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[6]-Gingerol isolated from ginger attenuates

sodium arsenite induced oxidative stress and
plays a corrective role in improving insulin
signaling in mice
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[6]-Gingerol isolated from ginger attenuates sodium arsenite induced oxidative

stress and plays a corrective role in improving insulin signaling in mice
Debrup Chakraborty, Avinaba Mukherjee, Sourav Sikdar, Avijit Paul, Samrat Ghosh,
Anisur Rahman Khuda-Bukhsh
Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India

a r t i c l e

i n f o

Article history:
Received 28 November 2011
Received in revised form
30 December 2011
Accepted 2 January 2012
Available online xxx
Keywords:
Sodium arsenite
[6]-Gingerol
Oxidative stress
Hyperglycemia
GLUT4
Insulin signaling

a b s t r a c t
Arsenic toxicity induces type 2 diabetes via stress mediated pathway. In this study, we attempt to reveal
how sodium arsenite (iAs) could induce stress mediated impaired insulin signaling in mice and if an
isolated active fraction of ginger, [6]-gingerol could attenuate the iAs intoxicated hyperglycemic condition of mice and bring about improvement in their impaired insulin signaling. [6]-Gingerol treatment
reduced elevated blood glucose level and oxidative stress by enhancing activity of super oxide dismutase
(SOD), catalase, glutathione peroxidase (GPx) and GSH. [6]-Gingerol also helped in increasing plasma
insulin level, brought down after iAs exposure. iAs treatment to primary cell culture of -cells and
hepatocytes in vitro produced cyto-degenerative effect and accumulated reactive oxygen species (ROS)
in pancreatic -cells and hepatocytes of mice. [6]-Gingerol appeared to inhibit/intervene iAs induced
cyto-degeneration of pancreatic -cells and hepatocytes, helped in scavenging the free radicals. The
over-expression of TNF and IL6 in iAs intoxicated mice was down-regulated by [6]-gingerol treatment.
iAs intoxication reduced expression levels of GLUT4, IRS-1, IRS-2, PI3K, AKT, PPAR signaling molecules;
[6]-gingerol mediated its action through enhancing the expressions of these signaling molecules, both
at protein and mRNA levels. Thus, our results suggest that [6]-gingerol possesses an anti-hyperglycemic
property and can improve impaired insulin signaling in arsenic intoxicated mice.
2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Arsenic is a naturally occurring heavy metal that is present in
food, soil and water. It is released in the environment from both
natural and man-made sources (Tchounwou et al., 1999). Inorganic
arsenic and their metabolites (both As+3 and As+5 forms) are known
to exert their toxic effects by a variety of mechanisms which may
lead to some serious health problems. Epidemiological data have
shown that chronic exposure of inorganic arsenical compounds to
humans are associated with liver injury, peripheral neuropathy and
an increased incidence of cancer of the lung, skin, and liver (Leonard
and Lauwerys, 1980). In East Asia alone, including Bangladesh, West
Bengal, India, Vietnam, Thailand and China, more than 30 million
people are chronically exposed to arsenic (Tseng et al., 1968). This
arsenic induced toxicity arises and sustains by generating stress
response through reactive oxygen species formation and antioxidant depletion (Jomova et al., 2011). According to a recent study,
sodium arsenite (iAs) is found to be associated with increased blood
glucose level in experimental rats (Yousef et al., 2008).

Corresponding author. Tel.: +91 33 25828750x315.

E-mail addresses: khudabukhsh 48@rediffmail.com, prof arkb@yahoo.co.in
(A.R. Khuda-Bukhsh).
0378-4274/$ see front matter 2012 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2012.01.002

Hydroarsenicism is a major public health problem since millions of people worldwide are exposed to arsenic by drinking of
contaminated water (Jones, 2007). Studies on mouse bone marrow
cells have predicted an increased level of chromosomal abnormality and micronucleus formation after treatment with arsenic
(Banerjee et al., 2007) and thereby have conrmed its cytotoxic and
cytodegenerative effects. One of the plausible modes of action of
arsenic toxicity is by oxidative stress since it can stimulate production of reactive oxygen species (ROS), resulting from an imbalance
between antioxidants and oxidants during arsenic metabolism
(Goering et al., 1999; Sun et al., 2006).
On the other hand arsenic has been recently proposed as
an additional risk factor for diabetes (Silbergeld et al., 2008;
Longnecker and Daniels, 2001). According to recent surveys it is
found that the occurrence of diabetes is signicantly higher in
arsenic-endemic villages in Taiwan and India than in the general
population (Zimmet, 1982; Wang et al., 1997; Belon et al., 2006).
The prevalence of diabetes mellitus was 2-fold higher in these areas
than in Taipei City and the Taiwan area in general.
From in vitro studies, the impairment of insulin secretion
et al., 2006) and the induction of oxidative stress
(Diaz-Villasenor
(Izquierdo-Vega et al., 2006) have been postulated for arsenicinduced type 2 diabetes. Induction of stress via generation of free
oxygen radicals and antioxidant depletion led into this process and

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D. Chakraborty et al. / Toxicology Letters 210 (2012) 3443

helped in the progression of insulin impairment that would result

into type 2 diabetes (Folli et al., 2011). A lack of insulin or insulin
resistance, or defects in the insulin signaling pathways is the cause
of type 2 diabetes mellitus, which is characterized by hyperglycemia
(Taylor, 1999). According to Walton et al. (2004), inorganic arsenic
could be an important inducer in this process and it accelerates
its effect by inhibiting insulin secretion due to degeneration of
pancreatic -cells and insulin-dependent glucose uptake, mainly
in peripheral tissues and liver (Navas-Acien et al., 2006), which
is predicted as a possible mechanism for arsenic-induced type 2
diabetes.
Therefore, in the present study we tried to investigate the mechanistic role of inorganic arsenic in the development of impaired
insulin responsiveness and also tried to evaluate the protective role
of [6]-gingerol, if any, to ameliorate this problem.
Ginger (Zingiber ofcinale roscoe, Zingiberaceae) is a medicinal
plant that has been widely used in Chinese, Ayurvedic and TibbUnani herbal medicines all over the world. Apart from its use
as a spice in many parts of the world, ginger has been medicinally used for a wide array of unrelated ailments that include
arthritis, rheumatism, sprains, muscular aches, pains, sore throats,
cramps, constipation, indigestion, vomiting, hypertension, dementia, fever, infectious diseases and helminthiasis (Badreldin et al.,
2008) since antiquity. Several studies have indicated that compounds found in ginger are effective in relief of symptoms from
chronic inammatory diseases and can suppress both the cyclooxygenase and lipoxygenase metabolites and arachidonic acid (Srivas,
1984; Kiuchi et al., 1992; Tjendraputra et al., 2001). Keeping this in
mind, we investigated the adverse effect of arsenic toxicity on the
development of type 2 diabetes (Navas-Acien et al., 2006). Besides,
we were also interested to see whether [6]-gingerol can effectively
improve insulin production through cell viability in pancreas and
impaired insulin responsiveness in liver from arsenic toxicity along
with oxidative stress, both in in vitro and in vivo systems.
We treated the iAs intoxicated mice with [6]-gingerol to see
whether it had any modulatory effects in maintaining the stress
balance, insulin production and on impaired insulin responsiveness
generated through elevation of ROS formation, and antioxidant
depletion by iAs induction. If it had any positive modulation, our
interest was also focused on examining the pathways, both biochemical and signaling, by which it could achieve this goal. We,
therefore, designed our experiments accordingly, in mice for in vivo
and on isolated pancreatic -cells and hepatocyte cells, for in vitro
studies.
2. Materials and methods
2.1. Drug
[6]-Gingerol was extracted from the freshly dried rhizomes of ginger (Z. ofcinale) purchased from the local market by following the method of Singh et al. (2009).
The extract of Z. ofcinale, had been obtained with chloroform. Chloroform fraction (35 g) was subjected to column chromatography over silica gel (230400 m
mesh) using hexane-ethyl acetate (9:1) mixture to yield the fraction rich in [6]gingerol (Fig. 1). The identity of [6]-gingerol was conrmed by comparison of the
data obtained from studies of nuclear magnetic resonance (NMR) 1 H, Fourier transform infrared spectroscopy (FTIR) and mass spectroscopy (Connell and Sutherland,
1969) with data of the present study. The mass spectra of the puried [6]-gingerol
has been shown in Fig. 1.

35

2.3. Chemicals
We purchased Insulin, GLUT4 (glucose transporter 4), TNF (tumor necrosis
factor ), IRS1 (insulin receptor substrate 1), IRS2 (insulin receptor substrate 2),
AKT (a protein kinase B), PI3K (phosphatidylinositol 3 kinase), PPAR (peroxisome proliferator-activated receptor gamma), GAPDH (glyceraldehyde 3-phosphate
dehydrogenase) primary monoclonal antibodies and FITC/ALP (uorescein isothiocyanate/alkaline phosphatase) conjugated secondary antibodies from Santa Cruz
Biotechnology, Inc. Santa Cruz, CA, USA. We procured M-mulv reverse transcriptase, Taq DNA polymerase, dNTPs (deoxyribonucleotide triphosphate) and
other RT-PCR (reverse transcriptase-polymerase chain reaction) reagents from
Biovision, 980 Linda Vista Avenue, Mountain View, California. We purchased
sodium arsenite, DCFHDA (dichlorodihydrouorescein diacetate), TPTZ (2,3,5triphenyltetrazolium chloride), MTT (thiazolyl blue tetrazolium bromide) from
SigmaAldrich, St. Louis, USA. We obtained ethylenediaminetetraaceticacid (EDTA),
nicotinamide adenine dinucleotide reduced (NADH), BCIP/NBT (5-bromo-4-chloro3-indolyl-phosphate/nitro blue tetrazolium), 5,5-dithiobis(2-nitrobenzoic acid)
[DTNB, (Ellmans reagent)], potassium dihydrogen phosphate (KH2 PO4 ), reduced
glutathione (GSH), sodium pyrophosphate, trichloroacetic acid (TCA), thiobarbituric
acid (TBA) from Sisco Research Laboratories Pvt. Ltd., Mumbai, India and glycocylated hemoglobin kit from Crest biosystems, India. We procured the synthetic
oligonucleotide primers used for RT-PCR from Bioserve Biotechnologies India Pvt.
Ltd. and RPMI 1640 (Roswell Park Memorial Institute) and FBS (fetal bovine serum)
from Invitrogen, Carlsbad, CA, USA.

2.4. Isolation of pancreatic -cell and hepatocytes from mice

We isolated mouse pancreatic -cells with collagenase as described previously
(Eto et al., 1999; Chang et al., 2003). We isolated the hepatocytes following the
method of Seqlen (1973). We centrifuged the single cell suspension at 500 g for 2 min
to obtain intact and homogeneous populations of both hepatocytes and -cells,
separately.

2.5. Cell viability assay

We incubated/intoxicated isolated hepatocyte cells with 10 M iAs (Das et al.,
et al., 2006) with 10 M iAs alone
2010) for 8 h and isolated -cells (Diaz-Villasenor
for 72 h. 1 h after the addition of iAs, we added different concentrations of [6]gingerol (25, 50, 75 and 100 g/ml, respectively) onto the cell. Then we incubated
the hepatocytes for the next 7 h and the -cells for the next 71 h. We measured
the cell viability by the MTT assay as described previously with some modications
(Mandal et al., 2009).

2.6. Intra-cellular ROS accumulation

To evaluate the level of intra-cellular ROS accumulation, we intoxicated the
isolated hepatocytes and pancreatic -cells (2 105 cells/well) with iAs at 10 M
concentration, incubated them with 50 and 75 g/ml doses of [6]-gingerol for the
same time intervals mentioned above. Photographs were taken in uorescence
microscope after staining with DCFHDA. For owcytometric analysis, after completion of the experimental period we collected the hepatocytes and -cells, xed
them in 70% chilled ethanol and further incubated with 10 M DCFHDA for 30 min
at room temperature and determined the intensity of DCFHDA uorescence by uorescence microscope and owcytometer with an excitation wave length of 480 nm
and an emission wavelength of 530 nm [FACS caliber, BD Bioscience, USA] (Ratha
et al., 2006).
For owcytometric analysis we incubated the hepatocytes rst with 10 M iAs
for 1 h. Then we added [6]-gingerol (at two different concentrations 50 and 75 g/ml,
respectively) to the medium for the next 7 h. We collected the hepatocytes, xed in
1% paraformaldehyde. Intra cellular GLUT4 content was analysed by owcytometer
[FACS caliber, BD Bioscience, USA] according to the method described in Chakraborty
et al. (2012). We used anti rabbit GLUT4 primary antibody and FITC conjugated anti
rabbit secondary antibody for this assay.

2.2. Animals

2.7. Determination of dose for iAs induced hepatic dysfunctions and

hyperglycemia in mice

We housed a large group of healthy inbred strains of Swiss albino mice (Mus musculus) (6/8 weeks: 25 g) for at least 14 days in an environmentally controlled room
(temperature, 24 2 C; humidity, 55 5%, 12-h light/dark cycle) in polypropylene cages, allowed free drinking water and basal diet ad libitum. We performed
all the experiments with the guidelines cleared by the Institutional Animal Ethics
Committee, University of Kalyani, West Bengal and under the supervision of the Animal Welfare Committee (Registration number: 892/ac/05/CPCSEA, dt. 11/05/2010),
Department of Zoology, University of Kalyani.

To establish the dose of iAs necessary for hepatic damage, we randomly allocated
mice into ve groups each consisting of six mice and they were treated as follows.
First group served as normal control (received water as vehicle). Remaining four
groups were treated with four different doses of NaAsO2 orally (2 mg/kg, 3 mg/kg,
4 mg/kg and 5 mg/kg body weight in distilled water for 12 weeks). Twenty-four
hours after the nal dose of iAs intoxication, ALT (alanine aminotransferase) and
AST (aspartate aminotransferase) (Stanton et al., 2005) and blood glucose levels
were measured using blood serum of all experimental mice.

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D. Chakraborty et al. / Toxicology Letters 210 (2012) 3443

Fig. 1. Mass spectra and structure of [6]-gingerol.

2.8. Animal treatment

After selection of the optimum dose of iAs (3 mg/kg), animals were randomized
and 36 mice were divided into six groups, consisting of six mice in each group and
they were treated for 15 weeks as follows:
Group 1: Normal control: animals received only water as vehicle.
Group 2: Arsenic control (iAs): animals received iAs at 3 mg/kg body weight once
daily for 12 weeks, orally.
Group 3: [6]-Gingerol treated group (iAs + [6]-gingerol): received iAs for 12 weeks
followed by [6]-gingerol administration at a dose of 50 mg/kg body weight in alcohol once daily for next 3 weeks.
Group 4: [6]-Gingerol treated group (iAs + [6]-gingerol): received iAs for 12 weeks
followed by [6]-gingerol administration at a dose of 75 mg/kg body weight in alcohol once daily for next 3 weeks.
Group 5: Alcohol treated group (iAs + alcohol): received iAs for 12 weeks followed
by alcohol administration (equal quantity as administered in groups 3 and 4) once
daily for next 3 weeks.
Group 6: [6]-Gingerol alone treated group: normal mice were treated with [6]gingerol (orally, 75 mg/kg body weight, once daily) for 3 weeks to see whether the
drug alone has any adverse effect on mice.
The animals were humanely sacriced under light ether anesthesia and livers
were collected.
We fed the drug orally to all the mice of different experimental groups through
gavage. No signicant changes in ALT, AST activity were found between iAs control
and alcohol (drug vehicle) treated groups. There were also no signicant differences of ALT and AST activities between normal control and [6]-gingerol alone
treated groups. Therefore, we excluded groups 5 and 6 from more in-depth studies. Mice showing blood glucose levels of more than 180 mg/dl were considered as
hyperglycemic animals and included in our experiments.

and estimated the total protein according to the method of Bradford using crystalline
bovine serum albumin (BSA) as standard (Bradford, 1976).
2.11. Determination of pancreatic and hepatic arsenic contents
The arsenic contents of liver tissues of all experimental animals were analyzed
according to the method described by Khuda-Bukhsh et al. (2005), using an atomic
absorption spectrophotometer (AAS).
2.12. Determination of in vivo antioxidant capacity
We determined antioxidant capacity of [6]-gingerol on hepatic tissues of all
experimental animals by FRAP (ferric reducing antioxidant potential) assay (Benzie
and Strain, 1999). We took the absorbance of the sample against reagent blank
(1.5 ml FRAP reagent + 50 l distilled water) at 593 nm (Manna et al., 2009).
2.13. Biochemical analysis of blood and activity of antioxidant markers in liver
We estimated the blood glucose level using a glucose estimation kit (Accu-chek
active), Roche diagnostics, Mannheim, Germany. We used liver tissue homogenates
for various enzymatic assays. We undertook spectrophotometric analysis of activity
of catalase (CAT) (Aebi, 1984), super oxide dismutase (SOD) (Fridovich, 1989), glutathione peroxidase (GPx), level of total glutathione (GSH) (Ellman, 1959) according
to the standard protocols.
2.14. Oral glucose tolerance test (OGTT)
An oral glucose tolerance test (OGTT) was performed on the last day of treatment
after overnight fasting. Blood was collected from the tail vein of mice at time 0, 60,
90 and 120 min after an oral glucose load of 3.0 g/kg of body weight. Only water was
provided inside the cages during the course of experiment.
2.15. ELISA for activity measurement of different antibodies

2.9. Collection of blood and tissue samples

At the end of the experimental period, we kept the animals on fast for 12 h
prior to being sacriced for determining glucose level in their fasting blood. We also
collected the pancreas and liver tissues from each experimental animal and stored
them at 80 C for further analysis.

We assayed the activity of plasma insulin and hepatic GLUT4 according to

the manufacturers protocol (Santa Cruz Biotechnology, Inc., USA) and quantied
them using an ELISA reader (Thermo Scientic, USA). We used pNPP (paranitrophenylphosphate) as a color developing agent and measured the color intensity
in 405 nm wave length.
2.16. Immunoblot analysis

2.10. Preparation of liver tissue homogenates

We collected the liver tissues from experimental mice, homogenized them in
lysis buffer using glass homogenizer and centrifuged at 12,000 g for 30 min at 4 C.
We collected the supernatant after centrifugation and used it for further experiment

We used the liver tissue homogenates for immunoblot analysis. For this we took
50 mg of tissue samples in 2 ml lysis buffer for protein extraction. We undertook SDSPAGE (12.5%) electrophoresis of equal amounts of lysate protein and transferred
them to polyvinyl diuoride (PVDF) membrane. After blocking with 3% BSA, we

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37

hepatocytes in uorescence microscope and found an elevated

ROS level at 8 h of iAs incubation. Therefore, we measured the
level of intracellular ROS by owcytometer after 8 h of treatment
(Fig. 3IP). Control cells showed the lowest percentage (9.4%) of
DCFHDA positive cells which increased up to 31.9% after iAs intoxication. Hepatocyte cells incubated with [6]-gingerol showed a
lesser amount of uorescence (23.5% and 17.6%, respectively), 8 h
after iAs-intoxication.
3.3. Role of [6]-gingerol on the intracellular GLUT4 content of iAs
intoxicated hepatocyte cells

Fig. 2. Effect of [6]-gingerol on the viability of iAs intoxicated hepatocytes and

pancreatic -cells. While the -cells were exposed to 10 M iAs and different concentrations of [6]-gingerol for 72 h, the hepatocytes were exposed for 8 h. The cell
viability was then detected by MTT assay. Each point expressed as mean SD (N = 6).
Signicance, *p < 0.05 vs. normal control group. Signicance, #p < 0.05 vs. iAs intoxicated group.
incubated the membranes with specic primary antibodies overnight at 4 C. We
further incubated membrane for 2 h with ALP conjugated secondary antibody. We
used BCIP-NBT for developing bound antibodies on the membrane and analysed
the band intensities densitometrically using image J software (Bhattacharyya et al.,
2009).
2.17. RNA extraction and semi-quantitative RT-PCR analysis
Total RNA from the experimental liver tissue were extracted and the gene
expressions were analyzed by semi-quantitative RT-PCR (reverse transcriptasepolymerase chain reaction) according to the method described by Chakraborty et al.
(2012). We measured the uorescence intensity of the bands on the agarose gel by
using the image J software.
2.18. Statistical analysis
Data were analyzed and signicance of the differences between the mean values
was determined by one-way ANOVA with Dunnetts post hoc tests, using SPSS 14
software. Statistical signicance was considered at *p < 0.05.

3. Results
3.1. Protective effect of [6]-gingerol against the cytotoxicity of iAs
Results of MTT assay (Fig. 2) revealed that a large number of
islets cells had been dead at 72 h intoxication with iAs, when compared with the control. Incubation of isolated pancreatic islets
cells with [6]-gingerol increased the cell viability from 59.03%
in the iAs-intoxicated control to 72.31% and 88.69%, respectively
(Fig. 2), in the 50 g/ml and 75 g/ml drug treated groups. Similarly, [6]-gingerol treated hepatocytes also showed reduced cell
death after iAs intoxication at 50 and 75 g/ml drug concentrations.
[6]-Gingerol treatment increased the cell viability from 71.15% in
the iAs-intoxicated control to 88.73% and 94.36%, respectively, at
the two doses of drug treatment. Therefore, we used 50 g/ml and
75 g/ml concentration of [6]-gingerol in all the subsequent in vitro
experiments.
3.2. Inhibitory activity of [6]-gingerol on intracellular ROS
generation in -cells and hepatocytes
We found an increased ROS production in pancreatic -cells
intoxicated with iAs in both uorescence microscopic and owcytometric studies (Fig. 3AH). Control cells showed the lowest
percentage (4.2%) of DCFHDA positive cells which increased up
to 26.4% after iAs intoxication. -cells incubated with [6]-gingerol
showed a lesser amount of uorescence (15.5% and 11.1%, respectively for 50 and 75 g/ml doses) at 72 h after iAs-intoxication.
Similarly, we also observed iAs induced ROS production in

Level of intracellular GLUT4 content (Fig. 3QT) was quantied

by means of owcytometer using anti-GLUT4 primary antibody and
FITC conjugated secondary antibody. Level of intracellular GLUT4
was found to increase in [6]-gingerol incubated hepatocytes (38.9%
and 43.0%, respectively, for 50 and 75 g/ml doses) than that of the
only iAs-intoxicated hepatocytes (31.3%).
3.4. Effect of iAs at various concentrations revealed by assays on
ALT, AST and blood glucose
In order to assess the hepatic damage and increased blood
glucose level, we applied different concentrations of iAs on experimental mice. Our results (Table 1) showed, signicantly increased
levels of ALT, AST and blood glucose of iAs intoxicated mice at a
dose of 3 mg/kg body weight after 12 weeks. Therefore, we had
chosen the concentration 3 mg/kg for iAs-induced hepatic damage
and hyperglycemia throughout the study.
3.5. Dose dependent study of [6]-gingerol
ALT and AST assays and blood glucose data were used to determine the optimum dose necessary for [6]-gingerol to protect liver
against iAs induced hepatic damages and increased blood glucose
levels that led to hyperglycemia. It is evident from our results that
iAs intoxication (at a dose of 3 mg/kg body weight, orally for 12
weeks) increased the ALT, AST and blood glucose levels which were
found to decrease signicantly by the treatment with [6]-gingerol
at doses of 50 mg/kg and 75 mg/kg body weight daily for 3 weeks
(Table 2). These doses were, therefore, chosen as optimum ones for
[6]-gingerol treatment throughout the study.
3.6. Effect of [6]-gingerol on iAs depositions in liver and pancreas
Arsenic was detectable in the liver and pancreas of mice of all
the groups that were treated with iAs, either alone or in combination with drugs. As compared to the positive control, the treatment
with the drug showed signicant reduction in the arsenic content,
deposited in both liver and pancreas tissues (Table 3).
3.7. Effect as revealed from oral glucose tolerance test (OGTT)
3 weeks of treatment with [6]-gingerol signicantly improved
the glucose tolerance level and inhibited rise in postprandial glucose level at both the time points, 90 and 120 min after glucose
load in mice, as compared to the iAs intoxicated hyperglycemic
mice (Fig. 4).
3.8. Anti-oxidative potentials of [6]-gingerol
Signicantly decreased activities of CAT, SOD, GPx and GSH were
found (Table 4) in liver of iAs intoxicated hyperglycemic mice, when
compared to drug fed mice.

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D. Chakraborty et al. / Toxicology Letters 210 (2012) 3443

Fig. 3. Dose dependent inhibitory effect of [6]-gingerol on iAs induced free radical accumulation. 1 h after incubation with iAs (10 M) cells were incubated with 50 and
75 g/ml doses of [6]-gingerol. -Cells and hepatocytes incubated for next 71 h and 7 h, respectively with drug. AD represents uorescence microscopic observations
and EH represents owcytometric analysis of ROS accumulation in -cells. On the other hand, IL represents uorescence microscopic observations and MP represents
owcytometric analysis of ROS accumulation in hepatocytes. A, E, I, M normal control cells, B, F, J, N 10 M iAs intoxicated cells, C, G, K, O iAs intoxicated cells, incubated
with 50 g/ml [6]-gingerol, D, H, L, P iAs intoxicated cells, incubated with 75 g/ml [6]-gingerol. The intra-cellular ROS was detected by DCFHDA method. QT represents
owcytometric analysis of intracellular GLUT4 content in hepatocytes. Q normal control cells, R 10 M iAs intoxicated cells, S iAs intoxicated cells, incubated with
50 g/ml [6]-gingerol, T iAs intoxicated cells, incubated with 75 g/ml [6]-gingerol.

Table 1
Effect of different concentrations of iAs on mice.

ALT (m/mg protein)

AST (m/mg protein)
Blood glucose (mg/dl)

Cont

2 mg/kg

3 mg/kg

4 mg/kg

5 mg/kg

11.06 3.10
5.33 0.203
92.5 2.12

16.6 3.109
6.55 1.66
132 5.65*

23.24 0.560*
14.007 0.509*
189.5 7.77*

25.04 0.66*
13.86 0.441*
186 8.48*

27.93 1.17*
17.32 2.68*
186 9.89*

Data are expressed as mean SD (N = 6).

*
p < 0.05 vs. normal control group.

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39

Table 2
Dose dependent Inuence of [6]-gingerol on iAs intoxicated hyperglycemic mice.

ALT (m/mg protein)

AST (m/mg protein)
Blood glucose (mg/dl)

Cont

iAs

11.06 3.109
5.33 0.203
92.5 2.12

23.24 0.56
14.007 0.51*
189.5 7.77*
*

25 mg/kg

50 mg/kg

75 mg/kg

21.87 1.37
12.78 1.08
182 8.48

15.55 1.35
8.68 0.71#
122.5 3.53#
#

13.51 0.45#
8.29 2.2#
110 2.82#

Data are expressed as mean SD (N = 6).

*
p < 0.05 vs. normal control group.
#
Signicance, p < 0.05 vs. iAs intoxicated group.
Table 3
Role of [6]-gingerol on arsenic deposition in pancreas and liver tissues of experimental animals expressed in nmol/g unit.

Pancreas
Liver

Cont

iAs

50 mg/kg

75 mg/kg

13.87 4.2
14.92 6.71

43.015 6.86*
49.27 7.73*

26.435 3.41
27.51 3.11#

23.669 3.74#
25.13 4.14#

Data are expressed as mean SD (N = 6).

*
p < 0.05 vs. normal control group.
#
Signicance, p < 0.05 vs. iAs intoxicated diabetic group.
Table 4
Role of [6]-gingerol on different anti-oxidant biomarkers of iAs intoxicated mice liver.
Cont
GHb (%)
SOD (m/mg protein)
Catalase (m/mg protein)
GSH (m/mg protein)
GPx (m/mg protein)
FRAP (%)

4.02
38.68
72.22
37.62
64.5
95.83

iAs

0.28
1.26
4.08
2.65
5.82
5.89

50 mg/kg

10
28.32
37.2032
21.92
48.36
54.16

0.19*
1.73*
2.97*
1.21*
1.44*
5.89*

7.05
32.19
52.16
28.53
54.16
67.91

0.27#
1.29
3.54#
1.4
1.59
1.76

75 mg/kg
5.32
35.35
58.547
32.29
58.18
79.16

0.97#
0.35#
0.95#
3.65#
2.36#
5.89#

Data are expressed as mean SD (N = 6).

*
p < 0.05 vs. normal control group.
#
Signicance, p < 0.05 vs. iAs intoxicated diabetic group.

3.9. Dose dependent study of [6]-gingerol by FRAP assay

Dose dependent effect of [6]-gingerol against iAs toxicity has
been represented in Table 4. The intracellular ferric reducing
antioxidant potential declining after iAs intoxication in animals was
found to increase signicantly with [6]-gingerol treatment at doses
of 50 mg/kg and 75 mg/kg bw, respectively.
3.10. Effect on plasma insulin and hepatic GLUT4 content
Under iAs intoxicated hyperglycemic condition, decreased levels of plasma insulin and hepatic GLUT4 were found. Administration

Fig. 4. Impact of [6]-gingerol (50 and 75 mg/kg) treatment on oral glucose tolerance
in iAs mice. Data were expressed as mean SD (N = 6), Ap < 0.05 cont vs. iAs intoxicated group and ap < 0.05 iAs vs. drug groups (A/a used to denote comperisons in
0 min interval groups, B/b used to denote comperisons in 60 min interval groups,
C/c used to denote comperisons in 90 min interval groups, D/d used to denote comperisons in 120 min interval groups).

of [6]-gingerol at both the doses increased the plasma insulin and

hepatic GLUT4 concentrations signicantly (Fig. 5).
3.11. Immunoblot analysis
Administration of the drug down regulated the expression of
TNF which was earlier found to increase in iAs intoxicated liver. In
addition, we observed that iAs intoxication reduced the expressions
of proteins related to insulin signaling, namely, Insulin, IRS1, IRS2,
AKT, PI3K, GLUT4 and PPAR (Figs. 67). However, expressions of
these proteins were signicantly up regulated after treatment with
[6]-gingerol.

Fig. 5. Effect of [6]-gingerol on plasma insulin and hepatic GLUT4 level in iAs intoxicated diabetic mice. Data were expressed in histogram as mean SD (N = 6). *p < 0.05
iAs-intoxicated vs. normal control group; signicance #p < 0.05 drug-fed vs. iAsintoxicated group.

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40

D. Chakraborty et al. / Toxicology Letters 210 (2012) 3443

Table 5
Primer sequences.
Primer name

Primer sequences

TNF

Fwd 5 -GCACAGAAAGCATGATCCGC-3
Rev 5 -CTTGGTGGTTTGCTACGACG-3
Fwd 5 -AACGATGATGCACTTGCAGA-3
Rev 5 -GAGCATTGGAAATTGGGGTA-3
Fwd 5 -AGAGTGGTGGAGTTGAGTTG-3
Rev 5 -GGTGTAACAGAAGCAGAAGC-3
Fwd 5 -GGATAATGGTGACTATACCGAGA-3
Rev 5 -CTCACATCGATGGCGATATAGTT-3
Fwd 5 -TTAAACGCGAAGGCAACGA-3
Rev 5 -CAGTCTCCTCCTGCTGCTGAT-3
Fwd 5 -CCTGGACTACCTGCACTCTCGGAA-3
Rev 5 -TTGCTTTCAGGGCTGCTCAAGAAGG-3
Fwd 5 -AAGATGGCCACGGAGAGAG-3
Rev 5 -GTGGGTTGTGGCAGTGAGTC-3
Fwd 5 -CCATGTTCGTCATGGGTGTGAACCA-3
Rev 5 -GCCAGTAGAGGCAGGGATGATGTTC-3
Fwd 5 -GCGGAGATCTCCAGTGATATC-3
Rev 5 -TCAGCGACTGGGACTTTTCT-3

IL6
IRS1
IRS2
PI3K
AKT
GLUT4
GAPDH
PPAR

Fig. 6. Immunoblot analysis of insulin, IRS1, TNF, IRS2, GLUT4, PI3K, AKT, PPAR
and GAPDH. GAPDH is used as an equal loading housekeeping gene. Lane 1. Normal
mice; Lane 2. iAs-intoxicated mice, Lane 3. iAs-intoxicated + [6]-gingerol 50 mg/kg
bw, Lane 4. iAs-intoxicated + [6]-gingerol 75 mg/kg bw. Signs indicate that a particular treatment has not () been or has been (+) given, respectively.

3.12. RT-PCR analysis

Results of RT-PCR conrmed that there was a signicant difference in the expressions of mRNA between the control and
the iAs-intoxicated groups and between the iAs and drug-treated
groups (Figs. 89). Results of RT-PCR analysis also supported
the results obtained from the western blot analysis. The primer
sequences of amplied genes are given in Table 5.
4. Discussion
Our experimental ndings showed that iAs intoxication can
signicantly reduce the pancreatic -cells and hepatocyte cell viability. But iAs intoxicated cells incubated with [6]-gingerol had the

potential to partially overcome the iAs induced toxicity and could

reduce the cell death, in vitro. It is reported that arsenic produces
hepatocyte dysfunction including the reduced cell viability (Das
et al., 2010). On the other hand, ROS also plays an important role
in insulin resistance which is a highly prevalent condition implicated in the development of diabetes. We assessed the changes
in free radical accumulation in -cells and hepatocytes and found
an increased level of ROS accumulation in cells intoxicated with
only iAs. On the other hand, treatment with [6]-gingerol along with
iAs intoxication showed lesser amount of ROS accumulation and
signicantly increased cell viability in both pancreatic -cells and
hepatocytes.
Recently researchers published that arsenic has a profound toxic
and cytodegenerative effects on pancreas and liver (Pi et al., 2002;
Santra et al., 2000). One of the major reasons behind arsenic induced
hepatotoxicity is the depletion of antioxidant defense mechanism
which is related to the reduction of anti-oxidative enzymes activity
including SOD, CAT, GPx and GSH (Das et al., 2010). Arsenic in different forms also increases the levels of free hydroxyl radicals, which
play an important role in the development of genotoxicity (Liu et al.,
2001). Therefore, any agent which by itself is non-toxic, but has
the ability to reduce oxidative stress will be desirable to antagonize ROS generation and also thereby genotoxicity. Our ndings

Fig. 7. Relative band intensities of immunoblots. The relative intensities of bands were determined using Image J software. The results shown in histograms are the average
SD (N = 6). Signicance *p < 0.05 iAs-intoxicated vs. normal control group; signicance #p < 0.05 drug-fed vs. iAs-intoxicated group.

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D. Chakraborty et al. / Toxicology Letters 210 (2012) 3443

Fig. 8. Reverse transcription polymerase chain reaction analysis of TNF, IL6,

IRS1, IRS2, PI3 K, AKT, PPAR and GAPDH. Lane 1. Normal mice; Lane 2. iAsintoxicated mice, Lane 3. iAs-intoxicated + [6]-gingerol 50 mg/kg bw, Lane 4.
iAs-intoxicated + [6]-gingerol 75 mg/kg bw. Signs indicate that a particular treatment has not () been or has been (+) given, respectively.

indicate that the administration of [6]-gingerol reduced the oxidative stress as revealed from the increased activity of antioxidant
biomarkers like CAT, SOD, GPx and GSH. On the other hand, it also
had an anti-hyperglycemic effect as the increased blood glucose
level due to iAs induction was found to decrease up to the normal
level after treatment with [6]-gingerol. In addition, mice treated
with [6]-gingerol also showed signicant improvement in oral glucose tolerance. GHb is an important index of diabetes management
and we observed a signicant fall in GHb after administration of
[6]-gingerol in iAs intoxicated hyperglycemic mice. The in vivo
antioxidant potential of [6]-gingerol in liver tissue was determined
by FRAP assay, which indicates the anti-oxidative potentials of

41

[6]-gingerol at two different concentrations. The analysis of AAS

data on arsenic content of liver and pancreas of arsenic intoxicated
mice before and after administration of [6]-gingerol conrmed the
ability of this drug to remove the accumulated arsenic content from
these organs.
According to several authors (Cemek et al., 2008; Singh et al.,
2009), a drug which has the combined anti-oxidative and antihyperglycemic properties can make the drug very effective as
an anti-diabetic drug. Pancreatic -cell dysfunction due to iAs
toxicity is the main reason behind impaired insulin secretion
which plays an important role in the onset of type 2 diabetes. In
in vivo set of experiment, treatment with both the doses (50 mg/kg
and 75 mg/kg bw) of [6]-gingerol was found to be associated
with increased plasma insulin concentration in iAs intoxicated
mice.
iAs has been shown to potentially alter some gene expressions
related to glucose homeostasis in body. Uptake of glucose molecule
inside the cell depends on the translocation of glucose transporter
4 (GLUT4), which plays a very important role in insulin signaling,
by the action of insulin molecule to the plasma membrane. Hence,
we targeted the intracellular GLUT4 content in isolated hepatocytes (control cells: 62.2%) and found that 10 M of iAs intoxication
lowers the level of GLUT4 content (31.3%), which was increased
(up to 38.9% and 43%) after incubation with [6]-gingerol. We also
assessed the mRNA and protein level expressions of GLUT4 in iAs
intoxicated mice, treated or un-treated with [6]-gingerol. Autophosphorylation of insulin receptor substrates and activation of
PI3K/AKT signaling pathway are required for the activation of down
steam signal cascade leading to glucose uptake and metabolism
in cell (Vijayakumar et al., 2005). Therefore we investigated the
expressions of IRS1, IRS2, p85 of PI3K and AKT in liver tissue of
experimental mice. Our results revealed that [6]-gingerol enhanced
the activity and expressions of these genes which were initially
down regulated in iAs intoxicated hyperglycemic mice. TNF- and
IL6 levels are closely associated with both hyper-insulinemia and
insulin resistance (Rondinone, 2006). Increased levels of TNF- and
IL6 are also associated with iAs exposure. Therefore, we examined
the changes of expressions of these two genes in iAs intoxicated
mice, and after treatment with [6]-gingerol, reduced expression
levels of the said genes were found. This would reveal that the drug
exerted its effect in combating impaired insulin responsiveness
through regulation of these genes. iAs intoxication can also down
regulate the transcription factor PPAR which is very closely related
to the activity of TNF- and IL6 (Gurnell, 2003). Our investigation

Fig. 9. Relative uorescence intensities of PCR bands. The relative intensities of bands were determined using Image J software. The results shown in histograms are the
average SD (N = 6). Signicance *p < 0.05 iAs-intoxicated vs. normal control group; signicance #p < 0.05 drug-fed vs. iAs-intoxicated group.

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42

D. Chakraborty et al. / Toxicology Letters 210 (2012) 3443

showed that treatment with [6]-gingerol up-regulated the expression of PPAR which was initially down regulated in iAs intoxicated
mice.
Effects of insulin are predominantly found in several organs and
tissues like skeletal muscles, liver, fat and brain. Variations in genes
encoding the insulin receptor substrates-1 and/or -2 (IRS-1, IRS2) and/or genes encoding the glucose transporter (GLUTs) proteins
have been found to have a dened role in the development of insulin
resistance. Our results also implicated the involvement of these
gene cascades as there was a down-regulation of the expressions of
IRS1, IRS2, PI3K, AKT and GLUT4 genes in the iAs intoxicated hyperglycemic mice. Treatment with [6]-gingerol showed up-regulation
of these genes, resulting in some improvement towards insulin
signaling/responsiveness in the experimental animals. This would
tempt one to suggest that [6]-gingerol has the efciency to activate
the downstream insulin signaling pathway as well.
5. Conclusion
In conclusion, we provided evidences that the isolated fraction
of ginger, [6]-gingerol has potentials of protecting animals from
arsenic induced oxidative stress and hyperglycemia by suitable
modulation of the insulin secretion from pancreas, and also modulating responsiveness of insulin in the liver of mice. Ginger being a
dietary component (a spice) could thus be recommended for regular use in the arsenic risk prone areas, because it has a benecial and
antagonistic effect of arsenic induced toxicity and hyperglycemia.
Conict of interest
None to declare.
Acknowledgements
The authors are grateful to Boiron Laboratories, Lyon, France for
partial nancial support of the work. Sincere thanks are due to Dr.
N. Boujedaini, Boiron Laboratories, for her encouragements. The
authors are thankful to Dr. P.K. Das, Former Director, Central Vector Control Research Centre, Puducherry, India for critically going
through the manuscript.
References
Aebi, H., 1984. Catalase in vitro. In: Packer, L. (Ed.), Methods in Enzymol, vol. 105. ,
pp. 121126.
Badreldin, H.A., Gerald, B., Musbah, O.T., Abderrahim, N., 2008. Some phytochemical, pharmacological and toxicological properties of ginger (Zingiber ofcinale
Roscoe): a review of recent research. Food Chem. Toxicol. 46, 409420.
Banerjee, P., Biswas, S.J., Belon, P., Khuda-Bukhsh, A.R., 2007. A potentized homeopathic drug, arsenicum album 200, can ameliorate genotoxicity induced by
repeated injections of arsenic trioxide in mice. J. Vet. Med. 54, 370376.
Belon, P., Banerjee, P., Choudhury, C.S., Banerjee, A., Biswas, J.S., Karmakar, R.S.,
Pathak, S., Guha, B., Chatterjee, S., Bhattacharjee, N., Das, K.J., Khuda-Bukhsh, A.R.,
2006. Can administration of potentized homeopathic remedy, arsenicum album,
alter antinuclear antibody (ANA) titer in people living in high-risk arsenic contaminated areas? I. A correlation with certain hematological parameters. eCAM
3, 99107.
Benzie, I.F.F., Strain, J.J., 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological uids and modied version for
simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299, 1527.
Bhattacharyya, S.S., Paul, S., Mandal, S.K., Banerjee, A., Boujedaini, N., Khuda-Bukhsh,
A.R., 2009. A synthetic coumarin (4-methyl-7 hydroxy coumarin) has anticancer potentials against DMBA-induced skin cancer in mice. Eur. J. Pharmacol.
614, 128136.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal.
Biochem. 72, 248254.
ukokuroglu,

Cemek, M., Kaga, S., Simsek, N., Buy

M.E., Konuk, M., 2008. Antihyperglycemic and antioxidative potential of Matricaria chamomilla L. in
streptozotocin-induced diabetic rats. Nat. Med. 62, 284293.

Chakraborty, D., Samadder, A., Dutta, S., Khuda-Bukhsh, A.R., 2012. Antihyperglycemic potentials of a threatened plant, Helonias dioica: antioxidative stress
responses and the signaling cascade. Exp. Biol. Med. 237, 6476.
Chang, I., Kim, S., Kim, J.Y., Cho, N., Kim, Y.H., Kim, H.S., Lee, M.K., Kim, K.W., Lee,
M.S., 2003. NF-k prevents cell death and autoimmune diabetes in NOD mice.
Diabetes 52, 11691175.
Connell, D., Sutherland, M., 1969. A reexamination of gingerol, shogaol, and
zingerone, the pungent principles of ginger. Aust. J. Chem. 22, 10331043.
Das, J., Ghosh, J., Manna, P., Sil, P.C., 2010. Protective role of taurine against arsenicinduced mitochondria-dependent hepatic apoptosis via the inhibition of PKCdJNK pathway. PLoS One 5, 119.

A., Sanchez-Soto, M.C., Cebrian, M.E., Ostrosky-Wegman, P., HiriDiaz-Villasenor,

art, M., 2006. Sodium arsenite impairs insulin secretion and transcription in
pancreatic beta-cells. Toxicol. Appl. Pharmacol. 214, 3034.
Ellman, G.L., 1959. Tissue sulfhydryl group. Arch. Biochem. Biophys. 82, 7077.
Eto, K., Tsubamoto, Y., Terauchi, Y., Sugiyama, T., Kishimoto, T., Takahashi, N.,
Yamauchi, N., Kubota, N., Murayama, S., Aizawa, T., Akanuma, Y., Aizawa, S.,
Kasai, H., Yazaki, Y., Kadowaki, T., 1999. Role of NADH shuttle system in glucoseinduced activation of mitochondrial metabolism and insulin secretion. Science
283, 981985.
Folli, F., Corradi, D., Fanti, P., Davalli, A., Paez, A., Giaccari, A., Perego, C., Muscogiuri,
G., 2011. The role of oxidative stress in the pathogenesis of type 2 diabetes
mellitus micro-and macrovascular complications: avenues for a mechanisticbased therapeutic approach. Curr. Diabet. Rev. 7, 313324.
Fridovich, I., 1989. Superoxide dismutase: an adaptation to a paramagnetic gas. J.
Biol. Chem. 264, 77617764.
Goering, P.L., Aposhian, H.V., Mass, M.J., Cebrian, M., Beck, B.D., Waalkes, M.P., 1999.
The enigma of arsenic carcinogenesis: role of metabolism. Toxicol. Sci. 49, 514.
Gurnell, M., 2003. PPAR gamma and metabolism: insights from the study of human
genetic variants. Clin. Endocrinol. (Oxf.) 59, 267277.
Izquierdo-Vega, J.A., Soto, C.A., Sanchez-Pena, L.C., De Vizcaya-Ruiz, A., Del Razo,
L.M., 2006. Diabetogenic effects and pancreatic oxidative damage in rats subchronically exposed to arsenite. Toxicol. Lett. 160, 135142.
Jomova, K., Jenisova, Z., Feszterova, M., Baros, S., Liska, J., Hudecova, D., Rhodes, C.J.,
Valko, M., 2011. Arsenic: toxicity, oxidative stress and human disease. J. Appl.
Toxicol. 31, 95107.
Jones, F.T., 2007. A broad view of arsenic. Poult. Sci. 86, 214.
Khuda-Bukhsh, A.R., Pathak, S., Guha, B., Karmakar, S.R., Das, J.K., Banerjee, P., Biswas,
S.J., Mukherjee, P., Bhattacharjee, N., Choudhury, S.C., Banerjee, A., Bhadra, S.,
Mallick, P., Chakrabarti, J., Mandal, B., 2005. Can homeopathic arsenic remedy
combat arsenic poisoning in humans exposed to groundwater arsenic contamination? A preliminary report on rst human trial. eCAM 2, 537548.
Kiuchi, F., Iwakami, S., Shibuya, M., Hanaoka, F., Sanakawa, U., 1992. Inhibition of
prostaglandin and leukotriene biosynthesis by gingerols and diarylheptanoids.
Chem. Pharm. Bull. 40, 387391.
Leonard, A., Lauwerys, R.R., 1980. Carcinogenicity, teratogenicity, and mutagenicity
of arsenic. Mutat. Res. 75, 4962.
Liu, G., Zhang, V., Bode, A.M., Ma, W.Y., Dong, Z., 2001. Phosphorylation of 4E-BP1
is mediated by the p38/MSK1 pathway in response to UVB irradiation. J. Biol.
Chem. 277, 88108816.
Longnecker, M.P., Daniels, J.L., 2001. Environmental contaminants as etiologic factors for diabetes. Environ. Health Perspect. 109, 871876.
Mandal, S.K., Biswas, R., Bhattacharyya, S.S., Paul, S., Dutta, S., Pathak, S., KhudaBukhsh, A.R., 2009. Lycopodine from Lycopodium clavatum extract inhibits
proliferation of HeLa cells through induction of apoptosis via caspase-3 activation. Eur. J. Pharmacol. 626, 18.
Manna, P., Sinha, M., Sil, P.C., 2009. Protective role of arjunolic acid in response to
streptozotocin-induced type-I diabetes via the mitochondrial dependent and
independent pathways. Toxicology 257, 5363.
Navas-Acien, A., Silbergeld, E.K., Streeter, R.A., Clark, J.M., Guallar, E., 2006. Arsenic
exposure and type 2 diabetes: a systematic review of the experimental and
epidemiologic evidence. Environ. Health Perspect. 114, 641648.
Pi, J., Yamauchi, H., Kumagai, Y., Sun, G., Yoshida, T., 2002. Evidence for induction
of oxidative stress caused by chronic exposure of Chinese residents to arsenic
contained in drinking water. Environ. Health Perspect. 110, 331336.
Ratha, J., Majumder, K.N., Mandal, S.K., Bera, R., Sarkar, C., Saha, B., Mandal, C., Saha,
K.D., Bhadra, R., 2006. A sphingolipid rich lipid fraction isolated from attenuated
donovani promastigote induces apoptosis in mouse and human melanoma cells
in vitro. Mol. Cell Biochem. 290, 113123.
Rondinone, C.M., 2006. Adipocyte-derived hormones, cytokines, and mediators.
Endocrine 29, 8190.
Santra, A., Maiti, A., Chowdhury, A., Guha mazumder, D.N., 2000. Oxidative stress
in liver of mice exposed to arsenic contaminated water. Indian J. Gastroenterol.
19, 112115.
Seqlen, P., 1973. Preparation of rat liver cells. II. Effects of ions and chelators on tissue
dispersion. Exp. Cell. Res. 76, 2530.
Silbergeld, E.K., Pastor-Barriuso, R., Guallar, E., Jama, P.H., 2008. Arsenic exposure
and prevalence of type 2 diabetes in US adults. JAMA 300, 814822.
Singh, A.B., Akanksha Singh, N., Maurya, R., Srivastava, A.K., 2009. Antihyperglycemic, lipid lowering and anti-oxidant properties of [6]-gingerol in
db/db mice. Int. J. Med. Med. Sci. 1, 536544.
Srivas, K.C., 1984. Effects of aqueous extracts of onion, garlic and ginger on platelet
aggregation and metabolism of arachidonic acid in the blood vascular system:
in vitro study. Prostaglandins Leukot. Med. 13, 227235.
Stanton, H., Rogerson, F.M., East, C.J., Golub, S.B., Lawlor, K.E., Meeker, C.T., Little, C.B.,
Last, K., Farmer, P.J., Campbell, I.K., Fourie, A.M., Fosang, A.J., 2005. ADAMTS5

Author's personal copy

D. Chakraborty et al. / Toxicology Letters 210 (2012) 3443
is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature 434,
648652.
Sun, X., Li, B., Li, X., Wang, Y., Xu, Y., Jin, Y., Piao, F., Sun, G., 2006. Effects of sodium
arsenite on catalase activity, gene and protein expression in HaCaT cells. Toxicology 20, 11391144.
Taylor, R., 1999. The nature of type 2 diabetes: the role for new agents. Int. J. Clin.
Pract. Suppl. 104, 1820.
Tchounwou, P.B., Wilson, B.A., Ishaque, A., 1999. Important considerations in the
development of public health advisories for arsenic and arsenic containing compounds in drinking water. Rev. Environ. Health 14, 119.
Tjendraputra, E., Tran, V.H., Liu-Brennan, D., Roufogalis, B.D., Duke, C.C., 2001. Effect
of ginger constituents and synthetic analogues on cyclooxygenase-2 enzyme in
intact cells. Bioorg. Chem. 29, 156163.
Tseng, W.P., Chu, H.M., How, S.W., Fong, J.M., Lin, C.S., Yeh, S., 1968. Prevalence of
skin cancer in an endemic area of chronic arsenicism in Taiwan. J. Natl. Cancer
Inst. 40, 453463.

43

Vijayakumar, M.V., Singh, S., Chhipa, R.R., Bhat, M.K., 2005. The hypoglycaemic activity of fenugreek seed extract is mediated through the
stimulation of an insulin signalling pathway. Br. J. Pharmacol. 146,
4148.
Walton, F.S., Harmon, A.W., Paul, D.S., Drobn, Z., Patel, Y.M., Styblo, M., 2004.
Inhibition of insulin-dependent glucose uptake by trivalent arsenicals: possible mechanism of arsenic-induced diabetes. Toxicol. Appl. Pharmacol. 198,
424433.
Wang, S.L., Pan, W.H., Hwu, C.M., Ho, L.T., Lo, C.H., Lin, S.L., Jong, Y.S., 1997. Incidence
of NIDDM and the effects of gender, obesity and hyperinsulinaemia in Taiwan.
Diabetologia 40, 14311438.
Yousef, M.I., El-Demerdash, F.M., Radwan, F.M.E., 2008. Sodium arsenite induced
biochemical perturbations in rats: ameliorating effect of curcumin. Food Chem.
Toxicol. 46, 35063511.
Zimmet, P., 1982. Type-2 non-insulin-dependent diabetes-an epidemiological
overview. Diabetologia 22, 399411.