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Toxicology Letters 212 (2012) 169– 179

Contents lists available at SciVerse ScienceDirect

Toxicology Letters

jou rn al h om epage: www.elsev ier .com/ locate / tox le t

race metals alter DNA repair and histone modification pathways concurrentlyn mouse embryonic stem cells

anket R. Gadhia, Anthony R. Calabro, Frank A. Barile ∗

t. John’s University, College of Pharmacy and Allied Health Professions, Department of Pharmaceutical Sciences, Toxicology Division, Queens, NY 11439, United States

i g h l i g h t s

Trace metals alter cell proliferation, differentiation and DNA repair concurrently in mouse embryonic stem cells.As, Cd, Hg, Ni preferentially decreased cell viability over total histone protein.Yet, As and Hg significantly decreased total histone protein production per cell.Metals suppress compensatory DNA repair mechanisms through epigenetic pathways.

r t i c l e i n f o

rticle history:eceived 16 January 2012eceived in revised form 11 May 2012ccepted 12 May 2012vailable online 19 May 2012

eywords:pigeneticistone modificationmbryonic stem cellsaracellular permeabilityetals

ytotoxicity

a b s t r a c t

Exposure to metals alters gene expression, changes transcription rates or interferes with DNA repairmechanisms. We tested a hypothesis to determine whether in vitro acute metal exposure, with or withoutrecovery, alters epigenetic pathways in mouse embryonic stem (mES) cells. We measured cell viability,total and histone protein production, changes in gene expression for differentiation and DNA repair, andhistone lysine mono-methylation (H3K27me1), in differentiated cells. Confluent differentiated culturesof mES cells were exposed to arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), lithium (Li), mercury(Hg), and nickel (Ni), for 1-h and 24-h, followed by a recovery period. The data demonstrate that maxi-mum cell death occurred during the first few hours of exposure at 24-h IC50 concentrations for all metals.Prolonged in vitro exposure to metals at low concentrations also inhibited protein production and cellproliferation. Subsequently, we determined that metals alter cell differentiation (Oct-4 and egfr) and DNArepair mechanisms (Rad-18, Top-3a and Ogg-1). Interestingly, As, Cd, Hg, and Ni decreased cell prolifera-tion to a greater extent than total histone protein production. Yet, at equivalent concentrations, As andHg significantly decreased total histone protein production per cell compared to respective controls,

suggesting suppression of repair or compensatory mechanisms involving histone pathways. And, acuteexposure to As, Cd, Hg and Ni decreased H3K27me1 residue, when compared to control cells. Becauseactivation of cellular differentiation, histone modification, and DNA repair are linked by common tran-scriptional pathways, and the data propose that metals alter these conduits, then it is reasonable toconclude that trace quantities of metals are capable of suppressing regulation of chromatin structure,

nd co

cellular differentiation, a

Abbreviations: As, arsenic; Cd, cadmium; Cu, copper; C-IV, collagen type IV; ECM,xtracellular matrix; egfr gene, epidermal growth factor receptor; FN, fibronectin;3K27me1, histone 3-lysine27-monomethylation; Hg, mercury; IC50, inhibitoryoncentration 50%; IS, internal standard; Li, lithium; LIF, leukemia inhibitory fac-or; MA, multicellular aggregates; MEF, mouse embryonic fibroblasts; mES, mousembryonic stem (cells); Ni, nickel; Oct-4 gene, Oct-4/POU domain transcriptionactor; Pb, lead; PP, paracellular permeability; TJ, tight junction; TMER, trans-

embrane electrical resistance.∗ Corresponding author. Tel.: +1 718 990 2640; fax: +1 718 990 1877.

E-mail address: barilef@stjohns.edu (F.A. Barile).

378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2012.05.013

ntrolled cell proliferation in mES cells.© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Mouse embryonic stem (mES) cells are derived from pluripotentcells of the early mammalian embryo. They are capable of unlim-ited, undifferentiated proliferation in vitro (Evans and Kaufman,1981; Simon and Lange, 2008). In vivo, pluripotency confers theability to differentiate to the three embryonic germ layers. Theircapacity to differentiate in vivo and in vitro has lead to the inten-sive study of the complex mechanisms surrounding cell growthand differentiation (Smith, 2001). Mouse ES cells undergo in vitro

differentiation with the formation of embryoid bodies (EBs) asan intermediate morphological step towards the differentiation ofepithelial-like cells (Martin et al., 1977). Addition of growth factors(GFs) to the medium manages culture conditions so as to control

1 gy Letters 212 (2012) 169– 179

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ifferentiation status to more specific cell types (Gritti et al., 1999;alabro et al., 2008). In addition, GFs such as leukemia inhibitoryactor (LIF), maintain the undifferentiated state, prevents initiationf spontaneous differentiation, and influences formation of intra-ellular contacts, such as tight junctions (TJs) (Furue et al., 2005). Forxample, �-actin serves as an anchor for the formation of TJs, andegulates cooperation between extracellular matrix (ECM) compo-ents and GFs in a variety of epithelial and stem cells (Calabro et al.,008; Gonzalez-Mariscal et al., 2003).

It is well accepted that metals fuel environmental havoc throughheir mutagenic, teratogenic and embryotoxic properties, therebyncreasing the potential for abnormal embryonic developmentFerm, 1976; Fishbein, 1984; Gopalakrishnan et al., 2008). In gen-ral, their mechanism is purported to interfere with differentiation,roliferation, gene expression and protein expression levels, par-icularly when acting as carcinogens or co-carcinogens. This theoryowever is not completely elucidated (Beyersmann, 2002; Rana,008; Salnikow and Zhitkovich, 2008). Consequently, this studyursues this theme by examining the effects of trace metals on stemell differentiation and DNA repair processes, in particular, by mon-toring expression of genes known to correspond with embryonictem cell differentiation (Oct-4 and Egfr).

Various structural and metabolic proteins are involved in trans-ort and movement of metals through biological membranes. For

nstance, cadmium gains entry into cells via ion channels (Goeringt al., 1994; Liu et al., 2008), by binding to circulating serum albu-in (Rossman, 2003) or divalent metal transport protein-1, or

y transmembrane diffusion or phagocytosis (Beyersmann, 2002;enkhaus and Salnikow, 2002; Liu et al., 2008). Metals also dic-

ate their own movement through the influence of proteins on ionimicry (Cousins et al., 2006; Kasprzak, 2002; Liu et al., 2008).

t is also clear that developmental disorders, neurological dis-ases, breast and prostate cancers, and reproductive syndromes,re triggered by means of environmental exposure to toxicants,articularly arsenic, and their interaction with the functional statef regulatory genes (Collman, 2011; Newnham and Ross, 2009).he mechanism however is only understood as far as the alterationf gene expression and/or protein regulation is apparent in con-unction with aberrant cell proliferation and differentiation. It maye inferred, therefore, that these alterations occur as a result ofpigenetic manipulation and chromatin remodeling, but the causalelationship may be time and/or tissue specific and may not beeversible, thus rendering an individual more vulnerable to pro-ression of a pathological condition in adult life (Heindel, 2005;aterland and Michels, 2007).Epigenetics involves heritable alterations in gene expression

hat are not coded in the DNA sequence (Jablonka and Lamb, 2002).hese changes are also capable of transgenerational inheritancehrough meiosis (Berger et al., 2009). Epigenetic changes have alsoeen linked to proliferative transformations, resulting in formationf cellular dysplasia and carcinoma in situ. Additionally, epigeneticilencing of genes is associated with various stages of cancerFeinberg et al., 2006; GrØNbÆK et al., 2007; Gupta and Massagué,006). It is unequivocal, therefore, that transcriptional regulation

s administered through important epigenetic pathways, dictatedrimarily by DNA methylation, RNA-mediated silencing, post-ranslational modification (PTM) of histones, replacement of coreistones by variants, and structural changes of the nucleosomeFeinberg et al., 2006; Jaenisch and Bird, 2003; Salnikow andhitkovich, 2008). Currently, it is understood that histone lysineethylation influences the structure of the nucleosome. In fact,

pecific histone lysine modifications, in combination with histone

TM, are associated with chromatin structure and functional out-ome (Völkel and Angrand, 2007). Because metals are recognizedo alter, bind to, or interrupt transcription and/or translationCalabro et al., 2011), we intend to provide further evidence for

Fig. 1. Multicellular aggregates (MAs) formed at onset of differentiation of mES cellsin culture.

the “missing link” between the interaction of metals and PTMs,with arsenic as representative, the net effect of which leads tochromosomal anomalies, abnormal cellular differentiation, andmetal-induced epigenetic toxicity.

2. Materials and methods

2.1. Cell culture

2.1.1. Mouse embryonic fibroblast (MEF) feeder cellsMEF feeder cells (CRL-1658TM; 3T3 Swiss mouse fibroblasts, American Type Cul-

ture Collection [ATCC], Rockville, MD, USA) were cultured in Dulbecco’s ModifiedEagle’s Medium (DMEM; high glucose formulation, 4.5 g/L, 22 mM), supplementedwith 10% newborn calf serum, 1.5 g/L sodium bicarbonate, 4 mM glutamine and1% antibiotic/antimycotic solution. Both mES and MEF cells were propagated inan atmosphere of 5% CO2 and 100% humidity at 37 ◦C. Maintenance cultures weregrown in T-75 flasks at a seeding density of 7.5 × 105 cells per flask.

2.1.2. Mouse embryonic stem (mES) cellsThe ES-D3 [D3] cell line (CRL-1934, ATCC) was propagated in a high glu-

cose formulation (DMEM + 4.5 g/L) containing 1% l-alanine-l-glutamine, 0.1 mM2-mercaptoethanol, 1% non-essential amino acids, 1% antibiotic/antimycotic solu-tion, 3.5 g/L sodium bicarbonate, and 15% heat inactivated fetal bovine serum(FBS). Undifferentiated cultures were maintained on mitomycin-C-inactivated(Sigma–Aldrich Corp., St. Louis, MO, USA) MEF feeder layers in growth mediumsupplemented with 1000 U/mL leukemia inhibitory factor (LIF, Millipore Corp., CA,USA). Stem cells form embryoid bodies (EBs) or multicellular aggregates (MA; Fig. 1).When LIF is removed from the growth medium, the cultures lose their globularmorphology upon differentiation (Barile, 2007). The cells were then washed withphosphate-buffered saline, and passaged onto collagen IV-coated (C-IV) 96-well cul-ture plates or T-75 tissue culture flasks. In each experiment, confluent differentiatedcultures of cells were exposed to only one metal—i.e. arsenic (As), cadmium (Cd),copper (Cu), lead (Pb), lithium (Li), mercury (Hg), and nickel (Ni) for 1-h followedby a recovery period, and 24-h. In some studies, only As was incorporated as a rep-resentative metal, primarily because of the similarity of results obtained with someof the other metals in preliminary experiments.

Morphology of stem and differentiated cells was documented using phase con-trast microscopy with the Leitz Labovert FS inverted phase contrast microscopeequipped with the SPOT® Insight 2.0 megapixel microscope camera (micrographsnot shown).

2.2. MTT assay

Mouse ES cells (5–10 × 103) were seeded onto 96-well C-IV-coated culture plates(BD BioCoatTM) and were allowed to reach confluence during which differentiationis induced. The MTT assay was performed as previously described (Calabro et al.,2011; Konsoula and Barile, 2007; Mosmann, 1983). Absorbance was read at 550 nmon the Opsys MR® absorbance microplate reader (Dynex Technologies, Chantilly,VA, USA). Cell viability is expressed as percentage of control cells.

In recovery experiments, cells were seeded and grown as above. Culture wellswere then treated with their respective metals for 1-h, washed with Hank’s BalancedSalt Solution, and incubated with growth media minus metal for an additional 23-h(recovery period).

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.3. NRU assay

NRU assay was performed as previously described (Borenfreund and Puerner,986; Konsoula and Barile, 2007). Mouse ES cells were seeded onto 96-well C-V-coated cell culture plates (BD BioCoatTM) and allowed to attain confluence andifferentiation. Cell viability is expressed as percentage of control groups.

Two exposure protocols, as noted with the MTT assay above, were used to deter-ine cell viability. Cells were exposed to increasing concentrations of metals for

1-h or 1-h, the latter of which was followed by a recovery period of 20-h. Bothxposure protocols required a total of 24-h incubation (21-h exposure + 3-h NRUncubation).

.4. Trans-membrane electrical resistance (TMER) measurements

Cultures containing MAs, and residual mitotically inhibited MEFs, wererypsinized and seeded onto 24-well plates fitted with Biocoat® porous 3.0 �m cul-ure inserts with or without extracellular matrix (ECM) component coating. ForMER measurements (� cm2), LIF was removed and mES cells were allowed todhere to the inserts for 24-h.

In the first series of experiments, mES cells were grown in media containing.034 mM As2O3 (day 1). Fresh medium containing As2O3 was changed daily untilay 12. In parallel sets of experiments, mES cells were grown in medium minuss2O3 for 6 days after which the metal was added on day 7. TMER was measuredaily as previously described (Calabro et al., 2008).

In a second set of parallel experiments, inserts were initially prepared asescribed above without As2O3 but in the presence of either epidermal growth fac-or (EGF, 400 ng/mL), or keratinocyte growth factor (KGF, 2 ng/mL). During this timeMER was measured daily.

.5. Gene expression

For gene expression studies, mRNA was extracted concurrently from aliquots ofepresentative sample wells and were assayed for expression of Rad-18, Ogg-1, andop-3a gene transcripts (Dynabeads® mRNA DirectTM, Invitrogen Corp., Carlsbad, CA,SA). Alternatively, Oct-4 and Egfr gene expression were calculated from total RNA

solated from sample wells (Micro-to-Midi Total RNA Isolation® , Invitrogen Corp.).Primers for corresponding genes were applied to detect the influence of metals

n gene expression involved in differentiation: Oct-4/POU5f1 (POU domain tran-cription factor for undifferentiated cells and trophectoderm layers); Egfr (epidermalrowth factor receptor) for differentiated cells (Ginis et al., 2004); Rad-18, Top-3and Ogg-1 DNA repair genes. Pairs of forward and reverse primer sequences for allranscripts were purchased from SuperArray Bioscience Corp. (Frederick, MD, USA).rimers were tested prior to their incorporation in the multiplex reaction, for sin-le discrete products with 96 bp (Oct-4/POU5f1), 117 bp (Rad-18), 81 bp (Top-3a) and6 bp (Ogg-1). RT-PCR amplification was performed as previously described (Calabrot al., 2008; Kebache et al., 2002) from a modification of the manufacturer’s proto-ol (Invitrogen Corp.) to allow for gene quantification, using 18S ribosomal internaltandard (IS; Ambion Corp., Austin, TX, USA). PCR products were separated on a 2%garose gel (UltraPureTM, Invitrogen Corp.) containing 0.5 �g/mL ethidium bromide.izes of the amplified bands were referenced against a 100 bp DNA ladder (Invitro-en Corp.) and quantified according to the integrated optical density of the bandsaptured on the UVP BioSpectrum® AC Imaging System with the OptiChem 600 HRamera. Bands were analyzed for corresponding genes and were compared to the ISpplied to the gel simultaneously and under identical conditions as the treatmentample, in order to provide an estimate of the relative changes in gene expressionetween and within samples. The final data are presented as % change of control.he information was then used to calculate the ratios of the intensities of the bandsnder various experimental conditions.

.6. Histone protein extraction

Histone protein was extracted (Epigentek® , Brooklyn, NY, USA) from confluent,ifferentiated cells seeded and cultured on C-IV-coated flasks as noted above. Briefly,ell pellets were suspended in cold triton extraction buffer (TEB; 107 cells/mL),entrifuged and re-suspended in cold 0.5 N HCl–10% glycerol (200 �L/107 cells) for0 min. After centrifugation, acetone (0.6 mL/107 cells) was added to the supernatantnd incubated overnight (−20 ◦C). Histone extracts were air dried; the pellet wasissolved in distilled water (30–50 �L/107 cells) and stored at −20 ◦C.

Extracted histone protein was quantified using the bicinchoninic acid (BCA) pro-ein assay (Sigma–Aldrich® , St. Louis, MO, USA) using bovine serum albumin as therotein standard. The amount of histone protein extracted per 104 control or treatedells per group were then statistically compared.

.7. Global histone H3-K27 mono-methyl (H3K27-me1) quantification

Histone H3-K27 mono-methyl (H3-K27-me1) quantification was performedsing a fluorimetric assay (Epigentek® , Brooklyn, NY, USA). Antibody buffer and cor-esponding detection antibody were incubated with the histone proteins (standardr sample) for 1–2 h at room temperature. Fluoro-developer and fluoro-enhancerolutions were added to the wells containing protein extracts, washed, and stored

ters 212 (2012) 169– 179 171

in the dark for 1–5 min. Developed fluorescence was measured on a microplatereader (Magellan Spectra-Fluorometer® , Tecan Group Ltd., Männedorf, Switzerland)at 530EX/590EM nm. Percent histone H3K27-me1 was calculated according to thefollowing formula:

% mono-methylation =[

RFU(sample-blank)RFU(control-blank)

]× 100

where RFU is the relative fluorescent units. Amount of protein was quantified byplotting RFU of treatment or control sample vs. the amount of standard; slope wascomputed as change in RFU/ng.

2.8. Statistical analysis

All experiments were performed three or four times, as noted in tables and fig-ures; sample means and SE were calculated from at least triplicate determinations(three or more wells) per group, each group based on concentrations and includebiological, process, and blank controls. IC50 values (mM/L) were extrapolated fromconcentration–effect curves using linear regression analysis. Cytotoxicity experi-ments (MTT, NRU) were plotted as % of control (cell viability) vs. log of concentration(mM), from which were calculated the coefficient of determination (R2) and slope(m). Levels of significance, between control sets and groups treated with IC25 and IC50

values of the metals, were calculated for the amount of histone protein extracted,for cell proliferation, and for percent H3K27-me1 (P < 0.05).

All results are expressed as mean ± standard error (SE). Group means for appro-priate experiments were subjected to one-way ANOVA (P < 0.05); post hoc testsinclude t-statistic (unpaired Student’s t-test with the more stringent equal variancesassumption), and the z-test (two-sample for means).

3. Results

3.1. Cell viability: MTT assay

Differentiated cells were exposed to increasing concentrationsof metals for 1-h and 24-h, and 1-h plus a 23-h recovery period.Absorbance values are plotted as “% of control” vs. “log of concentra-tion” (mM). IC50 values are extrapolated from regression analysis.Coefficient of correlation (R2) for each plot was computed fromthe regression curves—all values were greater than 0.90 (regres-sion calculation not shown). Cell viability data (Table 1) allow forthe interpretation of subtle differences in toxic phenomena whencells are exposed to different experimental protocols, particularlythose differences that reveal relative acute and cumulative toxi-city, as well as the ability to recover from toxic insult. Thus, Cd,Hg, and Ni uncover the highest toxicities for cell viability at 24-h(0.16 mM, 0.18 mM, 0.24 mM, respectively; P < 0.001). Indeed, 1-hexposures require higher doses than 24-h exposures for all metals.In addition, stem cells do not recover after 1-h exposure to Cd, Li, Ni,and Pb (shift of 1.70 mM to 0.57 mM, within a 23-h recovery periodfor CdCl2; Table 1), and viability barely improves with As and Cu.Thus the data suggest that these metals exert their toxicologicaldominance in the initial hours of exposure and do not afford thecells the benefit of recovery—toxicity therefore is cumulative andsustainable.

3.2. Cell viability: NRU assay

As with the MTT assay, confluent differentiated cells wereexposed to increasing concentrations of metals for 1-h, plus 23-hrecovery period, and 21-h (allowing 3-h assay incubation), repre-senting an incubation period of 24-h. All other parameters wereidentical as above. Unlike the MTT assay, NRU data (Table 1) revealthat this assay is not as sensitive as the former for detectingacutely toxic metals for cell viability. NRU IC50 values for Cd, Hg,and Ni are 0.22 mM, 0.51 mM, and 0.35 mM, respectively, and arehigher than corresponding MTT IC50s (Table 1). Since NRU IC50

values from “1-h exposure + 23-h recovery” are higher than 21-hexposures, we conclude that lysosomes are able to recover frommetal toxicity—cumulative toxicity is not apparent as compared tomitochondria.

172 S.R. Gadhia et al. / Toxicology Letters 212 (2012) 169– 179

Table 1Mean IC50 values (mM) calculated from MTT and NRU assays for indicated exposure times with recovery.

Metals MTT IC50s (mM) NRU IC50 (mM)

24-h exposure 1-h exposure* 1-h exposure; 23-h recovery* 21-h exposure 1-h exposure; 20-h recovery*

Arsenic trioxide 0.93 ± 0.082 1.81 ± 0.05 2.04 ± 0.08 1.02 ± 0.30 1.74 ± 0.007Cadmium chloride 0.16 ± 0.008 1.70 ± 0.22 0.57 ± 0.03 0.22 ± 0.09 1.35 ± 0.21Copper sulfate 0.93 ± 0.08 1.92 ± 0.06 2.62 ± 0.45 1.05 ± 0.17 2.71 ± 0.15Lead acetate 3.57 ± 0.27 6.39 ± 0.22 3.52 ± 0.12NS 3.48 ± 0.55 3.61 ± 0.13NS

Lithium sulfate 4.52 ± 0.38 10.37 ± 0.06 6.40 ± 0.25 4.95 ± 0.77 6.80 ± 0.12Mercuric chloride 0.24 ± 0.14 0.40 ± 0.02 1.28 ± 0.009 0.51 ± 0.04 0.89 ± 0.09Nickel chloride 0.18 ± 0.003 1.71 ± 0.19 1.59 ± 0.52 0.35 ± 0.01 0.59 ± 0.06

Each value represents the average of IC data calculated from 4 experiments, and were computed from the regression lines of the respective plots. IC values were analyzedb gnificr specti

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.3. Effect of metals on TJ formation

Mouse ES cells were grown on semi-permeable inserts, asescribed above, for 14 days in the presence of As2O3 (0.034 mMr 1/8 × IC50; Calabro et al., 2008). Cultures were exposed to theetal either on day 1 or day 7 of incubation (Fig. 2). Even at 12%

f IC50, the plots illustrate the ability of As to quickly prevent (day) or reverse (day 7) TMER values, an indicator of electrical resis-ance and TJ formation. Additionally, while FN maintains highestMER values by the end of the incubation period, neither FN nor-IV significantly affect TMER.

Fig. 3 illustrates the effect of As2O3 on cell proliferation in thebsence or presence of LIF or ECM. Despite the presence of LIF orCM, cell proliferation decreases at any rate. These results differrom gene expression for differentiation where ECM influences Oct-

and egfr genes (see below, Figs. 4 and 5, respectively).

.4. Effect of metals on gene expression

Figs. 4 and 5 summarize expression of Oct-4 and egfr mRNA, andorresponding TMER values, in mES cells grown in the presence of.034 mmol/L As2O3, administered on day 1 or day 7. As2O3 sig-ificantly reduces Oct-4 expression in mES cells in the presence ofN, as compared to cells maintained on C-IV. In almost all cases,ct-4 expression is reduced at each time period (except on day; Fig. 4). Conversely, egfr expression decreases when mES cellsre exposed to As2O3 on day 1 or day 7 in the presence of C-IVFig. 5). In addition, egfr expression increased with As, comparedo respective controls when grown on FN. Simultaneous measure-

ents of TMER during As2O3 exposure showed uniform inhibitoryffects on mES cell TJ formation, regardless of ECM component.hus, FN demonstrated the largest interaction with As, coordi-ating the decrease in Oct-4 expression with an increase in Egfrxpression.

In order to understand the role of DNA repair mechanismsriggered in response to metal-induced trauma, we studied theffects of trace metals on DNA repair gene expression in cells atifferent stages of differentiation. The results were unexpectedet notable—transcriptional changes are summarized in Table 2s tabulated from Fig. 6. Only Pb reduced expression of all threeenes. Almost all of the metals decreased Rad-18 and Ogg-1 genexpression after 1-h. Changes in Rad18 gene expression were moreronounced in cells exposed to As, Cu, Hg, and Pb (Fig. 6B), whiled and Mn showed no significant differences from untreated con-rol cells. Significant decreases in Ogg1 gene expression levelsere observed upon exposure of cells to Hg, Li, Mn, Ni and Pb

Fig. 6A). Only cells exposed to Cu and Pb exhibited a significantecrease in Top3a gene expression (Fig. 6C). Representative gelsrom RT-PCR analysis are shown from cells exposed to As, Li, and NiFig. 6D).

50

ant difference from corresponding MTT or NRU, 24-h or 21-h exposure (*P < 0.001),ve control).

3.5. Effect of metals on total histone protein (THP)

Cell proliferation and THP were determined from differentiatedmonolayers of cells exposed to IC25 and IC50 (24-h MTT) concentra-tions of As2O3, CdCl2, HgCl2, NiCl2 for 24-h. The results indicate thatall metals significantly decrease (P < 0.01) cell proliferation (Fig. 7).Under identical conditions, exposure to As2O3 and HgCl2 signif-icantly decreased THP only (P < 0.01), compared to correspondingcontrols. Among all metals, Cd exhibited the highest suppression ofcell proliferation (39% and 34% of control at IC25 and IC50 concen-trations, respectively; Fig. 7), whereas As presented with prevalentreductions in THP production (82% and 79% at IC25 and IC50 concen-trations, respectively). While the results suggest that trace metalsnon-specifically reduce cell proliferation, comparison of the ratiosof the decreases in THP produced vs. cell counts reveals that theremaining viable mES cells produce more THP per cell than cor-responding controls. Therefore, these metals apparently stimulatemES cell histone protein production per unit cell. Consequently,we speculate that the phenomenon is related to a hormesis effectpreviously demonstrated in human mammary cells (Schmidt et al.,2004). Furthermore, whereas these metals suppress THP producedper 104 cells, they activate histone protein production in otherwiseviable mES cells (Fig. 9).

3.6. Effect of metals on H3K27 mono-methylation

Twenty-four hour exposures to As2O3, CdCl2, HgCl2 and NiCl2significantly decreased H3K27 mono-methylation as a percent ofcontrol (% H3K27me1, Fig. 8). IC25 and IC50 values were derived from24-h MTT data. All of the metals significantly reduced %H3K27me1

beyond 50% (Fig. 8). Consequently, because H3K27me1 is linked toactivation of transcription, it is reasonable to conclude that tracemetals trigger transcriptional repression either by decreasing thetranscriptionally active states on promoter regions of the gene or byconverting transcriptionally active states of histones to repressivemarks, such as di- and tri-methyl states of H3K27.

4. Discussion

Embryonic stem cells have long been recognized as a modelfor in vitro mutagenicity and embryotoxicity studies. In particu-lar, mES cells possess developmental plasticity making them idealfor studying gene expression, DNA repair and epigenetic perturba-tions. More recently, mES cells are important for epigenetic studies,namely, genome-wide profiling of DNA methylation, histone mod-

ifications and DNA occupancy patterns of chromatin modifyingenzymes (Drab et al., 1997; Hemberger et al., 2009; Hembergerand Pedersen, 2010; Keller, 2005; Klug et al., 1996; Konsoula andBarile, 2005; McDonald et al., 1999; Potocnik et al., 1997).

S.R. Gadhia et al. / Toxicology Letters 212 (2012) 169– 179 173

Fig. 2. Transmembrane electrical resistance (TMER) of mES cells in culture after 14 days on fibronectin (FN) or collagen-IV (C-IV), in the absence or presence of arsenic (As)administered on day 1 or 7. Each bar represents the mean ± SE of three experiments.

Fig. 3. Effect of As, LIF and ECM on mES cell counts, administered on day 1 continuously for 12 days. Values are mean ± SE of three experiments.

174 S.R. Gadhia et al. / Toxicology Letters 212 (2012) 169– 179

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ig. 4. mRNA expression of Oct-4 gene from mES cells and their concurrent TMER vas shown as the ratio of band intensity resulting from RT-PCR for Oct-4 gene markerrom corresponding controls (P < 0.01), except A-day 12 and C-day 4.

Several growth factors, particularly IL-3, (Wiles and Keller,991), retinoic acid (Bain et al., 1995), and TGF�1 (Rohwedel et al.,994), have been shown to direct linear specific differentiationf mouse stem cells. EGF, and the related EGF family membermphiregulin, are mitogenic polypeptides that induce differen-iation into ectoderm and mesoderm (Gritti et al., 1999). In thebsence of LIF, a cytokine that inhibits differentiation, mES cellsreate intercellular contacts and initiate signaling and spontaneousifferentiation (Furue et al., 2005). Keller (2005) describes the

nduction of epithelial- or epidermal-specific gene expression and

ifferentiation using a combination of GFs plus ECM components

n human ES cells. Based on these reports, we explored the pos-ibility that mES cells could be stimulated to differentiate and

able 2ummary of changes in genes responsible for DNA repair as a result of 1 h exposure to m

Metals As Cd Cu

DNA repair gene IC50 (mg/mL)

0.02 0.02 0.25

Rad18 ↓ ≈ ↓

Ogg1 ↑ ≈ ≈

Top3a ≈ ≈ ↓

suppression; ↑ induction; ≈ no change.

ultured on FN or C-IV in the absence or presence of 0.034 mM/L As. Gene expressiones are mean ± SE of three experiments. All As treated groups significantly different

form confluent monolayers with significant intercellular contactsresulting in high resistance, when grown on porous inserts coatedwith ECM substrata. Moreover, the inclusion of mitogenic GFs inthe media, in the absence of MEF or LIF, could further programdifferentiation. We then examined how environmental toxicantscould influence linear specific differentiation.

Originally, we discovered that arsenic reduces cell proliferationregardless of the presence of LIF or ECM (Calabro et al., 2008). Yet LIFdominates only when there is no ECM. Thus, in the current study,we designed experiments to address the cytotoxic and epigenetic

effects of arsenic and other heavy metals on stem cell developmentand recovery under conditions that vary the times and concen-trations of exposure. In order to understand the mechanism of

etals.

Hg Li Mn Ni Pb

0.02 1.10 1.90 0.23 0.02

↓ ↓ ≈ ↓ ↓↓ ↓ ↓ ↓ ↓≈ ≈ ≈ ≈ ↓

S.R. Gadhia et al. / Toxicology Letters 212 (2012) 169– 179 175

Fig. 5. mRNA expression of egfr gene from mES cells and their concurrent TMER values, cultured on FN or C-IV in the absence or presence of 0.034 mM/L As administered ond resul

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t

ay 1 or day 7, until day 12. Gene expression is shown as the ratio of band intensity

rolonged cytotoxicity and recovery, we monitored mitochondrialnd lysosomal activities in the presence of one-tenth the IC50oncentration. Stem cell proliferation was significantly reducedfter 12 days of As exposure, indicating that not only are cytotoxiconsequences present but TJ integrity is also compromised. Thisas confirmed by the reduction of TMER when As was adminis-

ered either on day 1 or day 7. Thus As affects the formation of, asell as newly formed, TJs, respectively.

The mechanism by which metals influence cell differentiationas eluded most investigations. In conjunction with TMER and TJ

ormation, we examined the interaction of metals with ECM andheir influence on gene expression for differentiation. Oct-4 is a

ember of the POU gene family of transcription regulators whoseessation of expression leads to the loss of pluripotency (Niwa et al.,000; Pesce et al., 1998). Oct-4 is necessary for the maintenance ofhe “stem” cell state. Changes in the relative expression levels ofct-4 influence the pattern of other genes, such as egfr, which areeeded to induce specific phenotypical transformations in embry-nic stem cells (Pesce and Schöler, 2001). Egfr is a gene expressed byifferentiated epithelial-like cells (Ginis et al., 2004). Overall, whenES cells are grown on FN substrata, Oct-4 expression drops in the

resence of As, while egfr expression increases. Cells grown on C-V substrata produce opposite effects; i.e. egfr expression decreases

hile Oct-4 expression increases. Thus, As, with the help of FN,

ompels unwarranted and uncontrolled cellular differentiation—anffect remarkably aligned with cancer initiation and promotion.

DNA damage induced by metals is attributed in part throughhe production of ROS (Jimi et al., 2004; Orrenius, 2007; Rana,

ting from RT-PCR for egfr gene marker. Values are mean ± SE of three experiments.

2008; Silbergeld et al., 2000). The response of DNA repair genesin mES cells appears to be consistent with the mechanism oftoxicity for the metal and the corresponding repair pathway.For instance, Ogg1 is involved in base excision repair, espe-cially in reorganization of oxidized base pairs in DNA (Barnesand Lindahl, 2004; Klungland and Bjelland, 2007). The gene alsoencodes for the repair enzyme 8-oxoguanine DNA glycosylase-1,which is an integral part of an ROS-induced DNA repair sys-tem. The importance of Ogg1 repair enzyme is underlined bythe finding that the oxidized form of guanine (7, 8-dihydro-8-oxoguanine) stimulates GC- to TA-transversion type pointmutations that play a crucial role in the induction of cancer(Cheng et al., 1992). Because mammalian cells possess repairmechanisms that focus on elimination of ROS-induced gene insta-bility, and because metals have been implicated in generation ofROS, we also focused on other genes that signal ROS involve-ment.

Rad-18 is a RING type ubiquitin ligase that affects post-replication DNA repair in Saccharomyces cerevisiae (Bailly et al.,1997). Additionally, double strand breaks in G1 phase is influ-enced by Rad-18 (Watanabe et al., 2009). Consequently, as withOgg-1, it was not unusual to discover that almost all of the metalsdown-regulated Rad-18. Alternatively, Top-3a codes for DNAtopoisomerase IIIa which is responsible for DNA replication and

is an integral part of BLM-TOP3A-RMI1 protein complex that isassociated with Bloom syndrome—a condition resulting in highincidences of cancer in early life (Broberg et al., 2009). Thus,most of the metals (except Cu and Pb) did not significantly affect

176 S.R. Gadhia et al. / Toxicology Letters 212 (2012) 169– 179

Fig. 6. Summary of DNA repair gene expression after exposure to various metals for1-h. (A) Ogg1 gene expression from mES cells; (B) Rad18 gene expression from mEScells; (C) Top3a gene expression from mES cells; (D) Representative RT-PCR gels forexpression of DNA repair genes after exposure to various metals for 1-h. Lanes A,B and C correspond to untreated samples (controls) for Rad-18, Ogg-1 and Top-3a,respectively. Lanes D, E and F represent treatment groups for respective genes. LanesG, H & I are procedural controls. Values are mean ± SE of three experiments.

Fig. 7. Cell counts and total histone protein (THP) extraction data after treatment

with arsenic trioxide, cadmium chloride, mercuric chloride and nickel chloride atIC50 and IC25 concentrations. Values are mean ± SE of four experiments. For theirrespective controls, *P < 0.01, **P < 0.05.

transcriptional changes in Top3a gene—consistent with the factthat this gene is not involved with ROS-induced DNA damage.

Post-translational modifications (PTM), particularly covalentmodifications of histones, alter gene expression, cellular signaling,proliferation rate, and cell division—known biomarkers of cancerprogression (Berger, 2007). Thus, growing evidence suggests thatthe potential for gene expression in undifferentiated cells is regu-lated by epigenetic processes on DNA and chromatin in regulatoryand coding regions. In addition, because histone octamers formthe core of chromatin (Smith, 1991), PTM of histone proteins alsoaffect chromatin structure, thereby leading to altered transcriptionand gene expression (Krueger and Srivastava, 2006). In fact, it isnow well understood that epigenetic changes involve distortions,alterations, or influence on DNA methylation or demethylationpathways. Because this study targets a specific set of epigeneticpathways, the results of exposure to metals pose an important

link toward understanding the role of epigenetic toxicity in humandisease induction and/or progression.

Furthermore, histone modifications are broadly classifiedaccording to their ability to activate or repress transcription

Fig. 8. % H3K27 mono-methylation upon exposure to IC25 and IC50 concentrations ofarsenic trioxide, cadmium chloride, mercuric chloride and nickel chloride for 24-h.Values are mean ± SE of four experiments. For their respective controls, *P < 0.01.

gy Letters 212 (2012) 169– 179 177

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Fig. 9. Comparison of total histone protein (�g)/104 cells after exposure of mEScells to IC25 and IC50 concentrations of arsenic trioxide, cadmium chloride, mercuricchloride and nickel chloride. Values are mean ± SE of four experiments. For theirrespective controls, *P < 0.05.

Table 3Amount of total histone protein (�g) per 104 cells and as percent of control.

�g histone/104 cells: (1) % of control; (2) �gtotal histone protein/104 cells

Control1 100 ± 5.22 1.99 ± 0.1

Metal IC25 IC50

As2O3

1 113.4 ± 14.9 90.12 ± 23.42 2.3 ± 0.3 1.8 ± 0.5

CdCl21 219.9 ± 23.4* 167.7 ± 15.8*2 4.4 ± 0.4* 3.3 ± 0.3

HgCl21 78.4 ± 16.6 154.6 ± 58.12 1.6 ± 0.3 3.0 ± 1.2

NiCl21 184.4 ± 39.6* 217.9 ± 14.9*2 3.7 ± 0.8* 4.3 ± 0.3*

Each value represents the average of IC25 or IC50 data calculated from at least 3 exper-iments, and were computed from the regression line of the respective plots. Values

S.R. Gadhia et al. / Toxicolo

f genes. For instance, lysine residues are either mono-, di- orri-methylated whereas arginine residues are either mono- ori-methylated (Berger, 2007). The most common lysine methy-

ation sites include H3K4, H3K9, H3K27, H3K36 and H4K20.nterestingly, mono-methylation of H3K27 (H3K27me1) resultsn transcriptional activation while dimethyl and trimethyl statesH3K27me2/me3) are repressive (Akbarian and Huang, 2009;anwal and Gupta, 2010; Yu et al., 2008). In addition, enzymes suchs EZH2 and JMJD3, are reportedly over expressed in metastaticrostate cancer, and are responsible for methylation and demethy-

ation of H3K27enzyme, respectively. (Kanwal and Gupta, 2010).Methylation of lysine residues is catalyzed by histone lysine

ethyltransferases. The enzymes catalyze mono-methylation ofistones, characterized by an initial burst in mono-methylation

evels followed by slower conversion to di- and tri-methylationorms. Accordingly, mono-methylation is a pre-requisite for forma-ion of di- and tri-methylation states (Rea et al., 2000; Simon andange, 2008). In this study, exposure of confluent, differentiatedells to As, Cd, Hg and Ni for 24-h resulted in a significant decreasen the percentage of mono-methyl H3K27 compared to controlroups (Fig. 8). However, only As and Hg decrease H3K27me1 to

greater extent than they decrease cell proliferation. This suggestshat As and Hg selectively act on the former pathway. Thus because3K27me1 is involved in transcriptional activation, the decrease

uggests that As and Hg at low doses represses gene transcription.hereas stable marks, like H3K27me3, are dynamic and reversible,

3K27me1 serves as a substrate for H3K27me2 and/or -me3, whichre repressive marks. Our future studies will investigate the effectf trace metal exposure on the conversion to H3K27me2/me3tates, and by determining heritability of metal-induced histoneodifications in subsequent cell generations.Cytotoxicity studies suggest that Cd was most toxic, followed by

i, Hg, As, Cu, Pb and Li, respectively, with 24-h exposures. Whenells are exposed for 1-h only, IC50 values increased dramatically,ith the exception of Hg. This suggests that although metals are

apable of exerting their toxic effects in the first few hours of expo-ure, prolonged duration and accumulation of the chemical is alsomportant in establishing toxicity. When stem cells are exposedo the same metals for 1-h, followed by a 23-h recovery period,nly the cells exposed to low concentrations of metals were ableo recover and proliferate. To further characterize this effect, DNAepair gene expression was suppressed in almost all cases afterxposure to low concentrations of various metals for 1-h (Fig. 6nd Table 2), confirming the inability of repair pathways to over-urn metal insult. Interestingly, Schmidt et al. (2004) reported that

ost metals, including As, Cd, Cu, Hg, Li, Ni and Pb, demonstrate anormesis effect at low doses.

Considering the carcinogenic potential of most of the metalssed in this study, only As and Hg produced significant histonerotein depletion (P < 0.05). However, the effect on mES cell pro-

iferation was greater than that of histone protein depletion. Tonvestigate this effect, we calculated the amount of histone pro-uced per 104 cells. Of the metals used, Ni and Cd returned an

ncrease of about 200% in histone protein production comparedo their respective controls (P < 0.05; Fig. 9 and Table 3). Alterna-ively, As and Hg failed to show a significant increase in histonerotein production per unit cell, suggesting that Ni and Cd elicit aompensatory repair mechanism requiring the contribution of his-one proteins to ameliorate metal insult. Along with the effects on3K27me1, As and Hg disrupt total protein production and do not

nduce a compensatory repair mechanism.This study presents evidence that metals alter expression of

enes responsible for differentiation and DNA repair in mES cells.ct-4 is linked to transcriptionally-activating histone modifica-

ions, is associated with maintenance of pluripotency in ES cellsGidekel et al., 2003; Monk and Holding, 2001; Rajasekhar and

were analyzed by one-way ANOVA; data are statistically different from respectivecontrols (*P < 0.05, Students’ t-test and z-test).

Begemann, 2007; Reya et al., 2001), and is necessary for main-taining the undifferentiated state (Ginis et al., 2004). Thus, wesuggest that gene expression relies on systematic temporal factors;that is, arsenic appears to exert maximal effect in early stages ofmES cell differentiation. The metals are also capable of suppressingH3K27me1 and altering expression of DNA repair. This supportsour hypothesis that heavy metals in trace quantities are capable ofsuppressing pathways required for transcriptional activation, thusaffecting cell proliferation, differentiation, protein production andrecovery from DNA damage. In addition, because histone modifica-tions have been implicated in the discussion as epigenetic marks,this study supports the contention that these indicators serve aspotential therapeutic targets in an environment where exposure tovarious toxic metals is inevitable.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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78 S.R. Gadhia et al. / Toxicolo

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