sex-specific patterns and deregulation of endocrine pathways in the gene expression profiles of...

Sex-specic patterns and deregulation of endexpression proles of Bangladeshi adults exp

Th, NYrk, Unive

Article history:Received 2 January 2015Revised 25 February 2015Accepted 26 February 2015Available online xxxx

Keywords:Arsenic

her-Wolz et al., 2009).ce to indicate that Aspathogenesis by alter-he level of DNAmeth-1).docrine disruptor in-

Toxicology and Applied Pharmacology xxx (2015) xxxxxx

YTAAP-13325; No of Pages 9

Contents lists available at ScienceDirect

Toxicology and Appl

j ourna l homepage: www.e llung, bladder, liver and prostate cancers (Benbrahim-Tallaa and 2003; Davey et al., 2007; Flora, 2011; SchuhmacIn addition, there is a growing body of evidenacts through epigeneticmechanisms as part of itsing the abundance of histone modications and tylation (Reichard and Puga, 2010; Ren et al., 201

The evidence to suggest that As acts as an en

Abbreviations:AR, androgen receptor; As, arsenic; CVD, cardiovascular disease; ER, es-trogen receptor; FACT, Folic Acid Creatinine Trial; GR, glucocorticoid receptor; HEALS,HealthEffectsofArsenicLongitudinalStudy;HSD17B,17-Hydroxysteroiddehydrogenase;Introduction

Inorganic arsenic (iAs), a known human carcinogen, naturallycontaminates drinking water and currently affects approximately 150million people in at least 70 countries throughout the world (IARC,2004; Ravenscroft et al., 2009). Exposure to As is associated with skin,

Waalkes, 2008; Rahman et al., 2009), cardiovascular disease (Chenet al., 2013; Tseng et al., 2003), diabetes (Pan et al., 2013), and hyperten-sion (Chen et al., 1995). Themechanisms that underlie arsenic's diversepathogenesis remain unclear and are thought to include oxidativestress, endocrine disruption, alteration of cell signaling and proliferationprocesses, and impairment of DNA damage responses (Andrew et al.,HSD,hydroxysteroiddehydrogenase; SCAN, single channeicity protein 1; SR, steroid receptors; uAs, urinary As;wAs Corresponding author. Tel.: +1 845 731 3515; fax: + Correspondence to: M. Costa, Department of EnviroUniversity School of Medicine, 57 Old Forge Road, Tuxed305 7949; fax: +1 212 305 3857.

E-mail addresses:[email protected] (M.V. Gamble)(M. Costa).1 A. Muoz and Y. Chervona contributed equally to this

http://dx.doi.org/10.1016/j.taap.2015.02.0250041-008X/ 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Muoz, A., et al.,Bangladeshi adults exposed to arsenic contaHumansHSD17B-1Cardiovascular diseaseArsenic contamination of drinkingwater occurs globally and is associatedwith numerous diseases including skin,lung and bladder cancers, and cardiovascular disease. Recent research indicates that arsenicmay be an endocrinedisruptor. This studywas conducted to evaluate the nature of gene expression changes amongmales and femalesexposed to arsenic contaminated water in Bangladesh at high and low doses. Twenty-nine (55% male)Bangladeshi adults with water arsenic exposure ranging from 50 to 1000 g/L were selected from the FolicAcid Creatinine Trial. RNA was extracted from peripheral blood mononuclear cells for gene expression prolingusing Affymetrix 1.0 ST arrays. Differentially expressed genes were assessed between high and low exposuregroups for males and females separately and ndings were validated using quantitative real-time PCR. Therewere 534 and 645 differentially expressed genes (p b 0.05) in the peripheral blood mononuclear cells of malesand females, respectively, when high and low water arsenic exposure groups were compared. Only 43 genesoverlapped between the two sexes, with 29 changing in opposite directions. Despite the difference in gene setsboth males and females exhibited common biological changes including deregulation of 17-hydroxysteroid de-hydrogenase enzymes, deregulation of genes downstream of Sp1 (specicity protein 1) transcription factor, andprediction of estrogen receptor alpha as a key hub in cardiovascular networks. Arsenic-exposed adults exhibitsex-specic gene expression proles that implicate involvement of the endocrine system. Due to arsenic'spossible role as an endocrine disruptor, exposure thresholds for arsenic may require different parameters formales and females.

2015 Elsevier Inc. All rights reserved.aa r t i c l e i n f o b s t r a c tcontaminated drinking water

Alexandra Muoz a,1, Yana Chervona a,1, Megan Hall b,a New York University School of Medicine, Nelson Institute of Environmental Medicine, Tuxedob Department of Epidemiology, Mailman School of Public Health, Columbia University, New Yoc Department of Environmental Health Sciences, Mailman School of Public Health, Columbia Ul arraynormalization; Sp1, spec-, water As1 845 731 2118.nmental Medicine, New Yorko, NY 10987, USA. Tel.: +1 212

, [email protected]

work.

Sex-specic patterns and deminated..., Toxicol. Appl. Pharocrine pathways in the geneosed to arsenic

omas Kluz a, Mary V. Gamble c,, Max Costa a,, USASArsity, New York, USA

ied Pharmacology

sev ie r .com/ locate /ytaapcludes in vitro activation of steroid hormone receptors (Bodwell et al.,2006; Davey et al., 2007), animal models with altered fertility (Changet al., 2007; Jana et al., 2006) and sex-specic disease distributions(Waalkes et al., 2003), aswell as human studies indicating altered fertil-ity (Hsieh et al., 2008; Xu et al., 2012), sex-specic disease distributions(Aballay et al., 2012), and sex-linked changes in methylation proles(Pilsner et al., 2012). Previous research from our lab supports this

regulation of endocrine pathways in the gene expression proles ofmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.025

trend with the observed sex-specic changes in the abundance of his-tone modications in Bangladeshi adults exposed to As contaminateddrinking water (Chervona et al., 2012). Microarray studies that haveassessed gene expression changes in response to As contaminatedwater have evaluated changes with males and females pooled together(Andrew et al., 2008; Lu et al., 2001; Wu et al., 2003) or with only fe-males (Wu et al., 2003). However, given the potential endocrine activityof As, in particular its possible role as a xenoestrogen (Jana et al., 2006;Waalkes et al., 2004), division of sexes in assessing its impact may benecessary to understand its pathogenesis. Indeed a recent studyconducted among Flemish adults exposed to an array of toxins,including xenoestrogens, exhibited sex-specic transcriptomic re-sponses (De Coster et al., 2013) that were revealed when sexes wereevaluated separately.

Here we evaluate the gene expression proles of RNA from theperipheral blood mononuclear cells (PBMC) of Bangladeshi adults(n = 29) chronically exposed to As contaminated drinking water.Subjects with high water As exposure are compared to those with lowwater As exposure among males and females separately. To evaluatethe sex-specic proles we compare the areas of biological relevance

patterns of 29 study participants from the FACT clinical trial. Our study

and females (n = 13) separately. Among the males there were9 males with low exposure and 7 with high exposure. Among the fe-males there were 6 females with low exposure and 7 with highexposure. Please see Table 1 for other information regarding thedemographic features of the groups.

Sample collection and handling. Blood samples were obtained by veni-puncture, and collected in EDTA-containing vacutainer tubes, whichwere then placed in IsoRack/IsoPack cool packs (Brinkmann Instru-ments). Samples were transported in hand-carried coolers to the locallaboratory at the eld clinic in Araihazar, within 4 h of collection. Sam-pleswere centrifuged for 10min at 3000g at 4 C, and the plasma frac-tion was stored at 80 C. PBS was added to the remaining cellsfollowed by a coll-hypaque gradient extraction of PBMCs using stan-dard techniques. PBMCswere stored at80 C. Urine sampleswere col-lected with 50-mL acid-washed polypropylene tubes. Within 4 hsamples were transported from portable coolers to freezers (20 C)in the Araihazar laboratory, and were then hand carried on dry ice toNew York.

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2 A. Muoz et al. / Toxicology and Applied Pharmacology xxx (2015) xxxxxxpopulation is comprised of adults ranging in age from 26 to 53. Herethe effects of relatively low exposure (50200 g/L) to water arsenicare compared to high exposure (2501000 g/L) for males (n = 16)

Table 1General characteristics for the total study sample by sex and water As groupa.

Total sample(n = 29)

Males, low water Asb

(n = 9)

Age 40.0 7.2 42.6 6.4BMI, kg/m2 20.4 2.9 19.7 2.1Education, years 2.9 3.7 2.8 3.4Water As g/L 246.4 207.4 103.0 33.7Urinary As, g/L 264.1 349.3 116.8 76.4Urinary creatinine (mg/dL) 75.0 41.5 75.3 40.9Urinary As/mg creatinine 345.8 324.9 165.5 74.9Blood As (g/L) 11.4 10.3 6.8 3.1Male, % 55.2 Land ownership, % 37.9 44.4Television ownership, % 24.1 11.1Current cigarette smoking, % 44.8 77.8Current betel nut use, % 34.5 55.6

a Mean SD unless otherwise noted.b Water As 50 to 150 g/L.c Water As 232 to 500 g/L.d Water As 50 to 200 g/L.e Water As 250 to 1000 g/L.for each sex and evaluate to what extent the same genes are foundamong biologically relevant areas that are common to males andfemales.

Materials and methods

Study site and subject recruitment. The Health Effects of Arsenic Longitu-dinal Study (HEALS) cohort is part of Columbia University's SuperfundResearch Program and currently includes approximately 30,000 partic-ipants (Ahsan et al., 2006). The present study utilizes samples from asubset of 600 HEALS participants who were recruited for enrollmentinto the Folic Acid and Creatine Trial (FACT), a clinical trial that aimedto lower blood As concentrations with nutritional supplementationand therefore required that participants be drinking from wells withwater As concentrations greater than or equal to 50 g/L. Fieldworkwas completed in June 2011. Study participants were given a water l-tration system for removal of As at the time of enrollment. Participantswith renal or gastrointestinal diseases, those taking nutritional supple-ments, or who were pregnant and/or planning to become pregnantwere not included in the study. This study evaluates the gene expressionPlease cite this article as: Muoz, A., et al., Sex-specic patterns and deBangladeshi adults exposed to arsenic contaminated..., Toxicol. Appl. PharWater and urinary As. Water sample collection, sample handling, andanalysis for this study were performed as previously described(Chervona et al., 2012). The water As samples were analyzed atColumbia University's Lamont Doherty Earth Observatory by inductive-ly coupled mass spectrometry (ICPMS), with a detection limit of0.1 g/L. Total urinary As (uAs) concentrations were measured atColumbia University Trace Metals Core Lab by GFAA spectrometryusing an AAnalyst 600 graphite furnace system (PerkinElmer), as de-scribed (Nixon et al., 1991). This laboratory is part of a quality controlprogram run by the Institut de Sante Publique du Quebec, Canada.Intraclass correlation coefcients (ICCs) were 0.99 between the Colum-bia laboratory's values and samples calibrated at the Quebec laboratory.To correct for hydration status, urinary creatinine was analyzed using amethod based on the Jaffe reaction.

RNA isolation, amplication, and hybridization. Total RNA was extractedfrom each sample according to the TRIzol (Invitrogen) manufacturer'sprotocol, and puried using RNeasy Plus Micro Kit (Qiagen). Sensestrand cDNA probes were synthesized (amplied) using an AmbionWhole Transcript Expression Kit. Amplied single stranded cDNA wasfragmented and labeled using a GeneChip WT Terminal Labeling Kit(Affymetrix). The fragmented and labeled DNA underwent hybridiza-tion with Affymetrix GeneChip Human Gene 1.0 ST Array that contains28,869 well-annotated genes. Samples were processed in two batches

ales, high water Asc

n = 7)Females, low water Asd

(n = 6)Females, high water Ase

(n = 7)

1.7 6.8 36.7 9.6 37.7 5.68.2 1.1 23.8 2.1 20.6 3.0

5.0 4.5 4.0 3.854.5 136.2 116.7 61.9 433.9 274.256.3 597.3 113.3 99.7 390.6 281.78.0 38.8 74.5 54.7 91.9 33.950.2 450.7 178.9 186.7 416.3 250.10.7 15.3 7.1 7.2 11.7 7.3

2.9 33.3 28.64.3 33.3 42.95.7 0 02.9 16.7 14.3regulation of endocrine pathways in the gene expression proles ofmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.025

3A. Muoz et al. / Toxicology and Applied Pharmacology xxx (2015) xxxxxxwith representatives from each exposure category in each batch andwere randomized during processing.

Data analysis to identify differentially expressed genes. Gene expressionanalyses were performed using R. Gene expression data were importedand normalized using a single-sample method, single channel arraynormalization (SCAN) (Piccolo et al., 2012), which summarizes probesto Entrez IDs, in R 2.15.0 GUI 1.53 64-bit. Datawere processed to expres-sion sets using the Affymetrix package version 1.30.0. in R 2.15.1 GUI1.42 Leopard build 64-bit, and were ltered to remove negative expres-sion values as per SCAN protocol. Unsupervised hierarchical clusteringusing the Pearson correlation method was used to evaluate potentialbatch effects. Surrogate variable analysis was used to estimate sevensurrogate variables and performed in order to remove batch effectsand other sources of expression heterogeneity (Leek and Storey,2007). Seven surrogate variables were incorporated into the analysisand such incorporation was used in lieu of correcting for demographicfeatures of the population,whichwas not possible due to the small sam-ple size. Signicance of gene expression changes for males and femaleswere assessed separately between high and low water As exposuregroups using a gene-wise linear model approach with LIMMA 3.14.4(Smyth, 2004). LIMMA utilizes an empirical Bayes approach to generatemoderated t-statistics by taking into account the standard errors and es-timated log-fold changes. Probe summarization and ltration occurredprior to LIMMA analysis and therefore 11,623 genes were tested for sig-nicance using moderated t-statistics and a p-value threshold ofp b 0.05.

Heat maps were generated using the heatmap.2 function in R andunsupervised hierarchical clustering was governed by Pearson correla-tion method. Networks and functional analysis were generated usingIPA (Ingenuity Systems, www.ingenuity.com). Gene set enrichmentanalysis was performed using the molecular signature database(Broad Institute Gene Set Enrichment Analysis, http://www.broadinstitute.org/gsea). To evaluate the inuence of cigarette smoking,lists of differentially expressed geneswere evaluated for the presence ofknown biomarkers of cigarette smoking (van Leeuwen et al., 2005) andstatistical signicance of biomarker representationwas assessed using ahypergeometric distribution as previously described (Draghici et al.,2003). All microarray data is MIAME compliant and the raw data hasbeen deposited in NCBIs Gene Expression Omnibus (GEO), and assignedseries accession number GSE57711.

Quantitative real-time PCR. Gene expression changes were validatedusing quantitative real-time polymerase chain reaction (q-RT-PCR).Total RNA was extracted from each sample using TRIzol Reagent(Invitrogen), and converted to single stranded cDNA using SuperscriptIII (Invitrogen). q-RT-PCR analysis was performed using SYBR greenPCR system (Applied Biosystems) on ABI prism 7900HT system(Applied Biosystems). All q-RT-PCR reactions were performed in tripli-cate. Relative gene expression level, normalized to 18s rRNA expression,was presented asmedian relativemRNA values SD (n=9) for partic-ipants in low and high exposure groups. Statistical signicancewas test-ed using two-tailed, unpaired t-test. The expression of the target geneswas rst normalized to the expression of the house-keeping geneglyceraldehyde 3-phosphate dehydrogenase (GADPH).

Results

Demographic data

Study population characteristics are presented in Table 1 for thestudy population by water As exposure category (low vs. high). Briey,a total of 29 participants were selected for this study with 16males and13 females. Among the males the average age was 42.6 6.4 for the

males with low exposure and 41.7 6.8 for the males with high

Please cite this article as: Muoz, A., et al., Sex-specic patterns and deBangladeshi adults exposed to arsenic contaminated..., Toxicol. Appl. Pharexposure. Among the females the average age was 36.7 9.6 for fe-maleswith lowexposure and 37.75.6 for the femaleswith high expo-sure. The median water As concentration for the low exposure groupwas 103 g/L for males and 117 g/L for females (range 50200 g/L).For the high exposure group, the median water As concentration was355 g/L for males (range 250500 g /L) and 434 g/L for females(range 2321000 g/L).

Gene expression analysis

In the PBMCs of highly exposed males, there were 534 differentiallyexpressed genes (p b 0.05), when compared tomaleswith lowAs expo-sure, with 271 up-regulated and 264 down-regulated. For the highly ex-posed females there were 645 differentially expressed genes (p b 0.05)when compared to females with low water As exposure, with 303 up-regulated and 342 down-regulated. The unsupervised hierarchical clus-tering analysis for differentially expressed genes of males (Fig. 1A) andfemales indicates clear clustering based on water As exposure group(low vs. high) for males and females (Fig. 1B), with the exception ofone outlier for females, indicating that dose is driving clustering in ourlists. Only 43 genes overlapped between the two sexes, with 29 out of43 genes changing in opposite directions (Fig. 1C; Table 2). Biologicalrelevance of gene expression was assessed via network analysis usingIPA and top ranked networks and enrichment scores are presented inTable 3.

Differentially expressed genes were also evaluated for highly rankedgene signatures using GSEA database and the top ranked signaturesfrom all possible signatures to query are presented in Supplementarymaterial, Table S1. Cardiovascular disease was a primary factor in atop-ranked network for both males and females, and each networkwas comprised of unique gene lists that are presented in Supplementalmaterial, Tables S2 and S3, with only one gene in common (RBBP8(retinoblastoma-binding protein 8)) between the sexes. Genes respon-sive to Sp1 (specicity protein 1) transcription factor comprised a top-ranked gene signature for both males and females. Sp1 responsivegenes were evaluated for their similarity between the sexes. Maleshad 110 Sp1 regulated genes and females had 91, both with a 50/50split between up and down-regulated genes. There was little overlapbetween the lists with only 9 genes overlapping, which are presentedin Supplementary material, Table S4. Due to the fact that the majorityof males were smokers and no females were reported as smokers(Table 1), we screened the male and female gene sets for 26 genesthat exhibited 90% sensitivity in predicting smokers versus nonsmokersin order to evaluate whether smoking status was confounding the sex-effect (Lampe et al., 2004). For themales, three of the smoking biomark-er genes appeared on our gene lists, which was statistically signicant(p b 0.0295); and there were no smoking genes in the female genelists. The smoking genes found in the male gene set included (CYP1B1(cytochrome P450, family 1, subfamily B, polypeptide 1), EPB41L3(erythrocyte membrane protein band 4.1-like 3), and BRD3(bromodomain containing 3)). To further assess the relationship, weprobed differentially expressed genes of high males vs. high females,and 3 smoking genes were present in this gene set. While a comparisonof low males vs. low females, contained no smoking biomarker genes.Collectively these ndings indicate that smoking status is primarily aninuence in the high males, but its signature is not dominant as onlythree smoking biomarker genes are present (CYP1B1, EPB4IL3, IL1B)and clustering in heatmaps is driven by As dose not smoking status.

Real-time PCR validation

To validate the results obtained from themicroarray study, quantita-tive real-time PCR was performed on a subset of genes with biologicalrelevance. POLR2E (polymerase (RNA) II (DNA directed) polypeptideE, 25 kDa), CKS2 (CDC28 protein kinase regulatory subunit 2), and

TGM2 (transglutaminase 2 (C polypeptide, proteinglutamine


gamma-glutamyltransferase)) were validated from the male differen-tially expressed genes. E2F5 (E2F transcription factor 5, p130-binding)was validated from the female differentially expressed genes. Asshown in Table 4 the up- or down-regulated patterns for all genes ob-tained from real-time PCR were consistent with those obtained in themicroarray study.

POLR2E (polymerase (RNA) II (DNA directed) polypeptide E,25 kDa), CKS2 (CDC28 protein kinase regulatory subunit 2), and TGM2(transglutaminase 2 (C polypeptide, proteinglutaminegamma-glutamyltransferase)) were validated in themales. E2F5 (E2F transcrip-

4 A. Muoz et al. / Toxicology and Applied Pharmacology xxx (2015) xxxxxx

Please cite this article as: Muoz, A., et al., Sex-specic patterns and deBangladeshi adults exposed to arsenic contaminated..., Toxicol. Appl. Phartion factor 5, p130-binding) was validated in the females. Patterns offold change found in quantitative real-time PCR exhibit consistent di-rectionality to those obtained by microarray.

Discussion

Our ndings suggest that As exposure deregulates expression ofgenes involved in the immune system, heat shock response andDNA re-pair processes, which is consistent with previous human studies on thistopic. The novelty of our paper is that we nd evidence to support thenotion that As acts through endocrine associated pathways in bothsexes, and that such pathways involve mediation of responses throughsex-specic mechanisms as evidenced by the distinct gene sets formales and females.

Given that the exposure inclusion criteria of the FACT study designrequired that As levels in drinking water exceed 50 g/L, we have con-ducted a comparison of high and low exposure groups. This study de-sign is consistent with previous studies of this cohort that detectedchanges in the abundance of histone marks in response to high andlow levels of As contaminated drinking water (Chervona et al., 2012).Due to the comparison between high and low groups, the range of Asexposure represented within each group, and the compression of ex-pression values that occurs when the SCAN normalization protocol isemployed, our fold change values are tightly expressed with maximumvalues in the range of1.3. In turnwe focus on evaluating thebiologicalsignicance of the differentially expressed genes, highlighting genesand areas of biological activity that are deregulated across exposurecategories, and evaluating how such deregulation manifests differentlyin males and females.

Arsenic's ability to impact gene expression changes is consistentwith previous studies which demonstrated that As deregulates the ex-pression of heat shock, DNA repair, and immunoregulatory genes. Webriey mention such changes here to biologically validate our genelists and contextualize our ndings with other similar studies. Heatshock proteins are consistently found to be aberrantly expressed in re-sponse to As. Here, HSP1AB, HSPBP1, and HSFS are deregulated inmales, and HSPA13 is deregulated in females (Table 5). Our ndingsare consistent with Andrew et al. and Argos et al. who nd thatHSP1AB is deregulated in their male dominant and female never smokercohorts, respectively (Andrew et al., 2008; Argos et al., 2006). Arsenic'sability to interfere with DNA repair and promote carcinogenesis as a co-mutagen has been rmly established in experimental systems (Hartwiget al., 1997; Rossman et al., 2004).We nd that drinkingwater As expo-sure deregulates expression of DNA repair genes in both males and fe-males and includes genes that are involved in excision repair (LIG3,

Fig. 1. Gene expression proles of As exposed adults. Unsupervised hierarchical clusteranalysis and heatmap of differentially expressed genes (p b 0.05) for males (A) and fe-males (B) with chronic exposure to As contaminated drinking water at high (H) andlow (L) doses. C) Unsupervised hierarchical cluster analysis and heatmap of differentiallyexpressed genes represented amongmales and females (p b 0.05). Each row represents astatistically signicant gene determined via LIMMA. Samples (columns) are labeled ac-cording to the following codes with appended numbers corresponding to unique individ-uals: MLmales with lowwater As exposure, MHmales with highwater As exposure,

FL females with low water As exposure, FH females with high water As exposure.


Table 2Differentially expressed genes common to both malesa and femalesb.

Gene symbol Description Absolute fold change

Malesa Femalesb

SLC37A2 Solute carrier family 37 (glycerol-3-phosphate transporter), member 2 1.12987 1.05461ZNF429 Zinc nger protein 429 1.11314 1.09785ABCA1 ATP-binding cassette, sub-family A (ABC1), member 1 1.09686 1.11128KMO Kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) 1.09616 1.11952CISH Cytokine inducible SH2-containing protein 1.08392 1.10739TUBA1B Tubulin, alpha 1b 1.07894 1.06552PKIG Protein kinase (cAMP-dependent, catalytic) inhibitor gamma 1.07413 1.06864SEMA4A Sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4A 1.06561 1.08562CLCN4 Chloride channel, voltage-sensitive 4 1.06541 1.08737RBBP8 Retinoblastoma binding protein 8 1.06036 1.07956TMEM68 Transmembrane protein 68 1.05458 1.08187

5A. Muoz et al. / Toxicology and Applied Pharmacology xxx (2015) xxxxxxIFIH1 Interferon induced with helicase C domain 1GLB1 Galactosidase, beta 1BTF3P11 Basic transcription factor 3 pseudogene 11VKORC1L1 Vitamin K epoxide reductase complex, subunit 1-like 1NOL11 Nucleolar protein 11MRPS18B Mitochondrial ribosomal protein S18BSNORA29 Small nucleolar RNA, H/ACA box 29SMUG1), DNA repair regulation (RBBP8, MDC1), and mismatch repair(MSH2) (Table 5). Previous drinking water exposure studies havefound that As interferes with nucleotide excision repair (Andrew et al.,2003, 2006) as well with base excision repair and strand breaks (Wuet al., 2003). With respect to the immune system we nd that As

PRMT10 Protein arginine methyltransferase 10 (putative)HSF5 Heat shock transcription factor family member 5PQLC1 PQ loop repeat containing 1KRTAP5-1 Keratin associated protein 5-1MRPS22 Mitochondrial ribosomal protein S22MAD2L1BP MAD2L1 binding proteinSMAP1 Small ArfGAP 1MAP3K6 Mitogen-activated protein kinase kinase kinase 6ZFPL1 Zinc nger protein-like 1KBTBD3 Kelch repeat and BTB (POZ) domain containing 3RNF126 Ring nger protein 126PPP1R12B Protein phosphatase 1, regulatory subunit 12BRORA RAR-related orphan receptor ASLC11A2 Solute carrier family 11 (proton-coupled divalent metal ion transporterSNTB1 Syntrophin, beta 1 (dystrophin-associated protein A1, 59 kDa, basic comHERC2 HECT and RLD domain containing E3 ubiquitin protein ligase 2TNIK TRAF2 and NCK interacting kinaseATIC 5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMIL11RA Interleukin 11 receptor, alphaETS2 v-ets erythroblastosis virus E26 oncogene homolog 2 (avian)ACER2 Alkaline ceramidase 2CLYBL Citrate lyase beta likeCCDC144NL Coiled-coil domain containing 144 family, N-terminal likePRDM1 PR domain containing 1, with ZNF domainKIAA1324 KIAA1324

a 534 differentially expressed genes (p b 0.05) for males high water As vs. males low waterb 645 differentially expressed genes (p b 0.05) for females high water As vs. females low wac Expression values for males high water As relative to males low water As.d Expression values for females high water As relative to females low water As.

Table 3Top IPA networks for differentially expressed genes of males (males high water As vs. males lo

Males Fe

Network Score N

Post-translational modication, protein folding, antimicrobial response 75 CCardiovascular disease, congenital heart anomaly, developmental disorder 66 C

mAmino acid metabolism, molecular transport, small molecule biochemistry 62 MLipid metabolism, small molecule biochemistry, cell morphology 48 D

mCellular development, cellular growth and proliferation, hematological systemdevelopment

48 C

Please cite this article as: Muoz, A., et al., Sex-specic patterns and deBangladeshi adults exposed to arsenic contaminated..., Toxicol. Appl. Phar1.05346 1.109041.04594 1.040021.04533 1.042361.04435 1.045631.04418 1.059341.04346 1.033511.04276 1.04468deregulates a number of interleukins (IL) in both sexes (Table 5), thatthere was a viral-response like signature in the males (RSAD2 IFIT1IFIT3, IFIT5), and deregulation of major histocompatibility (MHC) classgenes (HLA-DRB6,HLA-B,HLA-G) in the females. Ourndings are consis-tent with previous studies, as Andrew et al. also found deregulation of

1.04159 1.048011.03604 1.042561.03523 1.040411.03504 1.033661.03337 1.047411.03253 1.046131.02688 1.03809

1.02623 1.038681.02681 1.0361.03002 1.037961.03488 1.045591.03503 1.06131.0373 1.05826

s), member 2 1.04141 1.05051ponent 1) 1.04204 1.06052

1.04328 1.049031.04349 1.04896

P cyclohydrolase 1.04567 1.048721.04633 1.10281.04934 1.066491.05118 1.066981.06358 1.073641.065 1.050191.06919 1.087341.09944c 1.14391d

As.ter As.

w water As) and females (females high water As vs. females low water As).

males

etwork Score

ardiovascular disease, cellular compromise, neurological disease 67ell death and survival, hematological system development and function, tissueorphology

42

olecular transport, RNA trafcking, cellular development 35NA replication, recombination, repair, cell morphology, cellular function andaintenance

25

ellular function and maintenance, cell death and survival, tissue morphology 24


MHC II class genes in their male dominant cohort (Andrew et al., 2006)and Wu et al. and Argos et al. report deregulation of interleukins. Ofthose IL deregulated, IL16 (females) was previously reported by Wuet al. in females exposed to As contaminated water in Taiwan where itexhibited dose-responsiveness (Wu et al., 2003) and IL1RN (males)was reported by Argos et al. to be down-regulated inwomenwith As in-duced skin lesions (Argos et al., 2006).

In addition to the areas discussed above, we observe that As deregu-lates expression of 17-hydroxysteroid dehydrogenases (17-HSDs),which are a family of enzymes involved in regulating steroid hormonesthroughout the body. Bothmales and females exhibited deregulation of

a HSD family member. In females 17-hydroxysteroid dehydrogenasetype 4 (HSD17B4) was deregulated. This enzyme can oxidize estradiolto estrone and has widespread constitutive expression with the highestlevels in the liver where it is involved in fatty acid metabolism and syn-thesis of bile acids (Moeller and Adamski, 2006). In males HSD17B10was deregulated, and it exhibits broad substrate specicity for biomole-cules including fatty acids, estrogens, androgens, corticosteroids andprogestins and is constitutively expressed in the liver, gonads, andbrain, where its regional expression levels may play a critical role inneurosteroidogenesis (Moeller and Adamski, 2006; Yang et al., 2005).HSD17B10 has been implicated in the development of Alzheimer's dis-ease (Moeller and Adamski, 2006), which is a highly ranked gene signa-ture for males and females (Supplementary material, Table S1).HSD17B10 is also utilized in the alternative pathway for biosynthesisof 5-dihydrotestosterone (5-DHT) from adiol (Yang et al., 2005).Deregulation of HSD17B10may help to explain why males exposed toAs exhibit reduced semen quality (Xu et al., 2012) and increased riskof erectile dysfunction (Hsieh et al., 2008). Animal models supportthis notion and consistently yield decreased levels of testicular enzymesincluding 17-HSD in conjunction with declined fertility parameters in

Table 4Validation of microarray results by quantitative real-time PCR.

Gene Affymetrix fold change Q-RT-PCR fold change

POLRE2 1.12 4.31CKS2 1.06 1.96TGM2 1.06 1.87E2F5 1.10 1.44

Table 5Biological validation of gene lists. Differentially expressed genes involved in heatshock response, DNA repair, and immune response for males and females.

Genesymbol

Description Foldchange

Heat shock

MalesHSP1AB Heat shock 70 kDa protein 1B 1.055HSPBP1 Heat shock 70 kDa biding protein, cytoplasmic chaperone 1 1.038HSF5 Heat shock transcription family member 5 1.036

FemalesHSPA13 Heat shock 70 kDa protein 13 1.054

DNA repair

MalesRBBP8 Retinoblastoma binding protein 8 1.060CDK7 Cyclin-dependent kinase 7 1.058POLD3 Polymerase (DNA-directed), delta 3, accessory subunit 1.036POLI Polymerase (DNA directed) iota), RPA3 (replication protein A3, 14 kDa 1.054RPA3 Replication protein A3, 14 kDa 1.030LIG3 Ligase III, DNA, ATP-dependent 1.027

FemalesRBBP8 Retinoblastoma binding protein 8 1.080MDC1 Mediator of DNA-damage checkpoint 1 1.041LIG4 Ligase IV, DNA, ATP-dependent 1.060MSH2 mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) 1.059SMUG1 Single-strand-selective monofunctional uracil-DNA glycosylase 1 1.069

e doteoteoteotecibvirusubur, a, alp-lik

6 A. Muoz et al. / Toxicology and Applied Pharmacology xxx (2015) xxxxxxImmune system

MalesRSAD2 S-adenosyl methioninIFIT1 Interferon-induced prIFIT3 Interferon-induced prIFIT5 Interferon-induced prIFI44L Interferon-induced prIFI6 Interferon, alpha-induMX1 Myxovirus (inuenzaIL23A Interleukin 23, alphaIL11RA Interleukin 11 receptoIL15RA Interleukin 5 receptorIL1RL1 Interleukin 1 receptor

IL1RN Interleukin 1 receptor anIL17C Interleukin 17C

FemalesIL11RA Interleukin 11 receptor, aIL13RA1 Interleukin 13 receptor, aIL18BP IL18 binding proteinIL16 Interleukin 16HLA-DRB6 Major histocompatibilityHLA-B Major histocompatibilityHLA-G Major histocompatibilityIFIH1 Interferon induced with h

Please cite this article as: Muoz, A., et al., Sex-specic patterns and deBangladeshi adults exposed to arsenic contaminated..., Toxicol. Appl. Pharomain containing protein 2 1.186in with tetratricopeptide repeats 1 1.207in with tetratricopeptide repeats 3 1.123in with tetratricopeptide repeats 5 1.095in 44-like 1.323le protein 6 1.201s) resistance 1, interferon-inducible protein p78 (mouse) 1.180nit p19 1.045lpha 1.046ha 1.047e 1 1.102tagonist 1.084

1.037

lpha 1.103lpha 1 1.089

1.0701.035

complex, class II, DR beta 6 (pseudogene) 1.083complex, class I, B 1.083complex, class I, G 1.129elicase C domain 1 1.109regulation of endocrine pathways in the gene expression proles ofmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.025

7A. Muoz et al. / Toxicology and Applied Pharmacology xxx (2015) xxxxxxboth rats and mice exposed to As contaminated drinking water (Changet al., 2007; Jana et al., 2006; Pant et al., 2004).

For both males and females cardiovascular disease was a top-rankednetwork, and this nding is consistent with strong epidemiological dataimplicating As in the progression and development of cardiovascular dis-ease (Argos et al., 2010; Chen et al., 2009). The specic nature of themaleand female cardiovascular disease networks was distinct as representedby their unique gene lists (Supplementary material, Tables S2 and S3),which had only one gene in common (RBBP8), and different secondarynetwork features. Such differences may arise via sex-specic actions ofsterol pathways as both networks share the involvement of the estrogenreceptor (ER) and luteinizing hormone (LH) as key hubs (Supplementarymaterial, Figs. S2 and S3). Our ndings suggest that while cardiovasculardisease is a common outcome of long-term As exposure, males and fe-malesmay have differentmechanisms that dominate in the pathogenesisleading to cardiovascular disease. Sex-specic pathogenesis of As-associated cardiovascular disease is also evident in recent work from theHEALS cohort study,whichnds a positive association inwomen only be-tween long-term As exposure and QT prolongation time, a measurementof the electrocardiogram that indicates increased risk for severe cardiacevents (Chen et al., 2013).

The top ranked gene signature for both males and females was genescontaining transcriptional binding sights for specicity protein 1 (Sp1)transcription factors. As mentioned in the results there were about 100genes regulated by Sp1 in gene sets of males and females with only 9genes overlapping between the genders (Supplemental material,Table S4). Sp1 can positively or negatively regulate gene expression(Li et al., 2004), is deregulated in numerous cancers and neurodegenera-tive disorders, and is involved in cell cycle regulation, angiogenesis, andsenescence (Chang and Hung, 2012), which are all events consistentwith As pathogenesis. It is unlikely that Sp1 deregulation occurs throughdirect interaction of As and Sp1 zinc ngers, because recent research indi-cates that trivalent As only binds zinc nger motifs with more than twocysteine residues, and does not bind to Sp1 which has two (Zhou et al.,2014).However, Sp1 activity ismodulatedbypost-translationalmodica-tions including acetylation, methylation, sumoylation, and ubiquitinationthat regulate Sp1 protein level, transactivation activity, and DNA bindingafnity (Chang and Hung, 2012). Arsenic may inuence Sp1 modica-tions as it has been shown to alter acetylation of histones (Chervonaet al., 2012), methylation of DNA (Ren et al., 2011), and ubiquitinationof proteins (Bredfeldt et al., 2004; Kirkpatrick et al., 2003). Suchmodica-tions can alter Sp1's ability to partner with steroid receptors (SR) includ-ing ER, themineralocorticoid receptor (MR), the glucocorticoid receptor(GR), and the androgen receptor (AR), and thereby alter the synergisticcouplings involved in transcription of Sp1 or SR responsive genes(Meinel et al., 2013; Ou et al., 2006; Porter et al., 1997; Yuan et al.,2005). In vitro models indicate that As inuences transcription of genesdownstreamof ER,MR, andGR, but themechanismdriving these changesis unknown (Bodwell et al., 2006), andwe suggest that post-translationalmodication of the Sp1 pool may be involved.

The gene expression proles observed inmales and females indicatethat similar channels mediate As impact in both sexes. However, thestriking difference in gene sets for the males and females, suggeststhat As may exert its inuence through sex-specic endocrine interac-tions, which could lead to sex-specic treatment or prevention strate-gies. Our data support the growing trend that As can be considered anendocrine disruptor and further study is needed to elucidate how thisinteractionmay occur (Bodwell et al., 2006; Davey et al., 2007). In addi-tion, our data support the perspective that toxic actions may be sex-specic and that evaluation of biomarkers and determination of safetythresholds may require separate analyses for males and females re-sponses (De Coster et al., 2013).

Given the nature of working with a human population, there are anumber of study limitations arising from demographic features of thestudy population that must be addressed before concluding. The

smoking status of the male cohort is one demographic feature that

Please cite this article as: Muoz, A., et al., Sex-specic patterns and deBangladeshi adults exposed to arsenic contaminated..., Toxicol. Appl. Pharmay be inuencing the presentation of our data. Due to the prevalenceof smoking among males in this region of Bangladesh, the majority ofmales were self-reported smokers while none of the females were re-ported as smokers. In order to evaluate the effect of smoking status onour results, we probed for detection of genes that are biomarkers forsmoking among multiple datasets and found limited detection of suchgenes. To further evaluate the inuence of smoking, we assessed the bi-ological validity of our gene lists to insure that our lists are representa-tive of a response to As exposure rather smoking status. As discussedwefound that deregulation of heat shock, DNA repair, and immunoregula-tory responses were consistent with changes observed in these areas insimilar studies (Andrew et al., 2003, 2006, 2008; Argos et al., 2006; Wuet al., 2003), indicating that the lists generated were a reection of Asexposure. The relevance of our gene lists is further supported by thefact that the unsupervised clustering in the heatmap (Fig. 1A) is drivenby As dose and not smoking status. Collectively, this analysis suggeststhat the sex-specic proles exhibited in our data are primarily areection of As exposure, which may to a limited extent be inuencedby smoking status of males. Another constraint of this study was thelimited sample size, which in combination with the fold-changecompression previously discussed, effectively inhibited our ability todetect differentially expressed genes using multiple hypothesiscorrection (i.e., FDR). Other epidemiologic studies conducting gene ex-pression analysis in human populations have also generated uncorrect-ed gene lists, which were utilized for pathway analysis in a similarmanner (De Coster et al., 2013; Hebels et al., 2011; Wang et al., 2005).The biological validation performed on our datasets again suggeststhat the gene lists generated here reect As exposure rather than ran-domly selected genes, given their consistency with previous gene ex-pression studies as well as their prediction of diseases well-known tobe associated with As exposure such as cardiovascular disease.

Conclusion

Our data indicate that As is inuencing gene expression proles in amanner consistent with previous human gene expression studies inwhich heat shock proteins, DNA repair processes, and immune responseswere deregulated. In addition, we observe sex-specic proles that maybedrivenby an interactionbetweenAs and the endocrine system. The ob-served deregulation of HSD enzymes, the presence of the ER and LH aspredicted hubs in the cardiovascular disease networks, and thederegulated Sp1 genes all implicate possible mechanisms throughwhich As may be exerting its multifactorial inuence. The involvementof steroid regulating enzymes, the prediction of steroid responsive hubs,and the cooperation between Sp1 and steroid receptors all indicate thatAsmay be acting through endocrinemediated channels and that such ef-fects require separate evaluation in males and females. These ndingssuggest that exposure thresholds may require different parameters formales and females and that further study is needed to fully understandthe impact and nature of sex differences in As pathogenesis.

Disclosure of potential conicts of interest

The authors have no actual or potential conicts of interest.

Acknowledgments

This work was supported by the following grant numbers: Y.Chervona and M. Gamble from P42 ES010349 and R01 ES17875. M.Costa by R01 ES023174, ES022935, ES000260, CA16087 and R01CA133595 from NCI; and RR029893 from National Center for ResearchResources; M. Gamble from R01 ES017875 NIEHS, R01 CA133595 fromNational Cancer Institute (NCI); M. Hall by R00ES018890. T. Kluz byES000260 NIEHS. A. Munoz by National Science Foundation Graduate Re-

search Fellowship under Grant number 1137475 and NIEHS 5 T32


8 A. Muoz et al. / Toxicology and Applied Pharmacology xxx (2015) xxxxxxES007324.We thankYuChen for the helpful comments and editing of themanuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.taap.2015.02.025.

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9A. Muoz et al. / Toxicology and Applied Pharmacology xxx (2015) xxxxxxPlease cite this article as: Muoz, A., et al., Sex-specic patterns and deBangladeshi adults exposed to arsenic contaminated..., Toxicol. Appl. Pharregulation of endocrine pathways in the gene expression proles ofmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.025

Sex-specific patterns and deregulation of endocrine pathways in the gene expression profiles of Bangladeshi adults exposed...IntroductionMaterials and methodsStudy site and subject recruitmentSample collection and handlingWater and urinary AsRNA isolation, amplification, and hybridizationData analysis to identify differentially expressed genesQuantitative real-time PCR

ResultsDemographic dataGene expression analysisReal-time PCR validation

DiscussionConclusionDisclosure of potential conflicts of interestAcknowledgmentsAppendix A. Supplementary dataReferences

sex-specific patterns and deregulation of endocrine pathways in the gene expression profiles of...

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