Chemico-Biological Interactions 194 (2011) 4047

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Chemico-Biological Interactions
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Protective role of sinapic acid against arsenic Induced toxicity in rats

L. Pari , A. Mohamed Jalaludeen
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar 608002, Tamil Nadu, India

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

Article history: Arsenic compounds are classied as toxicants and human carcinogens. Environmental exposure to
Received 7 June 2011 arsenic imposes a big health issue worldwide. Sinapic acid is a phenylpropanoid compound and is found
Received in revised form 5 August 2011 in various herbal materials and high-bran cereals. It has been reported that sinapic acid has antioxidant
Accepted 6 August 2011
efcacy as metal chelators due to the orientation of functional groups. However, it has not yet been
Available online 16 August 2011
examined in experimental animals. In light of this fact, the purpose of this study was to characterize
the protective role of sinapic acid against arsenic induced toxicity in rats. Rats were orally treated with
Keywords:
arsenic alone (5 mg/kg body weight (bw)/day) plus sinapic acid at different doses (10, 20 and 40 mg/kg
Arsenic
Sinapic acid
bw/day) for 30 days. Hepatotoxicity was measured by the increased activities of serum hepatospecic
Lipid peroxidation enzymes namely aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma glutamyl
Oxidative stress transferase, lactate dehydrogenase and total bilirubin along with increased elevation of lipid peroxidative
Liver markers, thiobarbituric acid reactive substances, lipid hydroperoxides, protein carbonyl content and
conjugated dienes. The toxic effect of arsenic was also indicated by signicantly decreased activities of
enzymatic antioxidants like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-trans-
ferase, glutathione reductase and glucose-6-phosphate dehydrogenase along with non-enzymatic antiox-
idant like reduced glutathione. Administration of sinapic acid exhibited signicant reversal of arsenic
induced toxicity in hepatic tissue. The effect at a dose of 40 mg/kg bw/day was more pronounced than
the other two doses (10 and 20 mg/kg bw/day). All these changes were supported by reduction of arsenic
concentration and histopathological observations of the liver. These results suggest that sinapic acid has a
protective effect over arsenic induced toxicity in rat.
2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Several studies have demonstrated that the liver is an important

Arsenic, one of the most harmful metalloids, is ubiquitous in the
earths crust and biosphere [1]. Besides the natural sources, re-
peated uses of arsenic as herbicides, insecticides and rodenticides
are drastically contaminating drinking water [2]. Episodes of ar-
senic poisoning caused by arsenic contaminated water have al-
ready been reported in both developing and developed countries
especially in India, Bangladesh and the United States [3,4]. Human
exposure to arsenic compounds mainly occur either in workplaces,
e.g. in smelting industries, coal red power plants, cosmetic indus-
tries, agriculture, etc. or through arsenic contaminated food and
drinking water [5,6].
Arsenic is the rst metalloid to be identied as a human carcin-
ogen. In addition epidemiological studies have revealed that
chronic exposure to arsenic has been linked with myriad of non-
cancer human diseases, such as diabetes, atherosclerosis, cardio-
vascular diseases and hyperkeratosis [79]. Since arsenic targets
ubiquitous enzyme reactions, it affects nearly all organ systems
in animals and humans [10].
Corresponding author. Tel.: +91 09345 168663; fax: +91 04144 238145.
E-mail address: jayampari@gmail.com (L. Pari).
target organ for arsenic toxicity and its importance as an organ for
arsenic biotransformation is well established in its enzymatic
reactions [1113]. The exact cellular mechanisms by which arsenic
produces hepatotoxicity in vivo are unknown, but the advance-
ment of research over the last decade demonstrated that oxidative
stress is the key contributor in arsenic induced hepatic injury as it
is known to produce reactive oxygen species, namely superoxide
(O  
2 ), hydroxyl ( OH), peroxyl radicals (ROO ) and hydrogen perox-
ide (H2O2) [1417]. Furthermore, arsenic exposure was shown to
depress the antioxidant defense system [18] leading to the oxida-
tive damage of cellular macromolecules including DNA, proteins
and lipids [19] that cause damage at the membrane, cell and tissue
levels which ultimately wreak havoc to the biological system [20].
Oxidative stress in the liver may lead to hepatocellular injuries, hy-
dropic, fatty degeneration, progressive brosis and more critical
consequences. Many investigators have conrmed that arsenic in-
duces hepatic oxidative stress and the use of antioxidants have re-
cently been considered as therapeutic agents to counteract liver
damages [21] in order to protect the cellular machinery from per-
oxidative injury inicted by ROS [22].
Sinapic acid is a cinnamic acid derivative, which possesses 3,5-
dimethoxyl and 4-hydroxyl substitutions in the phenyl group of

0009-2797/$ - see front matter 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2011.08.004
 L. Pari, A. Mohamed Jalaludeen / Chemico-Biological Interactions 194 (2011) 4047
cinnamic acid (Fig. 1). Sinapic acid is widely distributed in the
plant kingdom and is obtained from various sources such as rye,
fruits and vegetables [23,24]. Sinapic acid has already been phar-
macologically evaluated for its potent antioxidant [25,26], anxio-
lytic [27], anti-inammatory [28], peroxynitrite scavenging
effects [29] and neuroprotective effects [30]. Moreover, bioprocess-
ing of wheat bran in whole wheat bread increases the bioavailabil-
ity of phenolic acids in men and exerts antiinammatory effects
ex vivo [31].
This is the rst study attempting to explore whether sinapic
acid, when administered at different doses (10, 20 and 40 mg/
kg bw/day) ameliorate toxic effects of arsenic and bring hepatic
recovery in terms of biochemical and histological variables.
2. Materials and methods
2.1. Drug and chemicals
Sinapic acid, sodium arsenite, 2-thiobarbituric acid, butylated
hydroxytoluene, reduced glutathione, 2,20 -dipyridyl, xylenol or-
ange, 2,20 -dinitro phenyl hydrazine, c-glutamyl-p-nitroanilide,
5,50 -dithiobis-2-nitrobenzoic acid, trichloroacetic acid, phenazine
methosulphate, nitroblue tetrazolium, reduced nicotinamide ade-
nine dinucleotide, 1-chloro 2,4-dintrobenzene were obtained from
Sigma Chemical Co. (St. Louis, MO, USA). The rest of the chemicals
utilized were obtained from a local rm (Himedia Laboratories Ltd.,
Mumbai, India) and were of analytical grade.
2.2. Animals
Adult male albino rats of Wistar strain (190220 g) were used
for the experiment. The animals were housed in polypropylene
cages and maintained in 12-h light/12-h dark cycle, 50% humidity
and 25 2 C. The animals had free access to standard pellet diet
(M/S. Pranav Agro Industries Ltd., Bangalore, India) and water ad
libitum. This study was approved (Vide. No. 740, 2010) by Institu-
tional Animal Ethics Committee of Annamalai University and the
study was conducted in accordance with the Guide for the Care
and Use of Laboratory Animals.
2.3. Preparation of arsenic and sinapic acid
Arsenic dose selection in the present study was based on the
previous reports [32,33]. Arsenic as sodium arsenite at a dose of
5 mg/kg bw/day was dissolved in water and treated orally. Sinapic
acid was dissolved in corn oil and each rat received daily 1 ml at a
dose of 10, 20 and 40 mg/kg bw/day administrated orally just after
exposure to arsenic by intragastric intubation throughout the
experimental period.
2.4. Experimental time line
The animals were randomly divided into six groups of six rats in
each group. Group I: Normal control rats (vehicle treated), Group
II: Normal rats were orally administered with sinapic acid
(40 mg/kg bw/day), Group III: Normal rats received arsenic as
Fig. 1. Structure of sinapic acid.
41
sodium arsenite orally (5 mg/kg bw/day) and Group IVVI: Normal
rats received arsenic with co-administration of sinapic acid orally
at different doses (10, 20 and 40 mg/kg bw/day) for 30 days. Dur-
ing the experimental period, changes in body weight gain, food
and water consumption were measured everyday.
At the end of the experimental period, animals in different
groups were sacriced by cervical decapitation. Blood samples
were collected in two different tubes, i.e. one is heparinised, for
plasma and another without heparin for serum collection. Serum
and plasma were separated by centrifugation and used for various
biochemical estimations.
2.5. Preparation of tissue homogenate
Rats were anesthetized by ketamine hydrochloride (30 mg/
kg bw, intramuscularly) and the animals were sacriced by cervi-
cal decapitation. The liver was quickly excised, rinsed with saline,
blotted dry on lter paper, weighed and then 10% (w/v) tissue
homogenates were prepared in buffer (0.025 M TrisHCl buffer
(pH 7.4) using a tissue homogenizer (Model No: RQT 127A, Teon
Remi, India) at 8000g for few min.
2.6. Activities of serum marker enzymes
The activities of serum aspartate aminotransferase (AST), ala-
nine aminotransferase (ALT), alkaline phosphatase (ALP) and lac-
tate dehydrogenase (LDH) were assayed using commercially
available diagnostic kits (Sigma diagnostics (I) Pvt. Ltd., Baroda,
India).
Gamma glutamyl transferase (GGT) activity was estimated by
the method of Rosalki et al. [34]. 0.05 ml of serum was made up
to 0.5 ml by the addition of L-gamma-glutamyl-p-nitroanilide sub-
strate and was incubated at 37 C for 30 min. The standard tubes
taken at a concentration ranging from 0.1 to 0.4 lmol were also
incubated as above. The reaction was arrested by the addition of
2.5 ml of 10% acetic acid. Simultaneously, a control without serum
was also subjected to the above treatment and incubation except
that serum was added after arresting the reaction. The yellow color
developed after the addition of acetic acid was measured at
410 nm against the blank using spectrophotometer.
Based on van den Berg reaction, serum bilirubin was estimated
by the method of Malloy and Evelyn [35]. 0.2 ml of serum was di-
luted to 2 ml with distilled water in two tubes marked as test and
blank. To the test, 0.5 ml of the diazo reagent and to the blank,
0.5 ml of 1.5% HCl was added. Finally to both tubes, 2.5 ml of meth-
anol was added and the tubes were kept at room temperature for
30 min. The color developed was read at 540 nm. For a standard
curve, one in ve dilutions of stock standard in methanol was
made to obtain a solution containing 2 mg/100 ml.
2.7. Estimation of lipid peroxidation
Thiobarbituric acid reactive substances (TBARS) in tissues were
estimated by the method of Niehius and Samuelson [36]. The tis-
sue homogenate was prepared in TrisHCl buffer (pH 7.5). 1 ml
of the tissue homogenate was treated with 2 ml of TBATCAHCl
reagent and mixed thoroughly. The mixture was kept in a boiling
water bath for 15 min. After cooling, the tubes were centrifuged
for 10 min and the supernatant was taken for measurement. A ser-
ies of standard solutions were also treated in a similar manner. The
absorbance of chromophore was read at 535 nm against the re-
agent blank.
Lipid hydroperoxides (LOOH) in tissues were estimated by the
method of Jiang et al. [37]. 1.8 ml of the Fox reagent was mixed
with 0.2 ml of the tissue homogenate. Then incubated for 30 min
at room temperature and read at 560 nm.
42 L. Pari, A. Mohamed Jalaludeen / Chemico-Biological Interactions 194 (2011) 4047
Protein carbonyl content (PCC) in tissues was estimated by the
method of Levine et al. [38]. The tissue homogenate was centri-
fuged at 10,000g for 20 min to separate cytosol. To 0.5 ml of cyto-
solic fraction, 0.5 ml of TCA was added to precipitate the proteins.
After precipitation, the sample was treated with 0.5 ml of DNPH
and stand for 1 h in room temperature (vortex the samples every
1015 min). Then the pellet was washed thrice with 1 ml of etha-
nolethyl acetate mixture. The pellet was dissolved in 1 ml of gua-
nidine hydrochloride and the color developed was read at 366 nm.
Conjugated dienes (CD) were estimated by the method of Rao
and Recknagel [39]. To, 1.0 ml of tissue homogenate, 5.0 ml of chlo-
roformmethanol reagent (2:1v/v) was added, mixed thoroughly
and centrifuged for 5 min. To this, 1.5 ml of cyclohexane was added
and the absorbance was read at 233 nm against a cyclohexane
blank. The amount of CD formed was calculated using a molar
extinction coefcient of 2.52  104 cm1. The concentration of
CD was expressed as mmol/100 g tissue. In plasma, CD was mea-
sured the ratio of absorbance at 240 and 214 nm.
2.8. Assay of enzymatic and non-enzymatic antioxidants
Superoxide dismutase (SOD) activity was determined by the
method of Kakkar et al. [40]. 0.5 ml of tissue homogenate was di-
luted to 1 ml with water. Then added 2.5 ml of ethanol and
1.5 ml of chloroform (all the reagents were chilled). This mixture
was shaken for 1 min at 4 C and then centrifuged. The enzyme
activity in the supernatant was determined. The assay mixture
contained 1.2 ml of sodium pyrophosphate buffer (0.025 M, pH
8.3), 0.1 ml of 186 lM PMS, 0.3 ml of 30 lM NBT, 0.2 ml of
780 lM NADH, appropriately diluted enzyme preparation and
water in a total volume of 3 ml. Reaction was started by the addi-
tion of NADH. After incubation at 30 C for 90 s, the reaction was
stopped by the addition of 1 ml glacial acetic acid. The reaction
mixture was stirred vigorously and shaken with 4 ml of n-butanol.
The intensity of the chromogen in the butanol layer was measured
at 560 nm against butanol blank. A system devoid of enzyme
served as control.
The activity of catalase (CAT) was determined by the method of
Sinha [41]. To 0.9 ml of phosphate buffer, 0.1 ml of tissue homog-
enate and 0.4 ml of H2O2 were added. After 60 s, 2 ml of dichro-
mate acetic acid reagent was added. The tubes were kept in a
boiling water bath for 10 min and the color developed was read
at 620 nm.
The activity of glutathione peroxidase (GPx) was estimated by
the method of Rotruck et al. [42]. To 0.2 ml of Tris buffer, 0.2 ml
of EDTA, 0.1 ml of sodium azide and 0.5 ml of tissue homogenate
were added. To the mixture, 0.2 ml of glutathione followed by
0.1 ml of H2O2 was added. The contents were mixed well and incu-
bated at 37 C for 10 min along with a tube containing all the re-
agents except sample. After 10 min the reaction was arrested by
the addition of 0.5 ml of 10% TCA, centrifuged and the supernatant
was assayed for glutathione by the method of Ellman.
The glutathione-S-transferase (GST) activity was determined
spectrophotometrically by the method of Habig et al. [43]. The
reaction mixture contained 1 ml of phosphate buffer, 0.1 ml of
CDNB, 0.1 ml of tissue homogenate and 0.7 ml of distilled water.
The reaction mixture was incubated at 37 C for 5 min and then
the reaction was started by the addition of 0.1 ml of 30 mM gluta-
thione. The absorbance change was read at 340 nm for 5 min. Reac-
tion mixture without the enzyme was used as the blank.
Glutathione reductase (GR) that utilizes NADPH to convert
GSSG to the reduced form was assayed by the method of Horn
and Burns [44]. The reaction mixture containing 1 ml of phosphate
buffer, 0.5 ml of EDTA, 0.5 ml of GSSG and 0.2 ml of NADPH was
made up to 3 ml with water. After the addition of 0.1 ml of tissue
homogenate, the change in optical density at 340 nm was moni-
tored for 2 min at 30 s intervals.
Glucose-6-phosphate dehydrogenase (G6PD) was assayed by
the method of Beutler [45]. The incubation mixture contained
1 ml of buffer, 0.1 ml of MgCl2, 0.1 ml of NADP+, 0.5 ml of PMS,
0.4 ml of 2,6-dichlorophenol indophenol dye solution and the re-
quired amount of the enzyme extract. The mixture was allowed
to stand at room temperature for 10 min to permit the oxidation
of endogenous materials. The reaction was initiated by the addition
of 0.5 ml of glucose-6-phosphate. The absorbance was read at
640 nm against water blank at 1 min intervals for 35 min in a
spectrophotometer.
Protein was determined by the method of Lowry et al. [46].
0.5 ml of tissue homogenate was precipitated with 0.5 ml of 10%
TCA, centrifuged for 10 min, precipitate was dissolved in 1 ml of
0.1 N NaOH. 0.1 ml of aliquot was taken and made up to 1 ml with
distilled water. Then was added 4.5 ml of alkaline copper reagent
and allowed standing at room temperature for 10 min. After incu-
bation, 0.5 ml of FolinCiocalteau reagent was added and the blue
color developed was read after 20 min at 620 nm. A standard curve
was obtained using BSA.
Reduced glutathione (GSH) was determined by the method of
Ellman [47]. 0.5 ml of tissue homogenate was mixed with 10%
TCA in a 1:1 ratio and then centrifuged for 10 min at 5000 rpm.
The clear supernatant was then mixed with 2 ml of phosphate buf-
fer and 0.5 ml of DTNB. After incubation for 10 min, the absorbance
was measured at 412 nm. A reagent blank with 1 ml of distilled
water and standards containing 20100 lg glutathione were pro-
cessed similarly along with test samples.
2.9. Determination of arsenic concentration
For determination of arsenic in the liver, 1 g of tissues was di-
gested with nitric acid in a microwave oven. After digestion, ar-
senic was continuously preconcentrated and determined by
ame atomic absorption spectrophotometry. A Perkin-Elmer
5000 atomic absorption spectrometer furnished with arsenic hol-
low-cathode lamp (lamp current 4 mA) was used to determine
the arsenic concentration. The instrument was set at 228.8 nm
with a slit width of 0.5 nm. The acetylene ow rate was 2.0 l/min
and an airow rate of 17.0 l/min was employed to ensure an oxi-
dizing ame. Arsenic estimation is based on the concentration of
the element which will produce a signal/noise ratio of 3. Thus,
the detection limit considers both the signal amplitude and the
base line noise and is the lowest concentration which can be
clearly differentiated from zero. The specic limit of detection
(i.e. actual concentration limit of detection for arsenic analysis) is
about 2 mg/g tissue.
2.10. Histopathological studies
For qualitative analysis of liver histology, the tissue samples
were xed for 48 h in 10% formalinsaline and dehydrated by pass-
ing successfully in different mixture of ethyl alcohol, water,
cleaned in xylene and embedded in parafn. Sections of the tissues
(56 lm thick) were prepared by using a rotary microtone and
stained with hematoxylin and eosin dye, which was mounted in
a neutral deparafned xylene medium for microscopical observa-
tions. Six rats from each group were sacriced for analyzing the
hepatic histological examinations.
2.11. Statistical analysis
Data presented as means SD and subjected to statistical signif-
icance were evaluated by one way analysis of variance (ANOVA)
using SPSS Version 13.0 (SPSS, Cary, NC, USA) and the individual
 L. Pari, A. Mohamed Jalaludeen / Chemico-Biological Interactions 194 (2011) 4047 43

comparisons were obtained by Duncans multiple range test TBARS

(DMRT). Values were considered statistically signicant when 20 b
p < 0.05 [48].

nMol/g tissue
15
c
a a
3. Results 10

5
Table 1 depicts the effect of arsenic and sinapic acid on food and
water intake, body weight gain and organ-body weight ratio (%) in 0
control and experimental rats. In arsenic treated rats, water and Control Sinapic acid Arsenic Arsenic
(40mg/k.g) (5mg/k.g) (5mg/k.g) +
pellet diet consumption were signicantly (P < 0.05) decreased Sinapic
Groups
with decrease in body weight gain. A signicant (P < 0.05) increase acid(40mg/k.g)

in organ body weight ratio was noted in arsenic treated rats. No

signicant changes were observed between control and sinapic
acid treated rats. All these changes induced by arsenic intoxication
were signicantly (P < 0.05) improved on oral administration of
sinapic acid in a dose related manner.
Table 2 shows the levels of serum hepatic marker enzymes and
bilirubin in control and experimental rats. Oral administration of
arsenic caused abnormal liver function in rats. In arsenic treated
rats, the activities of serum hepatospecic enzymes, such as serum
aminotransferase, alanine aminotransferase, alkaline phosphatase,
lactate dehydrogenase, gamma glutamyl transferase and the level
of bilirubin were signicantly (P < 0.05) increased, when compared
with control rats. But, oral administration of sinapic acid (40 mg/
kg bw/day) to normal rats did not show any signicant (P < 0.05)
effect on hepatic markers. Treatment of sinapic acid (40 mg/
kg bw/day) with arsenic signicantly (P < 0.05) decreased the lev-
els of serum hepatic markers and bilirubin. Restoration of hepatic
marker enzymes and bilirubin was maximum in the higher dose le-
vel (40 mg/kg bw/day) of sinapic acid when compared to the other
two doses (10 and 20 mg/kg bw/day). Based on these ndings,
40 mg/kg bw of sinapic acid was xed as an effective dose and used
for further biochemical investigations.
The changes in the levels of hepatic lipid peroxidation, hydro-
peroxides, protein carbonyl content and conjugated dienes in con-
trol and experimental rats are shown in Figs. 25. The levels of
Fig. 2. Changes in the level of thiobarbituric acid reactive substance in control and
experimental rats. Values are given as mean SD for six rats in each group. Values
not sharing a common superscript letter (ac) differ signicantly at P < 0.05
(DMRT).
thiobarbituric acid reactive substances, lipid hydroperoxides, pro-
tein carbonyl content and conjugated dienes were signicantly in-
creased (p < 0.05) in arsenic-treated rats when compared with
normal control rats. Oral administration of sinapic acid (40 mg/
kg bw/day) along with arsenic signicantly lowered the levels of
TBARS, LOOH, PCC and CD in the liver of rats when compared to ar-
senic-treated rats.
Table 3 illustrates the activities of enzymatic antioxidants
namely superoxide dismutase, catalase, glutathione peroxidase,
glutathione-S-transferase, glutathione reductase and glucose-6-
phosphate dehydrogenase in the liver of control and experimental
rats. A signicant (P < 0.05) depletion in the activities of enzymatic
antioxidants in arsenic treated rats was observed. Treatment of
sinapic acid along with arsenic increased the levels of enzymatic
antioxidants in the liver.
Fig. 6 shows the changes in the levels of hepatic non-enzymatic
antioxidants namely reduced glutathione in the liver of control and
experimental rats. A signicant (P < 0.05) decrease in the levels of
reduced glutathione was noticed in rats treated with arsenic when

Table 1
Body weight, organ weight, food and water intake in control and experimental rats.

Groups Body weight (g) Organ-body weight

ratio (%)
Initial Final Food intake Water intake Liver
(g/100 g body weight/day) (mL/rat/day)
Control rats 191.00 19.24 211.00 21.35 13.34 1.05a 21.17 2.14a 2.97 0.28a
Normal rats + sinapic acid (40 mg/kg) 194.00 19.74 213.00 21.18 12.65 0.98a 19.28 1.96a 3.43 0.32a
Arsenic (5 mg/kg) 196.00 21.14 163.00 13.61 9.27 0.86b 13.69 1.34b 4.42 0.47b
Arsenic + sinapic acid (10 mg/kg) 199.00 19.18 182.00 17.24 9.63 0.87 bc 14.84 1.36bc 4.12 0.38b
Arsenic + sinapic acid (20 mg/kg) 200.00 21.80 187.00 16.93 9.44 0.79 c 14.06 1.72c 3.51 0.36bc
Arsenic + sinapic acid (40 mg/kg) 194.00 19.18 213.00 21.21 11.97 1.04d 18.46 1.72a 4.19 0.41ac

Values are given as mean SD from six rats in each group. Values not sharing a common superscript letter (ad) differ signicantly at P < 0.05 (DMRT).

Table 2
Changes in the activities of serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma glutamyl
transferase (GGT) and level of bilirubin in control and experimental rats.

Groups Control rats Normal rats + sinapic acid Arsenic Arsenic + sinapic acid Arsenic + sinapic acid Arsenic + sinapic acid
(40 mg/kg) (5 mg/kg) (10 mg/kg) (20 mg/kg) (40 mg/kg)
AST (IU/L) 60.80 4.44a 65.83 3.62a 108.20 7.17b 97.24 5.30c 84.39 3.65d 75.50 4.23e
ALT (IU/L) 24.95 1.96a 23.05 1.72a 48.51 3.72b 41.39 3.25c 37.79 1.83d 29.86 1.92e
ALP (IU/L) 76.61 5.83a 73.59 4.52a 122.92 9.50b 103.61 7.73c 93.93 5.83d 85.57 6.38e
LDH (IU/L) 112.79 9.81a 114.26 9.93a 157.23 13.26b 148.31 12.67c 139.45 11.83d 129.87 10.56e
GGT (IU/L) 0.62 0.05a 0.63 0.04a 0.98 0.09b 0.90 0.07c 0.82 0.06d 0.72 0.04e
Bilirubin (mg/dL) 0.45 0.03a 0.43 0.04a 0.94 0.07 b 0.86 0.06 c 0.79 0.05d 0.52 0.04e

Values are given as mean SD from six rats in each group. Values not sharing a common superscript letter (ae) differ signicantly at P < 0.05 (DMRT).
44 L. Pari, A. Mohamed Jalaludeen / Chemico-Biological Interactions 194 (2011) 4047

LHP 6 GSH
2
a a
c

nMol/g tissue
b
nMol/g tissue

1.5
c 4
a a
1 b
2
0.5

0 0
Control Sinapic acid Arsenic Arsenic Control Sinapic acid Arsenic Arsenic
(40mg/k.g) (5mg/k.g) (5mg/k.g) + (40mg/k.g) (5mg/k.g) (5mg/k.g) +
Sinapic Sinapic
Groups acid(40mg/k.g) Groups acid(40mg/k.g)

Fig. 3. Changes in the level of lipid hydroperoxides in control and experimental Fig. 6. Changes in the levels of reduced glutathione in control and experimental
rats. Values are given as mean SD for six rats in each group. Values not sharing a rats. Values are given as mean SD for six rats in each group. Values not sharing a
common superscript letter (ac) differ signicantly at p < 0.05 (DMRT). common superscript letter differ signicantly at p < 0.05 (DMRT).

CD Concentration of arsenic in liver

120 b 600
105 b
nMol/mg tissue

g/g wet tissue

90 a a 500
75 400
c
60 300
45
30 200
15 100 a a
0 0
Control Sinapic acid Arsenic Arsenic
(40mg/k.g) (5mg/k.g) (5mg/k.g) + Control Sinapic acid Arsenic Arsenic
Sinapic (40mg/k.g) (5mg/k.g) (5mg/k.g) +
Groups acid(40mg/k.g) Sinapic
Groups acid(40mg/k.g)

Fig. 4. Changes in the level of conjugated dienes in control and experimental rats.
Fig. 7. Concentration of arsenic in liver of control and experimental rats. Values are
Values are given as mean SD for six rats in each group. Values not sharing a
given as mean SD for six rats in each group. Values not sharing a common
common superscript letter (ac) differ signicantly at p < 0.05 (DMRT).
superscript letter differ signicantly at p < 0.05 (DMRT).

PCC cant increase in the concentration of arsenic in liver tissue. How-

8 b ever, sinapic acid restored the elevated levels signicantly to
7
within normal range in these animals when compared to their
6
respective control groups.
nMol/mg

5 c
4 a a Histopathological studies showed that arsenic administration
3 induces the pathological changes in the liver. The liver of control
2
rats (Fig. 8a) and sinapic acid (Fig. 8b) treated rats showed a nor-
1
0 mal architecture of the liver. Arsenic exposure resulted in changes
Control Sinapic acid Arsenic Arsenic in liver architecture as indicated by portal triad with mild inam-
(40mg/k.g) (5mg/k.g) (5mg/k.g) +
Sinapic mation, cell inltration, focal necrosis and giant cell formation
Groups acid(40mg/k.g) (Fig. 8c). Arsenic along with sinapic acid administration (Fig. 8d)
showed near normal hepatocytes with mild portal inammation.
Fig. 5. Changes in the level of protein carbonyl content in control and experimental
rats. Values are given as mean SD for six rats in each group. Values not sharing a
common superscript letter (ac) differ signicantly at p < 0.05 (DMRT). 4. Discussion

The outcome of the present study revealed the protective role of

compared to control rats. Treatment of sinapic acid (40 mg/kg bw/
sinapic acid against arsenic induced toxicity in rats. Reduction in
day) along with arsenic restored the levels of reduced glutathione
body weight is used as an indicator for the deterioration of the
to near normal.
rats general health status. In addition to body weight, we assessed
The concentration of arsenic in the liver has been depicted in
the changes in liver weight of the rats during the experimental
Fig. 7. Arsenic administration to normal rats resulted in a signi-
periods.

Table 3
Changes in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (GST), glutathione reductase (GR) and glucose-
6-phosphate dehydrogenase (G6PD) in control and experimental rats.

Groups Control rats Normal rats + sinapic acid (40 mg/kg) Arsenic (5 mg/kg) Arsenic + sinapic acid (40 mg/kg)
SOD (Units/mg protein) 7.83 0.51a 8.27 0.65a 5.28 0.42b 7.08 0.06c
CAT (lmol/(min mg protein)) 92.24 5.87a 96.17 5.28a 55.14 4.27b 78.32 5.87c
GPX (lg/(min mg protein)) 9.68 0.66a 9.81 0.69a 5.32 0.43b 8.89 0.70c
GST (lmol/(min mg protein)) 7.82 0.51a 8.23 0.57a 5.75 0.23b 7.12 0.42c
GR (nmol/(min mg protein)) 0.47 0.03a 0.49 0.04a 0.31 0.02b 0.40 0.03c
G6PD (nmol/(min mg protein)) 2.05 0.12a 2.13 0.17a 1.49 0.08b 1.81 0.10c

Values are given as mean SD from six rats in each group. Values not sharing a common superscript letter (ac) differ signicantly at P < 0.05 (DMRT).
 L. Pari, A. Mohamed Jalaludeen / Chemico-Biological Interactions 194 (2011) 4047
Fig. 8. Hematoxylin and eosin-stained sections of rat liver (40). (a) Liver section
from control rats showing normal appearance of liver cells. (b) Liver section from
sinapic acid treated rats showing normal appearance of liver cells. (c) Arsenic
exposed rats, showing portal triad with mild inammation, cell inltration, focal
necrosis and giant cell formation. (d) Liver section from arsenic and sinapic acid
treated rats, showing normal hepatocytes with mild portal inammation.
In the present study administration of arsenic at a dose of 5 mg/
kg bw induced hepatic toxicity as evidenced by the pathological
alterations with no mortality. It has been reported that arsenic
accumulation causes disturbances in the total body weight, abso-
lute and relative liver weights of rats may be due to regenerative
changes of arsenic in hepatic cells [31,32]. Our results are consis-
tent with these reports as arsenic exposed rats showed decreased
intake of water and food accompanied with retardation in growth
rate and alterations in organ-body weight ratio. All these morpho-
logical changes observed in arsenic intoxicated rats were attenu-
ated by treatment with sinapic acid.
Arsenic is known to cause hepatic tissue injury as reected from
a signicant increase in serum hepatic marker enzymes indicating
the cellular leakage and loss of functional integrity of hepatic
membrane architecture [49]. In the present study, arsenic intoxica-
tion caused a signicant increase in the activities of AST, ALT, ALP
and LDH, probably resulting from hepatocyte membrane damage.
GGT is known as an important liver function marker and has
been used as an index of liver dysfunction. Recent studies reported
that measurement of serum GGT might be useful in studying oxi-
dative stress-related tissues. The products of the GGT reaction
may themselves lead to increased free radical production, particu-
larly in the presence of iron [5052]. Previous studies showed that
increase in plasma total bilirubin concentrations was associated
with free radical production in rats treated with arsenic [5355].
Administration of sinapic acid attenuated arsenic-induced hepato-
toxicity as shown by the decreased levels of AST, ALT, ALP, LDH and
GGT and reduced level of serum bilirubin, thus offering protection
against arsenic toxicity by stabilizing the cell membrane of rat
hepatocytes. Administration of sinapic acid at a dose of 40 mg/kg
body weight more remarkably improved the alterations in hepatic
function indicator induced by arsenic when compared to two other
doses which exhibit partial effects on hepatic restoration. This sug-
gests that sinapic acid at 10 and 20 mg/kg body weight could be
low dose to exert the desired effects. Hence the chosen dose of sin-
apic acid at 40 mg/kg body weight was further analyzed for its abil-
ity to prevent iAs accumulation and oxidative stress markers in the
liver and it was found to be effective.
Arsenic acts as a pro-oxidant in biological systems and causes
lipid peroxidation which is a basic cellular deteriorating process
in the liver [56]. Profound free radical generation and enhanced li-
pid peroxidation are the dual facets of oxidative stress that initiate
45
the pathogenesis of arsenic-induced hepatotoxicity [57,58]. In
addition to lipid peroxidation, protein carbonylation also served
as a validated marker for protein oxidation particularly for the pro-
teins containing amino acid residues like lysine, arginine, proline,
threonine and glutamic acid. In the present investigation, there
was a signicant increased level of TBARS, LOOH, PCC and CD in
the liver of arsenic exposed rats which conrms the onset of oxida-
tive stress. Decreased levels of oxidative stress markers in arsenic
treated rats administered with sinapic acid revealed the radical
scavenging activity of sinapic acid which could be due to the pres-
ence of two methoxy groups and one hydroxyl group at 3rd, 4th
and 5th positions, respectively [59].
The elevation of lipid peroxidative markers observed in this
study might be an important factor in the impairment of the anti-
oxidant defense system. Antioxidant enzymes, such as SOD, CAT,
GST, GR and GPx are considered to be the rst line of cellular de-
fense against oxidative injury. Status of these antioxidant enzymes
is an appropriate indirect way to assess the prooxidantantioxi-
dant status in arsenic induced toxicity. Among them SOD and
CAT mutually function as important enzymes in the elimination
of ROS and RNS. SOD is an antioxidant enzyme which catalyzes
the dismutation of superoxide to H2O2 which in turn is removed
by catalase [60]. Thus, SOD can act as a primary defense against
superoxide anion and prevents further generation of free radicals.
Reduction in SOD activity in arsenic-exposed animals reects en-
hanced production of superoxide radical anions [61]. The increase
in superoxide radicals also inhibits catalase activity [62]. NADPH is
required for the activation of CAT from its inactivated form. Thus,
reduced activity of catalase in arsenic exposed animals may be
due to the insufcient supply of NADPH during arsenic metabolism
[63]. On the other hand, glutathione-related enzymes, such as GPx,
GR and GST function either directly or indirectly as antioxidants.
GST is a family of proteins involved in the detoxication process
by catalyzing the reaction of glutathione with toxicants to form
an S-substituted glutathione [64]. GPx is a selenium-containing en-
zyme, it is well established that arsenic interacts with essential
selenocysteine moiety of the enzyme to form insoluble and inac-
tive arsenicselenium complex [65,66] rendering it unavailable
and ultimately resulting in the inhibition of GPx activity or altering
the expression and synthesis of selenoproteins like GPx [67,68].
GST and GPx play principle function to reduce organic hydroperox-
ides within membranes and lipoproteins in the presence of GSH.
Therefore, decreased activities of GST and GPx with a concomitant
decrease in the activity of GSH-regenerating enzyme, GR suggest
the consumption of glutathione while protecting against the ar-
senic-induced oxidative stress, as they help to maintain cellular re-
dox status [69,70]. In the present study, it has been observed that
arsenic intoxicated rats signicantly reduced the activities of all
the antioxidant enzymes. Interestingly, the fact that sinapic acid
could markedly renew the impairment of antioxidant defense sys-
tem in the liver of arsenic-treated rats might be attributed to its
antioxidant and chelating properties which could be due to the ori-
entation of functional groups in the 3rd, 4th and 5th positions in
sinapic acid [25,71].
G6PD is an important enzyme of hexosemonophosphate (HMP)
shunt. It converts one molecule of glucose-6-phosphate into 6-
phosphogluconolactone in the presence of Mg2+, Mn2+ and Ca2+
ions and subsequently NADP+ is reduced to NADPH. A subsequent
reduction of the G6PD activity in arsenic induced rats showed im-
paired generation of NADPH which is required for the reduction of
GSSG to GSH.
Consequently, biochemical perturbations seem to be correlated
with the evidence of increased arsenic content associated with his-
tological changes in the liver. A different bio-kinetic pattern of ar-
senic distribution facilitates its accumulation in the liver and
causes oxidative stress. In our present study, hepatic arsenic
46 L. Pari, A. Mohamed Jalaludeen / Chemico-Biological Interactions 194 (2011) 4047
concentration was found to be signicantly increased in all the
experimental groups compared to control rats. Routine histological
studies on the liver have documented arsenic-induced changes
characterized by dilated sinusoids, vacuolization and the appear-
ance of hepatic cells with distorted nuclei [72]. The present inves-
tigation suggests that sodium arsenite intoxication produces
various pathological alterations in the liver, such as portal triad
with mild inammation, cell inltration, focal necrosis, and giant
cell formation. Administration of sinapic acid improved the histo-
logical alterations induced by arsenic and reduced the arsenic con-
tent in the liver which could be attributed to a free radical
scavenging activity as it prevents autoxidation of methyl linoleate,
indicating an increased antioxidant efcacy of phenolic acids with
methoxy substitutions [73] This may be due to a better electron-
donationg activity by a methoxy as compared with a hydroxyl
group. The presence of methoxy substitutions in the 3- and 5-posi-
tions, in sinapic acid, may impose a negative effect on the hydro-
gen-donating ability of the molecule, but the orientation of
functional groups in the 3-, 4-, and 5-positions enhances the anti-
oxidant efcacy of the molecules as metal chelators [71]. It con-
cludes that arsenic induced toxicity can be signicantly protected
by the co-administration of sinapic acid.
Conict of interest statement
None declared.
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