Molecular & Human Genetics Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, IndiaInfectious Diseases & Immunology Division, Indian Institute of Chemical Biology, Kolkata 700032, India
r t i c l e i n f o
rticle history:eceived 11 May 2010ccepted 7 July 2010vailable online 21 July 2010
eywords:yclinA
a b s t r a c t
Arsenic is a well-established human carcinogen; however molecular mechanisms to arsenic-inducedcarcinogenesis are complex and elusive. The present study identifies a potential biomarker of arsenicexposure, and redefines arsenic-induced signaling in stimulation of cell proliferation. The effect of arsenicexposure on gene expression was evaluated in PBMC of arsenic-exposed individuals selected from aseverely affected district of West Bengal, India. A novel, un-documented biomarker of arsenic exposure,CyclinA was identified by microarray analysis from the study. Non-transformed cell lines HaCat and
rkitogen activated protein kinase (MAPK)
eactive oxygen species (ROS)rsenic
Int407 when exposed to clinically achievable arsenic concentration showed significant increase of CyclinAsubstantiating the clinical data. An associated increase in S phase population of cells in cell cycle, indicativeof enhanced proliferation was also noticed. On further investigation of the pathway to arsenic-inducedproliferation, we observed that arsenic resulted: ROS generation; activated Erk signaling; stimulated AP-1activity, including immediate early genes, c-Jun and c-Fos. N-Acetyl-l-cysteine, a ROS quencher, blockedthe arsenic-induced effects. Our study underlines a previously undefined mechanism by which arsenic
sults
imparts its toxicity and re
. Introduction
Arsenic is a well-established human carcinogen (Cantor andubin, 2007; Huang et al., 2004). A close association and positiveorrelation exists between arsenic exposure and increased inci-ences of various forms of cancer, as documented from studies inifferent arsenic-endemic areas of the world (Chen et al., 2005;apio and Grosche, 2006). The worst sufferers are the millionsnhabiting the coastal zone of West Bengal, India and Bangladesh,xposed to exceedingly high arsenic concentrations through drink-ng water (Chakraborti et al., 2004). Arsenic was observed to have
clear predilection towards skin in these regions with primaryanifestations being premalignant skin lesions with propensity
f future cancer development (Ghosh et al., 2007, 2008; Morris,995). Given the consequences, elucidation of the precise molec-lar mechanisms of arsenic modus operandi is critical to ournderstanding. A few studies at the molecular level revealing the
[email protected] (K. Chaudhuri).1 Present address: Massachusetts Institute of Technology (MIT), Department ofiological Engineering, Room No. 56-654, 77, Massachusetts Avenue, Cambridge,A 02139-4307, USA.
basic biology of arsenic activity in different species have previ-ously been undertaken to unravel a handful of information (Nemecet al., 1998; Tchounwou et al., 2004), but despite all progress themolecular basis of arsenic-induced skin lesions and its successionto cancer is still poorly understood and is yet to be fully decipheredand characterized.
One important mechanism that facilitates cellular adaptationto chemical exposure is induction of specific gene expression(Denison et al., 1995; Yih et al., 2002). Gene expression being asensitive endpoint, microarray can be a useful monitor of exposureeffect and in possible identification of new biomarkers (Frueh et al.,2001). We therefore conducted a microarray-based gene expres-sion study in peripheral blood mononuclear cells (PBMC) amongindividuals chronically-exposed to arsenic possessing skin lesions.Our endeavor was to assess whether arsenic exposure is associ-ated with differential gene expression. The total set of differentiallyexpressed genes obtained from microarray is not discussed in thepresent study; instead, with clues from microarray analysis we pri-marily focused on elucidation of putative arsenic mode of action.
The results obtained from microarray study were supported within vitro studies, aimed at understanding exactly how arsenic affectsthe biological systems, and with an objective to identify genes thatcould be used as predictors or potential biomarkers of arsenic expo-sure.
To the best of our knowledge, this study is the first of itsind that specifically underscores and pinpoints a novel geneticarker of arsenic exposure that has implication in the process
f carcinogenesis and adds a new vista to the understanding andharacterization of molecular mechanisms of arsenic-induced car-inogenesis facilitating identification of potential molecular targetsor chemoprevention.
. Materials and methods
.1. Cell culture and treatments
Non-tumorigenic human keratinocyte, HaCaT and non-transformed humanmbryonic intestinal epithelial cells, Int407 (ATCC, USA) were maintained in Dul-ecco’s Modified Eagle’s Medium (Invitrogen, USA); PBMC, isolated from blood, inPMI-1640 medium (GIBCO, Grand Island, NY, USA), supplemented with 10% fetalovine serum and antibiotics (penicillin, 100 U/ml and streptomycin, 50 �g/ml)Gibco BRL, Gaithersburg, MD). Cells were cultured at 37 ◦C in 95% air/5% CO2
umidified incubators. Cells were exposed to sodium arsenite (NaAsO2) at 2 �Moncentration and incubated for specific time points. Specific inhibitors of Erk (extra-ellular signal-regulated kinase) PD98059 (50 �M), Jnk (jun N-terminal kinase)P600125 (20 �M), and p38 SB203580 (10 �M) (Sigma, St. Louis, MO, USA) weredded 30 min before NaAsO2 treatment.
.2. Measurement of cell viability
Cell viability was assayed using 3-[4,5-dimethylthiazol-2-yl]-2,5-iphenyltetrazolium bromide (MTT) dye (Sigma–Aldrich, USA). Briefly, 1 × 105
ells were seeded/well in 200 �l of medium in 24-well plates (Nunc, Denmark) andxposed to NaAsO2 for 24 h. MTT (1.2 mg/ml) was then added and incubated for 4 ht 37 ◦C. Optical density of dimethylsulfoxide (100 �l) dissolved precipitates werehen measured at 595 nm (EMax Precision Micro Plate Reader, Molecular Devices,SA).
.3. Analysis of cell cycle profile by propidium iodide (PI) staining
For cell cycle analysis, PBMC or NaAsO2-exposed cells (24 h) seeded at a densityf 1 × 104 were taken. Cells were then fixed in 70% ethanol for 24 h at 4 ◦C, cen-rifuged (1500 × g) and cell pellet resuspended in PBS (400 �l), RNaseA (10 mg/ml,0 �l) and PI (2 mg/ml, 10 �l). The mixture was incubated in dark at 37 ◦C for 30 minnd then acquired in FACSCalibur (fluorescence activated cell sorter, Becton Dick-nson, CA, USA).
.4. Analysis of apoptotic cells by annexinV-PI staining
For quantization of apoptotic cells, 2 × 105 cells were cultured in 6-well platesnd exposed to NaAsO2 for 24 h. Apoptosis was assessed by ApoAlert annexinV-FITCfluorescein isothiocyanate) and PI (BD Biosciences, Pharmingen, San Diego, USA),s described previously (Chowdhury et al., 2009). Apoptotic cells were determinedy FACSCalibur followed by analysis with CellQuestPro software (Becton Dickinson,an Jose, CA, USA).
.5. Evaluation of oxidative stress by H2DCFDA
ROS were detected using fluorescent probe 2′-7′-dichlorofluorescein-diacetateH2DCFDA) (Sigma–Aldrich, USA) (Miller et al., 2002). Briefly, cells were exposed toaAsO2 for 1 h, incubated with H2DCFDA (20 �M) for 30 min at 37 ◦C and fluores-ence monitored by flow cytometry at excitation (488 nm) and emission wavelength530 nm). The data was analyzed by CellQuestPro software and mean fluorescencentensity was calculated. N-Acetyl-l-cysteine (NAC) (5 mM) (Sigma, St. Louis, MO,SA), was applied 1 h before NaAsO2 treatment.
.6. RNA extraction, cDNA preparation and quantitative real-time PCR
Total RNA was extracted by Rneasy Mini Kit (Qiagen Inc., Valencia, CA,SA). cDNA was prepared using SuperscriptTM Synthesis System (Invitrogen) asescribed earlier (Sarkar and Chaudhuri, 2004). cDNA was subjected to Real-ime quantitative RT-PCR using the 7500 Fast Instrument (ABI, USA) with SYBRreen as a fluorescent reporter using SYBR Green JumpStartTM Taq ReadymixTM
Sigma–Aldrich, USA). The expression of cell cycle associated genes were analyzedith specific primers: CyclinA (forward-5′-TCCAAGAGGACCAGGAGAATATCA-3′
TTGTGTACAAAGCC-3′ and reverse 5′-GCTAGTCCAAAGTCTGCTAGCTTG-3′) anddc2 (forward 5′-CGTGGGGGAGCGGATTT-3′ and reverse 5′-CGGAGGGCGA-TATTGAGGA-3′). Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) (forward′-ATGGGGAAGGT GAAGGTCGG-3′ and reverse 5′-GGATGCTAAGCAGTTGGT-3′)as used as internal control. Threshold cycle number (CT), of triplicate reactions,as calculated and the mean CT of triplicate reactions was determined.
etters 198 (2010) 263–271
2.7. Western blot analysis
Equal amount of cell lysate (30 �l/lane) were subjected to immunoblot fol-lowing methods described previously (Chowdhury et al., 2009). The antibodiesused were anti-p38, anti-phospho-p38 (Thr180/Tyr182) (BD Biosciences, USA), andanti-Jnk, anti-phospho-Jnk (T183/Y185), anti-Erk-44/42, anti-phospho-Erk-44/42(Thr202/Tyr204), anti-CyclinA, anti-c-Jun and anti-c-Fos (Cell Signaling, MA, USA)and anti-mouse ˇ-actin (Sigma–Aldrich, USA). The secondary antibodies were alka-line phosphatase-conjugated goat anti-rabbit or rabbit anti-mouse IgG (Genei,India). The alkaline phosphatase-positive bands were visualized following previ-ously described methods (Chowdhury et al., 2009).
2.8. Analysis of protein localization by immuno-fluorescent staining
Cover slip cultured arsenite treated or untreated Int407 cells were fixed withmethanol at −20 ◦C for 15 min and rehydrated with PBS for 30 min. Non-specificbinding was blocked by incubating the cells in 3% BSA for 1 h. The cells were incu-bated overnight with anti-CyclinA (Cell Signaling, MA, USA) primary antibody (1:50)and incubated with FITC-conjugated secondary antibody (Genei, India) at dilution1:100 for 30 min. The fluorescence image of glycerine mounted was visualized andcaptured using Leica DM 3000 fluorescence microscope.
2.9. Constructs, transfection and luciferase assay
CyclinA luciferase reporter plasmid was obtained by cloning into the KpnIand HindIII (New England Biolabs, Beverly, MA) restriction sites of pGL3 basicmammalian vector (Promega, Madison, WI, USA) a 213-bp fragment of thehuman CyclinA promoter (from −165 to +48 bp relative to the most 3′ transcrip-tion initiation site). The amplified fragment was generated by PCR from humangenomic DNA with oligonucleotides 5′-CTCCGGTACCAGCCAGTTTGTTTCTC-3′ and5′-TGGCAAGCTTAAGACGCCCA GAGATG-3′ which also have KpnI and HindIII cuttingsites (Tessari et al., 2003).
HaCat and Int407 cells were transiently transfected with either CyclinA or AP-1-driven luciferase reporter plasmid (1 �g) (Chowdhury et al., 2009) (Stratagene, USA)using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and 46 h post-transienttransfection cells were treated with NaAsO2 (24 h for CyclinA and 1 h for AP-1-luciferase reporter plasmid) before they were harvested. The cells were then lysed inluciferase lysis buffer (Promega, Madison, USA) and luminescence was measured asrelative luciferase units (RLU) in luminometer (Junior LB9505, Berthold, Germany).
2.10. Study location and selection criteria for subjects
Four arsenic-affected administrative blocks (Gaighata, Habra, Deganga andBaduria) of West Bengal, were selected as the exposed study sites for arsenic sincethe magnitude of arsenic calamity in ground water of these regions has already beenwell documented (Basu et al., 2004). The un-exposed study area was Contai subdi-vision of East Midnapur district known to be unaffected by arsenic contamination(Ghosh et al., 2007). The exposure classification of the study participants for microar-ray was based on previous studies (Ahsan et al., 2007; Anawar et al., 2002; BeaneFreeman et al., 2004; Chakraborti et al., 2004) and included the following diverseparameters: arsenic concentration in toenails and hair; urine and drinking waterarsenic content (Karagas et al., 2000; Kile et al., 2005). Subjects with nail and hairarsenic level >0.5 �g/g; urine arsenic content above 1000 �g/l; blood arsenic level>30 �g/l and subjects with visible dermal lesions were preferentially selected fromexposed area; furthermore similar age, and sex matched individuals were consid-ered. Only study subjects who had a cell number above 15–20 × 106 were selectedfor microarray analysis because of limited samples of lymphocytes.
2.11. Sample collection and arsenic measurement
Approximately, 10 ml peripheral blood was collected, with prior consentusing heparin as an anticoagulant. Drinking water and urine (100 ml each), nails(∼250–500 mg) and hair (∼300–500 mg) samples were also collected for arsenicestimation from the same subjects. Arsenic estimation in the collected bio-sampleswas done by flow injection–hydride generation–atomic absorption spectrometry(FI–HG–AAS) (Das et al., 1995).
2.12. Separation of peripheral blood mononuclear cells from whole blood
10 ml of fresh heparinized blood was layered carefully and slowly over 3 mlof Ficoll/Hypaque (Sigma Diagnostics, USA) and centrifuged at room temperature(1000 rpm) for 45 min. The buffy coat layer containing PBMC at the interface wastaken out and cultured under tissue culture conditions for 24 h. Cultured cells werethereafter subjected to RNA extraction.
2.13. Microarray analysis
A total of 6 study subjects were shortlisted based on above-mentioned arsenicexposure criteria; RNA was isolated from cultured PBMC and pooled to form theexposed group. Pooled RNA from subjects (n = 6) with no manifest skin lesions in
R. Chowdhury et al. / Toxicology Letters 198 (2010) 263–271 265
Fig. 1. (A) Prevalence of dermatological manifestations in arsenic-exposed population. The abbreviations for the observed skin lesions are denoted as: RD, raindrop pigmen-tation; HK, palmer and palnter hyperkeratosis; HP, hyper-pigmentation; DP, de-pigmentation. Hyperkeratosis and raindrop pigmentation were found to be the most specificdiagnostic parameters of arsenic exposure and their prevalence was significantly high (double-asterisk (**) indicates significant difference, P < 0.05) compared to other formsof skin abnormalities. (B) Alteration in expression of CyclinA, Cdk2 and Cdc2 in PBMC. Cellular Cyclin and Cdk expression as verified by qRT-PCR. The expression, shown inhistogram, was estimated as fold change relative to un-exposed expression level and the values are normalized against GAPDH (control) expression. Standard deviation wasc in S pp try is pp ells in
aursflrcrasdo(
TAp
alculated from two to four replicate experiments. (C) Increased population of cellsropidium iodide (PI) and analyzed. A representative figure (n = 6) of flow cytomehase, S phase and G2/M phase are demarcated in the diagram. The percentage of c
rsenic-unaffected regions was set as control. The A260/A280 ratio was measuredsing the Picodrop Spectrophotometer (Cambridge, UK). All samples had A260/A280atio > 1.9. Using BD Atlas SmartTM Fluorescent Probe Amplification Kit (BD Bio-ciences, CA, USA), first-strand cDNA was prepared; PCR amplified and labeled withuorescent dye (Cy3-Control, Cy5-Exposed, Amersham Biosciences, UK). The fluo-escent labeled probes were hybridized to BD AtlasTM Glass Human 3.8II Microarrayhip. Signal intensity of spots on array was acquired using ScanArrayTM Lite Microar-
ay Scanner (PerkinElmer, MA, USA) at appropriate optical resolution. Image analysisnd quantification was done using QuantArray Microarray Analysis Software (ver-ion 3.00, PerkinElmer Life, MA, USA). A mean of absolute expression values wereeduced from technical duplicates of the arrays for expressed transcripts. A cut-ff of two-fold (up- or down-regulation) was used to define differential expressionexposed versus un-exposed) as fold change of a gene.
able 1rsenic content in drinking water and clinical samples of exposed and un-exposed popularevalence of skin lesions. The magnitude of severity was related to the concentration of
Population Incidence of skin lesion Arsenic
Drinkin
Un-exposed group (n = 210) 0 8.04
Exposed group (n = 451)Exposed to As < 1 �M (n = 45) 29 (64.4%) 65.74Exposed to 1 �M < As < 2 �M (n = 162) 106 (65.4%) 111.12Exposed to 2 �M < As < 3 �M (n = 110) 62 (56.6%) 181.29Exposed to As > 3 �M (n = 134) 99 (73.8%) 393.73
hase, as evaluated by flow cytometric analysis. Cultured PBMC were stained withresented (I) un-exposed group; (II) exposed skin-lesion group. Sub-G1 phase, G1S phase is denoted in the inset.
3. Results
3.1. Prevalence of dermatological manifestations
The arsenic concentration in tube-well water of arsenic-infestedvillages ranged up to ∼400 �g/l and 76% of the participants wereexposed to levels >100 �g/l WHO recommended limit is 10 �g/l
(WHO, 1996). The average drinking water arsenic concentrationwas 187.97 �g/l. Approximately, 65% of the exposed individualsshowed arsenic-induced manifestations of skin lesions. Hyperk-eratosis and raindrop pigmentation were found to be the most
tion. A positive relationship was apparent between water levels of arsenic and thearsenic in water as well as duration of exposure.
content (mean ± SD)
g water (�g/l) Nail (�g/g) Hair (�g/g) Urine (�g/l)
266 R. Chowdhury et al. / Toxicology Letters 198 (2010) 263–271
Fig. 2. (A) Dose-dependent cytotoxicity of sodium arsenite on HaCat and Int407 cells. 1 × 105 cells were seeded/well in 24-well plates and incubated overnight. The followingday the cells were treated with different doses of NaAsO2. After 24 h the number of cells was measured by MTT assay. Data are mean values from three independentexperiments. (B) Sodium arsenite induces increase in S and G2 M population of cells in vitro. A representative figure of flow cytometrically analyzed cell cycle distributionof HaCat & Int407 cells after treatment with 2 �M NaAsO2 for 24 h followed by staining with PI is displayed. The denotations used represents the following (I) un-exposedHaCat; (II) un-exposed Int407; (III) NaAsO2-treated HaCat and (IV) NaAsO2-treated Int407. The bar chart with standard deviations represents the percentage of cells in S andG2 M phase from thee independent experiments. A double-asterisk (**) indicates significant difference (P < 0.05) compared to NaAsO2-un-exposed cells. (C) Arsenic insultat clinically achievable dose is not associated with apoptosis. HaCat and Int407 cells were treated with 2 �M or 20 �M NaAsO2 for 24 h, harvested and then stained withannexinV/PI. Apoptotic cells were quantitated by flow cytometry. Dot plots represent PI fluorescent intensity (FL-2) against annexinV (FL-1). The denotations used representsthe following (I) un-exposed HaCat (II) un-exposed Int407 (III) 2 �M NaAsO2-treated HaCat (IV) 2 �M NaAsO2-treated Int407 (V) 20 �M NaAsO2-treated HaCat (VI) 20 �MNaAsO2-treated Int407. A representative figure from three independent experiments is given.
Fig. 3. (A) Arsenic induces increased expression of CyclinA in vitro. HaCat and Int407 cells were treated with 2 �M NaAsO2 for 24 h and the expression of CyclinA wasanalyzed by western blot. NAC was added 1 h before NaAsO2 treatment. ˇ-actin was used as a loading control. A representative figure from three independent experimentsis given. B. HaCat and Int407 cells were transfected with CyclinA promoter luciferase construct for 46 h followed by 2 �M NaAsO2 treatment for 24 h. NAC was added 1 hbefore NaAsO2 treatment. The symbols ** and ## indicates significant difference (P < 0.05) compared to NaAsO2-un-exposed and NaAsO2-treated cells, respectively. (C) Cellswere treated with 2 �M NaAsO2 for 24 h and localization of CyclinA was analyzed by anti-CyclinA primary antibody followed by incubation with FITC-tagged secondaryantibody. Immunofluorescence was visualized and captured using Leica DM 3000 fluorescence microscope. The accumulation of CyclinA in nucleus is marked by arrows. Arepresentative figure from three independent experiments is presented.
R. Chowdhury et al. / Toxicology Letters 198 (2010) 263–271 267
Fig. 4. (A) Arsenic induces AP-1 activity in vitro. HaCat and Int407 cells were transfected with pAP-1-luc vector and 46 h post-transfection cells were exposed to 2 �MNaAsO2 for 1 h and subjected to luciferase assay. NAC was added 1 h before NaAsO2 treatment. The symbols ** and ## indicates significant difference (P < 0.05) compared toNaAsO2-un-exposed and NaAsO2-treated cells, respectively. The bar chart represents mean ± SD from three independent experiments. (B) Arsenite induces c-Jun and c-Fosactivity. HaCat and Int407 cells were treated with 2 �M NaAsO2 for 1 h and expression of c-Jun and c-Fos was analyzed by western blot. NAC was added 1 h before NaAsO2
treatment. ˇ-actin was used as a loading control. The band intensities are representative figures from three independent experiments. (C) Flow cytometric determinationo HaCatt n forf * andN
spal[u
3r
efacttehtkmTrds
not apoptosis in HaCat and Int407 cells
It was apparent from microarray that arsenic exposure dis-rupts proper functioning of cell division. We exposed twonon-transformed cell lines (HaCat and Int407) to clinically achiev-
Table 2A list of over-expressed Cyclins and Cdks in the group (n = 6) of individualschronically-exposed to arsenic possessing skin lesions compared to un-exposedgroup obtained from the microarray analysis is represented. Only genes above acut-off of two-fold (up-regulation) is shown.
Gene name Fold change Gene ID Locus link GenBank
CyclinA 4.37 OH7888 8900 U66838
f DCF fluorescence by H2DCFDA as a measure of intracellular ROS generation inreatment. The vertical line through the histograms (a representative figure) is drawrom three independent experiments is represented as bar diagram. The symbols *aAsO2-treated cells, respectively.
pecific diagnostic parameters of arsenic exposure (Fig. 1A). A clearositive relationship was apparent between water levels of arsenicnd the prevalence of skin lesions (Table 1). A weak positive corre-ation was observed with arsenic exposure and arsenic level in haircorrelation coefficient (r = 0.3, p = 10−12), nail (r = 0.29, p = 10−10) orrine (r = 0.25, p = 10−7)].
.2. Evidence of arsenic-mediated deregulation of cell cycleegulatory genes
We conducted a microarray analysis to check whether arsenicxposure and arsenical skin-lesion status are associated with dif-erential expression of check point genes. In response to chronicrsenic exposure the most significant up-regulation amongst theellular Cyclins was in CyclinA (4.37-fold); a list of Cdks and Cyclinshat were up-regulated in PBMC of individuals chronically-exposedo arsenic, possessing skin lesions are presented in Table 2 (differ-ntial response observed in other genes from microarray analysisas not been discussed in this study). It was previously reportedhat CyclinA binds both Cdk2 and Cdc2, giving two distinct CyclinAinase activities, one appearing in S phase, the other in G2, thus aug-
enting proliferation of cells (Liu et al., 1998; Pagano et al., 1992).
he expression pattern of CyclinA validated through quantitativeeal-time PCR (qRT-PCR) (Fig. 1B) support the dose response trendepicted by the array data showing a ∼12.5-fold increase in expres-ion along with ∼13.6-fold increase in Cdk2 expression in exposed
and Int407 cells treated with NaAsO2 for 1 h. NAC was added 1 h before NaAsO2
comparison. The change in mean fluorescence intensity (MFI) ± standard deviation## indicates significant difference (P < 0.05) compared to NaAsO2-un-exposed and
group compared to un-exposed control. It epitomizes a putativechronic arsenic exposure induced activation of specific checkpointregulatory genes that plays part in uncontrolled cell proliferation.We thereafter conducted a cell cycle analysis using FACS in PBMC ofexposed group and compared it with control. PBMC from arsenic-exposed skin lesion manifestation group had ∼12% more cells inthe proliferative phase when compared to control (Fig. 1C).
3.3. Arsenic induces increase in S and G2M population of cells but
268 R. Chowdhury et al. / Toxicology Letters 198 (2010) 263–271
Fig. 5. (A) Arsenite induces Erk activity. Western blot analysis of phospho-p38, phospho-Jnk and phospho-Erk levels following NaAsO2 treatment for 1 h. The last lane oft lls. The inhibitors used were SB203580 (p38), SP600125 (Jnk) and PD98059 (Erk) applied3 om three independent experiments. (B) Arsenite-induced Erk activity is associated withi for 30 min before 2 �M NaAsO2 exposure for 24 h and then subjected to MTT assay. Dataa ignificant difference (P < 0.05) compared to only NaAsO2-treated cells.
aalvatt(
eiHactot(S(
toae2lnasc
he gel profile is representative of specific MAPK-inhibitors and NaAsO2-treated ce0 min before NaAsO2 treatment. The band intensities are representative figures fr
ncreased cellular division. HaCat and Int407 cells were pre-treated with PD98059re mean values from three independent experiments. The symbols ** indicates a s
ble concentration of arsenic (2 �M). Since human exposure torsenic is normally at a much lower level, we deliberately usedow arsenic concentrations in order to achieve results directly rele-ant to arsenic toxicity in humans (Hayashi et al., 2002). Arsenict 2 �M was non-toxic. NaAsO2 showed a sigmoidal curve inoxicity versus concentration; the LC50 values were determinedo be ∼39 �M and ∼37.5 �M for HaCat and Int407, respectivelyFig. 2A).
To gain further insights into whether low level of arsenitexposure possesses the potential to influence cell proliferationn vitro; cell cycle distribution was examined in NaAsO2-treatedaCat and Int407 cells by FACS following PI staining. There wassignificant increase in percentage of S and G2/M population of
ells following arsenic exposure when compared to untreated con-rol (Fig. 2B). In the absence of NaAsO2 treatment, ∼11% and 14%f cells were in the S phase in HaCat and Int407 cells, respec-ively (Fig. 2B). Arsenite at clinically achievable concentration2 �M) led to a significant increase in the number of cells in the
phase (25% and 33%), indicative of increased DNA synthesisFig. 2B).
The response to an environmental insult, in some cellypes, if the damage is irreparable can lead to inductionf cell death by apoptosis. Hence we investigated whetherrsenic at low concentrations induces apoptosis or not. How-ver, significant apoptosis induction was not observed at�M concentration (Fig. 2C). In contrast, however a higher
evel (20 �M) of NaAsO2 stimulated cell apoptosis predomi-antly in Int407 cells. The above observation indicates that thersenic-induced cellular insult is not associated with apopto-is in HaCat and Int407 cells at clinically achievable arseniconcentrations.
Fig. 6. A schematic representation of the proposed pathway of arsenic-inducedeffects.
3.4. Arsenic induces increased expression of cellular CyclinA invitro
To evaluate the effects of arsenic exposure on CyclinA expres-sion in vitro, HaCat and Int407 cells were exposed to NaAsO2 (2 �M)and monitored by qRT-PCR and immunoblot analysis. In corrobora-tion with our previous observation, low level of arsenite exposure,led to a ∼5.96 and ∼4.32-fold induction of CyclinA, in HaCat and
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nt407 cells, respectively at RNA level. The protein expression ofyclinA also significantly increased following treatment (Fig. 3A).f N-acetyl-l-cysteine (NAC), a ROS quencher was added, beforeaAsO2 treatment, there was a moderate attenuation of arsenic-ediated CyclinA protein expression (Fig. 3A); arsenic is known toediate its toxic effects by ROS generation (Chen et al., 1998; Flora
t al., 2007; Liu et al., 2001). Moreover, we observed an increase inyclinA promoter dependent luciferase activity, more significantly
n HaCat cells following NaAsO2 treatment (Fig. 3B). Cellular Cyclinsn association with CDKs is known to play a vital role in promot-ng cell proliferation (Santamaria and Ortega, 2006); our study thusroposes a novel putative marker of arsenic exposure that plays aart in arsenite-induced acceleration of cellular division.
To further validate our point sub-cellular distribution of CyclinAollowing arsenic exposure was analyzed by immunofluorescence.
representative figure from Int407 cells is presented (Fig. 3C).yclinA, in arsenic-exposed cells was found to be diffused through-ut the cell but was also more clearly present in the nucleus, thusrobably actively participating in cell cycle progression. This is ingreement with recent findings that suggest that CyclinA accumula-ion in the nucleus is needed for entry into or progression through Shase of the cell cycle (Pagano et al., 1992; Pines and Hunter, 1990).
.5. Arsenic induces enhanced AP-1 activity in vitro
AP-1 activation has been shown to be required for cell trans-ormation by stimulation of DNA synthesis (Young et al., 1999).he pAP-1-luc vector was transfected into HaCat and Int407 cells.rsenite clearly increased AP-1 dependent luciferase activity thus
ndicating and establishing its putative role in DNA synthesis or cellroliferation (Fig. 4A). Furthermore, we also observed an increasedxpression of AP-1 members, c-jun and c-fos on NaAsO2 treatmentFig. 4B). Furthermore, AP-1 is an important oxidation-sensitiveranscription factor and its activity can be induced by oxidativetress (Hsu et al., 2000). It is well known that ROS generationssociated with arsenic exposure plays a fundamental role in thenduction of its adverse health effects (Bode and Dong, 2002).herefore we investigated whether arsenic-mediated modulationf AP-1 activity is associated with alteration of redox balance. Webserved an elevated level of intracellular ROS in arsenic-exposedaCat and Int407 cells (more predominant in HaCat) (Fig. 5C). NACttenuated generation of ROS (Fig. 4).
.6. Arsenic-mediated activation of MAPK signaling in vitro
To decipher the signaling pathways activated by low level ofrsenite, proteins from NaAsO2-exposed HaCat and Int407 cellsere subjected to immunoblot analysis. Jnk (c-Jun N-terminal
inase) and p38 activity was found to be very weakly stimulatedFig. 5A). A common feature of major carcinogens is their ability tonhance Erk signaling pathway that is actively involved in cell pro-iferation (Johnson et al., 1996; Su and Karin, 1996). Our data alsohowed that at low (2 �M) arsenite concentration Erk is activated,uggesting that arsenite at low concentrations can act as an inducerf cell proliferation. To further validate the above HaCat and Int407ells were pre-treated with PD98059 followed by NaAsO2 exposure.D98059 is a highly selective inhibitor of MEK-1 activation, leadingo suppression of Erk MAP kinase phosphorylation. The cell viabil-ty was thereafter determined by MTT assay. PD98059 inhibitedrsenite-induced activation of cellular proliferation (Fig. 5B).
. Discussion
Millions of people globally are at risk from the detrimentalffects of arsenic exposure, with drinking water levels far exceed-ng the World Health Organization (WHO) guideline (Welch et al.,
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1999). Arsenic exposure is presently associated with a spectrum ofdreadful diseases including skin, urinary bladder, liver, kidney andrespiratory tract cancers (Benbrahim-Tallaa and Waalkes, 2008;Cantor and Lubin, 2007; Vahter, 2008). Though the harmful effectsof arsenic are well documented, yet the molecular mechanismsof arsenic-induced carcinogenesis are still elusive and have sincebeen under intensive investigation. Under these circumstances, thisstudy provides considerable insight into the mechanism involvedin arsenic-induced toxicity which can be appropriately utilized infuture to attenuate arsenic-induced toxic effects.
In this study, the effect of arsenic exposure on gene expressionwas assessed amongst individuals who have been chronically-exposed to arsenic with clinical evidence of arsenical skin lesions.Notably, a novel, potential genetic biomarker for arsenic exposureand arsenic-induced carcinogenesis, as in CyclinA was identified.Other differentially expressed genes were not discussed in thisstudy. This exploratory study thus identifies a candidate gene orputative marker of arsenic exposure, and provides novel investiga-tional targets.
The role of CyclinA in cell cycle regulation is obscure. Drosophilaembryos homozygous for CyclinA mutations undergo a block incell division (Lehner and O’Farrell, 1989). This demonstrates thatCyclinA function is not redundant. Recent studies demonstrate thatCyclinA function is needed both in S and G2 phases. During S phase,CyclinA interacts with specific factors needed for DNA replication;in G2, CyclinA involves in the activation of M phase Cdc2-Cyclin Bcomplex (Gautier et al., 1990; Minshull et al., 1990). We observedan increased expression of CyclinA in PBMC of arsenic-exposed skinmanifestation group. Transcription changes in CyclinA, as observedin our study have not previously been linked to arsenic carcino-genesis, but its association with carcinogenesis in other systemshowever suggests that CyclinA plays a major role in early stagesof arsenic-induced cellular proliferation and can be considered apotential novel biomarker.
Following this novel and exciting observation we exposed cul-tured non-transformed cells to clinically achievable concentrationof arsenic, monitored CyclinA level and tried to delineate a putativepathway of arsenic-induced carcinogenesis. Interestingly, arsenic-exposed cells in vitro also showed an increased CyclinA leveland apparent accumulation in the nucleus, as confirmed throughimmunoblot and immunofluorescence study, respectively (Fig. 3).Using flow cytometric analysis, we further demonstrated that 2 �Mof NaAsO2 exposure led to a significant increase in number of cells inS phase as compared with that of control (Fig. 2). Thus, a low level ofarsenite did appear to enhance cell proliferation by selectively up-regulating specific cyclins. Additionally, our data establishes thatlow level of NaAsO2 exposure induces AP-1 transactivation and isalso associated with over-expression of associated genes, such asc-Jun and c-Fos (Fig. 4). AP-1 activation has been previously shownto be required for tumor promoter-induced cell transformation(Young et al., 1999). Interestingly NAC, a ROS scavenger reduced theabove-mentioned arsenic-induced effects, indicating that arsenicprobably mediated its action by enhancing cellular ROS.
Arsenite is previously known to activate members of the MAPKfamily, which help to regulate the expression of onco-proteins andgrowth factors (Huang et al., 2001a,b, 1999a). Many studies previ-ously have demonstrated activation of MAP kinases in response tolethal concentrations of arsenic in several cell types in the range of50–500 �M (Samet et al., 1998; Wu et al., 1999). However, since theexposure of humans to arsenic is normally at a much lower levelin daily life, we exposed cells to lower concentration of arsenic.
We demonstrated that a low concentration of arsenite-induced Erkphosphorylation and its stimulation in vitro (Fig. 5). Erk mediatedsignaling has previously been linked with augmentation of cellproliferation (Huang et al., 1999a,b); whereas stress signals (e.g.ultraviolet irradiation, heat or synthesis inhibitors) activate the Jnk
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nd p38 MAPK pathways, resulting in growth arrest or apoptosisChowdhury et al., 2009; Kyriakis and Avruch, 1996). Furthermore,n addition of MEK inhibitor, PD98059, we observed a reduction inrsenic-induced cell viability (Fig. 5). Therefore, an activation of Erkignaling, following arsenic insult, marks a putative mechanism ofow arsenic induces cell proliferation.
. Conclusion
Given the implications of arsenic exposure on human health andhe known public health hazards of arsenic exposure our results aref immense significance. We postulate a new genetic biomarker forrsenic exposure, CyclinA, and also further underscore that there isrobust response that correlates with arsenic exposure that couldodulate MAPK-Erk pathway leading to cell proliferation (Fig. 6),
ltimately affecting health status.
onflicts of interest
None.
cknowledgements
The study is supported by grants (CMM-0003) and research fel-owships (R.C.) from Council of Scientific and Industrial ResearchCSIR), Government of India.
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