Food and Chemical Toxicology 48 (2010) 1644–1653

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Food and Chemical Toxicology

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Protective effects of silica hydride against carbon tetrachloride-inducedhepatotoxicity in mice

Yu-Wen Hsu a,1, Chia-Fang Tsai b,1, Wen-Chen Chuang c, Wen-Kang Chen d, Yung-Chyuan Ho a,Fung-Jou Lu e,f,*

a Department of Applied Chemistry, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Rd., Taichung City 402, Taiwanb School of Occupational Safety and Health, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Rd., Taichung City 402, Taiwanc Institute of Veterinary Pathobiology, National Chung Hsing University, No. 250, Kuo Kuang Rd., Taichung City 402, Taiwand National Tainan Institute of Nursing, No. 78, Sec. 2, Minzu Rd., Tainan City, Taiwane Institute of Medicine, College of Medicine, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Rd., Taichung City 402, Taiwanf Department of Nutrition, Chung Shan Medical University Hospital, No. 110, Sec. 1, Jianguo N. Rd., Taichung 402, Taiwan

a r t i c l e i n f o

Article history:Received 13 November 2009Accepted 23 March 2010

Keywords:Hepatoprotective effectsSilica hydrideCarbon tetrachlorideAntioxidant

0278-6915/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.fct.2010.03.039

Abbreviations: ALP, alkaline phosphatase; ALT, alaspartate aminotransferase; CCl4, carbon tetrachloridglutathione peroxidase; GSH-Rd, glutathione reductaMDA, malondialdehyde; ROS, reactive oxygen speciesTBA, Thiobarbituric acid; TG, triglyceride.

* Corresponding author at: Institute of Medicine,Shan Medical University, No. 110, Sec. 1, Jianguo N. RdTel.: +886 4 24730022x11872; fax: +886 4 23248189

E-mail address: fjlu@csmu.edu.tw (F.-J. Lu).1 Both authors contribute equally.

a b s t r a c t

The protective effects of MegaHydrate™ silica hydride against liver damage were evaluated by its atten-uation of carbon tetrachloride (CCl4)-induced hepatotoxicity in mice. Male ICR mice were orally treatedwith silica hydride (104, 208 and 520 mg/kg) or silymarin (200 mg/kg) daily, with administration of CCl4

(1 mL/kg, 20% CCl4 in olive oil) twice a week for eight weeks. The results showed that oral administrationof silica hydride significantly reduced the elevated serum levels of alanine aminotransferase (ALT), aspar-tate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride (TG), and cholesterol and the level ofmalondialdehyde (MDA) in the liver that were induced by CCl4 in mice. Moreover, the silica-hydridetreatment was also found to significantly increase the activities of superoxide dismutase (SOD), catalase,and glutathione peroxidase (GSH-Px), as well as increase the GSH content, in the liver. Liver histopathol-ogy also showed that silica hydride reduced the incidence of liver lesions induced by CCl4. The resultssuggest that silica hydride exhibits potent hepatoprotective effects on CCl4-induced liver damage in mice,likely due to both the increase of antioxidant-defense system activity and the inhibition of lipidperoxidation.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Liver diseases often progress from subclinical icteric hepatitis tonecroinflammatory hepatitis, fibrosis, cirrhosis, and hepatocellularcarcinoma (Vitaglione et al., 2004; Cristovao et al., 2007). Docu-mented evidence has been reported that reactive oxygen species(ROS), including singlet oxygen, superoxide, and hydroxyl radicals,are known to play an important role in liver-disease pathologyand progression (Vitaglione et al., 2004), and ROS have been provento be associated with carbon tetrachloride (CCl4)-induced

ll rights reserved.

anine aminotransferase; AST,e; GSH, glutathione; GSH-Px,se; H2O2, hydrogen peroxide;; SOD, superoxide dismutase;

College of Medicine, Chung., Taichung City 402, Taiwan..

hepatotoxicity (Slater and Sawyer, 1971). Hepatotoxins, such asethanol, acetaminophen, and CCl4, cause liver damage that is char-acterized by varying degrees of hepatocyte degeneration and celldeath (Wu et al., 1999). CCl4 has been commonly used as a hepato-toxin in experimental hepatopathy (Hsu et al., 2008; Geetha et al.,2008) because experimentally induced cirrhotic response in ani-mals by CCl4 has been shown to be superficially similar to humancirrhosis of the liver (Taira et al., 2004; Lee et al., 2007; Rudnickiet al., 2007). Therefore, CCl4-induced hepatic injury has been exten-sively used in animal models to evaluate the therapeutic potential ofdrugs and dietary antioxidants. The metabolites of CCl4, trichloro-methyl free radicals, are capable of binding to DNA, lipids, proteinsor carbohydrates and eventually lead to membrane-lipid peroxida-tion and finally to cell death (Recknagel et al., 1989; Weber et al.,2003; Basu, 2003). Many studies have reported that antioxidantsupplements are efficacious in preventing oxidative-stress-relatedliver pathologies due to particular interactions and synergisms(Bhathal et al., 1983; Vitaglione et al., 2004). Additionally, one ofthe major defense mechanisms for the prevention and treatmentof liver damage includes reducing the production of reactive

Y.-W. Hsu et al. / Food and Chemical Toxicology 48 (2010) 1644–1653 1645

metabolites using antioxidants (Wu et al., 1999; Bansal et al., 2005).Antioxidants appear to act against disease processes by elevatingthe levels of endogenous antioxidant enzymes, such as superoxidedismutase (SOD), catalase, and glutathione peroxidase (GSH-Px),thus decreasing lipid peroxidation (Aruoma, 1994; Wu et al.,1999; Bansal et al., 2005).

Silica hydride is a novel silica-based polymeric colloid that hasinterstitially enclosed hydride anions. The silica hydride synthesisprocess appears to cluster the organosilicate subunits into hydro-gen-bonded aggregates (Stephanson and Flanagan, 2003a,b). Inan aqueous environment, silica hydride is characterized by the fea-tures of stable release of the hydride ion for an extended length oftime, slightly alkaline pH, and low oxidation–reduction potential.Recent publications have shown that silica hydride is nontoxicand safe as a dietary supplement (Carlise, 1982; Purdy-Lloydet al., 2001; Stephanson and Flanagan, 2004a). Several studies havereported that silica hydride acts as an effective antioxidant that hasthe ability to scavenge free radicals and thus protect mammaliancells against strong oxidative stress (Stephanson et al., 2002;Stephanson and Flanagan, 2003a). Stephanson and Flanagan(2003b) have also shown that silica hydride decreased oxidativestress and effectively protected against ROS-induced pathologicalchanges. In an in vitro study, silica hydride increased the ratio of[NADH]/[NAD+] in the mitochondria of Chinese hamster ovary cellsas a function of increased cellular ATP production (Stephanson andFlanagan, 2004). In addition, a clinical study reported (Purdy-Lloydet al., 2001) that a silica mineral supplement effectively decreasedblood lactate concentrations after exercise. Altogether, these stud-ies suggest that silica hydride is an incredibly effective radicalscavenger and helps reduce oxidative stress due to its minimal sizeand high reduction potential.

Based on the excellent antioxidant potential of silica hydridefound in vitro, it was of interest to evaluate its protective effectsin vivo. However, it is not known if silica hydride can prevent oralleviate liver injury induced by CCl4, and the mechanisms by whichsilica hydride may protect against CCl4-induced hepatotoxicity areunclear. In the present study, male ICR mice were orally treatedwith silica hydride or silymarin (as a standard drug) daily accompa-nied by CCl4 administration twice a week for 8 weeks. Hepatic GSHand MDA levels, as well as serum activities of AST, ALT, and ALP andSOD, catalase, GSH-Px, and GSH-Rd levels in liver tissues, weremeasured to monitor liver injury. The extent of CCl4-induced liverinjury was also analyzed through histopathological observations.

2. Materials and methods

2.1. Chemicals

Silymarin was obtained from the Sigma Chemical Co. All other chemicals andreagents used were obtained from local sources and were of analytical grade.

2.2. Material

Commercially available preparations of MegaHydrate™ silica hydride from NewI Ten Rin Enterprise Co., Ltd. (Changhua City, Taiwan) were dissolved in distilledwater prior to use. The quality of silica hydride was described and provided bythe company. In accordance with the company-provided data, the constituents inthe silica hydride powder included potassium citrate, silica, potassium carbonate,oleic acid, vitamin C, and negative hydrogen ions.

2.3. Animals

Male ICR mice (20 ± 2 g) were obtained from the Animal Department of BioLAS-CO Taiwan Company and were quarantined and allowed to acclimate for a weekprior to experimentation. The animals were handled under standard laboratoryconditions of a 12-h light/dark cycle in a temperature- and humidity-controlledroom. Food and water were available ad libitum. Our Institutional Animal Careand Use Committee approved the protocols for the animal study, and the animalswere cared for in accordance with the institutional ethical guidelines.

2.4. Treatment

The animals were randomly divided into seven groups each consisting of tenmice. Group I served as the normal control and was given normal saline daily fora period of 8 weeks. Group II served as the vehicle control and was given olive oildaily for a period of 8 weeks. For inducing hepatotoxicity (in vivo), animals ofGroups III, IV, V, VI, and VII were orally administered 1 mL/kg body weight of carbontetrachloride (20% CCl4 in olive oil) twice a week for a period of eight weeks. AfterCCl4 intoxication, Group III served as the CCl4 control. Group IV served as the posi-tive control and was orally administered silymarin (200 mg/kg) daily for a period ofeight weeks. Groups V, VI and VII were orally administered silica hydride dissolvedin olive oil at doses of 104, 208, and 520 mg/kg, respectively, daily for a period of8 weeks. At the end of the experiment, the animals were sacrificed by cervical dis-location. Blood was collected into heparinized tubes (50 U/mL). Liver samples weredissected out and washed immediately with ice-cold saline to remove as muchblood as possible, and then they were immediately stored at �70 �C until analysis.An extra sample of each liver was excised and fixed in a 10% formalin solution forhistopathological analysis.

2.5. Measurement of serum ALT, AST, cholesterol and triglyceride (TG) levels

Liver damage was assessed by the estimation of serum activities of alanine ami-notransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase(ALP) using commercially available test kits from Randox Laboratories Ltd. (UK).The results were expressed as units/liter (IU/L). In addition, the serum levels of cho-lesterol and TG were estimated in the experimental animals using kits produced byRandox Laboratories Ltd. (UK).

2.6. Measurement of SOD, catalase, GSH-Px, GSH-Rd, and GSH in liver homogenate

Liver homogenates were prepared in cold Tris–HCl (5 mmol/L, containing2 mmol/L EDTA, pH 7.4) using a homogenizer. The unbroken cells and cell debriswere removed by centrifugation at 10,000g for 10 min at 4 �C. The supernatantwas used immediately for the assays for SOD, catalase, GSH-Px, GSH-Rd, andGSH. The activities of all of these enzymes were determined following the instruc-tions in the Randox Laboratories Ltd kit.

2.7. Measurement of lipid peroxidation

The quantitative measurement of lipid peroxidation was done by measuring theconcentration of thiobarbituric acid reactive substances (TBARS) in liver using themethod of Berton et al. (1998). The amount of malondialdehyde (MDA) formedwas quantitated by reaction with thiobarbituric acid (TBA) and used as an indexof lipid peroxidation. In brief, samples were mixed with TBA reagent consisting of0.375% TBA and 15% trichloroacetic acid in 0.25 N hydrochloric acid. The reactionmixtures were placed in a boiling-water bath for 30 min and centrifuged at1811g for 5 min. The supernatant was collected, and its absorbance was measuredat 535 nm with an ELISA plate reader (Quant, BioTek, Vermont, USA). The resultswere expressed as nmole/mg protein using the molar extinction coefficient of thechromophore (1.56 � 10�5 M�1 cm�1).

2.8. Assessment of liver damage

The livers were preserved in neutral buffered formalin and were processed forparaffin embedding, following the standard microtechniques. Four- to five-micronsections of livers, stained with hematoxylin and eosin for the estimation of hepato-cyte necrosis and vacuolization, as well as Masson trichrome stain and Sirius redstain for hepatocyte fibrosis, were observed under the microscope (IX71S8F-2,Olympus, Tokyo, Japan).

2.9. Statistical analysis

All values are expressed as means ± SD. Comparison between any two groupswas performed using one-way analysis of variance (ANOVA) followed by Tukey’smultiple comparison test using a computer program SPSS. Statistically significantdifferences between groups were defined as p < 0.05.

3. Results

3.1. Effect of silica hydride on CCl4-induced hepatotoxicity

The serum biochemical data for the evaluation of CCl4-inducedhepatotoxicity are summarized in Table 1. There was a significantelevation of serum ALT, AST, and ALP activities in the CCl4-treatedgroup as compared to the vehicle control (p < 0.05), indicatingCCl4-induced damage to the hepatic cells. However, treatment

Table 2Effects of silica hydride on liver SOD, catalase, GSH-Px, and GSH-Rd in CCl4-intoxicated mice.

Design of treatment SOD (Units/mgprotein)

Catalase (Units/mgprotein)

GSH-Px (nmoleNADPH/min/mg protein)

GSH-Rd (nmoleNADPH/min/mg protein)

Normal control 9.45 ± 0.59b 20.15 ± 1.12b 286.11 ± 22.89b 4.97 ± 0.48b

Vehicle control 8.93 ± 0.67b 19.93 ± 2.08b 289.09 ± 35.06b 4.99 ± 0.73b

CCl4 control (1 mL/kg) 6.42 ± 0.62a,c 12.24 ± 1.43a,c 185.13 ± 22.60a,c 4.31 ± 0.66a

Silymarin (200 mg/kg) + CCl4 8.56 ± 0.71b 16.40 ± 1.52a,b 240.40 ± 26.88b 4.41 ± 0.32Silica hydride (104 mg/kg) + CCl4 8.20 ± 0.37b 13.85 ± 1.50a,c 209.32 ± 22.58a 4.36 ± 0.65Silica hydride (208 mg/kg) + CCl4 8.74 ± 0.29b 15.15 ± 1.75b 268.15 ± 37.15b 4.79 ± 0.70Silica hydride (520 mg/kg) + CCl4 9.27 ± 0.77b 17.31 ± 1.35b 256.57 ± 29.28b 4.45 ± 0.81

Values are mean ± S.D., n = 10.a p < 0.05 compare with vehicle control.b p < 0.05 compare with CCl4.c p < 0.05 compare with silymarin.

Table 1Effects of silica hydride on serum ALT, AST, ALP, TG, and cholesterol in CCl4-intoxicated mice.

Design of treatment ALT (Units/L) AST (Units/L) ALP (Units/L) TG (mg/dl) Cholesterol (mg/dl)

Normal control 13.04 ± 1.20b,c 11.87 ± 1.63b,c 46.18 ± 8.56b 138.56 ± 10.82b,c 273.44 ± 26.13b

Vehicle control 15.13 ± 2.80b,c 13.39 ± 2.70b,c 48.39 ± 6.94b 147.77 ± 8.58b,c 274.37 ± 27.17b

CCl4 control (1 mL/kg) 213.13 ± 25.34a,c 65.13 ± 13.69a,c 70.29 ± 9.13a,c 236.43 ± 35.77a,c 336.04 ± 33.69c

Silymarin (200 mg/kg) + CCl4 101.85 ± 15.31a,b 51.04 ± 12.54a,b 57.78 ± 5.77b 168.66 ± 10.57a,b 293.13 ± 25.96b

Silica hydride (104 mg/kg) + CCl4 158.89 ± 19.52a,b,c 67.05 ± 7.51a,c 59.25 ± 6.98b 213.75 ± 23.37a,c 304.46 ± 35.99Silica hydride (208 mg/kg) + CCl4 135.84 ± 26.15a,b,c 56.45 ± 9.65a 52.07 ± 8.05b 213.20 ± 26.55a,c 290.90 ± 31.92b

Silica hydride (520 mg/kg) + CCl4 121.06 ± 27.46a,b 41.73 ± 8.40a,b 59.80 ± 4.44b 194.64 ± 20.54a,b 284.58 ± 38.01b

Values are mean ± S.D., n = 10.a p < 0.05 compare with vehicle control.b p < 0.05 compare with CCl4.c p < 0.05 compare with silymarin.

Table 3Effects of silica hydride on liver GSH and MDA-TBA in CCl4-intoxicated mice.

Design of treatment GSH (lmole/gwet weight)

MDA-TBA (nmole/mgprotein)

Normal control 12.40 ± 0.91b,c 6.47 ± 0.87b

Vehicle control 13.03 ± 0.84b,c 5.26 ± 0.83b

CCl4 control (1 mL/kg) 8.89 ± 0.66a,c 10.55 ± 0.58a,c

Silymarin (200 mg/kg) + CCl4 15.58 ± 0.99a,b 5.11 ± 0.54b

Silica hydride (104 mg/kg) + CCl4 11.65 ± 1.18b,c 9.59 ± 1.07a,c

Silica hydride (208 mg/kg) + CCl4 12.46 ± 1.28b,c 7.32 ± 0.82a,b,c

Silica hydride (520 mg/kg) + CCl4 13.99 ± 0.83b,c 7.59 ± 1.06a,b,c

Values are mean ± S.D., n = 10.a p < 0.05 compare with vehicle control.b p < 0.05 compare with CCl4.c p < 0.05 compare with silymarin.

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with silica hydride at a dose of 520 mg/kg significantly decreasedthe percentages of ALT, AST, and ALP by 43%, 35%, and 14%, respec-tively, compared with the CCl4-treated group. Furthermore, therewas a significant reduction (p < 0.05) of lipid parameters (TGand cholesterol) in serum, up to 17% in the silica hydride-treatedgroup at a dose of 520 mg/kg, but silica-hydride treatment at adose of 104 mg/kg did not significantly affect lipid parameterscompared to the CCl4-treated group. However, at the studied doses,silica hydride did not show a dose-dependent protective effectagainst CCl4-induced hepatotoxicity. The positive control treat-ment, silymarin at a dose of 200 mg/kg, also reduced the serumlevels of ALT, AST, and ALP (52%, 21% and 17%, respectively). Theseresults suggested the possibility that silica hydride provides pro-tection against CCl4-induced liver injury.

3.2. Hepatic antioxidant enzyme activities

SOD, catalase, GSH-Px and GSH-Rd were measured as an indexof antioxidant status of tissues. Significantly lower activities ofliver SOD, catalase, GSH-Px, and GSH-Rd were observed in CCl4-treated group as compared to the vehicle-control group. Therewere significant increases in SOD, catalase, and GSH-Px activitiesin the silica hydride-treated groups at doses of both 208 and520 mg/kg compared to the CCl4-treated control group (p < 0.05).The group treated with silica hydride at a dose of 104 mg/kgshowed a significantly increased (p < 0.05) SOD activity (by 21%),but the catalase and GSH-Px activities were not significantlyaffected compared to the CCl4-treated group. The GSH-Rd activitywas not different between the CCl4-treated group and the each sil-ica-hydride treatment groups (Table 2).

3.3. Effect of silica hydride on GSH level and lipid peroxidation

GSH acts as a nonenzymatic antioxidant in the detoxificationpathway that reduces the reactive toxic metabolites of CCl4. The

hepatic GSH levels in the mouse livers are shown in Table 3. Treat-ment with CCl4 significantly decreased the GSH levels in the liveras compared to the vehicle-control group. In contrast to the CCl4-treated group, mice treated with silica hydride at doses of 104,208, and 520 mg/kg showed significantly increased GSH levels,by 23%, 28%, and 36%, respectively; similar results were also foundwith the dose of 200 mg/kg of silymarin.

The MDA level is widely used as a marker of free-radical med-iated lipid peroxidation. The results of the MDA assays in the liversare also shown in Table 3. MDA levels in the CCl4-treated group(10.55 ± 0.58 nmol/mg protein) were significantly higher than inthe vehicle-control group (5.26 ± 0.83 nmol/mg protein, p < 0.05).Consistent with the liver levels of SOD, catalase and GSH-Px,administration of silica hydride significantly decreased CCl4-in-duced hepatic lipid peroxidation. The MDA levels in the silica hy-dride-treated group at doses of 208 and 520 mg/kg weresignificantly lower, by at least 28%, than that in the CCl4-treatedcontrol group (p < 0.05). Silymarin also inhibited the elevation ofMDA levels after CCl4 administration. These findings indicated that

Fig. 1. Effect of the silica hydride on hepatic morphological analysis in CCl4-intoxicated mice. Livers were sectioned and stained with hematoxylineosin by standardtechniques (200�). (A) Normal control, (B) vehicle control, (C) CCl4 control, (D) silymarin (200 mg/kg) + CCl4, (E) silica hydride (104 mg/kg) + CCl4, (F) silica hydride (208 mg/kg) + CCl4, (G) silica hydride (520 mg/kg) + CCl4.

Y.-W. Hsu et al. / Food and Chemical Toxicology 48 (2010) 1644–1653 1647

the free radicals being released in the liver were effectively scav-enged by silica hydride.

3.4. Histopathological examination

Histopathological studies also provided important evidencesupporting the biochemical analysis and liver antioxidant status.

In the normal control and vehicle-control animals, liver sectionsshowed normal hepatic cells, i.e., with a well-preserved cytoplasmand a prominent nucleus, nucleolus and central vein (Fig. 1A andB). The livers of CCl4-intoxicated mice revealed moderate to severehepatocellular vacuolization, hepatic necrosis and swelling,bile-duct hyperplasia, and increasing cellular mitosis (Fig. 1C).Compared with the lesions observed in the CCl4 control group,

Fig. 2. Histopathological changes of fibrosis occurred in CCl4-intoxication and prevention by the treatment with silica hydride (Masson Trichrome stain, 200X). (A) Normalcontrol, (B) vehicle control, (C) CCl4 control, (D) silymarin (200 mg/kg) + CCl4, (E) silica hydride (104 mg/kg) + CCl4, (F) silica hydride (208 mg/kg) + CCl4, (G) silica hydride(520 mg/kg) + CCl4.

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the lesions of the silymarin-treated mice were of a much milderdegree (Fig. 1D). These animals showed slight to moderate diffusenecrosis of hepatocytes and slight to mild hepatocellular vacuoli-zation, whereas a slight to mild degree of hepatocellular necrosisand slight to moderate degree of hepatocellular vacuolization wereobserved in the livers of silica hydride-treated mice at 104, 208,and 520 mg/kg (Fig. 1E–G).

Furthermore, histopathological changes (fibrosis) occurred inCCl4-intoxicated mouse livers, and their prevention by treatmentwith silica hydride was observed, as shown in Fig. 2 (Masson tri-chrome stain) and Fig. 3 (Sirius red stain). In the normal controland vehicle-control groups, liver sections showed normal hepaticcells without fibrosis (Figs. 2A and B and 3A and B). The livers ofmice treated with CCl4 showed numerous hepatic lobules

Fig. 3. Histopathological changes of fibrosis occurred in CCl4-intoxication and prevention by the treatment with silica hydride (Sirius red stain, 200�). (A) Normal control, (B)vehicle control, (C) CCl4 control, (D) silymarin (200 mg/kg) + CCl4, (E) silica hydride (104 mg/kg) + CCl4, (F) silica hydride (208 mg/kg) + CCl4, (G) silica hydride (520 mg/kg) + CCl4.

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surrounded by thick fibrotic tissue, resulting in the formation ofcontinuous fibrotic septa. The collagen of these fibrotic tissuesshowed a blue color when stained by Masson’s Trichrome(Fig. 2C) and a red color when stained by Sirius red (Fig. 3C). Thelesions of silymarin-treated mice were observed to a much milderdegree (Figs. 2D and 3D) than in the CCl4 control group. The groupsintoxicated with CCl4 and treated with 104, 208, and 520 mg/kg of

silica hydride showed mild to moderate degrees of fibrosis and theformation of incomplete septa from portal tract to central vein(Figs. 2E–G and 3E–G).

Histopathological examinations such as hepatocyte necrosis,vacuolization, and hepatocyte fibrosis are recorded and scored inTable 4. In this semi-quantitative assessment, all scores of histopa-thological examinations in the CCl4 control group were

Table 4Effects of silica hydride on hepatic histopathology of liver damage in mice treated with CCl4.

Parameter GradesA/scoreB

Design of treatment

Normalcontrol

Vehiclecontrol

CCl4 control(1 ml/kg)

Silymarin(200 mg/kg) + CCl4

Silica hydride(104 mg/kg) + CCl4

Silica hydride(208 mg/kg) + CCl4

Silica hydride(520 mg/kg) + CCl4

Hepatocyte necrosis � 10 10 0 0 0 0 0+ 0 0 0 4 5 8 7++ 0 0 3 3 5 2 3+++ 0 0 5 3 0 0 0++++ 0 0 2 0 0 0 0Score 0.00b,c 0.00b,c 2.90 ± 0.74a,c 1.90 ± 0.88a,b 1.50 ± 0.53a,b 1.20 ± 0.42a,b 1.30 ± 0.48a,b

Vacuolization � 10 10 0 0 0 0 0+ 0 0 0 6 3 3 4++ 0 0 6 4 5 7 4+++ 0 0 2 0 2 0 2++++ 0 0 2 0 0 0 0Score 0.00b,c 0.00b,c 2.60 ± 0.84a,c 1.40 ± 0.52a,b 1.90 ± 0.74a 1.70 ± 0.48a,b 1.80 ± 0.79a

Hepatocyte fibrosisC � 10 10 0 0 0 0 0+ 0 0 0 1 0 2 0++ 0 0 2 8 1 2 4+++ 0 0 4 1 8 5 4++++ 0 0 4 0 1 1 2Score 0.00b,c 0.00b,c 3.20 ± 0.79a,c 2.00 ± 0.47a,b 3.00 ± 0.47a,c 2.50 ± 0.97a 2.80 ± 0.79a

a p < 0.05 compare with vehicle control.b p < 0.05 compare with CCl4.c p < 0.05 compare with silymarin.A Grades are as follows: �, absent; +, trace (1–25%); ++, weak (26–50%); +++, moderate (50–75%); ++++, severe (75–100%).B The numerical score of histopathology were the result of adding the number per grade of affected mice and dividing by the total number of examined mice.C For estimation of fibrosis, livers were sectioned and stained with Masson Trichrome by standard techniques.

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significantly higher than that of the normal control (p < 0.05), indi-cating that CCl4-induced severe damage to the hepatic cells. All ofthe tested doses of silica hydride significantly decreased (p < 0.05)the scores of hepatocyte necrosis as compared to the CCl4 controlgroup; in contrast, mice treated with silica hydride at doses of104, 208, and 520 mg/kg showed reduced scores for vacuolizationand hepatocyte fibrosis, although these did not amount to statisti-cally significant differences, except for those treated with 208 mg/kg silica hydride with respect to vacuolization. The positive controldrug, silymarin, significantly reduced the scores for hepatocytenecrosis, vacuolization, and hepatocyte fibrosis compared to theCCl4 control group. When silica hydride-treated groups were com-pared to silymarin-treated groups, the overwhelming majority ofscores were not significantly difference (p > 0.05) between eachhistopathological examination.

According to the microscopic examinations, severe liver dam-age induced by CCl4 was remarkably reduced by the administrationof silica hydride, which was in good correlation with the results ofthe liver-functional parameters of the serum and hepatic antioxi-dant enzyme activities and hepatic lipid peroxidation.

4. Discussion

Several studies have reported that silica hydride possesses free-radical scavenging activities for species including singlet oxygen,hydroxyl radical, and superoxide radical and protects cells againststrong oxidative stress (Stephanson et al., 2002; Stephanson andFlanagan, 2003a). Stephanson et al. (2003) have also shown thatsilica hydride decreases oxidative stress and effectively protectsagainst ROS-induced pathological changes. In addition, a clinicalstudy reported (Purdy-Lloyd et al., 2001) that a silica mineral sup-plement was effective in decreasing the postexercise blood lactateconcentrations in bicycle-training subjects. Therefore, we consid-ered that silica hydride may be useful in the prevention of varioushepatic damages induced by oxidative stress. In the present study,the capability of silica hydride to protect against CCl4-induced hep-atotoxicity and oxidative stress were investigated.

Biotransformed metabolites of CCl4 formed by cytochromeP-450 2E1, including trichloromethyl radical (CCl3) and trichloro-methyl peroxyl radical (CCl3O2), have been demonstrated to initi-ate peroxidation (Recknagel et al., 1989) and affect liverpathogenesis (Recknagel, 1967). Both radicals have been shownto cause numerous cellular anomalies, such as DNA alteration, pro-tein damage, lipid peroxidation, cell necrosis, and liver fibrosis(Brattin et al., 1985; Recknagel et al., 1989; Basu, 2003). Several re-ports have indicated that an important mechanism in hepatopro-tective effects may be related to the capacities of antioxidants toscavenge reactive oxygen species (Naik and Panda, 2007; Tsaiet al., 2009) and/or alleviate CCl4-induced toxic effects by the pre-vention of lipid peroxidation (Hsu et al., 2009). In the presentstudy, we found that treatment with silica hydride significantlyprevented CCl4-induced liver damage as evidenced by decreasedserum activities of AST, ALT, and ALP and reduced serum concen-trations of TG and cholesterol.

The balance of intracellular ROS depends on both their produc-tion within cells during normal aerobic metabolism and theirremoval by the antioxidant-defense system that includes nonenzy-matic antioxidants (e.g., GSH, bilirubin, and vitamins E and C) andenzymatic antioxidants such as SOD, catalase, GSH-Px, and GSH-Rdin mammalian cells (Halliwell and Gutteridge, 1990; Sreelathaet al., 2009). Therefore, the enzymatic antioxidant activities and/or the inhibition of free-radical generation are important in termsof protecting the liver from CCl4-induced damage (Campo et al.,2001). A decrease in antioxidant enzyme activity is related to anincrease in free radical production in CCl4 toxicity. SOD convertsthe dismutation of superoxide anions into hydrogen peroxide(H2O2) (Reiter et al., 2000) and catalase decomposes H2O2 to oxy-gen and water. GSH-Px metabolizes H2O2 and hydroperoxides tonontoxic products and terminates the chain reaction of lipid perox-idation by removing lipid hydroperoxides from the cell membrane(Jung and Henke, 1996). GSH-Rd is involved in the detoxification ofa range of xenobiotic compounds by their conjugation with GSH(Baudrimont et al., 1997; Naik and Panda, 2007). These antioxidantenzymes are easily deprived of their activity by lipid peroxides or

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free radicals, resulting in their decreased activities in CCl4 toxicity(Szymonik-Lesiuk et al., 2003). The results of the present studyindicate that SOD, catalase, GSH-Px, and GSH-Rd activities weresignificantly decreased in the liver in response to CCl4 treatmentalone compared with normal control mice, suggesting increasedoxidative damage to the liver. In contrast, SOD, catalase, andGSH-Px levels were significantly elevated by administration of sil-ica hydride to CCl4-intoxicated mice, suggesting that it has theability to restore and/or maintain the activities of hepatic enzymesin CCl4-damaged liver. However, administration of silica hydride toCCl4-intoxicated mice could not significantly increase the activityof GSH-Rd. GSH-Rd and NADPH can rapidly reduce GSSG to formGSH, which is prone to oxidation to glutathione disulfide (GSSG)by xenobiotics. Mendieta-Wejebe et al., 2008 indicated that treat-ment with silica hydride elevated the amount of NADPH in rabbithepatic cells. Therefore, the mechanism by silica hydride elevatedthe GSH content may be mainly due to the increased the amountNADPH instead of an increase in the activity of GSH-Rd.

Previous studies on the mechanism of CCl4-induced hepatotox-icity showed that GSH acts as a nonenzymatic antioxidant that re-duces H2O2, hydroperoxides (ROOH), and xenobiotic toxicity(Kadiska et al., 2000). In particular, the amount of GSH depletionis substantially correlated with the degree of liver necrosis (Dam-bach et al., 2006). Therefore, it appears that GSH conjugation is crit-ically required for attenuating CCl4-induced liver injury. GSH iseasily oxidized to GSSG by xenobiotic compounds, and there mayadditionally be reaction with any of the selenium-containingGSH-Px isozymes that may subsequently result in the reductionof GSH levels. GSSG is either rapidly reduced by GSH-Rd and NADPHor utilized in the protein-folding process in the endoplasmic retic-ulum. Because of these recycling mechanisms, GSH is an extremelyefficient intracellular antioxidant for oxidative stress (Cantin et al.,2007). A study reported that silica hydride has the capability toregenerate some amount of NADPH from the reduction of NADP+

in rabbit hepatic cells (Mendieta-Wejebe et al., 2008), suggestingthat GSH content in the liver may be elevated by the administrationof silica hydride. In the present study, the hepatic content of GSHwas significantly decreased in CCl4-intoxicated mice comparedwith control mice. Conversely administration of silica hydride toCCl4-intoxicated mice significantly elevated GSH content in the li-ver compared to the untreated group, indicating that silica hydridecan protect against the CCl4-induced depletion of hepatic GSH.

CCl4 biotransformed metabolites have been demonstrated tocause lipid peroxidation (Recknagel et al., 1989), which is one ofthe principal mechanisms of CCl4-induced liver injury (Castoret al., 1974). Moreover, the initiation of oxidative stress relatedto various tissue injuries, cell death, and the progression of manyacute and chronic diseases is generally believed to be induced byincreased lipid peroxidation (Halliwell, 1997). Lipid peroxidationgenerates a variety of more-or-less stable toxic products and manyof these are aldehydes (Esterbauer, 1982; Brattin et al., 1985). MDAis the major reactive aldehyde that appears during the peroxida-tion of biological membrane polyunsaturated fatty acids (Vacaet al., 1988). An increase in MDA levels in the liver suggests en-hanced peroxidation leading to tissue damage and failure of theantioxidant-defense mechanisms to prevent the formation ofexcessive free radicals (Naik, 2003). In the present study, CCl4-in-duced toxicity caused an increase in liver MDA levels comparedto the normal control group. Treatment with silica hydride signif-icantly reversed these changes. The administration of silica hydridecaused a significant decrease in MDA levels compared to the CCl4-induced toxicity group.

Silymarin, an antioxidant flavonoid complex isolated from theseed of milk thistle (Silybum marianum, Compositae), has beenused to treat hepatotoxicity diseases in clinical practice for at leasttwo decades. Silymarin has powerful free-radical scavenging prop-

erties and regulates intracellular GSH levels (Kren and Walterova,2005; Shaker et al., 2010). The mechanism by which silymarin pre-vents against CCl4-induced lipid peroxidation and hepatotoxicity iseither by decreasing the metabolic activation of CCl4 or by acting asa chain-breaking antioxidant for scavenging free radicals, or by acombination of these effects (Lettéron et al., 1990). In fact, a con-siderable body of experimental work in animal models has re-ported that silymarin, as a positive control, reduced CCl4-inducedhepatotoxic effects by the prevention of lipid peroxidation (Wanget al., 2004; Naik and Panda, 2007; Shyu et al., 2008; Sreelathaet al., 2009). In the present study, silymarin acted as an effectivepositive control as evidenced by decreasing the ALT, AST, andALP serum levels and increasing the SOD and catalase, GSH-Pxactivities and GSH content in liver while decreasing the hepaticMDA content when compared with the CCl4 control group.

The intracellular antioxidant status is always maintained at anequilibrium in mammalian cells. Dietary supplementation with ex-tra natural antioxidants mainly assists the intracellular antioxi-dant-defense system in protecting cells and organs against ROS-induced oxidative damage. Indeed, allochthonous antioxidantsusually have no effect on normal intracellular antioxidant status,as evidenced by Mansour (2000) and Lee et al. (2004), who re-ported that the hepatic antioxidant status was not affected bytreatment with samples alone. Undoubtedly, silica hydride is aneffective antioxidant for protecting mammalian cells againststrong oxidative stress (Stephanson et al., 2002; Stephanson andFlanagan, 2003a). In the present study, we provide evidence thatsilica hydride has the ability to restore and/or maintain the levelsof antioxidant status in CCl4-damaged liver. In addition, our previ-ous study evaluating the safety of silica hydride showed thatadministration of silica hydride alone via gavage to ICR mice atdosage levels of 500, 1250, 2500, and 5000 mg/kg body weightdid not cause treatment-related adverse effects on liver weight,serum liver-function tests (AST, ALT, and ALP) or histopathologicalchanges of the liver. The results of the safety evaluation provideevidence that silica hydride is nontoxic (Tsai et al., submitted forpublication). Therefore, silica hydride alone has no effect on liverstructure, function, and/or antioxidant status.

In the histological examinations in this study, liver damageincluding hepatocyte necrosis and vacuolization was evaluatedby hematoxylin and eosin staining as well as Masson trichromeand Sirius red staining for hepatocyte fibrosis. In clinical diagnosisand experimental examination, liver fibrosis has often dependedupon microscopic detection of the collagen fibers, and Masson tri-chrome and Sirius red stains are generally the routine stainingtechniques for detecting collagen fibers (Marceau et al., 1999; Yanget al., 2008; Fang and Lin, 2008). Using routine staining techniquessuch as Sirius red and/or Masson’s trichrome has its advantages inthat experienced pathologists have reliably and consistently iden-tified liver damage in formalin-fixed, paraffin-embedded tissuesections. Collagen fibers stained with Masson’s trichrome show aconspicuous blue color, and when stained with Sirius red the fibersshow an obvious red color. In our histological examination forhepatocyte fibrosis, we not only observed collagen by Masson tri-chrome staining but also confirmed it by Sirius red staining.

Histological examination of CCl4-treated mouse liver showedsignificant hepatotoxicity characteristics, such as necrosis in hepa-tic lobules, vacuolization, Kupffer cells around the central vein, andhepatocyte fibrosis. However, treatment with silica hydride signif-icantly decreased these hepatotoxicity characteristics in mouse li-ver, suggesting that silica hydride provided protection againstCCl4-induced liver injury. The histological examinations of thehepatocyte fibrosis assessment also found that both staining meth-ods showed identical results in all liver sections, indicating thatthere was no difference between Masson trichrome stain and Siriusred stain with respect to histopathology. Through a semi-quantita-

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tive assessment (see Table 4), the results were in agreement withthe histological observations that administration of silica hydridesignificantly reduced hepatotoxicity in CCl4-induced mice.

Stephanson and Flanagan (2004a) reported that silica hydride inan aqueous environment has several unique characteristics, suchas the ability to stably release the hydride ion over an extendedlength of time, a slightly alkaline pH, and a low oxidation–reduc-tion potential, but this study used silica hydride dissolved in oliveoil. In fact, when silica hydride powder is dissolved in water thereis a small amount of carbon dioxide released to the aqueous envi-ronment. It is well known that stomach acid in mice causes releaseof CO2 from some otherwise nontoxic products and that the pres-sure can kill mice because mice and rats cannot burp, belch or re-lease gas from their stomachs. Therefore, it was routine in thelaboratory to add silica hydride to food-grade oil before testing.The silica hydride was absorbed without evolving CO2 gas. How-ever, the small amount of CO2 released from a silica hydride solu-tion is not a problem in humans. In the present study, dissolvingsilica hydride in olive oil not only retained the unique features ofsilica hydride in an aqueous environment but also solved the prob-lem of CO2 release. In addition, dissolving silica hydride in olive oilallowed the use of the same vehicle as for CCl4 and silymarin.

Silica hydride has been verified as an effective antioxidant thatplays an important role in protecting cells and organisms againstthe harmful effects of free radicals. The primary mechanism of ac-tion of this phenomenon appears to be the ability of silica hydrideto quench superoxide reactive species, hydroxyl radical speciesand singlet oxygen (Stephanson et al., 2002; Stephanson and Flan-agan, 2003a). In addition, silica hydride regenerated the NADPHproduced by the reduction of NADP+ and decreased the catalyticactivity of cytochrome P-450 in rabbit hepatic cells (Mendieta-Wejebe et al., 2008), suggesting that silica hydride protected hepa-tic cells against oxidative stress mediated by CCl4-biotransforma-tion metabolites. Therefore, silica hydride was expected toprotect against CCl4-induced liver damage.

In conclusion, the results of this study demonstrate that silica hy-dride was effective for the prevention of CCl4-induced hepatic dam-age in ICR mice. Our results show that the hepatoprotective effectsof silica hydride may be due to both an increase in the activity of theantioxidant-defense system and an inhibition of lipid peroxidation.This is the first report of the hepatoprotective effects of silica hy-dride in vivo. According to the results of the present study, silica hy-dride possesses a potent antioxidant activity. Oxidation is known tobe involved in the pathogenesis of many diseases in which treat-ment with silica hydride is claimed to be effective. The inhibitory ef-fects of dietary silica hydride may be useful as a hepatoprotectiveagent against chemical-induced hepatotoxicity in vivo.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors would like to thank Dr. Jiunn-Wang Liao (Instituteof Veterinary Pathobiology, National Chung Hsing University, Tai-chung City, Taiwan) for the help provided with the histologicalexaminations. This work was supported by New I Ten Rin Enter-prise Co. Ltd., Changhua City, Taiwan.

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