase-3 levels via immunohistochemistry. The protein expression of phosphorylated JNK in the AAP group significantly increased compared to that in the vehicle control group. In addition, the p-JNK/JNK ratio in the AAP group significantly

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increased compared to that in the vehicle control group. In contrast, pretreatment with DADS was associated with a significant decrease in p-JNK/JNK ratio compared to that in the AAP group (Fig. 3A and 3B). We have thus confirmed the effects of DADS on AAP-induced

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hepatocellular apoptotic changes. Few TUNEL- and caspase-3-positive cells were identified in the vehicle control and DADS groups (Fig. 3C-3F). However, an increase in the number of

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TUNEL- and caspase-3-positive cells was observed in livers from AAP-treated rats (Fig. 3G and 3H). In contrast, the number of TUNEL- and caspase-3-positive cells markedly decreased in the AAP + DADS group (Fig. 3I and 3J).

3.4. Effects of DADS on hepatic microsomal CYP2E1 expression

CYP2E1 is usually assumed the most active CYP in catalyzing the metabolism of AAP to hepatotoxic NAPQI. We investigated the effects of DADS on the expression of hepatic CYP2E1 protein levels via western blot analysis. Treatment with DADS at 50 and 100 mg/kg/day for 5

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days resulted in a dose-dependent decrease in CYP2E1 expression (Fig. 4A and 4B). Hepatic microsomal CYP2E1 protein levels in the AAP group significantly increased compared to those of the vehicle control group. However, CYP2E1 expression levels in the AAP + DADS group

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decreased significantly compared to those in the AAP group (Fig. 4A and 4C).

3.5. Effects of DADS on AAP-induced NF-κB and inflammatory mediators

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We also confirmed the effects of DADS on NF-κB and inflammatory mediator levels

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induced by AAP. In the AAP treatment group, levels of cytosolic NF-κB markedly decreased, whereas nuclear NF-κB levels increased compared to those of the vehicle control group (Fig. 5A). The ratio of nuclear/cytosolic NF-κB significantly increased compared to that in the vehicle control group (Fig. 5B). In addition, TNF-α, iNOS, and Cox-2 protein levels in the AAP group significantly increased compared to those in the vehicle control group (Fig. 5C-5E). In contrast,

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the AAP + DADS group showed a significant decrease in the ratio of nuclear/cytosolic NF-κB,

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as well as TNF-α, iNOS, and Cox-2 levels compared to those of the AAP group.

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4. Discussion

The aim of the present study was to investigate the protective effects of DADS on AAP-

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induced acute hepatotoxicity and elucidate the molecular mechanisms underlying these protective effects in rats. The results obtained in this study clearly show that pretreatment with DADS effectively attenuates acute hepatic injury by suppressing hepatic CYP2E1 expression,

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enhancing antioxidant enzymes activities, and inhibiting NF-κB activation.

Hepatic damage induced by AAP resulted in elevated levels of serum aminotransferases,

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which act as sensitive indicators (Amacher, 2002). Elevated levels of these enzymes in the circulation are an indication of cellular leakage and loss of functional integrity of the hepatocytes (Heidari et al., 2016). Consistent with previous reports (Nelson, 1990; Noh et al., 2013), AAP treatment caused acute liver damage with marked elevation of serum AST and ALT levels. The acute hepatotoxic effects observed in the livers from AAP-treated rats were consistent with

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corresponding histological changes, including hepatocellular degeneration and necrosis, vacuolation, sinusoidal dilation, and inflammatory cell infiltration. In contrast, pretreatment with DADS effectively suppressed the elevation of serum AST and ALT levels and attenuated the

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histological changes in the liver tissues, indicating that DADS confers protection against AAPinduced acute hepatic injury.

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It is generally accepted that the hepatotoxic mechanism of AAP is mediated by hepatic

microsomal CYPs, in particular by CYP2E1, which is responsible for the metabolism of AAP to the toxic metabolite NAPQI (Chen et al., 2003; Noh et al., 2013; Jiang et al., 2015). In addition, CYP2E1-null mice resist AAP-induced hepatic injury (Chen et al., 2008), suggesting that CYP2E1 plays a major role in the modulation of AAP-induced hepatic injury. In this study, pretreatment with DADS not only reduced CYP2E1 levels by itself, but also suppressed elevation of CYP2E1 expression induced by AAP treatment. According to Teyssier et al. (1999), DADS oxidized to allicin (diallyl thiosulfinate, DADSO) mainly mediated by CYP2E1, and

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allicin could be oxidized to a reactive species (possibly DADSO2) that should interact with the enzyme and should not be released from the active site of the enzyme. Thus, these findings indicate that the ameliorative effects of DADS on acute hepatic injury caused by AAP are closely

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correlated to the suppression of CYP2E1, which may involve a reduction in NAPQI formation. Previous reports have demonstrated that oxidative stress plays an important role in the development of AAP hepatotoxicity (Roberts et al., 1991; Hinson et al., 1998). The GSH enzyme system is an important factor as it initially traps NAPQI by adducting GSH. However, excessive

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formation of NAPQI causes GSH depletion and leads to lipid peroxidation, and depletion of

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antioxidant enzymes (Michael et al., 1999; Hinson et al., 2004). In this study, AAP treatment caused high levels of oxidative stress in hepatic tissues, as evidenced by an increase in the lipid peroxidation product MDA, depletion of GSH, and suppression of antioxidant enzymes activities. However, DADS effectively prevented elevation of MDA, depletion of GSH, and suppression of antioxidant enzymes activities. Many previous studies reported that antioxidants are effective in

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protecting from AAP-induced acute liver damage (Girish et al., 2009; Nagi et al., 2010). DADS enhances GSH levels and acts as a potent inducer of GST, and these effects seems to be due to synergy of thiols modulation of allicin and -SS-group of DADS (Rabinkov et al., 1998; Sheen et

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al., 2001; Okada et al., 2005). Thus, the results of previously mentioned studies and our study suggest that the protective effects of DADS may be due to its ability to suppress the depletion of

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GSH and to enhance the antioxidant enzymes activities. Oxidative stress-mediated JNK activation plays a central role in AAP-induced hepatic injury

(Saito et al., 2010; Shinohara et al., 2010). Studies with AAP overdose clearly demonstrated that prolonged activation of JNK promotes cell death (Zhou et al., 2008; Han et al., 2009). Furthermore, pharmacological inhibition of JNK or silencing expression of the JNK gene markedly attenuate hepatic injury after AAP overdose (Henderson et al., 2007; Hanawa et al., 2008). In this study, a marked elevation of phosphorylated JNK levels was observed in the hepatic tissues from AAP-treated rats. In addition to increased phosphorylation of JNK, AAP treatment caused extensive hepatocellular apoptotic changes. It has been reported that apoptosis

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is triggered by various physiological and pathological stimuli including oxidative damage (Elmore, 2007; Lee and Kang, 2016). However, pretreatment with DADS effectively suppressed phosphorylation of JNK, which was consistent with decreased apoptotic changes in the liver

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tissues. Based on these findings, inhibition of CYP2E1 and enhancement of antioxidant enzymes activities by DADS resulted in a blockade of JNK activation, which in turn enhanced cell survival against the toxic effects of AAP.

Oxidative stress has been shown to activate NF-κB during AAP-induced hepatotoxicity,

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suggesting that this transcription factors may be involved in the inflammatory response (Blazka

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et al., 1996; Dambach et al., 2006). AAP activates Kupffer cells and induces subsequent release of numerous inflammatory mediators and signaling molecules, which play a crucial role in the pathogenesis of AAP-induced hepatotoxicity (Oz and Chen, 2008; Fouad et al., 2012). Our findings confirmed the results of previous studies showing that AAP treatment activated NF-κB and induced inflammatory mediators, including TNF-α, iNOS, and Cox-2 in hepatic tissues. In

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contrast, DADS remarkably reduced the nuclear translocation of NF-κB and subsequent upregulation of inflammatory mediators induced by AAP treatment. In our previous study, DADS markedly suppressed expression of inflammatory mediators or cytokines by inhibiting NF-κB

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activation during carbon tetrachloride-induced hepatic injury, cyclophosphamide-induced hemorrhagic cystitis, and AAP-induced renal injury (Lee et al., 2014; Kim et al., 2015; Ko et al.,

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2017). Thus, these findings suggest that the anti-inflammatory effects of DADS may be due to inhibition of NF-κB activation, thus contributing to the protective effects of DADS against AAPinduced hepatotoxicity.

Collectively, the results of this study indicate that DADS effectively attenuates AAP-

induced acute hepatic injury in rats. The ameliorative effects of DADS may be related to its ability to reduce oxidative stress-mediated JNK activation by inhibiting CYP2E1 or by enhancing antioxidant enzymes activities, and to suppress inflammatory responses by inhibiting NF-κB activation.

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Conflict of Interest The authors declare no conflicts of interest.

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Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-

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2016R1D1A2B04936124) and Grant from the KRIBB Research Initiative Program,

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Republic of Korea (KGM2221723).

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Figure legends

Fig. 1. Effects of DADS on AAP-induced acute hepatic injury and histopathological alterations.

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Serum levels of (A) AST and (B) ALT in male rats treated with DADS (100 mg/kg/day) and/or AAP (4 g/kg). Livers from (C) vehicle control and (D) DADS treated rats showing normal appearance. (E) Liver form AAP-treated rats showing hepatocyte degeneration/necrosis (closed arrowheads), vacuolation (closed arrows), sinusoidal dilation (asterisks), and inflammatory cell **

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infiltration (open arrows). Values are presented as means ± SD (n = 6).

p < 0.01 compared

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with the vehicle control group; †† p < 0.01 compared with the AAP group.

Fig. 2. Effects of DADS on AAP-induced hepatic oxidative stress. Levels of (A) malondialdehyde, a marker of lipid peroxidation, (B) reduced glutathione, (C) glutathione reductase, (D) glutathione S-transferase, (E) superoxide dismutase, and (F) catalase. Values are

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presented as means ± SD (n = 6). ** p < 0.01 compared with the vehicle control group; †, †† p < 0.05, p < 0.01 compared with the AAP group.

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Fig. 3. Effects of DADS on AAP-induced activation of JNK and hepatocellular apoptosis. (A) Western blot analysis of JNK and p-JNK (loading control: β-actin). The bar graphs show (B) the

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relative ratio of p-JNK/JNK in hepatic tissues for vehicle control, DADS-, AAP-, and AAP + DADS-treated groups. Representative photographs of TUNEL assay and immunohistochemical analysis for caspase-3 performed on liver sections of (C, D) vehicle control rats, and rats treated with (E, F) DADS, (G, H) AAP, and (I, J) AAP + DADS (counterstained with hematoxylin). Black arrows point to TUNEL- and caspase-3-positive cells (×100). Bar = 50 µm. Values are presented as means ± SD (n = 6). compared with the AAP group.

**

p < 0.01 compared with the control group;

††

p < 0.01

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Fig. 4. Effects of DADS on hepatic microsomal CYP2E1 expression. (A) Western blot analysis of hepatic CYP2E1 (loading control: β-actin). The bar graphs show relative CYP2E1 protein levels from in livers from rats treated with (B) DADS alone (50 mg/kg/day or 100 mg/kg/day for

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5 days, upper) and (C) DADS (100 mg/kg/day) and/or AAP (4 g/kg, lower). Values are presented as means ± SD (n = 6). ** p < 0.01 compared with the control group; †† p < 0.01 compared with the AAP group.

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Fig. 5. Effects of DADS on AAP-induced NF-κB and inflammatory mediators. Western blot

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analysis of (A) hepatic nuclear/cytosolic NF-κB, TNF-α, Cox-2, and iNOS (loading control: βactin; cytosolic loading control: α-tubulin; nuclear loading control: Lamin B1). The bar graphs show (B) the relative ratio of nuclear/cytoplasmic NF-κB, (C) levels of TNF-α, (D) Cox-2, and (E) iNOS in hepatic tissues for vehicle control, DADS-, AAP-, and AAP + DADS-treated group. Values are presented as means ± SD (n = 6). ** p < 0.01 compared with the control group; †, †† p