Transcriptome analysis in human blood as a biomarker serum as described by Van Wouwe et al. (2004); p,p´-dichlorodiphenyldichloro- ethylene (p,p´-DDE) and non–dioxin-like polychlorinated biphenyls (PCBs) in serum as described by Covaci et al. (2002) and Gomara et al. (2002); 1-OH-pyrene (a metabolite of polycyclic aromatic hydrocarbons) and t,t-muconic acid (t,t-MA; a metabolite of ben- zene) in urine as described by Angerer and Schaller (1997, 1998). Smoking status was derived from questionnaires instead of cotinine measurements. Measurement of early biological effect and tumor markers. We evaluated the induction of DNA strand breaks as a measure for DNA damage using the alkaline COMET assay as described by Singh et al. (1988). We analyzed the slides (200 cells per individual) using an image analysis system from Metasystems (Altslussheim, Germany). Median percentages of DNA migration in the tail areas were deter- mined and used as a measure of DNA damage. As a positive control, one slide with nuclei from deep-frozen whole blood was added to each electrophoresis chamber. DNA migration in positive controls had to be > 30%. To inves- tigate the MN frequencies, we performed the cytokinesis-block micronucleus assay on whole blood cultures using standard procedures according to Fenech (2000). For each individual, we evaluated 1,000–2,200 cells for the presence of micronuclei using the Metafer automatic program (Metasystems). We meas- ured 8-hydroxydeoxyguanosine (8-OH-dG) in urine by means of ELISA using the competi- tive immunosorbent assay (Gentaur, Brussels, Belgium) according to the manufacturer’s instructions. Serum protein levels of the tumor marker p53 were analyzed using the enzyme immunometric assay and Titerzyme EIA p53 (Assay Designs, Ann Arbor, MI, USA). Carcino-embryonic antigen (CEA) and prostate-specific antigen (PSA) levels were measured using a solid-phase chemilumines- cent immunometric assay and Immulite 2000 (DPC, Los Angeles, CA, USA). Samples were analyzed for PSA within 24 hr after collection. Data analysis. Ct values [concentration of DNA molecules (in moles) multiplied by time] per subject and per gene were normal- ized by subtraction of the mean Ct value of ACTB and PPIA. Subsequently, we calculated ΔΔCt values per subject and per gene relative to the normalized pooled reference sample within each quantitative PCR run using the following formula: ΔΔCt = pooled reference [Ctgene – Ct(mean ACTB+PPIA)] – individual [Ctgene – Ct(mean ACTB+PPIA)]. This generated one expression value per gene per individual study participant. All gene expression data are reported as ΔΔCt values on log (base = 2) scale (Livak and Schmittgen 2001) and presented as average group values. Next to gene expression for the eight individual genes, an integrative expression value was calcu- lated as the mean of the eight individual genes. We performed statistical testing using SPSS 14.0 (SPPS Inc., Chicago, IL, USA). We used one-way analysis of variance (ANOVA) with post hoc Bonferroni correction to test for signif- icance of differences in gene expression between groups of individuals inhabiting different regions and between groups of individuals with different smoking status (current vs. former, current vs. never-smoker, and former vs. never- smoker) and other variables, as well as blood and urinary measures of exposure and effect biomarkers. For examination of intervariable correlations at the level of all individuals (no division based on region of inhabitance), we applied Pearson correlation analysis. p-Values < 0.05 are considered statistically signiﬁcant. Results Tables 3–5 describe the characteristics of the study population according to the region of inhabitance and of the population as a whole. For seven individuals (1.8%), the smoking Table 3. Characteristics of the study population by region of residence. Abbreviations: Fruit, fruit cultivation region; HCB, hexachlorobenzene. Individuals for whom smoking status was not known or reported are not included. amg/g creatinine. bμg/L. cng/g fat. dSum of PCBs 138, 153, and 180; expressed as ng/g fat in serum. epg TEQ/gr fat in serum. Environmental Health Perspectives • VOLUME 116 | NUMBER 11 | November 2008 Olen 39 58.9 ± 0.7 17 (44) 22 (56) 17 (43.6) 14 (35.9) 7 (17.9) 1 (2.6) Olen 0.86 (0.3–2.1) 0.72 (0.2–3.0) 43.58 (21.0–96.8) 0.32 (0.02–1.8) 82.74 (30.1–177.2) 0.14 (0.01–0.63) 445.8 (126.5–919.3) 1444.3 (126.7–7575.8) 16.84 (4.2–70.9) Gent 72 58.9 ± 0.6 38 (53) 34 (47) 38 (52.8) 23 (31.9) 11 (15.3) 0 (0) Gent 0.61 (0.2–2.2) 0.59 (0.1–3.4) 45.24 (15.7–106.9) 0.20 (0.01–1.2) 63.52 (20.7–216.7) 0.14 (0.01–0.58) 388.9 (152.4–949.5) 600.3 (61.8–4458.6) 20.08 (4.2–103.4) Incinerator 50 59.0 ± 0.6 25 (50) 25 (50) 24 (48) 15 (30) 11 (22) 0 (0) Incinerators 0.82 (0.2–6.5) 0.80 (0.2–1.8) 46.07 (9.3–99.6) 0.26 (0.01–1.8) 74.40 (14.6–242.7) 0.12 (0.01–0.61) 378.7 (42.3–710.7) 813.1 (1.1–5208.4) 20.65 (4.4–44.3) Rural 76 59.3 ± 0.5 42 (55.3) 34 (44.7) 43 (56.5) 20 (26.3) 12 (15.8) 1 (1.3) Rural 0.61 (0.1–1.7) 0.61 (0.1–2.4) 51.19 (18.1–133.2) 0.24 (0.01–1.3) 73.95 (20.3–242.6) 0.13 (0.01–1.34) 391.6 (75.5–765.4) 905.1 (30.8–3846.2) 25.80 (3.6–114.8) Canal 35 60.1 ± 0.6 16 (46) 19 (54) 17 (48.6) 12 (34.3) 6 (17.1) 0 (0) Canal 0.78 (0.3–1.7) 0.77 (0.3–2.0) 37.78 (12.0–106.6) 0.22 (0.01–1.0) 74.71 (27.5–188.7) 0.09 (0.01–0.36) 407.1 (211.5–1317.6) 1446.7 (89.0–8614.9) 19.58 (3.5–52.2) Characteristic Subjects (no.) Age (years) Sex [no. (%)] Males Females Smokers [no. (%)] Nonsmokers Former Current Unknown Antwerp 50 58.6 ± 0.6 30 (60) 20 (40) 19 (38) 16 (32) 15 (30) 0 (0) Cd (urine)a Cd (blood)b Pb (blood)b 1-OH-pyrenea HCBc t,t-MAa PCBsd p,p´-DDEc Dioxinse Antwerp 0.73 (0.2–2.2) 0.61 (0.1–1.7) 44.60 (10.7–181.5) 0.34 (0.02–2.3) 71.19 (22.9–189.0) 0.16 (0.01–0.59) 382.4 (183.1–875.4) 692.7 (11.4–3014.2) 28.85 (4.2–64.9) Harbor 41 57.1 ± 0.7 20 (48.8) 21 (51.2) 19 (46.3) 11 (26.8) 6 (14.6) 5 (12.2) Harbor 0.83 (0.3–2.4) 0.68 (0.2–1.6) 38.41 (12.7–74.1) 0.27 (0.01–2.2) 63.00 (13.7–157.3) 0.12 (0.01–0.50) 363.2 (62.1–600.0) 527.2 (1.8–1926.2) 17.68 (3.5–51.9) Fruit 35 57.0 ± 0.7 19 (54) 16 (46) 19 (54.3) 12 (34.3) 4 (11.4) 0 (0) Fruit 0.72 (0.3–1.9) 0.50 (0.1–1.8) 39.04 (2.5–129.6) 0.37 (0.02–2.0) 62.18 (12.6–125.8) 0.16 (0.01–0.65) 368.9 (67.4–1061.6) 1189.2 (1.8–16967.0) 41.67 (7.3–69.2) Fruit, fruit cultivation region. Values shown for age are mean ± SE. Table 4. Blood or urine concentrations [mean (range)] of environmental pollutants by region of residence. Total 398 59.2 ± 0.2 207 (52) 191 (48) 196 (49.2) 123 (30.9) 72 (18.1) 7 (1.8) Total 0.72 (0.1–6.5) 0.65 (0.1–3.4) 44.41 (2.5–181.5) 0.27 (0.01–2.3) 70.62 (12.6–242.7) 0.13 (0.01–1.34) 390.4 (42.3–1317.6) 900.8 (1.1–16967.0) 23.26 (3.5–114.8) 1521