Hindawi Publishing CorporationISRN HepatologyVolume 2013 Article ID 817693 13 pageshttpdxdoiorg1011552013817693

Research ArticleIn Vivo Effect of Arsenic Trioxide on Keap1-p62-Nrf2Signaling Pathway in Mouse Liver Expression of AntioxidantResponsive Element-Driven Genes Related toGlutathione Metabolism

Ritu Srivastava1 Archya Sengupta1 Sandip Mukherjee2

Sarmishtha Chatterjee2 Muthammal Sudarshan3 Anindita Chakraborty3

Shelley Bhattacharya2 and Ansuman Chattopadhyay1

1 Radiation Genetics and Chemical Mutagenesis Laboratory Department of Zoology Centre for Advanced StudiesVisva-Bharati University Santiniketan West Bengal 731235 India

2 Environmental Toxicology Laboratory Department of Zoology Centre for Advanced Studies Visva-Bharati UniversitySantiniketan West Bengal 731235 India

3 UGC-DAE Consortium for Scientific Research Kolkata Centre 3LB-8 Bidhan Nagar Kolkata West Bengal 700098 India

Correspondence should be addressed to Ansuman Chattopadhyay chansuman1rediffmailcom

Received 7 May 2013 Accepted 11 June 2013

Academic Editors J J Marin Z Mathe and Y Yano

Copyright copy 2013 Ritu Srivastava et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Arsenic is a Group I human carcinogen and chronic arsenic exposure through drinking water is a major threat to humanpopulation Liver is one of the major organs for the detoxification of arsenic The present study was carried out in mice in vivoafter arsenic treatment through drinking water at different doses and time of exposure Arsenic toxicity is found to be mediatedby reactive oxygen species Nuclear factor (erythroid-2 related) factor 2 (Nrf2)Keap1 (Kelch-like ECH-associated protein 1)ARE(antioxidant response element)mdashdriven target gene system protects cells against oxidative stress and maintains cellular oxidativehomeostasis Our result showed 04 ppm 2 ppm and 4 ppm arsenic trioxide treatment through drinking water for 30 days and 90days induced damages in the liver of Swiss albino mice as evidenced by histopathology disturbances in liver function induction ofheat shock protein 70 modulation of trace elements alteration in reduced glutathione level glutathione-s-transferase and catalaseactivity malondialdehyde production and induction of apoptosis Cellular Nrf2 protein level and mRNA level increased in alltreatment groups Keap1 protein as well as mRNA level decreased concomitantly in arsenic treatedmice Our study clearly indicatesthe important role of Nrf2 in activating ARE driven genes related to GSH metabolic pathway and also the adaptive responsemechanisms in arsenic induced hepatotoxicity

1 Introduction

Arsenic (As) a Group I human carcinogen is the majorsource of ground water contamination all over the worldThe permissible limit of As set by World Health Orga-nization (WHO) is 10 parts per billion (ppb) Howeverin many countries including India and Bangladesh peopleare consuming As through drinking water at much higherlevel Up to 50 ppm of As is reported in many states in theUSA [1] Fu et al [2] estimated that 13 million Americans

were exposed to more than 001 ppm of arsenic throughpublic water systems by 2006 According to the report of thePrevention and Treatment Academy of China this numberreached 1466 million in China [3] where in many placesindividuals were exposed up to a level of 1 ppm of As [4] InWest Bengal India As concentrations in some tube wells isas high as 34 ppm [5] Chronic arsenic exposure has becomea great concern than acute exposure mainly because of itscarcinogenic effects [6 7] Environmental exposure to arsenicis generally in the form of either arsenite (As3+) or arsenate

2 ISRN Hepatology

(As5+) which undergoes redox conversion where arsenite isthe predominant form in drinking water and is considered asthe major carcinogen in epidemiological studies [6 8]

Liver is one of the major target organs of arsenic tox-icity and carcinogenesis [9ndash11] When consumed throughdrinking water inorganic As species is converted into itsmethylated form within the liver and excreted out [12] Inexposed human populations chronic arsenic causes variety oftoxic effects in the liver and other organs and is associatedwith tumerogenesis [13] Gastrointestinal symptoms abnor-mal liver function and elevations of serum enzymes likealanine amino transferase (ALT) aspartate amino transferase(AST) and alkaline phosphatase (ALP) are reported afteracute or chronic exposure [14 15] Developments of portalhypertension and liver fibrosis have also been observedamong As-exposed populations [14 16] Histopathology andinduction of stress protein by arsenic have been reported inChanna punctatus [17] The roles of reactive oxygen species(ROS) and reactive nitrogen activity are known duringarsenic toxicity [18ndash20] but the exact mechanism for thegeneration of all these reactive species is yet to be elucidated[21] Glutathione (GSH) the major nonprotein thiol inmammalian cells is a well known free radical scavenger andreducing equivalent and plays a major protective role againstionizing radiation as well as chemical reagents generatingROS Liver is a rich source of GSH and a major site forarsenic detoxification through GSH-As conjugation pathway[9] Trivalent arsenicals readily react in vitro with GSH andcysteine [22] The binding of trivalent arsenic to critical thiolgroups causes GSH depletion affecting the status of otherantioxidants and thus inhibits important biochemical eventswhich could lead to toxicity [23] However the effect ofarsenic on GSH metabolic pathway in liver in vivo is not yetclearly known which prompted the present investigation

Cellular oxidative homeostasis is maintained by a tran-scriptional factor Nrf2 (Nuclear factor erythroid 2-relatedfactor 2) which does so through transcriptional upreg-ulation of an array of downstream genes such as glu-tathione s-transferase (GST) glutathione peroxidase (GPx)glutathione reductase (GR) 120574-gamma glutamyl cysteinesynthase (120574GCS) glutamate cysteine ligase (GCL) hemeoxygenase-1 (HO-1) andNAD(P)Hquinone oxidoreductase-1 (NQO1) [24ndash26] Studies on As-induced activation of Nrf2and its downstream genes have been reported in differentcell lines [20 27ndash32] Nrf2 plays a pivotal role in modulat-ing the expression of phase II detoxification enzymes andendogenous antioxidants Using Nrf2 knockout mice Jianget al [12] showed that Nrf2 protects against liver and bladderinjury in response to six weeks of arsenic exposure butthe detailed mechanism particularly its role on the GSH-metabolic pathway in vivo was not studied

In some parts of West Bengal India arsenic concentra-tion is reported to be as high as 34 ppm [5] Therefore weselected three doses (04 2 and 4 ppm of As

2O3) through

drinking water in mice to see their effect on body weightgain organ to body weight ratio and histopathology of liverafter chronic exposure for one to three months Level ofGSH activity of GST and catalase MDA production andexpression ofHsp70Nrf2 Keap1 (Kelch-like ECH-associated

protein 1) p62 andAREdriven genes for antioxidant enzymesinvolved in GSH-metabolic pathway were observed to under-stand the role of Nrf2 on As-induced hepatotoxicity

Trace element profile is a valuablemarker for health statusof animal body and any disturbance in the profile indicatesmalfunctioning of the normal metabolism Trace elementsare involved in almost every cellular biochemical processand inadequacy or imbalance in the level of trace elementconsequently affects a number of physiological functionsThemodulation of some important trace elements that mediateoxidative stress and are related to redox status of the cells suchas copper (Cu) zinc (Zn) iron (Fe) magnesium (Mg) andselenium (Se) was considered in the present study since theyare the key elements in cellular protection against As-inducedhepatic damages which could influence the Nrf2 mediatedantioxidant responses

2 Materials and Methods

21 Chemicals andReagents Arsenic trioxide (As2O3 molec-

ular weight 19784) was purchased from Sigma-Aldrich Corp(St Louis MO USA) Glutamate oxaloacetate transam-inase GOT (AST) and glutamate pyruvate transaminaseGPT (ALT) test kits were purchased from Span diagnos-tics Ltd Surat India Primary antibodies against Hsp 70Nrf2 gamma glutamyl cysteine synthase (120574GCS) glutathionereductase (GR) glutathione S-transferase (GST) p62 Keap1120573-actin were purchased from Santa Cruz BiotechnologyInc (Santa Cruz CA USA) Mouse anti-rabbit ALP conju-gated secondary antibody Hoechst (Bisbenzimide H 33342)BCIPNBT andTRI reagent for RNA isolationwere procuredfrom Sigma-Aldrich Corp (St Louis MO USA) Reversetranscriptase and all chemicals of PCR mix were purchasedfromFermentas (USA) All other chemicals usedwere of ana-lytical grade and purchased from Sisco Research laboratories(Mumbai India) and Merck (Darmstadt Germany)

22 Animals and Treatment Male Swiss albino mice aged 2-3 months weighing 25ndash30 g were maintained in communitycages in a temperature-controlled room at 20 plusmn 2∘C and12 hr light12 hr dark cycle They were fed standard mousediet procured from NMC Oil Mills Ltd Pune India andwere provided with water ad libitum Animal studies wereapproved by the Institutional Animal Ethics CommitteeVisva-Bharati University and were performed in accordancewith the ethical standards laid down in the 1964 Declarationof Helsinki and its later amendments Mice were dividedinto five groups with 6 mice per group as given below Allmice were sacrificed under anesthesia using light sodiumpentobarbital

Group I untreated animals (control)Group II 04 ppm As

2O3treated through drinking

water for 30 daysGroup III 2 ppm As

2O3treated through drinking

water for 30 daysGroup IV 4 ppm As

2O3treated through drinking

water for 30 days

ISRN Hepatology 3

Group V 4 ppm As2O3treated through drinking

water for 90 days

23 Measurement of Body Weight and Water ConsumptionThe body weight of all animals was recorded initially andalso during the course of the treatment Rate of waterconsumption and gain in body weight were recorded foreach individual mouse at certain time intervals during theexperiment

24 Determination of Organ to Body Weight Ratio Theweight of themicewas recorded before sacrificeThe liver wasdissected out carefully blotted free of blood and fresh weightwas recorded Organ to body weight ratio was calculated andcompared with the control mice

25 Histopathological Studies Portions of liver tissue of allanimals were fixed in Bouinrsquos fluid dehydrated throughgraded alcohol and embedded in paraffin and routine mic-rotomy was carried out to obtain 6120583m thick tissue sectionsSections were stained by routine hematoxylin-eosin (HE)technique and viewed under light microscope

26 Liver Function Tests Serum glutamate oxaloacetate tran-saminase (SGOT) (aspartate transaminase AST) and serumglutamate pyruvate transaminase (SGPT) (alanine transami-nase ALT) levelswere estimated following themanufacturerrsquosprotocol

27 Determination of Reduced Glutathione (GSH) Liver GSHwas measured following the method of Beutler et al [33]In brief liver tissue was quickly dissected out and blanchedin ice-cold isotonic saline A 10 homogenate was preparedfrom each tissue with ice-cold saline-EDTA at 4∘C Onemilliliter of freshly prepared 20 ice-cold trichloroacetic acid(TCA) was added to equal volume of homogenate and themixture was vortexed and allowed to stand for 10min in 4∘CThe mixture was then centrifuged at 5000 rpm for 5minThe clear supernatant was used as the GSH sample fromwhich 1mL of supernatant was taken andmixed with 3mL of03M disodium hydrogen phosphate buffer and 1mL of 551015840-dithiobis-2-nitro benzoic acid (DTNB) solution After 5minthe optical density of the samples was measured at 412 nmand results were expressed as 120583MGSHmg protein

28 Assay of Glutathione-s-Transferase (GST) GST activitywas assessed in the liver cytosolic fractions as described byHabig et al [34] using 1-chloro-24-dinitrobenzene (CDNB)(1mM final concentration) as substrate in the presence ofexcess GSH (5mM) The rate of CDNB conjugation wasestimated by direct spectrophotometry at 340 nm for 3minThe result was expressed as 120583M GS-CDNB formedminmgprotein

29 Assay ofThiobarbituric Acid Reactive Substances (TBARS)Level in Liver Lipid peroxidation products namely mal-ondialdehyde (MDA) was estimated in liver microsomesassuming high PUFA content of microsomal membranes

The level of lipid peroxidation as measured by TBARS wasdetermined according to the method of Buege and Aust [35]Briefly 1mL of microsomal sample was mixed with 2mL ofTBA-TCA-HCl mixture thoroughly and heated for 15min ina boiling water bath After cooling the flocculent precipitatewas removed by centrifugation at 1000 g for 10min Theabsorbance of the supernatant was determined at 535 nm andexpressed in terms of nMMDAmg protein

210 Catalase Assay Catalase activity was assayed followingthe procedure of Aebi [36] as modified by Kawamura [37]A 5 homogenate was prepared in 50mM phosphate buffer(pH 70) and centrifuged at 12500timesg for 30min at 4∘C Thesupernatant or the peroxisome-rich fraction was used as thesample The sample (20 120583L) was added to 980120583L of an assaybuffer containing 50mMTris-HCl (pH80) 9mMH

2O2 and

025mM EDTA to constitute the assay volume of 1mL Thedecrease in ΔODmin of that assay mixture was recorded at240 nm for 1min The results were expressed as unit catalaseactivitymg protein

211 Western Blot Analysis

2111 Sample Preparation Whole cell protein extracts andWestern blot analysis were performed as previously described[38] Liver homogenates (10 wv) were prepared in 50mMphosphate buffer (pH 75) and centrifuged at 10000 g for20min The cytosolic supernatant was collected very care-fully and the protein content of the sample was measuredfollowing the method of Lowry et al [39]

2112 Methods forWestern Blotting Protein (60 120583g) from thelysates of control and treated cells was resolved on 10 SDS-PAGE at a constant voltage (60V) for 25 h and then blottedonto a polyvinylidene fluoride (PVDF) membrane using asemidry trans blot apparatus (Bio-Rad Trans Blot SD CellUSA) The membranes were first incubated with primaryantibodies at a dilution of 1 1000 overnight at 4∘C followedby 2 h incubation with corresponding ALP-conjugated sec-ondary antibodies at 1 2000 (Sigma) dilutions with contin-uous rocking The immunoreactive bands were detected byusing 5-bromo-4chloro-3-indolylphosphatenitroblue tetra-zolium (BCIPNBT) Densitometric quantification was doneby ImageJ (NIH) software

212 Total RNA Extraction and RT-PCR Analysis Total RNAfrom liver tissues were extracted using TRI reagent Equalamounts of RNA (5 120583g) were reverse transcribed into cDNAusing the RevertAid reverse transcriptase (Fermentas) fol-lowing manufacturers protocol for RT-PCR The PCR wasperformed following the procedure as per the manufacturerrsquosinstruction for 35 cycles All test samples were amplifiedsimultaneously from equal volume of first strand cDNAwith the particular primer pair using a master PCR mixPCR reactions were run in a programmable thermal cycler(GeneAmp 9700 ABI) with simultaneous NTC (no tem-plate control) 120573-actin was amplified simultaneously as aninternal control Specific primers for Nrf2 and 120573-actin for

4 ISRN Hepatology

PCR amplification are Nrf2 forward 51015840-TCTCCTCGCTGG-AAAAAGAA-31015840 51015840-AATGTGCTGGCTGTGCTTTA-31015840 120573-actin forward 51015840-TGGAATCCTGTGGCATCCATGAAAC-31015840 120573-actin reverse 51015840-TAAAACGCAGCTCAGTAACAG-TCCG-31015840 [40] For amplification of Keap1 specific primerwas designed using Primer 3 software Keap1 forward 51015840-GTACGCTGCGAGTCCGAGGT-31015840 Keap1 reverse 51015840-GCC-ATTGCTCGGGTTGTAGG-31015840 The PCR products were runin 1 agarose gel and visualized in a gel documentationsystem (Gel Doc EZ Imager Bio-Rad) after staining withethidium bromide The densitometric quantification wasdone using ImageJ (NIH) software

213 Hoechst 33342 Staining Hepatocytes were isolated fromblanched liver by a two-step collagenase (Sigma AldrichUSA) digestion method [41] The isolated hepatocytes fromdifferent treatment groups were washed with PBS fixedwith 37 para-formaldehyde solution at room tempera-ture stained with bisBenzimide H 33342 trihydrochloride(Hoechst 33342 2mgmL) and visualized under fluorescencemicroscope (Dewinter Italy) within 30min of adding thestain

214 EDXRF Measurement Liver was dissected out carefullyusing stainless steel forceps and blotted free of blood Thesamples were not washed to avoid leaching of soluble ele-ments Tissues were freeze dried in a lyophilizer after fixing inliquid nitrogen and made into fine powder using mortar andpestle About 150mg pooled powdered samples were used tomake into pellets (1mm thick and 10mm diameter) using atabletop pelletiser (pressure 100 to 110 kgcm2 for 5 minutes)

The tissue samples were analyzed by Jordan Valley EX-3600 ED-XRF system All measurements were carried outin vacuum using different filters (between the source andsample) for optimum detection of elements for 600 s TheX-rays detection was done by using liquid nitrogen cooled125mm2 Si (Li) semiconductor detectors with the resolution148 eV at 59 KeV The X-ray fluorescence spectra were quan-titatively analyzed using Ex-Win software integrated with thesystem Standardization of the procedure was done usingNIST bovine liver standard SRM 1577b

215 Statistical Analysis All assays were repeated at leastthree times Data were analyzed by Studentrsquos 119905-test usingthe Sigma plot 80 statistical package Differences betweencontrol and experimental group(s) with a value of 119875 lt 005was considered as significant

3 Results

31Mortality and Clinical Observations Allmice were exam-ined daily for any clinical signs of toxicity There was nodeath in both control and treatment groups and no clinicalsymptoms were found to appear in any of the treatmentgroups

32 Change in Body Weight and Water Consumption RateThere was no significant difference in water consumption

Org

an to

bod

y w

eigh

t rat

io

Treatment groupsGr I Gr II Gr III Gr IV Gr V

7

6

5

4

3

2

1

0

Figure 1 Organ to body weight ratio of liver in different groups ofmice Groups II III IV and V were compared with group I Valuesare expressed as mean plusmn SEM

rate as well as change in body weight (weight gain) recordedbetween the control and treatment groups (data not shown)

33 Organ to Body Weight Ratio No significant difference inthe organ to body weight ratio of liver of any treatment groupwith the control group was observed (Figure 1)

34 Histopathology Liver appeared normal and healthy(Figure 2(a)) in the control group of mice Disorganization ofhepatic parenchyma and disruption in the epithelial lining ofthe central vein and vacuolar degeneration were commonlyobserved in all treatment groups Liver in Group II micedemonstrated sinusoidal dilation and vacuolar degenerationin the cytoplasm (Figure 2(b)) In group IIImice (treatedwith2 ppm of As

2O3) extensive vacuolar degeneration and loss of

integrity in the epithelial lining of the central vein were found(Figure 2(c)) Loss of typical organization of hepatic cord andvacuolar degeneration were seen in group IV mice (treatedwith 4 ppm of As

2O3for 30 days) (Figure 2(d)) In group

V mice treated with 4 ppm of As2O3for 90 days extensive

degeneration of epithelial lining of the central vein (thickarrow) loss of typical hepatic cord organization hepatocellu-lar degradation and infiltration of the nucleus (thin arrows)into the central vein were prominent (Figure 2(e))

35 Liver Function Test An overall increase was noted in theserum GOT (AST) and GPT (ALT) levels in all treatmentgroups against control (Figure 3) SGOT level increased sig-nificantly in group II (7794) group III (15379) and groupIV (11304) mice SGPT level also increased significantly ingroup II (11596) group III (9509) and group IV (15373)

36 GSH and GST Response and TBARS Production A dosedependent decrease in GSH level was recorded in all the30 days treatment groups of mice and the decrease wassignificant in group III (3229) and group IV (479) againstcontrol In group V mice after 4 ppm of As

2O3treatment for

90 days GSH level recovered which increased significantlyagainst control mice (1931) (Figure 4(a)) TBARS increased

ISRN Hepatology 5

CV

(a) (b)

(c)

(d) (e)

Figure 2 Changes in liver histology Microphotographs of liver sections (6 120583m) stained with hematoxylin and eosin (HE) The originalmagnificationtimes400 (a)Normal histological appearance of liver tissue of controlmice central vein (CV) (b)Group II vacuolar degenerations(thin arrows) sinusoidal dilation (thick arrow) (c) Group III disruption of epithelium lining (short thick arrows) of the central vein vacuolardegenerations (thin arrows) (d) Group IV vacuolar degeneration (thin arrow) loss of integrity in epithelium lining of the central vein (shortthick arrow) and loss of typical hepatic cords organization (e) Group V extensive degeneration of epithelial lining of the central vein (thickarrow) loss of typical hepatic cord organization hepatocellular degradation and infiltration of the nucleus into the central vein (thin arrow)

Enzy

me c

once

ntra

tion

(uni

t enz

yme

L se

rum

)

Serum GPTSerum GOT

Gr I Gr II Gr III Gr IV Gr VTreatment groups

300

250

200

150

100

50

0

lowast

lowast

lowast

lowast

lowast

lowast

Figure 3 SGPT and SGOT activity (unit enzymeL of serum) inthe serum of different groups of mice exposed to different doses ofAs2O3 Groups II III IV and Vwere compared with group I Values

are expressed as mean plusmn SEM lowastValues are statistically significant at(119875 lt 005)

significantly in group III (8656) group IV (8993)and group V (2684) mice GST activity also depicted asignificant increase in group III (7219) group IV (6213)and group V (3478) mice (Figures 4(b) and 4(c))

37 Catalase Activity Catalase activity increased significantlyin group II (55) and thereafter gradually decreased withsubsequent higher doses in group III (744) and group IV(2709) mice against control In group V catalase activityshowed an increase though the increase was not significantcompared to the control (Figure 4(d))

38 Heat Shock Protein (Hsp) 70 Expression Hsp 70 profileshowed increasing pattern of expression against control in allthe treatment groups Expression increased 125-folds 126-folds 132-folds and 154-folds in group II III IV andVmicerespectively as against the control group (Figures 5(a) and5(c))

39 Nrf2 Keap1 and p62 Protein Expression Nrf2 proteinlevels were detected in whole cell lysates as this can give anidea of relative Nrf2 levels in the nuclear fractions of arsenic

6 ISRN HepatologyG

SH120583

mol

mg

prot

ein

Gr I Gr II Gr III Gr IV Gr VTreatment groups

20

18

16

14

12

10

8

6

4

2

0

lowast

lowast

lowast

(a)

nM M

DA

pro

duce

dm

gpr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

35

3

25

2

15

1

0

lowastlowast

lowast

05

(b)

Gr I Gr II Gr III Gr IV Gr VTreatment groups

30

25

20

15

10

5

0

lowastlowast

lowast

nM D

NPG

prod

uced

min

mg

prot

ein

(c)

Uni

t enz

yme

activ

itym

inm

g pr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

lowast

lowast

800

700

600

500

400

300

200

100

0

(d)

Figure 4 (a) GSH content (b) MDA production (c) GST activity (d) catalase activity in liver of different groups of mice exposed to differentdoses of As

2O3 Groups II III IV andVwere comparedwithGroup I Values are expressed asmeanplusmn SEM lowastValues are statistically significant

at (119875 lt 005)

treated cells [20 42 43] We observed the induction of Nrf2protein for different doses of arsenic in all the treatmentgroups but the increase in the Nrf2 protein level was notconsistent with the increasing doses of arsenic treatmentThemaximum level of Nrf2 protein was recorded in group II(144-fold of control) mice which was found to decrease ingroup III (114-fold) mice Further in group IVmice the levelof Nrf2 protein increased to 136-folds of the control againshowing a decreasing pattern in group V (118-fold) mice(Figures 5(a) and 5(b)) Keap1 protein expression decreasedin all the treatment groups compared to the control groupLowest level of Keap1 protein was found in group V (Figures5(a) and 5(b)) mice p62 protein expression was also low inthe control group while elevated levels of protein expressionwere observed in all the treatment groups though the increasein protein level was not dose dependent The highest levelof p62 protein was observed in group II (18-fold of control)followed by group III and group IV mice where it decreasedgradually by 13-fold and 12-fold respectively In group V

mice however an elevation of 16 folds was recorded (Figures5(a) and 5(b))

310 120574GCS Protein GST and GR Protein Expression 120574GCSprotein level in group II mice showed a slight decreaseas compared to the control group while the protein levelincreased continuously in the subsequent groups by 103-fold 122-fold and 151-fold in group III IV and V micerespectively against control (Figures 5(a) and 5(c)) GSTprotein expression increased in all treatment groups with thehighest level recorded in groupV (123-fold) (Figures 5(a) and5(c)) Expression of GR protein also showed an increasingtrend in group II and III whereas in group IV and V micethe level gradually decreased and in group Vmice it reachedalmost the control level (Figures 5(a) and 5(c))

311 Nrf2 and Keap1 mRNA Expression Expression of Nrf2mRNA increased in all the treatment groups compared tothe control group Expression increased by 231-fold 221-fold

ISRN Hepatology 7

Hsp 70

Nrf2

Keap1

GST

GR

Gr I Gr II Gr III Gr IV Gr V

p62

M

120573-Actin

120574GCS

(a)

Nrf 2Keap1p62

20

18

16

14

12

10

08

06Gr I Gr II Gr III Gr IV Gr V

Rela

tive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

Treatment groups

(b)

GSTGRHsp 70

18

16

14

12

10

08Gr I Gr II Gr III Gr IV Gr V

Treatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

120574GCS

(c)Figure 5 (a) Western blotting (b) densitometric analysis of the Nrf2 protein Keap1 protein and p62 protein profile and (c) densitometricanalysis of 120574GCS protein GST protein GR protein and Hsp 70 protein profile of liver of mice treated with different doses of As

2O3

and 229-fold in group II group III and group IV micerespectively against control group ofmice In groupV (4 ppmof As2O3treated for 90 days) decrement of the expression

was noteworthy against the 30 days treatment groups (Figures6(a) and 6(b)) though the level was still higher than control(177-fold) Keap1 mRNA level decreased in all treatmentgroups Keap1 mRNA level detected was 081-fold that ofcontrol in group II 054 folds of the control in group III 061folds of the control in group IV and 042-fold of control ingroup V mice (Figures 6(a) and 6(b))

312 Detection of Apoptosis by Hoechst 33342 The hepato-cytes of mice in all the treatment groups exhibited con-densed and fragmented nuclei upon staining with Hoechst33342 which is an indicator of possible apoptotic cell death(Figure 7)

313 Modulation of Elements Concentration of Mg showedan increasing trend in all treatment groups and the increasewas significant in group III and group IV mice Group IImice showed significant increase and decrease respectivelyfor Cu and Zn whereas significant depletion in Se level wasobserved in group IV mice Iron concentration increased ingroup II group III and group IV with the highest level werefound in group IV mice In group V mice a reduction in theiron concentration was observed reaching almost the controlvalue (Table 1)

4 Discussion

According toWHOguideline the permissible limit of arsenicin drinking water is 10 ppb In some states in USA andChina people are exposed to more than 1 ppm of As throughtheir drinking water Higher arsenic contamination is mainly

8 ISRN Hepatology

Nrf2

Keap1

GrI Gr II Gr III Gr IV Gr V

120573-Actin

(a)

Nrf2Keap1

GrI Gr II Gr III Gr IV Gr VTreatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

30

25

20

15

10

05

00

(b)

Figure 6 (a) Nrf2 and Keap1 mRNA expressions in liver (b) Densitometric analysis of Nrf2 mRNA Keap1 mRNA in liver of mice treatedwith different doses of As

2O3

(a) (b) (c)

(d) (e) (f)

Figure 7 Mouse hepatocytes showing apoptosis (arrow) by Hoechst 33342 staining (a) (b) control (c) 04 ppm (d) 2 ppm and (e) 4 ppmAs2O3treatment for 30 days (f) 4 ppm arsenic trioxide treatment for 90 days

ISRN Hepatology 9

Table 1 Concentration of elements (mgkg) in whole-liver tissue following in vivo administration of different concentrations of As2O3 (ppm)in the drinking water of the mice for 30 and 90 days (Values are mean plusmn SEM)

Elements Group I Group II Group III Group IV Group VMg 35445 plusmn 5417 54729 plusmn 5888 67879 plusmn 2113

lowast57821 plusmn 4246

lowast46143 plusmn 2990

Cu 1541 plusmn 023 1749 plusmn 073lowast

1454 plusmn 072 1444 plusmn 047 1468 plusmn 059

Zn 10755 plusmn 157 9763 plusmn 397lowast

10377 plusmn 164 10762 plusmn 335 10226 plusmn 471

Se 1419 plusmn 134 1325 plusmn 055 1252 plusmn 189 843 plusmn 074lowast

1409 plusmn 113

Fe 50365 plusmn 3866 61029 plusmn 4599 56782 plusmn 2297 73421 plusmn 4494lowast

50689 plusmn 2822

lowastValues are statistically significant compared to control at (119875 lt 005)

found in ground water which is the most common sourceof drinking water The chronic exposure is the main causeof arsenic induced cancer development in human popula-tion and the mechanism of chronic arsenic toxicity is notfully known Therefore in our present experiment micewere treated with As through drinking water Accordingto Singh et al [44] arsenic administration decreased bodyweight gain in a dose dependant manner Santra et al [45]have reported significant increase in the liver weight ofBALBc mice after 12 months of arsenic treatment whereasin the present study there was no significant change in bodyweight gain rate of water consumption or the organ to bodyweight ratio of liver in any of the treatment groups

Liver is an important organ for various metabolic path-ways and effect of any chemical or xenobiotic appears pri-marily in the liver In case of arsenic metabolism liver is themain site of arsenic methylation [46]Themost commonwayof assertion of liver damage is to determine two pathophys-iological enzymes serum glutamate pyruvate transaminase(SGPT) and serum glutamate oxaloacetate transaminase(SGOT) In our findings there was a distinct pattern ofincreased SGOT and SGPT levels in all the treatment groupstreated for 30 days In case of As

2O3(4 ppm) treatment

for 90 days however reversal in the activities of both theenzymes occurred almost reaching basal level The reasonfor such reversal is difficult to explain since histopathologicalalterations as well as induction of apoptosis was observed inthe same treatment group The increased SGOT and SGPTlevels in rat after 45 days treatment of 10 ppm sodium arsenitewas also described by Tandan et al [47] whereas Santra et al[45] reported increased SGOT and SGPT levels after 12months of arsenic treatment at 32 ppm in mice Liver alter-ation in histopathological architecture was evident mainly inthe form of loss of integrity of central vein loss of hepaticcord organization and dose and time dependent vacuolationwhich corroborates with earlier studies [45 48 49] Thesestudies indicate that chronic exposure to different inorganicarsenic compounds (arsenite arsenic trioxide or arsenate)produces characteristic pathology in the liver including fattyinfiltration liver degeneration inflammatory cell infiltrationand focal necrosis Das Neves et al [50] have shown that evenacute exposure of sodium arsenite at a dose of 10mgkg bodyweight (intraperitoneally) for only 90min could producevarious histopathological alterations in liver

In the present study Hoechst 33342 stained hepatocytesclearly indicated fragmented nuclei in all the treatment

groups as a mark of apoptosis as reported earlier in fish liver[17]

Oxidative stress is a relatively new theory in assessmentof arsenic toxicity [51 52] Arsenic causes oxidative stress byproducing reactive oxygen species [20 53 54] which damageproteins Due to lipophilic nature arsenic also attaches tolipid thereby increasing the rate of lipid peroxidation [55]Cellular GSH plays an important role in mitigating arsenicinduced oxidative stress Arsenic being a strong electrophilepredominantly binds with nucleophilic SH group of GSHThus consequent depletion of GSHmay alter the redox statusof the cell and present a stressful and toxic situation Accord-ing to Bashir et al [56] acute treatment of sodium arseniteat doses of 63mgkg 105mgkg and 126mgkg of bodyweight for 24 h significantly decreased GSH content in liverfor all doses tested On the other hand a significant increasein lipid peroxidation and glutathione peroxidase activityalongwith significant decrease in the activity of GST catalaseand superoxide dismutase was observed at 105mgkg and126mgkg of As treatment Santra et al [45] reported thatchronic exposure to 32 ppm of arsenic significantly depletedGSHafter 6months of exposure catalase activity significantlydecreased after 9 months and GST activity significantlyreduced at 12 and 15 months of arsenic exposure In ourstudy GSH level showed decreasing trend in all the 30 daystreatment groups with highest depletion recorded at 4 ppmof As2O3treatment for 30 days while after 90 days treatment

with the same dose the GSH level increased significantlywith respect to both control and also the 30 days treatmentgroupsThis shows the upregulation ofGSHagainst long termarsenic treatment which is due to its adaptive response tooxidative stress [20] However a reverse trend was observedin TBARS production and GST activity in liver in group II(04 ppm As

2O3treatment for 30 days) there was almost no

change in TBARS and GST level against the control groupwhile in group III and group IV levels of both TBARS andGST activity increased significantly Interestingly in groupV (90 days treatment with 4 ppm As

2O3) both TBARS and

GST level decreased significantly with respect to group IVthat is 4 ppm As

2O3for 30 days though the levels were still

significantly higher than the control group The increasedGST activity might be responsible for the reduction in GSHwhile protecting against the arsenic-induced oxidative stressCatalase activity increased significantly in group II mice withrespect to control while at subsequent higher doses that is2 ppm and 4 ppm As

2O3for 30 days the activity decreased

10 ISRN Hepatology

Increased level of SGOT and SGPT

Histopathological alterationsModulation in trace element levels

Increased production of p62

Increased Nrf2 level

Decreased activity of Keap1

Increased production of Hsp 70

Active Nrf2

Gene transcription

GST

GCS

GR

GSH

p62

Alteration in the activity of GST

Modulation in GSH level

Apoptotic induction

Increased production of MDA

p62

Keap1Keap1

ubub

ub

ubInactive Nrf2

Cytosol Nucleus

Nuclear localiztion

Nrf2Nrf2

Nrf2

ARE

Oxidative stress

As

120574GCS GR and catalase

Figure 8 Schematic representation of the arsenic induced liver damages and induction of gene expression through the Keap1-Nrf2-AREsignaling pathway

and highest rate of depletion was observed after 4 ppmAs2O3

treatment for 30 days Further in group V (4 ppm As2O3for

90 days) the recovery in the catalase activity was observedand it increased significantly when compared to group IV(4 ppmAs

2O3treatment for 30 days) According toMittal and

Flora [57] catalase activity significantly decreased in liver andkidney with respect to control after treatment with 100 ppmsodium meta-arsenite in drinking water for 8 weeks

Trace elements play an important role in maintainingnormal homeostasis of the body Oxidative stress results fromchanges in the levels of trace elements It has been reportedthat increased Mg level is associated with the increasedproduction ofMDA in dementia patients [58] Our result alsoshowed an increasing pattern ofMg concentration alongwithincrease in liver TBARS level

Zn is an essential element required for growth andnormal development and is a constituent of more than 200enzymes one of which is a CuZn superoxide dismutase(CuZn SOD) CuZn SOD is a powerful antioxidant whichtransforms free radical O

2

∙ to H2O2 therefore reducing the

risk of formation of highly reactive hydroxyl radical HO∙ Cuis also an integral part of CuZn SOD enzyme DecreasedZn and Cu levels in tissues may result in reduction of CuZnSOD activity and subsequently accelerate the process of cellaging and death via oxidative damage [59] However free orincorrectly bound Cu+2 can catalyze the generation of the

most damaging radicals such as HO∙ resulting in a chemicalmodification of the protein alterations in protein structureand solubility and oxidative damage to surrounding tissue[60] Our findings also indicate significant modulation of Cuand Zn level in liver of group II mice

Selenium (Se) is a well known antagonist of arsenic toxic-ity [61] The most probable cause of such a protective effectof selenium is due to its ability to upregulate antioxidantenzymes like GSH peroxidase and thioredoxin reductasewhich protects against arsenic induced oxidative damage[62] Decrement in selenium level after As

2O3treatment was

reported byMolin et al [63 64] In the present study Se levelin liver decreased dose dependently recording maximumdepletion at 4 ppm of As

2O3for 30 days but in the group

treated for 90 days with 4 ppm As2O3 Se level increased and

almost reached the normal levelIron (Fe) catalyses the formation of reactive oxygen

species through the Fenton and Haber-Weiss reactions [65]which generate highly toxic hydroxyl radical and cause lipidperoxidation [66] According to Ahmad et al [67] body ironstores (serum ferritin and transferrin saturation) in the bodycan be used as an early investigative tool for assessing theoxidative stress in coronary heart disease In our study ironlevel increased in all the treatment groups except in group Vwhere the iron concentration is almost same to that of thecontrol group

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Diabetes ResearchJournal of

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Computational and Mathematical Methods in Medicine

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Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

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Volume 2013

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

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Oxidative Medicine and Cellular Longevity

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Volume 2013

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ObesityJournal of

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PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

2 ISRN Hepatology

(As5+) which undergoes redox conversion where arsenite isthe predominant form in drinking water and is considered asthe major carcinogen in epidemiological studies [6 8]

Liver is one of the major target organs of arsenic tox-icity and carcinogenesis [9ndash11] When consumed throughdrinking water inorganic As species is converted into itsmethylated form within the liver and excreted out [12] Inexposed human populations chronic arsenic causes variety oftoxic effects in the liver and other organs and is associatedwith tumerogenesis [13] Gastrointestinal symptoms abnor-mal liver function and elevations of serum enzymes likealanine amino transferase (ALT) aspartate amino transferase(AST) and alkaline phosphatase (ALP) are reported afteracute or chronic exposure [14 15] Developments of portalhypertension and liver fibrosis have also been observedamong As-exposed populations [14 16] Histopathology andinduction of stress protein by arsenic have been reported inChanna punctatus [17] The roles of reactive oxygen species(ROS) and reactive nitrogen activity are known duringarsenic toxicity [18ndash20] but the exact mechanism for thegeneration of all these reactive species is yet to be elucidated[21] Glutathione (GSH) the major nonprotein thiol inmammalian cells is a well known free radical scavenger andreducing equivalent and plays a major protective role againstionizing radiation as well as chemical reagents generatingROS Liver is a rich source of GSH and a major site forarsenic detoxification through GSH-As conjugation pathway[9] Trivalent arsenicals readily react in vitro with GSH andcysteine [22] The binding of trivalent arsenic to critical thiolgroups causes GSH depletion affecting the status of otherantioxidants and thus inhibits important biochemical eventswhich could lead to toxicity [23] However the effect ofarsenic on GSH metabolic pathway in liver in vivo is not yetclearly known which prompted the present investigation

Cellular oxidative homeostasis is maintained by a tran-scriptional factor Nrf2 (Nuclear factor erythroid 2-relatedfactor 2) which does so through transcriptional upreg-ulation of an array of downstream genes such as glu-tathione s-transferase (GST) glutathione peroxidase (GPx)glutathione reductase (GR) 120574-gamma glutamyl cysteinesynthase (120574GCS) glutamate cysteine ligase (GCL) hemeoxygenase-1 (HO-1) andNAD(P)Hquinone oxidoreductase-1 (NQO1) [24ndash26] Studies on As-induced activation of Nrf2and its downstream genes have been reported in differentcell lines [20 27ndash32] Nrf2 plays a pivotal role in modulat-ing the expression of phase II detoxification enzymes andendogenous antioxidants Using Nrf2 knockout mice Jianget al [12] showed that Nrf2 protects against liver and bladderinjury in response to six weeks of arsenic exposure butthe detailed mechanism particularly its role on the GSH-metabolic pathway in vivo was not studied

In some parts of West Bengal India arsenic concentra-tion is reported to be as high as 34 ppm [5] Therefore weselected three doses (04 2 and 4 ppm of As

2O3) through

drinking water in mice to see their effect on body weightgain organ to body weight ratio and histopathology of liverafter chronic exposure for one to three months Level ofGSH activity of GST and catalase MDA production andexpression ofHsp70Nrf2 Keap1 (Kelch-like ECH-associated

protein 1) p62 andAREdriven genes for antioxidant enzymesinvolved in GSH-metabolic pathway were observed to under-stand the role of Nrf2 on As-induced hepatotoxicity

Trace element profile is a valuablemarker for health statusof animal body and any disturbance in the profile indicatesmalfunctioning of the normal metabolism Trace elementsare involved in almost every cellular biochemical processand inadequacy or imbalance in the level of trace elementconsequently affects a number of physiological functionsThemodulation of some important trace elements that mediateoxidative stress and are related to redox status of the cells suchas copper (Cu) zinc (Zn) iron (Fe) magnesium (Mg) andselenium (Se) was considered in the present study since theyare the key elements in cellular protection against As-inducedhepatic damages which could influence the Nrf2 mediatedantioxidant responses

2 Materials and Methods

21 Chemicals andReagents Arsenic trioxide (As2O3 molec-

ular weight 19784) was purchased from Sigma-Aldrich Corp(St Louis MO USA) Glutamate oxaloacetate transam-inase GOT (AST) and glutamate pyruvate transaminaseGPT (ALT) test kits were purchased from Span diagnos-tics Ltd Surat India Primary antibodies against Hsp 70Nrf2 gamma glutamyl cysteine synthase (120574GCS) glutathionereductase (GR) glutathione S-transferase (GST) p62 Keap1120573-actin were purchased from Santa Cruz BiotechnologyInc (Santa Cruz CA USA) Mouse anti-rabbit ALP conju-gated secondary antibody Hoechst (Bisbenzimide H 33342)BCIPNBT andTRI reagent for RNA isolationwere procuredfrom Sigma-Aldrich Corp (St Louis MO USA) Reversetranscriptase and all chemicals of PCR mix were purchasedfromFermentas (USA) All other chemicals usedwere of ana-lytical grade and purchased from Sisco Research laboratories(Mumbai India) and Merck (Darmstadt Germany)

22 Animals and Treatment Male Swiss albino mice aged 2-3 months weighing 25ndash30 g were maintained in communitycages in a temperature-controlled room at 20 plusmn 2∘C and12 hr light12 hr dark cycle They were fed standard mousediet procured from NMC Oil Mills Ltd Pune India andwere provided with water ad libitum Animal studies wereapproved by the Institutional Animal Ethics CommitteeVisva-Bharati University and were performed in accordancewith the ethical standards laid down in the 1964 Declarationof Helsinki and its later amendments Mice were dividedinto five groups with 6 mice per group as given below Allmice were sacrificed under anesthesia using light sodiumpentobarbital

Group I untreated animals (control)Group II 04 ppm As

2O3treated through drinking

water for 30 daysGroup III 2 ppm As

2O3treated through drinking

water for 30 daysGroup IV 4 ppm As

2O3treated through drinking

water for 30 days

ISRN Hepatology 3

Group V 4 ppm As2O3treated through drinking

water for 90 days

23 Measurement of Body Weight and Water ConsumptionThe body weight of all animals was recorded initially andalso during the course of the treatment Rate of waterconsumption and gain in body weight were recorded foreach individual mouse at certain time intervals during theexperiment

24 Determination of Organ to Body Weight Ratio Theweight of themicewas recorded before sacrificeThe liver wasdissected out carefully blotted free of blood and fresh weightwas recorded Organ to body weight ratio was calculated andcompared with the control mice

25 Histopathological Studies Portions of liver tissue of allanimals were fixed in Bouinrsquos fluid dehydrated throughgraded alcohol and embedded in paraffin and routine mic-rotomy was carried out to obtain 6120583m thick tissue sectionsSections were stained by routine hematoxylin-eosin (HE)technique and viewed under light microscope

26 Liver Function Tests Serum glutamate oxaloacetate tran-saminase (SGOT) (aspartate transaminase AST) and serumglutamate pyruvate transaminase (SGPT) (alanine transami-nase ALT) levelswere estimated following themanufacturerrsquosprotocol

27 Determination of Reduced Glutathione (GSH) Liver GSHwas measured following the method of Beutler et al [33]In brief liver tissue was quickly dissected out and blanchedin ice-cold isotonic saline A 10 homogenate was preparedfrom each tissue with ice-cold saline-EDTA at 4∘C Onemilliliter of freshly prepared 20 ice-cold trichloroacetic acid(TCA) was added to equal volume of homogenate and themixture was vortexed and allowed to stand for 10min in 4∘CThe mixture was then centrifuged at 5000 rpm for 5minThe clear supernatant was used as the GSH sample fromwhich 1mL of supernatant was taken andmixed with 3mL of03M disodium hydrogen phosphate buffer and 1mL of 551015840-dithiobis-2-nitro benzoic acid (DTNB) solution After 5minthe optical density of the samples was measured at 412 nmand results were expressed as 120583MGSHmg protein

28 Assay of Glutathione-s-Transferase (GST) GST activitywas assessed in the liver cytosolic fractions as described byHabig et al [34] using 1-chloro-24-dinitrobenzene (CDNB)(1mM final concentration) as substrate in the presence ofexcess GSH (5mM) The rate of CDNB conjugation wasestimated by direct spectrophotometry at 340 nm for 3minThe result was expressed as 120583M GS-CDNB formedminmgprotein

29 Assay ofThiobarbituric Acid Reactive Substances (TBARS)Level in Liver Lipid peroxidation products namely mal-ondialdehyde (MDA) was estimated in liver microsomesassuming high PUFA content of microsomal membranes

The level of lipid peroxidation as measured by TBARS wasdetermined according to the method of Buege and Aust [35]Briefly 1mL of microsomal sample was mixed with 2mL ofTBA-TCA-HCl mixture thoroughly and heated for 15min ina boiling water bath After cooling the flocculent precipitatewas removed by centrifugation at 1000 g for 10min Theabsorbance of the supernatant was determined at 535 nm andexpressed in terms of nMMDAmg protein

210 Catalase Assay Catalase activity was assayed followingthe procedure of Aebi [36] as modified by Kawamura [37]A 5 homogenate was prepared in 50mM phosphate buffer(pH 70) and centrifuged at 12500timesg for 30min at 4∘C Thesupernatant or the peroxisome-rich fraction was used as thesample The sample (20 120583L) was added to 980120583L of an assaybuffer containing 50mMTris-HCl (pH80) 9mMH

2O2 and

025mM EDTA to constitute the assay volume of 1mL Thedecrease in ΔODmin of that assay mixture was recorded at240 nm for 1min The results were expressed as unit catalaseactivitymg protein

211 Western Blot Analysis

2111 Sample Preparation Whole cell protein extracts andWestern blot analysis were performed as previously described[38] Liver homogenates (10 wv) were prepared in 50mMphosphate buffer (pH 75) and centrifuged at 10000 g for20min The cytosolic supernatant was collected very care-fully and the protein content of the sample was measuredfollowing the method of Lowry et al [39]

2112 Methods forWestern Blotting Protein (60 120583g) from thelysates of control and treated cells was resolved on 10 SDS-PAGE at a constant voltage (60V) for 25 h and then blottedonto a polyvinylidene fluoride (PVDF) membrane using asemidry trans blot apparatus (Bio-Rad Trans Blot SD CellUSA) The membranes were first incubated with primaryantibodies at a dilution of 1 1000 overnight at 4∘C followedby 2 h incubation with corresponding ALP-conjugated sec-ondary antibodies at 1 2000 (Sigma) dilutions with contin-uous rocking The immunoreactive bands were detected byusing 5-bromo-4chloro-3-indolylphosphatenitroblue tetra-zolium (BCIPNBT) Densitometric quantification was doneby ImageJ (NIH) software

212 Total RNA Extraction and RT-PCR Analysis Total RNAfrom liver tissues were extracted using TRI reagent Equalamounts of RNA (5 120583g) were reverse transcribed into cDNAusing the RevertAid reverse transcriptase (Fermentas) fol-lowing manufacturers protocol for RT-PCR The PCR wasperformed following the procedure as per the manufacturerrsquosinstruction for 35 cycles All test samples were amplifiedsimultaneously from equal volume of first strand cDNAwith the particular primer pair using a master PCR mixPCR reactions were run in a programmable thermal cycler(GeneAmp 9700 ABI) with simultaneous NTC (no tem-plate control) 120573-actin was amplified simultaneously as aninternal control Specific primers for Nrf2 and 120573-actin for

4 ISRN Hepatology

PCR amplification are Nrf2 forward 51015840-TCTCCTCGCTGG-AAAAAGAA-31015840 51015840-AATGTGCTGGCTGTGCTTTA-31015840 120573-actin forward 51015840-TGGAATCCTGTGGCATCCATGAAAC-31015840 120573-actin reverse 51015840-TAAAACGCAGCTCAGTAACAG-TCCG-31015840 [40] For amplification of Keap1 specific primerwas designed using Primer 3 software Keap1 forward 51015840-GTACGCTGCGAGTCCGAGGT-31015840 Keap1 reverse 51015840-GCC-ATTGCTCGGGTTGTAGG-31015840 The PCR products were runin 1 agarose gel and visualized in a gel documentationsystem (Gel Doc EZ Imager Bio-Rad) after staining withethidium bromide The densitometric quantification wasdone using ImageJ (NIH) software

213 Hoechst 33342 Staining Hepatocytes were isolated fromblanched liver by a two-step collagenase (Sigma AldrichUSA) digestion method [41] The isolated hepatocytes fromdifferent treatment groups were washed with PBS fixedwith 37 para-formaldehyde solution at room tempera-ture stained with bisBenzimide H 33342 trihydrochloride(Hoechst 33342 2mgmL) and visualized under fluorescencemicroscope (Dewinter Italy) within 30min of adding thestain

214 EDXRF Measurement Liver was dissected out carefullyusing stainless steel forceps and blotted free of blood Thesamples were not washed to avoid leaching of soluble ele-ments Tissues were freeze dried in a lyophilizer after fixing inliquid nitrogen and made into fine powder using mortar andpestle About 150mg pooled powdered samples were used tomake into pellets (1mm thick and 10mm diameter) using atabletop pelletiser (pressure 100 to 110 kgcm2 for 5 minutes)

The tissue samples were analyzed by Jordan Valley EX-3600 ED-XRF system All measurements were carried outin vacuum using different filters (between the source andsample) for optimum detection of elements for 600 s TheX-rays detection was done by using liquid nitrogen cooled125mm2 Si (Li) semiconductor detectors with the resolution148 eV at 59 KeV The X-ray fluorescence spectra were quan-titatively analyzed using Ex-Win software integrated with thesystem Standardization of the procedure was done usingNIST bovine liver standard SRM 1577b

215 Statistical Analysis All assays were repeated at leastthree times Data were analyzed by Studentrsquos 119905-test usingthe Sigma plot 80 statistical package Differences betweencontrol and experimental group(s) with a value of 119875 lt 005was considered as significant

3 Results

31Mortality and Clinical Observations Allmice were exam-ined daily for any clinical signs of toxicity There was nodeath in both control and treatment groups and no clinicalsymptoms were found to appear in any of the treatmentgroups

32 Change in Body Weight and Water Consumption RateThere was no significant difference in water consumption

Org

an to

bod

y w

eigh

t rat

io

Treatment groupsGr I Gr II Gr III Gr IV Gr V

7

6

5

4

3

2

1

0

Figure 1 Organ to body weight ratio of liver in different groups ofmice Groups II III IV and V were compared with group I Valuesare expressed as mean plusmn SEM

rate as well as change in body weight (weight gain) recordedbetween the control and treatment groups (data not shown)

33 Organ to Body Weight Ratio No significant difference inthe organ to body weight ratio of liver of any treatment groupwith the control group was observed (Figure 1)

34 Histopathology Liver appeared normal and healthy(Figure 2(a)) in the control group of mice Disorganization ofhepatic parenchyma and disruption in the epithelial lining ofthe central vein and vacuolar degeneration were commonlyobserved in all treatment groups Liver in Group II micedemonstrated sinusoidal dilation and vacuolar degenerationin the cytoplasm (Figure 2(b)) In group IIImice (treatedwith2 ppm of As

2O3) extensive vacuolar degeneration and loss of

integrity in the epithelial lining of the central vein were found(Figure 2(c)) Loss of typical organization of hepatic cord andvacuolar degeneration were seen in group IV mice (treatedwith 4 ppm of As

2O3for 30 days) (Figure 2(d)) In group

V mice treated with 4 ppm of As2O3for 90 days extensive

degeneration of epithelial lining of the central vein (thickarrow) loss of typical hepatic cord organization hepatocellu-lar degradation and infiltration of the nucleus (thin arrows)into the central vein were prominent (Figure 2(e))

35 Liver Function Test An overall increase was noted in theserum GOT (AST) and GPT (ALT) levels in all treatmentgroups against control (Figure 3) SGOT level increased sig-nificantly in group II (7794) group III (15379) and groupIV (11304) mice SGPT level also increased significantly ingroup II (11596) group III (9509) and group IV (15373)

36 GSH and GST Response and TBARS Production A dosedependent decrease in GSH level was recorded in all the30 days treatment groups of mice and the decrease wassignificant in group III (3229) and group IV (479) againstcontrol In group V mice after 4 ppm of As

2O3treatment for

90 days GSH level recovered which increased significantlyagainst control mice (1931) (Figure 4(a)) TBARS increased

ISRN Hepatology 5

CV

(a) (b)

(c)

(d) (e)

Figure 2 Changes in liver histology Microphotographs of liver sections (6 120583m) stained with hematoxylin and eosin (HE) The originalmagnificationtimes400 (a)Normal histological appearance of liver tissue of controlmice central vein (CV) (b)Group II vacuolar degenerations(thin arrows) sinusoidal dilation (thick arrow) (c) Group III disruption of epithelium lining (short thick arrows) of the central vein vacuolardegenerations (thin arrows) (d) Group IV vacuolar degeneration (thin arrow) loss of integrity in epithelium lining of the central vein (shortthick arrow) and loss of typical hepatic cords organization (e) Group V extensive degeneration of epithelial lining of the central vein (thickarrow) loss of typical hepatic cord organization hepatocellular degradation and infiltration of the nucleus into the central vein (thin arrow)

Enzy

me c

once

ntra

tion

(uni

t enz

yme

L se

rum

)

Serum GPTSerum GOT

Gr I Gr II Gr III Gr IV Gr VTreatment groups

300

250

200

150

100

50

0

lowast

lowast

lowast

lowast

lowast

lowast

Figure 3 SGPT and SGOT activity (unit enzymeL of serum) inthe serum of different groups of mice exposed to different doses ofAs2O3 Groups II III IV and Vwere compared with group I Values

are expressed as mean plusmn SEM lowastValues are statistically significant at(119875 lt 005)

significantly in group III (8656) group IV (8993)and group V (2684) mice GST activity also depicted asignificant increase in group III (7219) group IV (6213)and group V (3478) mice (Figures 4(b) and 4(c))

37 Catalase Activity Catalase activity increased significantlyin group II (55) and thereafter gradually decreased withsubsequent higher doses in group III (744) and group IV(2709) mice against control In group V catalase activityshowed an increase though the increase was not significantcompared to the control (Figure 4(d))

38 Heat Shock Protein (Hsp) 70 Expression Hsp 70 profileshowed increasing pattern of expression against control in allthe treatment groups Expression increased 125-folds 126-folds 132-folds and 154-folds in group II III IV andVmicerespectively as against the control group (Figures 5(a) and5(c))

39 Nrf2 Keap1 and p62 Protein Expression Nrf2 proteinlevels were detected in whole cell lysates as this can give anidea of relative Nrf2 levels in the nuclear fractions of arsenic

6 ISRN HepatologyG

SH120583

mol

mg

prot

ein

Gr I Gr II Gr III Gr IV Gr VTreatment groups

20

18

16

14

12

10

8

6

4

2

0

lowast

lowast

lowast

(a)

nM M

DA

pro

duce

dm

gpr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

35

3

25

2

15

1

0

lowastlowast

lowast

05

(b)

Gr I Gr II Gr III Gr IV Gr VTreatment groups

30

25

20

15

10

5

0

lowastlowast

lowast

nM D

NPG

prod

uced

min

mg

prot

ein

(c)

Uni

t enz

yme

activ

itym

inm

g pr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

lowast

lowast

800

700

600

500

400

300

200

100

0

(d)

Figure 4 (a) GSH content (b) MDA production (c) GST activity (d) catalase activity in liver of different groups of mice exposed to differentdoses of As

2O3 Groups II III IV andVwere comparedwithGroup I Values are expressed asmeanplusmn SEM lowastValues are statistically significant

at (119875 lt 005)

treated cells [20 42 43] We observed the induction of Nrf2protein for different doses of arsenic in all the treatmentgroups but the increase in the Nrf2 protein level was notconsistent with the increasing doses of arsenic treatmentThemaximum level of Nrf2 protein was recorded in group II(144-fold of control) mice which was found to decrease ingroup III (114-fold) mice Further in group IVmice the levelof Nrf2 protein increased to 136-folds of the control againshowing a decreasing pattern in group V (118-fold) mice(Figures 5(a) and 5(b)) Keap1 protein expression decreasedin all the treatment groups compared to the control groupLowest level of Keap1 protein was found in group V (Figures5(a) and 5(b)) mice p62 protein expression was also low inthe control group while elevated levels of protein expressionwere observed in all the treatment groups though the increasein protein level was not dose dependent The highest levelof p62 protein was observed in group II (18-fold of control)followed by group III and group IV mice where it decreasedgradually by 13-fold and 12-fold respectively In group V

mice however an elevation of 16 folds was recorded (Figures5(a) and 5(b))

310 120574GCS Protein GST and GR Protein Expression 120574GCSprotein level in group II mice showed a slight decreaseas compared to the control group while the protein levelincreased continuously in the subsequent groups by 103-fold 122-fold and 151-fold in group III IV and V micerespectively against control (Figures 5(a) and 5(c)) GSTprotein expression increased in all treatment groups with thehighest level recorded in groupV (123-fold) (Figures 5(a) and5(c)) Expression of GR protein also showed an increasingtrend in group II and III whereas in group IV and V micethe level gradually decreased and in group Vmice it reachedalmost the control level (Figures 5(a) and 5(c))

311 Nrf2 and Keap1 mRNA Expression Expression of Nrf2mRNA increased in all the treatment groups compared tothe control group Expression increased by 231-fold 221-fold

ISRN Hepatology 7

Hsp 70

Nrf2

Keap1

GST

GR

Gr I Gr II Gr III Gr IV Gr V

p62

M

120573-Actin

120574GCS

(a)

Nrf 2Keap1p62

20

18

16

14

12

10

08

06Gr I Gr II Gr III Gr IV Gr V

Rela

tive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

Treatment groups

(b)

GSTGRHsp 70

18

16

14

12

10

08Gr I Gr II Gr III Gr IV Gr V

Treatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

120574GCS

(c)Figure 5 (a) Western blotting (b) densitometric analysis of the Nrf2 protein Keap1 protein and p62 protein profile and (c) densitometricanalysis of 120574GCS protein GST protein GR protein and Hsp 70 protein profile of liver of mice treated with different doses of As

2O3

and 229-fold in group II group III and group IV micerespectively against control group ofmice In groupV (4 ppmof As2O3treated for 90 days) decrement of the expression

was noteworthy against the 30 days treatment groups (Figures6(a) and 6(b)) though the level was still higher than control(177-fold) Keap1 mRNA level decreased in all treatmentgroups Keap1 mRNA level detected was 081-fold that ofcontrol in group II 054 folds of the control in group III 061folds of the control in group IV and 042-fold of control ingroup V mice (Figures 6(a) and 6(b))

312 Detection of Apoptosis by Hoechst 33342 The hepato-cytes of mice in all the treatment groups exhibited con-densed and fragmented nuclei upon staining with Hoechst33342 which is an indicator of possible apoptotic cell death(Figure 7)

313 Modulation of Elements Concentration of Mg showedan increasing trend in all treatment groups and the increasewas significant in group III and group IV mice Group IImice showed significant increase and decrease respectivelyfor Cu and Zn whereas significant depletion in Se level wasobserved in group IV mice Iron concentration increased ingroup II group III and group IV with the highest level werefound in group IV mice In group V mice a reduction in theiron concentration was observed reaching almost the controlvalue (Table 1)

4 Discussion

According toWHOguideline the permissible limit of arsenicin drinking water is 10 ppb In some states in USA andChina people are exposed to more than 1 ppm of As throughtheir drinking water Higher arsenic contamination is mainly

8 ISRN Hepatology

Nrf2

Keap1

GrI Gr II Gr III Gr IV Gr V

120573-Actin

(a)

Nrf2Keap1

GrI Gr II Gr III Gr IV Gr VTreatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

30

25

20

15

10

05

00

(b)

Figure 6 (a) Nrf2 and Keap1 mRNA expressions in liver (b) Densitometric analysis of Nrf2 mRNA Keap1 mRNA in liver of mice treatedwith different doses of As

2O3

(a) (b) (c)

(d) (e) (f)

Figure 7 Mouse hepatocytes showing apoptosis (arrow) by Hoechst 33342 staining (a) (b) control (c) 04 ppm (d) 2 ppm and (e) 4 ppmAs2O3treatment for 30 days (f) 4 ppm arsenic trioxide treatment for 90 days

ISRN Hepatology 9

Table 1 Concentration of elements (mgkg) in whole-liver tissue following in vivo administration of different concentrations of As2O3 (ppm)in the drinking water of the mice for 30 and 90 days (Values are mean plusmn SEM)

Elements Group I Group II Group III Group IV Group VMg 35445 plusmn 5417 54729 plusmn 5888 67879 plusmn 2113

lowast57821 plusmn 4246

lowast46143 plusmn 2990

Cu 1541 plusmn 023 1749 plusmn 073lowast

1454 plusmn 072 1444 plusmn 047 1468 plusmn 059

Zn 10755 plusmn 157 9763 plusmn 397lowast

10377 plusmn 164 10762 plusmn 335 10226 plusmn 471

Se 1419 plusmn 134 1325 plusmn 055 1252 plusmn 189 843 plusmn 074lowast

1409 plusmn 113

Fe 50365 plusmn 3866 61029 plusmn 4599 56782 plusmn 2297 73421 plusmn 4494lowast

50689 plusmn 2822

lowastValues are statistically significant compared to control at (119875 lt 005)

found in ground water which is the most common sourceof drinking water The chronic exposure is the main causeof arsenic induced cancer development in human popula-tion and the mechanism of chronic arsenic toxicity is notfully known Therefore in our present experiment micewere treated with As through drinking water Accordingto Singh et al [44] arsenic administration decreased bodyweight gain in a dose dependant manner Santra et al [45]have reported significant increase in the liver weight ofBALBc mice after 12 months of arsenic treatment whereasin the present study there was no significant change in bodyweight gain rate of water consumption or the organ to bodyweight ratio of liver in any of the treatment groups

Liver is an important organ for various metabolic path-ways and effect of any chemical or xenobiotic appears pri-marily in the liver In case of arsenic metabolism liver is themain site of arsenic methylation [46]Themost commonwayof assertion of liver damage is to determine two pathophys-iological enzymes serum glutamate pyruvate transaminase(SGPT) and serum glutamate oxaloacetate transaminase(SGOT) In our findings there was a distinct pattern ofincreased SGOT and SGPT levels in all the treatment groupstreated for 30 days In case of As

2O3(4 ppm) treatment

for 90 days however reversal in the activities of both theenzymes occurred almost reaching basal level The reasonfor such reversal is difficult to explain since histopathologicalalterations as well as induction of apoptosis was observed inthe same treatment group The increased SGOT and SGPTlevels in rat after 45 days treatment of 10 ppm sodium arsenitewas also described by Tandan et al [47] whereas Santra et al[45] reported increased SGOT and SGPT levels after 12months of arsenic treatment at 32 ppm in mice Liver alter-ation in histopathological architecture was evident mainly inthe form of loss of integrity of central vein loss of hepaticcord organization and dose and time dependent vacuolationwhich corroborates with earlier studies [45 48 49] Thesestudies indicate that chronic exposure to different inorganicarsenic compounds (arsenite arsenic trioxide or arsenate)produces characteristic pathology in the liver including fattyinfiltration liver degeneration inflammatory cell infiltrationand focal necrosis Das Neves et al [50] have shown that evenacute exposure of sodium arsenite at a dose of 10mgkg bodyweight (intraperitoneally) for only 90min could producevarious histopathological alterations in liver

In the present study Hoechst 33342 stained hepatocytesclearly indicated fragmented nuclei in all the treatment

groups as a mark of apoptosis as reported earlier in fish liver[17]

Oxidative stress is a relatively new theory in assessmentof arsenic toxicity [51 52] Arsenic causes oxidative stress byproducing reactive oxygen species [20 53 54] which damageproteins Due to lipophilic nature arsenic also attaches tolipid thereby increasing the rate of lipid peroxidation [55]Cellular GSH plays an important role in mitigating arsenicinduced oxidative stress Arsenic being a strong electrophilepredominantly binds with nucleophilic SH group of GSHThus consequent depletion of GSHmay alter the redox statusof the cell and present a stressful and toxic situation Accord-ing to Bashir et al [56] acute treatment of sodium arseniteat doses of 63mgkg 105mgkg and 126mgkg of bodyweight for 24 h significantly decreased GSH content in liverfor all doses tested On the other hand a significant increasein lipid peroxidation and glutathione peroxidase activityalongwith significant decrease in the activity of GST catalaseand superoxide dismutase was observed at 105mgkg and126mgkg of As treatment Santra et al [45] reported thatchronic exposure to 32 ppm of arsenic significantly depletedGSHafter 6months of exposure catalase activity significantlydecreased after 9 months and GST activity significantlyreduced at 12 and 15 months of arsenic exposure In ourstudy GSH level showed decreasing trend in all the 30 daystreatment groups with highest depletion recorded at 4 ppmof As2O3treatment for 30 days while after 90 days treatment

with the same dose the GSH level increased significantlywith respect to both control and also the 30 days treatmentgroupsThis shows the upregulation ofGSHagainst long termarsenic treatment which is due to its adaptive response tooxidative stress [20] However a reverse trend was observedin TBARS production and GST activity in liver in group II(04 ppm As

2O3treatment for 30 days) there was almost no

change in TBARS and GST level against the control groupwhile in group III and group IV levels of both TBARS andGST activity increased significantly Interestingly in groupV (90 days treatment with 4 ppm As

2O3) both TBARS and

GST level decreased significantly with respect to group IVthat is 4 ppm As

2O3for 30 days though the levels were still

significantly higher than the control group The increasedGST activity might be responsible for the reduction in GSHwhile protecting against the arsenic-induced oxidative stressCatalase activity increased significantly in group II mice withrespect to control while at subsequent higher doses that is2 ppm and 4 ppm As

2O3for 30 days the activity decreased

10 ISRN Hepatology

Increased level of SGOT and SGPT

Histopathological alterationsModulation in trace element levels

Increased production of p62

Increased Nrf2 level

Decreased activity of Keap1

Increased production of Hsp 70

Active Nrf2

Gene transcription

GST

GCS

GR

GSH

p62

Alteration in the activity of GST

Modulation in GSH level

Apoptotic induction

Increased production of MDA

p62

Keap1Keap1

ubub

ub

ubInactive Nrf2

Cytosol Nucleus

Nuclear localiztion

Nrf2Nrf2

Nrf2

ARE

Oxidative stress

As

120574GCS GR and catalase

Figure 8 Schematic representation of the arsenic induced liver damages and induction of gene expression through the Keap1-Nrf2-AREsignaling pathway

and highest rate of depletion was observed after 4 ppmAs2O3

treatment for 30 days Further in group V (4 ppm As2O3for

90 days) the recovery in the catalase activity was observedand it increased significantly when compared to group IV(4 ppmAs

2O3treatment for 30 days) According toMittal and

Flora [57] catalase activity significantly decreased in liver andkidney with respect to control after treatment with 100 ppmsodium meta-arsenite in drinking water for 8 weeks

Trace elements play an important role in maintainingnormal homeostasis of the body Oxidative stress results fromchanges in the levels of trace elements It has been reportedthat increased Mg level is associated with the increasedproduction ofMDA in dementia patients [58] Our result alsoshowed an increasing pattern ofMg concentration alongwithincrease in liver TBARS level

Zn is an essential element required for growth andnormal development and is a constituent of more than 200enzymes one of which is a CuZn superoxide dismutase(CuZn SOD) CuZn SOD is a powerful antioxidant whichtransforms free radical O

2

∙ to H2O2 therefore reducing the

risk of formation of highly reactive hydroxyl radical HO∙ Cuis also an integral part of CuZn SOD enzyme DecreasedZn and Cu levels in tissues may result in reduction of CuZnSOD activity and subsequently accelerate the process of cellaging and death via oxidative damage [59] However free orincorrectly bound Cu+2 can catalyze the generation of the

most damaging radicals such as HO∙ resulting in a chemicalmodification of the protein alterations in protein structureand solubility and oxidative damage to surrounding tissue[60] Our findings also indicate significant modulation of Cuand Zn level in liver of group II mice

Selenium (Se) is a well known antagonist of arsenic toxic-ity [61] The most probable cause of such a protective effectof selenium is due to its ability to upregulate antioxidantenzymes like GSH peroxidase and thioredoxin reductasewhich protects against arsenic induced oxidative damage[62] Decrement in selenium level after As

2O3treatment was

reported byMolin et al [63 64] In the present study Se levelin liver decreased dose dependently recording maximumdepletion at 4 ppm of As

2O3for 30 days but in the group

treated for 90 days with 4 ppm As2O3 Se level increased and

almost reached the normal levelIron (Fe) catalyses the formation of reactive oxygen

species through the Fenton and Haber-Weiss reactions [65]which generate highly toxic hydroxyl radical and cause lipidperoxidation [66] According to Ahmad et al [67] body ironstores (serum ferritin and transferrin saturation) in the bodycan be used as an early investigative tool for assessing theoxidative stress in coronary heart disease In our study ironlevel increased in all the treatment groups except in group Vwhere the iron concentration is almost same to that of thecontrol group

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN AIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Computational and Mathematical Methods in Medicine

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Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Biomarkers

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Volume 2013

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OncologyJournal of

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OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

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ObesityJournal of

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PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Hepatology 3

Group V 4 ppm As2O3treated through drinking

water for 90 days

23 Measurement of Body Weight and Water ConsumptionThe body weight of all animals was recorded initially andalso during the course of the treatment Rate of waterconsumption and gain in body weight were recorded foreach individual mouse at certain time intervals during theexperiment

24 Determination of Organ to Body Weight Ratio Theweight of themicewas recorded before sacrificeThe liver wasdissected out carefully blotted free of blood and fresh weightwas recorded Organ to body weight ratio was calculated andcompared with the control mice

25 Histopathological Studies Portions of liver tissue of allanimals were fixed in Bouinrsquos fluid dehydrated throughgraded alcohol and embedded in paraffin and routine mic-rotomy was carried out to obtain 6120583m thick tissue sectionsSections were stained by routine hematoxylin-eosin (HE)technique and viewed under light microscope

26 Liver Function Tests Serum glutamate oxaloacetate tran-saminase (SGOT) (aspartate transaminase AST) and serumglutamate pyruvate transaminase (SGPT) (alanine transami-nase ALT) levelswere estimated following themanufacturerrsquosprotocol

27 Determination of Reduced Glutathione (GSH) Liver GSHwas measured following the method of Beutler et al [33]In brief liver tissue was quickly dissected out and blanchedin ice-cold isotonic saline A 10 homogenate was preparedfrom each tissue with ice-cold saline-EDTA at 4∘C Onemilliliter of freshly prepared 20 ice-cold trichloroacetic acid(TCA) was added to equal volume of homogenate and themixture was vortexed and allowed to stand for 10min in 4∘CThe mixture was then centrifuged at 5000 rpm for 5minThe clear supernatant was used as the GSH sample fromwhich 1mL of supernatant was taken andmixed with 3mL of03M disodium hydrogen phosphate buffer and 1mL of 551015840-dithiobis-2-nitro benzoic acid (DTNB) solution After 5minthe optical density of the samples was measured at 412 nmand results were expressed as 120583MGSHmg protein

28 Assay of Glutathione-s-Transferase (GST) GST activitywas assessed in the liver cytosolic fractions as described byHabig et al [34] using 1-chloro-24-dinitrobenzene (CDNB)(1mM final concentration) as substrate in the presence ofexcess GSH (5mM) The rate of CDNB conjugation wasestimated by direct spectrophotometry at 340 nm for 3minThe result was expressed as 120583M GS-CDNB formedminmgprotein

29 Assay ofThiobarbituric Acid Reactive Substances (TBARS)Level in Liver Lipid peroxidation products namely mal-ondialdehyde (MDA) was estimated in liver microsomesassuming high PUFA content of microsomal membranes

The level of lipid peroxidation as measured by TBARS wasdetermined according to the method of Buege and Aust [35]Briefly 1mL of microsomal sample was mixed with 2mL ofTBA-TCA-HCl mixture thoroughly and heated for 15min ina boiling water bath After cooling the flocculent precipitatewas removed by centrifugation at 1000 g for 10min Theabsorbance of the supernatant was determined at 535 nm andexpressed in terms of nMMDAmg protein

210 Catalase Assay Catalase activity was assayed followingthe procedure of Aebi [36] as modified by Kawamura [37]A 5 homogenate was prepared in 50mM phosphate buffer(pH 70) and centrifuged at 12500timesg for 30min at 4∘C Thesupernatant or the peroxisome-rich fraction was used as thesample The sample (20 120583L) was added to 980120583L of an assaybuffer containing 50mMTris-HCl (pH80) 9mMH

2O2 and

025mM EDTA to constitute the assay volume of 1mL Thedecrease in ΔODmin of that assay mixture was recorded at240 nm for 1min The results were expressed as unit catalaseactivitymg protein

211 Western Blot Analysis

2111 Sample Preparation Whole cell protein extracts andWestern blot analysis were performed as previously described[38] Liver homogenates (10 wv) were prepared in 50mMphosphate buffer (pH 75) and centrifuged at 10000 g for20min The cytosolic supernatant was collected very care-fully and the protein content of the sample was measuredfollowing the method of Lowry et al [39]

2112 Methods forWestern Blotting Protein (60 120583g) from thelysates of control and treated cells was resolved on 10 SDS-PAGE at a constant voltage (60V) for 25 h and then blottedonto a polyvinylidene fluoride (PVDF) membrane using asemidry trans blot apparatus (Bio-Rad Trans Blot SD CellUSA) The membranes were first incubated with primaryantibodies at a dilution of 1 1000 overnight at 4∘C followedby 2 h incubation with corresponding ALP-conjugated sec-ondary antibodies at 1 2000 (Sigma) dilutions with contin-uous rocking The immunoreactive bands were detected byusing 5-bromo-4chloro-3-indolylphosphatenitroblue tetra-zolium (BCIPNBT) Densitometric quantification was doneby ImageJ (NIH) software

212 Total RNA Extraction and RT-PCR Analysis Total RNAfrom liver tissues were extracted using TRI reagent Equalamounts of RNA (5 120583g) were reverse transcribed into cDNAusing the RevertAid reverse transcriptase (Fermentas) fol-lowing manufacturers protocol for RT-PCR The PCR wasperformed following the procedure as per the manufacturerrsquosinstruction for 35 cycles All test samples were amplifiedsimultaneously from equal volume of first strand cDNAwith the particular primer pair using a master PCR mixPCR reactions were run in a programmable thermal cycler(GeneAmp 9700 ABI) with simultaneous NTC (no tem-plate control) 120573-actin was amplified simultaneously as aninternal control Specific primers for Nrf2 and 120573-actin for

4 ISRN Hepatology

PCR amplification are Nrf2 forward 51015840-TCTCCTCGCTGG-AAAAAGAA-31015840 51015840-AATGTGCTGGCTGTGCTTTA-31015840 120573-actin forward 51015840-TGGAATCCTGTGGCATCCATGAAAC-31015840 120573-actin reverse 51015840-TAAAACGCAGCTCAGTAACAG-TCCG-31015840 [40] For amplification of Keap1 specific primerwas designed using Primer 3 software Keap1 forward 51015840-GTACGCTGCGAGTCCGAGGT-31015840 Keap1 reverse 51015840-GCC-ATTGCTCGGGTTGTAGG-31015840 The PCR products were runin 1 agarose gel and visualized in a gel documentationsystem (Gel Doc EZ Imager Bio-Rad) after staining withethidium bromide The densitometric quantification wasdone using ImageJ (NIH) software

213 Hoechst 33342 Staining Hepatocytes were isolated fromblanched liver by a two-step collagenase (Sigma AldrichUSA) digestion method [41] The isolated hepatocytes fromdifferent treatment groups were washed with PBS fixedwith 37 para-formaldehyde solution at room tempera-ture stained with bisBenzimide H 33342 trihydrochloride(Hoechst 33342 2mgmL) and visualized under fluorescencemicroscope (Dewinter Italy) within 30min of adding thestain

214 EDXRF Measurement Liver was dissected out carefullyusing stainless steel forceps and blotted free of blood Thesamples were not washed to avoid leaching of soluble ele-ments Tissues were freeze dried in a lyophilizer after fixing inliquid nitrogen and made into fine powder using mortar andpestle About 150mg pooled powdered samples were used tomake into pellets (1mm thick and 10mm diameter) using atabletop pelletiser (pressure 100 to 110 kgcm2 for 5 minutes)

The tissue samples were analyzed by Jordan Valley EX-3600 ED-XRF system All measurements were carried outin vacuum using different filters (between the source andsample) for optimum detection of elements for 600 s TheX-rays detection was done by using liquid nitrogen cooled125mm2 Si (Li) semiconductor detectors with the resolution148 eV at 59 KeV The X-ray fluorescence spectra were quan-titatively analyzed using Ex-Win software integrated with thesystem Standardization of the procedure was done usingNIST bovine liver standard SRM 1577b

215 Statistical Analysis All assays were repeated at leastthree times Data were analyzed by Studentrsquos 119905-test usingthe Sigma plot 80 statistical package Differences betweencontrol and experimental group(s) with a value of 119875 lt 005was considered as significant

3 Results

31Mortality and Clinical Observations Allmice were exam-ined daily for any clinical signs of toxicity There was nodeath in both control and treatment groups and no clinicalsymptoms were found to appear in any of the treatmentgroups

32 Change in Body Weight and Water Consumption RateThere was no significant difference in water consumption

Org

an to

bod

y w

eigh

t rat

io

Treatment groupsGr I Gr II Gr III Gr IV Gr V

7

6

5

4

3

2

1

0

Figure 1 Organ to body weight ratio of liver in different groups ofmice Groups II III IV and V were compared with group I Valuesare expressed as mean plusmn SEM

rate as well as change in body weight (weight gain) recordedbetween the control and treatment groups (data not shown)

33 Organ to Body Weight Ratio No significant difference inthe organ to body weight ratio of liver of any treatment groupwith the control group was observed (Figure 1)

34 Histopathology Liver appeared normal and healthy(Figure 2(a)) in the control group of mice Disorganization ofhepatic parenchyma and disruption in the epithelial lining ofthe central vein and vacuolar degeneration were commonlyobserved in all treatment groups Liver in Group II micedemonstrated sinusoidal dilation and vacuolar degenerationin the cytoplasm (Figure 2(b)) In group IIImice (treatedwith2 ppm of As

2O3) extensive vacuolar degeneration and loss of

integrity in the epithelial lining of the central vein were found(Figure 2(c)) Loss of typical organization of hepatic cord andvacuolar degeneration were seen in group IV mice (treatedwith 4 ppm of As

2O3for 30 days) (Figure 2(d)) In group

V mice treated with 4 ppm of As2O3for 90 days extensive

degeneration of epithelial lining of the central vein (thickarrow) loss of typical hepatic cord organization hepatocellu-lar degradation and infiltration of the nucleus (thin arrows)into the central vein were prominent (Figure 2(e))

35 Liver Function Test An overall increase was noted in theserum GOT (AST) and GPT (ALT) levels in all treatmentgroups against control (Figure 3) SGOT level increased sig-nificantly in group II (7794) group III (15379) and groupIV (11304) mice SGPT level also increased significantly ingroup II (11596) group III (9509) and group IV (15373)

36 GSH and GST Response and TBARS Production A dosedependent decrease in GSH level was recorded in all the30 days treatment groups of mice and the decrease wassignificant in group III (3229) and group IV (479) againstcontrol In group V mice after 4 ppm of As

2O3treatment for

90 days GSH level recovered which increased significantlyagainst control mice (1931) (Figure 4(a)) TBARS increased

ISRN Hepatology 5

CV

(a) (b)

(c)

(d) (e)

Figure 2 Changes in liver histology Microphotographs of liver sections (6 120583m) stained with hematoxylin and eosin (HE) The originalmagnificationtimes400 (a)Normal histological appearance of liver tissue of controlmice central vein (CV) (b)Group II vacuolar degenerations(thin arrows) sinusoidal dilation (thick arrow) (c) Group III disruption of epithelium lining (short thick arrows) of the central vein vacuolardegenerations (thin arrows) (d) Group IV vacuolar degeneration (thin arrow) loss of integrity in epithelium lining of the central vein (shortthick arrow) and loss of typical hepatic cords organization (e) Group V extensive degeneration of epithelial lining of the central vein (thickarrow) loss of typical hepatic cord organization hepatocellular degradation and infiltration of the nucleus into the central vein (thin arrow)

Enzy

me c

once

ntra

tion

(uni

t enz

yme

L se

rum

)

Serum GPTSerum GOT

Gr I Gr II Gr III Gr IV Gr VTreatment groups

300

250

200

150

100

50

0

lowast

lowast

lowast

lowast

lowast

lowast

Figure 3 SGPT and SGOT activity (unit enzymeL of serum) inthe serum of different groups of mice exposed to different doses ofAs2O3 Groups II III IV and Vwere compared with group I Values

are expressed as mean plusmn SEM lowastValues are statistically significant at(119875 lt 005)

significantly in group III (8656) group IV (8993)and group V (2684) mice GST activity also depicted asignificant increase in group III (7219) group IV (6213)and group V (3478) mice (Figures 4(b) and 4(c))

37 Catalase Activity Catalase activity increased significantlyin group II (55) and thereafter gradually decreased withsubsequent higher doses in group III (744) and group IV(2709) mice against control In group V catalase activityshowed an increase though the increase was not significantcompared to the control (Figure 4(d))

38 Heat Shock Protein (Hsp) 70 Expression Hsp 70 profileshowed increasing pattern of expression against control in allthe treatment groups Expression increased 125-folds 126-folds 132-folds and 154-folds in group II III IV andVmicerespectively as against the control group (Figures 5(a) and5(c))

39 Nrf2 Keap1 and p62 Protein Expression Nrf2 proteinlevels were detected in whole cell lysates as this can give anidea of relative Nrf2 levels in the nuclear fractions of arsenic

6 ISRN HepatologyG

SH120583

mol

mg

prot

ein

Gr I Gr II Gr III Gr IV Gr VTreatment groups

20

18

16

14

12

10

8

6

4

2

0

lowast

lowast

lowast

(a)

nM M

DA

pro

duce

dm

gpr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

35

3

25

2

15

1

0

lowastlowast

lowast

05

(b)

Gr I Gr II Gr III Gr IV Gr VTreatment groups

30

25

20

15

10

5

0

lowastlowast

lowast

nM D

NPG

prod

uced

min

mg

prot

ein

(c)

Uni

t enz

yme

activ

itym

inm

g pr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

lowast

lowast

800

700

600

500

400

300

200

100

0

(d)

Figure 4 (a) GSH content (b) MDA production (c) GST activity (d) catalase activity in liver of different groups of mice exposed to differentdoses of As

2O3 Groups II III IV andVwere comparedwithGroup I Values are expressed asmeanplusmn SEM lowastValues are statistically significant

at (119875 lt 005)

treated cells [20 42 43] We observed the induction of Nrf2protein for different doses of arsenic in all the treatmentgroups but the increase in the Nrf2 protein level was notconsistent with the increasing doses of arsenic treatmentThemaximum level of Nrf2 protein was recorded in group II(144-fold of control) mice which was found to decrease ingroup III (114-fold) mice Further in group IVmice the levelof Nrf2 protein increased to 136-folds of the control againshowing a decreasing pattern in group V (118-fold) mice(Figures 5(a) and 5(b)) Keap1 protein expression decreasedin all the treatment groups compared to the control groupLowest level of Keap1 protein was found in group V (Figures5(a) and 5(b)) mice p62 protein expression was also low inthe control group while elevated levels of protein expressionwere observed in all the treatment groups though the increasein protein level was not dose dependent The highest levelof p62 protein was observed in group II (18-fold of control)followed by group III and group IV mice where it decreasedgradually by 13-fold and 12-fold respectively In group V

mice however an elevation of 16 folds was recorded (Figures5(a) and 5(b))

310 120574GCS Protein GST and GR Protein Expression 120574GCSprotein level in group II mice showed a slight decreaseas compared to the control group while the protein levelincreased continuously in the subsequent groups by 103-fold 122-fold and 151-fold in group III IV and V micerespectively against control (Figures 5(a) and 5(c)) GSTprotein expression increased in all treatment groups with thehighest level recorded in groupV (123-fold) (Figures 5(a) and5(c)) Expression of GR protein also showed an increasingtrend in group II and III whereas in group IV and V micethe level gradually decreased and in group Vmice it reachedalmost the control level (Figures 5(a) and 5(c))

311 Nrf2 and Keap1 mRNA Expression Expression of Nrf2mRNA increased in all the treatment groups compared tothe control group Expression increased by 231-fold 221-fold

ISRN Hepatology 7

Hsp 70

Nrf2

Keap1

GST

GR

Gr I Gr II Gr III Gr IV Gr V

p62

M

120573-Actin

120574GCS

(a)

Nrf 2Keap1p62

20

18

16

14

12

10

08

06Gr I Gr II Gr III Gr IV Gr V

Rela

tive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

Treatment groups

(b)

GSTGRHsp 70

18

16

14

12

10

08Gr I Gr II Gr III Gr IV Gr V

Treatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

120574GCS

(c)Figure 5 (a) Western blotting (b) densitometric analysis of the Nrf2 protein Keap1 protein and p62 protein profile and (c) densitometricanalysis of 120574GCS protein GST protein GR protein and Hsp 70 protein profile of liver of mice treated with different doses of As

2O3

and 229-fold in group II group III and group IV micerespectively against control group ofmice In groupV (4 ppmof As2O3treated for 90 days) decrement of the expression

was noteworthy against the 30 days treatment groups (Figures6(a) and 6(b)) though the level was still higher than control(177-fold) Keap1 mRNA level decreased in all treatmentgroups Keap1 mRNA level detected was 081-fold that ofcontrol in group II 054 folds of the control in group III 061folds of the control in group IV and 042-fold of control ingroup V mice (Figures 6(a) and 6(b))

312 Detection of Apoptosis by Hoechst 33342 The hepato-cytes of mice in all the treatment groups exhibited con-densed and fragmented nuclei upon staining with Hoechst33342 which is an indicator of possible apoptotic cell death(Figure 7)

313 Modulation of Elements Concentration of Mg showedan increasing trend in all treatment groups and the increasewas significant in group III and group IV mice Group IImice showed significant increase and decrease respectivelyfor Cu and Zn whereas significant depletion in Se level wasobserved in group IV mice Iron concentration increased ingroup II group III and group IV with the highest level werefound in group IV mice In group V mice a reduction in theiron concentration was observed reaching almost the controlvalue (Table 1)

4 Discussion

According toWHOguideline the permissible limit of arsenicin drinking water is 10 ppb In some states in USA andChina people are exposed to more than 1 ppm of As throughtheir drinking water Higher arsenic contamination is mainly

8 ISRN Hepatology

Nrf2

Keap1

GrI Gr II Gr III Gr IV Gr V

120573-Actin

(a)

Nrf2Keap1

GrI Gr II Gr III Gr IV Gr VTreatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

30

25

20

15

10

05

00

(b)

Figure 6 (a) Nrf2 and Keap1 mRNA expressions in liver (b) Densitometric analysis of Nrf2 mRNA Keap1 mRNA in liver of mice treatedwith different doses of As

2O3

(a) (b) (c)

(d) (e) (f)

Figure 7 Mouse hepatocytes showing apoptosis (arrow) by Hoechst 33342 staining (a) (b) control (c) 04 ppm (d) 2 ppm and (e) 4 ppmAs2O3treatment for 30 days (f) 4 ppm arsenic trioxide treatment for 90 days

ISRN Hepatology 9

Table 1 Concentration of elements (mgkg) in whole-liver tissue following in vivo administration of different concentrations of As2O3 (ppm)in the drinking water of the mice for 30 and 90 days (Values are mean plusmn SEM)

Elements Group I Group II Group III Group IV Group VMg 35445 plusmn 5417 54729 plusmn 5888 67879 plusmn 2113

lowast57821 plusmn 4246

lowast46143 plusmn 2990

Cu 1541 plusmn 023 1749 plusmn 073lowast

1454 plusmn 072 1444 plusmn 047 1468 plusmn 059

Zn 10755 plusmn 157 9763 plusmn 397lowast

10377 plusmn 164 10762 plusmn 335 10226 plusmn 471

Se 1419 plusmn 134 1325 plusmn 055 1252 plusmn 189 843 plusmn 074lowast

1409 plusmn 113

Fe 50365 plusmn 3866 61029 plusmn 4599 56782 plusmn 2297 73421 plusmn 4494lowast

50689 plusmn 2822

lowastValues are statistically significant compared to control at (119875 lt 005)

found in ground water which is the most common sourceof drinking water The chronic exposure is the main causeof arsenic induced cancer development in human popula-tion and the mechanism of chronic arsenic toxicity is notfully known Therefore in our present experiment micewere treated with As through drinking water Accordingto Singh et al [44] arsenic administration decreased bodyweight gain in a dose dependant manner Santra et al [45]have reported significant increase in the liver weight ofBALBc mice after 12 months of arsenic treatment whereasin the present study there was no significant change in bodyweight gain rate of water consumption or the organ to bodyweight ratio of liver in any of the treatment groups

Liver is an important organ for various metabolic path-ways and effect of any chemical or xenobiotic appears pri-marily in the liver In case of arsenic metabolism liver is themain site of arsenic methylation [46]Themost commonwayof assertion of liver damage is to determine two pathophys-iological enzymes serum glutamate pyruvate transaminase(SGPT) and serum glutamate oxaloacetate transaminase(SGOT) In our findings there was a distinct pattern ofincreased SGOT and SGPT levels in all the treatment groupstreated for 30 days In case of As

2O3(4 ppm) treatment

for 90 days however reversal in the activities of both theenzymes occurred almost reaching basal level The reasonfor such reversal is difficult to explain since histopathologicalalterations as well as induction of apoptosis was observed inthe same treatment group The increased SGOT and SGPTlevels in rat after 45 days treatment of 10 ppm sodium arsenitewas also described by Tandan et al [47] whereas Santra et al[45] reported increased SGOT and SGPT levels after 12months of arsenic treatment at 32 ppm in mice Liver alter-ation in histopathological architecture was evident mainly inthe form of loss of integrity of central vein loss of hepaticcord organization and dose and time dependent vacuolationwhich corroborates with earlier studies [45 48 49] Thesestudies indicate that chronic exposure to different inorganicarsenic compounds (arsenite arsenic trioxide or arsenate)produces characteristic pathology in the liver including fattyinfiltration liver degeneration inflammatory cell infiltrationand focal necrosis Das Neves et al [50] have shown that evenacute exposure of sodium arsenite at a dose of 10mgkg bodyweight (intraperitoneally) for only 90min could producevarious histopathological alterations in liver

In the present study Hoechst 33342 stained hepatocytesclearly indicated fragmented nuclei in all the treatment

groups as a mark of apoptosis as reported earlier in fish liver[17]

Oxidative stress is a relatively new theory in assessmentof arsenic toxicity [51 52] Arsenic causes oxidative stress byproducing reactive oxygen species [20 53 54] which damageproteins Due to lipophilic nature arsenic also attaches tolipid thereby increasing the rate of lipid peroxidation [55]Cellular GSH plays an important role in mitigating arsenicinduced oxidative stress Arsenic being a strong electrophilepredominantly binds with nucleophilic SH group of GSHThus consequent depletion of GSHmay alter the redox statusof the cell and present a stressful and toxic situation Accord-ing to Bashir et al [56] acute treatment of sodium arseniteat doses of 63mgkg 105mgkg and 126mgkg of bodyweight for 24 h significantly decreased GSH content in liverfor all doses tested On the other hand a significant increasein lipid peroxidation and glutathione peroxidase activityalongwith significant decrease in the activity of GST catalaseand superoxide dismutase was observed at 105mgkg and126mgkg of As treatment Santra et al [45] reported thatchronic exposure to 32 ppm of arsenic significantly depletedGSHafter 6months of exposure catalase activity significantlydecreased after 9 months and GST activity significantlyreduced at 12 and 15 months of arsenic exposure In ourstudy GSH level showed decreasing trend in all the 30 daystreatment groups with highest depletion recorded at 4 ppmof As2O3treatment for 30 days while after 90 days treatment

with the same dose the GSH level increased significantlywith respect to both control and also the 30 days treatmentgroupsThis shows the upregulation ofGSHagainst long termarsenic treatment which is due to its adaptive response tooxidative stress [20] However a reverse trend was observedin TBARS production and GST activity in liver in group II(04 ppm As

2O3treatment for 30 days) there was almost no

change in TBARS and GST level against the control groupwhile in group III and group IV levels of both TBARS andGST activity increased significantly Interestingly in groupV (90 days treatment with 4 ppm As

2O3) both TBARS and

GST level decreased significantly with respect to group IVthat is 4 ppm As

2O3for 30 days though the levels were still

significantly higher than the control group The increasedGST activity might be responsible for the reduction in GSHwhile protecting against the arsenic-induced oxidative stressCatalase activity increased significantly in group II mice withrespect to control while at subsequent higher doses that is2 ppm and 4 ppm As

2O3for 30 days the activity decreased

10 ISRN Hepatology

Increased level of SGOT and SGPT

Histopathological alterationsModulation in trace element levels

Increased production of p62

Increased Nrf2 level

Decreased activity of Keap1

Increased production of Hsp 70

Active Nrf2

Gene transcription

GST

GCS

GR

GSH

p62

Alteration in the activity of GST

Modulation in GSH level

Apoptotic induction

Increased production of MDA

p62

Keap1Keap1

ubub

ub

ubInactive Nrf2

Cytosol Nucleus

Nuclear localiztion

Nrf2Nrf2

Nrf2

ARE

Oxidative stress

As

120574GCS GR and catalase

Figure 8 Schematic representation of the arsenic induced liver damages and induction of gene expression through the Keap1-Nrf2-AREsignaling pathway

and highest rate of depletion was observed after 4 ppmAs2O3

treatment for 30 days Further in group V (4 ppm As2O3for

90 days) the recovery in the catalase activity was observedand it increased significantly when compared to group IV(4 ppmAs

2O3treatment for 30 days) According toMittal and

Flora [57] catalase activity significantly decreased in liver andkidney with respect to control after treatment with 100 ppmsodium meta-arsenite in drinking water for 8 weeks

Trace elements play an important role in maintainingnormal homeostasis of the body Oxidative stress results fromchanges in the levels of trace elements It has been reportedthat increased Mg level is associated with the increasedproduction ofMDA in dementia patients [58] Our result alsoshowed an increasing pattern ofMg concentration alongwithincrease in liver TBARS level

Zn is an essential element required for growth andnormal development and is a constituent of more than 200enzymes one of which is a CuZn superoxide dismutase(CuZn SOD) CuZn SOD is a powerful antioxidant whichtransforms free radical O

2

∙ to H2O2 therefore reducing the

risk of formation of highly reactive hydroxyl radical HO∙ Cuis also an integral part of CuZn SOD enzyme DecreasedZn and Cu levels in tissues may result in reduction of CuZnSOD activity and subsequently accelerate the process of cellaging and death via oxidative damage [59] However free orincorrectly bound Cu+2 can catalyze the generation of the

most damaging radicals such as HO∙ resulting in a chemicalmodification of the protein alterations in protein structureand solubility and oxidative damage to surrounding tissue[60] Our findings also indicate significant modulation of Cuand Zn level in liver of group II mice

Selenium (Se) is a well known antagonist of arsenic toxic-ity [61] The most probable cause of such a protective effectof selenium is due to its ability to upregulate antioxidantenzymes like GSH peroxidase and thioredoxin reductasewhich protects against arsenic induced oxidative damage[62] Decrement in selenium level after As

2O3treatment was

reported byMolin et al [63 64] In the present study Se levelin liver decreased dose dependently recording maximumdepletion at 4 ppm of As

2O3for 30 days but in the group

treated for 90 days with 4 ppm As2O3 Se level increased and

almost reached the normal levelIron (Fe) catalyses the formation of reactive oxygen

species through the Fenton and Haber-Weiss reactions [65]which generate highly toxic hydroxyl radical and cause lipidperoxidation [66] According to Ahmad et al [67] body ironstores (serum ferritin and transferrin saturation) in the bodycan be used as an early investigative tool for assessing theoxidative stress in coronary heart disease In our study ironlevel increased in all the treatment groups except in group Vwhere the iron concentration is almost same to that of thecontrol group

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

of

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN AIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Computational and Mathematical Methods in Medicine

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Biomarkers

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Anesthesiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ObesityJournal of

ISRN Allergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

4 ISRN Hepatology

PCR amplification are Nrf2 forward 51015840-TCTCCTCGCTGG-AAAAAGAA-31015840 51015840-AATGTGCTGGCTGTGCTTTA-31015840 120573-actin forward 51015840-TGGAATCCTGTGGCATCCATGAAAC-31015840 120573-actin reverse 51015840-TAAAACGCAGCTCAGTAACAG-TCCG-31015840 [40] For amplification of Keap1 specific primerwas designed using Primer 3 software Keap1 forward 51015840-GTACGCTGCGAGTCCGAGGT-31015840 Keap1 reverse 51015840-GCC-ATTGCTCGGGTTGTAGG-31015840 The PCR products were runin 1 agarose gel and visualized in a gel documentationsystem (Gel Doc EZ Imager Bio-Rad) after staining withethidium bromide The densitometric quantification wasdone using ImageJ (NIH) software

213 Hoechst 33342 Staining Hepatocytes were isolated fromblanched liver by a two-step collagenase (Sigma AldrichUSA) digestion method [41] The isolated hepatocytes fromdifferent treatment groups were washed with PBS fixedwith 37 para-formaldehyde solution at room tempera-ture stained with bisBenzimide H 33342 trihydrochloride(Hoechst 33342 2mgmL) and visualized under fluorescencemicroscope (Dewinter Italy) within 30min of adding thestain

214 EDXRF Measurement Liver was dissected out carefullyusing stainless steel forceps and blotted free of blood Thesamples were not washed to avoid leaching of soluble ele-ments Tissues were freeze dried in a lyophilizer after fixing inliquid nitrogen and made into fine powder using mortar andpestle About 150mg pooled powdered samples were used tomake into pellets (1mm thick and 10mm diameter) using atabletop pelletiser (pressure 100 to 110 kgcm2 for 5 minutes)

The tissue samples were analyzed by Jordan Valley EX-3600 ED-XRF system All measurements were carried outin vacuum using different filters (between the source andsample) for optimum detection of elements for 600 s TheX-rays detection was done by using liquid nitrogen cooled125mm2 Si (Li) semiconductor detectors with the resolution148 eV at 59 KeV The X-ray fluorescence spectra were quan-titatively analyzed using Ex-Win software integrated with thesystem Standardization of the procedure was done usingNIST bovine liver standard SRM 1577b

215 Statistical Analysis All assays were repeated at leastthree times Data were analyzed by Studentrsquos 119905-test usingthe Sigma plot 80 statistical package Differences betweencontrol and experimental group(s) with a value of 119875 lt 005was considered as significant

3 Results

31Mortality and Clinical Observations Allmice were exam-ined daily for any clinical signs of toxicity There was nodeath in both control and treatment groups and no clinicalsymptoms were found to appear in any of the treatmentgroups

32 Change in Body Weight and Water Consumption RateThere was no significant difference in water consumption

Org

an to

bod

y w

eigh

t rat

io

Treatment groupsGr I Gr II Gr III Gr IV Gr V

7

6

5

4

3

2

1

0

Figure 1 Organ to body weight ratio of liver in different groups ofmice Groups II III IV and V were compared with group I Valuesare expressed as mean plusmn SEM

rate as well as change in body weight (weight gain) recordedbetween the control and treatment groups (data not shown)

33 Organ to Body Weight Ratio No significant difference inthe organ to body weight ratio of liver of any treatment groupwith the control group was observed (Figure 1)

34 Histopathology Liver appeared normal and healthy(Figure 2(a)) in the control group of mice Disorganization ofhepatic parenchyma and disruption in the epithelial lining ofthe central vein and vacuolar degeneration were commonlyobserved in all treatment groups Liver in Group II micedemonstrated sinusoidal dilation and vacuolar degenerationin the cytoplasm (Figure 2(b)) In group IIImice (treatedwith2 ppm of As

2O3) extensive vacuolar degeneration and loss of

integrity in the epithelial lining of the central vein were found(Figure 2(c)) Loss of typical organization of hepatic cord andvacuolar degeneration were seen in group IV mice (treatedwith 4 ppm of As

2O3for 30 days) (Figure 2(d)) In group

V mice treated with 4 ppm of As2O3for 90 days extensive

degeneration of epithelial lining of the central vein (thickarrow) loss of typical hepatic cord organization hepatocellu-lar degradation and infiltration of the nucleus (thin arrows)into the central vein were prominent (Figure 2(e))

35 Liver Function Test An overall increase was noted in theserum GOT (AST) and GPT (ALT) levels in all treatmentgroups against control (Figure 3) SGOT level increased sig-nificantly in group II (7794) group III (15379) and groupIV (11304) mice SGPT level also increased significantly ingroup II (11596) group III (9509) and group IV (15373)

36 GSH and GST Response and TBARS Production A dosedependent decrease in GSH level was recorded in all the30 days treatment groups of mice and the decrease wassignificant in group III (3229) and group IV (479) againstcontrol In group V mice after 4 ppm of As

2O3treatment for

90 days GSH level recovered which increased significantlyagainst control mice (1931) (Figure 4(a)) TBARS increased

ISRN Hepatology 5

CV

(a) (b)

(c)

(d) (e)

Figure 2 Changes in liver histology Microphotographs of liver sections (6 120583m) stained with hematoxylin and eosin (HE) The originalmagnificationtimes400 (a)Normal histological appearance of liver tissue of controlmice central vein (CV) (b)Group II vacuolar degenerations(thin arrows) sinusoidal dilation (thick arrow) (c) Group III disruption of epithelium lining (short thick arrows) of the central vein vacuolardegenerations (thin arrows) (d) Group IV vacuolar degeneration (thin arrow) loss of integrity in epithelium lining of the central vein (shortthick arrow) and loss of typical hepatic cords organization (e) Group V extensive degeneration of epithelial lining of the central vein (thickarrow) loss of typical hepatic cord organization hepatocellular degradation and infiltration of the nucleus into the central vein (thin arrow)

Enzy

me c

once

ntra

tion

(uni

t enz

yme

L se

rum

)

Serum GPTSerum GOT

Gr I Gr II Gr III Gr IV Gr VTreatment groups

300

250

200

150

100

50

0

lowast

lowast

lowast

lowast

lowast

lowast

Figure 3 SGPT and SGOT activity (unit enzymeL of serum) inthe serum of different groups of mice exposed to different doses ofAs2O3 Groups II III IV and Vwere compared with group I Values

are expressed as mean plusmn SEM lowastValues are statistically significant at(119875 lt 005)

significantly in group III (8656) group IV (8993)and group V (2684) mice GST activity also depicted asignificant increase in group III (7219) group IV (6213)and group V (3478) mice (Figures 4(b) and 4(c))

37 Catalase Activity Catalase activity increased significantlyin group II (55) and thereafter gradually decreased withsubsequent higher doses in group III (744) and group IV(2709) mice against control In group V catalase activityshowed an increase though the increase was not significantcompared to the control (Figure 4(d))

38 Heat Shock Protein (Hsp) 70 Expression Hsp 70 profileshowed increasing pattern of expression against control in allthe treatment groups Expression increased 125-folds 126-folds 132-folds and 154-folds in group II III IV andVmicerespectively as against the control group (Figures 5(a) and5(c))

39 Nrf2 Keap1 and p62 Protein Expression Nrf2 proteinlevels were detected in whole cell lysates as this can give anidea of relative Nrf2 levels in the nuclear fractions of arsenic

6 ISRN HepatologyG

SH120583

mol

mg

prot

ein

Gr I Gr II Gr III Gr IV Gr VTreatment groups

20

18

16

14

12

10

8

6

4

2

0

lowast

lowast

lowast

(a)

nM M

DA

pro

duce

dm

gpr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

35

3

25

2

15

1

0

lowastlowast

lowast

05

(b)

Gr I Gr II Gr III Gr IV Gr VTreatment groups

30

25

20

15

10

5

0

lowastlowast

lowast

nM D

NPG

prod

uced

min

mg

prot

ein

(c)

Uni

t enz

yme

activ

itym

inm

g pr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

lowast

lowast

800

700

600

500

400

300

200

100

0

(d)

Figure 4 (a) GSH content (b) MDA production (c) GST activity (d) catalase activity in liver of different groups of mice exposed to differentdoses of As

2O3 Groups II III IV andVwere comparedwithGroup I Values are expressed asmeanplusmn SEM lowastValues are statistically significant

at (119875 lt 005)

treated cells [20 42 43] We observed the induction of Nrf2protein for different doses of arsenic in all the treatmentgroups but the increase in the Nrf2 protein level was notconsistent with the increasing doses of arsenic treatmentThemaximum level of Nrf2 protein was recorded in group II(144-fold of control) mice which was found to decrease ingroup III (114-fold) mice Further in group IVmice the levelof Nrf2 protein increased to 136-folds of the control againshowing a decreasing pattern in group V (118-fold) mice(Figures 5(a) and 5(b)) Keap1 protein expression decreasedin all the treatment groups compared to the control groupLowest level of Keap1 protein was found in group V (Figures5(a) and 5(b)) mice p62 protein expression was also low inthe control group while elevated levels of protein expressionwere observed in all the treatment groups though the increasein protein level was not dose dependent The highest levelof p62 protein was observed in group II (18-fold of control)followed by group III and group IV mice where it decreasedgradually by 13-fold and 12-fold respectively In group V

mice however an elevation of 16 folds was recorded (Figures5(a) and 5(b))

310 120574GCS Protein GST and GR Protein Expression 120574GCSprotein level in group II mice showed a slight decreaseas compared to the control group while the protein levelincreased continuously in the subsequent groups by 103-fold 122-fold and 151-fold in group III IV and V micerespectively against control (Figures 5(a) and 5(c)) GSTprotein expression increased in all treatment groups with thehighest level recorded in groupV (123-fold) (Figures 5(a) and5(c)) Expression of GR protein also showed an increasingtrend in group II and III whereas in group IV and V micethe level gradually decreased and in group Vmice it reachedalmost the control level (Figures 5(a) and 5(c))

311 Nrf2 and Keap1 mRNA Expression Expression of Nrf2mRNA increased in all the treatment groups compared tothe control group Expression increased by 231-fold 221-fold

ISRN Hepatology 7

Hsp 70

Nrf2

Keap1

GST

GR

Gr I Gr II Gr III Gr IV Gr V

p62

M

120573-Actin

120574GCS

(a)

Nrf 2Keap1p62

20

18

16

14

12

10

08

06Gr I Gr II Gr III Gr IV Gr V

Rela

tive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

Treatment groups

(b)

GSTGRHsp 70

18

16

14

12

10

08Gr I Gr II Gr III Gr IV Gr V

Treatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

120574GCS

(c)Figure 5 (a) Western blotting (b) densitometric analysis of the Nrf2 protein Keap1 protein and p62 protein profile and (c) densitometricanalysis of 120574GCS protein GST protein GR protein and Hsp 70 protein profile of liver of mice treated with different doses of As

2O3

and 229-fold in group II group III and group IV micerespectively against control group ofmice In groupV (4 ppmof As2O3treated for 90 days) decrement of the expression

was noteworthy against the 30 days treatment groups (Figures6(a) and 6(b)) though the level was still higher than control(177-fold) Keap1 mRNA level decreased in all treatmentgroups Keap1 mRNA level detected was 081-fold that ofcontrol in group II 054 folds of the control in group III 061folds of the control in group IV and 042-fold of control ingroup V mice (Figures 6(a) and 6(b))

312 Detection of Apoptosis by Hoechst 33342 The hepato-cytes of mice in all the treatment groups exhibited con-densed and fragmented nuclei upon staining with Hoechst33342 which is an indicator of possible apoptotic cell death(Figure 7)

313 Modulation of Elements Concentration of Mg showedan increasing trend in all treatment groups and the increasewas significant in group III and group IV mice Group IImice showed significant increase and decrease respectivelyfor Cu and Zn whereas significant depletion in Se level wasobserved in group IV mice Iron concentration increased ingroup II group III and group IV with the highest level werefound in group IV mice In group V mice a reduction in theiron concentration was observed reaching almost the controlvalue (Table 1)

4 Discussion

According toWHOguideline the permissible limit of arsenicin drinking water is 10 ppb In some states in USA andChina people are exposed to more than 1 ppm of As throughtheir drinking water Higher arsenic contamination is mainly

8 ISRN Hepatology

Nrf2

Keap1

GrI Gr II Gr III Gr IV Gr V

120573-Actin

(a)

Nrf2Keap1

GrI Gr II Gr III Gr IV Gr VTreatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

30

25

20

15

10

05

00

(b)

Figure 6 (a) Nrf2 and Keap1 mRNA expressions in liver (b) Densitometric analysis of Nrf2 mRNA Keap1 mRNA in liver of mice treatedwith different doses of As

2O3

(a) (b) (c)

(d) (e) (f)

Figure 7 Mouse hepatocytes showing apoptosis (arrow) by Hoechst 33342 staining (a) (b) control (c) 04 ppm (d) 2 ppm and (e) 4 ppmAs2O3treatment for 30 days (f) 4 ppm arsenic trioxide treatment for 90 days

ISRN Hepatology 9

Table 1 Concentration of elements (mgkg) in whole-liver tissue following in vivo administration of different concentrations of As2O3 (ppm)in the drinking water of the mice for 30 and 90 days (Values are mean plusmn SEM)

Elements Group I Group II Group III Group IV Group VMg 35445 plusmn 5417 54729 plusmn 5888 67879 plusmn 2113

lowast57821 plusmn 4246

lowast46143 plusmn 2990

Cu 1541 plusmn 023 1749 plusmn 073lowast

1454 plusmn 072 1444 plusmn 047 1468 plusmn 059

Zn 10755 plusmn 157 9763 plusmn 397lowast

10377 plusmn 164 10762 plusmn 335 10226 plusmn 471

Se 1419 plusmn 134 1325 plusmn 055 1252 plusmn 189 843 plusmn 074lowast

1409 plusmn 113

Fe 50365 plusmn 3866 61029 plusmn 4599 56782 plusmn 2297 73421 plusmn 4494lowast

50689 plusmn 2822

lowastValues are statistically significant compared to control at (119875 lt 005)

found in ground water which is the most common sourceof drinking water The chronic exposure is the main causeof arsenic induced cancer development in human popula-tion and the mechanism of chronic arsenic toxicity is notfully known Therefore in our present experiment micewere treated with As through drinking water Accordingto Singh et al [44] arsenic administration decreased bodyweight gain in a dose dependant manner Santra et al [45]have reported significant increase in the liver weight ofBALBc mice after 12 months of arsenic treatment whereasin the present study there was no significant change in bodyweight gain rate of water consumption or the organ to bodyweight ratio of liver in any of the treatment groups

Liver is an important organ for various metabolic path-ways and effect of any chemical or xenobiotic appears pri-marily in the liver In case of arsenic metabolism liver is themain site of arsenic methylation [46]Themost commonwayof assertion of liver damage is to determine two pathophys-iological enzymes serum glutamate pyruvate transaminase(SGPT) and serum glutamate oxaloacetate transaminase(SGOT) In our findings there was a distinct pattern ofincreased SGOT and SGPT levels in all the treatment groupstreated for 30 days In case of As

2O3(4 ppm) treatment

for 90 days however reversal in the activities of both theenzymes occurred almost reaching basal level The reasonfor such reversal is difficult to explain since histopathologicalalterations as well as induction of apoptosis was observed inthe same treatment group The increased SGOT and SGPTlevels in rat after 45 days treatment of 10 ppm sodium arsenitewas also described by Tandan et al [47] whereas Santra et al[45] reported increased SGOT and SGPT levels after 12months of arsenic treatment at 32 ppm in mice Liver alter-ation in histopathological architecture was evident mainly inthe form of loss of integrity of central vein loss of hepaticcord organization and dose and time dependent vacuolationwhich corroborates with earlier studies [45 48 49] Thesestudies indicate that chronic exposure to different inorganicarsenic compounds (arsenite arsenic trioxide or arsenate)produces characteristic pathology in the liver including fattyinfiltration liver degeneration inflammatory cell infiltrationand focal necrosis Das Neves et al [50] have shown that evenacute exposure of sodium arsenite at a dose of 10mgkg bodyweight (intraperitoneally) for only 90min could producevarious histopathological alterations in liver

In the present study Hoechst 33342 stained hepatocytesclearly indicated fragmented nuclei in all the treatment

groups as a mark of apoptosis as reported earlier in fish liver[17]

Oxidative stress is a relatively new theory in assessmentof arsenic toxicity [51 52] Arsenic causes oxidative stress byproducing reactive oxygen species [20 53 54] which damageproteins Due to lipophilic nature arsenic also attaches tolipid thereby increasing the rate of lipid peroxidation [55]Cellular GSH plays an important role in mitigating arsenicinduced oxidative stress Arsenic being a strong electrophilepredominantly binds with nucleophilic SH group of GSHThus consequent depletion of GSHmay alter the redox statusof the cell and present a stressful and toxic situation Accord-ing to Bashir et al [56] acute treatment of sodium arseniteat doses of 63mgkg 105mgkg and 126mgkg of bodyweight for 24 h significantly decreased GSH content in liverfor all doses tested On the other hand a significant increasein lipid peroxidation and glutathione peroxidase activityalongwith significant decrease in the activity of GST catalaseand superoxide dismutase was observed at 105mgkg and126mgkg of As treatment Santra et al [45] reported thatchronic exposure to 32 ppm of arsenic significantly depletedGSHafter 6months of exposure catalase activity significantlydecreased after 9 months and GST activity significantlyreduced at 12 and 15 months of arsenic exposure In ourstudy GSH level showed decreasing trend in all the 30 daystreatment groups with highest depletion recorded at 4 ppmof As2O3treatment for 30 days while after 90 days treatment

with the same dose the GSH level increased significantlywith respect to both control and also the 30 days treatmentgroupsThis shows the upregulation ofGSHagainst long termarsenic treatment which is due to its adaptive response tooxidative stress [20] However a reverse trend was observedin TBARS production and GST activity in liver in group II(04 ppm As

2O3treatment for 30 days) there was almost no

change in TBARS and GST level against the control groupwhile in group III and group IV levels of both TBARS andGST activity increased significantly Interestingly in groupV (90 days treatment with 4 ppm As

2O3) both TBARS and

GST level decreased significantly with respect to group IVthat is 4 ppm As

2O3for 30 days though the levels were still

significantly higher than the control group The increasedGST activity might be responsible for the reduction in GSHwhile protecting against the arsenic-induced oxidative stressCatalase activity increased significantly in group II mice withrespect to control while at subsequent higher doses that is2 ppm and 4 ppm As

2O3for 30 days the activity decreased

10 ISRN Hepatology

Increased level of SGOT and SGPT

Histopathological alterationsModulation in trace element levels

Increased production of p62

Increased Nrf2 level

Decreased activity of Keap1

Increased production of Hsp 70

Active Nrf2

Gene transcription

GST

GCS

GR

GSH

p62

Alteration in the activity of GST

Modulation in GSH level

Apoptotic induction

Increased production of MDA

p62

Keap1Keap1

ubub

ub

ubInactive Nrf2

Cytosol Nucleus

Nuclear localiztion

Nrf2Nrf2

Nrf2

ARE

Oxidative stress

As

120574GCS GR and catalase

Figure 8 Schematic representation of the arsenic induced liver damages and induction of gene expression through the Keap1-Nrf2-AREsignaling pathway

and highest rate of depletion was observed after 4 ppmAs2O3

treatment for 30 days Further in group V (4 ppm As2O3for

90 days) the recovery in the catalase activity was observedand it increased significantly when compared to group IV(4 ppmAs

2O3treatment for 30 days) According toMittal and

Flora [57] catalase activity significantly decreased in liver andkidney with respect to control after treatment with 100 ppmsodium meta-arsenite in drinking water for 8 weeks

Trace elements play an important role in maintainingnormal homeostasis of the body Oxidative stress results fromchanges in the levels of trace elements It has been reportedthat increased Mg level is associated with the increasedproduction ofMDA in dementia patients [58] Our result alsoshowed an increasing pattern ofMg concentration alongwithincrease in liver TBARS level

Zn is an essential element required for growth andnormal development and is a constituent of more than 200enzymes one of which is a CuZn superoxide dismutase(CuZn SOD) CuZn SOD is a powerful antioxidant whichtransforms free radical O

2

∙ to H2O2 therefore reducing the

risk of formation of highly reactive hydroxyl radical HO∙ Cuis also an integral part of CuZn SOD enzyme DecreasedZn and Cu levels in tissues may result in reduction of CuZnSOD activity and subsequently accelerate the process of cellaging and death via oxidative damage [59] However free orincorrectly bound Cu+2 can catalyze the generation of the

most damaging radicals such as HO∙ resulting in a chemicalmodification of the protein alterations in protein structureand solubility and oxidative damage to surrounding tissue[60] Our findings also indicate significant modulation of Cuand Zn level in liver of group II mice

Selenium (Se) is a well known antagonist of arsenic toxic-ity [61] The most probable cause of such a protective effectof selenium is due to its ability to upregulate antioxidantenzymes like GSH peroxidase and thioredoxin reductasewhich protects against arsenic induced oxidative damage[62] Decrement in selenium level after As

2O3treatment was

reported byMolin et al [63 64] In the present study Se levelin liver decreased dose dependently recording maximumdepletion at 4 ppm of As

2O3for 30 days but in the group

treated for 90 days with 4 ppm As2O3 Se level increased and

almost reached the normal levelIron (Fe) catalyses the formation of reactive oxygen

species through the Fenton and Haber-Weiss reactions [65]which generate highly toxic hydroxyl radical and cause lipidperoxidation [66] According to Ahmad et al [67] body ironstores (serum ferritin and transferrin saturation) in the bodycan be used as an early investigative tool for assessing theoxidative stress in coronary heart disease In our study ironlevel increased in all the treatment groups except in group Vwhere the iron concentration is almost same to that of thecontrol group

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

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Diabetes ResearchJournal of

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ISRN AIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Computational and Mathematical Methods in Medicine

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Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

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ISRN Biomarkers

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

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OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

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ObesityJournal of

ISRN Allergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Hepatology 5

CV

(a) (b)

(c)

(d) (e)

Figure 2 Changes in liver histology Microphotographs of liver sections (6 120583m) stained with hematoxylin and eosin (HE) The originalmagnificationtimes400 (a)Normal histological appearance of liver tissue of controlmice central vein (CV) (b)Group II vacuolar degenerations(thin arrows) sinusoidal dilation (thick arrow) (c) Group III disruption of epithelium lining (short thick arrows) of the central vein vacuolardegenerations (thin arrows) (d) Group IV vacuolar degeneration (thin arrow) loss of integrity in epithelium lining of the central vein (shortthick arrow) and loss of typical hepatic cords organization (e) Group V extensive degeneration of epithelial lining of the central vein (thickarrow) loss of typical hepatic cord organization hepatocellular degradation and infiltration of the nucleus into the central vein (thin arrow)

Enzy

me c

once

ntra

tion

(uni

t enz

yme

L se

rum

)

Serum GPTSerum GOT

Gr I Gr II Gr III Gr IV Gr VTreatment groups

300

250

200

150

100

50

0

lowast

lowast

lowast

lowast

lowast

lowast

Figure 3 SGPT and SGOT activity (unit enzymeL of serum) inthe serum of different groups of mice exposed to different doses ofAs2O3 Groups II III IV and Vwere compared with group I Values

are expressed as mean plusmn SEM lowastValues are statistically significant at(119875 lt 005)

significantly in group III (8656) group IV (8993)and group V (2684) mice GST activity also depicted asignificant increase in group III (7219) group IV (6213)and group V (3478) mice (Figures 4(b) and 4(c))

37 Catalase Activity Catalase activity increased significantlyin group II (55) and thereafter gradually decreased withsubsequent higher doses in group III (744) and group IV(2709) mice against control In group V catalase activityshowed an increase though the increase was not significantcompared to the control (Figure 4(d))

38 Heat Shock Protein (Hsp) 70 Expression Hsp 70 profileshowed increasing pattern of expression against control in allthe treatment groups Expression increased 125-folds 126-folds 132-folds and 154-folds in group II III IV andVmicerespectively as against the control group (Figures 5(a) and5(c))

39 Nrf2 Keap1 and p62 Protein Expression Nrf2 proteinlevels were detected in whole cell lysates as this can give anidea of relative Nrf2 levels in the nuclear fractions of arsenic

6 ISRN HepatologyG

SH120583

mol

mg

prot

ein

Gr I Gr II Gr III Gr IV Gr VTreatment groups

20

18

16

14

12

10

8

6

4

2

0

lowast

lowast

lowast

(a)

nM M

DA

pro

duce

dm

gpr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

35

3

25

2

15

1

0

lowastlowast

lowast

05

(b)

Gr I Gr II Gr III Gr IV Gr VTreatment groups

30

25

20

15

10

5

0

lowastlowast

lowast

nM D

NPG

prod

uced

min

mg

prot

ein

(c)

Uni

t enz

yme

activ

itym

inm

g pr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

lowast

lowast

800

700

600

500

400

300

200

100

0

(d)

Figure 4 (a) GSH content (b) MDA production (c) GST activity (d) catalase activity in liver of different groups of mice exposed to differentdoses of As

2O3 Groups II III IV andVwere comparedwithGroup I Values are expressed asmeanplusmn SEM lowastValues are statistically significant

at (119875 lt 005)

treated cells [20 42 43] We observed the induction of Nrf2protein for different doses of arsenic in all the treatmentgroups but the increase in the Nrf2 protein level was notconsistent with the increasing doses of arsenic treatmentThemaximum level of Nrf2 protein was recorded in group II(144-fold of control) mice which was found to decrease ingroup III (114-fold) mice Further in group IVmice the levelof Nrf2 protein increased to 136-folds of the control againshowing a decreasing pattern in group V (118-fold) mice(Figures 5(a) and 5(b)) Keap1 protein expression decreasedin all the treatment groups compared to the control groupLowest level of Keap1 protein was found in group V (Figures5(a) and 5(b)) mice p62 protein expression was also low inthe control group while elevated levels of protein expressionwere observed in all the treatment groups though the increasein protein level was not dose dependent The highest levelof p62 protein was observed in group II (18-fold of control)followed by group III and group IV mice where it decreasedgradually by 13-fold and 12-fold respectively In group V

mice however an elevation of 16 folds was recorded (Figures5(a) and 5(b))

310 120574GCS Protein GST and GR Protein Expression 120574GCSprotein level in group II mice showed a slight decreaseas compared to the control group while the protein levelincreased continuously in the subsequent groups by 103-fold 122-fold and 151-fold in group III IV and V micerespectively against control (Figures 5(a) and 5(c)) GSTprotein expression increased in all treatment groups with thehighest level recorded in groupV (123-fold) (Figures 5(a) and5(c)) Expression of GR protein also showed an increasingtrend in group II and III whereas in group IV and V micethe level gradually decreased and in group Vmice it reachedalmost the control level (Figures 5(a) and 5(c))

311 Nrf2 and Keap1 mRNA Expression Expression of Nrf2mRNA increased in all the treatment groups compared tothe control group Expression increased by 231-fold 221-fold

ISRN Hepatology 7

Hsp 70

Nrf2

Keap1

GST

GR

Gr I Gr II Gr III Gr IV Gr V

p62

M

120573-Actin

120574GCS

(a)

Nrf 2Keap1p62

20

18

16

14

12

10

08

06Gr I Gr II Gr III Gr IV Gr V

Rela

tive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

Treatment groups

(b)

GSTGRHsp 70

18

16

14

12

10

08Gr I Gr II Gr III Gr IV Gr V

Treatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

120574GCS

(c)Figure 5 (a) Western blotting (b) densitometric analysis of the Nrf2 protein Keap1 protein and p62 protein profile and (c) densitometricanalysis of 120574GCS protein GST protein GR protein and Hsp 70 protein profile of liver of mice treated with different doses of As

2O3

and 229-fold in group II group III and group IV micerespectively against control group ofmice In groupV (4 ppmof As2O3treated for 90 days) decrement of the expression

was noteworthy against the 30 days treatment groups (Figures6(a) and 6(b)) though the level was still higher than control(177-fold) Keap1 mRNA level decreased in all treatmentgroups Keap1 mRNA level detected was 081-fold that ofcontrol in group II 054 folds of the control in group III 061folds of the control in group IV and 042-fold of control ingroup V mice (Figures 6(a) and 6(b))

312 Detection of Apoptosis by Hoechst 33342 The hepato-cytes of mice in all the treatment groups exhibited con-densed and fragmented nuclei upon staining with Hoechst33342 which is an indicator of possible apoptotic cell death(Figure 7)

313 Modulation of Elements Concentration of Mg showedan increasing trend in all treatment groups and the increasewas significant in group III and group IV mice Group IImice showed significant increase and decrease respectivelyfor Cu and Zn whereas significant depletion in Se level wasobserved in group IV mice Iron concentration increased ingroup II group III and group IV with the highest level werefound in group IV mice In group V mice a reduction in theiron concentration was observed reaching almost the controlvalue (Table 1)

4 Discussion

According toWHOguideline the permissible limit of arsenicin drinking water is 10 ppb In some states in USA andChina people are exposed to more than 1 ppm of As throughtheir drinking water Higher arsenic contamination is mainly

8 ISRN Hepatology

Nrf2

Keap1

GrI Gr II Gr III Gr IV Gr V

120573-Actin

(a)

Nrf2Keap1

GrI Gr II Gr III Gr IV Gr VTreatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

30

25

20

15

10

05

00

(b)

Figure 6 (a) Nrf2 and Keap1 mRNA expressions in liver (b) Densitometric analysis of Nrf2 mRNA Keap1 mRNA in liver of mice treatedwith different doses of As

2O3

(a) (b) (c)

(d) (e) (f)

Figure 7 Mouse hepatocytes showing apoptosis (arrow) by Hoechst 33342 staining (a) (b) control (c) 04 ppm (d) 2 ppm and (e) 4 ppmAs2O3treatment for 30 days (f) 4 ppm arsenic trioxide treatment for 90 days

ISRN Hepatology 9

Table 1 Concentration of elements (mgkg) in whole-liver tissue following in vivo administration of different concentrations of As2O3 (ppm)in the drinking water of the mice for 30 and 90 days (Values are mean plusmn SEM)

Elements Group I Group II Group III Group IV Group VMg 35445 plusmn 5417 54729 plusmn 5888 67879 plusmn 2113

lowast57821 plusmn 4246

lowast46143 plusmn 2990

Cu 1541 plusmn 023 1749 plusmn 073lowast

1454 plusmn 072 1444 plusmn 047 1468 plusmn 059

Zn 10755 plusmn 157 9763 plusmn 397lowast

10377 plusmn 164 10762 plusmn 335 10226 plusmn 471

Se 1419 plusmn 134 1325 plusmn 055 1252 plusmn 189 843 plusmn 074lowast

1409 plusmn 113

Fe 50365 plusmn 3866 61029 plusmn 4599 56782 plusmn 2297 73421 plusmn 4494lowast

50689 plusmn 2822

lowastValues are statistically significant compared to control at (119875 lt 005)

found in ground water which is the most common sourceof drinking water The chronic exposure is the main causeof arsenic induced cancer development in human popula-tion and the mechanism of chronic arsenic toxicity is notfully known Therefore in our present experiment micewere treated with As through drinking water Accordingto Singh et al [44] arsenic administration decreased bodyweight gain in a dose dependant manner Santra et al [45]have reported significant increase in the liver weight ofBALBc mice after 12 months of arsenic treatment whereasin the present study there was no significant change in bodyweight gain rate of water consumption or the organ to bodyweight ratio of liver in any of the treatment groups

Liver is an important organ for various metabolic path-ways and effect of any chemical or xenobiotic appears pri-marily in the liver In case of arsenic metabolism liver is themain site of arsenic methylation [46]Themost commonwayof assertion of liver damage is to determine two pathophys-iological enzymes serum glutamate pyruvate transaminase(SGPT) and serum glutamate oxaloacetate transaminase(SGOT) In our findings there was a distinct pattern ofincreased SGOT and SGPT levels in all the treatment groupstreated for 30 days In case of As

2O3(4 ppm) treatment

for 90 days however reversal in the activities of both theenzymes occurred almost reaching basal level The reasonfor such reversal is difficult to explain since histopathologicalalterations as well as induction of apoptosis was observed inthe same treatment group The increased SGOT and SGPTlevels in rat after 45 days treatment of 10 ppm sodium arsenitewas also described by Tandan et al [47] whereas Santra et al[45] reported increased SGOT and SGPT levels after 12months of arsenic treatment at 32 ppm in mice Liver alter-ation in histopathological architecture was evident mainly inthe form of loss of integrity of central vein loss of hepaticcord organization and dose and time dependent vacuolationwhich corroborates with earlier studies [45 48 49] Thesestudies indicate that chronic exposure to different inorganicarsenic compounds (arsenite arsenic trioxide or arsenate)produces characteristic pathology in the liver including fattyinfiltration liver degeneration inflammatory cell infiltrationand focal necrosis Das Neves et al [50] have shown that evenacute exposure of sodium arsenite at a dose of 10mgkg bodyweight (intraperitoneally) for only 90min could producevarious histopathological alterations in liver

In the present study Hoechst 33342 stained hepatocytesclearly indicated fragmented nuclei in all the treatment

groups as a mark of apoptosis as reported earlier in fish liver[17]

Oxidative stress is a relatively new theory in assessmentof arsenic toxicity [51 52] Arsenic causes oxidative stress byproducing reactive oxygen species [20 53 54] which damageproteins Due to lipophilic nature arsenic also attaches tolipid thereby increasing the rate of lipid peroxidation [55]Cellular GSH plays an important role in mitigating arsenicinduced oxidative stress Arsenic being a strong electrophilepredominantly binds with nucleophilic SH group of GSHThus consequent depletion of GSHmay alter the redox statusof the cell and present a stressful and toxic situation Accord-ing to Bashir et al [56] acute treatment of sodium arseniteat doses of 63mgkg 105mgkg and 126mgkg of bodyweight for 24 h significantly decreased GSH content in liverfor all doses tested On the other hand a significant increasein lipid peroxidation and glutathione peroxidase activityalongwith significant decrease in the activity of GST catalaseand superoxide dismutase was observed at 105mgkg and126mgkg of As treatment Santra et al [45] reported thatchronic exposure to 32 ppm of arsenic significantly depletedGSHafter 6months of exposure catalase activity significantlydecreased after 9 months and GST activity significantlyreduced at 12 and 15 months of arsenic exposure In ourstudy GSH level showed decreasing trend in all the 30 daystreatment groups with highest depletion recorded at 4 ppmof As2O3treatment for 30 days while after 90 days treatment

with the same dose the GSH level increased significantlywith respect to both control and also the 30 days treatmentgroupsThis shows the upregulation ofGSHagainst long termarsenic treatment which is due to its adaptive response tooxidative stress [20] However a reverse trend was observedin TBARS production and GST activity in liver in group II(04 ppm As

2O3treatment for 30 days) there was almost no

change in TBARS and GST level against the control groupwhile in group III and group IV levels of both TBARS andGST activity increased significantly Interestingly in groupV (90 days treatment with 4 ppm As

2O3) both TBARS and

GST level decreased significantly with respect to group IVthat is 4 ppm As

2O3for 30 days though the levels were still

significantly higher than the control group The increasedGST activity might be responsible for the reduction in GSHwhile protecting against the arsenic-induced oxidative stressCatalase activity increased significantly in group II mice withrespect to control while at subsequent higher doses that is2 ppm and 4 ppm As

2O3for 30 days the activity decreased

10 ISRN Hepatology

Increased level of SGOT and SGPT

Histopathological alterationsModulation in trace element levels

Increased production of p62

Increased Nrf2 level

Decreased activity of Keap1

Increased production of Hsp 70

Active Nrf2

Gene transcription

GST

GCS

GR

GSH

p62

Alteration in the activity of GST

Modulation in GSH level

Apoptotic induction

Increased production of MDA

p62

Keap1Keap1

ubub

ub

ubInactive Nrf2

Cytosol Nucleus

Nuclear localiztion

Nrf2Nrf2

Nrf2

ARE

Oxidative stress

As

120574GCS GR and catalase

Figure 8 Schematic representation of the arsenic induced liver damages and induction of gene expression through the Keap1-Nrf2-AREsignaling pathway

and highest rate of depletion was observed after 4 ppmAs2O3

treatment for 30 days Further in group V (4 ppm As2O3for

90 days) the recovery in the catalase activity was observedand it increased significantly when compared to group IV(4 ppmAs

2O3treatment for 30 days) According toMittal and

Flora [57] catalase activity significantly decreased in liver andkidney with respect to control after treatment with 100 ppmsodium meta-arsenite in drinking water for 8 weeks

Trace elements play an important role in maintainingnormal homeostasis of the body Oxidative stress results fromchanges in the levels of trace elements It has been reportedthat increased Mg level is associated with the increasedproduction ofMDA in dementia patients [58] Our result alsoshowed an increasing pattern ofMg concentration alongwithincrease in liver TBARS level

Zn is an essential element required for growth andnormal development and is a constituent of more than 200enzymes one of which is a CuZn superoxide dismutase(CuZn SOD) CuZn SOD is a powerful antioxidant whichtransforms free radical O

2

∙ to H2O2 therefore reducing the

risk of formation of highly reactive hydroxyl radical HO∙ Cuis also an integral part of CuZn SOD enzyme DecreasedZn and Cu levels in tissues may result in reduction of CuZnSOD activity and subsequently accelerate the process of cellaging and death via oxidative damage [59] However free orincorrectly bound Cu+2 can catalyze the generation of the

most damaging radicals such as HO∙ resulting in a chemicalmodification of the protein alterations in protein structureand solubility and oxidative damage to surrounding tissue[60] Our findings also indicate significant modulation of Cuand Zn level in liver of group II mice

Selenium (Se) is a well known antagonist of arsenic toxic-ity [61] The most probable cause of such a protective effectof selenium is due to its ability to upregulate antioxidantenzymes like GSH peroxidase and thioredoxin reductasewhich protects against arsenic induced oxidative damage[62] Decrement in selenium level after As

2O3treatment was

reported byMolin et al [63 64] In the present study Se levelin liver decreased dose dependently recording maximumdepletion at 4 ppm of As

2O3for 30 days but in the group

treated for 90 days with 4 ppm As2O3 Se level increased and

almost reached the normal levelIron (Fe) catalyses the formation of reactive oxygen

species through the Fenton and Haber-Weiss reactions [65]which generate highly toxic hydroxyl radical and cause lipidperoxidation [66] According to Ahmad et al [67] body ironstores (serum ferritin and transferrin saturation) in the bodycan be used as an early investigative tool for assessing theoxidative stress in coronary heart disease In our study ironlevel increased in all the treatment groups except in group Vwhere the iron concentration is almost same to that of thecontrol group

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

of

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN AIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Computational and Mathematical Methods in Medicine

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Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

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Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ObesityJournal of

ISRN Allergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

6 ISRN HepatologyG

SH120583

mol

mg

prot

ein

Gr I Gr II Gr III Gr IV Gr VTreatment groups

20

18

16

14

12

10

8

6

4

2

0

lowast

lowast

lowast

(a)

nM M

DA

pro

duce

dm

gpr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

35

3

25

2

15

1

0

lowastlowast

lowast

05

(b)

Gr I Gr II Gr III Gr IV Gr VTreatment groups

30

25

20

15

10

5

0

lowastlowast

lowast

nM D

NPG

prod

uced

min

mg

prot

ein

(c)

Uni

t enz

yme

activ

itym

inm

g pr

otei

n

Gr I Gr II Gr III Gr IV Gr VTreatment groups

lowast

lowast

800

700

600

500

400

300

200

100

0

(d)

Figure 4 (a) GSH content (b) MDA production (c) GST activity (d) catalase activity in liver of different groups of mice exposed to differentdoses of As

2O3 Groups II III IV andVwere comparedwithGroup I Values are expressed asmeanplusmn SEM lowastValues are statistically significant

at (119875 lt 005)

treated cells [20 42 43] We observed the induction of Nrf2protein for different doses of arsenic in all the treatmentgroups but the increase in the Nrf2 protein level was notconsistent with the increasing doses of arsenic treatmentThemaximum level of Nrf2 protein was recorded in group II(144-fold of control) mice which was found to decrease ingroup III (114-fold) mice Further in group IVmice the levelof Nrf2 protein increased to 136-folds of the control againshowing a decreasing pattern in group V (118-fold) mice(Figures 5(a) and 5(b)) Keap1 protein expression decreasedin all the treatment groups compared to the control groupLowest level of Keap1 protein was found in group V (Figures5(a) and 5(b)) mice p62 protein expression was also low inthe control group while elevated levels of protein expressionwere observed in all the treatment groups though the increasein protein level was not dose dependent The highest levelof p62 protein was observed in group II (18-fold of control)followed by group III and group IV mice where it decreasedgradually by 13-fold and 12-fold respectively In group V

mice however an elevation of 16 folds was recorded (Figures5(a) and 5(b))

310 120574GCS Protein GST and GR Protein Expression 120574GCSprotein level in group II mice showed a slight decreaseas compared to the control group while the protein levelincreased continuously in the subsequent groups by 103-fold 122-fold and 151-fold in group III IV and V micerespectively against control (Figures 5(a) and 5(c)) GSTprotein expression increased in all treatment groups with thehighest level recorded in groupV (123-fold) (Figures 5(a) and5(c)) Expression of GR protein also showed an increasingtrend in group II and III whereas in group IV and V micethe level gradually decreased and in group Vmice it reachedalmost the control level (Figures 5(a) and 5(c))

311 Nrf2 and Keap1 mRNA Expression Expression of Nrf2mRNA increased in all the treatment groups compared tothe control group Expression increased by 231-fold 221-fold

ISRN Hepatology 7

Hsp 70

Nrf2

Keap1

GST

GR

Gr I Gr II Gr III Gr IV Gr V

p62

M

120573-Actin

120574GCS

(a)

Nrf 2Keap1p62

20

18

16

14

12

10

08

06Gr I Gr II Gr III Gr IV Gr V

Rela

tive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

Treatment groups

(b)

GSTGRHsp 70

18

16

14

12

10

08Gr I Gr II Gr III Gr IV Gr V

Treatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

120574GCS

(c)Figure 5 (a) Western blotting (b) densitometric analysis of the Nrf2 protein Keap1 protein and p62 protein profile and (c) densitometricanalysis of 120574GCS protein GST protein GR protein and Hsp 70 protein profile of liver of mice treated with different doses of As

2O3

and 229-fold in group II group III and group IV micerespectively against control group ofmice In groupV (4 ppmof As2O3treated for 90 days) decrement of the expression

was noteworthy against the 30 days treatment groups (Figures6(a) and 6(b)) though the level was still higher than control(177-fold) Keap1 mRNA level decreased in all treatmentgroups Keap1 mRNA level detected was 081-fold that ofcontrol in group II 054 folds of the control in group III 061folds of the control in group IV and 042-fold of control ingroup V mice (Figures 6(a) and 6(b))

312 Detection of Apoptosis by Hoechst 33342 The hepato-cytes of mice in all the treatment groups exhibited con-densed and fragmented nuclei upon staining with Hoechst33342 which is an indicator of possible apoptotic cell death(Figure 7)

313 Modulation of Elements Concentration of Mg showedan increasing trend in all treatment groups and the increasewas significant in group III and group IV mice Group IImice showed significant increase and decrease respectivelyfor Cu and Zn whereas significant depletion in Se level wasobserved in group IV mice Iron concentration increased ingroup II group III and group IV with the highest level werefound in group IV mice In group V mice a reduction in theiron concentration was observed reaching almost the controlvalue (Table 1)

4 Discussion

According toWHOguideline the permissible limit of arsenicin drinking water is 10 ppb In some states in USA andChina people are exposed to more than 1 ppm of As throughtheir drinking water Higher arsenic contamination is mainly

8 ISRN Hepatology

Nrf2

Keap1

GrI Gr II Gr III Gr IV Gr V

120573-Actin

(a)

Nrf2Keap1

GrI Gr II Gr III Gr IV Gr VTreatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

30

25

20

15

10

05

00

(b)

Figure 6 (a) Nrf2 and Keap1 mRNA expressions in liver (b) Densitometric analysis of Nrf2 mRNA Keap1 mRNA in liver of mice treatedwith different doses of As

2O3

(a) (b) (c)

(d) (e) (f)

Figure 7 Mouse hepatocytes showing apoptosis (arrow) by Hoechst 33342 staining (a) (b) control (c) 04 ppm (d) 2 ppm and (e) 4 ppmAs2O3treatment for 30 days (f) 4 ppm arsenic trioxide treatment for 90 days

ISRN Hepatology 9

Table 1 Concentration of elements (mgkg) in whole-liver tissue following in vivo administration of different concentrations of As2O3 (ppm)in the drinking water of the mice for 30 and 90 days (Values are mean plusmn SEM)

Elements Group I Group II Group III Group IV Group VMg 35445 plusmn 5417 54729 plusmn 5888 67879 plusmn 2113

lowast57821 plusmn 4246

lowast46143 plusmn 2990

Cu 1541 plusmn 023 1749 plusmn 073lowast

1454 plusmn 072 1444 plusmn 047 1468 plusmn 059

Zn 10755 plusmn 157 9763 plusmn 397lowast

10377 plusmn 164 10762 plusmn 335 10226 plusmn 471

Se 1419 plusmn 134 1325 plusmn 055 1252 plusmn 189 843 plusmn 074lowast

1409 plusmn 113

Fe 50365 plusmn 3866 61029 plusmn 4599 56782 plusmn 2297 73421 plusmn 4494lowast

50689 plusmn 2822

lowastValues are statistically significant compared to control at (119875 lt 005)

found in ground water which is the most common sourceof drinking water The chronic exposure is the main causeof arsenic induced cancer development in human popula-tion and the mechanism of chronic arsenic toxicity is notfully known Therefore in our present experiment micewere treated with As through drinking water Accordingto Singh et al [44] arsenic administration decreased bodyweight gain in a dose dependant manner Santra et al [45]have reported significant increase in the liver weight ofBALBc mice after 12 months of arsenic treatment whereasin the present study there was no significant change in bodyweight gain rate of water consumption or the organ to bodyweight ratio of liver in any of the treatment groups

Liver is an important organ for various metabolic path-ways and effect of any chemical or xenobiotic appears pri-marily in the liver In case of arsenic metabolism liver is themain site of arsenic methylation [46]Themost commonwayof assertion of liver damage is to determine two pathophys-iological enzymes serum glutamate pyruvate transaminase(SGPT) and serum glutamate oxaloacetate transaminase(SGOT) In our findings there was a distinct pattern ofincreased SGOT and SGPT levels in all the treatment groupstreated for 30 days In case of As

2O3(4 ppm) treatment

for 90 days however reversal in the activities of both theenzymes occurred almost reaching basal level The reasonfor such reversal is difficult to explain since histopathologicalalterations as well as induction of apoptosis was observed inthe same treatment group The increased SGOT and SGPTlevels in rat after 45 days treatment of 10 ppm sodium arsenitewas also described by Tandan et al [47] whereas Santra et al[45] reported increased SGOT and SGPT levels after 12months of arsenic treatment at 32 ppm in mice Liver alter-ation in histopathological architecture was evident mainly inthe form of loss of integrity of central vein loss of hepaticcord organization and dose and time dependent vacuolationwhich corroborates with earlier studies [45 48 49] Thesestudies indicate that chronic exposure to different inorganicarsenic compounds (arsenite arsenic trioxide or arsenate)produces characteristic pathology in the liver including fattyinfiltration liver degeneration inflammatory cell infiltrationand focal necrosis Das Neves et al [50] have shown that evenacute exposure of sodium arsenite at a dose of 10mgkg bodyweight (intraperitoneally) for only 90min could producevarious histopathological alterations in liver

In the present study Hoechst 33342 stained hepatocytesclearly indicated fragmented nuclei in all the treatment

groups as a mark of apoptosis as reported earlier in fish liver[17]

Oxidative stress is a relatively new theory in assessmentof arsenic toxicity [51 52] Arsenic causes oxidative stress byproducing reactive oxygen species [20 53 54] which damageproteins Due to lipophilic nature arsenic also attaches tolipid thereby increasing the rate of lipid peroxidation [55]Cellular GSH plays an important role in mitigating arsenicinduced oxidative stress Arsenic being a strong electrophilepredominantly binds with nucleophilic SH group of GSHThus consequent depletion of GSHmay alter the redox statusof the cell and present a stressful and toxic situation Accord-ing to Bashir et al [56] acute treatment of sodium arseniteat doses of 63mgkg 105mgkg and 126mgkg of bodyweight for 24 h significantly decreased GSH content in liverfor all doses tested On the other hand a significant increasein lipid peroxidation and glutathione peroxidase activityalongwith significant decrease in the activity of GST catalaseand superoxide dismutase was observed at 105mgkg and126mgkg of As treatment Santra et al [45] reported thatchronic exposure to 32 ppm of arsenic significantly depletedGSHafter 6months of exposure catalase activity significantlydecreased after 9 months and GST activity significantlyreduced at 12 and 15 months of arsenic exposure In ourstudy GSH level showed decreasing trend in all the 30 daystreatment groups with highest depletion recorded at 4 ppmof As2O3treatment for 30 days while after 90 days treatment

with the same dose the GSH level increased significantlywith respect to both control and also the 30 days treatmentgroupsThis shows the upregulation ofGSHagainst long termarsenic treatment which is due to its adaptive response tooxidative stress [20] However a reverse trend was observedin TBARS production and GST activity in liver in group II(04 ppm As

2O3treatment for 30 days) there was almost no

change in TBARS and GST level against the control groupwhile in group III and group IV levels of both TBARS andGST activity increased significantly Interestingly in groupV (90 days treatment with 4 ppm As

2O3) both TBARS and

GST level decreased significantly with respect to group IVthat is 4 ppm As

2O3for 30 days though the levels were still

significantly higher than the control group The increasedGST activity might be responsible for the reduction in GSHwhile protecting against the arsenic-induced oxidative stressCatalase activity increased significantly in group II mice withrespect to control while at subsequent higher doses that is2 ppm and 4 ppm As

2O3for 30 days the activity decreased

10 ISRN Hepatology

Increased level of SGOT and SGPT

Histopathological alterationsModulation in trace element levels

Increased production of p62

Increased Nrf2 level

Decreased activity of Keap1

Increased production of Hsp 70

Active Nrf2

Gene transcription

GST

GCS

GR

GSH

p62

Alteration in the activity of GST

Modulation in GSH level

Apoptotic induction

Increased production of MDA

p62

Keap1Keap1

ubub

ub

ubInactive Nrf2

Cytosol Nucleus

Nuclear localiztion

Nrf2Nrf2

Nrf2

ARE

Oxidative stress

As

120574GCS GR and catalase

Figure 8 Schematic representation of the arsenic induced liver damages and induction of gene expression through the Keap1-Nrf2-AREsignaling pathway

and highest rate of depletion was observed after 4 ppmAs2O3

treatment for 30 days Further in group V (4 ppm As2O3for

90 days) the recovery in the catalase activity was observedand it increased significantly when compared to group IV(4 ppmAs

2O3treatment for 30 days) According toMittal and

Flora [57] catalase activity significantly decreased in liver andkidney with respect to control after treatment with 100 ppmsodium meta-arsenite in drinking water for 8 weeks

Trace elements play an important role in maintainingnormal homeostasis of the body Oxidative stress results fromchanges in the levels of trace elements It has been reportedthat increased Mg level is associated with the increasedproduction ofMDA in dementia patients [58] Our result alsoshowed an increasing pattern ofMg concentration alongwithincrease in liver TBARS level

Zn is an essential element required for growth andnormal development and is a constituent of more than 200enzymes one of which is a CuZn superoxide dismutase(CuZn SOD) CuZn SOD is a powerful antioxidant whichtransforms free radical O

2

∙ to H2O2 therefore reducing the

risk of formation of highly reactive hydroxyl radical HO∙ Cuis also an integral part of CuZn SOD enzyme DecreasedZn and Cu levels in tissues may result in reduction of CuZnSOD activity and subsequently accelerate the process of cellaging and death via oxidative damage [59] However free orincorrectly bound Cu+2 can catalyze the generation of the

most damaging radicals such as HO∙ resulting in a chemicalmodification of the protein alterations in protein structureand solubility and oxidative damage to surrounding tissue[60] Our findings also indicate significant modulation of Cuand Zn level in liver of group II mice

Selenium (Se) is a well known antagonist of arsenic toxic-ity [61] The most probable cause of such a protective effectof selenium is due to its ability to upregulate antioxidantenzymes like GSH peroxidase and thioredoxin reductasewhich protects against arsenic induced oxidative damage[62] Decrement in selenium level after As

2O3treatment was

reported byMolin et al [63 64] In the present study Se levelin liver decreased dose dependently recording maximumdepletion at 4 ppm of As

2O3for 30 days but in the group

treated for 90 days with 4 ppm As2O3 Se level increased and

almost reached the normal levelIron (Fe) catalyses the formation of reactive oxygen

species through the Fenton and Haber-Weiss reactions [65]which generate highly toxic hydroxyl radical and cause lipidperoxidation [66] According to Ahmad et al [67] body ironstores (serum ferritin and transferrin saturation) in the bodycan be used as an early investigative tool for assessing theoxidative stress in coronary heart disease In our study ironlevel increased in all the treatment groups except in group Vwhere the iron concentration is almost same to that of thecontrol group

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

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ObesityJournal of

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PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Hepatology 7

Hsp 70

Nrf2

Keap1

GST

GR

Gr I Gr II Gr III Gr IV Gr V

p62

M

120573-Actin

120574GCS

(a)

Nrf 2Keap1p62

20

18

16

14

12

10

08

06Gr I Gr II Gr III Gr IV Gr V

Rela

tive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

Treatment groups

(b)

GSTGRHsp 70

18

16

14

12

10

08Gr I Gr II Gr III Gr IV Gr V

Treatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

120574GCS

(c)Figure 5 (a) Western blotting (b) densitometric analysis of the Nrf2 protein Keap1 protein and p62 protein profile and (c) densitometricanalysis of 120574GCS protein GST protein GR protein and Hsp 70 protein profile of liver of mice treated with different doses of As

2O3

and 229-fold in group II group III and group IV micerespectively against control group ofmice In groupV (4 ppmof As2O3treated for 90 days) decrement of the expression

was noteworthy against the 30 days treatment groups (Figures6(a) and 6(b)) though the level was still higher than control(177-fold) Keap1 mRNA level decreased in all treatmentgroups Keap1 mRNA level detected was 081-fold that ofcontrol in group II 054 folds of the control in group III 061folds of the control in group IV and 042-fold of control ingroup V mice (Figures 6(a) and 6(b))

312 Detection of Apoptosis by Hoechst 33342 The hepato-cytes of mice in all the treatment groups exhibited con-densed and fragmented nuclei upon staining with Hoechst33342 which is an indicator of possible apoptotic cell death(Figure 7)

313 Modulation of Elements Concentration of Mg showedan increasing trend in all treatment groups and the increasewas significant in group III and group IV mice Group IImice showed significant increase and decrease respectivelyfor Cu and Zn whereas significant depletion in Se level wasobserved in group IV mice Iron concentration increased ingroup II group III and group IV with the highest level werefound in group IV mice In group V mice a reduction in theiron concentration was observed reaching almost the controlvalue (Table 1)

4 Discussion

According toWHOguideline the permissible limit of arsenicin drinking water is 10 ppb In some states in USA andChina people are exposed to more than 1 ppm of As throughtheir drinking water Higher arsenic contamination is mainly

8 ISRN Hepatology

Nrf2

Keap1

GrI Gr II Gr III Gr IV Gr V

120573-Actin

(a)

Nrf2Keap1

GrI Gr II Gr III Gr IV Gr VTreatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

30

25

20

15

10

05

00

(b)

Figure 6 (a) Nrf2 and Keap1 mRNA expressions in liver (b) Densitometric analysis of Nrf2 mRNA Keap1 mRNA in liver of mice treatedwith different doses of As

2O3

(a) (b) (c)

(d) (e) (f)

Figure 7 Mouse hepatocytes showing apoptosis (arrow) by Hoechst 33342 staining (a) (b) control (c) 04 ppm (d) 2 ppm and (e) 4 ppmAs2O3treatment for 30 days (f) 4 ppm arsenic trioxide treatment for 90 days

ISRN Hepatology 9

Table 1 Concentration of elements (mgkg) in whole-liver tissue following in vivo administration of different concentrations of As2O3 (ppm)in the drinking water of the mice for 30 and 90 days (Values are mean plusmn SEM)

Elements Group I Group II Group III Group IV Group VMg 35445 plusmn 5417 54729 plusmn 5888 67879 plusmn 2113

lowast57821 plusmn 4246

lowast46143 plusmn 2990

Cu 1541 plusmn 023 1749 plusmn 073lowast

1454 plusmn 072 1444 plusmn 047 1468 plusmn 059

Zn 10755 plusmn 157 9763 plusmn 397lowast

10377 plusmn 164 10762 plusmn 335 10226 plusmn 471

Se 1419 plusmn 134 1325 plusmn 055 1252 plusmn 189 843 plusmn 074lowast

1409 plusmn 113

Fe 50365 plusmn 3866 61029 plusmn 4599 56782 plusmn 2297 73421 plusmn 4494lowast

50689 plusmn 2822

lowastValues are statistically significant compared to control at (119875 lt 005)

found in ground water which is the most common sourceof drinking water The chronic exposure is the main causeof arsenic induced cancer development in human popula-tion and the mechanism of chronic arsenic toxicity is notfully known Therefore in our present experiment micewere treated with As through drinking water Accordingto Singh et al [44] arsenic administration decreased bodyweight gain in a dose dependant manner Santra et al [45]have reported significant increase in the liver weight ofBALBc mice after 12 months of arsenic treatment whereasin the present study there was no significant change in bodyweight gain rate of water consumption or the organ to bodyweight ratio of liver in any of the treatment groups

Liver is an important organ for various metabolic path-ways and effect of any chemical or xenobiotic appears pri-marily in the liver In case of arsenic metabolism liver is themain site of arsenic methylation [46]Themost commonwayof assertion of liver damage is to determine two pathophys-iological enzymes serum glutamate pyruvate transaminase(SGPT) and serum glutamate oxaloacetate transaminase(SGOT) In our findings there was a distinct pattern ofincreased SGOT and SGPT levels in all the treatment groupstreated for 30 days In case of As

2O3(4 ppm) treatment

for 90 days however reversal in the activities of both theenzymes occurred almost reaching basal level The reasonfor such reversal is difficult to explain since histopathologicalalterations as well as induction of apoptosis was observed inthe same treatment group The increased SGOT and SGPTlevels in rat after 45 days treatment of 10 ppm sodium arsenitewas also described by Tandan et al [47] whereas Santra et al[45] reported increased SGOT and SGPT levels after 12months of arsenic treatment at 32 ppm in mice Liver alter-ation in histopathological architecture was evident mainly inthe form of loss of integrity of central vein loss of hepaticcord organization and dose and time dependent vacuolationwhich corroborates with earlier studies [45 48 49] Thesestudies indicate that chronic exposure to different inorganicarsenic compounds (arsenite arsenic trioxide or arsenate)produces characteristic pathology in the liver including fattyinfiltration liver degeneration inflammatory cell infiltrationand focal necrosis Das Neves et al [50] have shown that evenacute exposure of sodium arsenite at a dose of 10mgkg bodyweight (intraperitoneally) for only 90min could producevarious histopathological alterations in liver

In the present study Hoechst 33342 stained hepatocytesclearly indicated fragmented nuclei in all the treatment

groups as a mark of apoptosis as reported earlier in fish liver[17]

Oxidative stress is a relatively new theory in assessmentof arsenic toxicity [51 52] Arsenic causes oxidative stress byproducing reactive oxygen species [20 53 54] which damageproteins Due to lipophilic nature arsenic also attaches tolipid thereby increasing the rate of lipid peroxidation [55]Cellular GSH plays an important role in mitigating arsenicinduced oxidative stress Arsenic being a strong electrophilepredominantly binds with nucleophilic SH group of GSHThus consequent depletion of GSHmay alter the redox statusof the cell and present a stressful and toxic situation Accord-ing to Bashir et al [56] acute treatment of sodium arseniteat doses of 63mgkg 105mgkg and 126mgkg of bodyweight for 24 h significantly decreased GSH content in liverfor all doses tested On the other hand a significant increasein lipid peroxidation and glutathione peroxidase activityalongwith significant decrease in the activity of GST catalaseand superoxide dismutase was observed at 105mgkg and126mgkg of As treatment Santra et al [45] reported thatchronic exposure to 32 ppm of arsenic significantly depletedGSHafter 6months of exposure catalase activity significantlydecreased after 9 months and GST activity significantlyreduced at 12 and 15 months of arsenic exposure In ourstudy GSH level showed decreasing trend in all the 30 daystreatment groups with highest depletion recorded at 4 ppmof As2O3treatment for 30 days while after 90 days treatment

with the same dose the GSH level increased significantlywith respect to both control and also the 30 days treatmentgroupsThis shows the upregulation ofGSHagainst long termarsenic treatment which is due to its adaptive response tooxidative stress [20] However a reverse trend was observedin TBARS production and GST activity in liver in group II(04 ppm As

2O3treatment for 30 days) there was almost no

change in TBARS and GST level against the control groupwhile in group III and group IV levels of both TBARS andGST activity increased significantly Interestingly in groupV (90 days treatment with 4 ppm As

2O3) both TBARS and

GST level decreased significantly with respect to group IVthat is 4 ppm As

2O3for 30 days though the levels were still

significantly higher than the control group The increasedGST activity might be responsible for the reduction in GSHwhile protecting against the arsenic-induced oxidative stressCatalase activity increased significantly in group II mice withrespect to control while at subsequent higher doses that is2 ppm and 4 ppm As

2O3for 30 days the activity decreased

10 ISRN Hepatology

Increased level of SGOT and SGPT

Histopathological alterationsModulation in trace element levels

Increased production of p62

Increased Nrf2 level

Decreased activity of Keap1

Increased production of Hsp 70

Active Nrf2

Gene transcription

GST

GCS

GR

GSH

p62

Alteration in the activity of GST

Modulation in GSH level

Apoptotic induction

Increased production of MDA

p62

Keap1Keap1

ubub

ub

ubInactive Nrf2

Cytosol Nucleus

Nuclear localiztion

Nrf2Nrf2

Nrf2

ARE

Oxidative stress

As

120574GCS GR and catalase

Figure 8 Schematic representation of the arsenic induced liver damages and induction of gene expression through the Keap1-Nrf2-AREsignaling pathway

and highest rate of depletion was observed after 4 ppmAs2O3

treatment for 30 days Further in group V (4 ppm As2O3for

90 days) the recovery in the catalase activity was observedand it increased significantly when compared to group IV(4 ppmAs

2O3treatment for 30 days) According toMittal and

Flora [57] catalase activity significantly decreased in liver andkidney with respect to control after treatment with 100 ppmsodium meta-arsenite in drinking water for 8 weeks

Trace elements play an important role in maintainingnormal homeostasis of the body Oxidative stress results fromchanges in the levels of trace elements It has been reportedthat increased Mg level is associated with the increasedproduction ofMDA in dementia patients [58] Our result alsoshowed an increasing pattern ofMg concentration alongwithincrease in liver TBARS level

Zn is an essential element required for growth andnormal development and is a constituent of more than 200enzymes one of which is a CuZn superoxide dismutase(CuZn SOD) CuZn SOD is a powerful antioxidant whichtransforms free radical O

2

∙ to H2O2 therefore reducing the

risk of formation of highly reactive hydroxyl radical HO∙ Cuis also an integral part of CuZn SOD enzyme DecreasedZn and Cu levels in tissues may result in reduction of CuZnSOD activity and subsequently accelerate the process of cellaging and death via oxidative damage [59] However free orincorrectly bound Cu+2 can catalyze the generation of the

most damaging radicals such as HO∙ resulting in a chemicalmodification of the protein alterations in protein structureand solubility and oxidative damage to surrounding tissue[60] Our findings also indicate significant modulation of Cuand Zn level in liver of group II mice

Selenium (Se) is a well known antagonist of arsenic toxic-ity [61] The most probable cause of such a protective effectof selenium is due to its ability to upregulate antioxidantenzymes like GSH peroxidase and thioredoxin reductasewhich protects against arsenic induced oxidative damage[62] Decrement in selenium level after As

2O3treatment was

reported byMolin et al [63 64] In the present study Se levelin liver decreased dose dependently recording maximumdepletion at 4 ppm of As

2O3for 30 days but in the group

treated for 90 days with 4 ppm As2O3 Se level increased and

almost reached the normal levelIron (Fe) catalyses the formation of reactive oxygen

species through the Fenton and Haber-Weiss reactions [65]which generate highly toxic hydroxyl radical and cause lipidperoxidation [66] According to Ahmad et al [67] body ironstores (serum ferritin and transferrin saturation) in the bodycan be used as an early investigative tool for assessing theoxidative stress in coronary heart disease In our study ironlevel increased in all the treatment groups except in group Vwhere the iron concentration is almost same to that of thecontrol group

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

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Diabetes ResearchJournal of

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ISRN AIDS

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Computational and Mathematical Methods in Medicine

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Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

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OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

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ObesityJournal of

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PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

8 ISRN Hepatology

Nrf2

Keap1

GrI Gr II Gr III Gr IV Gr V

120573-Actin

(a)

Nrf2Keap1

GrI Gr II Gr III Gr IV Gr VTreatment groups

Relat

ive d

ensit

omet

ric v

alue

(fold

s of c

ontro

l)

30

25

20

15

10

05

00

(b)

Figure 6 (a) Nrf2 and Keap1 mRNA expressions in liver (b) Densitometric analysis of Nrf2 mRNA Keap1 mRNA in liver of mice treatedwith different doses of As

2O3

(a) (b) (c)

(d) (e) (f)

Figure 7 Mouse hepatocytes showing apoptosis (arrow) by Hoechst 33342 staining (a) (b) control (c) 04 ppm (d) 2 ppm and (e) 4 ppmAs2O3treatment for 30 days (f) 4 ppm arsenic trioxide treatment for 90 days

ISRN Hepatology 9

Table 1 Concentration of elements (mgkg) in whole-liver tissue following in vivo administration of different concentrations of As2O3 (ppm)in the drinking water of the mice for 30 and 90 days (Values are mean plusmn SEM)

Elements Group I Group II Group III Group IV Group VMg 35445 plusmn 5417 54729 plusmn 5888 67879 plusmn 2113

lowast57821 plusmn 4246

lowast46143 plusmn 2990

Cu 1541 plusmn 023 1749 plusmn 073lowast

1454 plusmn 072 1444 plusmn 047 1468 plusmn 059

Zn 10755 plusmn 157 9763 plusmn 397lowast

10377 plusmn 164 10762 plusmn 335 10226 plusmn 471

Se 1419 plusmn 134 1325 plusmn 055 1252 plusmn 189 843 plusmn 074lowast

1409 plusmn 113

Fe 50365 plusmn 3866 61029 plusmn 4599 56782 plusmn 2297 73421 plusmn 4494lowast

50689 plusmn 2822

lowastValues are statistically significant compared to control at (119875 lt 005)

found in ground water which is the most common sourceof drinking water The chronic exposure is the main causeof arsenic induced cancer development in human popula-tion and the mechanism of chronic arsenic toxicity is notfully known Therefore in our present experiment micewere treated with As through drinking water Accordingto Singh et al [44] arsenic administration decreased bodyweight gain in a dose dependant manner Santra et al [45]have reported significant increase in the liver weight ofBALBc mice after 12 months of arsenic treatment whereasin the present study there was no significant change in bodyweight gain rate of water consumption or the organ to bodyweight ratio of liver in any of the treatment groups

Liver is an important organ for various metabolic path-ways and effect of any chemical or xenobiotic appears pri-marily in the liver In case of arsenic metabolism liver is themain site of arsenic methylation [46]Themost commonwayof assertion of liver damage is to determine two pathophys-iological enzymes serum glutamate pyruvate transaminase(SGPT) and serum glutamate oxaloacetate transaminase(SGOT) In our findings there was a distinct pattern ofincreased SGOT and SGPT levels in all the treatment groupstreated for 30 days In case of As

2O3(4 ppm) treatment

for 90 days however reversal in the activities of both theenzymes occurred almost reaching basal level The reasonfor such reversal is difficult to explain since histopathologicalalterations as well as induction of apoptosis was observed inthe same treatment group The increased SGOT and SGPTlevels in rat after 45 days treatment of 10 ppm sodium arsenitewas also described by Tandan et al [47] whereas Santra et al[45] reported increased SGOT and SGPT levels after 12months of arsenic treatment at 32 ppm in mice Liver alter-ation in histopathological architecture was evident mainly inthe form of loss of integrity of central vein loss of hepaticcord organization and dose and time dependent vacuolationwhich corroborates with earlier studies [45 48 49] Thesestudies indicate that chronic exposure to different inorganicarsenic compounds (arsenite arsenic trioxide or arsenate)produces characteristic pathology in the liver including fattyinfiltration liver degeneration inflammatory cell infiltrationand focal necrosis Das Neves et al [50] have shown that evenacute exposure of sodium arsenite at a dose of 10mgkg bodyweight (intraperitoneally) for only 90min could producevarious histopathological alterations in liver

In the present study Hoechst 33342 stained hepatocytesclearly indicated fragmented nuclei in all the treatment

groups as a mark of apoptosis as reported earlier in fish liver[17]

Oxidative stress is a relatively new theory in assessmentof arsenic toxicity [51 52] Arsenic causes oxidative stress byproducing reactive oxygen species [20 53 54] which damageproteins Due to lipophilic nature arsenic also attaches tolipid thereby increasing the rate of lipid peroxidation [55]Cellular GSH plays an important role in mitigating arsenicinduced oxidative stress Arsenic being a strong electrophilepredominantly binds with nucleophilic SH group of GSHThus consequent depletion of GSHmay alter the redox statusof the cell and present a stressful and toxic situation Accord-ing to Bashir et al [56] acute treatment of sodium arseniteat doses of 63mgkg 105mgkg and 126mgkg of bodyweight for 24 h significantly decreased GSH content in liverfor all doses tested On the other hand a significant increasein lipid peroxidation and glutathione peroxidase activityalongwith significant decrease in the activity of GST catalaseand superoxide dismutase was observed at 105mgkg and126mgkg of As treatment Santra et al [45] reported thatchronic exposure to 32 ppm of arsenic significantly depletedGSHafter 6months of exposure catalase activity significantlydecreased after 9 months and GST activity significantlyreduced at 12 and 15 months of arsenic exposure In ourstudy GSH level showed decreasing trend in all the 30 daystreatment groups with highest depletion recorded at 4 ppmof As2O3treatment for 30 days while after 90 days treatment

with the same dose the GSH level increased significantlywith respect to both control and also the 30 days treatmentgroupsThis shows the upregulation ofGSHagainst long termarsenic treatment which is due to its adaptive response tooxidative stress [20] However a reverse trend was observedin TBARS production and GST activity in liver in group II(04 ppm As

2O3treatment for 30 days) there was almost no

change in TBARS and GST level against the control groupwhile in group III and group IV levels of both TBARS andGST activity increased significantly Interestingly in groupV (90 days treatment with 4 ppm As

2O3) both TBARS and

GST level decreased significantly with respect to group IVthat is 4 ppm As

2O3for 30 days though the levels were still

significantly higher than the control group The increasedGST activity might be responsible for the reduction in GSHwhile protecting against the arsenic-induced oxidative stressCatalase activity increased significantly in group II mice withrespect to control while at subsequent higher doses that is2 ppm and 4 ppm As

2O3for 30 days the activity decreased

10 ISRN Hepatology

Increased level of SGOT and SGPT

Histopathological alterationsModulation in trace element levels

Increased production of p62

Increased Nrf2 level

Decreased activity of Keap1

Increased production of Hsp 70

Active Nrf2

Gene transcription

GST

GCS

GR

GSH

p62

Alteration in the activity of GST

Modulation in GSH level

Apoptotic induction

Increased production of MDA

p62

Keap1Keap1

ubub

ub

ubInactive Nrf2

Cytosol Nucleus

Nuclear localiztion

Nrf2Nrf2

Nrf2

ARE

Oxidative stress

As

120574GCS GR and catalase

Figure 8 Schematic representation of the arsenic induced liver damages and induction of gene expression through the Keap1-Nrf2-AREsignaling pathway

and highest rate of depletion was observed after 4 ppmAs2O3

treatment for 30 days Further in group V (4 ppm As2O3for

90 days) the recovery in the catalase activity was observedand it increased significantly when compared to group IV(4 ppmAs

2O3treatment for 30 days) According toMittal and

Flora [57] catalase activity significantly decreased in liver andkidney with respect to control after treatment with 100 ppmsodium meta-arsenite in drinking water for 8 weeks

Trace elements play an important role in maintainingnormal homeostasis of the body Oxidative stress results fromchanges in the levels of trace elements It has been reportedthat increased Mg level is associated with the increasedproduction ofMDA in dementia patients [58] Our result alsoshowed an increasing pattern ofMg concentration alongwithincrease in liver TBARS level

Zn is an essential element required for growth andnormal development and is a constituent of more than 200enzymes one of which is a CuZn superoxide dismutase(CuZn SOD) CuZn SOD is a powerful antioxidant whichtransforms free radical O

2

∙ to H2O2 therefore reducing the

risk of formation of highly reactive hydroxyl radical HO∙ Cuis also an integral part of CuZn SOD enzyme DecreasedZn and Cu levels in tissues may result in reduction of CuZnSOD activity and subsequently accelerate the process of cellaging and death via oxidative damage [59] However free orincorrectly bound Cu+2 can catalyze the generation of the

most damaging radicals such as HO∙ resulting in a chemicalmodification of the protein alterations in protein structureand solubility and oxidative damage to surrounding tissue[60] Our findings also indicate significant modulation of Cuand Zn level in liver of group II mice

Selenium (Se) is a well known antagonist of arsenic toxic-ity [61] The most probable cause of such a protective effectof selenium is due to its ability to upregulate antioxidantenzymes like GSH peroxidase and thioredoxin reductasewhich protects against arsenic induced oxidative damage[62] Decrement in selenium level after As

2O3treatment was

reported byMolin et al [63 64] In the present study Se levelin liver decreased dose dependently recording maximumdepletion at 4 ppm of As

2O3for 30 days but in the group

treated for 90 days with 4 ppm As2O3 Se level increased and

almost reached the normal levelIron (Fe) catalyses the formation of reactive oxygen

species through the Fenton and Haber-Weiss reactions [65]which generate highly toxic hydroxyl radical and cause lipidperoxidation [66] According to Ahmad et al [67] body ironstores (serum ferritin and transferrin saturation) in the bodycan be used as an early investigative tool for assessing theoxidative stress in coronary heart disease In our study ironlevel increased in all the treatment groups except in group Vwhere the iron concentration is almost same to that of thecontrol group

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

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ISRN AIDS

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Computational and Mathematical Methods in Medicine

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Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

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Volume 2013

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

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Oxidative Medicine and Cellular Longevity

ISRN Addiction

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International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

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ObesityJournal of

ISRN Allergy

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PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Hepatology 9

Table 1 Concentration of elements (mgkg) in whole-liver tissue following in vivo administration of different concentrations of As2O3 (ppm)in the drinking water of the mice for 30 and 90 days (Values are mean plusmn SEM)

Elements Group I Group II Group III Group IV Group VMg 35445 plusmn 5417 54729 plusmn 5888 67879 plusmn 2113

lowast57821 plusmn 4246

lowast46143 plusmn 2990

Cu 1541 plusmn 023 1749 plusmn 073lowast

1454 plusmn 072 1444 plusmn 047 1468 plusmn 059

Zn 10755 plusmn 157 9763 plusmn 397lowast

10377 plusmn 164 10762 plusmn 335 10226 plusmn 471

Se 1419 plusmn 134 1325 plusmn 055 1252 plusmn 189 843 plusmn 074lowast

1409 plusmn 113

Fe 50365 plusmn 3866 61029 plusmn 4599 56782 plusmn 2297 73421 plusmn 4494lowast

50689 plusmn 2822

lowastValues are statistically significant compared to control at (119875 lt 005)

found in ground water which is the most common sourceof drinking water The chronic exposure is the main causeof arsenic induced cancer development in human popula-tion and the mechanism of chronic arsenic toxicity is notfully known Therefore in our present experiment micewere treated with As through drinking water Accordingto Singh et al [44] arsenic administration decreased bodyweight gain in a dose dependant manner Santra et al [45]have reported significant increase in the liver weight ofBALBc mice after 12 months of arsenic treatment whereasin the present study there was no significant change in bodyweight gain rate of water consumption or the organ to bodyweight ratio of liver in any of the treatment groups

Liver is an important organ for various metabolic path-ways and effect of any chemical or xenobiotic appears pri-marily in the liver In case of arsenic metabolism liver is themain site of arsenic methylation [46]Themost commonwayof assertion of liver damage is to determine two pathophys-iological enzymes serum glutamate pyruvate transaminase(SGPT) and serum glutamate oxaloacetate transaminase(SGOT) In our findings there was a distinct pattern ofincreased SGOT and SGPT levels in all the treatment groupstreated for 30 days In case of As

2O3(4 ppm) treatment

for 90 days however reversal in the activities of both theenzymes occurred almost reaching basal level The reasonfor such reversal is difficult to explain since histopathologicalalterations as well as induction of apoptosis was observed inthe same treatment group The increased SGOT and SGPTlevels in rat after 45 days treatment of 10 ppm sodium arsenitewas also described by Tandan et al [47] whereas Santra et al[45] reported increased SGOT and SGPT levels after 12months of arsenic treatment at 32 ppm in mice Liver alter-ation in histopathological architecture was evident mainly inthe form of loss of integrity of central vein loss of hepaticcord organization and dose and time dependent vacuolationwhich corroborates with earlier studies [45 48 49] Thesestudies indicate that chronic exposure to different inorganicarsenic compounds (arsenite arsenic trioxide or arsenate)produces characteristic pathology in the liver including fattyinfiltration liver degeneration inflammatory cell infiltrationand focal necrosis Das Neves et al [50] have shown that evenacute exposure of sodium arsenite at a dose of 10mgkg bodyweight (intraperitoneally) for only 90min could producevarious histopathological alterations in liver

In the present study Hoechst 33342 stained hepatocytesclearly indicated fragmented nuclei in all the treatment

groups as a mark of apoptosis as reported earlier in fish liver[17]

Oxidative stress is a relatively new theory in assessmentof arsenic toxicity [51 52] Arsenic causes oxidative stress byproducing reactive oxygen species [20 53 54] which damageproteins Due to lipophilic nature arsenic also attaches tolipid thereby increasing the rate of lipid peroxidation [55]Cellular GSH plays an important role in mitigating arsenicinduced oxidative stress Arsenic being a strong electrophilepredominantly binds with nucleophilic SH group of GSHThus consequent depletion of GSHmay alter the redox statusof the cell and present a stressful and toxic situation Accord-ing to Bashir et al [56] acute treatment of sodium arseniteat doses of 63mgkg 105mgkg and 126mgkg of bodyweight for 24 h significantly decreased GSH content in liverfor all doses tested On the other hand a significant increasein lipid peroxidation and glutathione peroxidase activityalongwith significant decrease in the activity of GST catalaseand superoxide dismutase was observed at 105mgkg and126mgkg of As treatment Santra et al [45] reported thatchronic exposure to 32 ppm of arsenic significantly depletedGSHafter 6months of exposure catalase activity significantlydecreased after 9 months and GST activity significantlyreduced at 12 and 15 months of arsenic exposure In ourstudy GSH level showed decreasing trend in all the 30 daystreatment groups with highest depletion recorded at 4 ppmof As2O3treatment for 30 days while after 90 days treatment

with the same dose the GSH level increased significantlywith respect to both control and also the 30 days treatmentgroupsThis shows the upregulation ofGSHagainst long termarsenic treatment which is due to its adaptive response tooxidative stress [20] However a reverse trend was observedin TBARS production and GST activity in liver in group II(04 ppm As

2O3treatment for 30 days) there was almost no

change in TBARS and GST level against the control groupwhile in group III and group IV levels of both TBARS andGST activity increased significantly Interestingly in groupV (90 days treatment with 4 ppm As

2O3) both TBARS and

GST level decreased significantly with respect to group IVthat is 4 ppm As

2O3for 30 days though the levels were still

significantly higher than the control group The increasedGST activity might be responsible for the reduction in GSHwhile protecting against the arsenic-induced oxidative stressCatalase activity increased significantly in group II mice withrespect to control while at subsequent higher doses that is2 ppm and 4 ppm As

2O3for 30 days the activity decreased

10 ISRN Hepatology

Increased level of SGOT and SGPT

Histopathological alterationsModulation in trace element levels

Increased production of p62

Increased Nrf2 level

Decreased activity of Keap1

Increased production of Hsp 70

Active Nrf2

Gene transcription

GST

GCS

GR

GSH

p62

Alteration in the activity of GST

Modulation in GSH level

Apoptotic induction

Increased production of MDA

p62

Keap1Keap1

ubub

ub

ubInactive Nrf2

Cytosol Nucleus

Nuclear localiztion

Nrf2Nrf2

Nrf2

ARE

Oxidative stress

As

120574GCS GR and catalase

Figure 8 Schematic representation of the arsenic induced liver damages and induction of gene expression through the Keap1-Nrf2-AREsignaling pathway

and highest rate of depletion was observed after 4 ppmAs2O3

treatment for 30 days Further in group V (4 ppm As2O3for

90 days) the recovery in the catalase activity was observedand it increased significantly when compared to group IV(4 ppmAs

2O3treatment for 30 days) According toMittal and

Flora [57] catalase activity significantly decreased in liver andkidney with respect to control after treatment with 100 ppmsodium meta-arsenite in drinking water for 8 weeks

Trace elements play an important role in maintainingnormal homeostasis of the body Oxidative stress results fromchanges in the levels of trace elements It has been reportedthat increased Mg level is associated with the increasedproduction ofMDA in dementia patients [58] Our result alsoshowed an increasing pattern ofMg concentration alongwithincrease in liver TBARS level

Zn is an essential element required for growth andnormal development and is a constituent of more than 200enzymes one of which is a CuZn superoxide dismutase(CuZn SOD) CuZn SOD is a powerful antioxidant whichtransforms free radical O

2

∙ to H2O2 therefore reducing the

risk of formation of highly reactive hydroxyl radical HO∙ Cuis also an integral part of CuZn SOD enzyme DecreasedZn and Cu levels in tissues may result in reduction of CuZnSOD activity and subsequently accelerate the process of cellaging and death via oxidative damage [59] However free orincorrectly bound Cu+2 can catalyze the generation of the

most damaging radicals such as HO∙ resulting in a chemicalmodification of the protein alterations in protein structureand solubility and oxidative damage to surrounding tissue[60] Our findings also indicate significant modulation of Cuand Zn level in liver of group II mice

Selenium (Se) is a well known antagonist of arsenic toxic-ity [61] The most probable cause of such a protective effectof selenium is due to its ability to upregulate antioxidantenzymes like GSH peroxidase and thioredoxin reductasewhich protects against arsenic induced oxidative damage[62] Decrement in selenium level after As

2O3treatment was

reported byMolin et al [63 64] In the present study Se levelin liver decreased dose dependently recording maximumdepletion at 4 ppm of As

2O3for 30 days but in the group

treated for 90 days with 4 ppm As2O3 Se level increased and

almost reached the normal levelIron (Fe) catalyses the formation of reactive oxygen

species through the Fenton and Haber-Weiss reactions [65]which generate highly toxic hydroxyl radical and cause lipidperoxidation [66] According to Ahmad et al [67] body ironstores (serum ferritin and transferrin saturation) in the bodycan be used as an early investigative tool for assessing theoxidative stress in coronary heart disease In our study ironlevel increased in all the treatment groups except in group Vwhere the iron concentration is almost same to that of thecontrol group

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

of

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN AIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Computational and Mathematical Methods in Medicine

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Biomarkers

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Anesthesiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ObesityJournal of

ISRN Allergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

10 ISRN Hepatology

Increased level of SGOT and SGPT

Histopathological alterationsModulation in trace element levels

Increased production of p62

Increased Nrf2 level

Decreased activity of Keap1

Increased production of Hsp 70

Active Nrf2

Gene transcription

GST

GCS

GR

GSH

p62

Alteration in the activity of GST

Modulation in GSH level

Apoptotic induction

Increased production of MDA

p62

Keap1Keap1

ubub

ub

ubInactive Nrf2

Cytosol Nucleus

Nuclear localiztion

Nrf2Nrf2

Nrf2

ARE

Oxidative stress

As

120574GCS GR and catalase

Figure 8 Schematic representation of the arsenic induced liver damages and induction of gene expression through the Keap1-Nrf2-AREsignaling pathway

and highest rate of depletion was observed after 4 ppmAs2O3

treatment for 30 days Further in group V (4 ppm As2O3for

90 days) the recovery in the catalase activity was observedand it increased significantly when compared to group IV(4 ppmAs

2O3treatment for 30 days) According toMittal and

Flora [57] catalase activity significantly decreased in liver andkidney with respect to control after treatment with 100 ppmsodium meta-arsenite in drinking water for 8 weeks

Trace elements play an important role in maintainingnormal homeostasis of the body Oxidative stress results fromchanges in the levels of trace elements It has been reportedthat increased Mg level is associated with the increasedproduction ofMDA in dementia patients [58] Our result alsoshowed an increasing pattern ofMg concentration alongwithincrease in liver TBARS level

Zn is an essential element required for growth andnormal development and is a constituent of more than 200enzymes one of which is a CuZn superoxide dismutase(CuZn SOD) CuZn SOD is a powerful antioxidant whichtransforms free radical O

2

∙ to H2O2 therefore reducing the

risk of formation of highly reactive hydroxyl radical HO∙ Cuis also an integral part of CuZn SOD enzyme DecreasedZn and Cu levels in tissues may result in reduction of CuZnSOD activity and subsequently accelerate the process of cellaging and death via oxidative damage [59] However free orincorrectly bound Cu+2 can catalyze the generation of the

most damaging radicals such as HO∙ resulting in a chemicalmodification of the protein alterations in protein structureand solubility and oxidative damage to surrounding tissue[60] Our findings also indicate significant modulation of Cuand Zn level in liver of group II mice

Selenium (Se) is a well known antagonist of arsenic toxic-ity [61] The most probable cause of such a protective effectof selenium is due to its ability to upregulate antioxidantenzymes like GSH peroxidase and thioredoxin reductasewhich protects against arsenic induced oxidative damage[62] Decrement in selenium level after As

2O3treatment was

reported byMolin et al [63 64] In the present study Se levelin liver decreased dose dependently recording maximumdepletion at 4 ppm of As

2O3for 30 days but in the group

treated for 90 days with 4 ppm As2O3 Se level increased and

almost reached the normal levelIron (Fe) catalyses the formation of reactive oxygen

species through the Fenton and Haber-Weiss reactions [65]which generate highly toxic hydroxyl radical and cause lipidperoxidation [66] According to Ahmad et al [67] body ironstores (serum ferritin and transferrin saturation) in the bodycan be used as an early investigative tool for assessing theoxidative stress in coronary heart disease In our study ironlevel increased in all the treatment groups except in group Vwhere the iron concentration is almost same to that of thecontrol group

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

of

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN AIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Computational and Mathematical Methods in Medicine

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Biomarkers

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Anesthesiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ObesityJournal of

ISRN Allergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Hepatology 11

Heat shock proteins (Hsps) are expressed in tissues inresponse to a harmful stress situation or adverse life con-ditions Roy and Bhattacharya [17] first reported aboutincreased Hsp 70 in the liver of Channa punctatus a freshwater teleost Similar induction of Hsp 70 protein was alsofound in the present study in all the treatment groups whichcorroborates earlier finding

Mammalian cells cope with xenobiotics by adaptivedefense mechanisms to maintain cellular homeostasis andphysiological functions [20] Nrf2Keap1ARE driven targetgene system is one such mechanism [28] In a recent studyLi et al [20] reported induction of Nrf2 protein in Changhuman hepatocytes They observed that 5 and 10 120583molL ofsodiumarsenite increased theNrf2 protein levels significantlyat 6 and 12 h and a decrease thereafter They also observedthat 5 to 25 120583molL of arsenic could increase the Nrf2 proteinlevels significantly but 50 120583molL of arsenic did not havesimilar effect They opined that this resulted due to thecytotoxicity caused at higher dose of arsenic

Our observation on the Nrf2 protein induction also didnot show a pattern of consistent increase within 30 daystreatment groups The maximum induction was observed at04 ppm arsenic treatment which decreased thereafter Nrf2protein level after 30 days treatment with 4 ppm of arsenicwas higher than that of 90 days treatment group supportingthe theory of adaptive response mechanism [20]The patternof Nrf2 mRNA level also matched with the correspondingprotein level We also monitored the expression patterns oftwo important proteins Keap1 and p62 which are closelyrelated to the expression and transfer of Nrf2 protein fromcytoplasm to nucleus and the activation of ARE driven geneslike 120574GCS GR and GST involved in glutathionemetabolismKeap1 (Kelch-like ECH-associated protein 1) plays a vitalrole in the localization of Nrf2 protein within cytoplasmby binding and thereby inhibiting the Nrf2 activity andp62 plays its role by docking the Keap1 protein through amotif called Keap1 interacting region (KIR) thereby blockingbinding between Keap1 and Nrf2 As a result Nrf2 protein isseparated from Keap1 following its migration to the nucleusInterestingly induction of p62 results from oxidative stressand is mediated by Nrf2 which binds to the ARE containingcis-element of p62Therefore p62SQSTM1 (sequestosome 1)is a target gene for Nrf2 which creates a positive feedbackloop by inducing ARE driven gene transcription [68] In thepresent study we report synchronization of Keap1 and p62levels with the Nrf2 protein level accompanied by inductionof the downstream genes GCS GR and GST involved inGSH metabolic pathway The role of Nrf2 signaling pathwayin cellular protection after arsenic exposure is schematicallyrepresented in the Figure 8 To the best of our knowledgethis is the first report on the effect of arsenic on Keap1-P62-Nrf2 signaling pathway with respect to expression of the AREdriven genes related to GSH metabolic pathway in mouseliver in vivo The expression pattern of the GCS GR andGST are in agreement with the Nrf2 mediated antioxidativeand adaptive response mechanisms against arsenic induceddamages in liver

5 Conclusion

The present study clearly indicates that treatment of arsenictrioxide through drinkingwater inmice in vivo induces hepa-totoxicity as evidenced by oxidative stress and histopathologi-cal changes with concomitant effect on normal liver functionand activates the Keap1p62Nrf2 signaling pathway leadingto activation of downstream ARE driven genes related toGSHmetabolism involved in protecting cells against arsenicinduced oxidative stress and activates the adaptive responsemechanism The alterations in the expressions of the ARE-driven genes might help in understanding the mechanism ofchronic arsenic induced hepatotoxicity in mammals includ-ing human beings facing serious threats in severe arsenicendemic regions all over the world

Conflict of Interests

The authors declare that there are no conflict of interests

Acknowledgments

The authors are grateful to University Grants Commission(UGC) for the support under Centre for Advanced Studies(CAS) Scheme Phase II and UGC-Department of AtomicEnergy (DAE)-Consortium for Scientific Research (CSR)KolkataCentre for partially funding the presentwork (ProjectSanction no UGC-DAE-CSR-KCCRS2009TE-061544 toAC) Ritu Srivastava is grateful to UGC-DAE-CSR Kolkatafor research fellowship Archya Sengupta is thankful to WestBengal State Council for Science and Technology (WBDST)Kolkata for research fellowship Sandip Mukherjee grate-fully acknowledges UGC for meritorious fellowship andSarmishtha Chatterjee thankfully acknowledges NationalAcademyof Science India (NASI) for Senior Research Fellow-ship Professor Shelley Bhattacharya gratefully acknowledgesNASI for the Senior Scientist Platinum Jubilee Fellowship

References

[1] P B Tchounwou A K Patlolla and J A Centeno ldquoCarcin-ogenic and systemic health effects associated with arsenicexposure a critical reviewrdquo Toxicologic Pathology vol 31 no 6pp 575ndash588 2003

[2] J Fu C G Woods E Yehuda-Shnaidman et al ldquoLow-levelarsenic impairs glucose-stimulated insulin secretion in pan-creatic beta cells involvement of cellular adaptive response tooxidative stressrdquo Environmental Health Perspectives vol 118 no6 pp 864ndash870 2010

[3] G Sun X Li J Pi et al ldquoCurrent research problems of chronicarsenicosis in Chinardquo Journal of Health Population and Nutri-tion vol 24 no 2 pp 176ndash181 2006

[4] G Sun Y Xu X Li Y Jin B Li and X Sun ldquoUrinaryarsenic metabolites in children and adults exposed to arsenicin drinking water in inner Mongolia Chinardquo EnvironmentalHealth Perspectives vol 115 no 4 pp 648ndash652 2007

[5] D N G Mazumder R Haque N Ghosh et al ldquoArsenic levelsin drinking water and the prevalence of skin lesions in WestBengal Indiardquo International Journal of Epidemiology vol 27 no5 pp 871ndash877 1998

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

of

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN AIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Computational and Mathematical Methods in Medicine

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Biomarkers

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Anesthesiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ObesityJournal of

ISRN Allergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

12 ISRN Hepatology

[6] World Health Organization International Agency for Researchon Cancer Some Metals and Metallic Compounds IARC Mono-graphs on the Evaluation of Carcinogenic Risk of Chemicals toMan vol 23 WHO Press IARC Lyon France 1980

[7] ZWang and T G Rossman ldquoThe carcinogenicity of arsenicrdquo inToxicology of Metals W C Louis Ed pp 219ndash227 CRC PressBoca Raton Fla USA 1996

[8] H Tinwell S C Stephens and J Ashby ldquoArsenite as theprobable active species in the human carcinogenicity of arsenicmouse micronucleus assays on Na and K arsenite orpimentand Fowlerrsquos solutionrdquo Environmental Health Perspectives vol95 pp 205ndash210 1991

[9] J Liu andM PWaalkes ldquoLiver is a target of arsenic carcinogen-esisrdquo Toxicological Sciences vol 105 no 1 pp 24ndash32 2008

[10] J Liu L Yu E J Tokar et al ldquoArsenic-induced aberrant geneexpression in fetal mouse primary liver-cell culturesrdquo Annals ofthe New York Academy of Sciences vol 1140 pp 368ndash375 2008

[11] J Wu J Liu M P Waalkes et al ldquoHigh dietary fat exacerbatesarsenic-induced liver fibrosis in micerdquo Experimental Biologyand Medicine vol 233 no 3 pp 377ndash384 2008

[12] T Jiang Z Huang J Y Chan and D D Zhang ldquoNrf2 protectsagainst As(III)-induced damage in mouse liver and bladderrdquoToxicology and Applied Pharmacology vol 240 no 1 pp 8ndash142009

[13] National Research Council and National Academy of SciencesArsenic in Drinking Water National Academy Press Washing-ton DC USA 1999

[14] D N G Mazumder ldquoEffect of chronic intake of arsenic-contaminated water on liverrdquo Toxicology and Applied Pharma-cology vol 206 no 2 pp 169ndash175 2005

[15] Y Xu Y Wang Q Zheng et al ldquoClinical manifestations andarsenic methylation after a rare subacute arsenic poisoningaccidentrdquo Toxicological Sciences vol 103 no 2 pp 278ndash2842008

[16] A Santra J Das Gupta B K De B Roy and D N GMazumder ldquoHepaticmanifestations in chronic arsenic toxicityrdquoIndian Journal of Gastroenterology vol 18 no 4 pp 152ndash1551999

[17] S Roy and S Bhattacharya ldquoArsenic-induced histopathologyand synthesis of stress proteins in liver and kidney of Channapunctatusrdquo Ecotoxicology and Environmental Safety vol 65 no2 pp 218ndash229 2006

[18] H Shi X Shi and K J Liu ldquoOxidative mechanism of arsenictoxicity and carcinogenesisrdquo Molecular and Cellular Biochem-istry vol 255 no 1-2 pp 67ndash78 2004

[19] S Das A Santra S Lahiri and D N Guha Mazumder ldquoImpli-cations of oxidative stress and hepatic cytokine (TNF-120572 andIL-6) response in the pathogenesis of hepatic collagenesis inchronic arsenic toxicityrdquo Toxicology and Applied Pharmacologyvol 204 no 1 pp 18ndash26 2005

[20] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 13 no 4 2011

[21] K Jomova Z Jenisova M Feszterova et al ldquoArsenic toxicityoxidative stress and human diseaserdquo Journal of Applied Toxicol-ogy vol 31 no 2 pp 95ndash107 2011

[22] M Delnomdedieu M M Basti J D Otvos and D J ThomasldquoReduction and binding of arsenate and dimethylarsinate byglutathione a magnetic resonance studyrdquo Chemico-BiologicalInteractions vol 90 no 2 pp 139ndash155 1994

[23] M F Hughes ldquoArsenic toxicity and potential mechanisms ofactionrdquo Toxicology Letters vol 133 no 1 pp 1ndash16 2002

[24] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[25] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[26] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[27] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[28] X He M G Chen G X Lin and Q Ma ldquoArsenic inducesNAD(P)H-quinone oxidoreductase I by disrupting the Nrf2times Keap1 times Cul3 complex and recruiting Nrf2 times Maf to theantioxidant response element enhancerrdquoThe Journal of Biologi-cal Chemistry vol 281 no 33 pp 23620ndash23631 2006

[29] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[30] X-J Wang Z Sun W Chen Y Li N F Villeneuve and D DZhang ldquoActivation of Nrf2 by arsenite and monomethylarson-ous acid is independent of Keap1-C151 enhanced Keap1-Cul3interactionrdquo Toxicology and Applied Pharmacology vol 230 no3 pp 383ndash389 2008

[31] H Endo Y Sugioka Y Nakagi Y Saijo and T Yoshida ldquoAnovel role of the NRF2 transcription factor in the regulationof arsenite-mediated keratin 16 gene expression in humankeratinocytesrdquo Environmental Health Perspectives vol 116 no7 pp 873ndash879 2008

[32] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[33] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[34] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[35] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology S Fleisher and L Packer Eds pp 302ndash310 Academic Press New York NY USA 1978

[36] H Aebi ldquoCatalase in vitrordquo in Methods in Enzymology LPacker Ed pp 121ndash126 Academic Press Orlando Fla USA1984

[37] N Kawamura ldquoCatalaserdquo in Experimental Protocols for ReactiveOxygen and Nitrogen Species J M C Gutteridge and NTaniguchi Eds pp 77ndash78 Oxford University Press New YorkNY USA 1999

[38] A Chattopadhyay S Podder S Agarwal and S BhattacharyaldquoFluoride-induced histopathology and synthesis of stress pro-tein in liver and kidney of micerdquo Archives of Toxicology vol 85no 4 pp 327ndash335 2011

[39] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

of

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN AIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Computational and Mathematical Methods in Medicine

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Biomarkers

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Anesthesiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ObesityJournal of

ISRN Allergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Hepatology 13

[40] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004

[41] K Shimano M Satake A Okaya et al ldquoHepatic oval cells havethe side population phenotype defined by expression of ATP-binding cassette transporter ABCG2BCRP1rdquoAmerican Journalof Pathology vol 163 no 1 pp 3ndash9 2003

[42] J Aono T Yanagawa K Itoh et al ldquoActivation of Nrf2 andaccumulation of ubiquitinated A170 by arsenic in osteoblastsrdquoBiochemical and Biophysical Research Communications vol 305no 2 pp 271ndash277 2003

[43] H Harada R Sugimoto A Watanabe et al ldquoDifferentialroles for Nrf2 and AP-1 in upregulation of HO-1 expressionby arsenite in murine embryonic fibroblastsrdquo Free RadicalResearch vol 42 no 4 pp 297ndash304 2008

[44] N Singh D Kumar K Lal S Raisuddin and A P SahuldquoAdverse health effects due to arsenic exposure modificationby dietary supplementation of jaggery in micerdquo Toxicology andApplied Pharmacology vol 242 no 3 pp 247ndash255 2010

[45] A Santra A Maiti S Das S Lahiri S K Charkaborty andD N Guha Mazumder ldquoHepatic damage caused by chronicarsenic toxicity in experimental animalsrdquo Journal of Toxicologyvol 38 no 4 pp 395ndash405 2000

[46] Z Drobna F S Walton D S Paul W Xing D J Thomas andM Styblo ldquoMetabolism of arsenic in human liver the role ofmembrane transportersrdquo Archives of Toxicology vol 84 pp 3ndash16 2010

[47] N Tandan M Roy and S Roy ldquoAmeliorative potential ofPsidium guajava on hemato-biochemical alterations in arsenic-exposed wistar ratsrdquo Toxicology International vol 19 pp 121ndash124 2012

[48] S J S Flora S C Pant P R Malhotra and GM Kannan ldquoBio-chemical and histopathological changes in arsenic-intoxicatedrats coexposed to ethanolrdquo Alcohol vol 14 no 6 pp 563ndash5681997

[49] R Ferzand J A Gadahi S Saleha and Q Ali ldquoHistological andhaematological disturbance caused by arsenic toxicity in micemodelrdquo Pakistan Journal of Biological Sciences vol 11 no 11 pp1405ndash1413 2008

[50] R N P Das Neves F Carvalho M Carvalho et al ldquoProtectiveactivity of hesperidin and lipoic acid against sodium arseniteacute toxicity in micerdquo Toxicologic Pathology vol 32 no 5 pp527ndash535 2004

[51] K T Kitchin ldquoRecent advances in arsenic carcinogenesismodes of action animalmodel systems andmethylated arsenicmetabolitesrdquo Toxicology and Applied Pharmacology vol 172 no3 pp 249ndash261 2001

[52] S J S Flora S Bhadauria S C Pant andR KDhaked ldquoArsenicinduced blood and brain oxidative stress and its response tosome thiol chelators in ratsrdquo Life Sciences vol 77 no 18 pp2324ndash2337 2005

[53] S J S Flora S Bhadauria GMKannan andN Singh ldquoArsenicinduced oxidative stress and the role of antioxidant supple-mentation during chelation a reviewrdquo Journal of EnvironmentalBiology vol 28 no 2 pp 333ndash347 2007

[54] M E Vahter ldquoInteractions between arsenic-induced toxicityand nutrition in early liferdquo Journal of Nutrition vol 137 no 12pp 2798ndash2804 2007

[55] E O Farombi O A Adelowo and Y R Ajimoko ldquoBiomarkersof oxidative stress and heavy metal levels as indicators of

environmental pollution in African cat fish (Clarias gariepinus)fromNigeria Ogun Riverrdquo International Journal of Environmen-tal Research and Public Health vol 4 no 2 pp 158ndash165 2007

[56] S Bashir Y Sharma M Irshad S D Gupta and T D DograldquoArsenic-induced cell death in liver and brain of experimentalratsrdquo Basic and Clinical Pharmacology and Toxicology vol 98no 1 pp 38ndash43 2006

[57] M Mittal and S J S Flora ldquoEffects of individual and combinedexposure to sodium arsenite and sodium fluoride on tissue oxi-dative stress arsenic and fluoride levels inmalemicerdquoChemico-Biological Interactions vol 162 no 2 pp 128ndash139 2006

[58] C-H Guo W-S Ko P-C Chen G-S W Hsu C-Y Lin andC-L Wang ldquoAlterations in trace elements and oxidative stressin uremic patients with dementiardquo Biological Trace ElementResearch vol 131 no 1 pp 13ndash24 2009

[59] Z Y Zhang N Q Liu F L Li et al ldquoCharacterization ofFe Cu and Zn in organs of PDAPP transgenic mice by XRFspectrometryrdquo X-Ray Spectrometry vol 35 no 4 pp 253ndash2562006

[60] T Kowalik-Jankowska M Ruta-Dolejsz K Wisniewska LLankiewicz and H Kozlowski ldquoPossible involvement of Cop-per(II) in Alzheimer diseaserdquo Environmental Health Perspec-tives vol 110 no 5 pp 869ndash870 2002

[61] O A Levander ldquoMetabolic interrelationships between arsenicand seleniumrdquo Environmental Health Perspectives vol 19 pp159ndash164 1977

[62] T G Rossman and A N Uddin ldquoSelenium prevents spon-taneous and arsenite-induced mutagenesisrdquo InternationalCongress Series vol 1275 pp 173ndash179 2004

[63] Y Molin P Frisk and N-G Ilback ldquoSequential effects of dailyarsenic trioxide treatment on essential and nonessential traceelements in tissues in micerdquo Anti-Cancer Drugs vol 19 no 8pp 812ndash818 2008

[64] Y Molin P Frisk and N-G Ilback ldquoArsenic trioxide affects thetrace element balance in tissues in infected and healthy micedifferentlyrdquo Anticancer Research vol 29 no 1 pp 83ndash90 2009

[65] M Haidari E Javadi A Sanati M Hajilooi and J GhanbilildquoAssociation of increased ferritin with premature coronarystenosis in menrdquo Clinical Chemistry vol 47 no 9 pp 1666ndash1672 2001

[66] Z Durackova L Bergendi A Liptakova and J Muchova ldquoFreeradicals derived from oxygen andmedicinerdquo BratislavaMedicalJournal vol 94 pp 419ndash434 1993

[67] M Ahmad M A Khan and A S Khan ldquoOxidative stress andlevel of-iron indices in coronary heart disease patientsrdquo Journalof Ayub Medical College Abbottabad vol 21 no 2 pp 56ndash592009

[68] A Jain T Lamark E Sjoslashttem et al ldquop62SQSTM1 is a targetgene for transcription factor NRF2 and creates a positivefeedback loop by inducing antioxidant response element-drivengene transcriptionrdquo Journal of Biological Chemistry vol 285 no29 pp 22576ndash22591 2010

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

of

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN AIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Computational and Mathematical Methods in Medicine

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Biomarkers

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Anesthesiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ObesityJournal of

ISRN Allergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

of

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN AIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Computational and Mathematical Methods in Medicine

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Biomarkers

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

ISRN Addiction

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Anesthesiology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom

OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ObesityJournal of

ISRN Allergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013