Article pubs.acs.org/est
Dose-Response Relationships of Polycyclic Aromatic Hydrocarbons Exposure and Oxidative Damage to DNA and Lipid in Coke Oven Workers Dan Kuang,† Wangzhen Zhang,‡ Qifei Deng,† Xiao Zhang,† Kun Huang,† Lei Guan,† Die Hu,† Tangchun Wu,† and Huan Guo†,* †
Department of Occupational and Environmental Health and Ministry of Education Key Lab for Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China ‡ Institute of Industrial Health, Wuhan Iron & Steel (group) Corporation, Wuhan 430070, China S Supporting Information *
ABSTRACT: Polycyclic aromatic hydrocarbons (PAHs) are known to induce reactive oxygen species and oxidative stress, but the dose−response relationships between exposure to PAHs and oxidative stress levels have not been established. In this study, we recruited 1333 male coke oven workers, monitored the levels of environmental PAHs, and measured internal PAH exposure biomarkers including 12 urinary PAH metabolites and plasma benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydotetrol-albumin (BPDE-Alb) adducts, as well as the two oxidative biomarkers urinary 8-hydroxydeoxyguanosine (8-OHdG) and 8-iso-prostaglandin-F2α (8-iso-PGF2α). We found that the total concentration of urinary PAH metabolites and plasma BPDE-Alb adducts were both significantly associated with increased 8OHdG and 8-iso-PGF2α in both smokers and nonsmokers (all p < 0.05). This exposure-response effect was also observed for most PAH metabolites (all ptrend < 0.01), except for 4-hydroxyphenanthrene and 8-OHdG (ptrend = 0.108). Furthermore, it was shown that only urinary 1-hydroxypyrene has a significant positive association with both 8-OHdG and 8-iso-PGF2α after a Bonferroni correction (p < 0.005). Our results indicated that urinary ΣOH-PAHs and plasma BPDE-Alb adducts can result in significant dose-related increases in oxidative damage to DNA and lipids. Furthermore, when a multianalyte method is unavailable, our findings demonstrate that urinary 1-hydroxypyrene is a useful biomarker for evaluating total PAHs exposure and assessing oxidative damage in coke oven workers.
■
INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are a large family of environmental pollutants with different properties of toxicity and biological effects. The dramatic energy consumption worldwide has led to an unprecedented release of the incomplete combustion of fuels, causing the continuous emission of PAHs into the atmosphere. Additionally, individuals may be exposed to high levels of PAHs in certain occupational environments. It has been reported that air pollution is associated with an increased risk of cancers and cardiopulmonary diseases and that these associations are mainly due to PAH exposure.1−3 PAHs are the predominant contaminants of coke oven emissions because of the incomplete combustion of coal. As a typical population who have been long-term exposed to PAHs, coke oven workers have significantly elevated risks of developing cancer and cardiovascular events.4,5 The currently available epidemiological and laboratory data indicate that oxidative stress plays a central role in both the carcinogenic potential and cardiovascular health effects of PAHs,6−8 but the dose−response relationships have not been clarified. © XXXX American Chemical Society
PAHs can be metabolized by the cytochrome P450 (CYP) enzymes to generate active semiquinones,9 which are known free radical intermediates and can go through redox cycling and generate reactive oxygen species (ROS).10 The ROS can then cause oxidative modification of DNA and lipids in the body.10 As one of the predominant forms of oxidative lesions in DNA, 8-hydroxydeoxyguanosine (8-OHdG) is a critical biomarker for oxidative DNA damage11,12 and 8-iso-prostaglandin-F2α (8-isoPGF2α) is a reliable biomarker of lipid peroxidation.13−15 Combined, determination of 8-OHdG and 8-iso-PGF2α can be used to represent the global oxidative levels in the human body. Air monitoring of PAH concentrations near the workplace can be used to represent occupational inhaled exposure levels for a specific group of workers. However, PAH internal doses, which reflect all sources of exposure, may vary dramatically among individuals. Urinary monohydroxy PAHs (OH-PAHs) Received: December 12, 2012 Revised: May 22, 2013 Accepted: June 7, 2013
A
dx.doi.org/10.1021/es401639x | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
OHNa, 2-hydroxyfluorene (2-OHFlu), 9-OHFlu, 1-hydroxyphenanthrene (1-OHPh), 2-OHPh, 3-OHPh, 4-OHPh, 9OHPh, 1-OHP, 6-hydroxychrysene (6-OHChr), and 3hydroxybenzo[a]pyrene (3-OHBaP)]. The standard materials (purity ≥98%) were purchased from Sigma-Aldrich (1-OHNa, 2-OHNa, 2-OHFlu, 9-OHFlu, 9-OHPh, 1-OHP, and 3OHBaP) (Munich, Germany), Dr. Ehrenstorfer (1-, 2-, 3-, and 4-OHPh) (Augsburg, Germany), and AccuStandard (6OHChr) (New Haven, CT); and the organic solvents (nhexane, and acetonitrile) (HPLC grade purity) were obtained from Sigma-Aldrich. The internal standards [2H9]1-hydroxypyrene (1-OHP-d9) and [2H7]1-hydroxynaphthalene (1OHNa-d7) were purchased from the Toronto Research Chemicals Inc. (Ontario, Canada) and C/D/N isotopes Inc. (Quebec, Canada), respectively, and were diluted by acetonitrile to a mixture internal standard solution containing 5 mg/L 1-OHP-d9 and 5 mg/L 1-OHNa-d7. The analyses were performed using the Agilent 5975B/ 6890N GC/MS System (Agilent, Santa Clara, CA) based on the method described previously with some modifications.24 3.0 mL of urine from each subject was extracted for measurement and each sample was determined in triplicate. The 3.0 mL urine sample, 1 mL of acetate acid buffer (0.5M, PH 5.0), 20 μL of internal standard solution, and 20 μL of β-Glucuronidase/ sulphatase (Sigma-Aldrich, Munich, Germany) were added to a 10 mL silanized glass vial, followed by incubation at 37 °C overnight. 1.5 mg of MgSO4·7H2O was then added to saturate the hydrolyzed urine samples. Samples were extracted twice with 1.5 mL n-hexane and were centrifuged each time at 1500 rpm for 10 min to facilitate phase separation. The organic extracts were then evaporated with a gentle stream of nitrogen, and the residue was reconstituted with 100 μL BSTFA and incubated at 90 °C for 45 min. After derivation, samples were cooled to room temperature and rapidly transferred into a 2 mL vial containing a 400 μL conical insert.1 μL of final extracted volume of each sample was injected on the GC/MS system. The MS was operated with electron impact ionization at an ionization voltage of 70 eV. High-purity helium (>99.99%) was used as a carrier gas with a constant flow of 1.0 mL/min. The temperature programs were as follows: 60 °C for 3 min, 10 °C/ min to 150 °C hold for 3 min, 10 °C/min to 210 °C hold for 5 min, and then 10 °C/min to 310 °C hold for 4 min. The identification and quantification of urinary PAH metabolites were based on retention time, mass-to-charge ratio and peak area using a linear regression curve obtained from separate internal standard solutions. We successfully detected ten urinary PAH metabolites, except for 6-hydroxychrysene and 3-hydroxybenzo[a]pyrene whose concentrations were less than the limits of detection (LOD). The LOD for the urinary PAH metabolites ranged from 0.1 μg/L to 1.4 μg/L, and the concentrations of those samples below LOD were substituted as 50% of the LOD value. The concentrations of urinary creatinine were measured according to Jaffe’s colorimetric method on an automated clinical chemistry analyzer. The concentrations of urinary PAH metabolites were calibrated by levels of urinary creatinine and defined as μg/mmol creatinine. Determination of Plasma BPDE-Alb Adducts. The concentrations of plasma BPDE-Alb adducts were detected by using the ELISA method developed by Ming et al with minor modifications.25 We first coated the 96-well plates with 5 μg/mL rabbit antimouse IgG-Fc antibody (Jackson-Immunoresearch, West Grove, PA) in 50 μL 0.1 M carbonate-
are a class of PAH metabolites used as biomarkers to assess human exposure to environmental PAHs.16 Urinary 1-OHP is a commonly used short-time biomarker of PAH exposure, but it alone cannot reflect the overall internal PAH metabolites,17,18 and hydroxylated nathalene and hydroxylated phenanthrenes in urine are suggested to be good surrogate biomarkers of occupational PAH exposure in asphalt and diesel industries.19 One previous study evaluating the associations between PAH exposure and oxidative stress investigated urinary 1-OHP and 8-OHdG in 78 workers of a coke oven plant,20 but no studies have evaluated the urinary profiles of PAH metabolites in coke oven workers. Benzo[a]pyrene (B[a]P) is the most well-studied carcinogen in PAH mixtures, and it can be metabolized into the ultimate carcinogen benzo[a]pyrene-r-7, t-8, t-9,c-10-tetrahydotetrol (BPDE), which then binds with albumin, forming BPDE-Alb adducts. This adduct is a widely accepted biomarker for long-term exposure to carcinogenic PAHs.21 In the present study, we aimed to investigate the potential dose- response relationships of PAH exposure with oxidative damage to DNA and lipid in coke oven workers. We measured environmental PAHs in the different workplaces, measured internal PAHs exposure biomarkers, including urinary PAH metabolites and plasma BPDE-Alb adducts, and evaluated the oxidative damage levels to DNA (urinary 8-OHdG) and lipid (8-iso-PGF2α) in 1333 male coke oven workers.
■
MATERIALS AND METHODS Airborne PAH Monitoring. For the different working sites of the coke oven plant, we set up 5 monitors in the office areas, 5 monitors at the adjacent workplaces, 16 monitors at the side and bottom of the coke oven, and 15 monitors at the top of the coke oven. We collected the corresponding airborne samples and determined the content of particulate matter (PM) as described previously.22 Furthermore, the measurements of 16 PAHs were from the collected PM and were analyzed by HPLC according to Method 5506 of the U.S. National Institute for Occupational Safety and Health.23 Subjects and Sample Collection. A total of 1333 male workers who worked for at least one year at the top, side, bottom, or adjacent workplaces of coke ovens, or in the office of this coke oven plant in Wuhan (Hubei, China), were included in this study. All participants gave their written informed consent and information on demographic characteristics, health status, body weight, and height, smoking status, alcohol consumption, occupational location, and employment time were collected via an interviewing using a standardized occupational questionnaire. Those who had smoked less than one cigarette per day for less than one year over their entire lifetime were defined as nonsmokers; otherwise, subjects were defined as smokers. 5 mL of venous blood was obtained from each subject in the morning. Plasma samples were separated from 4 mL heparinised whole blood by centrifugation and stored at −80 °C until laboratory examination of BPDE-Alb adduct. In addition, we collected 20 mL of urine from each worker in sterile sample cups at the start (preshift) and end (postshift) of each workshift and stored the urine samples at −80 °C until laboratory examination. The research protocol was approved by the Ethics and Human Subject Committee of Tongji Medical College. Determination of Urinary PAH Metabolites. A gas chromatography/mass spectrometry (GC/MS) method was used to measure the concentrations of twelve urinary PAH metabolites, including 1-hydroxynaphthalene (1-OHNa), 2B
dx.doi.org/10.1021/es401639x | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
Table 1. General Characteristics for Workers in Different Exposure Groups exposure groups
a
variables
control group (n = 384)
low (n = 545)
intermediate (n = 325)
high (n = 79)
p-value
age (years, mean ± SD) years worked (years, mean ± SD) workshift, n (%) preshift postshift current smokers, n (%) yes no pack-years (years, mean ± SD) alcohol use, n (%) yes no body mass index (kg/m2, mean ± SD)
43.45 ± 7.95 22.32 ± 9.21
42.20 ± 9.16 20.58 ± 10.84
42.35 ± 7.99 21.08 ± 9.23
42.08 ± 7.98 20.88 ± 9.35
0.128a 0.067a
