van Leeuwen et al. exposure to certain environmental carcinogens, biomarkers of early biological effect (DNA strand breaks and MN frequencies), and serum levels of several tumor markers. The present study was conducted as part of the Flanders Environment and Health Study (FLEHS; Flemish Centre of Expertise on Environment and Health 2007). FLEHS is a multicenter program with the goal of researching the impact of the environment on human health across the general Flanders population. Materials and Methods Study population. The study population con- sisted of 398 subjects from eight different regions of residence in Flanders, Belgium (Table 2). Participants were recruited within several communities or sectors of communities in each of the eight regions of interest, based on random sampling. Inclusion criteria were age 50–65 years, living in the region > 5 years, and being able to complete questionnaires in Dutch. Prior to blood collection, informed consent was obtained from all individuals. Study protocols were approved by the Institutional Review Board/Ethical Committee of Antwerp University. Participants completed a questionnaire covering age, sex, and smoking habits, among other items, and they donated a blood and urine sample for measurement of the biomarkers. The questionnaire was based partly on a questionnaire used in the pilot phase of the FLEHS project (Staessen et al. 2001) and supplemented with specific ques- tions concerning the current study. Gene expression analysis. For gene expres- sion analyses, blood samples of 2.5 mL were collected from each subject into PAXgene Blood RNA vacutainer tubes (PreAnalytix; Cytochrome P450 1B1 (CYP1B1) Activating transcription factor 4 (ATF4) Superoxide dismutase 2 (SOD2) Mitogen-activated protein kinase 14 (MAPK14) Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1) PTEN-induced putative kinase-1 (PINK1) Diacylglycerol O-acyltransferase homolog 2 (mouse) (DGAT2) Tigger transposable element derived 3 (TIGD3) NM_000104 NM_001675 NM_000636 NM_001315 NM_001511 NM_032409 NM_032564 NM_145719 aNational Center for Biotechnology Information (2008). 1520 Table 1. Overview of genes monitored for expression in blood cells. Gene name (abbreviation)a GenBank accession no.a Qiagen, Hilden, Germany), a system that accounts for immediate ex vivo preservation of blood RNA. Total RNA was isolated and purified using the PAXgene Blood RNA kit (PreAnalytix) according to the manufacturer’s instructions. cDNA was synthesized from 2 µg total RNA using the BioRad iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manu- facturer’s instructions. Aliquots were used for quantitative PCR on the BioRad MyiQ iCycler Single Color quantitative detection system using iQ SYBR Green Supermix (both from Bio-Rad) according to the manufac- turer’s instructions. Reactions were initiated for 3 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 45 sec at 60°C. After each run, we performed a melting curve analysis starting at 60°C with stepwise temperature elevations of 0.5°C every 10 sec to check for nonspecific products. We included β-actin (ACTB) and cyclophillin A (PPIA) as refer- ence genes (internal controls). Primers were as follows: ACTB, 5´-CCTGGCACCCAGCA- CAAT-3´ (forward) and 5´-GCCGATCCA- CACGGAGTACT-3´ (reverse); and PPIA, 5 ´ - T T C C T G C T T T C A C A G A A T T ATTCC-3´ (forward) and 5´-GCCACCAG- TGCCATTATGG-3´ (reverse). These genes perform best in terms of most stable expres- sion and best resemblance to microarray- derived results in our previous analyses (data not shown). All reactions were performed in duplicate. In each run, negative controls (not containing template) and positive controls (a dilution series of a pooled sample, consisting of cDNA reverse-transcribed from total RNA of 20 randomly selected subjects) were included to estimate PCR efficiency. Primer sequences are shown in Table 1. Exposure analysis. We measured whole blood, serum, or urine levels of multiple envi- ronmental carcinogens or their metabolites by various methods: heavy metals (cadmium and lead) in whole blood as described by Schroijen et al. (2008); dioxins and furans in Table 2. Environmental characteristics of the study regions. Region Antwerp Harbor Fruit Olen Gent Incinerators Rural Canal Population (no.) 404,241 130,064 95,829 68,068 213,025 56,405 153,770 64,763 Description City and suburbs with exclusion of harbor area Harbor areas of Antwerp and Gent combined (petrochemical and steel industries) Eight municipalities with > 10 ha/km2 apple and pear cultivation Inﬂuenced by the presence of a nonferro industrial company City and suburbs with exclusion of harbor area Surroundings of 11 incinerators; municipalities with their center < 6 km from an incinerator (or < 12 km northeast of incinerator) Twenty-four municipalities with population density < 250 persons/km2; no registered emission source; > 5% industry and no highway in the territory Presence of six chemical companies (BP-Chembel, Exxon Mobil, Dow Chemical, Borealis, T.C. Ham, Tessenderlo Chemie-LVM) VOLUME 116 | NUMBER 11 | November 2008 • Environmental Health Perspectives Fruit, fruit cultivation region. Biological summarya Catalysis of many reactions involved in drug and xenobiotic metabolism (e.g., metabolism of procarcinogens) Encodes a transcription factor that belongs to a family of DNA-binding proteins, including the AP-1 and CREB families Associated with oxidative stress; converts superoxide to hydrogen peroxide and diatomic oxygen Activated by various environmental stressors and proinﬂammatory cytokines; integration point for multiple biochemical signals and involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation, and development Regulates cell trafﬁcking of various types of leukocytes and has a role in development, homeostasis, and function of the immune system Encodes a serine/threonine protein kinase that localizes to mitochondria; it is thought to protect cells from stress-induced mitochondrial dysfunction Responsible for triglyceride synthesis Belongs to the tigger subfamily of the pogo superfamily of DNA-mediated transposons in humans; exact function of gene is not known Primers 5´-AGTGCAGGCAGAATTGGATCA-3´ (forward) 5´-GCGCATGGCTTCATAAAGGA-3´ (reverse) 5´-CTCCAGCGACAAGGCTAAGG-3´ (forward) 5´-GTTGTTGGAGGGACTGACCAA-3´ (reverse) 5´-ATCAGGATCCACTGCAAGGAA-3´ (forward) 5´-CGTGCTCCCACACATCAATC-3´ (reverse) 5´-TGAAGACTGTGAGCTGAAGATTCTG-3´ (forward) 5´-CCACGTAGCCTGTCATTTCATC-3´ (reverse) 5´-CCACTGCGCCCAAACC-3´ (forward) 5´-GCAGGATTGAGGCAAGCTTT-3´ (reverse) 5´-AGCAGTCACTTACAGAAAATCCAAGA-3´ (forward) 5´-GGTGAAGGCGCGGAGAA-3´ (reverse) 5´-GCACAGAGGCCACAGAAGTG-3´ (forward) 5´-CCCTCAACACAGGCATTCG-3´ (reverse) 5´-GTGCTGGAACTCCTGGATGAG-3´ (forward) 5´-TTGCAGATGCGCGAGATCT-3´ (reverse)