Effect of S-allylcysteine on oxidant-antioxidant status during N-methyl-N'-nitro-N-nitrosoguanidine and saturated sodium chloride-induced gastric carcinogenesis in Wistar rats.

Abstract

We investigated the chemopreventive effect of S-allylcysteine (SAC), a water-soluble garlic constituent against gastric carcinogenesis induced in male Wistar rats by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and saturated sodium chloride (S-NaCl). The animals were divided into four groups of six animals. Rats in groups 1 and 2 were administered MNNG (200 mg/kg body weight) on days 0 and 14 as well as S-NaCl (1 mL/rat) three days during weeks 0 to 3, and thereafter placed on basal diet until the end of the experiment. Rats in group 2 in addition received SAC (200 mg/kg body weight) three times per week starting on the day following the first exposure to MNNG and continued until the end of the experimental period. Group 3 animals were given SAC alone as in group 2. Group 4 animals received basal diet and tap water throughout the experiment and served as the untreated control. The animals were sacrificed after an experimental period of 21 weeks. Measurement of lipid peroxidation and antioxidants of the glutathione redox cycle in the stomach tissue, liver and venous blood was used to monitor the chemopreventive potential of SAC. All animals that received MNNG and S-NaCl alone, developed tumours, identified histologically as squamous cell carcinomas. In the tumour tissue, diminished lipid peroxidation was accompanied by increase in reduced glutathione (GSH) and GSH-dependent enzymes, whereas in the liver and circulation, enhanced lipid peroxidation was associated with antioxidant depletion. Administration of SAC suppressed the incidence of MNNG+S-NaCl-induced gastric tumours as revealed by the absence of carcinomas. SAC ameliorated MNNG-induced decreased susceptibility of the gastric mucosa to lipid peroxidation, whilst simultaneously increasing the antioxidant status. In the liver and blood, SAC reduced the extent of lipid peroxidation and significantly enhanced antioxidant activities. We suggest that SAC exerts its chemopreventive effects by modulating lipid peroxidation and enhancing GSH-dependent antioxidants in the target organ as well as in the liver and blood.

Full text

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Asia Pacific J Clin Nutr 2003;12 (4): 488-494 488?
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Original Article

Effect of S-allylcysteine on oxidant-antioxidant
status during N-methyl-N’-nitro-N-nitrosoguanidine
and saturated sodium chloride-induced gastric
carcinogenesis in Wistar rats

Balaiya Velmurugan MPhil, Vaidhyanathan Bhuvaneswari MPhil and
Siddavaram Nagini PhD*


Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar-608 002, Tamil
Nadu, India.
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We investigated the chemopreventive effect of S-allylcysteine (SAC), a water-soluble garlic constituent against
gastric carcinogenesis induced in male Wistar rats by N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and
saturated sodium chloride (S-NaCl). The animals were divided into four groups of six animals. Rats in groups 1
and 2 were administered MNNG (200 mg/kg body weight) on days 0 and 14 as well as S-NaCl (1mL/rat) three
days during weeks 0 to 3, and thereafter placed on basal diet until the end of the experiment. Rats in group 2 in
addition received SAC (200 mg/kg body weight) three times per week starting on the day following the first
exposure to MNNG and continued until the end of the experimental period. Group 3 animals were given SAC
alone as in group 2. Group 4 animals received basal diet and tap water throughout the experiment and served as
the untreated control. The animals were sacrificed after an experimental period of 21 weeks. Measurement of
lipid peroxidation and antioxidants of the glutathione redox cycle in the stomach tissue, liver and venous blood
was used to monitor the chemopreventive potential of SAC. All animals that received MNNG and S-NaCl
alone, developed tumours, identified histologically as squamous cell carcinomas. In the tumour tissue,
diminished lipid peroxidation was accompanied by increase in reduced glutathione (GSH) and GSH-dependent
enzymes, whereas in the liver and circulation, enhanced lipid peroxidation was associated with antioxidant
depletion. Administration of SAC suppressed the incidence of MNNG+S-NaCl-induced gastric tumours as
revealed by the absence of carcinomas. SAC ameliorated MNNG-induced decreased susceptibility of the gastric
mucosa to lipid peroxidation, whilst simultaneously increasing the antioxidant status. In the liver and blood,
SAC reduced the extent of lipid peroxidation and significantly enhanced antioxidant activities. We suggest that
SAC exerts its chemopreventive effects by modulating lipid peroxidation and enhancing GSH-dependent
antioxidants in the target organ as well as in the liver and blood.

Key Words: gastric carcinogenesis, N-methyl-N’-nitro-N-nitrosoguanidine, chemoprevention, garlic, S-allylcysteine,
lipid peroxidation, antioxidants.
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Introduction
Chemoprevention by dietary constituents has emerged as a
cost-effective approach to control the incidence of gastric
cancer, the second most common malignancy worldwide,
and a major cause of mortality in Chennai, India.1,2 Gastric
cancer induced by the administration of N-methyl-N’-nitro-
N-nitrosoguanidine (MNNG) in Wistar rats which shows
similarities to human gastric tumours is an ideal model for
investigating the development of stomach cancer and the
effects of intervention by chemopreventive agents.3 In
previous reports from this laboratory, we demonstrated the
protective effects of neem leaf, garlic and lycopene in the
MNNG model.4,5
Garlic (Allium sativum Linn) has been extensively used as
an effective chemopreventive agent in a wide range of mali-
gnancies. Previously, we reported the antioxidant, hepato-
protective and anticarcinogenic effects of garlic in 7,12-

dimethylbenz[a]anthracene
buccal pouch (HBP) carcinogenesis as well as in MNNG-
induced rat stomach carcinogenesis.6,7 Recently, we
demonstrated induction of cellular differentiation by garlic
in the HBP model.8 S-Allylcysteine (SAC), a naturally
occurring, non-toxic, water-soluble, organosulfur com-
pound is considered as one of the important biologically
active constituents of garlic.9 SAC is recognized to
possess anti-inflammatory and antioxidant properties.10,11
(DMBA)-induced hamster

Correspondence address: Dr S Nagini, Dept. of Biochemistry
Faculty of Science, Annamalai University, Annamalainagar-
608 002, Tamil Nadu, India
Tel: +91-4144-238343 (Off)/238124 (Res);
Fax:+91-4144-238145/238080
E-mail: s_nagini@yahoo.com, nrgopal@satyam.net.in
Accepted 17 March 2003

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489 ?????????????? B Velmurugan, V Bhuvaneswari and S Nagini


SAC has come under extensive study in the light of its
anticancer effects both in vitro and in vivo.12 Recently,
we demonstrated a positive correlation between the anti-
oxidant properties of SAC and its chemopreventive effi-
cacy against DMBA-induced HBP carcinogenesis.13 We
have also documented the apoptosis-inducing effects of
SAC in the HBP model.14 Many phytochemicals are
known to exert their anticarcinogenic effects by
scavenging oxygen free radicals (OFR) and modulating
host antioxidant defence mechanisms.15 Previously, we
demonstrated that assay of OFR-induced lipid peroxi-
ation and antioxidant enzymes in the liver and blood in
addition to the target organ is a reliable method for
screening putative chemopreventive agents. 6,13,16,17 In the
present study, we have evaluated the chemopreventive
potential of SAC by measurement of lipid peroxidation
and the antioxidant status with respect to the glutathione
redox cycle in the stomach tissue, liver and venous blood
during MNNG+S-NaCl-induced gastric carcinogenesis.
?
??????????????????????
Animals
All the experiments were carried out with male Wistar
rats aged 6-8 weeks obtained from the Central Animal
House, Annamalai University, India. They were housed
six in a polypropylene cage and provided food and water
ad libitum. The animals were maintained under standard
conditions of temperature and humidity with an alter-
nating 12 hours light/dark cycle. All animals were fed
standard pellet diet (Mysore Snack Feed Ltd, Mysore,
India) and maintained in accordance with the guidelines
of the National Institute of Nutrition, Indian Council of
Medical Research, Hyderabad, India, and approved by the
ethical committee of Annamalai University.
Chemicals
Bovine serum albumin, 2-thiobarbituric acid, trichloro-
acetic acid, 2,4-dinitrophenylhydrazine (DNPH), reduced
glutathione (GSH), 5,5’-dithiobis(2-nitrobenzoic acid)
(DTNB), 1-chloro-2,4-dinitrobenzene (CDNB) and fla-
vine adenine dinucleotide (FAD) were purchased from
Sigma Chemical Company, St. Louis, USA. MNNG of
purity ≥97% was obtained from Fluka-Chemika-
Biochemika, Buchs, Switzerland. SAC of purity 99.9%
kindly provided by Wakunaga Pharmaceutical Co. Ltd.
(Hiroshima, Japan), was dissolved in distilled water
before use. All other reagents used were of analytical
grade.
Treatment schedule
Animals were randomized into experimental and control
groups and divided into four groups of six. The experi-
mental protocol for the present study is shown in Fig. 1.
Rats in group 1 were given MNNG (200 mg/kg body
weight) by intragastric intubation on days 0 and 14 as
well as S-NaCl (1mL/rat) every 3 days during weeks 0 to
3 (six times; on days 3,7,10,14,17,21).18 Rats in group 2
administered MNNG+S-NaCl as in group 1, in addition
received intragastric intubation of SAC (200 mg/kg body
weight) three times per week starting on the day
following the first exposure to MNNG (day 1) and
continued until the end of the experimental period.19
Group 3 animals were given SAC alone as in group 2 but















3













without MNNG and S-NaCl. Group 4 received basal diet
and tap water throughout the experiment and served as the
untreated control. All animals had free access to food and
water.
The experiment was terminated at 21 weeks and all
animals were killed by cervical dislocation after an
overnight fast. Fresh tissues were used for estimations.
Biochemical estimations were carried out in stomach
tissue, liver and blood samples of experimental and
control animals. For histopathological examination,
tissues were fixed in 10% formalin, embedded in paraffin
and 2-3 µm sections were cut on a rotary microtome and
stained with haematoxylin and eosin.
Preparation of hemolysate
Blood samples were collected in heparinised tubes and
plasma was separated by centrifugation at 1000 g for 15
minutes. After centrifugation, the buffy coat was removed
and the packed cells washed thrice with physiological
saline. 0.5 mL of erythrocytes was lysed with 4.5 mL of
hypotonic phosphate buffer, pH 7.4. The hemolysate was
separated by centrifuging at 2500 g for 15 min at 2°C.
Biochemical methodology
Lipid peroxidation as evidenced by the formation of
thiobarbituric acid reactive substances (TBARS) was
assayed in tissues by the method described by Ohkawa et
al.,20 in the plasma by the method of Yagi21 and in
erythrocytes by the method of Buege and Aust.22 The
pink coloured chromogen formed by the reaction of
2-thiobarbituric acid with the breakdown products of lipid
peroxidation was read at 535 nm. Reduced glutathione
(GSH) was determined by the method of Anderson23
based on the development of a yellow colour when DTNB
** ** **
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?????????????????????????????????????????????????????????????????????????????????????????????????????????????
** ** **
????????????????????????????????????????????????????????????????????????????????????????????????????????????????
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?????????????
?????????????
?????????????
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Week 1 2 3 4 5 21
Group
1
2
4
MNNG+S-NaCl
MNNG+S-NaCl +SAC
SAC
Control
SAC (200 mg/kg
body weight)
Basal diet and
tap water ?
MNNG 200mg/kg body weight i.g twice for 2 weeks.
* Saturated sodium chloride solution (S-NaCl) (1mL/rat i.g), six times
during weeks 1-3.
Figure 1. Experimental protocol?

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Chemoprevention of gastric carcinogenesis by S-allylcysteine 490
is added to compounds containing sulfhydryl groups.
Glutathione peroxidase (GPx) activity was assayed by the
method of Rotruck et al.,24 with modifications. A known
amount of enzyme preparation was incubated with
hydrogen peroxide in the presence of GSH for 10
minutes. The amount of hydrogen peroxide utilized was
determined by estimating GSH content by the method of
Anderson.23 The activity of glutathione S-transferase
(GST) was determined as described by Habig et al.,25 by
following the increase in absorbance at 340 nm using
CDNB as substrate. Glutathione reductase (GR) activity
was determined by the method of Carlberg and
Mannervik26 using oxidized glutathione as substrate and
FAD as cofactor.
The protein content was estimated by the method of
Lowry et al.27 Plasma ascorbic acid was estimated by the
method of Omaye et al.28 This involves oxidation of
ascorbic acid by copper followed by treatment with
DNPH to form the derivative bis 2,4-dinitrophenyl-
hydrazone that undergoes rearrangement to form a
product with an absorption maximum at 520 nm. Plasma
vitamin E was measured by the method of Baker et al.,29
on the basis of the reduction of ferric ions to ferrous ions
by α-tocopherol and the formation of a red coloured
complex with 2,2’-dipyridyl at 520 nm. Haemoglobin in
erythrocytes and hemolysate was measured according to
the method of Drabkin and Austin.30 Blood was diluted
in an alkaline medium containing potassium cyanide and
potassium ferricyanide. Haemoglobin oxidized to methe-
moglobin combines with cyanide to form cyan-
methemoglobin, which was measured at 540 nm.
Statistical analysis
Statistical analysis on the incidence of lesions was
performed using Fisher’s exact probability test. The body
weight and biochemical parameters were analysed using
ANOVA and the group means were compared by
Duncan’s multiple range test (DMRT). The results were
considered statistically significant if the P<0.05.


Table 1. Body weight, tumour incidence and histopathological changes in each group during MNNG + S-NaCl-
induced gastric carcinogenesis (mean ± SD; N = 6)












a Significantly different from group 4 P<0.05; b Significantly different from group 1 P<0.05 (Duncan’s multiple range test);
Parantheses – percentage of lesions



Results

Gross observations
Table 1 shows the mean body weights and the incidence
of gastric cancer in control and experimental animals in
each group. Rats in group 1 (MNNG+S-NaCl) showed a
tendency to be lower in body weight gain during the
experiment and the mean final body weights were
decreased compared with all other groups. Multiple
friable chalky white nodules with small cauliflower-like
growth were seen in the forestomach of group 1 animals.
The forestomach of group 2 animals revealed fewer and
smaller nodules without cauliflower like growth. No
obvious changes were observed in groups 3 and 4.
Histopathological observations
The incidence of gastric tumours in group 1 was 100 per
cent. No malignant neoplasms were observed in the stomach
in groups 2 through 4. Forestomach tumours induced by
MNNG+S-NaCl were squamous cell carcinomas with a
number of epithelial and keratin pearls. The cells showed
increased nuclear/cytoplasmic ratio, nuclear pleomorphism
and hyperchromatism (Fig. 2). One of the six animals treated









































Body weight Group Treatment
Initial Final
Hyperplasia Dysplasia Carcinoma
1. MNNG + S-NaCl
126.3 ± 4.2 249.7 ± 9.2a
6 (100) 6(100) 6 (100)
2. MNNG + S-NaCl + SAC
125.1 ± 3.8 270.5 ± 13.6b
3(50) 1(16.6) 0
3. SAC
123.7 ± 4.7 290.4 ± 10.3
- - 0
4. Control
121.4 ± 4.6 284.1 ± 10.4
- - 0
?
Figure 2. Photomicrograph showing squamous cell carcinoma with
extensive infiltration: Group 1 (H and E X 50)

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491 ?????????????? B Velmurugan, V Bhuvaneswari and S Nagini




































with MNNG and SAC (group 2) showed severe dysplasia
while the remaining exhibited normal keratinised stratified
squamous epithelium with mild dysplasia and hyperplasia of
lining epithelium (Fig. 3). The forestomach of rats in groups
3 and 4 showed normal lining of keratinised stratified
squamous epithelium with a limiting ridge demarcating the
forestomach from the glandular stomach lined by mucous
glands (Fig. 4,5).
Biochemical findings
Table 2 shows TBARS in the stomach tumour tissue in
group 1 to be the lowest among all groups. TBARS in
groups 2 and 3 were significantly higher than those in
group 1. In the liver and blood, TBARS were significantly































increased in group 1 compared with group 4. In groups 2
and 3, the values were significantly lower than in group 1.
Table 3 shows markedly higher levels of GSH and
activities of GPx, GST and GR in stomach tissues in
group 1 animals than in group 4. The levels were raised
in group 2 compared with groups 1 and 4. In group 3, the
levels were significantly higher compared with group 4.
The levels of antioxidants in the livers of control and
experimental animals in each group are presented in
Table 4. The levels of GSH and the activities of GSH-
dependent enzymes (GPx, GST and GR) in liver tissues
were significantly lower in group 1 compared to group 4.
The antioxidant levels in groups 2 and 3 were higher than
those of group 1.
Table 5 shows levels of GSH, vitamin C and vitamin E
in plasma and activities of GSH-dependent enzymes in
erythrocyte lysate of control and experimental animals in
each group. In group 1, the levels of GSH and the
activities of GSH-dependent enzymes as well as vitamin C
and vitamin E were found to be depleted compared to
group 4. The concentrations of GSH, vitamin C and
vitamin E and the activities of GPx, GST and GR in groups
2 and 3 were significantly higher compared to group 1.

Discussion
Effect of MNNG + S-NaCl on target tissue
Cell proliferation, which plays a key role in cancer
development, is associated with changes in OFR-induced
lipid peroxidation and the status of antioxidants that use
GSH as a substrate. Lipid peroxidation involved in the
regulation of cell division, is reported to be inversely














Table 2. TBARS in stomach, liver and blood in each group during MNNG+S-NaCl-induced gastric carcinogenesis
(mean ± SD; N = 6)

Group Treatment Stomach TBARS
(nmols/100mg protein)
Liver TBARS
(nmols/100mg protein)
Plasma TBARS
(nmols/ml)
Erythrocyte TBARS
(pmol/mg Hb)
1. MNNG + S-NaCl
89.2 ± 6.20d
202.8 ± 17.49a 5.01 ± 0.31a 3.78 ± 0.30a
2. MNNG + S-NaCl +
SAC
110.0 ± 9.41c
170.2 ± 12.82b 3.97 ± 0.14b 2.63 ± 0.15b
3. SAC
123.1 ± 9.20b
125.9 ± 10.57d 2.51 ± 0.13d 0.83 ± 0.08d
4. Control
134.6 ± 8.02a
139.9 ± 11.64c 2.96 ± 0.13c 1.41 ± 0.13c
Values not sharing a common superscript letter differ significantly at P<0.05 (Duncan’s multiple range test)

Figure 3. Photomicrograph showing stratified squamous
epithelial layer with mild dysplasia and invasion: Group 2 (H
and E X 50)
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Table 3. Glutathione concentration and glutathione-dependent enzyme activities in stomach tissues of rats in each group
during MNNG and S-NaCl-induced gastric carcinogenesis (mean ± SD; N = 6)

Group Treatment GSH (UA) GPx (UB) GST (UC) GR (UD)
1. MNNG + S-NaCl
0.73 ± 0.07b 21.38 ± 1.50b 1.63 ± 0.17b 46.20 ± 4.11b
2. MNNG + S-NaCl +
SAC
0.94 ± 0.08a
30.07 ± 2.04a
2.20 ± 0.18a 58.38 ± 4.02a
3. SAC
0.38 ± 0.03c
19.31 ± 1.35c 1.36 ± 0.15c 34.36 ± 2.31c
4. Control
0.30 ± 0.03d
13.59 ± 1.06d 0.93 ± 0.08d 26.18 ± 1.90d
Values not sharing a common superscript letter differ significantly at P<0.05 (Duncan’s multiple range test); Amg/g tissue; Bµmols of GSH
utilized/min/g protein; Cµmols of CDNB-GSH conjugate formed/min/mg protein; Dµmols of NADPH oxidized/h/mg protein.

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Chemoprevention of gastric carcinogenesis by S-allylcysteine 492




















related to cell proliferation with highly proliferating
tumours showing low levels of lipid peroxidation
compared to their normal counterparts.31 GSH, an
important non-protein thiol in conjunction with GPx, and
GR, plays a regulatory role in cell proliferation.32
Obrador et al.,33 observed a positive correlation between
enhanced synthesis of GSH and high rates of cell
proliferation in tumours. We postulate that diminished
lipid peroxidation combined with enhanced GSH-
dependent antioxidant capacity of MNNG+S-NaCl
induced gastric tumours facilitates cell proliferation
offering a selective growth advantage to tumour cells over
their surrounding normal cells.

Effect of MNNG + S-NaCl on liver and blood
In contrast to decreased lipid peroxidation in tumour
tissues, we found enhanced lipid peroxidation associated
with antioxidant depletion in the liver and blood of
tumour bearing animals. We observed similar results
during 4-nitroquinoline-1-oxide-induced rat tongue carci-
nogenesis and DMBA-induced HBP carcinogenesis in
addition to experimental rat stomach carcinoge-
nesis.6,17,34,35 These findings are also consistent with the
observation that lipid peroxidation is decreased in rapidly
proliferating tumours compared
counterparts.36,37 Thus the tumour and host tissue appear
to comprise two separate metabolic compartments with
respect to their susceptibility to lipid peroxidation.














d µmoles of NADPH oxidized/h/mg protein.
to their normal




















MNNG is known to produce toxic and highly diffusible
OFR capable of producing deleterious effects in the liver
and blood.38 Free radical-mediated oxidative stress has
been implicated in the hepatotoxic effects of MNNG.39
The erythrocytes are also highly susceptible to oxidative
damage due to the high content of iron and polyun-
saturated fatty acids and their role as oxygen
transporters.40 Thus enhanced lipid peroxidation in the
liver and blood of tumour-bearing rats reflects over-
production of OFR in these tissues exacerbated by a
compromised GSH redox cycle.
The liver, which contains high amounts of GSH
supplies it to various extrahepatic tissues including the
erythrocytes and plasma. Tumour cells have been
reported to sequester essential antioxidants such as GSH
to meet the demands of a growing tumour.5 The
deficiency of antioxidants in the liver and blood of
tumour-bearing animals may be ascribed to increased
utilization to scavenge lipid peroxides as well as
sequestration by tumour cells.
Effect of SAC on MNNG + S-NaCl-induced oxidant-
antioxidant changes
Administration of SAC significantly suppressed the
incidence of MNNG and S-NaCl-induced gastric cancer
as revealed by the absence of carcinomas. The results of
the present study substantiate the anticarcinogenic activity
of SAC reported by us as well as by other workers. SAC














Figure 4. Photomicrograph of the stomach showing
hyperkeratosis and papillomatosis: Group 3 (H and E X 50)
?
Table 4. Glutathione concentration and glutathione-dependent enzyme activities in liver tissues of rats in each group
during MNNG and S-NaCl-induced gastric carcinogenesis (mean ± SD; N= 6)
Group Treatment GSH (UA) GPx (UB) GST (UC) GR (UD)
1. MNNG + S-NaCl
0.35 ± 0.02d 7.56 ± 0.52d
0.93 ± 0.05d
20.55 ± 1.49d
2. MNNG + S-NaCl + SAC
0.42 ± 0.02c
10.39 ± 1.09c
1.67 ± 0.13c
27.51 ± 1.58c
3. SAC
0.58 ± 0.03a
18.04 ± 1.51a
2.26 ± 0.14a
37.34 ± 1.41a
4. Control
0.53 ± 0.04b
14.28 ± 1.50b
1.91 ± 0.13b
31.57 ± 1.65b
Values not sharing a common superscript letter differ significantly at P<0.05 (Duncan’s multiple range test)
A mg/g tissue; B- µmoles of GSH utilized/min/g protein; C µmoles of CDNB-GSH conjugate formed/min/mg protein.
?
?
Figure 5. Photomicrograph of the stomach showing normal
keratinised stratified squamous epithelium: Group 4
(H and E X 50)