Introduction
Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the atmosphere from motor vehicle emissions, tobacco smoke, and incomplete combustion of coal, oil, gas, and wood. Human exposure to PAHs occurs primarily through dietary intake, environmental tobacco smoke, and air pollution. Chronic exposure to PAHs has been associated with various diseases, including obesity, cardiovascular disease, diabetes, and several cancers (e.g., breast, lung, and bladder cancer). PAHs are lipophilic and persist in the body for extended periods, making long-term exposure particularly harmful [1-5]. The International Agency for Research on Cancer (IARC) classifies benzo[a]pyrene (BaP) as Group 1 carcinogen (Carcinogenic to humans), while dibenzo[a,h]anthracene (DBA) is classified as Group 2A (Probably carcinogenic to humans). Other PAHs, such as naphthalene (Na), chrysene (Chr), benzo[a]anthracene (BaA), benzo[k]fluoranthene (BkF), and benzo[b]fluoranthene (BbF) are categorized as Group 2B (Possibly carcinogenic to humans) [6].
Upon entering the body, PAHs are metabolized into hydroxy-PAHs (OH-PAHs) via cytochrome P450 enzymes. The low-molecular-weight OH-PAHs (2-3 benzene rings) are excreted primarily in the urine, whereas high-molecular-weight OH-PAHs (4 or more benzene rings) are excreted through the feces [2, 7, 8].
Pyrene, one of the most representative PAHs, is metabolized and excreted in urine as 1-hydroxypyrene (1-OHP), a well-established biomarker for PAH exposure [2,9,10]. However, 1-OHP is insufficient for assessing carcinogenic risk, as it is not carcinogenic [11]. The metabolite of BaP, 3-hydroxybenzo[a]pyrene (3-OH-BaP), is recognized as a more accurate biomarker for PAH-related carcinogenic exposure but occurs at such low levels in urine that detection is challenging [11].
PAH exposure has also been associated with increased oxidative stress, as evidenced by elevated levels of oxidative damage markers such as malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) [12-14]. These markers assess lipid peroxidation and DNA damage, respectively, both of which are linked to the generation of reactive oxidative species (ROS) following PAH exposure [12,13].
There are various pollutants in the air, and PAHs are present in both particulate and gaseous forms [14-15]. In particular, PAHs are also emitted during vehicle operation, and depending on the type of fuel, gasoline has been reported to contribute higher molecular weight PAHs and diesel to lower molecular weight PAHs [16]. Given that transportation workers, particularly taxi drivers, spend extended periods in traffic, they are at heightened risk of exposure to vehicular emissions and associated pollutants, including PAHs. [18].
Exposure to PAHs has been reported in a variety of occupations, including asphalt workers [7,19], coke oven workers [20], hair salon workers [21], firefighters [22-23], heavy truck drivers [24], transportation workers [25], coal miners [26-27], and waste incinerator workers [28]. However, transportation workers (especially taxi drivers) drive in urban centers with high air pollution and spend more than 10 hours a day on the road, but there is a lack of research on occupational exposure. This study focuses on the assessment of urinary PAHs metabolites, oxidative stress markers, and smoking indicators among taxi drivers and a control group, aiming to provide insights into occupational exposure and its health implications. While several studies have previously investigated PAH exposure in taxi drivers through both urinary biomonitoring and air concentration measurements in different countries (e.g., Italy, Denmark, and Taiwan) [29-31], this study is the first to evaluate occupational exposure to PAHs in taxi drivers in Korea. Our study also uniquely incorporates oxidative stress biomarkers (malondialdehyde, MDA, and 8-hydroxy-2'-deoxyguanosine, 8-OHdG) to assess the broader health impact of PAH exposure, providing novel insights into the potential oxidative damage associated with occupational PAH exposure among Korean transportation workers.
Materials and Methods
Chemicals and reagents
1-naphthol (1-OH-Na), 2-naphthol (2-OH-Na), 2-hydroxyfluorene (2-OH-Flu), 9-hydroxyphenanthrene (9-OHPhe), 13C6-2-OH-Na, 8-OHdG, MDA, COT, COT-d3 were purchased from Sigma-Aldrich (Milwaukee, WI, USA). 3-hydroxyfluorene (3-OH-Flu), 1-hydroxyphenanthrene (1-OH-Phe), 2-hydroxyphenanthrene (2-OH-Phe), 3-hydroxyphenanthrene (3-OH-Phe), 4-hydroxyphenanthrene (4-OH-Phe), 13C6-3-OH-Phe, 13C6-1-OHP, 15N5-8OHdG, 1,1,3,3-tetraethoxypropane (MDA-d3, 96-98%) were obtained Cambridge Isotope Laboratories (Andover, MA, USA). 3-hydroxyfluoranthene (3-OH-Fluoran), 1-OHP, 6-hydroxy chrysene (6-OH-Chr), 3-OH-BaP, 3-hydroxydibenzo[a,h]anthracene (3-OH-DBA), 3-hydroxybenzo[j]fluoranthene (3-OH-BjF), 1-hydroxyindeno[1,2,3-cd]pyrene (1-OH-IP), 13C6-3-OH-Chr were supplied by Midwest Research Institute (Kansas Ciry, MO, USA). 1-hydroxybenzo[a]anthracene (1-OH-BaA), 2-hydroxybenzo[a]anthracene (2-OH-BaA), 3-hydroxybenzo[a]anthracene (3-OH-BaA), 2-hydroxymethylbenzo[a]anthracene (2-OHM-BaA) were purchased from Toronto Research Chemicals (North York, ON, CA). 13C6-2-OH-Flu was obtained from BOC Science (Shirley, NY, USA). Ammonium formate, formic acid, acetic acid, β-glucuronidase/aryl sulfatase, 2,4-dinitrophenylhydrazine (DNPH) were purchased from Sigma-Aldrich (Milwaukee, WI, USA). Hexane and ethyl acetate were purchased from Burdick & Jackson (Muskegon, MI, USA). Water, methanol and acetonitrile (ACN) were purchased from J.T Baker (Phillipsburg, NJ, USA).
Study population and sample collection
Participants were recruited between July and October 2021 from the Seoul, Gyeonggi, and Ulsan regions. The study included 19 male taxi drivers and 46 controls (male = 18, female = 28). Only male taxi drivers who had been working for a minimum of 5 years in urban centers were included to ensure adequate exposure to PAHs through occupational settings. Participants with chronic diseases such as cancer or cardiovascular conditions were excluded, as were individuals with previous occupations that might have increased PAH exposure. Controls were selected from non-driving occupations, such as office workers and housewives, to match the environmental exposure levels of taxi drivers without occupational PAH exposure. Urine samples were collected in the morning and stored at -20 °C until analysis. All participants signed informed consent forms and completed a questionnaire capturing demographic data, socioeconomic status, lifestyle behaviors (e.g., smoking, alcohol consumption), diet, and occupational and residential exposures. This study was approved by the Eulji University Institutional Review Board (EUIRB2021-041).
Measurements of OH-PAHs
OH-PAHs in urine were analyzed using a modified version of the method developed by Park et al (2015) [32]. Briefly, 1 mL urine sample was spiked with internal standards (IS) and β-glucuronidase/sulfatase and incubated for 16 hours at 37°C. The reaction products were purified by solid phase extraction (SPE) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The details of the instrumental analysis conditions are available in Supplementary Table S1 - S4
Measurements of MDA
MDA was measured in urine using the following protocol: 100 μL of IS and 500 μL of 2,4-dinitrophenylhydrazine (DNPH) solution (0.05 g DNPH in 4N HCl) were added to 1 mL of urine and incubated at 37°C for one hour. The supernatant was extracted with hexane, concentrated, reconstituted with 50 % acetonitrile (ACN), and analyzed by LC-MS/MS. Instrumental conditions and additional sample preparation details are provided in Supplementary Fig. S1, and Tables S5 - S6.
Measurements of Cotinine and 8-OHdG
For simultaneous analysis of cotinine and 8-OHdG, 100 μL of urine was mixed with 100 μL of IS mobile phase A, purified by SPE, and quantified by LC-MS/MS. Sample preparation steps are summarized in Supplementary Tables S7 - S8.
Quality assurance (QA) and quality control (QC)
In this study, strict quality assurance (QA) and quality control (QC) measures were implemented to ensure data reliability. OH-PAHs were quantified using linear regression analysis weighted by 1/x, and synthetic urine was prepared to match the matrix of the urine samples. Blank samples were analyzed between calibration points to check for carryover. Calibration curves were analyzed at the beginning of each batch, and quality control standards were included every 20 samples. Internal standards were employed throughout the analysis to account for any sample loss or fluctuations in instrument sensitivity, ensuring the reproducibility of results.
Statistical analysis
Statistical analysis was performed for substances with a detection rate of 70 % or higher. For results below the limit of detection (LOD), the value was replaced with LOD/√2. Spearman correlation analysis was conducted to assess relationships between different urinary substances. Non-parametric tests (Kruskal-Wallis test) were applied to evaluate differences in urinary concentrations of OH-PAHs, oxidative damage markers, and cotinine between taxi drivers and controls. Additionally, chi-square tests were used to compare demographic and behavioral characteristics with substance exposure levels. A p-value of < 0.05 was considered statistically significant for all tests, which were performed using SAS software.
Results and Discussion
Subject Characteristics
The characteristics of the study population are shown in Table 1. The study included a total of 65 subjects: 19 male taxi drivers and 46 controls (18 male, 28 female). The mean age of the taxi drivers was 60.5 years, while the mean age of the control group was 54.1 years. Among the taxi drivers, 63.2 % were smokers, compared to 22.2 % in the male control group and 3.6 % in the female control group.
All taxi drivers reported wearing masks while working, compared to approximately 60 % of the control group. The majority of taxi drivers (57.9 %) reported working more than five days per week, while controls were not queried on their weekly working hours. Additional lifestyle and dietary data, such as alcohol consumption and recent intake of baked or delivery food, were recorded but showed no significant association with urinary PAH metabolite levels.
Urinary levels of OH-PAHs, Cotinine and 8-OHdG
The predominant OH-PAHs detected in urine were 1-hydroxynaphthalene (1-OH-Na, 96.9 %), 2-hydroxynaphthalene (2-OH-Na, 90.8 %), 2-hydroxyfluorene (2-OH-Flu, 86.2 %), 1-OHP (80.0 %), and 3-hydroxyphenanthrene (3-OH-Phe, 70.8 %). In contrast, polymeric PAHs such as benzo[a]anthracene (BaA), methyl benzo[a]anthracene, benzo[a]pyrene (BaP), dibenzo[a,h]anthracene (DBA), benzo[j]fluoranthene (BjF), and indeno [1,2,3-cd]pyrene are not suitable for metabolomic analysis in urine because they are primarily excreted in feces rather than urine [8,12]. The median PAHs metabolite concentrations in the urine of taxi drivers were 2-OH-Na (3.440 ng/mL), 1-OH-Na (1.770 ng/mL), 2-OH-Flu (0.110 ng/mL), 3-OH-Phe (0.046 ng/mL), and 1-OHP (0.044 ng/mL), while PAHs metabolite concentrations in the control group (male and female) were 2-OH-Na (1. 698 and 0.675 ng/mL), 1-OH-Na (0.666 and 0.229 ng/mL), 2-OHFlu (0.067 and 0.023 ng/mL), 1-OHP (0.045 and 0.018 ng/mL), and 3-OH-Phe (0.025 and 0.008 ng/mL) (Table 2). The difference in urinary concentrations of OH-PAHs between taxi drivers and controls (males) was not significant, while the difference in concentrations between taxi drivers and controls (females) was significant for 1-OH-Na (p = 0.024), 2-OH-Na (p = 0.004), 2-OH-Flu (p = 0.001), 3-OH-Phe (p = 0.004), and 1-OHP (p = 0.006). The difference in concentrations between men and women in the control group was significant for 2-OH-Na (p = 0.016), 2-OH-Flu (p = 0.007), and 1-OHP (p = 0.024) (Fig. 1).
In this study, the median concentration of OH-PAHs in the urine of taxi drivers and general men was also higher in taxi drivers, but did not reach statistical significance. This is due to the small sample size and the fact that taxi drivers are overwhelmingly smokers, while the control group is predominantly non-smokers.
More than half of the taxi drivers were smokers. The urinary OH-PAHs concentrations of taxi drivers were 2.32 times lower for 2-OH-Na and 5.20 times lower for 1-OHP compared to smokers in the KoNEHS (2015-2017). In the control group, 2-OH-NA was 2.13-3.27 times lower and 1-OHP was 4.06-5.63 times lower compared to men, women, and non-smokers (Fig. 2) [33]. This is likely due to the decreased exposure to PAHs through breathing as masks became mandatory after the COVID-19 outbreak in 2019.
Urinary oxidative damage markers (MDA, 8-OHdG) were detected in more than 90 % of all groups, while cotinine, a marker of smoking, was detected in 84.2% of taxi drivers and less than 30% of controls. The median MDA was 25.50 ng/mL in taxi drivers, 25.75 ng/mL in controls (men), and 19.20 ng/mL in controls (women), with a significant difference between men and women in the control group (p=0.031). Median 8-OHdG was 6.02 ng/mL in taxi drivers, 5.79 ng/mL in controls (male), and 3.50 ng/mL in controls (female), with a significant difference between taxi drivers and controls (female) (p=0.035). The median cotinine concentration was 378.00 ng/mL in taxi drivers and <LOD in controls (male and female) (Fig. 3).
The significantly elevated levels of 8-OHdG (6.02 ng/mL) and MDA (25.50 ng/mL) in taxi drivers, compared to female controls (3.50 ng/mL and 19.20 ng/mL, respectively), indicate increased oxidative damage. 8-OHdG is a well-established biomarker for DNA damage, and the observed levels suggest a heightened risk of genotoxicity in taxi drivers, likely due to prolonged exposure to traffic pollutants. Elevated MDA concentrations reflect increased lipid peroxidation, which is linked to cell membrane damage and inflammatory responses. These findings support the hypothesis that occupational exposure to PAHs not only increases the risk of carcinogenesis but also enhances oxidative stress, contributing to other health outcomes such as cardiovascular diseases.
It is important that taxi drivers and similar transportation workers are likely at risk for cumulative PAH exposure over extended periods. The findings support the need for occupational safety policies, such as enhanced ventilation systems in vehicles, the use of high-efficiency air filters, and stricter emission control regulations in urban areas.
Correlation between substances
Spearman correlation analyses were performed to determine substance-specific correlations between urinary OH-PAHs, oxidative damage markers, and smoking markers. OH-PAHs showed statistically significant positive correlations with correlation coefficients ranging from 0.745 (1-OH-Na vs 2-OH-Na) to 0.770 (1-OHP vs 3-OH-Phe) (p < 0.01). The levels of oxidative damage markers MDA and 8-OHdG were also significantly positively correlated (r = 0.589, p < 0.01), and significant positive correlations were also found between MDA and the five OH-PAHs and 8-OHdG and the five OH-PAHs (p < 0.01). Cotinine showed significant positive correlations with four OH-PAHs except 1-OH-Na. However, there was no significant correlation between cotinine and oxidative damage markers (MDA and 8-OHdG) (Table S9).
Due to the small number of subjects in this study, a chi-square test was performed based on the median (Table S10). Taxi drivers and controls showed significant differences in exposure to all OH-PAHs, oxidative damage markers, and cotinine except 1-OH-Na. Exposure was higher in men than women, and higher in those with higher BMI. Smoking status was associated with significant differences in cotinine concentrations for all OH-PAHs except 1-OH-Na, but not for oxidative damage markers. Exposure to 1-OHP was higher among those wearing masks, likely due to taxi drivers working more than 12 hours with masks. For 3-OH-Phe, the closer the residence was to the road, the higher the exposure. These results are consistent with previous studies that have shown significant differences in the levels of 2-OH-Na and 1-OHP in KoNEHS urine by gender, age, smoking, alcohol consumption, and distance from neighboring roads [33].
We have conducted additional stratified analysis by smoking status which was classified into two groups based on urinary cotinine levels (i.e., ≤1.53 vs. >1.53 ng/mL) rather than just answer to the questionnaire. We observed basically similar, although the difference became smaller, pattern of results among non-smoking subjects with low urinary cotinine concentration as all subjects including smokers (Table S9).
Discussion
The results of this study provide compelling evidence that taxi drivers, who spend prolonged periods in high-traffic urban environments, experience elevated exposure to polycyclic aromatic hydrocarbons (PAHs). Higher concentrations of OH-PAHs in the urine of taxi drivers compared to controls, particularly female controls, suggest that vehicular emissions are a significant source of PAH exposure for this occupational group. These findings align with previous studies that have reported higher levels of 1-OHP and other PAH metabolites in transportation workers, including bus drivers and truck drivers [24, 30].
Previous studies have reported urinary PAH metabolite levels in taxi drivers from various countries, including Italy [29] and Taiwan [31], using similar biomonitoring approaches. However, our study is the first to investigate PAH exposure in Korean taxi drivers and to include a detailed analysis of oxidative stress biomarkers (MDA and 8-OHdG) alongside PAH metabolites. This integrative approach allows for a more comprehensive evaluation of the potential health impacts, distinguishing our findings from those of previous research.
The median concentrations of 2-OH-Na and 1-OH-Na in taxi drivers (3.440 ng/mL and 1.770 ng/mL, respectively) were significantly higher than those observed in male and female controls. These elevated levels suggest greater exposure to lower molecular weight PAHs, which are primarily derived from vehicle emissions and traffic-related pollutants. Although low molecular weight PAHs are less carcinogenic than high molecular weight PAHs, their high urinary excretion indicates ongoing exposure and potential cumulative effects, particularly in occupational settings.
Although many other factors other than consumption of baked food, delivery food, and alcohol were evaluated for the association with PAH metabolites, no apparent association was observed for them. The other factors include a number of variables related with characteristics of the residence such as traffic, type of house, and facility near house, and characteristics of public transportation.
The significantly elevated levels of oxidative stress markers—malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)—in taxi drivers indicate that exposure to PAHs is contributing to increased oxidative damage. This oxidative stress may enhance the risk of several chronic diseases, including cardiovascular disease and cancer, as oxidative damage to lipids, proteins, and DNA has been implicated in the pathogenesis of these conditions(EAHT_OH-PAHs_Taxi_241008).
Furthermore, our study found a strong positive correlation between urinary OH-PAHs and oxidative stress markers. These correlations suggest that exposure to PAHs, particularly in occupational settings, can induce significant biological stress, which may contribute to adverse health outcomes. Interestingly, smoking was identified as a major confounder, with cotinine levels strongly correlated with several OH-PAHs. However, no significant correlation was observed between cotinine and oxidative damage markers, indicating that while smoking increases PAH exposure, the additional oxidative stress may be more directly related to occupational exposure.
Comparison with Previous Studies
Several previous studies have investigated PAH exposure in various occupational groups. For example, a study of bus drivers in Denmark found elevated levels of 1-OHP compared to non-transportation workers, similar to our findings in taxi drivers [30]. Another study of heavy vehicle drivers in Taiwan reported higher urinary levels of PAH metabolites among drivers who smoked, reinforcing the findings of this study [24].
A study by Chuang et al. (2003) [34] found that 8-OHdG is generally elevated in smokers, with drivers having higher levels than non-drivers. Bortey-Sam et al. (2017) [12] showed that urinary MDA and 8-OHdG concentrations were higher in men than in women, and this difference may be influenced by several factors, including lifestyle and genetic factors that affect metabolic processes.
We also compared our findings with reference values from previous epidemiological studies. The urinary 8-OHdG levels in this study were comparable to those reported in other high-exposure occupational groups (e.g., asphalt workers and coke oven workers), indicating that taxi drivers may have similar levels of oxidative stress and associated health risks.
However, unlike some studies that reported significant differences between smokers and non-smokers in terms of oxidative stress markers, our study did not find a direct association between cotinine and markers such as MDA and 8-OHdG. This may be due to the mandatory mask-wearing implemented during the COVID-19 pandemic, which could have mitigated some of the air pollution exposure for both smokers and non-smokers.
Limitations
This study has several limitations that should be considered when interpreting the results. First, the relatively small sample size, particularly in the taxi driver group, may limit the generalizability of the findings. Additionally, the control group included both males and females, while the taxi driver group was exclusively male, potentially introducing gender-related bias into the results. Further research with larger, gender-balanced sample sizes is needed to confirm these findings. Another limitation is the potential for selection bias, as participation in the study was voluntary. Individuals with existing health concerns may have been more likely to participate, potentially skewing the results.
Conclusions
This study provides valuable insights into the occupational exposure of taxi drivers to polycyclic aromatic hydrocarbons (PAHs) and its association with oxidative stress. Taxi drivers were found to have significantly higher levels of urinary OH-PAHs and oxidative stress markers compared to controls, particularly female controls. The findings highlight the importance of occupational safety measures, such as enhanced ventilation systems in vehicles, improved air quality monitoring, and the use of high-efficiency air filters, to reduce exposure to PAHs. Further studies are warranted to explore the long-term health implications of chronic PAH exposure in transportation workers and to develop targeted interventions to mitigate these risks. The reason for the sex imbalance among the subjects of this study (i.e., taxi drivers and controls) may be due to the fact that almost all taxi drivers in Korea are men. Therefore, Given that our study has limitations in terms of sample size, sex imbalance in subject selection, and multiple analysis with adjustment for comprehensive potential confounding factors, further studies with more sound study design may be warranted to elucidate the extent of occupational exposure to PAHs among taxi drivers and its association with adverse health effects.
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