This study was conducted after review and approval by the Chungbuk National University Bioethics Committee (IRB). The study subjects were 65 Cheongju residents who were recruited from a group expected to be occupationally exposed to DEP. Among them, 16 were Yakurt delivery and sales females, 8 were bus terminal workers, 37 were street sweepers, and 4 were office workers. They signed the consent form after being informed of the study in detail and provided blood and 24 h urine samples.

A direct interview was conducted using a questionnaire that included questions on demographic characteristics, working environments, dietary patterns, and diesel car use.

The subjects were divided into two groups. Those with 1-NP exposure above average were classified into the high exposure group, and those with below average exposure were classified into the low exposure group.

Air was sampled at a flow rate of 3 L/min for 24 h with a personal air sampler (Apex standard, SN0376420 Casella CEL, Bedford, England) with a filter holder attached to the collars of the subjects. PTFE filters with a pore size of 2 µm were used. The filters were placed in a flask and mixed with dichloromethane (2 mL). The flask was shaken and treated with ultrasonic waves to extract PAHs and nitro-PAHs. The extracts were evaporated to dryness and the residue was redissolved in acetonitrile. Aliquots of the solution were then injected into a two-dimensional high-performance liquid chromatography (HPLC) system with a fluorescence detector (FD) for the quantification of PAHs and nitro-PAHs [14]. The injected sample was eluted through a clean-up column (Cosmosil, 5NPE, 150 × 4.6 mm i.d., 5 μm, Nacalai Tesque, Kyoto, Japan) with guard column 1 (10 × 4.6 mm i.d.), and nitro-PAHs were reduced to their amino-derivatives using a reduction column (NPpak-RS, 10 × 4.6 mm i.d., JASCO, Tokyo, Japan) at 80 °C. The mobile phase for the clean-up and reduction columns was an ethanol/acetate buffer (pH 5.5) (95/5, v/v) at a flow rate of 0.2 mL/min. A fraction of the amino derivatives and unchanged PAHs eluted from the reduction column with the mobile phase were mixed with 30 mM ascorbic acid at a flow rate of 1.6 mL/min and then trapped on the concentration column (Spheri-5 RP-18, 30 × 4.6 mm i.d. 5 μm, Perkin Elmer, MA, USA). The concentrated fraction was passed through two separation columns (Inertsil ODS-P, 250 × 4.6 mm i.d., 5 μm, GL Sciences, Tokyo, Japan) with a guard column (10 × 4.6 mm i.d.) in tandem. All columns, except the reduction column, were maintained at 20 °C. A programmed gradient elution of the separation columns was performed using 10 mM imidazole buffer (pH 7.6) as eluent A and acetonitrile as eluent B. Finally, the separated analytes were detected with their optimum excitation and emission wavelengths by the dual-channel FD. Eighteen nitro-PAHs, including 1,3-, 1,6-, and 1,8-dinitropyrene (1,3-, 1,6- and 1,8-DNP), 2-nitrofluorene (2-NF), 9-nitrophenathrene (9-NPh), 2- and 9-nitroanthracene (2- and 9-NA), 1-, 2-, and 3-nitrofluoranthrene (1-, 2- and 3-NFR), 1-, 2-, and 4-nitropyrene (1-, 2-, and 4-NP), 7-nitrobenz[a]anthracene (7-NBaA), 6-nitrochrysene (6-NC), 6-nitrobenz[a]pyrene (6-NBaP), 1-nitroperylene (1-NPer), and 3-nitroperylene (3-NPer), were measured as analytes (Fig. S1).

Urinary 1-AP levels were measured from 2 h urine samples. One milliliter of 10 M hydrochloric acid was added to each 10 mL urine sample, and the urine was stirred in a 90-°C water bath for 2 h. After adjusting the pH to 7.0–8.0, the supernatant was extracted with Sep-Pak cartridge (C18, 3 mL, 200 mg, Waters, Milford, UK). Before extraction, it was pre-conditioned with 5 mL of methanol and 5 mL of water. After loading the sample, it was washed with 5 mL of 20% methanol in water and then extracted with 4 mL of 100% methanol. The extract was dried with N2 gas and dissolved in 200 μL of methanol. The extract was analyzed by injecting 20 μL into the HPLC system equipped with a fluorescence detector (Shimadzu, RF-20A, Kyoto, Japan). A reverse phase-amide column (Ascentis RP-Amide, 25 cm × 4.6 mm, 5 μm, SUPELCO, Bellefonte, PA, USA) was used. The mobile phase was composed of methanol and 50 mM sodium acetate buffer pH 7.2 (80:20, v/v) at a flow rate of 1.0 mL/min, and the excitation and emission wavelengths were 254 nm and 425 nm, respectively (Fig. 1). Urinary 1-AP concentration was corrected using the urinary creatinine level.

Chromatograms for 1-aminopyrene (a 1-aminopyrene standard 10 ng/ml, b urine sample)

Statistical analysis was performed using IBM SPSS Statistics 24.0 (IBM Corp., Armonk, NY, USA). The concentrations of nitro-PAH in the air and 1-AP in urine were analyzed for outliers by plotting a scatterplot. To prevent statistical significance distortion by extreme values, the confirmed outliers were replaced with mean + 2 × standard deviation values. The difference between the two groups in the concentration of nitro-PAH in air and the concentration of nitro-PAH metabolites in urine was compared using the Student’s t test or Mann–Whitney test, and the comparison between the three groups was analyzed using one-way ANOVA or the Kruskal–Wallis test. The relationship between urinary 1-AP concentration and the exposure levels of nitro-PAHs, including 1-NP, was statistically tested using Pearson’s correlation analysis and a general linear model. All statistical analyses were determined to be statistically significant at a significance level of < 0.05.