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International Journal of Hygiene and Environmental Health 218 (2015) 564–574

Contents lists available at ScienceDirect

International Journal of Hygiene andEnvironmental Health

jou rn al hom ep age: www.elsev ier .com/ locate / i jheh

hronic low level arsenic exposure evokes inflammatory responsesnd DNA damage

austav Dutta, Priyanka Prasad, Dona Sinha ∗

eceptor Biology and Tumor Metastasis, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700026, India

r t i c l e i n f o

rticle history:eceived 14 February 2015eceived in revised form 8 June 2015ccepted 10 June 2015

eywords:ow arsenic exposureroundwaterD14

nflammationxidative DNA damage

a b s t r a c t

The cross-sectional study investigated the impact of chronic low level arsenic (As) exposure (11–50 �g/L)on CD14 expression and other inflammatory responses in rural women of West Bengal enrolled fromcontrol (As level <10 �g/L; N, 131) and exposed area (As level 11–50 �g/L, N, 142). Atomic absorp-tion spectroscopy revealed that As level in groundwater was higher in endemic areas (22.93 ± 10. 1 vs.1.61 ± 0.15, P < 0.0001) and showed a positive correlation [Pearsons r, 0.9281; 95% confidence interval,0.8192–0.9724] with As content in nails of the exposed women. Flow cytometric analysis showed that CD14 expression on monocytes was significantly higher (P < 0.001) in exposed women and positively corre-lated with groundwater As [Pearsons r, 0.9191; 95% confidence interval, 0.7584–0.9745]. Leucocytes andairway cells of As exposed women exhibited up regulation of an inflammatory mediator, tumor necrosisfactor-� (TNF-�) and transcription factor, nuclear factor-�B (NF-�B) (P < 0.0001). Plasma pro inflamma-tory cytokines like – TNF-�, interleukins (ILs) – IL-6, IL-8, IL-12 were elevated whereas anti-inflammatorycytokine IL-10 was depleted in the exposed women. Sputa of the exposed women had elevated activity of
inflammatory markers – MMP-2 and MMP-9 whereas sera were observed with only increased activity ofMMP-9. Airway cells of the exposed women had exacerbated DNA damage than control. Level of oxida-tive DNA adducts like 8-hydroxy-2′-deoxyguanosine (8OHdG) were also enhanced in plasma of exposedwomen. Therefore it might be indicated that low level As exposure elicited a pro-inflammatory profilewhich might have been contributed in part by CD14 expressing monocytes and prolong persistence ofpulmonary and systemic inflammation might have promoted oxidative DNA damage in the rural women.
© 2015 Elsevier GmbH. All rights reserved.

. Introduction

The environmental hazard of groundwater arsenic (As) con-amination has engulfed nine out of nineteen districts (Dey et al.,014) of West Bengal, India since the first report of this problem

n the lower Ganga basin of the state (Garai et al., 1984). Longerm exposure to inorganic As has been associated with severalypes of skin lesions such as rain drop pigmentation, hypopigmen-ation, hyperpigmentation, keratosis and hyperkeratosis (Guha

azumder, 2003) and other clinical complications like cardiovas-
ular diseases (States et al., 2009) diabetes mellitus (Tseng, 2004),europathies (Ghosh et al., 2007) and liver diseases (Das et al.,012). Skin and several types of internal cancers including bladder,idney, liver, prostate, and lung have been reported with chronic
∗ Corresponding author. Tel.: +91 33 2476 5101/02/22x327;ax: +91 33 2475 7606.

E-mail address: [email protected] (D. Sinha).

ttp://dx.doi.org/10.1016/j.ijheh.2015.06.003438-4639/© 2015 Elsevier GmbH. All rights reserved.

exposure to As (Martinez et al., 2011). Considering this, the WorldHealth Organization (WHO) and the U.S. Environmental ProtectionAgency (USEPA) has set the maximum contaminant level of inor-ganic As in drinking water as 10 �g/L (WHO, 2012; USEPA website).In India, Bureau of Indian Standards (BIS) has set the desirabledrinking water standards for As at 10 �g/L but the legally enforce-able standard is 50 �g/L if alternative sources are unavailable (BIS,2009).

Inflammation is a protective response for cellular or tissuedamage against various pathogens, damaged cells, or toxic insults(Ferrero-Miliani et al., 2007). Long persistence of inflammation orchronic inflammation may lead to the occurrence of different typesof diseases including malignancies (Coussens and Werb, 2002).Toll like receptors (TLRs) activate inflammatory responses, innate
immune responses, and adaptive immune responses to combat theeffect of pathogens and irritants. CD14 serves as a receptor involvedin the activation of the proinflammatory signal transduction path-way which occurs through the activation of TLR (reviewedby Kallapura et al., 2014). CD14, a glycophosphotidylinositol

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GPI)-anchored protein is present predominantly on the surfacef monocytes, macrophages and neutrophils, at lower levels onpithelial cells, endothelial cells and fibroblasts and also in solubleorm without GPI in serum (Anas et al., 2010). CD14 is known tolay a pivotal role in varied inflammatory disorders linked withetal toxicity (Mokgobu et al., 2015), pathogens (Beutler and

ietschel, 2003; Parajuli et al., 2012), sepsis (Huber-Lang et al.,014), stress (Miller et al., 2014) and cancer (Khaled et al., 2014;ellami et al., 2014).

CD14/TLR4 mediated signaling cascade for activation of tran-cription factor nuclear factor-�B (NF-�B) and subsequent releasef cytokines and other proinflammatory mediators, by immuneells like monocytes and macrophages have been reported dur-ng gram negative bacterial infection (Jabaut and Ckless, 2012;i and Verma, 2002). Activated NF-�B triggers the expression ofRNA of various pro-inflammatory mediators including tumor

ecrosis factor-� (TNF-�), interleukins (ILs), adhesion moleculesnd enzymes, such as cyclooxygenase 2 (COX-2) and inducibleitric oxide synthase (iNOS), which are implicated in disease pro-ess (Adams and Hamilton, 1984; Zhao et al., 2013; Lee et al.,012). TNF-� stimulates leukocytes and vascular endothelial cellso release other cytokines (as well as additional TNF-�), to expressell-surface adhesion molecules and to increase arachidonic acidurnover. However, the unregulated release of TNF-� into the cir-ulation results in circulatory dysfunction, increased endothelialermeability and inflammation of different organs (Hensley et al.,000).

Respiratory tract or lung diseases are characterized by chronicnflammation which is usually accompanied with increased expres-ion of cytokines, chemokines, and other inflammatory targetolecules like matrix metalloproteinase-9 (MMP-9), intercellular

dhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1VCAM-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase2 (cPLA2) (Lee and Yang, 2013). It is suggested that oxidative stresslay a crucial role in the pathophysiology of inflammation (Sies,986; Liaudet et al., 2009; Nathan and Cunningham-Bussel, 2013).eutrophils and macrophages intercept and kill pathogens contain-

ng variety of pathogen-associated molecular patterns (PAMPs),rimarily by production of reactive redox species, via induc-ion of the NADPH oxidase system (Robinson, 2008; Slauch,011). Alveolar epithelial type II cells of lungs release inflam-atory mediators and cytokines/chemokines, like IL-1ˇ, IL-6,

L-8, and TNF-� in response to oxidative stress (Lee and Yang,012).

Arsenicals were reported with induction of pro inflammatoryytokines (TNF-�, IL6, IL8, IL1�) in intestinal cells, Caco-2 anduman bronchial epithelial cells, HBE (Calatayud et al., 2014; Xut al., 2013). Arsenic exposure increased susceptibility of C57BL/6ice to respiratory infections and inflammation (Ramsey et al.,

013). Arsenic was also found to be associated with inflammationnduced alteration of metabolic pathway in fatty livers of C57BL/6J

ice (Shi et al., 2014). Evidence suggests that As has inductive effectn CD14 expression of human macrophages (Lemarie et al., 2006).rsenic promulgates oxidative stress, immune dysfunction and tis-ue inflammatory responses, which may lead to dysregulation ofF�B signaling pathway (Hunt et al., 2014). However epidemiolog-

cal studies regarding the impact of low level chronic As exposureith respect to Indian scenario are scarce. Our present work is

he continuation of our previous study where we have reportednduction of various pro inflammatory and inflammatory markersn chronic low level As exposed population (Sinha et al., 2014). In
his study we tried to investigate whether chronic low level Asxposure (11–50 �g/L) has any effect on the expression of CD14nd related inflammatory responses in the rural women of Westengal.
Environmental Health 218 (2015) 564–574 565

2. Materials and methods

2.1. Materials

All the reagents were of analytical grade. Milli Q (MilliporeElix 5, BM9JN2559) water was used throughout the study. Arsenicstandard for AAS and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was obtained from Sigma–Aldrich Chemicals Pvt. Ltd, USA.Gelatin sepharose 4B beads were purchased from GE HealthcareBio-Sciences AB (Uppsala, Sweden). The anti CD14 antibody andinflammatory enzyme-linked immunosorbent assay (ELISA) kitswere obtained from BD Biosciences (San Jose, CA). All other primaryantibodies and secondary antibodies were purchased from SantaCruz Biotechnologies (Santa Cruz, CA). Super Signal West FemtoMaximum sensitivity substrate for enhanced chemiluminiscencewas purchased from Thermo Scientific (Rockford, IL). OxiSelectTM

Oxidative DNA Damage ELISA Kit was purchased from Cell Biolabs,Inc. (San Diego, CA). All other chemicals were obtained from SiscoResearch Laboratories, India.

2.2. Participants and study areas

Two hundred and seventy three adult never-smoking women inthe age group of 22–45 year were enrolled from villages Narayan-pur of Nadia district and Boria of Diamond Harbor district in WestBengal. The participants were subdivided into 2 groups on the basisof As in drinking water from the tube wells: (i) control (n = 131) –from Boria, with relatively low level of As (<10 �g/L), (ii) exposed(n = 142) from Narayanpur, where the groundwater As level wasin the range of 11–50 �g/L. They were invited to participatein this study through village panchayats (local administra-tion), and nongovernment organizations after obtaining informedconsent.

2.3. Inclusion and exclusion criteria

Inclusion criteria were (i) drinking water from the village tubewells for the past 10 years or more; (ii) non-smoker and non-chewer of tobacco, betel nut/betel quid, and non-consumer ofalcoholic beverages. Exclusion criteria were (i) having As-relatedskin lesions like palmar and plantar keratosis, hyperkeratosis, rain-drop pigmentation, (ii) under medication, (iii) having a past historyof malignancy, (iv) pregnant and lactating women. The study proto-col was approved by the Ethics Committee of Chittaranjan NationalCancer Institute.

Demographic and socio-economic data and water samples werecollected at the time of field visits. Arsenic induced skin lesionstatus was evaluated by a dermatologist. The study protocol wasapproved by the Institutional Ethics Committee of ChittaranjanNational Cancer Institute, Kolkata. Informed consent was obtainedfrom all the participants before enrollment for this study. The workwas carried out in accordance with The Code of Ethics of the WorldMedical Association (Declaration of Helsinki).

2.4. Collection of samples

Water samples from tube wells were collected in clean cen-trifuge tubes with 0.1% conc. HCl. Nail samples were collected fromparticipants in zip locked plastic pouches and were coded.

Blood samples (3 ml) were collected from antecubital vein invacutainer tubes with and without K2EDTA as anticoagulant for
plasma and serum samples of the participants.
The early morning sputum was collected from each subject forthree consecutive days. The participants were requested to rinsetheir mouth with sterile normal saline (0.9% NaCl) and to cough

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igorously. The sputum was collected with 5 ml PBS in a sterileontainer.

.5. Determination of arsenic concentration in nails by atomicbsorption spectroscopy (AAS) using vapour veneration assemblyethod (VGA)

The procedure followed was same as mentioned in our previoustudies (Sinha et al., 2014; Das et al., 2014)

.6. Determination of arsenic concentration in nails by atomicbsorption spectroscopy (AAS) using vapour generation assemblyethod (VGA) following microwave digestion

The nails were subjected to microwave digestion following theethod of Samanta et al. (2004). Nails weighing in between 0.1 and

.5 g were taken in teflon vessels and pre-digested for 20 min withitric acid and hydrogen peroxide in ratio 3:1. Subsequently theamples were digested in microwave digestion system (MARS 5,EM Corporation, USA) with 100% efficiency at 160 ◦C with 20 minolding time and 15 min ramping time. The cooled and digestedail samples were diluted to a volume of 10 ml and stored at 4 ◦Cntil further analysis of arsenic content using AAS-VGA.

For the purpose of analysis, a stock solution of 1 mg/ml1000 ppm) was prepared from As standard for AAS in milliQ waterith 0.1% HCl. Subsequently standards were prepared with dilu-

ion of the stock solution. During standard preparation nails werere-digested along with As in the range of 1, 5, 10 and 20 ppb foralibration before use. The AAS-VGA (Agilent VGA 77) was usedor estimation of arsenic. Instrument was calibrated using 5 M HCln the acid channel and 0.6% NaBH4 and 0.5% NaOH in the reduc-ion channel in the working range of 01–50 ppb As standard. Forre reduction experiment sample was prepared in 5 M HCl and 20%I was added and allowed to react up to 45 min at room temper-ture. For direct analysis unknown samples were diluted varyingoncentrations and pre reduced in the same way as standards. Atandard curve was obtained with the prepared range of standardsnd unknown samples were analyzed with respect to the standardurve with the help of the software Spectra AA. Samples in triplicateere analyzed on AAS and the mean of each sample was calculated

n the basis of three readings.

.7. Hematology

Total and differential count of blood cells was done by standardrocedures (Dacie and Lewis, 1975).

.8. Assessment of CD14 expression with flow cytometry

Whole blood (25 �l) was diluted with PBS and thereafter therythrocytes were lysed with 2 ml of 1× FACS lysing solutionBD Pharmingen, USA). Subsequently samples were centrifuged at00 × g for 5 min. The residual leucocytes were diluted with PBSto obtain a cell count in the order of 106 cells) and incubatedith fluorescein isothicyanate (FITC)-conjugated anti-human CD14onoclonal antibody [20 �l/106 cells] for 20 min, in dark, at room

emperature. The cell suspension was centrifuged at 300 × g for min and the cell-pellet was washed with ice-cold PBS containing.1% sodium azide and resuspended in 500 �l of 1% paraformalde-yde in PBS. Finally 15,000 events were acquired per sample

n a flow cytometer (FACS Caliber, BD, San Jose, CA) using Cell
uest software (BD, USA) equipped with a 488 nm argon lasernd a 525 ± 10 nm band pass emission filter. Fluorescence wasetermined with FL1H channel. Results were recorded as meanuorescence intensity (MFI), which represents the cell surfaceeceptor density.
Environmental Health 218 (2015) 564–574

2.9. Separation of leucocytes

Whole blood was mixed with Solution A [NH4Cl (0.87%) in10 mM Tris–HCl, pH 7.2] in the ratio of 1:3, kept in ice for 30 min,centrifuged for 20 min at 400 × g. The supernatant was discardedand the process was repeated with the pellets. Subsequently thepellet was suspended in Solution B [0.25 mM mesoinositol, 1 mMMgCl2 in 10 mM Na2PO4 buffer, pH 7.2] and centrifuged at 120 × gfor 6 min. The final pellet obtained was that of leucocytes.

2.10. Preparation of sputum extract

Sputa were centrifuged at 1000 × g for 5–6 min. The non trans-parent viscous part containing airway cells was used for ELISA,detection of ROS generation, and comet assay whereas the trans-parent supernatant was used for zymography. The viscous part ofthe sputum containing airway cells was again centrifuged with PBSat 7000 × g for 5–6 min to obtain the pellet of airway cells (pri-marily comprising of neutrophils, eosionophils, leucocytes, alveolarmacrophages and non squamous epithelial cells).

2.11. Preparation of cell lysate and protein estimation

The final pellet of leucocytes/airway cells was lysed with RIPAbuffer [Tris–HCl 50 mM, pH 7.5; NaCl 150 mM; NP40 10%; sodiumdeoxycholate 0.5%; SDS 0.1%; protease inhibitor cocktail; NaF0.01%; Na3VO4 0.01%] at −80 ◦C for 1 h. Subsequently, it was cen-trifugation at 12,074 × g for 15 min.

Protein estimation of cell supernatant obtained from lysate ofleucocytes/airway cells was done according to Lowry’s method(Lowry et al., 1951).

2.12. Detection of protein expression with ELISA

The wells of microtitre plate were coated with leukocytelysate/lysate of airway cells (10 �g) and kept overnight at 4 ◦C.Next day wells were blocked with blocking buffer (1% BSA in PBS)for 1 h at 37 ◦C, washed with wash buffer (0.5% NP-40 and 0.5%BSA dissolved in PBS), incubated with primary antibody – anti-NF-�B/TNF-� monoclonal antibodies (1:1000 dilution) at 37 ◦C for 1 hand subsequently with respective horse radish peroxidase conju-gated secondary antibody solution (1:1000 dilution) at 37 ◦C for 1 h.Finally substrate 3,3′,5,5′-tetramethylbenzidine (in darkness) wasadded till color developed. Reaction was stopped with H2SO4 (1 M)and reading was taken in ELISA plate reader [Infinite M200, TECAN,Mannedrof, Switzerland] at 450 nm.

2.13. Western blot analysis

Briefly protein (50 �g) obtained from leukocyte extracts wassubjected to SDS-PAGE (7.5%) and blotted onto nitrocellulose mem-branes, blocked using 5% BSA in TBS-T (Tris buffered saline withTween-20; 50 mM Tris, 150 mM NaCl, 0.05% Tween-20), incubatedwith anti-NF-�B/TNF-� monoclonal antibodies (1:1000 dilution)for 90 min at 37 ◦C, washed with TBS-T and incubated with respec-tive alkaline phosphatase coupled secondary antibody for 90 minat 37 ◦C (1:1000). Bands were visualized using enhanced chemilu-miniscence method.

2.14. Measurement of the level of pro-inflammatory and antiinflammatory mediators

The measurement of tumor necrosis factor alpha (TNF-�),interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12) and

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Table 1Demographic survey of the control and chronic low As-exposed population.

Parameter Control (n = 131) As-exposed (n = 142) P value*

Age in year, mean ± SD 39 ± 4 38 ± 6 0.109Body mass index

(kg/m2), mean ± SD22.7 ± 3.5 22.3 ± 4.2 0.395

Years of schooling,mean ± SD

8 ± 4 7 ± 5 0.07

Occupation (%)Household work only 40.2 37.9 0.954House-hold + agriculturalwork

59.8 62.1 0.97

Cooking fuel use at home (%)LPG, kerosene 16.4 13.8 0.874Biomass 83.6 86.2 0.976

Members in family,mean ± SD

4 ± 4 4 ± 5 1.00

Family income per 83 ± 7 72 ± 13 <0.0001


nterleukin-10 (IL-10) in blood plasma was done with ELISA kitsccording to manufacturer’s protocol.

.15. Gelatin zymography

Gelatin zymography was performed with sputa/sera obtainedrom As-exposed and control area. MMPs were concentrated withelatin sepharose 4B beads for 2 h at 4 ◦C. The beads were washed× with Tris-buffered saline with Tween-20 (TBST) and suspended

n 1× sample buffer (0.075 g Tris, 0.2 g SDS in 10 ml water, pH 6.8).he suspension was incubated for 30 min at 37 ◦C then subjected toymography on 10% SDS-PAGE co-polymerized with 0.1% gelatin.els were washed in 2.5% Triton-X100 for 30 min to remove SDSnd then incubated overnight in reaction buffer (50 mM Tris–HClH 7, 4.5 mM CaCl2, 0.2 M NaCl). After incubation, the gel wastained with 0.5% Coomassie Blue in 30% methanol and 10% glacialcetic acid. The gel was subsequently destained with 30% methanolnd 10% glacial acetic acid and the bands for gelatinase A (MMP-2)nd gelatinase B (MMP-9) were visualized in a gel documentationystem (Bio-Rad, Molecular Image Chemi DocTM XRS+ with ImageabTM Software).

.16. Quantification of the results

Bands of zymography and western blots were quantitated usingmage J Launcher (version 1.4.3.67).

.17. Assessment of ROS generation

ROS generation in sputum cells was measured by flow cytome-ry. Cells were diluted with 1.0 ml of Hank’s balanced salt solutionHBSS) containing NaCl (0.15 M) and HEPES (5 mM), pH 7.35. There-fter DCFH-DA (0.5 mM) solution in dimethyl formamide wasdded to the cell suspension and incubated at 37 ◦C for 30 min inarkness. Finally the samples were washed in ice-cold phosphate-uffered saline and 15,000 events were acquired in flow cytometersing Cell Quest software equipped with a 488 nm argon laser and

525 ± 10 nm band pass emission filter. Fluorescence was deter-ined with FL-1H channel. Respiratory burst and generation of ROS

esulted in green fluorescence that was expressed as MFI.

.18. Evaluation of DNA damage by comet assay

DNA damage (single strand breaks) was measured by alka-ine single cell gel electrophoresis (SCGE) or comet assay (Singht al., 1988). Briefly, airway cells (1 × 104) were suspended in.6% (w/v) low melting agarose and layered over a frosted micro-copic slide previously coated with a layer of 0.75% normal meltinggarose to ensure firm gripping. The slides were then kept at 4 ◦Cor solidification. Subsequently slides were immersed in a lysisuffer of pH 10 and left overnight for lysis of cell membrane anduclear membrane. Next day, slides were pre soaked for 20 min inlectrophoresis buffer (alkaline solution of 300 mM NaOH, 1 mMa2EDTA; pH 13.0) in order to unwind DNA. Electrophoresis was

hen carried out for 20 min (300 mA, 20 V). Slides were then washedhrice with neutralizing buffer (Tris 0.4 M, pH 7.5), stained withthidium bromide (final concentration 40 �g/ml) and examinednder a fluorescence microscope (Leica DM 4000B, Germany).omet tail moment was calculated using software Komet 5.5.

.19. Determination of oxidative DNA damage

The quantitative measurement of 8-OHdG was done with ELISAit according to manufacturer’s protocol. In brief the unknown 8-HdG plasma samples or 8-OHdG standards were first added to an

month in US$,mean ± SD

*P value was calculated with Students t test and Chi square test.

8-OHdG/BSA conjugate preabsorbed microplate. After a brief incu-bation, an anti-8-OHdG monoclonal antibody was added, followedby an HRP conjugated secondary antibody. The 8-OHdG contentof the control and As-exposed plasma samples was determinedby comparison with predetermined 8-OHdG standard curve. Thekit had an 8-OHdG detection sensitivity range of 100 pg/mL to20 ng/mL.

2.20. Statistical evaluation of data

Statistical package SPSS windows version 10.0 (SPSS, Chicago,IL) was used for data entry and analysis. The statistical signifi-cance between As exposed and control groups were determinedusing the one way ANOVA followed by Student t-test, where signif-icance level was set at 0.05 and Chi square test. Analysis of variance,regression analysis and test of significance were applied. Results areexpressed as mean ± SD (standard deviation). Univariate, bivari-ate and multivariate analyses were done according to the merit ofthe data variables. Odd’s ratio and correlation coefficient was mea-sured to find the degree of association between the variables underconsideration.

3. Results

3.1. Demographic characteristics of the study population

The socioeconomic and demographic parameters of the con-trol and exposed group of women have been depicted in Table 1.There was no significant difference between As exposed and con-trol population with respect to age, body mass index (BMI), years ofschooling, occupation, household work, household and agriculturalwork together, cooking fuels used at home, LPG, biomass and mem-bers in family. However family income per month had a significantdifference between exposed and control women.

3.2. Arsenic level in water

We had collected 25 groundwater samples from tube wells ofNarayanpur and 20 from Boria. The samples were detected for Ascontent by AAS-VGA method. The results showed that As concen-
tration in groundwater of Narayanpur had a mean of 22.93 ± 10.1(SD) �g/L and the median value was 23.94 �g/L. In Boria, the meanAs levels was 1.16 ± 0.15 �g/L and the median value was 1.22 �g/L.The mean value of the groundwater As contamination at Narayan-pur was significantly higher (P < 0.0001; Fig. 1a) than the mean As

568 K. Dutta et al. / International Journal of Hygiene and Environmental Health 218 (2015) 564–574

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ig. 1. Comparison of As content in the groundwater from chronic low level As-expndividuals from chronic low level As-exposed area and control area (b); correlatio

omen (c).

evel of Boria. Thus, from here onwards we will refer to the areaith <10 �g/L of As level in groundwater as control area and the

ubjects enrolled from these places as control subjects. Similarly,e refer to the area with the As level in between 11 and 50 �g/L of

s chronically low As-exposed area and subjects enrolled from thisrea as exposed group.

.3. Arsenic in nails

Few nail samples were analyzed at random from exposed andontrol group to have an estimate of the individualized chronic Asxposure. The As level in the nail samples of the control people (N,6)as 2.15 ± 1.27 �g/g. In contrast, the mean As concentration in theail samples of the chronic low arsenic exposed people (N,12) wasignificantly higher 61.37 ± 21.99 �g/g (P = 0.0002; Fig. 1b).

The As level in groundwater showed a positive correlation withs content in nails where Pearsons r was 0.9281 and 95% confidence

nterval (CI) was 0.8192–0.9724 (Fig. 1c). The correlation coefficientas highly significant P < 0.001. The regression coefficient (R2) was

.8614.

rea [As – 11–50 �g/L] and control area (a); comparison of As content in the nails ofrsenic content in groundwater with the arsenic content in the nails of the exposed

3.4. Effect of low level arsenic exposure on CD14 expression

The flow cytometric analysis representing the difference inmean fluorescence intensity (MFI) of CD14 expressing mono-cytes of chronically low As-exposed women and control hasbeen depicted in Fig. 2a. The MFI of CD14 positive monocytesof the chronically low As-exposed group was significantly higher(187.8 ± 42.15, P < 0.0001) than the control 95.38 ± 44.52 (Fig. 2b).The difference in the percentage of CD14 expressing monocyteswas not statistically significant (P = 0.0899) between the groups.This percentage of monocytes has been reported based on theparent gate of monocytes in FSC/SSC plot. It is not percentageof monocytes obtained from differential count. The differentialwhite blood count varied from 2 to 3% of PBMCs in controlpopulation (As <10 �g/L) and 3 to 5% of PBMCs in exposed pop-
ulation (As 11–50 �g/L). The gating strategy with the percentageof CD14+ monocytes has been shown in the Supplementary Figs. 1and 2.
Supplementary Fig. 1 related to this article can be found, in theonline version, at http://dx.doi.org/10.1016/j.ijheh.2015.06.003

K. Dutta et al. / International Journal of Hygiene and Environmental Health 218 (2015) 564–574 569

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of TNF-� and NF-�� (P < 0.0001) expression in leucocytes (Fig. 3c)and sputa (Fig. 3d) of the exposed women with respect to control.Chronic low level As exposure (11–50 �g/L) was found to be posi-

ig. 2. Flow cytometric analysis of CD14 expression in monocytes. Representative fiow As-exposed [As – 11–50 �g/L] women than control (a); MFI of CD14 in chronicf arsenic content in groundwater with CD14+ monocytes of exposed women (c).

Supplementary Fig. 2 material related to this article can beound, in the online version, at http://dx.doi.org/10.1016/j.ijheh.015.06.003

However the total number of monocytes/�l varied signifi-antly (P < 0.05) between control (196 ± 23) and exposed groups243 ± 37). The total number of monocytes was 23.97% higher inxposed than control which was calculated as follows

(Mean of total no. of monocytes for exposed) − (Mean of total no. of

Mean of total no. of monocytes for control

The As level in groundwater showed a positive correlationith CD 14 expression on monocytes of chronically low As-

xposed women, where Pearsons r was 0.9191 and 95% CI was.7584–0.9745. The correlation coefficient was highly significant

< 0.001and R2 was 0.8448 (Fig. 2c).

.5. Pro inflammatory signaling

The Western blot analyses (Fig. 3a) as well as quantitativessessment of the band intensities (Fig. 3b) exhibited that bothF-�B and TNF-� were considerably up-regulated in the chronic

of FACS analysis showing pronounced expression of CD14 in monocytes of chronics-exposed women was significantly higher (P < 0.001) than control (b); correlation

ocytes for control) × 100 = 243 − 196/196 × 100 = 23.97

low As-exposed leucocytes (N, 35) compared to that of the control(N, 23). The mean band intensities reflected significant up regu-lation of NF-�B and TNF-� (P < 0.05). The ELISA results reflecteda similar modulation of protein profile with significant increase

tively associated with up regulated expression of NF-�B {plasma:[OR = 2.59, 95% CI: 1.55–4.30]; sputa:[OR = 2.25, 95% CI: 1.38–3.66];leucocytes: [OR = 4.8, 95% CI: 1.45–15.86]} and TNF-� {plasma:[OR = 2.01, 95% CI: 1.19–3.39]; sputa:[OR = 2.11, 95% CI: 1.29–3.43];leucocytes: [OR = 6.29, 95% CI: 1.57–25.09]}.


Fig. 3. Signaling related with pro-inflammatory responses. The pronounced expression ocontrol as depicted by representative figure of western blot (a); mean band intensities (asof leucocytes obtained from ELISA (c); mean OD of sputum derived airway cells obtained

Table 2Comparison of pro and anti-inflammatory circulating mediators in control and As-exposed women.

Group Control (N, 131) As-exposed (N,142)

Plasma TNF-� (pg/ml) 9.3 ± 3.2 14.2 ± 8.2*

Plasma IL-8 (pg/ml) 15.3 ± 3.8 26.3 ± 3.3*

Plasma IL-6 (pg/ml) 5.6 ± 1.9 9.4 ± 2.4*

Plasma IL-10 (pg/ml) 3.2 ± 0.3 2.5 ± 0.6*

Plasma IL-12 (pg/ml) 15.1 ± 2.8 24.6 ± 5.9*

V

3

�t6rtcmiac(6

3.8. ROS generation, DNA damage and oxidative DNA adducts

alues are mean ± SD.* P < 0.001 compared with control in unpaired Student’s t test.

.6. Circulating pro-inflammatory cytokines

The mean concentration of the pro-inflammatory cytokine TNF- in blood plasma of As-exposed subjects was 52.7% higher than

hat of control (P < 0.001). Similarly, the levels of plasma IL-8, IL- and IL-12 were 71.8%, 67.8%, and 62.9% higher than the control,espectively (P < 0.001; Table 2). In contrast, the plasma level ofhe anti-inflammatory cytokine IL-10 was 21.8% lower than theontrol (P < 0.001). Collectively, the results suggest systemic inflam-ation in women who were chronically exposed to low level of As

n drinking water. After controlling for age, occupation, education
nd family income as potential confounders, a strong positive asso-iation was found between As in drinking water with plasma TNF-�OR = 2.66, 95% CI: 1.60–4.43), IL-8 (OR = 2.61, 95% CI: 1.55–4.38), IL-
(OR = 2.45, 95% CI: 1.50–4.00), IL-12 (OR = 1.66, 95% CI: 1.60–3.43)

f NF-�B and TNF-� in leucocytes of low As-exposed [As – 11–50 �g/L] women than calculated by Image J software) obtained from western blot analyses (b); mean OD

from ELISA (d).

and a negative association between As in water and plasma IL-10(OR = 2.61, 95% CI: 1.55–4.38).

3.7. Activity of inflammatory gelatinases

The gelatin zymography elicited enhanced activity of MMP-2and MMP-9 in sputa of chronic low As-exposed women than con-trol. The representative zymograms showing the increased activityof the gelatinases in the sputa of the exposed than control groupshave been depicted in Fig. 4a and b respectively. The mean banddensities of MMP-2 was 7.94 ± 6.02 in control vs 13.6 ± 6.9 in lowAs-exposed group [P < 0.001] and that of MMP-9 was 12.18 ± 7.76in control vs 23.28 ± 11.26 in sputa of low As-exposed women[P < 0.001] (Fig. 4c). However with sera we did not observe any sig-nificant band of MMP-2. Elevated activity of MMP-9 was observedwhich was reflected with a mean band density of 11.71 ± 3.47in control vs 38.04 ± 13.67 in sera of low As-exposed women[P < 0.001] (Fig. 4d). The activity of the gelatinases showed a strongpositive association {sputa: [MMP-2, OR = 3.47, 95% CI: 2.08–5.8;MMP-9, OR = 3.2, 95% CI: 1.95–5.26]; sera: [MMP-9, OR = 1.88, 95%CI: 1.16–3.04]} with groundwater As.

Chronic low As exposure was found to enhance the ROS gen-eration (233.2 ± 44.7) in sputa compared to control (26.3 ± 15.2)[P = 0.001; Fig. 5a]. The DNA damage of the As-exposed airway cells

K. Dutta et al. / International Journal of Hygiene and Environmental Health 218 (2015) 564–574 571

F sed w( tensitw sed w

wTi(glTdoc

4

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htWi

ig. 4. Enhanced activity of gelatinases in low level arsenic [As – 11–50 �g/L] expoMMP-9) in sputa of control (a) and exposed women (b); enhanced mean band inomen than control (c); enhanced mean band intensities of MMP-9 in sera of expo

as observed to be exacerbated in comparison to control (Fig. 5b).he mean comet tail moment of the airway cells was 3.7 ± 2.5 �mn control vs 6.8 ± 3.4 �m in low As-exposed women [P = 0.0008]Fig. 5c). The mean plasma 8OHdG level 33.13 ± 12.10, in the controlroup, was significantly elevated [P = 0.0074] than the mean 8OHdGevel 122.58 ± 73.53, in the chronic low As-exposed group, (Fig. 5d).he increase in ROS generation [OR = 1.65, 95% CI: 1.01–2.68], DNAamage [OR = 1.65, 95% CI: 1.01–2.68] and enhancement in the levelf 8OHdG [OR = 1.89, 95% CI: 1.14–3.13] exhibited a positive asso-iation with groundwater As.

. Discussion

Arsenic in groundwater is a worldwide public health concern.espite of the numerous efforts for reducing the levels of Asxposure it is still a dreadful threat for many countries includ-ng Bangladesh, China, Chile, Argentina, Australia, Mexico, Taiwan,ietnam, south west United States of America and India. In all the 19istricts of West Bengal, India, analyses of 140,150 hand tube-wellater samples revealed that 48.1% tube-wells had As concentra-

ions above 10 �g/L (the WHO guideline value) and 23.8% above0 �g/L (the standard value of As in many developing countries)Chakraborti et al., 2009).

People exposed to chronic low level As (11–50 �g/L) may notave characteristic As-related skin lesions like palmar and plan-ar keratosis and hyperkeratosis but may be sub clinically affected.

e have previously reported that such low level As exposure maynflict pulmonary and systemic inflammation (Sinha et al., 2014),

omen in comparison to control. Activity of gelatinase A (MMP-2) and gelatinase Bies (as calculated by Image J software) of MMP-2 and MMP-9 in sputa of exposedomen than control (d).

lung function decrement in adult never smoker men (Das et al.,2014) and depression in women (Mukherjee et al., 2014). This studymay be regarded as an addition to our existing knowledge of chroniclow level As related systemic inflammation where we have shownthe probable role of CD14 in pro inflammatory signaling and DNAdamage.

CD14 is a multifunctional high-affinity receptor for endotox-ins, lipopolysaccharides and other bacterial wall components. It hasbeen implicated in the development and maturation of the innateimmune system. CD14 has been reported to induce pro inflam-matory cytokine expression by crosslinking TLR4 to downstreamenzymes that activate NF-�B and AP-1 (Akira and Takeda, 2004).TNF is one of the best described pro inflammatory cytokines whichis related with host innate immunity, inflammation, and apoptosis(Surbatovic et al., 2013). TNF-� exerts it is inflammatory responsespartially via activation of NF-�B, which in turn influences iNOS,cytokines and adhesive molecules (Xu et al., 1998). Increased secre-tion of inflammatory biomarkers like IL-8, TNF-�, and TGF-� hasbeen related with As exposure in urothelial cells (Liu et al., 2014).Elevated levels of CD14 and several other inflammatory cytokineswere observed to be elevated in isolated lymphocytes from chronicAs exposed population reported with atherosclerosis (Wu et al.,2003). In congruence with the above studies we observed that thelow As exposed women had elevated expression of CD14 on mono-
cytes which showed a positive correlation with As in groundwaterof that area. The women exhibited a strong positive correlation ofAs content of the nails with the groundwater As. Moreover theselow As exposed women elicited pronounced expression of TNF-� and NF-�B in their leucocytes, sera and sputa. They were also


F tion ie cantlyt = 0.00( expos

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ig. 5. Oxidative stress induced by chronic low level As-exposure. Mean ROS generanhanced (P < 0.0001) than normal [As < 10 �g/L] (a); mean 8OHdG level was signifihan normal [As < 10 �g/L] (b); mean comet tail moment was significantly higher (P

c); comet assay showing enhanced DNA damage in airway cells of chronic low As-

bserved with elevated levels of plasma pro-inflammatory medi-tors including TNF-�, IL-6, IL-8, IL-12 as well as decreased levelf plasma anti inflammatory cytokine IL-10 which suggested thatD14, might have at least in part, contributed toward inflamma-ion. This pro-inflammatory response might have been due to bysterile”/non-pathogenic triggers like As.

Several reports suggest that inflammatory cytokines are theost potential inducer of matrix metalloproteinases (MMPs).MP-2 and MMP-9 have been related with airway inflammation

Mautino et al., 1997; Yang et al., 2005). Asthma patients wereeported with increased activity of MMP-2 and MMP-9 and higherevels of tissue inhibitor of metalloproteinase-1 (TIMP-1) in theirputum (Cataldo et al., 2000). During airway inflammation transac-ivation of NF-�B was reported to be associated with TNF-� induced

MP-9 gene expression (Lin et al., 2008). Our study also revealedhat women who were chronically exposed to low level of As hadncreased activity of MMP-2 and MMP-9 in their sputa along withncreased expression of NF-�B and TNF-�. MMP-9 activity was alsoncreased in the sera of these exposed women.

Arsenic exposure has been well established as a potent inducerf oxidative stress which induces genotoxicity (Kesari et al., 2012;ie et al., 2014) and oxidative DNA adducts (Hinhumpatch et al.,013; Pei et al., 2013). In this aspect our findings also confirmedhat chronic low level As exposure induced ROS generation andNA damage in airway cells, and high levels of 8OHdG in plasma

amples of exposed women.Oxidants also promote inflammation by activating NF-�B or

P-1, which orchestrates the expression of multiple inflamma-ory genes. ROS have been shown to induce gene expression of
nflammatory mediators, such as IL-1 and TNF-� (Rahman and
acNee, 2000; Rahman et al., 2001). Oxidative tissue damage cane also translated into an inflammatory response via recognition ofxidized phospholipids by the pattern recognition receptor TLR2,

n airway cells of chronic low As-exposed [As, 11–50 �g/L] women was significantly higher (P = 0.0074) in plasma of chronic low As-exposed [As, 11–50 �g/L] women

08) in chronic low As-exposed [As – 11–50 �g/L] women than normal [As < 10 �g/L]ed [As – 11–50 �g/L] woman than normal [As < 10 �g/L] (d).

thus providing evidence for a role of the innate immune sys-tem in recognition of an oxidatively modulated microenvironment(Kadl et al., 2011). In addition, oxidative stress has been regardedas contributor of proteinase–antiproteinase imbalance, both byinactivating antiproteinases, such as �1-antitrypsin and secretoryleukocyte proteinase inhibitor, and by activating proteinases, suchas MMP-9 (Lee and Yang, 2012). Thus inflammation and oxida-tive stress form an interwoven network where persistence of anyone factor would act as a feedback loop for the other. Moreoverprolonged inflammation initiated by macrophage subtypes, mastcells, myeloid progenitors, and neutrophils which may lead also totumor-promoting activities (Hanahan and Weinberg, 2011). Sincethe sputa were observed with elevated inflammatory markers theywere investigated for DNA damage which was found to be enhancedin the airway cells. The systemic inflammation was also observedto be associated with increased level of 8OHdG. Therefore chroniclow level As exposure which was linked with pulmonary and sys-temic inflammation may not only lead to deleterious consequencesof oxidative stress and continue the cycle of inflammation but inthe long run may also increase the risk of cancer among these ruralwomen.

In summary, we found that chronic low level As exposuremay lead to pronounced expression of CD14 on the surface ofmonocytes. This in turn might up regulate pro inflammatorysignaling molecules like TNF-� and NF-�B to enhance inflammatoryresponses and oxidative stress. However, this study has some limi-tations. First, it is a cross-sectional study which limits our inferenceon the causal direction. Second, we did not know the As levels in theparticipants’ drinking water over a period of time. So how much As a
woman has actually ingested in the past 10 or 15 years is only spec-ulative. Third we have not used any other marker for monocytes likeCD91 and we have not looked for subsets of monocytes like – classi-cal CD14++CD16−, intermediate CD14++CD16+, and non-classical

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D14+CD16++ monocytes (Ziegler-Heitbrock et al., 2010) and theirole during inflammation. Fourth we did not explore the possibilityf other co-minerals and metals in the water samples contributingo health outcomes. Moreover sources of As from food chain haveot been measured in this study. Fifth, there are several other envi-onmental pollutants like agricultural pesticides that may causenflammation. Despite these shortcomings, our sample size wasarge enough to indicate that low level As exposure elicited a pro-nflammatory profile which might have been partially contributedy CD14 expressing monocytes and prolong persistence of inflam-ation might have promoted oxidative DNA damage in the ruralomen. In essence, these findings suggest that the people exposed

o As contaminated (11–50 �g/L) groundwater may be sufferingrom the deleterious consequences of inflammation and oxidativetress for which mass awareness and supply of safe water is theeed of the hour.

onflict of interest

The authors declare no conflict of interest.

cknowledgements

The authors are thankful to Indian Council of Medical Researchor providing the fellowship grant of PP (3/1/3/JRF-2012/HRD-21).he authors are indebted to Prof. (Dr.) Jaydip Biswas, Direc-or, Chittaranjan National Cancer Institute, Kolkata for providinghe infrastructural facilities. The authors would like to thank Dr.ipankar Chakraborti, Research Director, School of Environmentaltudies, Jadavpur University, Kolkata for his guidance during thersenic analyses.

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