Food and Chemical Toxicology 50 (2012) 3306–3312

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Protective effect of allicin against acrylamide-induced hepatocyte damage

in vitro and in vivo
Lulu Zhang, Huanjie Zhang 1, Yutian Miao 1, Sijia Wu, Haiqing Ye, Yuan Yuan ⇑
College of Quartermaster Technology, Jilin University, Changchun 130062, China

a r t i c l e i n f o a b s t r a c t

Article history: Acrylamide (AA) is known to be a neurotoxic, genotoxic, and carcinogenic compound. The presence of AA
Available online 13 June 2012 in foods causes public health concerns. In our previous study, we found that allicin can effectively reduce
AA content in Maillard model system. However, there has been no report on whether allicin can reduce
Keywords: AA-induced toxicity in vitro and in vivo. In our present study, we evaluated the protective effect of allicin
Allicin against AA-induced hepatocyte damage in cultured mouse primary hepatocytes and mouse liver. Our
Acrylamide date suggested that allicin significantly decreased the levels of maleic dialdehyde (MDA) and 8-
Hepatocyte damage
hydroxy-desoxyguanosine (8-OHdG) both in vitro and in vivo study. Allicin also markedly increased
Mice
the activity of total superoxide dismutase (SOD) and level of glutathione (GSH). The in vitro study
revealed that 15 lM allicin was the optimum concentration for inhibiting AA-induced hepatocyte dam-
age. The in vivo study revealed that 20 mg/kg b.w./day allicin was the optimum dose for inhibiting AA-
induced hepatocyte damage. The protective effects of allicin against AA-induced hepatocyte damage
may be due to its ability to scavenge free radicals and its effective recovery of the antioxidative defense
system, and its ability to block the epoxidation process of AA to GA by inhibiting P450 enzyme.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction
In April 2002, researchers from the Swedish National Food
Administration (SNFA) and Stockholm University announced that
carbohydrate-rich foods heated or fried at high temperatures, such
as fried potato products, contained relatively high levels of acryl-
amide (AA). AA is known to be neurotoxic, genotoxic and carcino-
genic. It is classified by the International Agency for Research on
Cancer (IARC) as a probable human carcinogen (IARC, 1994). AA
can undergo oxidative biotransformation by cytochrome P450
2E1 (CYP2E1), the resulting metabolite is an epoxide derivative
glycidamide (GA), which is more reactive toward DNA and proteins
to form adducts than AA (Sumner et al., 1999; Besaratinia and Pfe-
ifer, 2007). Thus the toxicity of AA to humans needs to be studied
to control or reduce. The protective effect of natural antioxidants
against the toxicity of AA has gained global attention not only be-
cause of new biologically active molecules that can be isolated and
identification for the pharmaceutical industry, but also because of
the emerging public interest in using crude plant extracts (Dra-
gland et al., 2003). The fact that human diet can be supplemented
with antioxidants via infusion intake is an additional convenient.
Indeed nature antioxidants have been successfully applied to re-
duce the AA-induced toxicity both in vitro and in vivo. Blasiak
⇑ Corresponding author. Tel.: +86 431 87836376.
E-mail address: yuanyuan1024@gmail.com (Y. Yuan).
1
The author should be regarded as joint first author.
et al. (2004) reported that free radicals participate in the DNA-
damaging action of AA and antioxidants, such as N-tert-butyl-al-
pha-phenylnitrone (PBN) spin traps, and vitamins C and E decrease
the potential of AA to damage DNA. Zhang et al. (2009) showed
that hydroxytyrosol (HT), a strong antioxidant compound ex-
tracted from extra virgin olive oil, have significant protective abil-
ity against AA-induced genotoxicity in a human hepatoma cell line,
HepG2. Cao et al. (2008) found that antioxidants extracted from
curcumin attenuated AA-induced cytotoxicity and genotoxicity in
HepG2 cells. Zamorano-Ponce et al. (2006) observed that the
administration of the antioxidant of aloysia triphylla exerts its pre-
ventive action against DNA damage induced by AA in vivo. Alturfan
et al. (2012) found that the antioxidant resveratrol ameliorates oxi-
dative DNA damage and protects against AA-induced oxidative
stress in rats.
Allicin is a natural compound present in garlic powder and
freshly crushed garlic, but not in garlic oil or oil macerated garlic
(Lawson et al., 1992). Allicin is extensively used as a dietary spice
and herbal medicine for inflammatory diseases. Allicin is a natural
antioxidant, that not only scavenges oxygen free radicals and hy-
droxyl radicals, but also prevents the lipid peroxidation of liver
homogenates induced by hydroxyl radicals. The mechanism of
the antioxidative activity of allicin may involve its ability to scav-
enge oxygen free radicals (Prasad et al., 1995; Yin and Cheng, 1998;
Chung, 2006). In our previous study, we found that allicin can
reduce AA formation both in Maillard model systems and in a food
matrix. The results demonstrated that allicin can effectively reduce

0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2012.05.060
 L. Zhang et al. / Food and Chemical Toxicology 50 (2012) 3306–3312
AA formation and achieve a maximum reduction rate of >50% for
the use of allicin at a concentration of 0.0375% in an asparagine/
fructose model system (Yuan et al., 2010). Although allicin can
effectively reduce AA formation, its protective effect on the body
against AA-induced toxicity remains unclear. In the study of Tau-
bert et al. (2006), the formation of AA epoxide metabolite, GA,
was diminished in the presence of the CYP2E1 inhibitor diallyl sul-
fide (DAS), which is a specific ingredient of garlic. Since allicin is
the main ingredient of garlic, is it possible to reduce the toxicity
of AA? Base on the current observations and our previous study,
it stimulated our interest in the investigation of the protective ef-
fects of allicin as a natural antioxidant against AA-induced toxicity
in vitro and in vivo in hepatocytes of mice. The activity of SOD, as
well as the levels of 8-OHdG, GSH, and MDA in AA-treated mouse
hepatocytes in the absence or presence of allicin was determined
to test the protective effect of allicin against AA-induced toxicity.
2. Materials and methods
2.1. Materials
AA (2-propene amide) (CAS 79-06-1, purity >99.8%), dimethyl sulfoxide
(DMSO), allicin and 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide
(MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Collagenase IV
was obtained from Gibco-Invitrogen (Carlsbad, CA). DNase I was purchased from
Applichem (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM),
fetal bovine serum (FBS), penicillin and streptomycin for cell culture were pur-
chased from Gibco BRL Co. Ltd. (Grand Island, NY, USA). Commercial kit of MDA,
GSH, and total SOD, as well as a mouse 8-OHdG ELISA kit were obtained from Nan-
jing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals and re-
agents were of the highest quality available. For the in vitro study, a stock solution
of 15 mM for allicin was prepared in DMSO and stored at 20 °C. For each experi-
ment, the stock solution of allicin was diluted with DMEM to obtain a series of con-
centrations (15, 30 and 60 lM). The final DMSO concentration was 0.1% (V/V). The
dose of AA in vitro study was selected based on its effectiveness in inducing geno-
toxicity in HepG2 cells (Koyama et al., 2006). For the in vivo study, allicin was also
dissolved in normal saline to give a final concentration of 0.5, 1 and 2 mg/mL,
respectively. The dosing volume of allicin was determined based on the body
weight of each animal i.e., 0.2 mL for a 20 g mouse. The dose of AA in vivo
study was selected based on its effectiveness in inducing clastogenicity in mice
(Abramsson-Zetterberg, 2003; Doerge et al., 2005). All AA solutions were prepared
in water.
2.2. Animals
Males BALB/c mice weighing 20 ± 5 g were provided by the Laboratory Animals
Center of Jilin University (Changchun, China). The experiments were carried out in
accordance with the Guideline for Animal Experimentation of Jilin University
(ChangChun, China). The animals were housed (eight mice per cage) in an air-con-
ditioned room at 22 ± 2 °C and 30% ± 10% relative humidity. The general conditions
of the animals were observed for 7 days during the quarantine and acclimation per-
iod to confirm that there were no abnormalities.
2.3. In vitro study
2.3.1. Primary culture of hepatocytes and treatment of cells
Hepatocytes were obtained from liver tissue of BALB/c mouse (male, 5–7 weeks
old) via the two-step collagenase perfusion technique according to the method of
Seglen (1976) with some modifications. The liver was perfused at 7 mL/min via
the portal vein for 10 min with a pre-perfusion buffer (Grey’s balanced salt solution,
GBSS) without Ca2 + containing the following: NaCl 7.0 g, KCl 0.37 g, MgSO4 7H2O
70 mg, Na2HPO412H2O3O2 mg, NaHCO32.27 g, KH2PO430 mg, MgCl26H2O
210 mg, Glucose 1.0 g, and EDTA 0.744 g (pH 7.4). After perfusion, the livers were
excised and pelleted into small pieces in 0.1% collagenase IV and 0.05% DNAase I
solution in DMEM and then incubated at 37 °C for 30 min. Primary liver cell suspen-
sions were centrifuged two times at 50g for 1 min. The pellet was discarded after
each centrifugation, and the supernatant was again centrifuged two times at 50g
for 2 min. The cell pellet was suspended in DMEM with 10% FBS, penicillin
(100 IU/mL), and streptomycin (100 mg/mL) (RPMI-FBS) and maintained at 37 °C
in a humidified atmosphere in a 5% CO2 incubator. Hepatocytes were plated onto
6-well plates (105 cells/well) and incubated for 1 h. The cells were divided into five
groups, including the control group, the AA group, and three allicin groups with dif-
ferent concentrations. For the control group, hepatocytes were cultured in the base
culture medium (DMEM) for 24 h. For the allicin groups, the cells were incubated
with allicin at 37 °C for 2 h with different concentrations at 3.75, 7.5, 15 lM (final
concentration after dilution). Then these allicin-treated cells were incubated with
3307
3.5 mM AA (final concentration after dilution) for other 24 h. For the AA group, cells
were cultured in the base culture medium supplemented with 3.5 mM of AA for
24 h. The cells in all groups were finally collected and washed twice with cold
PBS, suspended in 1 mL of PBS and used for future biochemical analyses.
2.3.2. Cell viability assay
Cell viability was evaluated by the MTT assay according to the method of Cao
et al. (2008) with some modifications. About 100 lL of cells were plated at a density
of 1  105 cells/ml in 96-well plates at 37 °C in a 5% CO2 incubator for 1 h. The cells
were then treated with 50 lL of allicin with different final concentrations (0, 3.75,
7.5, 15 lM) for 2 h, added 0 mM or 3.5 mM AA 50 lL for 24 h. After treatment, 20 lL
of MTT (5 mg/mL) was added to each well, and the cells were further incubated for
an additional 4 h. The supernatant was removed, and the formation of formazan
was resolved with 150 lL/well of DMSO. The optical density was measured at
570 nm on a microplate reader (Tecan, Austria).
2.4. In vivo study
2.4.1. Experimental design
After a quarantine period of 7 days, 40 mice were randomly divided into five
groups each consisting of eight animals. Group I was treated with saline by oral ga-
vage for 14 consecutive days and was denoted as the control group. Group II was
intragastrically given saline for 7 consecutive days. On day 8, after the oral gavage
saline, the mice were intraperitoneal injected with AA solution (50 mg/kg b.w./day)
for another 7 days and denoted as the Group AA. Groups III, IV, and V were intragas-
trically given allicin (5, 10, and 20 mg/kg b.w./day), respectively, for 7 days (once
daily) (Li et al., 2001). On day 8, Groups III–V were intragastrically given allicin
(5, 10 and 20 mg/kg b.w./day) and intraperitoneal injected with a single dose of
AA (50 mg/kg b.w./day) for another 7 days. The body weight of the animals was
weighted every day and the dose of AA and allicin was administrated according
to the body weight of the animals. On day 15, the animals were sacrificed within
24 h of the last treatment and the liver was removed. The weight of the liver was
measured immediately after the removal from the animal body. After weighing
the weight of the body and liver, the coefficients of liver weight to body weight
were calculated as the ratio of tissue wet weight (in grams) to body weight (in
grams). And then, the livers were washed thoroughly with ice-cold normal saline.
The tissues were homogenized with 10% pre-chilled normal saline in a tissue
homogenizer, and then centrifuged at 2500g for 10 min at 4 °C. The supernatant
was used for subsequent biochemical analyses.
3. Biochemical analysis
The activity of SOD, levels of MDA, GSH, and 8-OHdG, protein
content in the liver, as well as protein content in the cell culture
supernatant were determined by commercial kits (Nanjing Jianch-
eng Bioengineering Institute, Nanjing, China) according to the
manufacturer’s instructions. In vitro study, each measurement
was carried out five independent experiments. In vivo study, each
measurement was carried out eight independent experiments.
3.1. Determination of protein
Protein contents were determined based on the instructions of a
Coomassie (Bradford) protein assay kit using 0.563 g/L bovine ser-
um albumin as the standard. About 50 lL of the protein sample or
cell suspension was mixed with 3 mL of Coomassie brilliant blue
solution. After 10 min of incubation, the absorbance was deter-
mined at a wavelength of 595 nm. The protein concentration was
expressed as gram per liter.
3.2. Determination of SOD activity
Total SOD activity was assayed by detecting the superoxide rad-
icals generated by xanthine oxidase and hypoxanthine according to
the manufacturer’s instructions. The reduction–oxidation reaction
of hypoxanthine and xanthine oxidase produces superoxide, which
oxidizes hydroxylamine by reacting with the reagent with a pur-
ple-red color. Given that superoxide can be consumed by SOD, dif-
ferent degrees of SOD activity can exhibit different shades of color.
About 30 lL of tissue homogenate or cell suspension was mixed
with 3.3 mL of reaction solution containing nitroblue tetrazolium
chloride and incubated at 37 °C for 40 min. The absorbance was
3308 L. Zhang et al. / Food and Chemical Toxicology 50 (2012) 3306–3312
measured at 560 nm. SOD activity was expressed as unit per milli-
gram of protein.
3.3. Determination of GSH content
Liver GSH levels were measured by colorimetric end point assay
using dithionitrobenzoic acid (DTNB) method according to the
manufacturer’s instructions. DTNB reacts with hydrosulfuryl com-
pound to form yellow compound, which can determined by colon-
metry. The content of GSH is reflected the intensity of the yellow
color. Briefly, the standard and testing sample wells were set,
and 100 lL of standard solution was added to the standard wells.
The concentrations of the standard solution were 0, 5, 10, 20, 50,
and 100 ng/mL. Then, 100 lL of liver homogenate or cell suspen-
sion was added to the testing sample wells. Finally, 100 lL of buf-
fer liquid and 25 lL of color reagents were added to these wells.
After thoroughly mixing and incubating for 5 min, the absorbance
was determined at a wavelength 405 nm. The GSH concentration
was expressed as nanomole per milligram of protein.
3.4. Determination of the MDA level
MDA is a biomarker of lipid peroxidation that has been widely
associated with food rancidity as well as many human diseases.
The MDA level was examined by detecting the reaction of MDA
with thiobarbituric acid (TBA), followed by UV-visible detection
according to the manufacturer’s instructions. Breifly, about
100 lL of tissue homogenate or cell suspension was mixed with
4.1 mL of work solution containing thiobarbituric acid. The mix-
ture was incubated at 95 °C for 40 min and cooled down using
tap water. The samples were centrifuged at 2000g for 10 min,
and the absorbance of the supernatant was measured at 532 nm.
The concentration of MDA was expressed as nanomole per milli-
gram of protein.
3.5. Determination of the 8-OHdG level
The supernatant from the liver homogenate was used to detect
the level of 8-OHdG using a commercial ELISA kit following the
instructions of the manufacturer. The samples which were treated
with streptavidin-horseradish peroxidase (HRP) were added to
plate wells precoated with mouse monoclonal anti-8-OHdG anti-
body. Then a substrate containing 3,30 ,5,50 -tetramethylbenzidine
(TMB) was added, the color of the substrate turned into blue by
the catalyzation of the peroxidase. The reaction was terminated
by the addition of sulphuric acid. The blue color of the substrate
was changed to yellow by the addition of sulphuric acid and the
intensity of the color was measured at 450 nm using a sepctroph-
otoneter. Briefly, standard and testing sample wells were prepared.
About 50 lL of standard solution was added to the standard wells
and precoated with mouse monoclonal anti-8-OHdG antibody. The
concentrations of the standard solution were 0, 3, 6, 12, 24, and
48 ng/mL. About 10 lL of biological chemical and 50 lL of liver
homogenate or cell suspension were added to the testing sample
well and were precoated with mouse monoclonal anti-8-OHdG
antibody. These wells were treated with HRP at 37 °C for 60 min.
The wells were then washed five times. A substrate containing
TMB was added, and the wells were incubated at 37 °C for another
15 min in the dark. The reaction was terminated by adding 50 lL of
the stop solution to each well. The absorbance was measured at
450 nm. The level of 8-OHdG was expressed as nanogram per
milligram of protein.
3.6. Statistical analysis
Statistical analysis was performed using SPSS 11.5 software
(Chicago, USA). The significance of difference was calculated by
one-way ANOVA test, and the results with p < 0.05 were consid-
ered to be statistically significant. Graphs were drawn with Origin-
Pro 7.5 software (OriginLab Corporation, Northampton, MA, USA).
4. Results
4.1. In vitro study
4.1.1. Effects of allicin on AA cytotoxicity in hepatocytes
The protective effect of allicin on the cytotoxicity of AA was
shown in Fig. 1. Cells treated with 3.5 mM AA showed evidently re-
duced cell viability. The cytotoxicity presented by allicin alone
with a concentration of 0, 3.75, 7.5, 15 lM (final concentration
after dilution) showed there was no significant difference between
each allicin concentration. The cytotoxic effect was significantly
attenuated by pretreatment with allicin compared with the AA
group (p < 0.05). There was no significant difference between the
hepatic cytotoxicity of the control and allicin (7.5, 15 lM) groups.
4.1.2. Protective effects of allicin on AA-induced cultured mouse
primary hepatocytes damage in vitro
The activity of SOD and level of GSH in mouse hepatocytes were
evaluated, and the results were shown in Table 1. Compared with
the normal group, treatment with 1 mg/mL of AA alone for 24 h
significantly decreased the activity of SOD and level of GSH
(p < 0.05). When pretreated with allicin at different concentrations
(3.75, 7.5, 15 lM), the activity of SOD and level of GSH depletion
was effectively inhibited in the hepatocytes treated with AA
(p < 0.05). The level of intracellular MDA and 8-OHdG were evalu-
ated, and the results were shown in Table 1. Compared with the
control group, the production of MDA and 8-OHdG increased in
the hepatocytes treated with 3.5 mM AA alone for 24 h (p < 0.05).
Pretreatment with allicin (3.75, 7.5, 15 lM) significantly inhibited
the formation of MDA and 8-OHdG in the hepatocytes treated with
AA (p < 0.05). The activity of SOD as well as the levels of GSH, MDA,
Allicin only
1.3 Allicin+AA (3.5mM)
1.2
*
1.1 *
1.0 *
0.9 #
Cell viability (OD)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0microM 3.75microM 7.5microM 15microM
Allicin concentration (microM)
Fig. 1. Effect of allicin on AA-induced cytotoxicity in cultured mouse primary
hepatocytes. Cells were preincubated with allicin for 2 h and incubated with AA for
24 h. The bar represented the mean values ± S.D. of five independent experiments
(n = 5). (⁄) indicates statistically significant differences between the group treated
with AA (p < 0.05). (#) indicates statistically significant differences with control
(p < 0.05).
 L. Zhang et al. / Food and Chemical Toxicology 50 (2012) 3306–3312 3309

Table 1
Effects of allicin on activity of SOD, levels of GSH, MDA, and 8-OHdG in cultured mouse primary hepatocytes treated with AA.

Groups SOD (U/mg protein) GSH (nmol/mg protein) MDA (nmol/mg protein) 8-OHdG (ng/mg protein)
Control 13.38 ± 1.15⁄ 33.82 ± 1.74⁄ 1.04 ± 0.16⁄ 0.33 ± 0.038⁄
AA (3.5 mM) 6.78 ± 0.79# 10.30 ± 1.62# 3.23 ± 0.47# 0.65 ± 0.040#
Allicin (3.75 lM) + AA (3.5 mM) 10.54 ± 1.03⁄# 19.52 ± 0.96⁄# 2.33 ± 0.29⁄# 0.41 ± 0.036⁄#
Allicin (7.5 lM) + AA (3.5 mM) 12.50 ± 1.23⁄ 30.44 ± 1.84⁄ 1.77 ± 0.21⁄ 0.38 ± 0.027⁄
Allicin (15 lM) + AA (3.5 mM) 12.72 ± 0.56⁄ 31.67 ± 1.86⁄ 1.42 ± 0.28⁄ 0.34 ± 0.057⁄

All values are means ± SD (n = 5). (⁄) indicates statistically significant differences between the group treated with AA (p < 0.05). (#) indicates statistically significant
differences with control (p < 0.05).

7 coefficient significantly increased in the AA-treated group com-

#
pared with the control group (p < 0.05). The liver weight coefficient
* markedly decreased in mice orally treated with allicin compared
6
* * with the AA group (p < 0.05).
Liver weight coefficient (%)

5
4 To determine the effect of allicin on oxidative damage in the li-
3
2
1 We also found that SOD activity and the level gradually increased
0 was no significant difference between the hepatic SOD activities
Control g/kg b.w./day g/kg b.w./day g/kg b.w./day g/kg b.w./day
0m 5m 10 m 20 m
Allicin(mg/kg b.w./day)+AA(50mg/kg b.w./day)
Fig. 2. The effects of allicin on liver weight coefficient changes of AA-treated mice.
Data are presented as the mean ± SD for 8 animals in each group (n = 8). (⁄)
indicates statistically significant differences between the group treated with AA
(p < 0.05). (#) indicates statistically significant differences with control (p < 0.05).
and 8-OHdG of the control group did not significantly differ from
those of the allicin (7.5, 15 lM) groups (p > 0.05). These results
suggest that 15 lM allicin is the best concentration for inhibiting
AA-induced oxidative stress in mouse hepatocytes.
4.2. In vivo study
4.2.1. Body weight and coefficients of liver weight
The effects of allicin on the changes of the body weight of AA-
treated mice illustrated in Table 1. The animals were weighed
every day among the whole experiments, and three records about
the body weight of the animals were listed in Table 1. The result
showed that the mice were gaining body weight during the treat-
ment, but there were no significant difference between the control
group and the treatment group. At the last week, the intake of the
food and water were significantly decreased in AA-treated group.
The effects of allicin on liver weight coefficient changes of AA-trea-
ted mice were also calculated as shown in Fig. 2. The liver weight
4.2.2. Protective effects of allicin on AA-induced liver damage in mice
ver of AA-treated mice, the activity of the major antioxidant en-
zyme SOD and the GSH level were determined. As shown in
Table 3, AA induced a significant decrease in the activities of SOD
and level of GSH compared with the control group (p < 0.05). Liver
SOD activity and the GSH level significantly increased in the groups
administered with allicin compared with the AA group (p < 0.05).
with the constant increase in the concentration of allicin. There
of the control and allicin (10 and 20 mg/kg) groups. Hence, allicin
can inhibit the AA-induced decrease in SOD activity and GSH level
in mouse liver.
The MDA levels were also determined in the mouse livers. As
shown in Table 2, the MDA level significantly increased in the
AA-treated group compared with the control group (p < 0.05).
The MDA level markedly decreased in mice orally treated with alli-
cin compared with the AA group (p < 0.05). There was no signifi-
cant difference between the hepatic levels of MDA in the control
and allicin (10 and 20 mg/kg) groups.
8-OHdG is an important oxidative DNA lesion formed by the
oxidation of the C-8 position of 20 -deoxyguanosine. 8-OHdG is
commonly used as a biomarker of oxidative DNA damage (Toyok-
uni et al., 1995; Pilger and Rudiger, 2006; Singh et al., 2007; Chen
et al., 2010). As shown in Table 3, the level of 8-OHdG in the liver
increased significantly in the AA-treated group compared with the
control group (p < 0.05). Mice orally treated with allicin showed
significantly lower levels of 8-OHdG compared with AA-treated
mice (p < 0.05). There were no significant difference between the
hepatic level of 8-OHdG in the control and allicin (20 mg/kg)
group.
5. Discussion
The liver is an important organ for the detoxification and depo-
sition of endogenous and exodogenous substances. The disease of

Table 2
Effects of allicin on body weight changes of AA-treated mice.

Groups Mice body weight (g) Mice body weight gain (g)
Day 1 Day 8 Day 15 The fist week The last week Two weeks
Control 22.24 ± 0.52 24.28 ± 0.63 26.53 ± 1.05 2.08 ± 0.15 2.25 ± 0.42 4.29 ± 0.47
AA (50 mg/kg b.w./day) 22.13 ± 1.65 24.19 ± 1.24 25.24 ± 1.78 2.06 ± 0.48 1.05 ± 0.34 2.21 ± 0.69
Allicin (5 mg/kg b.w./day) + AA (50 mg/kg b.w./day) 22.23 ± 1.63 24.43 ± 2.42 26.10 ± 2.57 2.20 ± 1.32 1.67 ± 0.28 3.87 ± 1.05
Allicin (10 mg/kg b.w./day) + AA (50 mg/kg b.w./day) 22.18 ± 2.74 24.69 ± 3.27 26.28 ± 3.36 2.61 ± 2.12 1.59 ± 0.72 4.2 ± 1.07
Allicin (20 mg/kg b.w./day) + AA (50 mg/kg b.w./day) 23.70 ± 1.47 24.48 ± 2.25 25.27 ± 2.16 2.78 ± 0.92 1.79 ± 0.71 4.57 ± 0.81

All values are means ± SD (n = 8).

3310 L. Zhang et al. / Food and Chemical Toxicology 50 (2012) 3306–3312
Table 3
Effects of allicin on activity of SOD, levels of GSH, MDA, and 8-OHdG in the liver of AA-treated mice.
Groups SOD (U/mg protein)
Control 107.40 ± 8.67⁄
AA (50 mg/kg b.w./day) 59.42 ± 1.60#
Allicin (5 mg/kg b.w./day) + AA (50 mg/kg b.w./day) 61.23 ± 5.00#
Allicin (10 mg/kg b.w./day)) + AA (50 mg/kg b.w./day) 94.82 ± 5.11⁄
Allicin (20 mg/kg b.w./day) + AA (50 mg/kg b.w./day) 106.79 ± 1.36⁄
All values are means ± SD (n = 8). (⁄) indicates statistically significant differences between the group treated with AA (p < 0.05). (#) indicates statistically significant
differences with control (p < 0.05).
liver is considered to be a serious health problem. Based on this
fact, mice liver was selected as the research objective in our pres-
ent study.
Some studies have demonstrated that an important mechanism
of hepato-protective effects may be related to the antioxidant
capacity of scavenging reactive oxygen species (ROS) (Naik and
Panda, 2007; MehmetÇik et al., 2008). Allicin is a major component
of garlic organosulfur and its antioxidant properties have been
confirmed by previous studies (Okada et al., 2005; Leelarungrayub
et al., 2006). Garlic organosulfur compounds also prevent toxicity
induced by cyanide, sodium nitrite, carbon tetrachloride, ethanol,
and sodium arsenite (Elsaid and Elkomy, 2006; Elsaid and Helal,
2006; Hussein et al., 2007; Kodai et al., 2007; Chowdhury et al.,
2008). The toxicity mechanism of all of these antitoxic activities in-
volves oxidative stress and impairment in the antioxidant defense
system (Veena et al., 2010). The cytotoxicity and genotoxicity of AA
result in damage to the oxidative defense system of cells, leading to
the release of ROS (Zamorano-Ponce et al., 2006; Liping et al.,
2007). AA can also induce oxidative stress; thus, allicin can be as-
sumed as able to prevent AA-induced toxicity (Catalgol et al., 2009;
Shuming et al., 2009). Our results suggest that allicin has signifi-
cant protective effect against AA-induced hepatocyte damage both
in vitro and in vivo.
Oxidative stress has been proven to be involved in mutation,
chromosomal aberration, tumor promotion, and cancer develop-
ment. Oxidative stress has also been considered as an important
mechanism of indirect genotoxicity (Speit et al., 2002). Antioxidant
enzymes such as SOD can protect cellular compounds against dam-
age induced by free radicals. Therefore, the activities of these en-
zymes have been used to assess oxidative stress in cells (Liu
et al. 2010a,b). In the present study, we found that allicin can
markedly increase the activities of SOD in AA-treated cultured
mouse primary hepatocytes (Tables 1 and 3). Allicin can restore
the activity of antioxidant enzymes and possibly reduce the gener-
ation of free radicals in vitro and in vivo.
GSH plays an important role in protecting several tissues and
cell lines against injuries by oxidants and reactive electrophiles
(Shan et al., 1990). Srivastava et al. (1986) reported that AA can in-
hibit gluthathione S-transferase activity and deplete GSH, consis-
tent with the present results. Park et al. (2002) reported that AA
itself, but not oxidative P450 metabolites of AA, appeared to be in-
volved in AA-induced cellular transformation and that GSH was in-
volved in AA-induced morphological transformation. Our data
show that allicin pretreatment significantly inhibits the AA-in-
duced depletion of hepatic GSH (Table 1). The results of the
in vivo study also suggested that mice orally administered with alli-
cin can increase GSH levels as compared to AA group (Table 3).
These findings are consistent with the proposal of Xie et al.
(2008) that the intake of tea polyphenols and diallyl trisulfide can
remarkably increase GSH S-transferase activity and GSH content.
Elevated liver MDA levels imply that enhanced peroxidation
causes tissue damage and the breakdown of antioxidant defense
mechanisms, thus preventing the formation of superabundant free
radicals (Hsu et al., 2009). The MDA level is widely used as a mar-
ker of free radical-mediated lipid peroxidation damage (Cuzzocrea
GSH (nmol/mg protein) MDA (nmol/mg protein) 8-OHdG (ng/mgprotein)
5.40 ± 0.64⁄ 1.90 ± 0.25⁄ 0.51 ± 0.019⁄
2.09 ± 0.66# 6.20 ± 0.65# 0.73 ± 0.033#
3.46 ± 0.51# 3.00 ± 0.35⁄# 0.71 ± 0.009#
4.34 ± 0.34⁄ 2.36 ± 0.28⁄ 0.61 ± 0.027⁄#
6.20 ± 0.36⁄ 2.13 ± 0.42⁄ 0.54 ± 0.014⁄
and Reiter, 2001; Aksu et al., 2007). In the current study, oral treat-
ment or pretreatment with allicin caused significantly decreased
MDA levels compared with the AA-treated group (Table 1, Table
2), which suggest that allicin can protect against AA-induced lipid
peroxidation in mice.
8-OHdG is a marker of oxidative damage, and mutations may
arise from the formation of 8-OHdG involving GC ? TA transver-
sions (Kasai, 1997; Moriya, 1993). Liping et al. (2007) showed that
AA treatment induces significantly increased intracellular genera-
tion of ROS and 8-OHdG formation. In the present research, the level
of 8-OHdG in the AA-treated group significantly increased com-
pared with the control group, in agreement with the findings of Lip-
ing et al. (2007). The present study also demonstrated that allicin
can significantly decrease the level of 8-OHdG in AA-treated liver
cell in vitro and in vivo (Tables 1 and 3). We also found that the high-
est dose of allicin (20 mg/kg b.w./day) had the best protective effect
(p < 0.05). The protective effect of a low allicin dosage (5 mg/kg) was
not significant compared with the AA group (p > 0.05).
Our observations confirmed the genotoxic capacity of AA, as
previously demonstrated in animal models and cells by other
researchers using other biomarkers as indicators for genetic dam-
age (Adler et al., 1988; Besaratinia and Pfeifer, 2004; Zamorano-
Ponce et al. 2006). AA not only decreased the GSH content and
SOD activity, but also increased the levels of MDA and 8-OHdG.
Besides, AA widespread presence has been uncovered in a range
of processed foods, leading to dietary exposure estimates of
0.38 lg/kg b.w./day in China, equivalent to 26.6 lg/kg of AA day
for an average 70 kg human male (Chen et al., 2008), which is rel-
atively low compared with the result reported by the Food and
Agricultural Organization/World Health Organization (FAO/
WHO). Our present study provides evidence that the toxicity of
AA was partially counteracted by the dietary allicin. The results
underline the important role of dietary antioxidants such as garlic
for nutritional prevention of certain pathological states linked to
oxidative stress.
AA is metabolized via epoxidation to glycidamide (GA) by the
cytochrome P450 (CYP 2E1) in vivo (Sumner et al., 1999; Besarat-
inia and Pfeifer, 2007; Settels et al., 2008). GA is believed to be
more toxic than AA, and is involved in the carcinogenic and muta-
genic effects of AA (Ghanayem et al., 2005). In our present study,
allicin demonstrated a significant protective effect against AA-in-
duced hepatocyte damage both in vitro and in vivo, the probable
mechanism may be because of the following two factors. The first
one involves blocking the epoxidation process of AA by inhibiting
P450 enzyme. Taubert et al. (2006) proved that allicin can inhibit
the cytochrome P450 2E1-dependent bioactivation of AA to GA.
The second one involves the reduction of oxidative stress in vivo.
In the present study, we found that allicin can reduce oxidative
stress by regulating the levels of MDA, 8-OHdG, SOD, and GSH.
6. Conclusion
In conclusion, the results of this study demonstrate for the first
time that allicin is effective in preventing AA-induced hepatocyte
 L. Zhang et al. / Food and Chemical Toxicology 50 (2012) 3306–3312
damage in vitro and in vivo. The protective effects of allicin may be
due to its ability to scavenge free radicals and its effective recovery
of the antioxidative defense system, and block the epoxidation pro-
cess of AA to GA by inhibiting P450 enzyme. The optimum allicin
dose for inhibiting AA-induced hepatocyte damage is 15 lM
in vitro and 20 mg/kg b.w./day in vivo. This is important when con-
sidering the possibility of using allicin or garlic as a dietary supple-
ment in diets for a beneficial application in the chemoprevention
the genotoxicity of AA.
Conflict of Interest
The authors declare that there are no conflicts of interest in the
present study.
Acknowledgements
This work was supported by the Fund of ‘‘National Natural Sci-
ence Fundation (31000750)’’, China Postdoctoral Science Founda-
tion Special Funded Project (201104527), National High
Technology Research and Development Program of China (‘‘863’’
Project, 2011AA100806), and Fund for Distinguished Young Schol-
ars of Heping Campus of Jilin University. Accordingly, the authors
gratefully acknowledge the fund supports.
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