Toxicology Letters 123 (2001) 115–124 www.elsevier.com/locate/toxlet Effects of genetic polymorphisms of CYP1A1, CYP2E1, GSTM1, and GSTT1 on the urinary levels of 1-hydroxypyrene and 2-naphthol in aircraft maintenance workers Chae-Yong Lee a, Jong-Young Lee a, Jong-Won Kang b, Heon Kim b,* a Department of Pre6enti6e Medicine, School of Medicine, Kyungbuk National Uni6ersity, 101 Dongin-dong 2 Ga, Jung-gu, Taegu 700-422, South Korea b Department of Pre6enti6e Medicine, College of Medicine, Chungbuk National Uni6ersity, 48 San Kaeshin-dong, Hungdok-gu, Cheongju-si, Chungbuk 361-763, South Korea Received 8 January 2001; received in revised form 17 May 2001; accepted 18 May 2001 Abstract This study was undertaken to investigate the effects of genetic polymorphisms of the cytochrome P450 1A1 (CYP1A1) and 2E1 (CYP2E1), and glutathione S-transferases mu (GSTM1) and theta (GSTT1) on urinary 1-hydroxypyrene and 2-naphthol levels, and to estimate the level of exposure to polycyclic aromatic hydrocarbons (PAHs) in aircraft maintenance workers. In 218 Korean aircraft maintenance workers, the geometric means of urinary 1-hydroxypyrene and 2-naphthol were 0.32 and 3.25 mmol/mol creatinine, respectively. These urinary concentrations were approximately at the upper limit of the general population. Mean urinary 2-naphthol concentrations were significantly different between smokers and non-smokers. CYP1A1 and GSTM1 were statistically significant in analyses on both 1-hydroxypyrene and 2-naphthol levels among smokers. The results suggest that smoking has more profound effects on urinary PAH metabolites than does genetic polymorphisms in this population, and that CYP1A1 and GSTM1 activity might be related to the metabolism of 1-hydroxypyrene and 2-naphthol. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: 1-Hydroxypyrene; 2-Naphthol; Polycyclic aromatic hydrocarbons; Aircraft maintenance workers; Cytochrome P450; Glutathione S-transferase 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are natural components of most fossil fuels. They are * Corresponding author. Tel.: +82-43-261-2864; fax: +82- 43-274-2965. formed by pyrolysis, incomplete combustion, or E-mail address: [email protected] (H. high-temperature processing of organic materials Kim). such as crude oil, coal, coke, or other industrial 0378-4274/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-4274(01)00374-5 116 C.-Y. Lee et al. / Toxicology Letters 123 (2001) 115–124 carbon compounds (Harrison, 1997; Schwarz- P-450 (CYP), glutathione S-transferases (GSTs) Miller et al., 1998). Epidemiological studies have and urinary concentrations of the PAH metabo- shown an increased incidence of cancer among lites (Øvrebø et al., 1998; Merlo et al., 1998; workers exposed to PAHs (Mastrangelo et al., Poirier et al., 1998; Yang et al., 1999). 1996). Concentrations of urinary PAH metabolites are Aircraft maintenance workers are exposed to jet also influenced by behavioral factors, such as fuel and jet engine exhausts. Jet and diesel fuels smoking and consumption of foods containing are composed of hydrocarbons from the middle PAHs (Van Rooij et al., 1994; Merlo et al., 1998; distillate fraction in the kerosene range, and the Yang et al., 1999). When occupational exposure toxicity of both resembles that of kerosene decreases, other sources of PAHs, such as food, (Cavender, 1996). Although jet fuel (kerosene) tobacco smoking, environmental air pollution and differs from motor fuel (petrol and diesel), exhaust drinking water, become relatively more important emissions from the jet engines are essentially simi- (Hemminki et al., 1997). In the present study, lar to those of internal combustion engines lifestyle factors, including smoking and consump- (Makker and Ayres, 1999; Tunnicliffe et al., 1999). tion of grilled foods, were also considered. Studies on workers exposed to diesel exhaust and The aim of this study was to investigate the on engine repair workers have shown higher levels effects of genetic polymorphisms of CYP1A1, of 1-hydroxypyrene (1-OHP) than in controls CYP2E1, GSTM1 and GSTT1, and lifestyle fac- (Granella and Clonfero, 1993; Nielsen et al., 1996; tors on the levels of urinary 1-OHP and 2-NAP, Karahalil et al., 1998). These reports and a study and to estimate the levels of exposure to PAHs in reporting a higher urinary 1-OHP concentration in aircraft maintenance workers. nautical engine room workers (Moen et al., 1996) lead to the supposition that aircraft maintenance workers are exposed to significant amounts of 2. Materials and methods PAHs. However, data on PAH exposure in air- craft maintenance workers are scarce. 2.1. Study subjects and sample collection As the metabolism of PAHs is a complex pro- cess with high individual variation, markers re- The subjects were 218 male aircraft maintenance lated to exposure and metabolic capacity are workers who worked at an air base in Korea. A useful in assessment of exposure and risk (Hem- self-completed questionnaire was used to obtain minki et al., 1997). Assessment of urinary 1-OHP information about age, work duration, depart- levels may be a useful and cost effective indicator ment of workplace, amounts and frequencies of of exposure to PAHs (Jongeneelen, 1997), and cigarette smoking, duration of smoking, and urinary 2-naphthol (2-NAP) concentrations have amounts and frequencies of consumption of been suggested to be a good route-specific grilled meat and fish. biomarker for airborne PAHs (Jansen et al., 1995; Peripheral blood was collected for analysis of Yang et al., 1999; Kim et al., 2001). the genetic polymorphisms of metabolic enzymes, Inter-individual differences in the activity of and urine samples were taken for the analysis of enzymes involved in the metabolism and detoxifi- metabolites of PAHs. The specimens were kept at cation of PAHs may cause substantial variability −20 °C until analyzed. in the biological response to the chemicals. Ge- netic polymorphisms of the enzymes have been 2.2. Analysis of urinary 1-hydroxypyrene and suggested to explain inter-individual differences in 2-naphthol the rate of metabolism and activation/deactivation of PAH-derived carcinogens (Alexandrie et al., Urinary 1-OHP and 2-NAP were determined 2000). There have been some recent reports on the using a high performance liquid chromatography relationship between genetic polymorphisms of system. Urinary 1-OHP was analyzed according to bio-transformation enzymes, such as cytochrome the method developed by Jongeneelen et al. C.-Y. Lee et al. / Toxicology Letters 123 (2001) 115–124 117 (1987), and 2-NAP was analyzed according to the concentrations were log-normally distributed, geo- method of Kim et al. (1999). 1-Hydroxypyrene and metric means and geometric standard deviations b-glucuronidase with sulfatase activity were pur- were presented. Statistical methods included Stu- chased from Sigma (St. Louis, MO), and 2-NAP dent’s t test and Pearson correlation analysis for the was from Aldrich (Germany). The HPLC system continuous variables and Spearman correlation used was a Shimadzu (Kyoto, Japan) model LC- analysis for the variables measured in ordinal scale. 19AD, consisting of a pump (SPD-M10A), an After stratification of smoking status, a multiple automatic injector (SIL-10A), a fluorescence detec- regression model was estimated for each stratum. tor (RF-10AXL), and a CBM-10A Chromatopac In multiple regression models for non-smokers, the integrator. The columns used were a 150 mm genotypes of CYP1A1, CYP2E1, GSTM1, and reverse-phase column (TSK-GEL ODS-80TM, GSTT1 were included, and in the models for TOSOH, Tokyo, Japan) for 1-OHP and a 250 mm smokers, average smoking amounts and number of reverse-phase column (Shim-pack CLC-ODS(M), cigarettes on the day of sampling were added to Shimadzu) for 2-NAP. The mobile phases used those four genotypes. were acetonitrile:water (56:44) for 1-OHP and acetonitrile:water (40:60) for 2-NAP, at a flow-rate of 1 ml/min. The retention times were 13.8 min for 3. Results 1-OHP and 16.9 min for 2-NAP. The excitation/ emission wavelengths for 1-OHP were 242/388 nm, 3.1. Characteristics of subjects and those for 2-NAP were 227/355 nm. Results are expressed as means9SDs. The mean 2.3. Determination of the genotypes age of the subjects was 32.17910.3 years, and the mean work duration was 11.2999.6 years. The Genomic DNA was isolated from blood samples numbers of workers in the base department and in using a DNA purification kit (Promega, Madison, the line maintenance department were 141 (64.4%) WI). The A4889G polymorphism in exon 7 of and 78 (35.6%), respectively. Characteristics of CYP1A1 that results in an Ile (CYP1A1*1A) to Val their smoking and consumption of grilled food are (CYP1A1*2C) amino acid replacement at residue shown in Table 1. 462 was determined by the PCR-restriction frag- ment length polymorphism method described by 3.2. Urinary concentration of PAH metabolites Oyama et al. (1995). Genotypes of CYP1A1 were classified into the *1A/*1A, *1A/*2C, and *2C/ There were no differences in the urinary 1-OHP *2C. Genotypes of CYP2E1 were classified into the and 2-NAP concentrations between workers in the predominant homozygote alleles (*1A/*1A), the base maintenance department and in the line heterozygote alleles (*1A/*5B), and the rare ho- maintenance department. Smoking was an impor- mozygote alleles (*5B/*5B) (Kawamoto et al., tant determinant of urinary concentrations of PAH 1995). A multiplex polymerase chain reaction metabolites. The urinary 2-NAP concentration of (PCR) method was used to detect the presence or smokers was significantly higher than that of non- absence of the GSTM1 and GSTT1 genes in smokers (Table 2). genomic DNA samples (Chen et al., 1996). PCR Table 2 shows the urinary concentrations of amplification for both GSTM1 and GSTT1 was 1-OHP and 2-NAP from this study and some carried out using b-globin as an amplification previous studies.