Article pubs.acs.org/est
Polycyclic Aromatic Hydrocarbons-Associated MicroRNAs and Their Interactions with the Environment: Influences on Oxidative DNA Damage and Lipid Peroxidation in Coke Oven Workers Qifei Deng,†,‡ Xiayun Dai,† Huan Guo,† Suli Huang,† Dan Kuang,† Jing Feng,† Tian Wang,† Wangzhen Zhang,§ Kun Huang,† Die Hu,† Huaxin Deng,† Xiaomin Zhang,† and Tangchun Wu*,† †
Key Laboratory of Environment and Health, Ministry of Education & Ministry of Environmental Protection, and State Key Laboratory of Environmental Health (Incubating), School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China ‡ Department of Preventive Medicine, School of Public Health, Sun Yat-sen University, Guangzhou 510080, Guangdong, China § Institute of Industrial Health, Wuhan Iron and Steel Corporation, Wuhan 430080, Hubei, China ABSTRACT: We previously identified five polycyclic aromatic hydrocarbons (PAHs)-associated microRNAs (miRNAs) and found they were associated with chromosome damage. As oxidative damage is the common contributory cause of various PAHs-related diseases, we further investigated the influences of these miRNAs and their interactions with environmental factors on oxidative DNA damage and lipid peroxidation. We measured PAHs internal exposure biomarkers [urinary monohydroxy-PAHs (OH-PAHs) and plasma benzo[a]pyrene-r-7,t-8,t9,c-10-tetrahydotetrol-albumin (BPDE-Alb) adducts], the expression levels of PAHs-associated plasma miRNAs (miR-24-3p, miR-27a-3p, miR-142-5p, miR-285p, and miR-150-5p), and urinary biomarkers of oxidative DNA damage [8-hydroxydeoxyguanosine (8-OH-dG)] and lipid peroxidation [8-iso-prostaglandinF2α (8-iso-PGF2α)] in 365 healthy male coke oven workers. These miRNAs were associated with a dose−response increase in 8-OH-dG (β > 0), and with a dose− response decrease in 8-iso-PGF2α (β < 0), especially in workers with lower PAHs exposure levels, in nonsmokers, and in nondrinkers. These miRNAs interacted antagonistically with ΣOH-PAHs and BPDE-Alb adducts (βinteraction < 0) and synergistically with drinking status (βinteraction > 0) to influence 8-OH-dG, while they interacted synergistically with BPDE-Alb adducts (βinteraction > 0) and antagonistically with smoking status (βinteraction < 0) to influence 8-iso-PGF2α. Our results suggested that miRNAs and their interactions with environmental factors might be novel mechanisms mediating the effects of PAHs exposure on oxidative DNA damage and lipid peroxidation.
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INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are a large class of toxic organic compounds that are mainly generated through incomplete combustion or pyrolysis of carbon-containing materials. PAHs are widely distributed in the environment, especially in certain occupational environments such as coke plants, severely threatening the health of exposed workers. An increasing number of epidemiological studies have suggested that long-term exposure to PAHs-rich emissions is associated with considerably higher risks of cardiopulmonary diseases (such as ischemic heart disease and chronic obstructive pulmonary disease)1,2 and cancers (such as lung cancer and skin cancer)3 in exposed workers. Thus, adverse health outcomes of PAHs exposure are the major public health concern in the exposed working population. PAHs may exert their deleterious effects partly by the induction of oxidative stress. After entering the organism, PAHs are metabolized by cytochrome P450 enzymes and epoxide hydrolase to generate trans-dihydrodiols and then oxidized to radical-cations and o-quinones, which can enter redox © 2014 American Chemical Society
cycles to release reactive oxygen species (ROS) and induce oxidative stress. In the presence of oxidative stress, ROS can attack DNA and lipids and cause oxidative DNA damage and lipid peroxidation,4,5 which are generally regarded as significant contributory factors in the pathogenesis of various PAHs-related diseases.6−8 Thus, monitoring the biomarkers of oxidative DNA damage and lipid peroxidation could be a reliable way to determine an individual’s oxidative status and risks of PAHs-related diseases.9 We have shown previously that in coke oven workers (the typical PAHs-exposed occupational population), PAHs exposure can result in significant dose-related increases in oxidative DNA damage and lipid peroxidation, as reflected by 8-hydroxydeoxyguanosine (8-OH-dG) and 8-iso-prostaglandin-F2α (8-isoPGF2α), respectively.10 However, the underlying mechanisms still need to be investigated. Received: Revised: Accepted: Published: 4120
December 12, 2013 February 18, 2014 February 20, 2014 February 20, 2014 dx.doi.org/10.1021/es4055516 | Environ. Sci. Technol. 2014, 48, 4120−4128
Environmental Science & Technology
Article
worker at the start (preshift) or end (postshift) of workshift. In addition, we also collected ∼4 mL heparin-anticoagulated venous whole blood and ∼1 mL EDTA-anticoagulated venous whole blood from each worker. The study was approved by the Medical Ethics Committee of the School of Public Health, Tongji Medical College. Measurements of Urinary Creatinine and OH-PAHs. The concentration of urinary creatinine was determined by a Randox Daytona fully automated clinical chemistry analyzer (Furuno electric Co., Ltd., Japan) to adjust concentrations of urinary analytes for variations in hydration status.22 The concentrations of 12 urinary OH-PAHs [1-OHNAP, 2-OHNAP, 2hydroxyfluorene (2-OHFLU), 9-OHFLU, 1-OHPHE, 2-OHPHE, 3-OHPHE, 4-OHPHE, 9-OHPHE, 1-hydroxypyrene (1-OHPYR), 6-hydroxychrysene (6-OHCHR), and 3-hydroxybenzo[a]pyrene (3-OHBaP)] were determined by gas chromatography−mass spectrometry (GC−MS) described in detail previously.10 Finally, 10 OH-PAHs (except for 6-OHCHR and 3-OHBaP) were successfully detected, with the percentages of samples at concentrations above the limits of quantification (LOQ) higher than 90% or nearly 100% (data not shown). Default values for ten OH-PAHs were replaced with 50% of the LOQ. The molar concentrations for urinary OH-PAHs were calibrated by urinary creatinine and expressed as μmol/mmol creatinine. Determination of Plasma BPDE-Alb Adducts. The concentrations of BPDE-Alb adducts in heparin-anticoagulated plasma were detected by sandwich enzyme-linked immunosorbent assay (ELISA) described in detail previously.10 The concentrations of BPDE-Alb adducts were calibrated by plasma albumin and expressed as ng/mg albumin. The default values were replaced with 50% of the LOQ, which was approximately 1 ng/mg albumin. RNA Isolation and Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) Assay. Total RNAs were isolated from EDTA-anticoagulated plasma with mirVana PARIS miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer’s protocol. Briefly, we mixed 200 μL plasma for each participant with denaturing solution, and then added synthetic Cenorhabditis elegans miRNA cel-miR-39 (RNA oligonucleotides synthesized by Qiagen) into the denatured plasma to normalize the sample-to-sample variation in the isolation step. The denatured plasma was then extracted twice with Acid-Phenol: Chloroform to remove other cellular components and leave a semipure RNA sample. The RNA sample was further purified with a glass-fiber filter and eluted with a low ionic-strength solution. We mixed the total RNA with the reverse transcription (RT) master mix from the commercial TaqMan miRNA Reverse Transcription Kit (Applied BioSystems, Foster City, CA), and then performed RT reactions with ABI Prism 7900HT Sequence Detection System (Applied BioSystems, Foster City, CA) at 16 °C for 30 min, 42 °C for 30 min, 85 °C for 5 min, and finally held at 4 °C. The RT products were diluted and mixed with qPCR reaction mix from the commercial TaqMan microRNA assays kit (Applied BioSystems, Foster City, CA). Then we performed real-time PCR at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15s and 60 °C for 1 min. All RT reactions and PCR reactions, including no-template controls, were run in duplicate. The expression levels of miRNAs were normalized to cel-miR-39 and calculated by the eq 2−ΔCt, in which ΔCt = cycle threshold (Ct) miRNA − Ct (cel‑miR‑39).18 Urinary 8-OH-dG and 8-iso-PGF2α Measurement. The measurement of urinary 8-OH-dG was carried out with HPLC method described previously.10,23,24 Briefly, 8-OH-dG in 2 mL urine specimen of each participant was extracted and enriched
PAHs exposure has been shown to modulate the expression patterns of genes involved in oxidative stress response.11,12 These modulations of gene expression may arise from altered expression patterns of microRNAs (miRNAs),13,14 which function as posttranscription regulators of gene expression by base pairing with the 3′-untranslated regions of target mRNAs and leading to either translational repression or degradation of target mRNAs.15 miRNAs play crucial roles in a broad range of physiologic and pathologic processes, including oxidative stress regulation.16,17 In our previous two-stage study of coke oven workers,18 we first screened and compared hundreds of miRNAs in plasma pools of two different PAHs exposure groups (discovery stage), and then validated several miRNAs in 365 coke oven workers to identify PAHs-associated miRNAs (validation stage). We have successfully identified five PAHs-associated miRNAs: the expression levels of miR-24-3p, miR27a-3p, miR-142-5p, and miR-28-5p elicited concentration-dependent decreases for urinary 4-hydroxyphenanthrene (4-OHPHE) and/or plasma benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydotetrolalbumin (BPDE-Alb) adducts, whereas miR-150-5p elicited a concentration-dependent increase for urinary 1-hydroxynaphthalene (1-OHNAP), 2-OHNAP, 2-OHPHE, and the sum of urinary monohydroxy-PAHs (ΣOH-PAHs). We also found these five miRNAs were all associated with a dose−response increase in chromosome damage.18 However, their dose−response relationships with oxidative DNA damage and lipid peroxidation still need to be clarified. Moreover, accumulated evidence has shown that miRNAs can interact with environmental factors to affect phenotypes and diseases.19,20 Thus, in the present study, we sought to further evaluate the potential influences of five PAHs-associated miRNAs and their interactions with environmental factors on the regulation of oxidative DNA damage and lipid peroxidation in coke oven workers.
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MATERIALS AND METHODS Study Subjects. In the validation stage of our previous study which identified five PAHs-associated miRNAs,18 we enrolled 365 healthy male coke oven workers in the coke oven plant of a steel company located in southern China. These workers had been working in their current workplaces (i.e., at the top, side, bottom, and adjacent workplaces of coke ovens, or in offices) for at least one year. They were between the ages of 20−60 years old and had no self-reported history of disease or recent medication use. We set up multiple monitors in their workplaces and collected the airborne samples in workers’ breathing zone with an average flow rate of 2.0 L/min for 6 h. We determined the concentrations of 16 PAHs in the collected airborne samples by high-performance liquid chromatography (HPLC).10,21 On the basis of the mean concentrations of total PAHs in each workplace, we classified 241 individuals working in offices or at adjunct workplaces as the control group, and 124 individuals working at the top, side, or bottom of the coke oven as the exposed group.18 After written informed consent was obtained, we administered a standardized occupational questionnaire for each worker to collect information on demographic characteristics, lifestyle (such as smoking and drinking), and detailed occupational experience. Individuals who had smoked an average of less than 1 cigarette per day for less than 1 year in their lifetime were defined as nonsmokers; otherwise, they were considered smokers. Individuals who had drunk alcoholic beverages less than once a week for less than 1 year in their lifetime were defined as nondrinkers; otherwise, they were considered drinkers. After the interview, we collected ∼20 mL morning urine from each 4121
dx.doi.org/10.1021/es4055516 | Environ. Sci. Technol. 2014, 48, 4120−4128
Environmental Science & Technology
Article
Table 1. General Characteristics, PAHs Internal Exposure Levels, Oxidative Damage Levels, and miRNA Expression Levels of Coke Oven Workers in Different Workplaces variablesa General characteristics age (years) smoking status, yes/no (%yes) pack-years of smoking drinking status, yes/no (%yes) working years (years) workshift, preshift/postshift (%preshift) BMI PAHs internal exposure biomarkersb 1-OHNAP (×10−2) 2-OHNAP (×10−2) 2-OHFLU (×10−3) 9-OHFLU (×10−3) 1-OHPHE (×10−3) 2-OHPHE (×10−3) 3-OHPHE (×10−3) 4-OHPHE (×10−3) 9-OHPHE (×10−3) 1-OHPYR (×10−2) ΣOH-PAHs (×10−2) BPDE-Alb Oxidative damage biomarkersb 8-OH-dG 8-iso-PGF2α PAHs-associated miRNA miR-24-3p miR-27a-3p miR-142-5p (×10−2) miR-28-5p (×10−2) miR-150-5p (×10−2)
control (n = 241)
exposed (n = 124)
P-value
41.98 ± 8.41 158/83 (65.6) 11.54 ± 14.35 107/134 (44.4) 20.62 ± 9.90 169/72 (70.1) 24.28 ± 3.13
42.48 ± 7.55 94/30 (75.8) 15.47 ± 16.32 53/71 (42.7) 21.55 ± 8.96 45/79 (36.3) 23.74 ± 3.29
0.579c 0.045c 0.019c 0.763c 0.385c
