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Toxicology Letters
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6 AUTHORS, INCLUDING:
Debrup Chakraborty
Avinaba Mukherjee
Jadavpur University
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Samrat Ghosh
University of Kalyani
University of Kalyani
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Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
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).
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
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.
2.2. Animals
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.
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
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
37
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
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.
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*
39
Table 2
Dose dependent Inuence of [6]-gingerol on iAs intoxicated hyperglycemic mice.
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#
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#
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#
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).
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.
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.
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.
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
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.
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,
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.
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.