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[6]-Gingerolisolatedfromgingerattenuatessodiumarseniteinducedoxidativestressandplaysacorrectiveroleinimprovinginsulin...

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Toxicology Letters 210 (2012) 34– 43

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[6]-Gingerol isolated from ginger attenuates sodium arsenite induced oxidativestress 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 2011Received in revised form30 December 2011Accepted 2 January 2012Available online xxx

Keywords:Sodium arsenite[6]-GingerolOxidative stressHyperglycemiaGLUT4Insulin 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 revealhow sodium arsenite (iAs) could induce stress mediated impaired insulin signaling in mice and if anisolated active fraction of ginger, [6]-gingerol could attenuate the iAs intoxicated hyperglycemic condi-tion of mice and bring about improvement in their impaired insulin signaling. [6]-Gingerol treatmentreduced 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 plasmainsulin level, brought down after iAs exposure. iAs treatment to primary cell culture of �-cells andhepatocytes 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 inducedcyto-degeneration of pancreatic �-cells and hepatocytes, helped in scavenging the free radicals. Theover-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, bothat protein and mRNA levels. Thus, our results suggest that [6]-gingerol possesses an anti-hyperglycemicproperty 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 infood, soil and water. It is released in the environment from bothnatural and man-made sources (Tchounwou et al., 1999). Inorganicarsenic and their metabolites (both As+3 and As+5 forms) are knownto exert their toxic effects by a variety of mechanisms which maylead to some serious health problems. Epidemiological data haveshown that chronic exposure of inorganic arsenical compounds tohumans are associated with liver injury, peripheral neuropathy andan increased incidence of cancer of the lung, skin, and liver (Leonardand Lauwerys, 1980). In East Asia alone, including Bangladesh, WestBengal, India, Vietnam, Thailand and China, more than 30 millionpeople are chronically exposed to arsenic (Tseng et al., 1968). Thisarsenic induced toxicity arises and sustains by generating stressresponse through reactive oxygen species formation and antioxi-dant depletion (Jomova et al., 2011). According to a recent study,sodium arsenite (iAs) is found to be associated with increased bloodglucose 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).

Hydroarsenicism is a major public health problem since mil-lions of people worldwide are exposed to arsenic by drinking ofcontaminated water (Jones, 2007). Studies on mouse bone marrowcells have predicted an increased level of chromosomal abnor-mality and micronucleus formation after treatment with arsenic(Banerjee et al., 2007) and thereby have confirmed its cytotoxic andcytodegenerative effects. One of the plausible modes of action ofarsenic toxicity is by oxidative stress since it can stimulate produc-tion of reactive oxygen species (ROS), resulting from an imbalancebetween antioxidants and oxidants during arsenic metabolism(Goering et al., 1999; Sun et al., 2006).

On the other hand arsenic has been recently proposed asan additional risk factor for diabetes (Silbergeld et al., 2008;Longnecker and Daniels, 2001). According to recent surveys it isfound that the occurrence of diabetes is significantly higher inarsenic-endemic villages in Taiwan and India than in the generalpopulation (Zimmet, 1982; Wang et al., 1997; Belon et al., 2006).The prevalence of diabetes mellitus was 2-fold higher in these areasthan in Taipei City and the Taiwan area in general.

From in vitro studies, the impairment of insulin secretion(Diaz-Villasenor et al., 2006) and the induction of oxidative stress(Izquierdo-Vega et al., 2006) have been postulated for arsenic-induced type 2 diabetes. Induction of stress via generation of freeoxygen radicals and antioxidant depletion led into this process and

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D. Chakraborty et al. / Toxicology Letters 210 (2012) 34– 43 35

helped in the progression of insulin impairment that would resultinto type 2 diabetes (Folli et al., 2011). A lack of insulin or insulinresistance, or defects in the insulin signaling pathways is the causeof type 2 diabetes mellitus, which is characterized by hyperglycemia(Taylor, 1999). According to Walton et al. (2004), inorganic arseniccould be an important inducer in this process and it acceleratesits effect by inhibiting insulin secretion due to degeneration ofpancreatic �-cells and insulin-dependent glucose uptake, mainlyin peripheral tissues and liver (Navas-Acien et al., 2006), whichis predicted as a possible mechanism for arsenic-induced type 2diabetes.

Therefore, in the present study we tried to investigate the mech-anistic role of inorganic arsenic in the development of impairedinsulin responsiveness and also tried to evaluate the protective roleof [6]-gingerol, if any, to ameliorate this problem.

Ginger (Zingiber officinale roscoe, Zingiberaceae) is a medicinalplant that has been widely used in Chinese, Ayurvedic and Tibb-Unani herbal medicines all over the world. Apart from its useas a spice in many parts of the world, ginger has been medic-inally used for a wide array of unrelated ailments that includearthritis, rheumatism, sprains, muscular aches, pains, sore throats,cramps, constipation, indigestion, vomiting, hypertension, demen-tia, fever, infectious diseases and helminthiasis (Badreldin et al.,2008) since antiquity. Several studies have indicated that com-pounds found in ginger are effective in relief of symptoms fromchronic inflammatory diseases and can suppress both the cyclooxy-genase and lipoxygenase metabolites and arachidonic acid (Srivas,1984; Kiuchi et al., 1992; Tjendraputra et al., 2001). Keeping this inmind, we investigated the adverse effect of arsenic toxicity on thedevelopment of type 2 diabetes (Navas-Acien et al., 2006). Besides,we were also interested to see whether [6]-gingerol can effectivelyimprove insulin production through � cell viability in pancreas andimpaired insulin responsiveness in liver from arsenic toxicity alongwith oxidative stress, both in in vitro and in vivo systems.

We treated the iAs intoxicated mice with [6]-gingerol to seewhether it had any modulatory effects in maintaining the stressbalance, insulin production and on impaired insulin responsivenessgenerated through elevation of ROS formation, and antioxidantdepletion by iAs induction. If it had any positive modulation, ourinterest was also focused on examining the pathways, both bio-chemical and signaling, by which it could achieve this goal. We,therefore, designed our experiments accordingly, in mice for in vivoand on isolated pancreatic �-cells and hepatocyte cells, for in vitrostudies.

2. Materials and methods

2.1. Drug

[6]-Gingerol was extracted from the freshly dried rhizomes of ginger (Z. offici-nale) purchased from the local market by following the method of Singh et al. (2009).The extract of Z. officinale, had been obtained with chloroform. Chloroform frac-tion (35 g) was subjected to column chromatography over silica gel (230–400 �mmesh) using hexane-ethyl acetate (9:1) mixture to yield the fraction rich in [6]-gingerol (Fig. 1). The identity of [6]-gingerol was confirmed by comparison of thedata obtained from studies of nuclear magnetic resonance (NMR) 1H, Fourier trans-form infrared spectroscopy (FTIR) and mass spectroscopy (Connell and Sutherland,1969) with data of the present study. The mass spectra of the purified [6]-gingerolhas been shown in Fig. 1.

2.2. Animals

We housed a large group of healthy inbred strains of Swiss albino mice (Mus mus-culus) (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 polypropy-lene cages, allowed free drinking water and basal diet ad libitum. We performedall the experiments with the guidelines cleared by the Institutional Animal EthicsCommittee, University of Kalyani, West Bengal and under the supervision of the Ani-mal Welfare Committee (Registration number: 892/ac/05/CPCSEA, dt. 11/05/2010),Department of Zoology, University of Kalyani.

2.3. Chemicals

We purchased Insulin, GLUT4 (glucose transporter 4), TNF� (tumor necrosisfactor �), IRS1 (insulin receptor substrate 1), IRS2 (insulin receptor substrate 2),AKT (a protein kinase B), PI3K (phosphatidylinositol 3 kinase), PPAR� (peroxi-some proliferator-activated receptor gamma), GAPDH (glyceraldehyde 3-phosphatedehydrogenase) primary monoclonal antibodies and FITC/ALP (fluorescein isothio-cyanate/alkaline phosphatase) conjugated secondary antibodies from Santa CruzBiotechnology, Inc. Santa Cruz, CA, USA. We procured M-mulv reverse tran-scriptase, Taq DNA polymerase, dNTPs (deoxyribonucleotide triphosphate) andother RT-PCR (reverse transcriptase-polymerase chain reaction) reagents fromBiovision, 980 Linda Vista Avenue, Mountain View, California. We purchasedsodium arsenite, DCFHDA (dichlorodihydrofluorescein diacetate), TPTZ (2,3,5-triphenyltetrazolium chloride), MTT (thiazolyl blue tetrazolium bromide) fromSigma–Aldrich, St. Louis, USA. We obtained ethylenediaminetetraaceticacid (EDTA),nicotinamide adenine dinucleotide reduced (NADH), BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium), 5,5-dithiobis(2-nitrobenzoic acid)[DTNB, (Ellman’s reagent)], potassium dihydrogen phosphate (KH2PO4), reducedglutathione (GSH), sodium pyrophosphate, trichloroacetic acid (TCA), thiobarbituricacid (TBA) from Sisco Research Laboratories Pvt. Ltd., Mumbai, India and glyco-cylated hemoglobin kit from Crest biosystems, India. We procured the syntheticoligonucleotide 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 ˇ-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 themethod of Seqlen (1973). We centrifuged the single cell suspension at 500 g for 2 minto 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.,2010) for 8 h and isolated �-cells (Diaz-Villasenor et al., 2006) with 10 �M iAs alonefor 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 incubatedthe hepatocytes for the next 7 h and the �-cells for the next 71 h. We measuredthe cell viability by the MTT assay as described previously with some modifications(Mandal et al., 2009).

2.6. Intra-cellular ROS accumulation

To evaluate the level of intra-cellular ROS accumulation, we intoxicated theisolated hepatocytes and pancreatic �-cells (2 × 105 cells/well) with iAs at 10 �Mconcentration, incubated them with 50 and 75 �g/ml doses of [6]-gingerol for thesame time intervals mentioned above. Photographs were taken in fluorescencemicroscope after staining with DCFHDA. For flowcytometric analysis, after com-pletion of the experimental period we collected the hepatocytes and �-cells, fixedthem in 70% chilled ethanol and further incubated with 10 �M DCFHDA for 30 minat room temperature and determined the intensity of DCFHDA fluorescence by flu-orescence microscope and flowcytometer with an excitation wave length of 480 nmand an emission wavelength of 530 nm [FACS caliber, BD Bioscience, USA] (Rathaet al., 2006).

For flowcytometric analysis we incubated the hepatocytes first with 10 �M iAsfor 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, fixed in1% paraformaldehyde. Intra cellular GLUT4 content was analysed by flowcytometer[FACS caliber, BD Bioscience, USA] according to the method described in Chakrabortyet al. (2012). We used anti rabbit GLUT4 primary antibody and FITC conjugated antirabbit secondary antibody for this assay.

2.7. Determination of dose for iAs induced hepatic dysfunctions andhyperglycemia in mice

To establish the dose of iAs necessary for hepatic damage, we randomly allocatedmice into five groups each consisting of six mice and they were treated as follows.First group served as normal control (received water as vehicle). Remaining fourgroups 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-fourhours after the final dose of iAs intoxication, ALT (alanine aminotransferase) andAST (aspartate aminotransferase) (Stanton et al., 2005) and blood glucose levelswere measured using blood serum of all experimental mice.

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36 D. Chakraborty et al. / Toxicology Letters 210 (2012) 34– 43

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 randomizedand 36 mice were divided into six groups, consisting of six mice in each group andthey 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 oncedaily for 12 weeks, orally.Group 3: [6]-Gingerol treated group (iAs + [6]-gingerol): received iAs for 12 weeksfollowed by [6]-gingerol administration at a dose of 50 mg/kg body weight in alco-hol once daily for next 3 weeks.Group 4: [6]-Gingerol treated group (iAs + [6]-gingerol): received iAs for 12 weeksfollowed by [6]-gingerol administration at a dose of 75 mg/kg body weight in alco-hol once daily for next 3 weeks.Group 5: Alcohol treated group (iAs + alcohol): received iAs for 12 weeks followedby alcohol administration (equal quantity as administered in groups 3 and 4) oncedaily 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 thedrug alone has any adverse effect on mice.

The animals were humanely sacrificed under light ether anesthesia and liverswere collected.

We fed the drug orally to all the mice of different experimental groups throughgavage. No significant changes in ALT, AST activity were found between iAs controland alcohol (drug vehicle) treated groups. There were also no significant differ-ences of ALT and AST activities between normal control and [6]-gingerol alonetreated groups. Therefore, we excluded groups 5 and 6 from more in-depth stud-ies. Mice showing blood glucose levels of more than 180 mg/dl were considered ashyperglycemic animals and included in our experiments.

2.9. Collection of blood and tissue samples

At the end of the experimental period, we kept the animals on fast for 12 hprior to being sacrificed for determining glucose level in their fasting blood. We alsocollected the pancreas and liver tissues from each experimental animal and storedthem at −80 ◦C for further analysis.

2.10. Preparation of liver tissue homogenates

We collected the liver tissues from experimental mice, homogenized them inlysis 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

and estimated the total protein according to the method of Bradford using crystallinebovine 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 analyzedaccording to the method described by Khuda-Bukhsh et al. (2005), using an atomicabsorption spectrophotometer (AAS).

2.12. Determination of in vivo antioxidant capacity

We determined antioxidant capacity of [6]-gingerol on hepatic tissues of allexperimental animals by FRAP (ferric reducing antioxidant potential) assay (Benzieand 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-chekactive), Roche diagnostics, Mannheim, Germany. We used liver tissue homogenatesfor various enzymatic assays. We undertook spectrophotometric analysis of activityof catalase (CAT) (Aebi, 1984), super oxide dismutase (SOD) (Fridovich, 1989), glu-tathione peroxidase (GPx), level of total glutathione (GSH) (Ellman, 1959) accordingto the standard protocols.

2.14. Oral glucose tolerance test (OGTT)

An oral glucose tolerance test (OGTT) was performed on the last day of treatmentafter 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 wasprovided inside the cages during the course of experiment.

2.15. ELISA for activity measurement of different antibodies

We assayed the activity of plasma insulin and hepatic GLUT4 according tothe manufacturer’s protocol (Santa Cruz Biotechnology, Inc., USA) and quantifiedthem using an ELISA reader (Thermo Scientific, USA). We used pNPP (para-nitrophenylphosphate) as a color developing agent and measured the color intensityin 405 nm wave length.

2.16. Immunoblot analysis

We used the liver tissue homogenates for immunoblot analysis. For this we took50 mg of tissue samples in 2 ml lysis buffer for protein extraction. We undertook SDS-PAGE (12.5%) electrophoresis of equal amounts of lysate protein and transferredthem to polyvinyl difluoride (PVDF) membrane. After blocking with 3% BSA, we

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Fig. 2. Effect of [6]-gingerol on the viability of iAs intoxicated hepatocytes andpancreatic �-cells. While the �-cells were exposed to 10 �M iAs and different con-centrations of [6]-gingerol for 72 h, the hepatocytes were exposed for 8 h. The cellviability was then detected by MTT assay. Each point expressed as mean ± SD (N = 6).Significance, *p < 0.05 vs. normal control group. Significance, #p < 0.05 vs. iAs intox-icated group.

incubated the membranes with specific primary antibodies overnight at 4 ◦C. Wefurther incubated membrane for 2 h with ALP conjugated secondary antibody. Weused BCIP-NBT for developing bound antibodies on the membrane and analysedthe 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 geneexpressions were analyzed by semi-quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) according to the method described by Chakraborty et al.(2012). We measured the fluorescence intensity of the bands on the agarose gel byusing the ‘image J’ software.

2.18. Statistical analysis

Data were analyzed and significance of the differences between the mean valueswas determined by one-way ANOVA with Dunnett’s post hoc tests, using SPSS 14software. Statistical significance 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 ofislets cells had been dead at 72 h intoxication with iAs, when com-pared with the control. Incubation of isolated pancreatic isletscells 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. Sim-ilarly, [6]-gingerol treated hepatocytes also showed reduced celldeath after iAs intoxication at 50 and 75 �g/ml drug concentrations.[6]-Gingerol treatment increased the cell viability from 71.15% inthe iAs-intoxicated control to 88.73% and 94.36%, respectively, atthe two doses of drug treatment. Therefore, we used 50 �g/ml and75 �g/ml concentration of [6]-gingerol in all the subsequent in vitroexperiments.

3.2. Inhibitory activity of [6]-gingerol on intracellular ROSgeneration in ˇ-cells and hepatocytes

We found an increased ROS production in pancreatic �-cellsintoxicated with iAs in both fluorescence microscopic and flow-cytometric studies (Fig. 3A–H). Control cells showed the lowestpercentage (4.2%) of DCFHDA positive cells which increased upto 26.4% after iAs intoxication. �-cells incubated with [6]-gingerolshowed a lesser amount of fluorescence (15.5% and 11.1%, respec-tively for 50 and 75 �g/ml doses) at 72 h after iAs-intoxication.Similarly, we also observed iAs induced ROS production in

hepatocytes in fluorescence microscope and found an elevatedROS level at 8 h of iAs incubation. Therefore, we measured thelevel of intracellular ROS by flowcytometer after 8 h of treatment(Fig. 3I–P). Control cells showed the lowest percentage (9.4%) ofDCFHDA positive cells which increased up to 31.9% after iAs intox-ication. Hepatocyte cells incubated with [6]-gingerol showed alesser amount of fluorescence (23.5% and 17.6%, respectively), 8 hafter iAs-intoxication.

3.3. Role of [6]-gingerol on the intracellular GLUT4 content of iAsintoxicated hepatocyte cells

Level of intracellular GLUT4 content (Fig. 3Q–T) was quantifiedby means of flowcytometer using anti-GLUT4 primary antibody andFITC conjugated secondary antibody. Level of intracellular GLUT4was found to increase in [6]-gingerol incubated hepatocytes (38.9%and 43.0%, respectively, for 50 and 75 �g/ml doses) than that of theonly iAs-intoxicated hepatocytes (31.3%).

3.4. Effect of iAs at various concentrations revealed by assays onALT, AST and blood glucose

In order to assess the hepatic damage and increased bloodglucose level, we applied different concentrations of iAs on exper-imental mice. Our results (Table 1) showed, significantly increasedlevels of ALT, AST and blood glucose of iAs intoxicated mice at adose of 3 mg/kg body weight after 12 weeks. Therefore, we hadchosen the concentration 3 mg/kg for iAs-induced hepatic damageand hyperglycemia throughout the study.

3.5. Dose dependent study of [6]-gingerol

ALT and AST assays and blood glucose data were used to deter-mine the optimum dose necessary for [6]-gingerol to protect liveragainst iAs induced hepatic damages and increased blood glucoselevels that led to hyperglycemia. It is evident from our results thatiAs intoxication (at a dose of 3 mg/kg body weight, orally for 12weeks) increased the ALT, AST and blood glucose levels which werefound to decrease significantly by the treatment with [6]-gingerolat 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 allthe groups that were treated with iAs, either alone or in combina-tion with drugs. As compared to the positive control, the treatmentwith the drug showed significant 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 significantly improvedthe glucose tolerance level and inhibited rise in postprandial glu-cose level at both the time points, 90 and 120 min after glucoseload in mice, as compared to the iAs intoxicated hyperglycemicmice (Fig. 4).

3.8. Anti-oxidative potentials of [6]-gingerol

Significantly decreased activities of CAT, SOD, GPx and GSH werefound (Table 4) in liver of iAs intoxicated hyperglycemic mice, whencompared to drug fed mice.

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38 D. Chakraborty et al. / Toxicology Letters 210 (2012) 34– 43

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 and75 �g/ml doses of [6]-gingerol. �-Cells and hepatocytes incubated for next 71 h and 7 h, respectively with drug. A–D represents fluorescence microscopic observationsand E–H represents flowcytometric analysis of ROS accumulation in �-cells. On the other hand, I–L represents fluorescence microscopic observations and M–P representsflowcytometric 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, incubatedwith 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. Q–T representsflowcytometric analysis of intracellular GLUT4 content in hepatocytes. Q – normal control cells, R – 10 �M iAs intoxicated cells, S – iAs intoxicated cells, incubated with50 �g/ml [6]-gingerol, T – iAs intoxicated cells, incubated with 75 �g/ml [6]-gingerol.

Table 1Effect of different concentrations of iAs on mice.

Cont 2 mg/kg 3 mg/kg 4 mg/kg 5 mg/kg

ALT (�m/mg protein) 11.06 ± 3.10 16.6 ± 3.109 23.24 ± 0.560* 25.04 ± 0.66* 27.93 ± 1.17*

AST (�m/mg protein) 5.33 ± 0.203 6.55 ± 1.66 14.007 ± 0.509* 13.86 ± 0.441* 17.32 ± 2.68*

Blood glucose (mg/dl) 92.5 ± 2.12 132 ± 5.65* 189.5 ± 7.77* 186 ± 8.48* 186 ± 9.89*

Data are expressed as mean ± SD (N = 6).* p < 0.05 vs. normal control group.

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D. Chakraborty et al. / Toxicology Letters 210 (2012) 34– 43 39

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

Cont iAs 25 mg/kg 50 mg/kg 75 mg/kg

ALT (�m/mg protein) 11.06 ± 3.109 23.24 ± 0.56* 21.87 ± 1.37 15.55 ± 1.35# 13.51 ± 0.45#

AST (�m/mg protein) 5.33 ± 0.203 14.007 ± 0.51* 12.78 ± 1.08 8.68 ± 0.71# 8.29 ± 2.2#

Blood glucose (mg/dl) 92.5 ± 2.12 189.5 ± 7.77* 182 ± 8.48 122.5 ± 3.53# 110 ± 2.82#

Data are expressed as mean ± SD (N = 6).* p < 0.05 vs. normal control group.# Significance, p < 0.05 vs. iAs intoxicated group.

Table 3Role of [6]-gingerol on arsenic deposition in pancreas and liver tissues of experimental animals expressed in nmol/g unit.

Cont iAs 50 mg/kg 75 mg/kg

Pancreas 13.87 ± 4.2 43.015 ± 6.86* 26.435 ± 3.41 23.669 ± 3.74#

Liver 14.92 ± 6.71 49.27 ± 7.73* 27.51 ± 3.11# 25.13 ± 4.14#

Data are expressed as mean ± SD (N = 6).* p < 0.05 vs. normal control group.# Significance, p < 0.05 vs. iAs intoxicated diabetic group.

Table 4Role of [6]-gingerol on different anti-oxidant biomarkers of iAs intoxicated mice liver.

Cont iAs 50 mg/kg 75 mg/kg

GHb (%) 4.02 ± 0.28 10 ± 0.19* 7.05 ± 0.27# 5.32 ± 0.97#

SOD (�m/mg protein) 38.68 ± 1.26 28.32 ± 1.73* 32.19 ± 1.29 35.35 ± 0.35#

Catalase (�m/mg protein) 72.22 ± 4.08 37.2032 ± 2.97* 52.16 ± 3.54# 58.547 ± 0.95#

GSH (�m/mg protein) 37.62 ± 2.65 21.92 ± 1.21* 28.53 ± 1.4 32.29 ± 3.65#

GPx (�m/mg protein) 64.5 ± 5.82 48.36 ± 1.44* 54.16 ± 1.59 58.18 ± 2.36#

FRAP (%) 95.83 ± 5.89 54.16 ± 5.89* 67.91 ± 1.76 79.16 ± 5.89#

Data are expressed as mean ± SD (N = 6).* p < 0.05 vs. normal control group.# Significance, 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 hasbeen represented in Table 4. The intracellular ferric reducingantioxidant potential declining after iAs intoxication in animals wasfound to increase significantly with [6]-gingerol treatment at dosesof 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 lev-els of plasma insulin and hepatic GLUT4 were found. Administration

Fig. 4. Impact of [6]-gingerol (50 and 75 mg/kg) treatment on oral glucose tolerancein iAs mice. Data were expressed as mean ± SD (N = 6), Ap < 0.05 cont vs. iAs intox-icated group and ap < 0.05 iAs vs. drug groups (A/a used to denote comperisons in0 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 com-perisons in 120 min interval groups).

of [6]-gingerol at both the doses increased the plasma insulin andhepatic GLUT4 concentrations significantly (Fig. 5).

3.11. Immunoblot analysis

Administration of the drug down regulated the expression ofTNF� which was earlier found to increase in iAs intoxicated liver. Inaddition, we observed that iAs intoxication reduced the expressionsof proteins related to insulin signaling, namely, Insulin, IRS1, IRS2,AKT, PI3K, GLUT4 and PPAR� (Figs. 6–7). However, expressions ofthese proteins were significantly up regulated after treatment with[6]-gingerol.

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

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40 D. Chakraborty et al. / Toxicology Letters 210 (2012) 34– 43

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. Normalmice; Lane 2. iAs-intoxicated mice, Lane 3. iAs-intoxicated + [6]-gingerol 50 mg/kgbw, Lane 4. iAs-intoxicated + [6]-gingerol 75 mg/kg bw. Signs indicate that a partic-ular treatment has not (−) been or has been (+) given, respectively.

3.12. RT-PCR analysis

Results of RT-PCR confirmed that there was a significant dif-ference in the expressions of mRNA between the control andthe iAs-intoxicated groups and between the iAs and drug-treatedgroups (Figs. 8–9). Results of RT-PCR analysis also supportedthe results obtained from the western blot analysis. The primersequences of amplified genes are given in Table 5.

4. Discussion

Our experimental findings showed that iAs intoxication cansignificantly reduce the pancreatic �-cells and hepatocyte cell via-bility. But iAs intoxicated cells incubated with [6]-gingerol had the

Table 5Primer sequences.

Primer name Primer sequences

TNF� Fwd 5′-GCACAGAAAGCATGATCCGC-3′

Rev 5′-CTTGGTGGTTTGCTACGACG-3′

IL6 Fwd 5′-AACGATGATGCACTTGCAGA-3′

Rev 5′-GAGCATTGGAAATTGGGGTA-3′

IRS1 Fwd 5′-AGAGTGGTGGAGTTGAGTTG-3′

Rev 5′-GGTGTAACAGAAGCAGAAGC-3′

IRS2 Fwd 5′-GGATAATGGTGACTATACCGAGA-3′

Rev 5′-CTCACATCGATGGCGATATAGTT-3′

PI3K Fwd 5′-TTAAACGCGAAGGCAACGA-3′

Rev 5′-CAGTCTCCTCCTGCTGCTGAT-3AKT Fwd 5′-CCTGGACTACCTGCACTCTCGGAA-3′

Rev 5′-TTGCTTTCAGGGCTGCTCAAGAAGG-3′

GLUT4 Fwd 5′-AAGATGGCCACGGAGAGAG-3′

Rev 5′-GTGGGTTGTGGCAGTGAGTC-3′

GAPDH Fwd 5′-CCATGTTCGTCATGGGTGTGAACCA-3′

Rev 5′-GCCAGTAGAGGCAGGGATGATGTTC-3′

PPAR� Fwd 5′-GCGGAGATCTCCAGTGATATC-3′

Rev 5′-TCAGCGACTGGGACTTTTCT-3′

potential to partially overcome the iAs induced toxicity and couldreduce the cell death, in vitro. It is reported that arsenic produceshepatocyte dysfunction including the reduced cell viability (Daset al., 2010). On the other hand, ROS also plays an important rolein insulin resistance which is a highly prevalent condition impli-cated in the development of diabetes. We assessed the changesin free radical accumulation in �-cells and hepatocytes and foundan increased level of ROS accumulation in cells intoxicated withonly iAs. On the other hand, treatment with [6]-gingerol along withiAs intoxication showed lesser amount of ROS accumulation andsignificantly increased cell viability in both pancreatic �-cells andhepatocytes.

Recently researchers published that arsenic has a profound toxicand cytodegenerative effects on pancreas and liver (Pi et al., 2002;Santra et al., 2000). One of the major reasons behind arsenic inducedhepatotoxicity is the depletion of antioxidant defense mechanismwhich is related to the reduction of anti-oxidative enzymes’ activityincluding SOD, CAT, GPx and GSH (Das et al., 2010). Arsenic in differ-ent forms also increases the levels of free hydroxyl radicals, whichplay an important role in the development of genotoxicity (Liu et al.,2001). Therefore, any agent which by itself is non-toxic, but hasthe ability to reduce oxidative stress will be desirable to antago-nize ROS generation and also thereby genotoxicity. Our findings

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). Significance *p < 0.05 iAs-intoxicated vs. normal control group; significance #p < 0.05 drug-fed vs. iAs-intoxicated group.

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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. 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 treat-ment has not (−) been or has been (+) given, respectively.

indicate that the administration of [6]-gingerol reduced the oxida-tive stress as revealed from the increased activity of antioxidantbiomarkers like CAT, SOD, GPx and GSH. On the other hand, it alsohad an anti-hyperglycemic effect as the increased blood glucoselevel due to iAs induction was found to decrease up to the normallevel after treatment with [6]-gingerol. In addition, mice treatedwith [6]-gingerol also showed significant improvement in oral glu-cose tolerance. GHb is an important index of diabetes managementand we observed a significant fall in GHb after administration of[6]-gingerol in iAs intoxicated hyperglycemic mice. The in vivoantioxidant potential of [6]-gingerol in liver tissue was determinedby FRAP assay, which indicates the anti-oxidative potentials of

[6]-gingerol at two different concentrations. The analysis of AASdata on arsenic content of liver and pancreas of arsenic intoxicatedmice before and after administration of [6]-gingerol confirmed theability of this drug to remove the accumulated arsenic content fromthese organs.

According to several authors (Cemek et al., 2008; Singh et al.,2009), a drug which has the combined anti-oxidative and anti-hyperglycemic properties can make the drug very effective asan anti-diabetic drug. Pancreatic �-cell dysfunction due to iAstoxicity is the main reason behind impaired insulin secretionwhich plays an important role in the onset of type 2 diabetes. Inin vivo set of experiment, treatment with both the doses (50 mg/kgand 75 mg/kg bw) of [6]-gingerol was found to be associatedwith increased plasma insulin concentration in iAs intoxicatedmice.

iAs has been shown to potentially alter some gene expressionsrelated to glucose homeostasis in body. Uptake of glucose moleculeinside the cell depends on the translocation of glucose transporter4 (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 hepato-cytes (control cells: 62.2%) and found that 10 �M of iAs intoxicationlowers the level of GLUT4 content (31.3%), which was increased(up to 38.9% and 43%) after incubation with [6]-gingerol. We alsoassessed the mRNA and protein level expressions of GLUT4 in iAsintoxicated mice, treated or un-treated with [6]-gingerol. Auto-phosphorylation of insulin receptor substrates and activation ofPI3K/AKT signaling pathway are required for the activation of downsteam signal cascade leading to glucose uptake and metabolismin cell (Vijayakumar et al., 2005). Therefore we investigated theexpressions of IRS1, IRS2, p85 of PI3K and AKT in liver tissue ofexperimental mice. Our results revealed that [6]-gingerol enhancedthe activity and expressions of these genes which were initiallydown regulated in iAs intoxicated hyperglycemic mice. TNF-� andIL6 levels are closely associated with both hyper-insulinemia andinsulin resistance (Rondinone, 2006). Increased levels of TNF-� andIL6 are also associated with iAs exposure. Therefore, we examinedthe changes of expressions of these two genes in iAs intoxicatedmice, and after treatment with [6]-gingerol, reduced expressionlevels of the said genes were found. This would reveal that the drugexerted its effect in combating impaired insulin responsivenessthrough regulation of these genes. iAs intoxication can also downregulate the transcription factor PPAR� which is very closely relatedto the activity of TNF-� and IL6 (Gurnell, 2003). Our investigation

Fig. 9. Relative fluorescence intensities of PCR bands. The relative intensities of bands were determined using ‘Image J’ software. The results shown in histograms are theaverage ±SD (N = 6). Significance *p < 0.05 iAs-intoxicated vs. normal control group; significance #p < 0.05 drug-fed vs. iAs-intoxicated group.

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42 D. Chakraborty et al. / Toxicology Letters 210 (2012) 34– 43

showed that treatment with [6]-gingerol up-regulated the expres-sion of PPAR� which was initially down regulated in iAs intoxicatedmice.

Effects of insulin are predominantly found in several organs andtissues like skeletal muscles, liver, fat and brain. Variations in genesencoding the insulin receptor substrates-1 and/or -2 (IRS-1, IRS-2) and/or genes encoding the glucose transporter (GLUTs) proteinshave been found to have a defined role in the development of insulinresistance. Our results also implicated the involvement of thesegene cascades as there was a down-regulation of the expressions ofIRS1, IRS2, PI3K, AKT and GLUT4 genes in the iAs intoxicated hyper-glycemic mice. Treatment with [6]-gingerol showed up-regulationof these genes, resulting in some improvement towards insulinsignaling/responsiveness in the experimental animals. This wouldtempt one to suggest that [6]-gingerol has the efficiency to activatethe downstream insulin signaling pathway as well.

5. Conclusion

In conclusion, we provided evidences that the isolated fractionof ginger, [6]-gingerol has potentials of protecting animals fromarsenic induced oxidative stress and hyperglycemia by suitablemodulation of the insulin secretion from pancreas, and also modu-lating responsiveness of insulin in the liver of mice. Ginger being adietary component (a spice) could thus be recommended for regu-lar use in the arsenic risk prone areas, because it has a beneficial andantagonistic effect of arsenic induced toxicity and hyperglycemia.

Conflict of interest

None to declare.

Acknowledgements

The authors are grateful to Boiron Laboratories, Lyon, France forpartial financial support of the work. Sincere thanks are due to Dr.N. Boujedaini, Boiron Laboratories, for her encouragements. Theauthors are thankful to Dr. P.K. Das, Former Director, Central Vec-tor Control Research Centre, Puducherry, India for critically goingthrough the manuscript.

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