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. 2009 Nov 5;31(2):216–221. doi: 10.1093/carcin/bgp278

Shorter telomere length in peripheral blood lymphocytes of workers exposed to polycyclic aromatic hydrocarbons

Sofia Pavanello 1,*, Angela-C Pesatori 1, Laura Dioni 1, Mirjam Hoxha 1, Valentina Bollati 1, Ewa Siwinska 2, Danuta Mielzyńska 3, Claudia Bolognesi 4, Pier-Alberto Bertazzi 1, Andrea Baccarelli 1
PMCID: PMC3491668  PMID: 19892797

Abstract

Shorter telomere length (TL) in peripheral blood lymphocytes (PBLs) is predictive of lung cancer risk. Polycyclic aromatic hydrocarbons (PAHs) are established lung carcinogens that cause chromosome instability. Whether PAH exposure and its molecular effects are linked with shorter TL has never been evaluated. In the present study, we investigated the effect of chronic exposure to PAHs on TL measured in PBLs of Polish male non-current smoking cokeoven workers and matched controls. PAH exposure and molecular effects were characterized using measures of internal dose (urinary 1-pyrenol), effective dose [anti-benzo[a]pyrene diolepoxide (anti-BPDE)–DNA adduct], genetic instability (micronuclei, MN) and DNA methylation [p53 promoter and Alu and long interspersed nuclear element-1 (LINE-1) repetitive elements, as surrogate measures of global methylation] in PBLs. TL was measured by real-time polymerase chain reaction. Cokeoven workers were heavily exposed to PAHs (79% exceeded the urinary 1-pyrenol biological exposure index) and exhibited lower TL (P = 0.038) than controls, as well as higher levels of genetic and chromosomal alterations [i.e. anti-BPDE–DNA adduct and MN (P < 0.0001)] and epigenetic changes [i.e. p53 gene-specific promoter and global methylation (P ≤ 0.001)]. TL decreased with longer duration of work as cokeoven worker (P = 0.039) and in all subjects with higher levels of anti-BPDE–DNA adduct (P = 0.042), p53 hypomethylation (P = 0.005) and MN (P = 0.009). In multivariate analysis, years of work in cokery (P = 0.008) and p53 hypomethylation (P = 0.001) were the principal determinants of shorter TL. Our results indicate that shorter TL is associated with chronic PAH exposure. The interrelations with other genetic and epigenetic mechanisms in our data suggest that shorter TL could be a central event in PAH carcinogenesis.

Introduction

The identification of markers predictive of lung cancer and the assessment of their frequency in subjects at risk are a priority for preventive medicine (1). Genomic instability and/or epigenetic changes are hallmarks of cancer, including that of the lung. However, the mechanisms through which lung carcinogens produce such alterations are still largely unknown (2).

Telomere dysfunction due to excessive shortening is a key element leading to chromosomal instability and an early event in carcinogenesis (3). Telomeres are DNA repeat sequences (TTAGGG)n that, together with associated proteins, form a sheltering complex that caps chromosomal ends and protects their integrity (4). Due to limitation of DNA polymerase (telomerase), genetic stability is gradually lost as telomeres shorten with each round of cell division (4,5). In addition, telomere erosion can even be further accelerated by external stressors. Telomeres, as triple-guanine-containing sequences, are highly sensitive to damage by oxidative stress (6), alkylation (7) or ultraviolet irradiation (8). The resulting damaged telomeric bases by double-strand breaks and/or interference with replication fork can induce a reduction in telomere length (TL) (9).

In the present study, we investigated cokeoven workers with high exposure to polycyclic aromatic hydrocarbons (PAHs), a class of lung carcinogens that are also a widespread exposure in the general population due to tobacco smoking, diet and environmental pollution (10). Benzo[a]pyrene (BP), which has been used as a tracer of PAH mixtures, after its metabolic activation forms stable DNA adducts prevalently at guanine nucleotides [anti-benzo[a]pyrene diolepoxide (anti-BPDE)–DNA)] (11). This specific stereoselective binding may result in genetic mutations, including mutational ‘hotspots’ in the pulmonary p53 gene, that are critical to BP tumorigenesis (12). P53, a key factor in the DNA damage-signalling pathway (13), is also frequently epigenetically altered in smoking/PAH-associated lung cancer (for a review see ref. 14). P53 hypomethylation is associated with DNA double-strand breaks and chromosomal instability (15,16). Global DNA hypermethylation following in vitro chronic exposure to BP was observed in mouse embryonic fibroblasts and has been associated to genetic mutations as a consequence of genetic instability (17,18).

Peripheral blood lymphocytes (PBLs), which can be easily and non-invasively obtained from human subjects, have been shown to undergo several of the genetic and molecular modifications induced by PAHs in the lung. In particular, PAH(BP)–DNA adducts and DNA methylation levels in PBLs are correlated with levels in the lung (19–21). High levels of DNA adducts (22) and micronuclei (MN) (23), shorter TL (24,25) and p53 hypomethylation (26) in PBLs have all been shown to predict lung cancer risk. DNA methylation changes of the p53 promoter or of global methylation content [estimated in Alu and long interspersed nuclear element-1 (LINE-1) repeats] detectable in PBLs have been related to anti-BPDE–DNA adduct and MN formations in subjects exposed to high levels of PAHs (27). Additionally, high PAH exposure through metabolic activation—via aldo-keto reductase and/or manganese superoxide dismutase (MnSOD) (28)—produces oxidative stress (reactive oxygen species) that forms high levels of oxidized guanine in PBLs (29) and can represent another source of damage to telomeres. However, no studies have addressed whether PAH(BP) exposure alters telomeres length in PBLs from individuals at high risk of lung cancer due to high levels of PAH exposure.

In this study, we examine the effect of PAH(BP) exposure on TL in PBLs of non-current smoking Polish male cokeoven workers and matched controls to identify novel molecular pathways, which may mediate lung cancer risk. This study population has been characterized with measures of internal dose (urinary 1-pyrenol), effective biological dose (anti-BPDE–DNA adduct), early biological effects (MN) and DNA methylation (p53 promoter methylation and global methylation estimated in Alu and LINE-1 repeats). We hypothesized that individuals with high effective biological dose, as reflected in high levels of anti-BPDE–DNA adduct, would show higher genomic instability measured by TL and MN formation. Since DNA methylation modifications, mainly global DNA and p53 promoter methylation, have been proposed as additional mechanisms associated with genetic instability induced by PAHs (27), we also evaluated whether [PAH(BP)]-induced TL was in turn associated with these epigenetic changes in PBLs.

Materials and methods

Study design

The study subjects (n = 92) were all males non-current smokers and included 48 cokeoven workers from three polish cokeries and 44 controls matched by gender and ethnicity, who had been part of a group of 94 study subjects examined in our previous work (30) for which DNA was still available (enrolment from January through May 2006). Cokeoven workers all had tasks (i.e. charging, coking and pushing operations at the cokeoven battery section) involving exposure to high levels of PAHs. We excluded individuals whose work involved exposure to benzene (i.e. workers in by-product operations). Controls were clerks recruited during their periodic check-ups at the Institute of Occupational Medicine and Environmental Health in Sosnowiec (Poland). All subjects were non-current smokers, defined as either never-smokers or former smokers who had stopped smoking at least 1 year before sample collection. Urine analysis of nicotine and its metabolites (31) confirmed that all subjects were non-exposed to tobacco smoke as they had urine nicotine concentrations <0.01 mg/mmol creatinine.

PAH exposure levels were assessed by measuring 1-pyrenol in a urine sample (50 ml) collected from each of the workers at the end of their work shift (after at least 3 consecutive working days) and in the late afternoon from controls. At the same time as urine was collected, blood samples were taken and placed in ethylenediaminetetraacetic acid-containing tubes (20 ml) and evacuated heparin-containing tubes (10 ml) for adduct and MN analysis, as described previously (30,32). At the Institute of Occupational Medicine and Environmental Health in Sosnowiec: (i) PBL cultures for MN detection were prepared and MN analyses performed; (ii) PBLs for adduct analyses were isolated in Ficoll separating solution (Seromed, Berlin, Germany) within 4 h after blood collection and kept frozen at −80°C until they were sent to the Department of Environmental Medicine and Public Health in Padova (Italy), where DNA was extracted. Data on other possible elevated non-occupational PAH exposure (diet and environment) and consumption of fruit and vegetables were collected by means of a questionnaire, as described previously (33). Subjects with high dietary intake of PAHs were those who reported consumption of PAH-rich meals (charcoaled meat and pizza) more than once a week; subjects with indoor exposure to PAHs were those who used wood or coal heating at home; subjects with environmental exposure to PAHs were those identified by a high index based on the location of their residence that was based on intense local traffic and presence of industries in the area. This study was reviewed by the appropriate Ethics Committee of the Institute of Occupational Medicine and Environmental Health in Sosnowiec. All participants gave their written informed consent.

Analysis of exposure to PAHs

1-Pyrenol in urine samples was determined by high-performance liquid chromatography–fluorescence as described previously (34), following the original method of Jongeneelen (35). 1-Pyrenol levels in each urine sample were expressed per mole urinary creatinine, determined according to the Boehringer Mannheim (Mannheim, Germany) colorimetric test, based on the reaction of creatinine with picrate in an alkaline medium. Anti-BPDE–DNA adduct formation was detected, after DNA isolation with a Promega Wizard genomic DNA purification kit (Promega, Milano, Italy), by high-performance liquid chromatography/fluorescence analysis of BP-tetrol-I-1 (r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene) released after acid hydrolysis of DNA samples, as described previously (33). The detection threshold of BP-tetrol-I-1 was 0.25 pg (signal/noise > 3) so that, with 100 μg DNA, this assay can measure 0.25 adducts per 108 nucleotides (1 fmol/μg DNA = 30 adducts per 108 nucleotides) (33). The mean coefficient of variation (CV) for analyses of standard curve repeated five times on five different days was 10%. The highest CV value was 5.70% for those samples (n = 8) with >200 μg DNA repeated twice. MN analysis was performed on coded slides scored by light microscopy at ×400 magnification, as described previously (30,32). To exclude artefacts, the identification of MN was confirmed at ×1000 magnification in 10% of samples. The scoring of bi-, tri- and tetra-nucleate cells and MN was done and the cytokinesis block proliferation index was calculated as being equal in both groups (P = 0.599). MN levels were verified on 20 slides (two slides per subject) previously scored at the Institute of Occupational Medicine and Environmental Health in Sosnowiec by blind scoring at the Environmental Carcinogenesis Unit, Istituto Nazionale per La Ricerca sul Cancro (IST, Genova, Italy). The results of this intercalibration exercise demonstrated a good correlation between Mn levels scored in the two laboratories (analysis of variance, F = 20.9, P < 0.001). Intraclass correlation coefficient was 0.79 (95% confidence interval = 0.55–0.91).

Analysis of DNA methylation

DNA methylation was quantified using bisulphite-polymerase chain reaction (PCR) and pyrosequencing (27,36). In brief, the samples were bisulphite treated by EZ-96 DNA Methylation-Gold Kit™ (Zymo Research, Orange, CA) and PCR amplified. Analyses of DNA methylation in Alu and LINE-1 repetitive element and p53 gene were performed using previously published methods (27,36). For each reaction, a 50 μl PCR was conducted in 50 μl of GoTaq Green Master mix (Promega, Madison, WI), 1 pmol of the forward primer, 1 pmol of the reverse primer, 50 ng of bisulphite-treated genomic DNA and water. One of the forward or reverse primers was biotin labelled depending on the proximity to CG sites to be quantified and used to purify the final PCR product using Sepharose beads. The PCR product was bound to Streptavidin Sepharose HP (Amersham Biosciences, Uppsala, Sweden) and the Sepharose beads containing the immobilized PCR product were purified using the Pyrosequencing Vacuum Prep Tool (Pyrosequencing, Westborough, MA). Methylation quantification was performed using the provided software. The degree of methylation was expressed for each considered DNA locus as percentage of methylated cytosines divided by the sum of methylated and unmethylated cytosines. We used built-in controls to verify bisulphite conversion efficiency. Every sample was tested three times for each marker to confirm the reproducibility of our results. CV obtained from duplicate runs was 0.7, 1.6 and 1.8% for LINE-1, Alu and p53, respectively.

TL measurement by quantitative PCR

TL was measured in PBL DNA using the real-time quantitative PCR method developed by Cawthon (37). This method measures the relative TL in genomic DNA by determining the ratio of telomere repeat copy number (T) to single copy gene (S) copy number (T:S ratio) in experimental samples relative to a reference pool sample (38). The single copy gene used in this study was human β-globin (hbg). The analysis was conducted as described previously (39), with some minor modifications, mainly regarding the use of a MICROLAB STARlet Robot (Hamilton Life Science Robotics; Hamilton Bonaduz AG, Bonaduz, Switzerland) for transferring in a 384-well plate a reduced reaction mix (5 μl) with 2 μl (2 ng/μl) DNA and an ‘eight-point standard curve’. This standard curve was generated from a serially diluted DNA pool (obtained from 20 DNA samples randomly selected from the samples tested in the present study), ranging from 30 to 0.234 ng in each plate, so that relative quantities of T and S (in nanograms) could be determined from it. The average of the three T measurements was divided by the average of the three S measurements to calculate the average T:S ratio.

As we already described (39), to increase PCR reproducibility (40) for every reaction, we prepared a 25 μl mixture of DNA sample (2 ng/μl) containing Escherichia coli DNA (15 ng/μl) used as a DNA carrier; these mixtures were heated to 96°C for 10 min and cooled to room temperature. PCR primer sets for telomere and hbg and PCR mix composition were described previously (39). The PCR runs were performed in triplicate on a 7900HT Fast Real Time PCR System (Applied Biosystems, Foster City, CA). The thermal cycling profile for both amplicons began with a 95°C incubation for 3 min to activate the hot-start iTaq DNA polymerase. The telomere PCR conditions were 25 cycles at 95°C for 15 s and 54°C for 60 s. The hgb PCR conditions were 35 cycles at 95°C for 15 s, 58°C for 1 s and 72°C for 15 s. After PCR amplification, the specificity of the product was confirmed by dissociation curve analysis. To test the reproducibility of TL measurements, we amplified telomere (T) and hbg (S) in 15 samples that were replicated three times on each of three different days. The within-sample CV for the average T:S ratio over the 3 consecutive days was 8.7%, which was similar to the CV reported for the original protocol (37).

Statistical analysis

Statistical comparisons were made between various groups using the non-parametric Mann–Whitney U-test or the chi-square test. Univariate linear regression analysis was used to test the influence of years of work, age, PAH internal dose (urinary 1-pyrenol), anti-BPDE–DNA adduct and p53 and global DNA methylation levels (independent variables) on TL (dependent variable) of cokeoven workers. Multivariate linear regression analysis was used to assess the influence of age, years of work, urinary 1-pyrenol, anti-BPDE–DNA adduct and p53 and global DNA methylation levels (independent variables, all fitted in the same model) on TL (dependent variable) of cokeoven workers and the entire study population. All statistical tests were two sided and were performed with Statsdirect Statistical software (Ashwell, Herts, UK).

Results

The study population of 48 cokeoven workers and 44 controls showed in Table I were all non-current smoking males, who were in the same age range, similar in dietary behaviour (PAH-rich meals and daily consumption of fruit and vegetables), as well as for their environmental or indoor exposure to PAHs (Table I). Cokeoven workers had a median of 11 working years and were heavily exposed to PAHs, as their urinary 1-pyrenol concentrations were ∼30 times higher than in controls and in 79% of the workers versus 0% of the controls urinary 1-pyrenol exceeded the biological exposure index (BEI) of 2.28 μmol/mol creatinine proposed by Jongeneelen (35). Cokeoven workers exhibited significantly lower TL in PBLs compared with controls, as well as significantly higher levels of genotoxic alterations as reflected in higher anti-BPDE–DNA adduct and MN. DNA methylation levels (global and p53 promoter methylation changes) were also significantly different between workers and controls. Decrease in TL in PBLs of cokeoven workers was significantly associated, in univariate linear regression analysis (data not showed), with years of work as cokeoven workers (P = 0.039), but not with age, urinary 1-pyrenol and anti-BPDE–DNA adduct. Table II shows the distribution of TL in the whole study population (n = 92) according to increasing quartile levels of 1-pyrenol, anti-BPDE–DNA adduct and DNA methylation. Individuals with higher anti-BPDE–DNA adduct and p53 hypomethylation revealed significantly shorter TL. Likewise, frequency of subjects with TL >1.08 (TL median value of all subjects) significantly decreased with increasing anti-BPDE–DNA adduct and p53 hypomethylation levels. These associations did not reach statistical significance when evaluated separately in the groups of 48 exposed and 44 non-exposed workers.

Table I.

Characteristics of Polish male cokeoven workers and non-exposed controls

Cokeoven workers Controls P-values
Number of subjects 48 44
Age (years)
    Median (range) 36 (20–59) 38 (21–58) 0.81 Z = 0.25
Dieta 4 1 0.22
Fruit and vegetablesb 27 21 0.44
Environmental exposurec 16 15 0.99
Indoor exposured 25 18 0.31
Nicotine plus metabolites (mg/mmol creatinine)
    n (%) positive subjectse 0 (0) 0 (0)
Years of work in the cokery (years)
    Median (range) 11 (1–40) 0 (0)
PAH exposure (1-pyrenol μmol/mol creatinine)f
    Median (range) 3.09 (0.41–7.48) 0.09 (0.01–0.40) <0.001 Z = 8.25
    n (%) with urinary post-shift excretion of 1-pyrenol ≥2.28 μmol/mol creatinine 38 (79) 0 (0) <0.001
Genotoxic alterations
    Anti-BPDE–DNA (adducts per 108 nucleotides)
     Median (range)g 5.06 (0.90–12.24) 0.21 (0.125–5.56) <0.0001 Z = 8.13
    MN per 1000 binucleate cells
        Median (range) 4 (1–11) 1 (0–4) <0.0001 Z = 6.47
Epigenetic alterations
    Global
        LINE-1 (%mC)h 80.3 (56.9–83.7) 75.7 (55.7–85.7) <0.0001 Z = 4.42
        Alu (%mC) 23.6 (21.8–24.5) 22.9 (20.6–24.4) 0.001 Z = 3.29
    Gene specific
        p53 (%mC)h 11.8 (5.63–25.1) 18.6 (6.85–46.2) 0.0004 Z = 3.55
Relative TL
    Median (range) 0.99 (0.31–3.00) 1.20 (0.43–2.12) 0.038 Z = 2.08
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Statistical comparison: Mann–Whitney U-test between cokeoven workers versus controls; age, 1-pyrenol μmol/mol creatinine, anti-BPDE–DNA (adducts per 108 nucleotides) and MN (MN per 1000 binucleate cells). Chi-square test, cokeoven workers versus controls: diet, fruit and vegetables, environmental exposure, indoor exposure and 1-pyrenol ≥2.28 μmol/mol creatinine.

a

Number of subjects with charcoaled meat consumption more than or equal to once a week.

b

Number of subjects with daily consumption of fruit or vegetables.

c

Number of subjects with high environmental exposure from residence in town, intense traffic and presence of industries near home (see Materials and Methods).

d

Number of subjects with wood- or coal-based heating at home.

e

Subjects with values higher than threshold limit of assay (0.01 mg/mmol creatinine).

f

PAH exposure evaluated by urinary excretion of 1-pyrenol.

g

A value of 0.125 adducts per 108 nucleotides was assigned to subjects with non-detectable adducts.

h

Methylated cytosine percentage.

Table II.

Distribution of TL (N = 92) according to increasing quartile levels of 1-pyrenol, anti-BPDE–DNA adduct and DNA methylation

Subjects
 TL
 Chi2 for trend; Pb
n Median (range)a n (%) subjects with TL >1.08 (T:S)
PAH exposure
    1-Pyrenol (μmol/mol creatinine)
        ≤0.10 23 1.21 (0.43–2.12) 15 (65)
        >0.10 ≤ 0.58 23 1.17 (0.54–2.03) 14 (61)
        >0.58 ≤ 3.35 23 0.99 (0.42–3.01) 10 (43)
        >3.35 23 1.01 (0.31–1.79) 8 (35) Chi2 = 5.70; P = 0.13
Biological effective dose
    Anti-BPDE–DNA (adducts per 108 nucleotides)
        ≤0.125 28 1.10 (0.54–2.03) 16 (57)
        >0.125 ≤ 2.47 18 1.43 (0.43–2.12) 13 (72)
        >2.47 ≤ 5.16 23 0.98 (0.40–1.52) 7 (30)
        >5.16 23 1.07 (0.31–3.01)* 11 (48) Chi2 = 7.65; P = 0.054
Global methylation (%mC)
    LINE-1
        ≤75.0 23 1.13 (0.58–2.00) 15 (65)
        >75.0 ≤ 79.6 23 1.06 (0.43–2.03) 10 (44)
        >79.6 ≤ 80.8 23 1.10 (0.31–1.79) 13 (57)
        >80.8 22 0.99 (0.43–3.01) 9 (41) Chi2 = 3.54; P = 0.32
    Alu
        ≤22.7 24 1.27 (0.59–2.03) 15 (63)
        >22.7 ≤ 23.4 23 1.07 (0.32–1.96) 11 (48)
        >23.4 ≤ 23.8 21 0.99 (0.42–3.01) 7 (33)
        >23.8 23 1.18 (0.43–2.12) 13 (57) Chi2 = 4.26; P = 0.24
Gene-specific methylation (%mC)
    p53
        ≤9.98 21 1.07 (0.43–1.95) 10 (47)
        >9.98 ≤ 13.95 24 0.98 (0.31–1.79) 8 (33)
        >13.95 ≤ 20.4 22 1.01 (0.40–1.96) 10 (46)
        >20.4 22 1.44 (0.43–2.12)** 17 (77) Chi2 = 9.43; P = 0.02
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a

Kruskal–Wallis test: anti-BPDE–DNA (adducts per 108 nucleotides).*P = 0.042 and p53 methylation **P = 0.005.

b

Chi-square (Chi2) test for trend was performed on n (%)subjects with TL >1.08 (median value of all workers)

Linear multiple regression analysis (Table III) conducted on data from the entire study population (n = 92) showed that years of work and p53 hypomethylation were the only independent significant determinants of lower TL. In cokeoven workers (n = 48), the decrease in TL was significantly associated with years of work but not with age, urinary 1-pyrenol and anti-BPDE–DNA adduct, p53 promoter and global (LINE-1 and Alu) DNA methylation. Linear multiple regression analysis (data not showed) showed that in controls (n = 44), there was a negative association of TL with p53 promoter hypomethylation (P = 0.009), but not with age (P = 0.489), urinary 1-pyrenol (P = 0.753) and anti-BPDE–DNA adduct (P = 0.954) and global (LINE-1 and Alu, P = 852 and P = 0.560) DNA methylation. However, the overall test of significance (analysis of variance) did not reach statistical significance (F = 1.173 and P = 0.343).

Table III.

Linear multiple regression analysis of age, years of work, 1-pyrenol, anti-BPDE–DNA adduct and DNA methylation levels on TL in all subjects and cokeoven workers

Subjects Age (years) Years of work in the cokery (years) 1-Pyrenol (μmol/mol creatinine) Adduct
 DNA methylation
Anti-BPDE per 108 bases p53 (%mC) LINE-1 (%mC) Alu (%mC)
All (n = 92)
    ba 0.006 −0.016 −0.002 0.037 0.020 −0.0019 0.009
    SE(b) 0.005 0.006 0.345 0.025 0.006 0.009 0.054
    r2 (%) 1.59 8.53 0.004 2.75 12.3 0.06 0.03
    Pc 0.259 0.008 0.954 0.137 0.001 0.828 0.868
Cokeoven workers (n = 48)b
    ba 0.013 −0.027 0.013 0.032 0.022 −0.014 0.245
    SE(b) 0.011 0.087 0.042 0.031 0.014 0.018 0.127
    r2 (%) 3.31 13.7 0.25 2.76 5.76 1.57 8.96
    Pc 0.260 0.018 0.757 0.304 0.130 0.441 0.061
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Overall test of significance (analysis of variance) for all and cokeoven workers: F = 3.56, P = 0.002 and F = 2.20, P = 0.054.

a

Coefficient of regression (b), standard error (SE(b)) and partial explained variance (r2) were estimated for each term included in the model.

b

TL as ln T/S.

c

Significant P values are bold.

Table IV shows MN levels categorized according to the median value of TL (≤1.08 versus >1.08 TL) in PBLs of workers, controls and all subjects. Higher MN levels were found in subjects with shorter TL. In addition, the percentage of subjects with MN >2 (median value of all subjects) was significantly higher in workers and all subjects with shorter TL than in those with longer telomeres.

Table IV.

MN levels according to TL in PBLs of study population

MN
TL N Median (range) n (%) with MN >2a
Cokeoven workers >1.08 19 2 (1–11) 8 (42)
≤1.08 29 4 (1–9) 22 (76)c*
Controls >1.08 28 0 (0–4) 3 (11)
≤1.08 16 1 (0–3) 2 (13)
All >1.08 46 1 (0–11) 11 (24)
≤1.08 46 3 (0–9)b** 24 (52)c**
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a

Number and frequency (%) of subject with MN >2 median levels of MN.

b

Mann–Whitney U-test: Z = 2.59, **P = 0.009.

c

Chi-square test: Chi2 = 5.58; *P = 0.018 and Chi2 = 7.79; **P = 0.005.

Discussion

In this study, we found that cokeoven workers with very high levels of PAH exposure, as indicated by measures of internal dose (urinary 1-pyrenol) and effective biological dose (anti-BPDE–DNA adduct), had significant shorter TL in PBLs compared with controls. Cokeoven workers also exhibited higher genomic instability, as reflected in MN number, and DNA methylation alterations both in the global content and in the p53 promoter. Moreover, telomeres significantly shortened with years of work in exposed workers. Shorter telomeres in PBLs have been shown to predict the risk of lung and other cancers (24,25). Our findings extend these observations by demonstrating that shorter telomeres may also occur in healthy subjects who are occupationally exposed to high levels (high BEI) of carcinogenic PAH(BP) and are, therefore, at higher risk for lung cancer. In our study, 79% of the PAH-exposed workers exceeded the BEI proposed by Jongeneelen (35) for urinary 1-pyrenol. At this value (2.28 μmol/mol creatinine), corresponding to the post-shift excretion value with environmental exposure to the airborne threshold limit value of coal tar pitch volatiles [i.e. 0.2 mg/m3 of ‘benzene soluble matter’, American Conference of Governmental Industrial Health (41)], cokeoven workers have been shown a 30% increased risk of lung cancer (35). Since telomeres play a key role in the maintenance of chromosome integrity and stability, our results indicate that decrease in TL of PBLs may represent an additional indicator of genetic instability induced by chronically high PAH exposure and confirm, with this new marker, that workers are at higher lung cancer risk than controls.

Loss of telomere sequence in circulating blood lymphocytes has been related with aging (42) and cumulative oxidative stress resulting from environmental exposures, including smoking (43,44), diet (43), life stress (45) and chronic inflammation (44). The chances that the association with shorter TL could depend on factors other than PAH exposure were minimized because no subjects were affected by chronic diseases, all study subjects were non-current smokers and exposed individuals were similar to controls for age, gender and ethnicity. In addition, we evaluated several other potential confounding factors, including dietary PAHs, indoor PAH exposure and environmental PAH exposure, that showed no differences between the two groups. However, we cannot exclude that different socio-economic status or different working conditions might have contributed, along with PAH exposure (for which we have supplied a measure of internal dose and biologically effective dose, i.e. the specific promutagenic anti-BPDE–DNA), in the reduction of TL that we observed in PBLs of cokeoven workers. Epel et al. (45) showed that individuals with high levels of psychological stress exhibited shorter telomeres. We cannot exclude that life stress related with differences in socio-economic and/or working conditions contributed to the decrease in TL of cokeoven workers. In our study, we did not find a statistically significant association between age and TL in PBLs. This lack of association is in line with other studies that used PBLs from healthy subjects (24,44,46), whereas investigations that used unfractionated blood leucocytes more consistently showed an age-related decrease in TL (39,42,47). The difference between studies using PBLs and those using unfractionated leucocytes could be ascribed to the longer half-life of blood lymphocytes, which therefore have less mitotic cycles over time compared with granulocytes, the other major white blood cell subtype that accounts for ∼50 to 80% of circulating leucocytes. Moreover, loss in TL is most pronounced in childhood with a more gradual attrition in midlife (42). We have analyzed TL in subjects with a limited age span (20–59 years), which also limited our capacity to evaluate the association between age and TL. In addition, study subjects of the same age generally display a large variation in TL. Thus, a wider age range, as well as a larger sample size, might be necessary to detect a significant correlation between TL and age among both exposed and control subjects.

In our study, we found that telomeres length decreases in association with increasing levels of anti-BPDE–DNA adduct. This finding suggests that adduct generation may have a direct role in shortening TL. BP is a strong clastogen, primarily through the action of one of the four metabolically generated optical BPDE isomers, the (+)-anti-BPDE. (+)-Anti-BPDE shows stereoselective binding to the exocyclic N2 of guanine nucleotides, which is also considered the primary essential damaging event in BP carcinogenicity (48,49). Telomeres, as triple-G-containing sequences, may represent a sensitive target for damage by (+)-anti-BPDE. Double-strand breaks and interference with replication fork by bulky-damaged telomeric bases may directly induce telomere shortening (8,9). However, other genotoxic coexposure or other mode of action of PAH(BP) on telomere shortening may be hypothesized. Although PAHs are still considered the primary genotoxic carcinogens produced by coal combustion emissions (for a comprehensive review see ref. 10 and references therein), the presence of some metals in coke emissions, or rather reactive oxygen species, produced through PAH (28) or metal (50) metabolism, might have contributed along with BP exposure in determining the lower TL observed in our study. As an additional mechanism, alterations of proteins in the telomere-sheltering complex, induced by PAHs and/or by anti-BPDE adduct itself, could be considered as an alternative event accounting for shorter TL.

Our results also suggest a relevant role of p53 promoter hypomethylation, which has been previously shown to induce activation of p53 expression (51) in PAH-related attrition of PBL TL. P53 has a key role in cellular responses to telomere dysfunction (13) and may be activated concurrently with telomere shortening and the associated genomic instability, leading to growth arrest and/or apoptosis (13). The p53 hypomethylation we found in PBLs can be appreciated in the light of the findings of a previous study on smoking lung cancer patients showing that p53 hypomethylation in PBLs was associated with lung cancer risk (26). Our finding adds further evidence indicating molecular mechanisms that may contribute to increase lung cancer risk in individuals occupationally exposed to high levels (>BEI) of carcinogenic PAH(BP).

We found that all study subjects and workers with shorter telomeres had significantly higher MN levels, which represent an efficient measure of chromosomal breakage and loss. Our data suggest a role for telomere erosion in MN formation, consistent with the known relation between telomere integrity and chromosomal protection. That kinetochore and spindle protein may be altered by anti-BPDE itself and/or PAHs could be an alternate hypothesis to explain increase in MN frequencies. Moreover, the concordant relationship between shorter telomeres and MN formation, both indicative of chromosome instability, suggests that these findings, taken together, may indicate higher cancer risk of the subjects carrying such alterations in PBLs.

The present study was carefully designed to minimize potential confounding and increase the capability to reveal PAH effects by selecting study subjects who were all non-current smoking males living in the same residential area. In addition, our study had reliable measurements of PAH(BP) internal and biological effective doses (27,30,34). By evaluating several other non-occupational potential sources of PAH exposure, we were able to confirm the specific association of genetic and epigenetic changes with occupational PAH exposure, thus excluding effects from other possible non-occupational genotoxic exposures. Finally, the results of this study appear to be biologically plausible and the direction of the effects is consistent with the available literature data on telomere shortening mechanisms.

In conclusion, cokeoven workers exposed to high levels of PAHs, in addition to higher levels of genotoxic alterations (i.e. anti-BPDE–DNA adduct and MN), exhibited significant shorter TL in PBLs. Subjects with shorter TL showed higher MN, a finding suggestive of chromosome instability. TL was independently associated with years worked as a cokeoven worker and to DNA methylation in the p53 gene. These features were found in PBLs of subjects chronically exposed to PAHs, thus suggesting that these events could be determined to identify subjects at high cancer risk. However, whether shorter TL mediates the risk of lung cancer determined by high-level PAH exposure should be determined in future investigations.

Funding

CARIPLO Foundation (2007-5469); Italian Association for Research on Cancer 2008; Italian Ministry of University and Research (PRIN2007-2S2HT8).

Acknowledgments

Conflict of Interest statement: None declared.

Glossary

Abbreviations

anti-BPDE

anti-benzo[a]pyrene diolepoxide

BEI

biological exposure index

BP

benzo[a]pyrene

CV

coefficient of variation

LINE-1

long interspersed nuclear element-1

MN

micronuclei

PAH

polycyclic aromatic hydrocarbon

PBL

peripheral blood lymphocyte

PCR

polymerase chain reaction

TL

telomere length

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