Mi

ARa

b

c

a

ARRAA

KAACMM

1

bimcet[

aodabd

M0

1h

Mutation Research 759 (2014) 51– 55

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

jou rn al h om ep age: www.elsev ier .com/ locate /gentoxC om mu n i ty add ress : www.elsev ier .com/ locate /mutres

icronucleus frequency in copper-mine workers exposed to arsenics modulated by the AS3MT Met287Thr polymorphism

lba Hernándeza,b,1, Leiliane Paivaa,1, Amadeu Creusa, Domingo Quinterosc,icard Marcosa,b,∗

Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, SpainCIBER Epidemiología y Salud Pública, ISCIII, SpainDepartamento de Salud Ocupacional, División Codelco Norte, Corporación del Cobre de Chile, Chile

r t i c l e i n f o

rticle history:eceived 4 May 2013eceived in revised form 6 August 2013ccepted 28 September 2013vailable online 17 December 2013

eywords:S3MTrsenic

a b s t r a c t

Arsenic(III)methyltransferase (AS3MT) has been demonstrated to be the key enzyme in the metabolism ofarsenic as it catalyses the methylation of arsenite and monomethylarsonic acid (MMA) to form methylatedarsenic species, which have higher toxic and genotoxic potential than the parent compounds. The aimof this study is to evaluate if genetic variation in the AS3MT gene influences arsenic-induced cytogeneticdamage, measured by the micronucleus (MN) assay. AS3MT Met287Thr allele frequencies and MN valueswere determined for 207 subjects working in the copper-mine industry, who were exposed to variablelevels of arsenic. The urinary arsenic profile was used as individual biomarker of arsenic exposure. Resultsindicate that the MN frequencies found in peripheral blood lymphocytes of the exposed population poorly

ooper-mine workerset287Thr polymorphismicronucleus

correlate with the levels of total arsenic content in urine. Nevertheless, when workers were classifiedaccording to their AS3MT Met287Thr genotypes, significantly higher MN values were observed for thosecarrying the variant allele [odds ratio (OR), 3.4 (1.6–5.2); P = 0.0003)]. To our knowledge, these resultsare the first to show that genetic variation in AS3MT, especially the Met287Thr polymorphism, may playa role in modulating the levels of arsenic-induced cytogenetic damage among individuals chronically

exposed to arsenic.

. Introduction

Arsenic is a ubiquitous element present in the environment fromoth natural and anthropogenic sources such as extracting, min-

ng and smelting of copper minerals. Inorganic arsenic (i-As) is theajor environmental form and it is known to be a potent human

arcinogen. Epidemiological data clearly reported that chronicxposure to this compound induces a significant increase of cer-ain types of cancer, particularly of bladder, kidney, skin and lung1–3].

After arsenic intake, a certain proportion of the compound isccumulated in different parts of the body. Approximately 40–60%f the ingested arsenic is eliminated through urine within 1 or 2ays, and urinary arsenic speciation is, therefore, the most reli-

ble measure of individual arsenic biotransformation ability. Thisiotransformation is mediated by the enzyme arsenic (+3 oxi-ation state) methyltransferase (AS3MT) [4,5], and the resulting

∗ Corresponding author at: Grup de Mutagènesi, Departament de Genètica i deicrobiologia, Universitat Autònoma de Barcelona, Edifici Cn, Campus de Bellaterra,

8193 Cerdanyola del Vallès, Spain.E-mail address: ricard.marcos@uab.es (R. Marcos).

1 These authors contributed equally to this work.

383-5718/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.mrgentox.2013.09.010

© 2013 Elsevier B.V. All rights reserved.

organic methylated products, monomethylarsonic acid (MMA) anddimethylarsinic acid (DMA), are rapidly excreted via the urine alongwith their inorganic parent species [6,7].

Although genetic variation in AS3MT has been associated withchanges in arsenic biotransformation [8–10] and with skin patholo-gies related to exposure to arsenic [11], no association witharsenic-induced cytogenetic damage has been proposed so far inchronically exposed human populations. In this context, the aim ofthis study is to evaluate if individual genotypes affecting the AS3MTgene are able to modulate the level of cytogenetic damage presentin a group of copper-mine workers exposed to different levels ofarsenic, dependent on their working place. The level of individualgenetic damage was evaluated in peripheral blood lymphocytesby measuring the frequency of micronuclei as the biomarker ofgenotoxic effect.

In spite of the genotoxic risk attributed to exposure to arsenic[1,12], in a previous study [13] we failed to found increased levels ofmicronuclei when copper mining workers were studied and clas-sified according to the working place setting. Thus, this work aims

to demonstrate, in the same population, whether arsenic is ableto induce genotoxic damage at the chromosome level, when theability of the exposed individuals to properly metabolize arsenic istaken into account. As we have previously demonstrated [8,9], the

5 tion Re

Mltro

2

2

AAoifoUscdst

2

ttBum1p

btLAti(aciqtwb

2

vsSt(csidib

2 A. Hernández et al. / Muta

et287Thr polymorphism in the AS3MT gene significantly modu-ates the urinary profile for arsenic metabolites; it is for this reasonhat we propose the use of this polymorphism to study not only itsole in arsenic metabolism, but also in the modulation of the levelsf arsenic-induced genotoxic damage.

. Materials and methods

.1. Study population

The recruited population comprised a total of 207 non-merindian Chilean men working in the copper industry in thentofagasta region (in the northern part of Chile), where high levelsf arsenic were found to be in direct correlation with mining activ-ties. One hundred and five of them were working in the smeltingacilities, while the others were involved in tasks with lower levelsf exposure. The study was approved by the Ethics Committee of theniversitat Autònoma de Barcelona. Previous to the sampling, all

ubjects gave informed consent and blood and urine samples wereollected and further manipulated in accordance with ethical stan-ards. All participants were healthy volunteers and completed atandardized questionnaire covering standard demographic ques-ions including ethnicity, habits, medical and family history.

.2. Lymphocyte culture and MN assay

Blood samples were obtained from each subject by venipunc-ure, collected in heparinized vacutainers, and sent directly tohe Laboratory of Mutagenesis at the Autonomous University ofarcelona in Bellaterra (Spain). Lymphocyte cultures were setp by adding 0.5 mL of whole blood to 4.5 mL of RPMI 1640edium supplemented with 15% heat-inactivated fetal calf serum,

% antibiotics (penicillin and streptomycin) and 1% l-glutamine (allrovided by Gibco Life Technologies, Paisley, UK).

For the micronucleus (MN) assay, lymphocytes were stimulatedy 1% of phytohaemagglutinin (Gibco) at 37 ◦C and, after 44 h ofhe culture set-up, 6 �g/mL of cytochalasin B (Cyt-B, Sigma, St.ouis, MO, USA) were added to the cultures to arrest cytokinesis.fter 72 h of incubation, cultures were harvested by centrifuga-

ion at 120 × g for 8 min. Next, blood cultures were washed oncen RPMI 1640 medium followed by a mild hypotonic treatment2–3 min in 0.075 M KCl at 4 ◦C). Cells were centrifuged and fixed in

methanol/acetic acid (3:1 v/v) solution. Two or more slides wereoded and stained with 10% Giemsa (Merck, Darmstadt, Germany)n phosphate buffer (pH 6.8) for 10 min. To determine the fre-uency of bi-nucleated cells with micronuclei (BNMN) and theotal number of micronuclei, a total of 1000 bi-nucleated cells withell-preserved cytoplasm (500 per replicate) for each subject were

lindly scored on coded slides.

.3. Urine collection and detection of arsenic species

Approximately 125 mL of urine were obtained from each indi-idual in vials containing 0.01 M sodium azide. Samples weretored frozen for further analysis at the National Institute of Healthciences in Tokyo. Determination of the levels of total arsenic,rivalent inorganic arsenic (AsIII), pentavalent inorganic arsenicAsV), MMA and DMA were obtained by high-performance liquidhromatography combined with inductively coupled plasma mass-pectrometry (HPLC-ICP/MS). The experimental protocol used was

ncapable to detect methylated trivalent arsenicals. A detailedescription of the analytical protocol for measurement of arsenic

n urine, including reagents, instrumentation and procedures haseen described previously [14,15].

search 759 (2014) 51– 55

2.4. SNP analyses

DNA was isolated from peripheral blood samples (9 mL) col-lected into ethylenediaminetetraacetic acid (EDTA) tubes by usinga standard phenol-chloroform extraction method. DNA sampleswere dissolved in 25 �L of 10 mM Tris/0.2 mM EDTA (pH 7.5) andpreserved at −20 ◦C at a final concentration of 1 �g/mL until anal-ysis.

Primers and specific conditions for DNA amplification bythe polymerase chain reaction (PCR) are described elsewhere[15]. Briefly, a 300-bp region of the AS3MT exon 9 containingthe Met287Thr polymorphism was amplified with the forwardprimer 5′-TGAGCAAGGCAACAACTGG-3′ and the reverse primer 5′-CAGAAAAATGGGAGGCAATG-3′. The amplification was carried outin a 25-�L reaction mixture containing 40 ng of DNA, 0.2 mM ofeach dNTP, 2 mM MgCl2, 1X PCR buffer, 1 U Taq DNA Polymerase(Promega, Madison, Wisconsin, USA) and 0.25 mM of each primeron an iCycler Thermal Cycler (Bio-Rad Laboratories, Richmond,California, USA) with the following cycling program: initial dena-turation at 95 ◦C for 2 min, 35 cycles of 95 ◦C for 30 s, 62 ◦C for 30 s,72 ◦C for 30 s and a final extension at 72 ◦C for 1 min.

The clean-up of PCR products for cycle sequencing was donewith the NucleoSpin Extract columns (Macherey-Nagel GmbH &Co., Düren, Germany) following the instructions of the manufac-turer. The sequence analysis was further carried out on a 3730xlDNA analyzer (Applied Biosystems, Foster City, California, USA)at the Macrogen sequencing service (Macrogen Inc., Seoul, Korea)with the sequencing primers, 5′-TGAGCAAGGCAACAACTGG-3′ and5′-TGAGCAAGGCAACAACTGG-3′.

2.5. Statistical analysis

Statistical computations were performed with the SPSS ver-sion 14.0 (SPSS Inc., Chicago, IL, USA) and the SNPStats web-toolsoftware (http://bioinfo.iconcologia.net/snpstats/). Specific geno-type frequencies, allele frequencies and goodness-of-fit tests forHardy–Weinberg equilibrium were calculated.

To test the hypothesis of an association between the polymor-phism and the urinary arsenic profile, multivariate methods on thebasis of logistic regression analyses were used. Odds ratios (OR) and95% confidence intervals were calculated for the genotypes carryingthe variant allele, compared with the homozygous genotype for thenormal allele (the allele with greater frequency among the group),which was set as the reference. All analyses were adjusted for ageand total arsenic exposure, and possible risk factors (alcohol, smok-ing, tea and coffee consumption) were excluded as confoundersbecause they were found to be independent of the variables stud-ied. Probabilities were derived from likelihood ratio tests, and asignificance level of 5% (two-sided analysis of variance) was usedfor the analyses.

3. Results

3.1. Descriptive of the population

The copper-mine workers analyzed in the study were 207 non-Amerindian Chilean men from the North of Chile exposed to arsenicfor a period of 19.78 ± 0.60 years (mean ± SEM). The total aver-age urinary arsenic content of the population was 91.26 ± 6.36 ppb(mean ± SEM), ranging from 1.40 ppb to 625.20 ppb, dependent ontheir occupational setting. The average age of the population was

46.30 ± 0.46 years. Of the subjects, 81.20% indicated to drink alcoholwith a mean consumption of 74.29 ± 5.29 g per week, 38.20% weretobacco smokers, although with a low mean value of 6.06 ± 0.59cigarettes per day, 86.50% were tea drinkers (mean of 2.28 ± 0.10

A. Hernández et al. / Mutation Research 759 (2014) 51– 55 53

Table 1Urinary arsenic profile of the study population.

Years of exposurea As totala (ppb) % AsVa % AsIIIa % MMAa % DMAa

Overall population, n = 207 19.78 ± 0.60 91.26 ± 6.36 5.22 ± 0.39 12.01 ± 0.89 13.20 ± 0.53 69.56 ± 0.97Group 1, n = 69 17.20 ± 1.14 21.2 ± 1.4 4.35 ± 0.73 11.36 ± 0.89 11.78 ± 1.12 72.5 ± 1.78Group 2, n = 69 21.69 ± 0.97 70.3 ± 1.8 5.22 ± 0.39 12.01 ± 0.89 13.20 ± 0.53 69.56 ± 0.97Group 3, n = 69 20.35 ± 0.94 182.6 ± 12.7 4.68 ± 0.60 16.19 ± 2.21 13.65 ± 0.80 65.46 ± 1.83

AsIII , trivalent inorganic arsenic; AsV, pentavalent inorganic arsenic; DMA, dimethylarsinic acid; MMA, monomethylarsonic acid; ppb, parts per billion. Groups 1, 2 and 3correspond to individuals with low, moderate and high levels of arsenic in urine, respectively.

a Mean ± S.E.

Table 2Micronucleus formation in the study population.

Overallpopulationn = 207

Group 1n = 69

Group 2n = 69

Group 3n = 69

As exposurea (ppb) 91.3 ± 6.4 (1.4–625.2) 21.2 ± 1.4 (1.4–42.1) 70.3 ± 1.8 (42.8–93.4) 182.6 ± 12.7 (95.1–625.2)BNMNa 8.6 ± 0.4 8.3 ± 0.7 9.7 ± 0.7 8.1 ± 0.7

G rsenic in urine, respectively.

cs

3

pvwtttl

otfAIu1[tuda

3

iedtteimM

3

ga

Table 3Influence of genetic variation in the observed cytogenetic damage.

BNMN F P value

Corrected model 15.72 0.000As exposure level (total As) 5.164 0.024AS3MT Met287Thr polymorphism 5.267 0.006

The influence of the polymorphisms appeared to be significant(P = 0.006) also in the linear univariate model shown in Table 3.A logistic regression was also carried out to better determine the

roups 1, 2 and 3 correspond to individuals with low, moderate and high levels of aa Mean ± SEM per 1000 BNMN (bi-nucleated cells with micronuclei).

ups per day), whereas only the 33.80% affirmed to be coffee con-umers (mean of 2.07 ± 0.19 cups per day).

.2. SNP analysis and urinary arsenic profiles

The study population was genotyped for the Met287Thr (T/C)olymorphism at the AS3MT gene. Results indicate that 172 indi-iduals were homozygous for the wild-type allele (T/T, 83.0%), 34ere heterozygous (T/C 16.0%), and only 2 were homozygous for

he variant allele (C/C 1.0%). The frequency of the variant allele washerefore of 0.09 in the study population, and the genotype dis-ribution was found to be in agreement with Hardy–Weinberg’saw.

The levels of total arsenic, inorganic arsenic (AsV and AsIII) andrganic arsenic metabolites (MMA and DMA) were determined inhe urine of the subjects and results are given in Table 1. The valuesor the overall population, measured in percentage, were as follows:sV 5.2 ± 0.4, AsIII 12.0 ± 0.9, MMA 13.2 ± 0.5 and DMA 69.6 ± 1.0.

n general, the urinary arsenic speciation profile of the study pop-lation was in agreement with what is considered as standard:0–30% of inorganic arsenic, 10–20% of MMA and 60–80% of DMA6]. The participants were divided in tertiles of exposure accordingo their individual urinary arsenic content and Table 1 shows theirrinary arsenic species characteristics. There were no significantifferences between the subgroups of exposure regarding habitsnd lifestyle (data not shown).

.3. Cytogenetic damage

Table 2 shows the frequency of arsenic-induced MN observedn the overall population, as well as in the three subgroups ofxposure. Although group 2 presented higher levels of MN, theifference did not attain statistical significance. In fact, only a close-o-significant correlation was found between the level of MN andotal arsenic in urine (P = 0.054). This slight influence of the level ofxposure on the cytogenetic damage of the subjects is also reflectedn the association model presented in Table 3. Also, as expected, the

odel showed age as a variable strongly influencing the individualN frequency.

.4. Associations between MN and AS3MT polymorphisms

We were particularly interested to investigate whether commonenetic variants affecting the functionality of the AS3MT enzymere associated with an increase in the level of MN damage induced

Age 50.976 0.000

R2 for the corrected model = 0.352.

by chronic arsenic exposure. As shown in Fig. 1, when the fre-quencies of MN were established according to the genotypes ofthe subjects, a clear increase in the values of MN was observeddepending on the number of variant alleles present: T/T (8.1 ± 0.4),T/C (10.6 ± 1.9), C/C (14.5 ± 2.5).

Fig. 1. Micronucleus formation – given as number of micronuclei per 1000 bi-nucleated cells – in individuals with the AS3MT Met287Thr genotypes.

54 A. Hernández et al. / Mutation Research 759 (2014) 51– 55

Table 4Effect of AS3MT Met287Thr polymorphism on the MN frequency.

Met287Thr (T/C) n BNMNa OR (95% CI)b P value

Population n = 207 T/T T/C + C/C 17235

8.1 ± 0.410.9 ± 1.0

0.003.4 (1.6–5.2)

0.0003

eqabplovm

4

bosdaqoinafsttei

c[blbot[tdtac

laSAclpcai[

b

a Mean ± SE.b OR adjusted for age and total arsenic exposure.

ffect of the AS3MT Met287Thr (T/C) polymorphism on the MN fre-uency and the results are indicated in Table 4. As observed, afterdjustment of the odds ratios (OR) for age and total urinary arsenic,oth variables were found to influence the MN frequency in ouropulation. The dominant model (where individuals carrying at

east one variant allele were included in the same category) was thene that better fit with the data. As indicated in the Table, the indi-iduals carrying at least one variant allele show 3.4-fold (1.6–5.2)ore MN than those homozygous for wild type alleles (P = 0.0003).

. Discussion

Chronic exposure to the environmental contaminant arsenic haseen associated with increases in human cancer incidence, mainlyf skin, lung, bladder, and kidney [1–3]. Most of the biomonitoringtudies carried out in human populations exposed to arsenic haveemonstrated positive correlations between the levels of exposurend the increase in biomarkers of effect such as micronucleus fre-uency and chromosomal aberrations [16–18]. Previous studies ofur group investigated whether occupational exposure to arsenicn a Chilean population lead to increases in the levels of cytoge-etic damage, measured by the sister-chromatid exchange (SCE)nd micronucleus (MN) frequencies, showing only a slight increaseor the SCE biomarker [13,19]. However, our group also demon-trated that the comet assay may be a more sensitive technique forhe detection of low-dose effects of arsenic [15]. Thus, we were ableo demonstrate a positive association between the level of arsenicxposure and the genetic damage measured as percentage of DNAn tail (P < 0.001) in a chronically exposed Mexican population [15].

It is generally accepted that the metabolism of arsenic plays arucial role in the genotoxic risk associated with human exposure10]. Indeed, different authors have found significant associationsetween the methylating ability and an increased risk of skin

esions and cancer, bladder cancer and hypertension in severaliomonitoring studies [20–23]. Methylated arsenic species in anxidative state of +3 have been demonstrated to be more cyto-oxic and genotoxic than the inorganic forms arsenate and arsenite24,25]. In fact, MMAIII is accepted as the arsenic metabolite withhe highest toxic and genotoxic potential [24]. Methylated arsenicerivatives are also more potent inhibitors of glutathione reduc-ase [26], thioredoxin reductase [27] and pyruvate reductase thanrsenite [28], and are known to possess DNA-damaging capabilities,lastogenic capabilities [29,30], and to induce aneuploidy [31].

The arsenic methyltransferase (AS3MT) enzyme is well estab-ished as the key enzyme in the bio-transformation pathway ofrsenic as it catalyzes the methylation of arsenite and MMA, with-adenosyl methionine as the methyl donor [32–35]. The humanS3MT gene encoding this 375-amino acid protein is located onhromosome 10q24.33, is organized in 11 exons and has a totalength of approximately 32 kb. Although several AS3MT polymor-hisms have been described so far, the most relevant variant is a T/Change localized in exon 9 leading to a Met/Thr amino acid changet position 287 of the protein. The mutated protein demonstrated

ncreased levels of both enzyme activity and immunoreactivity36].

Our group was the first to demonstrate a positive associationetween the AS3MT Met287Thr polymorphism and the percent

of MMA excreted in the urine of exposed subjects [8,9]. In thesestudies, exposed individuals with the variant allele presented a>4% higher level of MMA in urine than those homozygous for thewild-type allele (P = 0.0007). Other authors were capable of corrob-orating the effect of the polymorphism on the percent of MMA ina different human population [11]. The study also indicated thatMet287Thr influences the susceptibility to premalignant arsenic-induced skin lesions, as individuals carrying the C (TC + CC) allele(Thr) were found to be at higher risk [odds ratio = 4.28; 95% confi-dence interval (1.0–18.5)].

Despite the demonstrated influence of the AS3MT Met287Thrpolymorphism in MMA excretion and arsenic-associated risk,only one biomonitoring study has been conducted linking thearsenic-induced cytogenetic damage and the genetic variabilityaffecting the arsenic metabolizing enzymes [15]. In that study, theAS3MT Met287Thr polymorphism was found to be associated withincreased levels of genotoxic DNA damage, but only among exposedchildren.

In the present work, a positive association between the AS3MTchange and the frequency of MN has been found. This finding wouldagree with the known role of this enzyme in arsenic methylation. Inthis sense, it is easy to assume that those individuals carrying thevariant allele will accumulate higher amounts of the more toxicMMAIII and, consequently, present a higher frequency of cytoge-netic damage.

Overall, this biomonitoring study is the first to show that AS3MTMet287Thr polymorphism may modulate the levels of cytogeneticdamage in a large population chronically exposed to arsenic. Thiswould agree with the known higher levels of MMA present in theurine of carriers of the variant allele and also with its known associ-ation with cancer incidence [37] and premalignant arsenic-inducedskin lesions [11].

Conflict of interest statement

The authors report no conflict of interests and are responsiblefor the content and writing of the paper.

Acknowledgements

We wish to thank all volunteers who participated in this study.This work was partly supported by a contract with the Corpo-ración del Cobre de Chile and by grants from the Generalitat deCatalunya (2009SGR-725) and the Spanish Ministry of Economiay Competitividad (SAF2011-23146). During this work, L. Paiva,and A. Hernández were supported by grants from the Generalitatde Catalunya and the Spanish Ministry of Education and Science,respectively.

References

[1] A. Basu, J. Mahata, S. Gupta, A.K. Giri, Genetic toxicology of a paradoxical humancarcinogen, arsenic: a review, Mutat. Res. 488 (2001) 171–194.

[2] S. Tapio, B. Grosche, Arsenic in the aetiology of cancer, Mutat. Res. 612 (2006)215–246.

[3] P. Bhattacharjee, D. Chatterjee, K.K. Singh, A.K. Giri, Systems biology approachesto evaluate arsenic toxicity and carcinogenicity: an overview, Int. J. Hyg. Envi-ron. Health 216 (2013) 574–586.

tion Re

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

methyltransferase (AS3MT) pharmacogenetics: gene resequencing and func-tional genomics studies, J. Biol. Chem. 281 (2006) 7364–7373.

A. Hernández et al. / Muta

[4] D.J. Thomas, Molecular processes in cellular arsenic metabolism, Toxicol. Appl.Pharmacol. 222 (2007) 365–373.

[5] D. Sumi, S. Himeno, Role of arsenic (+3 oxidation state) methyltransferase inarsenic metabolism and toxicity, Biol. Pharm. Bull. 35 (2012) 1870–1875.

[6] M. Vahter, Mechanisms of arsenic biotransformation, Toxicology 181–182(2002) 211–217.

[7] T. Watanabe, S. Hirano, Metabolism of arsenic and its toxicological relevance,Arch. Toxicol. 87 (2013) 969–979.

[8] A. Hernández, N. Xamena, C. Sekaran, H. Tokunaga, A. Sampayo-Reyes, D. Quin-teros, A. Creus, R. Marcos, High arsenic metabolic efficiency in AS3MT287Thrallele carriers, Pharmacogenet. Genomics 18 (2008) 349–355.

[9] A. Hernández, N. Xamena, J. Surrallés, C. Sekaran, H. Tokunaga, D. Quinteros, A.Creus, R. Marcos, Role of the Met(287)Thr polymorphism in the AS3MT gene onthe metabolic arsenic profile, Mutat. Res. 637 (2008) 80–92.

10] A. Hernández, R. Marcos, Genetic variations associated with interindividualsensitivity in the response to arsenic exposure, Pharmacogenomics 9 (2008)1113–1132.

11] O.L. Valenzuela, Z. Drobná, E. Hernández-Castellanos, L.C. Sánchez-Pena, G.G.García-Vargas, V.H. Borja-Aburto, M. Styblo, L.M. Del Razo, Association ofAS3MT polymorphisms and the risk of premalignant arsenic skin lesions, Tox-icol. Appl. Pharmacol. 239 (2009) 200–207.

12] F. Faita, L. Cori, F. Bianchi, M.G. Andreassi, Arsenic-induced genotoxicity andgenetic susceptibility to arsenic-related pathologies, Int. J. Environ. Res. PublicHealth 10 (2013) 1527–1546.

13] L. Paiva, V. Martínez, A. Creus, D. Quinteros, R. Marcos, Evaluation of micro-nucleus frequencies in blood lymphocytes from smelting plant workersexposed to arsenic, Environ. Mol. Mutagen. 49 (2008) 200–205.

14] R. Marcos, V. Martínez, A. Hernández, A. Creus, C. Sekaran, H. Toku-naga, D. Quinteros, Metabolic profile in workers occupationally exposed toarsenic: role of GST polymorphisms, J. Occup. Environ. Med. 48 (2006) 334–341.

15] A. Sampayo-Reyes, A. Hernández, N. El-Yamani, C. López-Campos, E. Mayet-Machado, C.B. Rincón-Castaneda, M.L. Limones-Aguilar, J.E. López-Campos,M.B. De León, S. González-Hernández, D. Hinojosa-Garza, R. Marcos, Arsenicinduces DNA damage in environmentally exposed Mexican children and adults.Influence of GSTO1 and AS3MT polymorphisms, Toxicol. Sci. 117 (2010)63–71.

16] A. Basu, J. Mahata, A.K. Roy, J.N. Sarkar, G. Poddar, A.K. Nandy, P.K. Sarkar, P.K.Dutta, A. Banerjee, M. Das, K. Ray, S. Roychaudhury, A.T. Natarajan, R. Nils-son, A.K. Giri, Enhanced frequency of micronuclei in individuals exposed toarsenic through drinking water in West Bengal, India, Mutat. Res. 516 (2002)29–40.

17] A. Basu, P. Ghosh, M. Das, A. Banerjee, K. Ray, A.K. Giri, Micronuclei as biomark-ers of carcinogen exposure in populations exposed to arsenic through drinkingwater in West Bengal, India: a comparative study in three cell types, CancerEpidemiol. Biomarkers Prev. 13 (2004) 820–827.

18] T. Chakraborty, U. Das, S. Poddar, B. Sengupta, M. De, Micronuclei and chromo-somal aberrations as biomarkers: a study in an arsenic exposed population inwest Bengal, India, Bull. Environ. Contam. Toxicol. 76 (2006) 970–976.

19] L. Paiva, V. Martínez, A. Creus, D. Quinteros, R. Marcos, Sister chromatidexchange analysis in smelting plant workers exposed to arsenic, Environ. Mol.Mutagen. 47 (2006) 230–235.

20] R.C. Yu, K.H. Hsu, C.J. Chen, J.R. Froines, Arsenic methylation capacity and skincancer, Cancer Epidemiol. Biomarkers Prev. 9 (2000) 1259–1262.

[

search 759 (2014) 51– 55 55

21] C.J. Chen, S.L. Wang, J.M. Chiou, C.H. Tseng, H.Y. Chiou, Y.M. Hsueh, S.Y. Chen,M.M. Wu, M.S. Lai, Arsenic and diabetes and hypertension in human popula-tions: a review, Toxicol. Appl. Pharmacol. 222 (2007) 298–304.

22] K.M. McCarty, Y.C. Chen, Q. Quamruzzaman, M. Rahman, G. Mahiuddin, Y.M.Hsueh, L. Su, T. Smith, L. Ryan, D.C. Christiani, Arsenic methylation, GSTT1,GSTM1, GSTP1 polymorphisms, and skin lesions, Environ. Health Perspect. 115(2007) 341–345.

23] Y.S. Pu, S.M. Yang, Y.K. Huang, C.J. Chung, S.K. Huang, A.W. Chiu, M.H. Yang, C.J.Chen, Y.M. Hsueh, Urinary arsenic profile affects the risk of urothelial carcinomaeven at low arsenic exposure, Toxicol. Appl. Pharmacol. 218 (2007) 99–106.

24] M.J. Mass, A. Tennant, B.C. Roop, W.R. Cullen, M. Styblo, D.J. Thomas, A.D. Kliger-man, Methylated trivalent arsenic species are genotoxic, Chem. Res. Toxicol. 14(2001) 355–361.

25] J.S. Petrick, F. Ayala-Fierro, W.R. Cullen, D.E. Carter, H.H. Aposhian, Monomethy-larsonous acid (MMA(III)) is more toxic than arsenite in Chang humanhepatocytes, Toxicol. Appl. Pharmacol. 163 (2000) 203–207.

26] D. Chouchane, E.T. Snow, In vitro effect of arsenical compounds on glutathione-related enzymes, Chem. Res. Toxicol. 14 (2001) 517–522.

27] S. Lin, L.M. Del Razo, M. Styblo, C. Wang, W.R. Cullen, D.J. Thomas, Arsenicalsinhibit thioredoxin reductase in cultured rat hepatocytes, Chem. Res. Toxicol.14 (2001) 305–311.

28] J.S. Petrick, B. Jagadish, E.A. Mash, H.V. Aposhian, Monomethylarsonous acid(MMA super(III)) and arsenite: LD sub(50) in hamsters and in vitro inhibitionof pyruvate dehydrogenase, Chem. Res. Toxicol. 14 (2001) 651–656.

29] A.D. Kligerman, C.L. Doerr, A.H. Tennant, Oxidation and methylation statusdetermine the effects of arsenic on the mitotic apparatus, Mol. Cell. Biochem.279 (2005) 113–121.

30] T. Schwerdtle, I. Walter, I. Mackiw, A. Hartwig, Induction of oxidative DNA dam-age by arsenite and its trivalent and pentavalent methylated metabolites incultured human cells and isolated DNA, Carcinogenesis 24 (2003) 967–974.

31] R. Colognato, F. Coppede, J. Ponti, E. Sabbioni, L. Migliore, Genotoxicityinduced by arsenic compounds in peripheral human lymphocytes analysed bycytokinesis-block micronucleus assay, Mutagenesis 22 (2007) 255–261.

32] S. Lin, Q. Shi, F.B. Nix, M. Styblo, M.A. Beck, K.M. Herbin-Davis, L.L.Hall, J.B. Simeonsson, D.J. Thomas, A novel S-adenosyl-l-methionine:arsenic(III) methyltransferase from rat liver cytosol, J. Biol. Chem. 277 (2002)10795–10803.

33] J. Li, S.B. Waters, Z. Drobna, V. Devesa, M. Styblo, D.J. Thomas, Arsenic (+3 oxida-tion state) methyltransferase and the inorganic arsenic methylation phenotype,Toxicol. Appl. Pharmacol. 204 (2005) 164–169.

34] D.J. Thomas, S.B. Waters, M. Styblo, Elucidating the pathway for arsenic meth-ylation, Toxicol. Appl. Pharmacol. 198 (2004) 319–326.

35] Z. Drobna, W. Xing, D.J. Thomas, M. Styblo, shRNA silencing of AS3MT expres-sion minimizes arsenic methylation capacity of HepG2 cells, Chem. Res. Toxicol.19 (2006) 894–898.

36] T.C. Wood, O.E. Salavagionne, B. Mukherjee, L. Wang, A.F. Klumpp, B.A. Thomae,B.W. Eckloff, D.J. Schaid, E.D. Wieben, R.M. Weinshilboum, Human arsenic

37] C.J. Chung, Y.M. Hsueh, C.H. Bai, Y.K. Huang, Y.L. Huang, M.H. Yang, C.J. Chen,Polymorphisms in arsenic metabolism genes, urinary arsenic methylation pro-file and cancer, Cancer Causes Control 20 (2009) 1653–1661.