Objectives Diesel engine exhaust (DEE) is a ubiquitous environmental pollutant and is carcinogenic to humans. To seek early and sensitive biomarkers for prediction of adverse health effects, we analysed the components of DEE particles, and examined the genetic and oxidative damages in DEE-exposed workers.
Methods 101 male diesel engine testing workers who were constantly exposed to DEE and 106 matched controls were enrolled in the present study. The components of DEE were analysed, including fine particulate matter (PM2.5), element carbon (EC), nitrogen dioxide (NO2), sulfur dioxide (SO2) and polycyclic aromatic hydrocarbons (PAHs). Postshift urine samples were collected and analysed for 1-hydroxypyrene (1-OHP), an internal exposure marker for DEE. Levels of DNA strand breaks and oxidised purines, defined as formamidopyrimidine-DNA glycosylase (FPG) sites in leucocytes, were measured by medium throughput Comet assay. Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) was also used to determine the level of oxidative stress.
Results We found higher levels of PM2.5, EC, NO2, SO2 and PAHs in the diesel engine testing workshop and significantly higher urinary 1-OHP concentrations in exposed subjects (p<0.001). Compared with controls, the levels of parameters in normal Comet and FPG-Comet assay were all significantly higher in DEE-exposed workers (p<0.001), and in a dose-dependent and time-dependent manner. There were no significant differences between DEE-exposed workers and controls in regard to leucocyte FPG sensitive sites and urinary 8-OHdG levels.
Conclusions These findings suggest that DEE exposure mainly induces DNA damage, which might be used as an early biomarker for risk assessment of DEE exposure.
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What this paper adds
Diesel engine exhaust (DEE) is carcinogenic to humans and is associated with an increased risk of lung cancer. To date, the effect and mechanism by which chronic exposure to DEE induces human lung cancer remains unclear.
Most evidence supporting the relationship between DEE exposure and lung cancer risk has been from occupational-exposed workers, including miners, those working on the railroad, in docks and on tunnel construction, and professional drivers. However, few studies have involved diesel engine testing workers.
DEE exposure was systematically assessed by fine particulate matter, element carbon, nitrogen dioxide, sulfur dioxide and polycyclic aromatic hydrocarbons (PAHs) in the workplace, and from urine PAHs metabolites in participants. We found diesel engine testing workers to be unique and stable subjects, and to have relatively high DEE exposure.
We found DEE exposure mainly induces primary DNA damage, which might contribute to lung cancer development. DNA damage may be used as an early biomarker for risk assessment on DEE exposure.
Diesel engine exhaust (DEE) was classified as carcinogenic to humans (group 1) by the International Agency for Research on Cancer (IARC) in 2012.1 The IARC also noted that large populations all over the world are exposed to DEE in daily life, either through their occupation or from ambient air. To date, evidence supporting the relationship between DEE exposure and lung cancer risk has been from studies on occupational-exposed workers, including miners, those working on the railroad, in docks and on tunnel construction, and professional drivers, etc.2–5 In addition to the occupational exposure aforementioned, diesel engine testing workers in engine manufacturing factories are specifically exposed to high levels of DEE during working hours. They are responsible for the testing and adjusting of engine assemblies to optimise conditions for starting, accelerating and decelerating, and for stable running of the engine.
DEE contains a complex of carbonaceous nuclei and a vast number of inorganic and organic compounds, including polycyclic aromatic hydrocarbons (PAHs) and PAH derivatives.6 Elemental carbon (EC) has often been regarded as a surrogate of DEE exposure. Besides, nitrogen dioxide (NO2) and sulfur dioxide (SO2) have also been found at high concentrations in DEE.7 Although the internal markers for evaluation of DEE exposure remain undefined, urinary 1-hydroxypyrene (1-OHP) has proven to be the most comprehensive biomarker for PAH exposure8 and DEE exposure in occupational population-based studies.9 ,10
Primary DNA damage is considered to be a key initial event in carcinogenesis. The Comet (single cell gel electrophoresis) assay has become the preferred test for qualitatively and quantitatively assessing DNA damage in small numbers of cells in vitro and human biomonitoring studies.11 Oxidative stress may also be implicated in carcinogenesis, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) is the most frequently measured biomarker of oxidative stress.12 Formamidopyrimidine-DNA glycosylase (FPG) is most often used in the endonucleases modified Comet assay to evaluate oxidative DNA damage. We found that the changes in FPG-Comet assay are correlated to the cellular reactive oxygen species (ROS) induced by genetic toxicants.13 For biomonitoring purposes, enzymatic detection of DNA oxidised products by Comet assay is an easier and more feasible approach.14 ,15
A few studies were conducted to investigate DNA damage among workers for genotoxicity assessment of DEE, but their findings are not consistent. For instance, one study evaluated 47 shale-oil miners in Denmark and found that DNA damage in frozen blood samples from workers with diesel-powered equipment was significantly higher than in the control group.16 Another study of 39 tunnel construction workers in Italy found no significant differences in the level of primary and oxidative DNA damage between tunnel construction workers and controls.5 To evaluate the relationship between long term, stable high levels of DEE exposure and DNA damage, we conducted a cross-sectional molecular epidemiology study to determine the DNA damage and oxidative damage ex vivo in diesel engine testing workers in China. On the basis of the comprehensive evaluation of DEE exposure and medium throughput methods on detecting biomarkers, our results might contribute to cancer prevention and risk assessment of DEE exposure.
Materials and methods
Identification of study factories
We selected one factory that produces diesel engine for heavy trucks; in 2011–2012, its average production was approximately 9000 machines per month. Monitoring of fine particulate matter (PM2.5) and EC levels was undertaken in the factory during an initial screening. There was high DEE exposure in the engine testing department, where the engine testing workers tested and tuned diesel engines after assembly. No other definite genotoxicants (eg, trichloroethylene, formaldehyde, etc) were found in the testing department by field survey. Although personal protective equipment was provided, most testing workers did not wear their masks or respirators consistently during work. According to the monitoring data, we divided the workers into low and high DEE-exposed groups. The mean EC level was 64.4 and 173.5 μg/m3 in the low DEE-exposed and high DEE-exposed groups, respectively. We chose a control group of workers from a water plant in the same city.
Characteristics of study subjects
Recruited DEE-exposed workers were required to meet the following two criteria: (1) they were male engine testing workers who spent most of their working hours testing the engines and (2) they had worked in the same area of the factory for at least 1 year. Participants with a history of cancer or kidney diseases, and those with previous exposure to a known carcinogen, were excluded from both DEE-exposed and control groups in this study. The workers of the control group operate pumps at a pump station without processing chlorine for disinfection of water. We selected 101 DEE-exposed workers and 106 controls who were matched by age within 5 years and smoking status to the DEE-exposed group in the present study. All participants were interviewed by trained personnel using a detailed questionnaire that included demographic information, educational level, smoking history, alcohol consumption, occupational history of exposure and personal medical history. The participation rates were approximately 90% for DEE-exposed workers as well as for controls. The study was approved by the Research Ethics Committee of the National Institute for Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, and informed consent was obtained from each participant.
A peripheral blood sample was collected in a heparin vacutainer tube for Comet assay, and another blood sample was collected in an EDTA vacutainer tube for complete blood cell count assay. The sample tubes were stored away from light, in a cooler box with frozen gel packs and transported to the laboratory within 3 h of collection. A spot urine sample was obtained from each participant at the end of his work shift, maintained at −70°C, and used for the detection of 1-OHP and 8-OHdG.
DEE exposure was assessed by the static air sampling method, including PM2.5, PM2.5 related EC, NO2, SO2 and airborne PAHs in the working environment of the engine testing department and in the control factory. We used an air particle sampler (TH150-DII, Wuhan, China) for PM2.5 mass collection on preweighed polytetrafluoroethylene (PTFE) filters (90 mm, Millipore) and carbon was collected onto a 37 mm quartz fibre filter using cyclones with a PM2.5 cut point. The PM2.5 filters were analysed for PAH constituents according to the guideline of NIOSH No.58. EC was determined by a Carbon Analyser (DRI2001A, Atmoslytic Inc) according to NIOSH 5040. An UMEX-200 passive sampler (SKC Inc) was used for NO2 and SO2 collection and analysis. Exposure assessment was performed in 12 samples collected from the environment of DEE-exposed workers and 6 samples from that of the controls.
Measurement of urinary 1-OHP
We determined urinary 1-OHP using the high performance liquid chromatography mass spectrometry (HPLC-MS)/MS method, described previously.17 Briefly, we used 2 mL of urine from each sample, in which 1.0 mL of sodium acetate buffer (0.5 mol/L, pH=5.0) was added, and the samples were incubated with β-glucuronidases (Sigma–Aldrich) in the dark. Naphthol-d8 (100 µg/mL, Sigma–Aldrich) was then added as the internal standard. Subsequently, 1-OHP was extracted from the urine, using 4 mL dichloromethane. The extracts were evaporated to dryness by nitrogen before being dissolved in 1.5 mL HPLC solvent and analysed by ultra-fast liquid chromatography (LC-20AD, Shimadzu, Kyoto, Japan) coupled with an integrated triple quadrupled mass spectrometer (ABI3200, Applied Biosystems). The detection limit of urinary 1-OHP was 0.2 μg/L. The 1-OHP concentration was adjusted by its corresponding level of urinary creatinine.
Determination of DNA strand breaks
All the experiments were performed by skilled research personnel, to minimise interoperator variation. DNA damage in leucocytes was determined by the alkaline Comet assay, as described previously,18 with a slight modification. Briefly, 5 μL of whole blood was mixed with 200 μL molten 1% (w/v) low-melting-point agarose, and 30 μL cell suspensions were pipetted into the wells of a 20-well Comet Slide (Trevigen Inc). After accelerating gelling at 4°C, the slides were submerged in cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH=10.0, with fresh 10% dimethyl sulfoxide and 1% Triton X-100) for 2.5 h at 4°C. After lysis, the slides were immersed in buffer F (40 mM hydroxyethyl) piperazene-N′-(2-ethane sulfonic acid), 0.1 M KCl, 0.5 mM EDTA and bovine serum albumin 0.2 mg/mL, pH=8.0) twice. Subsequently, the slides were transferred to an electrophoresis system (Trevigen) and covered with alkaline solution (1 mmol/L Na2 EDTA and 0.3 mol/L NaOH, pH ≥13.0) for 20 min. DNA electrophoresis was run at 300 mA, 1 V/cm, for 25 min. DNA unwinding and electrophoresis were performed in a cold unit at 4°C. We mixed the samples of DEE-exposed workers and controls in each batch of assays. The experiment was performed under dim light to prevent additional DNA damage. After electrophoresis, the slides were neutralised with 0.4 M Tris-HCl, pH=7.5. The slides were dehydrated with ethanol for 10 min, and allowed to dry at air temperature until staining.
Slides were stained with 5 μg/mL ethidium bromide for 20 min. Observations were made at ×200 magnification under a fluorescent microscope (Olympus BX50), through a CCD camera connected to a computer-based image analysis system (CometScore V1.5, TriTek Corporation, Sumerduck, Virginia, USA). A total of 100 cells from each participant were examined randomly. To quantify DNA damage, the parameters of Olive tail moment (OTM) and % Tail DNA were evaluated. The variation in the Comet experiment was evaluated by a master internal control sample, a fresh blood sample from one fixed human volunteer for each event, and the values of coefficients of variation for each assay were less than 10%.
FPG-modified Comet assay (FPG-Comet)
The effect of FPG enzyme was validated by our former assay.13 We performed the FPG-Comet assay to assess the oxidative DNA damage in all participants. In brief, following lysis, the slides were also immersed in buffer F twice (for 5 min, one at a time) at room temperature. The slides were then incubated for 45 min at 37°C with 30 μL buffer F, FPG (dilutions 1:1000 with buffer F, 0.25 U/well), in a humidified atmosphere. The control gels with buffer alone provide the background of DNA-strand breaks. The FPG-treated gels revealed strand breaks and oxidative lesions recognised and cleaved by the enzymatic treatment. We used the measure of net FPG sensitive sites (net level of % Tail DNA) for evaluating oxidised DNA. Net FPG sensitive sites were calculated by subtracting the % Tail DNA of buffer incubation from the % Tail DNA of FPG incubation.
Measurement of urinary 8-OHdG
Urinary 8-OHdG was analysed using HPLC-MS/MS,19 with minor modifications. Briefly, after centrifugation at 12 000 rpm for 5 min, 100 μL of urine was diluted with 10 mM NaCOOH buffer (pH 7.5). The urine solution contained 7.5 ng of (13C10, 15N5)-8-OHdG as an internal standard. Samples were purified using hydrophilic-lipophilic balance (HLB) cartridges (60 mg, 3 cc, Waters). The extracts were dried under nitrogen and the residue was dissolved in 150 μL of methanol: 0.1% aqueous formic acid (FA) (5:95 v/v). A total of 10 µL of the sample was applied for analysis by HPLC-MS/MS (Agilent 6410B) using a 2.1×75 mm C18 analytical column (Supelco 569254-U) with gradient elution. The mobile phases were 0.1% FA and MeOH with 0.1% FA. The following m/z ion combinations (precursor<–product) were monitored, and the multiple reaction monitoring (MRM) transitions were used as follows: 284 <−168 for 8-OHdG and 287<−171 for (13C10, 15N5)-8-OHdG. The calibration was performed by plotting peak area ratios of 8-OHdG to (13C10, 15N5)-8-OHdG, measured at each nominal concentration. The concentration of urinary 8-OHdG was calculated by extrapolating the peak area of the sample from standard solutions of 0, 1.5, 3, 6, 12 and 24 μg/L. The concentration of urinary 8-OHdG was also adjusted by its corresponding concentration of urinary creatinine.
Statistical comparisons for parameters of DNA damage and oxidative DNA damage were performed between various groups using the Mann-Whitney U and Kruskal-Wallis tests. Medians and IQR were calculated for urinary 1-OHP, and for net FPG sensitive sites in DNA of leucocytes in exposed workers and controls. We divided exposed subjects into short-term, medium-term and long-term DEE-exposed groups by tertile of diesel engine testing years for duration–response analysis, which were ≤6.5, 7–9.0 and >9.0 years, respectively. Linear regression using the natural logarithm (ln) of data derived from the 1-OHP, OTM and % Tail DNA was used to test for differences between workers exposed to DEE and controls. All statistical models were adjusted for matching variables, leucocyte counts, age (as a continuous variable) and smoking status (non-current or current). Potential confounders previously shown to influence one or more of the end points in this report were also included in the final models, those being, body mass index (BMI) and number of cigarettes/day. Correlation between DNA damage parameters and urinary 1-OHP concentration were analysed using Spearman correlation. All statistics were two sided and performed using SPSS software (V.20, IBM SPSS, Chicago, Illinois, USA).
DEE exposure assessment
The exposure assessment of DEE is shown in table 1. Compared to controls, there were higher levels of particles, gas pollutants and carcinogenic PAHs from DEE in the engine testing department. The mean PM2.5 and EC was 3.14 and 9.66 times higher than that of the control group (p<0.001), respectively. The levels of NO2 and SO2 were 7.48 and 40 times higher than the control group (p<0.001), respectively. The PAHs levels were much higher in the workplace of exposed workers: 141.6 times higher than the levels of the control area (p<0.001). Therefore, diesel engine testing workers could be unique subjects for DEE-exposure study.
Subject characteristics and internal PAH exposure
The average age of DEE-exposed workers was 32.09±8.95. Their age, BMI, smoking status and alcohol drinking status were matched to the controls (table 2). Most were exposed long term to DEE with an average duration of 8.31 years (range, 1–37 years).
Urinary 1-OHP is representative of internal PAH and DEE exposure.9 The median of urinary 1-OHP in DEE-exposed workers was 2.96-fold higher than that of controls (2.19 vs 0.74, p<0.001) (table 2). There was no cigarette smoking effect on 1-OHP levels in DEE-exposed workers (figure 1A). However, a higher 1-OHP level was found in current smokers in the control group, but with no statistical significance (figure 1A).
DNA damage was correlated to DEE exposure
As shown in table 2, OTM and % Tail DNA measured in the regular Comet assay increased 3.4-fold and 2.2-fold, respectively, in DEE-exposed workers and those in the control group (both p<0.001). The level of OTM and % Tail DNA in the FPG-Comet assay was 2.8 and 2.5 times higher than that of the control (both p<0.001). Furthermore, smoking status did not show a significant influence on these findings (figure 1B).
Compared with the control group, the parameters of two kinds of Comet assay in the low and high DEE-exposed groups were increased, with a good dose–effect relationship (p<0.001). The significant differences were maintained after adjustment for age, BMI and number of cigarettes/day (table 3). In all participants, we found a significant correlation between urinary 1-OHP levels and the degree of DNA damage (All p<0.001, see online supplementary table S1).
DEE exposure and oxidative DNA damage
As shown in table 2, there was no significant difference between DEE-exposed workers and controls in terms of net FPG sensitive sites. In addition, we did not find a correlation between urinary 1-OHP levels and net FPG sensitive sites in any of the participants (see online supplementary table S1). Although the levels of net FPG sensitive sites appeared slightly higher in the high DEE-exposed group than controls (table 3), we did not observe a dose–response relationship (p>0.05).
To validate the changes after oxidative DNA damage in the DEE-exposed population, we examined the levels of 8-OHdG in urine. In line with what we observed in the FPG-Comet assay, there was no significant difference in urinary 8-OHdG levels between the two groups by a linear regression analysis (p>0.05). Moreover, urinary 1-OHP levels were not correlated with the 8-OHdG levels in any of the participants (see online supplementary table S1). Compared to controls, the level of urinary 8-OHdG was not significantly different in low and high level of DEE exposure groups. In addition, no significant effect was observed between smoking status and oxidative DNA damage (figure 1C, D).
DEE exposure duration and DNA damage
For the exposure duration–response analysis, we divided the DEE-exposed subjects into short-term, medium-term and long-term-exposed groups by tertile of engine testing years. The levels of OTM and % Tail DNA in the normal Comet assay were higher in the long-term-exposed group than in the short-term and medium-term groups, with significant differences compared with the controls (figure 2A, B). A significant time–response relationship was found in regard to OTM changes (figure 2A). For oxidative DNA damage measurement, urinary 8-OHdG levels decreased in all exposure groups and significantly decreased in the medium-term group (figure 2C). Net FPG sensitive sites significantly decreased in the long-term group (figure 2D).
In order to identify early and sensitive biomarkers for risk assessment, it is critical to determine the mechanism of DEE-induced carcinogenesis. In this study, we selected a group of diesel engine testing workers who were specifically exposed to a high level of DEE. High levels of PM2.5, EC, NO2, SO2 and PAHs were detected in their workshop areas. For example, the EC level as a marker of DEE exposure in occupational settings7 was 10 times higher in the workshop of the exposure group as compared with the area of the control group, and was similar to the level from the underground miner study,4 and at least 10 times higher than the level from the truck driver study.2 The exposed subjects in our study are a unique population, stably exposed to DEE in their work process and area. To the best of our knowledge, this is the first molecular epidemiology study to investigate the effect of DEE on DNA damage and oxidative DNA damage in a large number of diesel engine testing workers. The results indicate that DEE exposure can induce primary DNA damage, which heightens concerns about its lung cancer potential from occupational and environmental exposure.
Recently, DEE exposure was associated with the development of lung cancer in a nested case–control study and in a cohort study.4 ,20 PAH is one of the most important carcinogenic components in DEE. We confirmed that the excretion of 1-OHP is a potential biomarker of DEE exposure, which is consistent with previous DEE population studies.9 ,10 ,21 Similar to studies on drivers and fuel attendants,22 we did not find a correlation between cigarette smoking and levels of urinary 1-OHP in occupational DEE-exposed workers.
The alkaline Comet assay sensitively detects a range of DNA lesions as well as alkaline sensitive sites.11 ,23 We applied the medium throughput Comet assay using commercial slides, which provide good reproducibility in our present study. Both in the normal and in the FPG-Comet assay, we found the levels of DNA strand breaks including OTM and % Tail DNA were significantly higher in DEE-exposed workers than those in the provincial controls. We also observed elevated DNA damage levels with higher exposure levels and increasing years of DEE exposure. For related DEE-exposed population studies, the outcome of the present study was in agreement with those on genomic damage in underground shale-oil miners from Denmark16 and in a traffic policeman study.24 Repeated exposure to DEE particles has also been associated with elevated levels of DNA damage in animal experimental models and cell culture systems.25 These results indicate that DEE-exposed workers may be more prone to the initiation of genetic damage, such as DNA strand breaks, which may be the earliest step in carcinogenesis.
We evaluated oxidative DNA damage using two assays with FPG enzyme-modified Comet assay in leucocytes and 8-OHdG in urine. The urinary 8-OHdG level of the control group in the present study is similar to that of control subjects in other Chinese studies.26 ,27 While there were lower levels of ‘net FPG sensitive sites’ and urinary 8-OHdG in DEE-exposed workers, there was no significant difference between the exposed subjects and unexposed controls. It is suggested that oxidative DNA damage did not occur and did not have an accumulative effect in long-term DEE-exposed workers, although there was a difference between the two measurements. The reason for this discrepancy might be that the amount of urinary 8-OHdG involves the repair products of oxidised guanine in DNA and the cellular pool from the whole body.28 ,29 In addition to 8-OHdG, the results of the Comet assay combined with enzymes of excision repair reflect a broad spectrum of oxidised purines and pyrimidines from leucocyte DNA.11 ,30 Intracellular ROS production is the key factor for oxidative damage. Our finding was supported by a recent in vitro study that found no obvious increase of ROS generation and 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1) sensitive sites in human lung epithelial cells and macrophages exposed to heavy-duty DEE particles.31 It is plausible that oxidative damage occurred following short-term DEE exposure. Another short-term study on 14 healthy men involved the inhalation of diluted DEE. The authors found changes in gene expression in peripheral blood mononuclear cells, which was attributed to oxidative stress and protein degradation on DEE exposure.32 Our findings are also consistent with the experiment on mice exposed to DEE by inhalation, in which DEE-induced oxidative DNA damage in terms of 8-OHdG in the lung tissue after a single exposure, whereas oxidative DNA damage was not found after repeated exposures.33 The unchanged levels of oxidative DNA damage after long-term DEE exposure might be due to the increased antioxidant defence mechanisms. Some studies found the rate of repair of 8-OHdG to be mediated by elevated gene expression of OGG1.33 However, to support that conclusion, other oxidative markers and antioxidant systems should be investigated in future studies.
Cigarette smoking showed different effects in studies pertaining to DEE-induced carcinogenesis.2 ,20 In the present study, we did not find that smoking coupled with DEE exposure has any measurable effects on urinary 1-OHP levels, DNA damage and oxidative stress. Although these measurements were minimally elevated in smokers from the control group, no significant changes were found in the exposed workers. We did not find a significant correlation between urinary 1-OHP and DNA damage parameters in the DEE-exposed populations. This suggests that DNA damage might also be induced by particles in DEE.
There are some factors hampering investigation of the adverse health effects of DEE in the human population. Most people are not solely exposed to DEE in the general environment and workplace; the vast majority are exposed to a mixture of pollutants from other sources in their working environments, such as minerals, chemicals and dust. On the basis of the present technical conditions, it is difficult to give an appropriate quantitative exposure assessment on DEE. In this study, we selected a large number of workers who had been regularly exposed to high and simple DEE for a prolonged period of time. They had been working in the same department, and their DEE sources were identical and basic, thus DEE concentrations did not fluctuate much. Therefore, the effects of an alternative exposure route were believed to be minimal. Second, the DEE exposure conditions in the workplace were systematically assessed, including PM2.5, EC, NO2, SO2 and PAH levels. We also used 1-OHP to evaluate the internal exposure of DEE chemicals. Investigations on exposure–response associations with measures of DEE exposure by average exposure/duration can give greater weight in the evaluation of carcinogenicity. Third, the exposed and control groups were well matched, and all the participants lived in the same geographic region. These two groups had comparable demographic and socioeconomic characteristics, and neither group took routine antioxidant supplementation (such as vitamin C, etc). Different dietary and environmental factors, other than DEE exposure, are unlikely to explain the observed results. Additionally, we evaluated oxidative DNA damage using Comet and chemical analysis methods, which further support our findings.
In summary, we investigated the effects of DEE on both DNA damage and oxidative DNA damage in simple and steadily exposed subjects, and found adverse effects of DEE pollutants on DNA. These findings provide important molecular epidemiological evidence for the association between DEE and lung cancer. DNA damage may be used as an earlier biomarker of effect for occupational and environmental DEE exposure. Future studies should aim at systemically evaluating personal EC and chemical exposure, and their association with genetic damages in diesel engine testing workers.
The authors would like to thank the members of Henan Institute of Occupational Medicine (Zhengzhou, China) for assistance with sample collection and instrumental support.
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Contributors YZ, HD and SY designed the research. HD, XJ, XL and QZ carried out the Comet assay. HD and YD set the protocols in the laboratory and performed statistical analysis. LM, SW and CH performed the urinary 8-OHdG and 1-hydroxypyrene analysis. HW and YN conducted recruitment of the study subjects and made the exposure assessment. HD, WG, WC and YZ participated in drafting and editing of the final manuscript. All the authors have read and approved the final version of this manuscript.
Funding This work was supported by a Key Program of National Natural Science Foundation of China (NSFC 81130050), NSFC 81172642, NSFC 81428021, and a National Key Technology Research and Development Program (2014BAI12B02).
Competing interests None declared.
Patient consent Obtained.
Ethics approval This study was approved by the Research Ethics Committee of the National Institute for Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention.
Provenance and peer review Not commissioned; externally peer reviewed.
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