Hepatic Protective Effects of S -Allyl-L-Cysteine (SAC) in Rats with Carbon Tetrachloride (CCl 4 ) Induced Liver Injury

S-allyl-L-cysteine (SAC) is an organosulfur compound derived from aged garlic extract (AGE). Studies have reported that AGE possesses bioprotective capacity, including antidiabetic, antimicrobial, antioxidant, and antitumor effects. The present study examined the protective effects of SAC against carbon tetrachloride (CCl 4 ) induced hepatotoxicity in rats. Ten male Wistar rats aged 11 - 12 weeks were randomly divided into two groups (five rats/group) as control and SAC groups. All rats had ad libitum access to water, and the SAC group received water containing SAC intragastrically (200 mg/kg) once daily for five consecutive weeks. In the fifth experimental week, 50% CCl 4 in olive oil (1 mL/kg) was


Introduction
Oxidative damages to hepatic cells are common sequelae of many liver diseases, including hepatic steatosis, chronic hepatitis, cholestasis, and liver cirrhosis [1]. Oxidative stress, a consequence of an imbalance between pro and antioxidants in cells and tissues, is generally defined as excess formation or insufficient removal of damaging molecules, such as reactive oxygen species (ROS). ROS have been implicated in pathophysiological changes in the liver, particularly in viral infections, cholestasis, and alcohol abuse [2]. Excessive alcohol consumption generates ROS in the liver where alcohol is metabolized, disrupting the liver's cellular antioxidant system [3] [4]. ROS can bind to cellular macromolecules, including proteins, lipids, and DNA, leading to physiological dysfunction.
Carbon tetrachloride (CCl 4 ) has been frequently used to produce xenobiotic induced and free radical mediated hepatotoxicity in animal models [5]. Its toxicity requires bioactivation to produce reactive metabolic intermediates using a trichloromethyl radical like 3 CCl  or peroxy trichloromethyl radicals ( 3 CCl OO  ) by mixed function oxidase cytochrome P450 (CYP450) in hepatic microsomes [6]. These free radicals can bind to polyunsaturated fatty acids (PUFAs) and form alkoxy ( R  ) and peroxy radicals ( ROO  ), which serially generate lipid peroxide, damage cell membranes, and disrupt antioxidant enzyme activity and antioxidative substrate content, resulting in hepatic injury [7]. Although several isoforms of CYP450 can metabolize CCl 4 , the focus has been on catalase and cytochrome P4502E1 (CYP2E1), which are ethanol inducible [8] [9]. The histopathological features of CCl 4 induced hepatotoxicity are fatty liver, vacuolar degeneration, inflammation, necrosis, fibrosis, and cirrhosis [10] [11]. Mechanisms for preventing hepatotoxicity induced by oxidative stress involve blocking the chain reaction of oxidation, improving phase II antioxidant enzyme activity, and increasing levels of antioxidant substances.
Garlic (Allium sativum Lynn.) has been used as a spice and as a folk medicine for centuries [12]. In recent years, studies have reported that garlic possesses bioprotective attributes, such as antidiabetic, antimicrobial, antioxidant, antithrombotic, anticancer, and cholesterol lowering effects [13] [14] [15] [16]. The health benefits of garlic derive from a diversity of its constituents, and it is administered in different preparations depending on use. Among its many components, S-allyl-L-cysteine (SAC) is a naturally occurring, water soluble, nontoxic, and odorless organosulfur compound made from aged garlic extract (AGE). AGE is produced by aging for up to 20 months to reduce garlic's harsh taste and odor [17]. Pharmacokinetic studies of SAC, the most abundant organosulfur compound in AGE (6.1 ± 2.7 mg/g dry extract), have revealed that SAC is easily absorbed in the rat gastrointestinal tract and is distributed to the plasma, liver, kidneys, lungs, and heart with a bioavailability of 98% [17]. Furthermore, SAC is 30 fold less toxic than other garlic components, such as allicin and diallyl disulfide, and relatively inexpensive and easy to synthesize [17]. SAC has been reported to prevent amyloidogenesis, atherosclerosis, glycation, and cholesterol synthesis and to reduce mortality via a decreased incidence of stroke [18] [19] [20] [21]. SAC was also found to exert dose dependent inhibition of nuclear factor kappa B (NF-KB) activation in human T lymphocytes (Jurkat cells) induced by tumor necrosis factor α (TNF-α) and hydrogen peroxide (H 2 O 2 ) [22].
SAC also mediates chemopreventive effects in carcinogenesis [23] and has been shown to be the active compound responsible for mitigating oxidative stress [24] [25] [26] [27] [28]. However, evidence of potential hepato-protective effects of SAC remains insufficient. In the present study, we examined the hepatoprotective properties of SAC against CCl 4 induced hepatotoxicity in a rat model.

Chemicals
SAC was obtained from Tokyo Chemical Industry Co., Ltd. (Japan) at a purity of more than 98.0%, as shown by gas chromatography. The water soluble test compound SAC was dissolved in tap water. CCl 4 (Sigma-Aldrich Co., USA) was diluted in olive oil (Sigma-Aldrich Co., USA) to produce 50% CCl 4 . All other chemicals and solvents were of the highest grade commercially available.

Experimental Animals
Male Wistar rats (11 -12 weeks old) weighing 353.06 ± 22.09 g were housed in conventional cages with ad libitum access to water and a standard rodent chow diet. Animals were housed in a controlled environment at a temperature of 22˚C ± 3˚C and a relative humidity of 50% ± 10% with a 12/12h light/dark cycle. Animals were randomly divided into two groups: the control group received 50% CCl 4 in olive oil (1 mL/kg) intraperitoneally three times in the fifth week; the SAC group was treated intragastrically with water containing SAC (200 mg/kg) once daily for the first four weeks and the same amount of CCl 4 in olive oil was administered at the same frequency in the fifth (Figure 1). Animals were sacrificed at 24 hours after the last CCl 4 dose and liver tissues were excised for histopathological, immunohistochemical and antioxidant analyses. All experimental procedures were performed in accordance with the National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals.

Determination of Antioxidant Enzymes
Superoxide dismutase (SOD) activity was measured according to the method described by Beauchamp and Fridovich [29]. An adequate amount of liver supernatant was mixed with the reaction mixtures containing 0.1 mM ethylenediaminetetraacetic acid (EDTA), 25 mM nitroblue tetrazolium (NBT), 0.1 mM xanthine, 50 mM sodium carbonate buffer (pH 10.2), and distilled water to a final volume of 3 mL. 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 was defined as the amount of enzyme required to reduce NBT by 50%. The specific activity of SOD is expressed as unit/mg for each supernatant. Catalase activity was measured according to the method described by Aebi [30]. One unit of catalase was defined as the amount of enzyme required to decompose 1.0 μM of H 2 O 2 in 1 min. The reaction was initiated by the addition of 1.0 mL of freshly prepared 20 mM H 2 O 2 . The decomposition rate of H 2 O 2 was measured spectrophotometrically at 240 nm at 1 min. Enzyme activity is expressed as unit/mg.

Determination of Glutathione Levels
Glutathione (GSH) and glutathione disulfide (GSSG) were prepared in 0.1 M sodium phosphate containing 0.005 M EDTA buffer (pH 8.0) and kept on ice until used. O-phthalaldehyde (OPT) solution was prepared in reagent-grade absolute methanol just prior to use. Liver samples were homogenized on ice with a homogenizer in a solution containing 3.75 mL of phosphate EDTA buffer and 1 mL of 25% H 3 PO 4 for protein precipitation. Total homogenate was centrifuged at 4˚C at 100,000 g for 20 min, and the supernatant was then decanted to assess GSH and GSSG levels. Determination of GSH values was performed as described by Hissin and Hilf [31]. An aliquot of 4.5 mL of phosphate-EDTA buffer (pH 8.0) was added to 0.5 mL of the supernatant for a final assay mixture of 2.0 mL containing 100 μL of diluted tissue supernatant, 1.8 mL of phosphate EDTA buffer, and 100 μL of OPT solution containing 100 μg of OPT. After mixing and incubation at room temperature for 15 min, the solution was transferred to a cuvette. Fluorescence at 420 nm was assessed, with activation at 350 nm. In the GSSG assay, a 0.5 mL portion of the supernatant generated above was incubated at room temperature with 200 μL of 0.04 M N-ethylmaleimida (NEM) for 30 min to interact with GSH in the tissues. An aliquot of 4.3 mL of 0.1 N NaOH was added to the mixture, and a 100 µL portion thereof was used to measure GSSG as described above, using 0.1 M NaOH as a diluent rather than phosphate-EDTA buffer. Results are expressed as nM/mg.

Determination of Lipid Peroxidation
Liver homogenate was assayed for lipid peroxidation level using a lipid peroxidation (LPO)-586 assay kit (OxisResearch, Portland, OR, USA) according to the manufacturer's instructions. This assay is based on the reaction of a chromogenic reagent, N-methyl-2-phenyl indole, with malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) at 45˚C. One molecule of either MDA or HAE reacts with two molecules of chromogenic reagent to yield a stable chromophore with maximal absorbance at 586 nm. Aliquots of 200 µL of samples were mixed with 650 µL of chromogenic reagent and 150 µL of methanesulfonic acid. Preparations were incubated at 45˚C for 60 min and then centrifuged at 15,000 g for 10 min. The supernatant was transferred to a cuvette and its absorbance was measured at 586 nm. MDA and MDA + HAE values were calculated using an MDA standard curve. The detection limit for the assay was 0.1 nmol/mL in the final reaction medium. Results are expressed as nM/mg.

Determination of ROS Generation
Intracellular production of ROS was measured with 2',7'-dichlorofluorescein diacetate (H 2 DCF-DA) (Molecular Probes, Eugene, OR, USA). This nonpolar compound is converted to the polar, membrane-impermeable derivative H 2 DCF by esterases upon cellular uptake. H 2 DCF is nonfluorescent but is rapidly oxidized to the highly fluorescent DCF by intracellular H 2 O 2 and other peroxides [32] [33]. Stocks of H 2 DCF-DA were made in absolute ethanol at a concentration of 12.5 mM and stored at −80˚C in the dark under argon. The 25 μM H 2 DCF-DA was added to the liver homogenate at a final concentration of 250 μM. After 30 min incubation, the homogenate was collected in a microcentrifuge, and the supernatant was removed and diluted 50-fold. Fluorescence was measured with excitation and emission wavelengths of 485 nm and 535 nm, respectively. Results are expressed as fluorescence/min/mg.

Histopathology
Liver tissue blocks were fixed in 10% neutral buffered formalin and processed routinely. Paraffin embedded liver sections were cut into 4 -5 µm slices. Liver tissue blocks were then stained with hematoxylin and eosin (H&E) for histopathological analysis.

Immunohistochemistry
Liver sections were deparaffinized in xylene and toluene, rehydrated in a series of graded alcohol concentrations, incubated in 3% hydrogen peroxide (H 2 O 2 ) in methanol for 30 min and microwaved at 750 W for 10 min in 10 mmol/L citrate buffer (pH 6.0). Tissue sections were washed with PBS. After blocking with rabbit serum (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temper-ature, sections were immunostained using anti-catalase and anti-cytochrome P4502E1 (CYP2E1) antibodies (Chemicon International, Inc., USA) at a dilution of 1:800. Antigen antibody complexes were visualized with an avidin biotin peroxidase complex solution using an ABC kit (Vector Laboratories, Burlingame, CA, USA). The sections were subsequently rinsed in distilled water and counterstained with Mayer's hematoxylin.

Histomorphometry
Histomorphometry was performed with a computerized analysis system (Image-Pro Plus, Media Cybernetics, Silver Spring, MD). Areas of the tissue sections to be measured were captured by a microscope connected to the system. Calibration was performed according to the instructions accompanying the software. The area of hepatic steatosis on H&E slides and the distribution area of CYP2E1 in immunohistochemistry were measured. Results are expressed as percentages of stained area.

Statistical Analysis
All data are presented as mean ± SD. Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by Student's t-test. A p-value of less than 0.05 was considered statistically significant.

Body Weight of Experimental Rats
The rats in the SAC group did not show abnormal behavior, such as decreased water intake or food consumption, and their body weights did not change significantly (p < 0.05) from the control group prior to CCl 4 treatment. After CCl 4 dosing, rats in the control group showed a slight reduction in body weight, although this reduction was not significantly different (p > 0.05) from the SAC group ( Figure 2).

SAC Effects on Antioxidant Enzymes
We measured superoxide dismutase (SOD) and catalase activities as indicators of oxidative stress. Hepatic SOD activity was 0.41 ± 0.09 unit/mg in the control group, while that in the SAC group increased to 0.46 ± 0.10 unit/mg. Catalase activity in liver tissue in the control group was 1.71 ± 0.52 unit/mg, but treatment with SAC resulted in an increase thereof to 3.69 ± 0.26 unit/mg (p < 0.05) over the control group (Figure 3).

SAC Effects on Glutathione Levels
Glutathione is the first line of defense against free radicals. Levels of glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG ratio, and total liver glutathione (GSH + GSSG) are shown in Figure 4. GSH in the control group was 1.37 ± 0.24 nM/mg, while that in the SAC group increased to 2.14 ± 0.38 nM/mg (p < 0.05). GSSG was 0.94 ± 0.18 nM/mg in controls and decreased to 0.84 ± 0.10 nM/mg in the SAC group (p < 0.05). The GSH/GSSG ratio of 1.83 ± 0.59 in the control group was lower than the 2.75 ± 0.56 in the SAC group. Total glutathione (GSH + GSSG) in the control group was 2.31 ± 0.20 nM/mg, while that in the SAC group increased to 2.98 ± 0.35 nM/mg (p < 0.05) ( Figure 4).

SAC Effects on Lipid Peroxidation and ROS Generation
Lipid peroxidation is critically implicated in CCl 4 -induced hepatotoxicity. Malondialdehyde (MDA) and 4-hydroxyalkenals (HAE), end products of lipid peroxidation, are common markers thereof. MDA and MDA + HAE contents in the SAC group were lower than those in the control group ( Figure 5), suggesting that CCl 4 -induced oxidative stress was slightly mitigated by SAC treatment. ROS generation was measured to examine SAC effects on radical scavenging during hepatic CCl 4 toxicity. Figure 5 shows the amount of fluorescent dichlorofluorescein (DCF) in the liver tissues of rats from the SAC and control groups as an index of ROS formation. DCF content in the SAC group was slightly diminished compared to the control group, indicating that SAC attenuated CCl 4 induced free radicals.

Histopathological Change after SAC Treatment
Histopathological changes in liver sections after CCl 4 administration were assessed in control and SAC groups ( Figure 6). Typical intense fatty hepatocytes surrounding central veins were observed in CCl 4 -treated rats (Figure 6(A) and Figure 6(C)). Centrilobular necrosis, inflammatory cell infiltration, and ballooning degeneration were also observed ( Figure 6(A) and Figure 6(C)). These hepatic histological changes were markedly reduced by treatment with SAC ( Figure 6(B) and Figure 6(D)). SAC administration also significantly decreased lipid droplets and areas of lipid laden cells.

Immunohistochemical Changes after SAC Treatment
Immunohistochemical changes to CYP2E1 levels were observed after CCl 4 administration in liver sections in both control and SAC groups. CYP2E1 expression in the control group was found at the injured hepatocytes around central veins in a pattern similar to hepatic steatosis (Figure 7(A) and Figure 7(C)), demonstrating that histological expression of CYP2E1 in the SAC group was repressed in CCl 4 induced hepatocyte damage (Figure 7(B) and Figure 7(D)).

Hepatic Histomorphometry after SAC Treatment
The percentages of hepatic steatosis area and CYP2E1 distribution area are shown in Figure 6 and Figure 7. The SAC group showed less hepatic steatosis and less hepatic CYP2E1 than the control group (p < 0.05, both) ( Figure 6 and Figure 7).

Discussion
Studies have shown that various components of garlic preparations exhibit a wide range of physiological, biochemical, and pharmacological effects [13] [16].
Garlic (Allium sativum Lynn.) has long been considered a valuable spice by many cultures and is still used today for its medicinal properties. Aged garlic extract (AGE) is produced by aging for up to 20 months, reducing garlic's harsh taste and odor [17]. SAC is a major water soluble organosulfur compound derived from AGE whose bioprotective capacity has been described in many reports. Its bioavailability has been well established in animal models as it has been shown to be easily absorbed and evenly distributed [17]. CCl 4 treatment has been shown to cause high levels of oxidative damage by significant increases in lipid peroxidation products, ROS generation, and hepatotoxic features, and by significant decreases in GSH concentration and antioxidant enzyme activity. In this study, treatment with SAC was associated with a significant protective effect against CCl 4 induced acute hepatic injury in rats. Superoxide dismutase (SOD) and catalase are major antioxidant enzymes that catalyze ROS in most cells; these enzymes are critical for the elimination of ROS via reduction of xenobiotics in liver tissue and are easily inactivated by lipid peroxide or ROS. SOD is an extremely effective antioxidant enzyme responsible for catalytic dismutation of highly reactive and potentially toxic superoxide radicals ( 2 O −  ) to hydrogen peroxide (H 2 O 2 ) [34]. SOD is most abundant in the liver and catalyzes H 2 O 2 to oxygen (O 2 ) and water (H 2 O) [35]. In this study, SOD and catalase activities were significantly increased in the SAC treated group (Figure 3), suggesting that SAC could restore or activate SOD and catalase enzymes in CCl 4 damaged liver tissue.
GSH (γ-glutamylcysteinylglycine) is an intracellular and extracellular nonenzymatic antioxidant that acts in conjunction with enzymatic processes to reduce H 2 O 2 and lipid hydroperoxides [36] [37]. It has also been theorized to contribute to the conservation of normal cell structure and function via reduction and detoxification reactions. The reduced form of GSH helps detoxify reactive, toxic metabolites of CCl 4 [5]. In the present study, treatment with SAC was shown to significantly ameliorate CCl 4 induced depletion of reduced GSH and elevation of GSSG, an oxidized form of GSH, in rat livers (Figure 4). Total GSH + GSSG levels were significantly elevated and GSH/GSSG ratios were increased in the SAC treated group compared with controls ( Figure 4). Glutathione results pointed to both the restoration of GSH and elimination of GSSG by antioxidant SAC effects in CCl 4 treated rats. Specially, thiol group-containing agents such as cysteine (Cys), thioproline, 2-mercaptopropionylglycine, captopril, and N-acetylcysteine have been reported to possess antioxidant capacity based on an association with increased amounts of reduced GSH [38]- [44]. SAC possesses a Cys structure, but without the SH residue, and may exert its protective activity by supplying Cys to hepatocytes.
Lipid peroxidation is an ROS mediated mechanism implicated in the pathogenesis of liver injuries in experimental animals and humans [45] [46]. MDA and HAE are major reactive aldehydes that present during the decomposition of PUFAs in biological membranes and are reliable markers of lipid peroxidation [6]. Hepatic contents of MDA and HAE are often assessed as indicators of liver tissue damage involving a series of chain reactions. Lipid peroxidation of hepatocyte membranes is a principal cause of CCl 4 induced hepatotoxicity that is mediated by the production of free radical derivatives of CCl 4 [5] [29]. In this study, amounts of hepatic MDA and MDA + HAE in the SAC group were reduced compared with the control group ( Figure 5), suggesting that treatment with SAC could reduce hepatic lipid peroxidation induced by CCl 4 in rats. ROS production was examined to evaluate the scavenging of free radicals by SAC and decreased concentrations of hepatic ROS in the SAC group indicate that SAC could scavenge free radicals produced by CCl 4 in rats ( Figure 5).
Hepatocellular steatosis, inflammation, necrosis, and vacuolar degeneration induced by CCl 4 were more pronounced in the control group compared with the SAC group ( Figure 6). Liver tissue damaged by CCl 4 was accompanied by CYP2E1 expression, as evidenced by immunohistochemistry, in a pattern similar to hepatic fatty changes around central veins in the control group; this pattern was less prevalent in the SAC treated group (Figure 7) and could indicate antioxidant action by SAC. Histomorphometry of liver sections with H&E staining and immunostained for CYP2E1 revealed that SAC treatment significantly reduced the average percentages of steatosis and CYP2E1 distribution areas ( Figure 6 and Figure 7). Although a previous study found that SAC did not inhibit hepatic CYP2E1 protein expression nor CYP2E1 mRNA levels [47], in this study, SAC treatment was associated with significantly reduced immunohistochemical CYP2E1 expression in liver tissue compared to controls ( Figure 6 and Figure 7). This activity suggests SAC as a potential inhibitor of P4502E1.

Conclusion
In conclusion, this study demonstrated that SAC treatment was associated with the alleviation of CCl 4 induced oxidative liver injury, accompanied by an elevation in antioxidant enzyme expression and action, and increased levels of reduced glutathione (GSH) together with decreased oxidized glutathione (GSSG) content, inhibition of lipid peroxidation, scavenging of free radicals, and CYP2E1 mediated hepatic steatosis induced by CCl 4 . Taken together, our findings indicate that SAC could be an effective hepatoprotective dietary supplement. Future studies are needed, however, to explore the potential of SAC to reduce hepatotoxicity in humans.