Analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine and DNA strand breaks in white blood cells of occupationally exposed workers: comparison with ambient monitoring, urinary metabolites and enzyme polymorphisms

B. Marczynski1,5, H.-P. Rihs1, B. Rossbach2, J. Hölzer3, J. Angerer2, M. Scherenberg4, G. Hoffmann4, T. Brüning1 and M. Wilhelm1,3

1 Research Institute of Occupational Medicine at the Ruhr-University Bochum, Bürkle-de-la-Camp-Platz 1, 44789 Bochum,
2 Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine of the University Erlangen-Nuremberg, Schillerstr. 25/29, 91054 Erlangen,
3 Department of Hygiene, Social and Environmental Medicine, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum and
4 Arbeitsmedizinischer Dienst der Bau-BG Rheinland und Westfalen, Hofkamp 84, 42095 Wuppertal, Germany

Abstract

The relationship between biomarkers of effect (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo, HPLC system) and tail extent moment (comet assay)), markers of external and internal exposure, and biomarkers of susceptibility was evaluated for coke-oven and graphite-electrode-producing plant workers exposed to polycyclic aromatic hydrocarbons (PAHs). Mean 8-oxodGuo levels in white blood cells (WBC) of exposed workers were between 1.38 times (coke-oven, n = 20; P < 0.01) and 2.15 times (graphite-electrode-producing plant, n = 30; P < 0.01) higher than levels found in control samples (mean ± SD 0.52 ± 0.16 8-oxodGuo/105 dGuo, n = 47). The mean tail extent moment in lymphocytes was 1.38 times higher for coke-oven workers (n = 19; P = 0.09) and 3.13 times higher for graphite-electrode-producing plant workers (n = 29; P < 0.01) when compared with controls (mean ± SD 2.54 ± 0.68, n = 32). Elevated tail extent moments (>3.73) were found in the majority (84%) of PAH-exposed workers showing increased DNA adduct levels (>0.78 8-oxodGuo/105 dGuo). However, no association (P > 0.05) was found between DNA damage (8-oxodGuo/105 dGuo or tail extent moment) in WBC of all PAH-exposed workers and either benzo[a]pyrene levels or the sum of 16 PAH levels in the air at work place. Furthermore, no relation (P > 0.05) could be established between DNA damage in WBC and biomarkers of internal exposure (1-hydroxypyrene (1-OHP) and sum of five hydroxyphenanthrenes (OHPHs)). Higher exposure to airborne pyrene and phenanthrene led to increasing concentrations of the metabolites 1-OHP (P < 0.01) and the sum of five OHPHs (P < 0.01) in the urine of PAH-exposed workers. The polymorphisms of genes CYP1A1, GSTM1, GSTT1 and GSTP1 (biomarkers of susceptibility) showed no association with biomarkers of effect. In conclusion, both biomarkers of effect may be appropriate for further surveillance studies of workers under PAH exposure.

Abbreviations: acn, acetonitrile; AP, apurinic/apyrimidinic; B[a]P, benzo[a] pyrene; BPDE, benzo[a]pyrene diolepoxide; CYP, cytochrome P450; GST, glutathione S-transferase; 1-OHP, 1-hydroxypyrene; OHPH, hydroxyphenanthrene; 8-oxodGuo, 8-oxo-7,8-dihydro-2'-deoxyguanosine; PAH, polycyclic aromatic hydrocarbons; PCR–RFLP, polymerase chain reaction–restriction fragment lengths polymorphism; ROS, reactive oxygen species; rS, Spearman rank correlation coefficient; LOD, limit of detection; SD, standard deviation; WBC, white blood cells; XAD-2, polystyrene/divinyl benzene-based polymer

Introduction

Workers in graphite-electrode manufacturing as well as in coke-oven plants are exposed to polycyclic aromatic hydrocarbons (PAHs) by inhalation of volatile PAHs and PAHs bound to respiratory particulate matter. An additional uptake of PAHs is caused by dermal contact with PAH-containing materials (1). Epidemiological studies have shown an increase in cancer incidence among workers exposed to PAHs, especially for the risk of developing lung, skin, bladder and prostate cancer (2,3). 1-Hydroxypyrene (1-OHP) and monohydroxylated metabolites of phenanthrene have been used as suitable variables for the biological monitoring of exposure to PAHs in various studies (4).

An increasing number of evidences demonstrate the involvement of reactive oxygen species (ROS) in the production of tumors by PAHs (5,6). These oxygen species may lead to the formation of oxidative DNA damage. DNA damage (adducts and strand breaks) represents an early, detectable and critical step in the chemical carcinogenesis process and thus, may serve as an internal dosimeter for carcinogens (for review see ref. 7). The induction of oxidative stress has been suggested as a possible mechanism of non-genotoxic chemical carcinogenesis and has been shown to participate in all stages of the carcinogenesis process, namely initiation, promotion and progression (8).

The spectrum of oxidation products involving DNA includes strand breaks, AP (apurinic/apyrimidinic) sites, and oxidized bases. Within the latter group, emphasis has been focused on 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a major product with a clear mutagenic potential (9). At this stage, there is little information describing the levels of strand breaks and of oxidized bases in DNA from workers exposed to PAHs (10–12).

The question of whether PAH carcinogenesis is modulated by host polymorphisms is currently under extensive investigation. The enzymes of interest in the context of exposure to PAHs are CYP1A1, GSTM1 and GSTP1. They are specially involved in the activation of PAHs to reactive epoxides (CYP1A1) as well as in their inactivation through hydrolysis or conjugation with glutathione (GSTM1/GSTP1).

Monoxygenation (mediated by the NADPH-dependent cytochrome P450 reductase) and one-electron oxidation (possibly mediated by the cytochrome P450 or by peroxidative reactions) are currently considered the two main reactions for the activation of benzo[a]pyrene (B[a]P) and PAH in general. Since one-electron oxidation and radical cation reactions can lead to unstable adducts (which represent the great majority of total DNA adducts in rodents), the mutagenic and carcinogenic role of radical-DNA interactions and DNA depurination is under discussion (last reviewed by Canova et al. (13)). More complex dose–response relationships have been found through parallel detection of oxidative DNA lesions (8-oxodGuo) and of CYP1A-immunopositive protein levels (13).

Glutathione S-transferase (GST) catalyzes the conjugation of diol epoxides, a major pathway of PAH metabolism. GST as well as glutathione (GSH) are therefore important in cellular protection against the below mentioned genotoxic compounds. The efficiency of the detoxification system is determined by different endogenous and exogenous factors among which the GSH level (14,15), and particularly the amount and nature of the GST isoenzymes might play a major role.

Taking into account the present knowledge about PAH metabolism pathways, we performed a cross-sectional study to analyze the levels of 8-oxodGuo adducts and the formation of strand breaks in DNA of white blood cells (WBCs) of workers occupationally exposed to PAHs in a coke-oven and in a graphite-electrode-producing plant. These results were compared with data obtained from ambient (16 PAHs in the air of the workplace) and biological monitoring (urinary metabolites of pyrene 1-OHP and sum of five metabolites of phenanthrene, 1-,2+9-,3-,4-hydroxyphenanthrenes (OHPHs)). Additionally, the influence of genetic polymorphism phase I (CYP1A1) and phase II enzymes (GSTM1, GSTT1, GSTP1), known to be involved in the metabolism of certain PAHs, was examined.

Materials and methods

Study groups
Workers occupationally exposed to PAHs in a coke-oven plant (n = 20) and in a graphite-electrode-producing plant (n = 30) were studied and compared with healthy control subjects (n = 47 for DNA adducts and n = 32 for comet assay). All exposed workers were males with a mean age of 37.4 years (range: 22–58 years). All control subjects were also males (mean age: 38.0 years, range: 23–58 years) and not occupationally exposed to PAHs (Table IGo).


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Table I. Demographic data of the exposed and control groups sampled (all males)
 
Prior to blood sampling, a questionnaire to elicit the workplace description, smoking habits, medical history, age, diet and use of personal protection devices was performed for each exposed worker. Questionnaires were completed in a personal interview with the company physician. Smoking status differed between exposed workers and controls: 40% of the controls were smokers and 60% non-smokers, whereas 68% of the exposed workers were smokers, 18% ex-smokers and 14% non-smokers. All workers used protective gloves and took a shower immediately after work. 95% of the PAH-exposed workers from the coke-oven plant used personal respiratory protection devices, compared with only 14% in the graphite-electrode-producing plant.

The study was approved by the ethical commission of the Ruhr-University of Bochum and was conducted in accordance with the principles for human experience as defined by the Helsinki Declaration.

Determination of PAHs in the air
Personal air sampling in the workers' breathing zone was carried out according to the Method 5506 published by the National Institute for Occupational Safety and Health (NIOSH; ref. 16). Particulate bound PAH and PAH vapors were collected on glass fibre or teflon filters and filled with XAD-2 (a polystyrene/divinyl benzene-based polymer) in sorbent tubes. Until the analysis of the 16 PAHs (namely acenaphthene, acenaphthylene, anthracene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[ghi] perylene, benzo[a]pyrene, chrysene, dibenz[ah]anthracene, fluoranthene, fluorene, indeno[1,2,3-cd]pyrene, naphthalene, phenanthrene, and pyrene) could be performed, the filters and sorbent tubes were stored at –20°C. The filters were then extracted ultrasonically (60 min), mixed with 5 ml acetonitrile and shaken for 30 min. Subsequently, XAD-2 material from sorbent tubes was extracted, mixed with both acetonitrile (2 ml) and dichloromethane (2 ml), and shaken again. The original filter extract and the extracts combined to the XAD-2 material were exposed to a gentle stream of nitrogen to evaporate until dried. The residues were redissolved with actetonitrile, added to a 25 µl aliquot, and then analyzed by high performance liquid chromatography with diode array detection (HPLC/DAD). The HPLC/DAD analysis was carried out by an HPLC-System from Merck (Pump L-6200, autosampler L-7200, column oven L-7350, diode array detector L-7450, computer with D-7000 HPLC-System Manager) using a reversed phase column (LiChrospher PAH 250 x 3 mm) and a water/acetronitrile (w/acn) gradient for separation at 30°C (0.5 min: 60% acn, 5–20 min: 60–100% acn, 20–30 min: 100% acn, flow 0,5 ml/min). To detect the analytes, the wavelengths 250, 266 and 286 nm were monitored. The detection limits for a 2 h sampling period varied between 7 and 513 ng/m3.

Determination of PAH metabolites in urine
The determination of 1-, 2 + 9-, 3-, 4-hydroxyphenanthrenes and 1-hydroxypyrene post-shift urine samples of the workers was carried out using a modified HPLC method developed by Lintelmann and Angerer (17). According to this method, 1 ml urine was diluted with 9 ml water, buffered with 12 ml acetate-buffer (0.1 M, pH 4.9) and hydrolyzed with 80 µl of ß-glucuronidase/arylsulfatase for 16 h at 37°C in a waterbath. In order to separate the solution from particles, it was centrifuged at 3000 r.p.m. for 10 min. From the supernatant 15 ml were transferred into a sampler vial from which 3 ml aliquot was injected into an HPLC system with an autosampler. The HPLC system used is described elsewhere in detail (17). The metabolites were enriched on a special precolumn consisting of copper phthalocyanine modified silica gel, separated on a RP-C18 (LiChrospher PAH 250 x 4 mm) column and quantified by fluorescence detection. The metabolites 2- and 9-hydroxyphenanthrene could not be separated by HPLC. The two co-eluting metabolites were quantified using a calibration curve of 2-hydroxyphenanthrene. The detection limit of the method ranged between 24 and 96 ng metabolite/l urine.

Creatinine measurements
Urinary creatinine was determined photometrically as picrate, according to the Jaffé method (18).

Determination of cotinine in urine
Urinary cotinine was determined by gas chromatography with nitrogen-specific detection after a liquid/liquid extraction of the urine samples, according to the procedure described by Scherer et al. (19).

Alkaline single-cell gel electrophoresis (Comet assay)
Alkaline single-cell gel electrophoresis was used to study DNA strand breaks and alkali-labile sites. The previously published protocol (20,21) has been modified according to Pouget et al. (22) as follows. Heparinized venous blood for the lymphocyte preparation was collected. Lymphocytes were isolated by the standard method of centrifugation on a Ficoll density gradient. 7 ml of whole blood from each subject was diluted 1:1 with a RPMI 1640 solution (pH 7.3) and kept on ice for 15 min. Lymphocytes were separated by centrifugation over 7 ml Lymphoprep at 200 g for 30 min. Buffy coats were removed and washed twice with RPMI 1640. Lymphocytes suspended in the RPMI solution were counted in a hemocytometer and ~2 x 104 cells were used immediately for the comet assay. Cell viability, determined using the trypan blue exclusion technique, was constantly found to be over 96%.

As usual, 100 µl of 1% standard agarose dissolved in PBS buffer was taken and allowed to solidify onto a microscope slide, kept at room temperature in a dry atmosphere. Another 10 µl of the lymphocyte suspension was mixed with 75 µl of 1.2% low melting-point agarose maintained at 37°C. Subsequently, the resulting solution was coated on the first layer after removal of the cover glass. All the subsequent steps were performed under red light to prevent the occurrence of additional DNA damage. The slides were then placed on ice for 15 min to allow the gel to solidify. Cover glasses were removed and the slides were immersed for 80 min at 4°C in a lysis buffer (1% triton X-100, 10% DMSO, 2.5 mM NaCl, 100 mM Na2EDTA, 10 mM Tris, sodium lauroylsarcosinate 10%, pH 10).

To conduct the electrophoresis, cover glasses were removed and the slides were transferred to a horizontal electrophoresis tank and kept covered with an alkaline solution (1 mM Na2EDTA, 300 mM NaOH, pH 13) at 4°C for 40 min. Thereafter, the electrophoresis was performed at 25 V–300 mA for 45 min at 4°C. The slides were then washed three times with 0.4 M of Tris–HCl, pH 7.4, and the nuclei were stained using 45 µl of 0.5 mg/ml ethidium bromide. The slides were placed at 4°C in a humidified air-tight container to prevent from drying. Under these conditions, slides could be kept for several days prior to analysis.

The analysis was performed with a fluorescence microscope (Olympus, BX60F-3, Olympus Optical Tokyo, Japan). Using the computer image analysis software Komet version 3.1 (Kinetic Imaging, Liverpool, UK), each slide (50 cells per slide, using two different slides prepared for one subject) was examined at x20 magnification under a fluorescence microscope equipped with both an excitation filter at 515–560 nm and a barrier filter at 590 nm. Fifty `comets' (100–150 cells/blood sample) were randomly selected from each slide avoiding the edges and damaged parts of the gel as well as the apparently dead cells (comets without a distinct `comet head') and the superimposed comets.

An image analyzing program automatically calculated the total area of each tail, its absolute average intensity, and its distance to the centre position of the head. From these data the program could calculate several indicators of DNA damage, from which we have selected the tail extent moment. The tail extent moment was defined as the percentage of the DNA fraction in the tail and the length of the tail. This parameter was used to estimate the DNA break frequency, as it could express the migration of the various DNA fragments forming the tail and estimates the relative amounts of DNA, as one figure.

Determination of DNA-adducts (8-oxodGuo)
Whole blood samples (9 ml) were collected in EDTA-treated tubes and immediately frozen at –20°C. DNA from white blood cells (WBC) was isolated and frozen at –80°C. DNA extraction and 8-oxodGuo adduct isolation were carried out using the procedure from Marczynski et al. (23), with the modifications described recently (22). The isolated DNA was dissolved in 200 µl of 10 mM sodium acetate, pH 5.0. The following day, the DNA was incubated at 95°C for 5 min and placed on ice for 10 min. 20 µl of 1 mM deferoxamine mesylate was added and the denaturated DNA was digested with 20 µg of nuclease P1 for 30 min at 37°C, followed by 20 µl Tris–HCl, pH 7.5 and by 1.2 U alkaline phosphatase at 37°C for 60 min. The resulting hydrolysates were centrifuged for 30 min using a Microcon YM-3 filter (Millipore, Corporation, Bedford, USA) in order to separate the nucleosides from the enzymes.

For the analysis of nucleosides in WBC DNA, a Shimadzu HPLC/UV apparatus connected to a Coulochem II (model 5200) electrochemical detector (ESA, Chelmsford, MA) was used. The analysis was carried out blind. The presence of 8-oxodGuo adducts in WBC DNA was detected according to the methods proposed by Floyd et al. (24) and Pouget et al. (22). The HPLC (with SIL-10A auto injector and sample cooler), set at a flow rate of 0.8 ml/min, was used to introduce 20 µl of DNA hydrolysate into a column (C18, 4.6 mm in diameter, 250 mm in length; Grom, Herrenberg-Kayh, Germany) in a CTO-10A oven at 37°C. The eluent consisted of 50 mM monosodium phosphate in 8% methanol, pH 5.1. The determination of normal nucleosides was performed at 290 nm with a UV detector (SPD-10A). The oxidation potentials of the analytical cell (model 5011; ESA) of the electrochemical detector (EC) were set at 150 mV and 350 mV for electrodes 1 and 2, respectively; whereas the potential of the guard cell was set at 400 mV. For the recording and integration of the UV and EC responses, the integrator (CR 5A) was used. Analyses were routinely run twice to three times to minimize instrumental errors.

DNA isolation and identification of CYP1A1, GSTM1, GSTT1 and GSTP1 genotypes
DNA was prepared from lymphocytes of frozen whole blood samples using the commercial PUREGENE DNA isolation kit (Biozym, Hessisch-Oldendorf, Germany). The Msp I polymorphism in the 3'-flanking region of the nucleotide 6235 from the CYP1A1 gene was identified by the PCR–RFLP method described by Hayashi et al. (25), but modified according to Wu et al. (26). The Ile462Val polymorphism in exon 7 of the CYP1A1 gene resulting in an Ile à Val change at amino acid residue position 462 was analyzed using the PCR–RFLP method described by Oyama et al. (27).

The genotyping of GSTM1 and GSTT1 was carried out as described by Hirvonen et al. (28), using ß-globin as positive control to verify the presence of amplificable DNA. The analysis of the GSTP1 polymorphism resulting in an IleVal substitution at residue 104 in exon 5 was performed as described by Watson et al. (29), whereas the AlaVal substitution at residue 115 in exon 6 was analyzed by the method of Saarikoski et al. (30).

Statistical analyses
All data were analyzed using the statistical software package SAS v. 8 (SAS Institute, Cary, NC). For our statistical calculations, results lower than the analytical limit of detection (LOD) have been set to half of the detection limit (1/2 LOD).

Data description: Minima, lower and upper quartiles, medians, arithmetic means and maxima of 8-oxodGuo/105 dGuo and tail extent moment in different groups of exposure and controls are presented in box and whisker plots. Scatter diagrams are used to visualize possible associations between 8-oxodGuo/105 dGuo or tail extent moment and 1-hydroxypyrene and hydroxyphenanthrene concentrations in the urine of the exposed workers, B[a]P and sum of EPA-PAHs in the air at the workplace, respectively. Frequency distributions of 8-oxodGuo/105 dGuo and tail extent moment levels were unimodal and skewed to the right. In order to obtain an acceptable fit to the normal distribution, 8-oxodGuo/105 dGuo and tail extent moment were log-transformed.

Data analysis: Differences of log(8-oxodGuo/105 dGuo) and log(tail extent moment) between workers from the different groups (coke-oven, graphite-electrode-producing plant and controls) were tested for statistical significance with an analysis of variance (ANOVA), followed by pairwise multiple comparison tests (Gabriel's procedure).

Non-parametric tests (Wilcoxon–Mann–Whitney, Kruskal–Wallis) were used to evaluate the differences between 8-oxodGuo/105 dGuo and tail extent moment in the subgroups defined by different genotypes. P < 0.05 was set as the criterion for the significance of a test.

Spearman rank correlation coefficients (rS) were calculated to evaluate the closeness of relationship between two continuous variables (8-oxodGuo/105 dGuo or tail extent moment and 1-hydroxypyrene, hydroxyphenanthrenes, B[a]P and other PAHs).

Results

8-OxodGuo in white blood cells
The 8-oxodGuo/105 dGuo ratio in white blood cells was significantly higher in each PAH-exposed group than in unexposed controls (coke-oven plant: mean ± SD 0.72 ± 0.13, median 0.69, n = 20, P < 0.01; workers in a graphite-electrode-producing plant: mean ± SD 1.12 ± 0.29, median 1.08, n = 30, P < 0.01; controls: mean ± SD 0.52 ± 0.17, median 0.50, n = 47). The results are shown as box and whisker-plots (Figure 1Go).



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Fig. 1. 8-oxodGuo adduct level in white blood cell DNA of PAH-exposed workers and controls (#P < 0.01, significantly different from controls).

 
DNA strand breaks in lymphocytes
The frequency of DNA strand breaks was measured as tail extent moment. The mean tail extent moment in lymphocytes of the graphite-electrode-producing plant workers (n = 29) was 7.95 ± 3.34 (median 7.99; P < 0.01), in comparison with 2.54 ± 0.68 (median 2.50) for the controls (n = 32). The corresponding value for the coke-oven workers (n = 19) was 3.50 ± 1.72 (median 2.83), which revealed to be not significant (P = 0.09, Figure 2Go) compared with the control group.



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Fig. 2. Tail extent moment in the lymphocyte DNA of PAH-exposed workers and controls (#P < 0.01, significantly different from controls).

 
Association between 8-oxodGuo adducts and DNA strand breaks
Figure 3Go shows the scatterplot of 8-oxodGuo/105 dGuo and tail extent moments for all subjects. For the majority (84%) of subjects with increased DNA adduct levels (i.e. levels >0.78 8-oxodG/105 dGuo; above 95th percentile, referring to the control group), elevated tail extent moments >3.73 (above 95th percentile, referring to the control group) were found. The Spearman rank correlation coefficient (rS) attributed to 8-oxodGuo/105 dGuo and tail extent moment was 0.64 (P < 0.01) for the whole study group (n = 72). This significant inter-group correlation is due to parallel increases from 8-oxodGuo/105 dGuo values and from tail extent moments occurring among the groups. However, the within-group correlation coefficients did not show such a tendency: coke-oven workers rS = –0.15, P = 0.55 (n = 19); graphite-electrode-producing plant workers rS = 0.15, P = 0.45 (n = 29) and control group: rS = –0.51, P = 0.01 (n = 24).



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Fig. 3. Tail extent moment in the lymphocyte DNA in relation to 8-oxodGuo/105 dGuo in white blood cell DNA of PAH-exposed workers and controls (rS = 0.64, P < 0.01).

 
Effect markers in relation to smoking and to age
Influence of smoking was assessed by medical history and by measurement of cotinine in the urine of the PAH exposed workers. Rank correlation calculations revealed no statistically significant relation between urinary cotinine levels (µg/g creatinine) and the production of 8-oxodGuo/105 dGuo adducts (rS = 0.01, P = 0.95, n = 50) nor to DNA strand breaks (tail extent moment, rS = –0.07, P = 0.64, n = 48).

Proportion of smokers and non-smokers differed between PAH-exposed workers and the control group (Table IGo). Taking into consideration the suspected role of smoking as a confounding factor, correlation coefficients for 8-oxodGuo adduct levels and DNA strand breaks between PAH-exposed groups and controls were separately calculated, with restriction to smokers (coke-oven plant: 15 smokers, graphite-electrode-producing plant: 19 smokers, controls: 22 smokers). The 8-oxodG/105 dGuo ratio and tail extent moment were significantly (P < 0.05) higher for smokers working in the graphite-electrode-producing plant and in the coke-oven plant than for the smokers of the control group.

For the comet assay, the frequency of DNA strand breaks can be expressed in several ways. Commonly used parameters are tail extent moment and Olive tail moment (product of the amount of DNA in the tail and the mean distance of migration in the tail). In our study, both appeared strongly related (Spearman correlation coefficient rS = 0.956, P < 0.01, n =80, data not shown). In fact, no clear increase of tail extent moment with age could be detected, neither in PAH-exposed workers (rS = 0.083, P = 0.58, n = 47) nor in controls (rS =–0.137, P = 0.45, n = 32). The same applied to 8-oxodGuo/105 dGuo (exposed: rS = 0.193, P = 0.18, n =49; controls: rS = 0.247, P = 0.09, n = 47).

Comparison of ambient monitoring with biological monitoring
Personal monitoring devices were used to evaluate the ambient exposure of the workers to 16 PAHs, including B[a]P, during an 8 h working shift. The B[a]P and sum of 16 PAHs concentrations in the air at the workplace of coke-oven workers ranged from 0.12 (1/2 LOD) to 16.26 (mean: 2.77) µg/m3 and from 4.51 to 316.45 (mean: 54.26) µg/m3 respectively. Corresponding ranges among graphite-electrode-producing plant workers were 0.02 (1/2 LOD) to 46.22 (mean: 2.77) µg/m3 for B[a]P and 0.97 to 1848.37 (mean: 143.08) µg/m3 for the sum of 16 PAHs. Increasing exposure of pyrene and phenanthrene in the ambient air at the workplace was accompanied by rising concentrations of 1-OHP and OHPHs in the urine of the workers (pyrene/1-OHP rS = 0.47; P < 0.01, n = 39; phenanthrene/OHPHs rS = 0.65; P < 0.01, n = 39) (data not shown).

Correlation between markers of effect and markers of external exposure
Rank correlation calculations did not reveal any obvious association between airborne B[a]P concentrations and DNA adduct levels (rS = –0.146, P = 0.38; n = 39) or with the formation of DNA strand breaks (rS = –0.293; P = 0.08, n = 37). The same applies to the concentration of the sum of PAHs in the air at the workplace (DNA adduct levels: rS =0.102, P = 0.54, n = 39, tail extent moment: rS = –0.104; P = 0.54, n = 37) (data not shown).

Urinary levels of PAH-metabolites
Higher concentrations of 1-OHP and the sum of five OHPHs were found in the urine of the workers in the graphite-electrode-producing plant in comparison with workers in the coke-oven plant (1-OHP: P < 0.01, sum of hydroxyphenanthrenes: P = 0.37, boxplots shown in Figure 4Go).



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Fig. 4. Concentrations of PAH-metabolites in urine of PAH-exposed workers. Boxplots: (A) 1-hydroxypyrene, P < 0.01; (B) sum of hydroxyphenanthrenes, P = 0.37. In comparison, 1-OHP-levels in urine of unexposed adults in Germany are usually <0.5 µg/g creatinine (4).

 
Correlation between markers of effect and biomarkers of internal exposure
Referring to the biological monitoring performed in this study, no clear association could be found between the concentrations of 1-OHP in urine and DNA adduct levels (rS = 0.200, P =0.17, n = 50) and with the amount of DNA strand breaks (rS = 0.054; P = 0.72, n = 48). Concentrations of the sum of five OHPHs in urine were not related to DNA adduct levels (rS = –0.002, P = 0.99, n = 50) nor to the production of DNA strand breaks (rS = –0.153; P = 0.30, n = 48) (data not shown).

Correlation coefficients between effect markers and ambient and biomonitoring data
Spearman rank correlation coefficients were calculated for 8-oxodGuo and tail extent moments respectively, in relation to ambient and biomonitoring data. The results are presented in Figure 5Go. The correlation coefficients appeared significant only for fluorene (8-oxodGuo/105 dGuo: rS = 0.44, P < 0.01) and acenaphthylene (8-oxodGuo/105 dGuo: rS = –0.56; tail extent moment: rS = –0.56, P < 0.01).



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Fig. 5. Spearman correlation coefficients between 8-oxodGuo adduct levels and tail extent moment, ambient and biomonitoring results (biomonitoring data are based on creatinine concentrations in urine). Results lower than the analytical LOD have been set to half of the detection limit (1/2 LOD) for calculation of the correlation coefficients. The number of values below the detection limit is shown in parentheses after behind the respective label.

 
Biomarkers of susceptibility
DNA damage parameters determined by 8-oxodGuo adducts and tail extent moments (Table IIGo) showed no significant associations with the absence of GSTM1 (GSTM1*0) and GSTT1 (GSTT1*0) as well as with the amino acid residues changes in exon 5 and 6 of the GSTP1 gene. The same applies to CYP1A1 gene, although the sole sample with the CYP1A1*1/*2B variant showed an increased value of 10.22 for tail extent moment. The corresponding DNA adduct value was 0.90 8-oxodGuo/105 dGuo.


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Table II. Comparison of DNA damage, determined by measurement of 8-oxodGuo and of DNA strand breaks and alkali-labile sites (tail extent moment), with different enzyme polymorphisms
 
Discussion

The purpose of the present investigation was to determine the degree of PAH-related oxidative DNA damage and DNA strand breaks (markers of effect) in WBC of occupationally exposed workers and to see whether an association exists between DNA damage, on the one hand, and ambient monitoring, biological monitoring (markers of exposure) and genetic polymorphisms of biotransformation enzymes (markers of susceptibility), on the other hand.

In WBC DNA of PAH-exposed workers, clearly elevated 8-oxodGuo adduct levels were found together with increased DNA strand break frequency (Figure 3Go). Up to now, the measurement of 8-oxodGuo in WBCs has never been used as a biomarker for occupational PAH exposure. The comet assay has only been employed in a few studies assessing genotoxic effects of PAH-exposed workers (10–12).

Our results are in line with the ones obtained by Popp et al. (11), who found significantly more DNA strand breaks (alkaline filter elution assay) in coke-oven workers than in controls. However, in their study, the DNA adduct rate (32P-postlabeling assay) was not significantly increased. Carstensen et al. (12) did not observe any significant difference between potroom workers and referents, with respect to comet parameters (tail inertia).

In all PAH-exposed workers with elevated DNA adduct levels (i.e. levels >1.08 8-oxodGuo/105 dGuo; n = 15), increased DNA strand breaks (tail extent moment >3.5, n ={uparrow}15) were also observed (Figure 3Go), whereby the relationship between DNA adduct levels and strand breaks differed among the two exposure groups (Figure 3Go).

The results based on alkaline single-cell gel electrophoresis include DNA strand breaks, but also base modifications (31), as the oxidized purine bases (8-oxodGuo and others) and pyrimidine bases could be converted into additional DNA single-strand breaks (32). The higher content of 8-oxodGuo in the cells was expected to lead to a higher formation of DNA strand breaks, although the steady-state level of damage could have been modulated by DNA repair. On a steady-state level, the contribution of repair through enzyme-mediated DNA cleavage at the site of oxidized bases is very little with respect to the overall formation of DNA strand breaks (22,33–35). The level of ·OH-induced base damage stays in the same range as the extent of radical reactions leading to DNA strand cleavage (35–37). However, the origin of the direct strand breaks and alkali-labile sites that may include modified sugar and base residues is difficult to establish using the alkaline comet assay and obviously, this strongly depends on the DNA modifying agent.

A computation of rank correlation coefficients revealed no significant association between 8-oxodGuo and tail extent moment in workers of the coke-oven and the graphite-electrode-producing plant. The data of this study suggest that the workers in the coke-oven and graphite-electrode-producing plant are exposed to DNA modifying agents (presumably a mixture of various compounds), which lead rather independently to DNA strand breaks and to the formation of 8-oxodGuo adducts. The formation of DNA double strand breaks does not seem to be very much influenced by base excision repair; otherwise a positive association of 8-oxodGuo and tail extent moment would have been expected. However, this hypothesis has to be proven by further investigations conducted in larger populations of PAH-exposed workers. There is also no clear explanation for the significant negative relationship we observed between 8-oxodGuo and tail extent moment among controls.

Smoking is assumed to influence both the formation of DNA strand breaks (38) and the level of oxidative adducts (8-oxodGuo) (39). Therefore, distinct analyses in smokers and non-smokers were performed. No significant correlation was observed between urinary cotinine on the one hand, and DNA adducts and tail extent moment on the other hand (Table IGo). Neither smoking habits nor age affected significantly the frequencies of DNA strand breaks and the 8-oxodGuo adduct levels (Table IGo).

Positive correlations between the concentrations of pyrene and phenanthrene in the air at the workplace and of their metabolites 1-OHP and OHPH respectively, in urine of PAH-exposed workers were observed. There was no obvious association between the increased oxidative DNA damage and the concentrations of B[a]P as well as the sum of PAHs in the air at the workplace. Furthermore, we did not find a correlation between oxidative DNA damage and the concentration of 1-OHP and the sum of five OHPHs in urine.

One reason for the absence of significant correlations in these cases could be the fact that the compared markers of exposure and the markers of effect are parameters of different time slots. Since air sampling displayed the exposure within the sampling period, the biological monitoring of urinary metabolites mirrors the exposure during the last workshift (and some days before). In contrast, biomarkers of effect represent a much longer period of exposure. An analysis of the results from ambient and biological monitoring, not conducted on an individual but on a collective basis, should provide for each plant typical mean values responsible for external and internal exposure. These mean values should not be subjected to larger day to day fluctuations. In that way, a comparison between the long-term biomarkers of effect and the short term markers of exposure should be more likely.

In our study, we observed higher levels of DNA damage (biomarkers of effect) in the graphite-electrode-producing plant workers group (Figures 1 and 2GoGo), for which we also obtained the highest urinary concentrations of 1-OHP and sum of hydroxyphenanthrenes (biomarkers of exposure, Figure 4Go). Taking into consideration that the dermal uptake of PAH by coke-oven workers may also play an important role (40), exposure data obtained by personal air sampling should be deemed to be a crude estimate of exposure. Differences concerning the wearing of protection devices, especially clothes and filters, are also factors influencing the degree of individual exposure.

Detailed comparison of the levels of both biomarkers of effect with the concentration of every 16 different PAH compounds in the air at the workplace revealed that only fluorene was significantly associated with an increase of the 8-oxodGuo levels. In contrast, acenaphthylene was associated with a decrease in the formation of 8-oxodGuo adducts and DNA strand breaks (Figure 5Go). As only seven from the 39 concentration values were above the detection limit, these observations must be interpreted with caution. Until now, there is no rationale to support such observations. However, acenaphthylene can function as an aromatic hydrocarbon-responsive receptor (AHR)-independent inducer of the cytochrome P450 (CYP) 1A2 and 1B1, as previously observed in knock-out mice liver (41).

CYP1A1 metabolizes certain PAHs whereas its two polymorphisms (identified in this study) are thought to be associated with large inter-individual differences in the arylhydrocarbon hydrolase activity (reviewed by Autrup (42)). Among workers who are highly exposed to PAHs in the air, a higher level of urinary 1-OHP was observed (43) (in individuals identified as homozygous for the MspI allele *2A/*2A, a rare variant not present in this study group). This combination more than *1/*2A followed by *1/*2B was also associated with signficantly higher levels of benzo[a]pyrene diolepoxide (BPDE)-DNA adducts in lung and blood cells of either smokers (43) or foundry workers (44). Furthermore, the presence of at least one CYP1A1 MspI variant allele (*1/*2A) was reported to be associated with an increased risk of squamous cell carcinoma of the lung (45). Regarding the DNA damage determined by 8-oxodGuo measurements as well as by comet assay for *1/*2A variants in comparison with the *1/*1 variants, no significant differences in both PAH-exposed groups were found. Only the CYP1A1*1/*2B variant in both PAH-exposed groups, carrying additionally the exon 7 mutation, displayed an increased value in the comet assay (10.22) as well as an increased DNA adduct level (0.90 8-oxodGuo/105 dGuo) (Table IIGo). Additionally, in five workers with GSTM1, CYP1A1 (*1/*1), GSTP1 (exon 5 I/I), GSTP1 (exon 6 A/A) and GSTT 1/*1, respectively, the highest median values of 13.22 (tail extent moment) and of 1.14 8-oxodGuo/105 dGuo have been found.

It is well known that the GST isoform GSTM1 is involved in the detoxification of BPDE, a carcinogenic compound of the PAH metabolism. In our study (Table IIGo), no evidence for the influence of GSTM1*0 on the DNA damage determined by comet assay or 8-oxodGuo/105 dGuo measurement was observed. Additionally, neither in coke-oven workers nor in graphite-electrode plant workers a significant association between polymorphisms of GSTP1 (the most frequent GST isoform in the human lung) and the frequency of DNA damage could be found.

Binkova et al. (10) showed an association between the ambient polyaromatic hydrocarbon concentration and the DNA damage determined by comet assay. This association appeared to be influenced by the phase II metabolizing enzyme GSTM1, since the absence of GSTM1 (GSTM1*0) was linked to the highest level of DNA damage. The effect of air pollution was later questioned in a further study conducted on a larger sample of 542 people. In that study, Sram et al. (46) reported that people from a region with a low level of pollution and people from a highly industrialized district had similar levels of DNA damage, even once the statistical analysis was adjusted for GSTM1 polymorphism, smoking, and ethnic background. Carstensen et al. (12) showed that 79 subjects with a GSTM1 null genotype had a higher concentration of 8-oxodGuo in urine than the 61 subjects with at least one copy of the GSTM1 gene. However, whether the levels of 8-oxodGuo in urine are directly comparable with those in WBC DNA is still a matter of debate.

The results we obtained indicate that PAH exposure creates oxidative DNA damage, as measured by the production of one type of oxidative base modification, the 8-oxodGuo, and the production of DNA strand breaks and alkali-labile sites. The oxidative DNA damage resulting from oxidative stress is the product of hydroxyl radical attacks on DNA produced through the Fenton reaction of H2O2 with traces of reduced transition metals (such as Fe2+ and Cu+) bound to DNA (47). H2O2 is the only ROS that can easily reach the nuclear DNA since it is able to cross the plasma and the nuclear membranes. Several carcinogenic PAHs (such as B[a]P) are involved in the dose- and time-dependent production of H2O2 (6). This oxidant is closely associated with tumor promotion (6,48).

The presence of 8-oxodGuo reveals a lower fidelity in the replication process and enhances the probability of adenine incorporation into the complementary strand, giving rise to G to T transversions (49,50). As a consequence, mutations will predominantly occur at G clusters. The carcinogenic risk is therefore substantially enhanced since several human hot spot codons of the p53 tumor suppressor gene as well as H-ras proto-oncogene contain GG sequences (51,52).

With the measurement of 8-oxodGuo levels only a part of the oxidative DNA damage is detectable, so the total oxidative DNA damage might be much greater in PAH-exposed workers. The formation of 8-oxodGuo adducts is of particular interest, since they cause, similar to B[a]P-DNA adducts, GT transversion mutations (53,54). The effects of DNA damage (8-oxodGuo and strand breaks and B[a]P-DNA adducts) may be cumulative.

It was estimated that the DNA of cells treated in culture with B[a]P contain an ~20-fold higher level of oxidized bases than B[a]P-base adducts (55). Our results show between 5- to 35-fold higher 8-oxodGuo levels, when compared with anti-BPDE-DNA adduct levels found in coke-oven workers (the 95th percentile in controls was 4.4 adducts/108 nucleotides and the highest adduct level in coke oven workers was 48.5 adducts/108 nucleotides, Pavanello et al. (56)). Despite the numerous repair enzymes and antioxidant defenses, the presence of oxidative damage might impair the removal of PAH-DNA adducts and could be responsible for their known persistence (57). In a recent study, Peluso et al. (58) reported that a diet rich in vegetables, fruits and cereals was associated with a reduction in white blood cell PAH–DNA adducts. Several PAHs are known to provide initiating (carcinogen-DNA base adducts) and/or promoting (oxidized bases) lesions (48,57). These initiating and promoting processes may explain PAH-induced carcinogenesis.

The mechanisms by which PAHs produce cancer in humans is unclear. Studies examining DNA binding and adduct formation have shown a very low level of DNA binding for some PAH metabolites. Further in vivo studies inquiring into PAH genotoxicity have yielded inconclusive results. The DNA adduct formation induced by some PAHs does not appear to be the only mechanism leading to cancer formation. An additional non-genotoxic mode of action had to be suggested to explain the PAH carcinogenicity. Our findings tend to show that complete carcinogens such as PAHs, exert their biological effects not only through DNA damage (equated with the initiation step of carcinogenesis), but also through production of reactive oxygen species (associated with the promotion phase).

In conclusion, this study provides evidence that PAH exposure can result in oxidative WBC DNA damage. These findings reveal that both biomarkers of internal and external exposure may be appropriate for further surveillance studies of workers with high PAH exposure. Attempts to elucidate the relationship between ambient, biological and biochemical effect markers are limited by the complexity of the subject, by interindividual differences and by the influence of various confounding factors. Biomarker studies with a large number of PAH-exposed workers will be necessary to improve statistical power and to establish differences in susceptibility to oxidative DNA damage.

Notes

5 To whom correspondence should be addressed Email: marczynski{at}bgfa.ruhr-uni-bochum.de Back

Acknowledgments

We thank Beate Chilian, Anja Bracht and Simone Höhler-Wefers for expert technical support. We thank Louise Lajoie-Junge for the text revision.

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Received October 5, 2001; revised November 9, 2001; accepted November 12, 2001.