Hossain et al. any medication at the time of the study: one being treated for gastritis and two for hyper- tension. No women reported any history of or current malignancy. We measured two biomarkers of cadmium exposure: blood cadmium, reflecting ongoing exposure, and urinary cadmium, reflecting long-term exposure (Järup and Åkesson 2009). Both oral and written informed consent was provided by all the study participants. The study was approved by the Ministry of Health in Salta, Argentina, and the Regional Ethical committee at Karolinska Institute, Sweden. Blood and urine collection. All biologi- cal samples were nonfasting spot samples collected during the daytime at local health clinics or the hospital in San Antonio de los Cobres. Peripheral blood for DNA extraction (n = 202) was collected in K2EDTA tubes (Vacuette®; Vacuette Bio One, Frickenhausen, Greiner, Germany), blood for element analysis (n = 202) in lithium heparin tubes (Vacuette Bio One), and blood for RNA extraction (n = 122) in PAXgene Blood RNA Tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Spot urine samples were collected and pro- cessed as described previously (Concha et al. 1998). Blood, plasma, and urine samples were kept at –20°C before and after the transport (with cooling blocks) to Sweden for analysis. Analysis of elements in blood and urine. We measured cadmium in blood and urine, and selenium and zinc in blood, because these elements may influence DNA methylation or one-carbon metabolism (Hong et al. 2000; Pilsner et al. 2011; Smeester et al. 2011). Cadmium, selenium, and zinc were measured using inductively coupled plasma mass spec- trometry (ICP-MS; model 7500ce; Agilent Technologies, Waldbronn, Germany) with a collision/reaction cell system to remove poten- tial interferences, particularly interference from molybdenum in the cadmium analysis (Concha et al. 2010; Kippler et al. 2009). The limit of detection (LOD; 3 × SD of blank values) was 0.001 µg/L for urinary cadmium, and no sample had a cadmium concentration below the LOD. For blood measurements, the LOD was 0.011, 0.081, and 0.49 µg/L for cadmium, selenium, and zinc, respectively, and no samples were below the LOD for any of the compounds measured. Analytical accu- racy was controlled by analyzing commer- cially available reference materials (Concha et al. 2010; Kippler et al. 2009). Because the study area is known to have varying concen- trations of arsenic in the drinking water, and because arsenic may alter DNA methylation (Chanda et al. 2006; Smeester et al. 2011), we also assessed exposure to arsenic by measuring arsenic metabolites (inorganic arsenic, methyl- arsonic acid, and dimethylarsinic acid) in urine using HPLC/ICP-MS (Engström et al. 2010). Additionally, the study area was recently found 880 to have elevated concentrations of boron, lith- ium, and cesium in the drinking water, and we measured these elements in urine by ICP-MS (Concha et al. 2010). To control for variation in urine dilution, we adjusted the cadmium and arsenic concentrations in urine to the mean specific gravity of urine (1.020 g/mL) (Nermell et al. 2008). We measured the speci- fic gravity of the urine samples with a digital refractometer (model RD 712 clinical refrac- tometer; EUROMEX, Arnhem, Holland). Polymerase chain reaction (PCR) and pyrosequencing. DNA was isolated using the QIAamp® DNA Blood Midi kit (Qiagen, Hilden, Germany). We used the EpiTect® kit (Qiagen) to treat 1 µg of DNA (50 ng/µL) with bisulfite. Bisulfite treatment of DNA converts unmethylated cytosine residues into uracil, leaving the methylated cytosine unchanged; these changes can be quantified by pyrosequencing. The bisufite-treated DNA was stored at –20°C until used for PCR. We used the bisulfite-treated DNA (0.6–1 µL) in a 15- to 25-µL PCR reaction using the Pyromark PCR kit (Qiagen). One of the PCR primers was biotinylated, and the PCR product was purified using Streptavidin Sepharose High Performance beads (Amersham Biosciences, Uppsala, Sweden). The Sepharose beads with the immobilized PCR products were purified, washed, and denatured with 0.2 M NaOH and washed again using a vacuum prep tool (Pyrosequencing Inc., Westborough, MA, USA). After annealing 12 µL of the 0.3-µmol/L pyrosequencing primer to the purified single-stranded PCR product, we per- formed pyrosequencing using the PSQ HS96 Pyrosequencing System (Qiagen). The degree of methylation was expressed as the percent- age of methylated cytosines over the sum of methylated and unmethylated cytosines. We verified bisulfite conversion using non-CpG (cytosine–guanine dinucleotide) cytosine resi- dues as built-in controls, and complete conver- sion of cytosine at a non-CpG site indicated successful bisulfite conversion. LINE­1 , p16, and MLH1 methylation. As a surrogate measure of genome-wide methylation, we measured peripheral blood DNA methylation in the long interspersed nuclear element-1 (LINE-1) retrotransposable elements that make up about 17% of human DNA (Lander et al. 2001). We also measured methylation of CpG islands in p16 [cyclin- dependent kinase inhibitor 2A (CDKN2A)], a tumor suppressor gene that is often hyper- methylated in tumors and may be relevant in cadmium-induced tumorigenesis (Benbrahim- Tallaa et al. 2007), and in the MLH1 (mutL homolog 1) gene, a component of the DNA mismatch repair pathway. Commercially available kits (Qiagen) were used to measure the methylation of four CpG sites in LINE-1 [positions 305–331 of GenBank accession no. X58075 (National Library of Medicine 2012a)], seven CpG sites in p16 (+148 to +174 in exon 1; GenBank accession no. L27211), and four CpG sites in MLH1 (–209 to –188; GenBank accession no. U07418) following the manufacturer’s instructions. A single PCR fragment spanning a part of each genetic element was amplified, and the degree of methylation was analyzed in a single pyrosequencing reaction using 20 µL PCR product for LINE-1, 3 µL for p16, and 4 µL for MLH1. The samples were analyzed as singlets for LINE-1 and as triplicates for p16 and MLH1. We selected 10% of the samples at random and reassayed them. The repeatability of the method, expressed as the variation in coefficients, was 2.0%, 36.7%, and 26.4%, for LINE-1, p16, and MLH1 methylation, respectively. Previous reports have validated pyrosequencing as an effective technique for measuring LINE-1 methylation at the population level (Schernhammer et al. 2010; Tarantini et al. 2009). G e n o t y p i n g a n d g e n e e x p r e s s i o n . Genotyping for polymorphisms of DNA methyltransferase-1 [(cytosine-5-)-methyl- transferase 1] (DNMT1 rs10854076 and rs2228611, one from each linkage disequilib- rium block in the gene) and DNA (cytosine- 5-)-methyltransferase 3 beta (DNMT3B rs2424913 and rs2424932) was performed on DNA from peripheral blood with iPLEX GOLD SNP genotyping (Sequenom Inc., San Diego, CA, USA) the Swegene DNA facility at Malmö University Hospital (Malmö, Sweden), as described previously (Engström K et al. 2011). DNMT1 rs2228611 [minor allele frequency (MAF) = 40%] is a synonymous amino acid exchange, whereas rs10854076 (MAF = 35%) is situated in an intron. DNMT3B rs2424913 (MAF = 5%) and rs2424932 (MAF = 6%) are both situ- ated in introns. The functional impact of these polymorphisms on DNA methylation, if any, is unknown. RNA was extracted using the PAXgene Blood RNA kit (PreAnalytiX, Hombrechtikon, Switzerland) and stored at –80°C until isola- tion. We evaluated RNA concentration and purity using a NanoDrop spectrophotome- ter (NANODROP 1000; Thermo Scientific, Wilmington, DE, USA), and we evaluated the RNA integrity number (RIN) using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). The results indicated good RNA quality (RIN > 7.5) for all of the sam- ples. Of 122 available PAX tubes for RNA extraction, 90 samples were selected for gene expression analysis based on their RNA quality and the amount of RNA left for analysis [for descriptive characteristics of this subset of the study population, see Supplemental Material, Table 1 (http://dx.doi.org/10.1289/ehp. 1104600)]. Of the 90 individuals selected, ovlume 120 | number 6 | June 2012 • Environmental Health Perspectives