Materials and Methods
Study subjects and sample collection
This cross-sectional study was conducted in Tehran, between 2013 and 2014. Tehran, the capital of Iran and one of the most crowded cities in the Middle East, with a population of around ten million, is located 35° 34–35° 50′ latitude and 51° 08–51° 37′ longitude (Naddafi, Jabbari et al. 2011, Kamani, Hoseini et al. 2014). The study population consisted in 222 randomly chosen subjects which were recruited by conducting door knock surveys from 10 a.m. to 8 p.m. It should be noted that from the timing of sampling, the first urine samples, which could be the most concentrated were not included anyway. Samples were normalized with the creatinine concentration to account for any dilution effect. The sampling procedure was started with the selection of random points weighted based on the population plotted on Tehran population density layer using Arc GIS software (Fig. 1), and followed by choosing randomly one home address with respect to the points. To collect the required spot urine samples, a resident per each address was randomly chosen to participate in this study; after receiving the consent of each chosen resident, a sample was taken from each one. All urine samples were collected in 70 mL urine bottles which were wrapped in aluminum foil to avoid sun light exposure and labeled with the subject specifications. Following collection, the samples were kept in a portable cool box contained icepacks (at about 2–8 ºC) and delivered to laboratory. It is noteworthy that the collected urine samples were divided on-site into three aliquots. Two of the aliquots were used to immediate analyzing the concentration of urinary creatinine and cotinine; and, the third one was kept frozen (−20 ◦C) for thereafter measuring of OH-PAHs.
All subjects were asked to fill a detailed questionnaire composed of seven parts (including demographic information, occupational exposure, smoking history/exposure, cooking-related appliances, activities, residential environment characteristics, and consumed foodstuff during the last week). Furthermore, the required data and information in this study was collected by conducting face-to-face interviews with the participants. The study protocol was approved and coded (24039) by the Ethics Committee of Tehran University of Medical Sciences.
Laboratory analysis of PAH metabolites
Six OH-PAH metabolites including 1-Hydroxy pyrene (1-OHP), 1-Hydroxy naphthalene (1-NAP), 2-Hydroxy naphthalene (2-NAP),2-Hydroxyfluorene (2-FLUO), 9-Hydroxyfluorene (2-FLUO), and 9-Hydroxyphenanthrene (9-PHEN) were measured in urine samples using enzymatic hydrolysis, followed by automated solid phase extraction (SPE), and quantified by gas chromatography/mass spectrometry (GC/MS) according to the method developed by Romanoff et al., 2006 with slight modification. Briefly, urine samples (3 mL) were transferred to test tubes (16mm×100 mm) and spiked with 10 µL of the internal standard solution (naphthalene-d8 and phenanthrene-d10).Next, sodium acetate buffer (0.1 M; pH 5.5; 5mL) and β-glucuronidase/arylsulfatase enzyme (10 µL, Helix pomatia, H-1; Roche Diagnostic) were added to each sample. The samples were then tightly sealed with Teflon-lined screw caps, gently mixed and then incubated at 37 ◦C for 17–18 h (overnight)at 210 rpm. After enzymatic hydrolysis, the samples were mixed, allowed to equilibrate and then applied to a SPE column packed with 0.5 g RP-C18 (Applied Separation, USA) using an Extraction Manifold (20 positions, 13×75 mm tubes, Waters) connected to a vacuum pump. Cartridges were conditioned with 3 ml methanol and 3 ml purified water, and then samples were injected (1 mL/min) to the column. After washing with purified water (3 mL, 10 mL/min) and methanol/sodium acetate buffer (3 mL, 4:6 v/v, pH 5.5, 10 mL/min), the cartridges were dried by applying a constant flow of nitrogen for 10 min and the final extract was eluted with 3 mL dichloromethane (0.5 mL/min). The sample extracts were spiked with dodecane (5 µL), and evaporated to about 5 µL under a stream of nitrogen at 40 ◦C and the residues were dissolved in 100 µL toluene. The extracts were derivatized to their trimethylsilylated derivatives by adding MSTFA (20 µL) and incubating (60 ◦C) for 30 min(Romanoff, Li et al. 2006). All samples were analyzed on a gas chromatography (GC, Agilent 7890N, Agilent Co.) equipped with a mass spectrometry (MSAgilent 5975C, Agilent Co.). A 30 m×0.25 μm×0.5 μm fused silica (DB-5MS) capillary column (Agilent Co., USA) in selected ion monitoring (SIM) mode was used. The injection volume was 2.0 μL in the splitless mode at 290 °C, and helium with a purity of 99.99 % was used as a carrier gas atconstant flow rate of 1 mL/min. Urinary creatinine was measured photometrically according to the Jaffe's method and then, the measured concentrations of OH-PAHs were normalized with the one determined for the urinary creatinine.
Quality control
Anonymous urine samples were collected from multiple volunteers, pooled, and diluted with purified water (4:1 v/v) to reduce endogenous levels of the metabolites. After pressure filtering through a 0.45-µm filter capsule, the urine was divided into three pools; QC low, QC medium, and QC high, spiked with a mixture of six OH-PAH standards at 100, 500, and 1000ng/L, respectively. The pools were homogenized at room temperature overnight and subsequently aliquoted into test tubes (3 mL in each tube) and stored at −70 ◦C until used.
For each batch of 20 samples during the study, one water blank, one QC low, one QC medium, and one QC high were processed. The analytical blank values for each target metabolites were significantly lower than those of samples. The recoveries of target analytes spiked to the samples ranged from 75 to 128%. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as the concentrations equivalent to three and ten times the noise of the quantifier ion for a blank sample. The LOD and LOQ of each OH-PAH are reported in Table 1. All glassware used during extraction steps was ultrasound-cleaned, soaked in 20%nitric acid (overnight soaking),and rinsed with purified water prior to being baked in an oven(5 h at 180 °C) to remove any background contamination.
Statistical analysis
All statistical analyses were performed using R statistical software. The distributions of different biomarkers were assessed for normality (Kolmogorov–Smirnov test). Urinary concentrations (mean, median, SD, and geometric mean) of different metabolites were reported according to age, gender, smoking status, dietary habits, etc. Non-parametric Mann-Whitney U tests and Kruskal-Wallis H tests were used to compare the results in different groups. Generalized linear model (GLM) was employed to evaluate the association between urinary OH-PAHs concentrations and independent variables gathered by questionnaire. A p-value of 0.05 was considered statistically significant.