Reduced nucleotide excision repair and GSTM1-null genotypes influence anti-B[a]PDE–DNA adduct levels in mononuclear white blood cells of highly PAH-exposed coke oven workers

Sofia Pavanello1,3, Alessandra Pulliero1, Ewa Siwinska2, Danuta Mielzynska2 and Erminio Clonfero1

1 Occupational Health Section, Department of Environmental Medicine and Public Health, University of Padova, Via Giustiniani 2, 35128 Padova, Italy and 2 Institute of Occupational Health, Sosnowiec, Poland

3 To whom correspondence should be addressed Email: sofia.pavanello{at}unipd.it


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is important to identify the potential genetic-susceptible factors that are able to modulate individual responses to exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). In the present study we evaluated the influence of four polymorphisms of nucleotide excision repair (NER) genes [xeroderma pigmentosum-C (XPC)-PAT +/–, xeroderma pigmentosum-A (XPA) 5' non-coding region-A23G, XPD-exon 23 A35931C Lys751Gln, xeroderma pigmentosum-D (XPD)-exon 10 G23591A Asp312Asn] and that of glutathione S-transferase µ1 (GSTM1-active or -null) on benzo[a]pyrene diol epoxide (B[a]PDE)–DNA adduct levels from the lympho-monocyte fraction (LMF) of highly PAH benzo[a]pyrene (B[a]P)-exposed Polish coke oven workers (n = 67, 67% current smokers) with individual urinary post-shift excretion of 1-pyrenol exceeding the proposed biological exposure index (BEI) (2.28 µmol/mol creatinine). The bulky (±)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-B[a]PDE)–DNA adduct levels were detected by high-performance liquid chromatography (HPLC)/fluorescence analysis and genotypes by polymerase chain reaction. We found that workers with the low DNA repair capacity of XPC-PAT+/+ and XPA-A23A genotypes had significantly increased anti-B[a]PDE–DNA adduct levels (Mann–Whitney U-test, z = 2.24, P = 0.02 and z = 2.65, P = 0.01). Moreover, DNA adducts were also raised in workers without GSTM1 activity (GSTM1-null genotype) (Mann–Whitney U-test, z = 2.25, P = 0.0246). Workers with unfavourable XPC-PAT+/+ and XPA-A23A NER genotypes, alone (~65% of workers) or combined with GSTM1-null genotype (~75% of workers) were in the tertile with the highest adduct level, i.e. >4.11 adducts/108 nt ({chi}2 = 5.85, P = 0.0156 and {chi}2 = 5.40, P = 0.01). The increase in anti-B[a]PDE–DNA adduct levels (ln values) was significantly related in a multiple linear regression analysis to PAH exposure (i.e. urinary post-shift excretion of 1-pyrenol) (t = 2.61, P = 0.0115), lack of GSTM1 activity (t = 2.41, P = 0.0192) and to low DNA repair capacity of the XPC-PAT+/+ genotype (t = 2.34, P = 0.0226). The influence of the XPA-A23A genotype was not evident in this statistical analysis, and no associations with XPD polymorphisms, dietary habits or tobacco smoking were found. The modulation of anti-B[a]PDE–DNA adducts in the LMF by GSTM1-null and some low-activity NER genotypes may be considered as a potential genetic susceptibility factor capable of modulating individual responses to PAH (B[a]P) genotoxic exposure and the consequent risk of cancer in coke oven workers.

Abbreviations: anti-B[a]PDE, (±)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene; B[a]P, benzo[a]pyrene; B[a]P-tetrol I-1, r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; GSTM1, glutathione S-transferase µ 1; HPLC, high-performance liquid chromatography; LMF, lymphocyte plus monocyte fraction; NER, nucleotide excision repair, PAHs, polycyclic aromatic hydrocarbons; PCR, polymerase chain reaction; XPA, xeroderma pigmentosum-A; XPC, xeroderma pigmentosum-C; XPD, xeroderma pigmentosum-D


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Considerable inter-individual variations in response to genotoxic exposure to polycyclic aromatic hydrocarbons (PAHs), and the consequent risk of cancer, exist in humans. Individual susceptibility on a genetic basis and possible gene–exposure interaction may play an important role. Thus, identification of genetic characteristics, which contribute to the individual susceptibility to PAH-induced cancer is valuable in molecular epidemiological studies.

Coke oven workers have among the highest exposure to coal-tar-pitch volatiles rich in PAHs and particularly benzo[a]pyrene (B[a]P). A higher incidence of cancer, especially of the lung, is reported for workers with high, long-term exposure to coke oven emissions (1). Occupational and environmental (mainly smoking habits) PAH (B[a]P) exposure may be detected by DNA–adduct dosimetry in white blood cells, easily obtained from human subjects. In several investigations, the levels of PAH bulky-DNA adducts detected by the 32P post-labelling method (for a comprehensive review see ref. 2) or analysis of specific B[a]PDE–DNA adducts (3) has been shown to be associated with external PAH exposure especially when measurements were made in the longer-living cells (lympho-monocyte fraction). Unlike other biomarkers of PAH exposure (e.g. single urinary PAH metabolites), DNA–adduct measurement in lymphocyte plus monocyte fraction (LMF) is considered a marker of the effective dose, replacing that of the target tissue (i.e. lung). Various studies indicate that polyaromatic–DNA adduct levels in LMF and lung tissue or alveolar macrophages of smokers are significantly correlated (see ref. 4 for a comprehensive review and refs 5,6 for recent articles). In animal experiments, after B[a]P i.p. administration, B[a]P–DNA adduct levels in the blood and lungs were found to be correlated with the occurrence of lung cancer (7).

Significant inter-individual variations in B[a]P–DNA binding in LMF among subjects with comparable exposure have also been reported (8). B[a]P, the most potent and best studied carcinogen of PAH mixtures, must be activated to exert its biological effect. Phase I activation by cytochrome P450 1A1 and 3A4, myeloperoxidase (MPO) and epoxidohydrolase (EPXH) generates the ultimate carcinogenic metabolite, anti-B[a]P diol epoxide (B[a]PDE) (9). Conjugation reactions (Phase II or detoxification), catalysed by glutathione S-transferase µ and {pi} (GSTM1 and P1) (10) may prevent stable DNA binding to the N2 exocyclic guanine, which is critical in the carcinogenic process of B[a]P (11). Finally, bulky (±)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-B[a]PDE)–DNA adducts may be removed specifically by nucleotide excision repair (NER) (12). Thus, B[a]PDE–DNA adduct levels in LMF reflect not only the intensity of exposure, but also the individual PAH (B[a]P) metabolism and DNA lesion repair capability, these activities occurring in LMF and target tissues (10,13).

The genetic polymorphism of PAH metabolism exists and it is mainly that of GSTM1 that has been demonstrated to influence the levels of polyaromatic or B[a]PDE–DNA adducts in LMF in both occupational and environmental exposure (14,15). More recently, some polymorphisms in NER genes have also been identified but less information exists on their influence on polyaromatic–DNA adducts forming in subjects exposed to PAHs (1619). Using the host-cell reactivation assay, which directly measures the kinetics of DNA repair capacity in human lymphocytes, the functional relevance of some NER polymorphisms has been studied in vitro (for a recent review, see ref. 20). The suboptimal DNA repair capacity of B[a]PDE-induced DNA damage has been associated with the presence of some mutated genes (2123). The present study evaluated the influence of four polymorphisms of NER genes XPD-Asp312Asn and Lys751Gln, XPC-polyAT deletion>insertion of 83 bp in intron 9 (PAT–/+) and XPA (5' non-coding region-A23G), together with GSTM1 on anti-B[a]PDE–DNA adduct levels detected by high-performance liquid chromatography (HPLC)/fluorescence analysis, from the LMF of coke oven workers occupationally exposed to comparably high levels of B[a]P. Our aim was to find potential susceptibility factors on a genetic basis capable of modulating individual responses to PAH (B[a]P) genotoxic exposure and the consequent risk of cancer in coke oven workers.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
A total of 67 male coke oven workers from two Polish plants, in which workers are periodically biomonitored for PAH exposure by urinary 1-pyrenol analysis, were enrolled in this study. For each subject, data regarding age, tobacco smoking and charcoaled meat consumption, environmental exposure to PAHs, personal behaviour and the possible use of coal tar-based ointment, shampoo or soap, were collected by means of a questionnaire administered by trained interviewers. All participants gave their informed consent. A high dietary intake of PAHs was considered to be the consumption of charcoaled meat more than once a week. This study was reviewed by the appropriate Ethics Committee of the Institute of Occupational Medicine and Environmental Health in Sosnowiec (Poland).

Sample collection
Urine samples (50 ml) were collected at the end of the work-shift, after at least 3 consecutive days of work for 1-pyrenol determination (see ref. 3), following the original method (24). Subjects with urinary 1-pyrenol levels exceeding the proposed biological exposure index (BEI) value (2.28 µmol/mol creatinine) (25) were asked to give 20-ml blood samples. Within 4 h of blood collection, mononuclear LMF were isolated in Ficoll separating solution (Seromed, Berlin, Germany) (26). LMF were kept frozen at –80°C until their transport to the Department of Environmental Medicine and Public Health in Padova (Italy) for further analysis.

DNA from cells was isolated as described previously (26). This yielded DNA free of RNA or protein contamination, as checked by the 260/230 and 260/280 nm absorbance ratios of DNA, which were always about 2.3 and 1.7, respectively.

HPLC/fluorescence analysis of anti-B[a]PDE–DNA adducts
Anti-B[a]PDE–DNA adducts were detected by HPLC/fluorescence analysis of anti-B[a]P-tetrols (r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene) released after acid hydrolysis of DNA samples. HPLC/fluorescence analysis of anti-B[a]PDE–DNA adducts was carried out as already described (27), and is identical to that reported by Alexandrov et al. (28), with some modifications, mainly regarding the entire automation of HPLC analysis. Briefly, a total amount of at least 100 µg of DNA from LMF was used for each analysis. DNA samples were dissolved in 0.1 N HCl, and acid hydrolysis was carried out at 90°C for 4 h. HPLC analysis was performed with a Waters 717 Autosampler coupled to a Waters Millipore automated gradient controller and a 510 model solvent delivery system. B[a]P-tetrols were concentrated by 10 min isocratic elution of 20% methanol in H2O over a pre-column module containing 10 µm C18 reverse-phase material (Supelguard LC-18, 2 cm length, Bellefonte, PA) at 0.5 ml/min. The concentrated material was then automatically switched by means of a switching valve (Waters) to a Lichrosorb RP pre-packed 5-µm C18 reverse-phase analytical column (250 x 4.0 mm, Merck, Darmstadt, Germany). The column was eluted isocratically with 55% methanol in water at a flow rate of 1 ml/min. Eluant fluorescence was continuously monitored on a Perkin Elmer Series 200 fluorimeter. Fluorescence excitation and emission wavelengths were set at 344 and 398 nm. B[a]P-tetrol-I-1 was determined by comparison with a standard curve generated from the fluorescence areas of an authentic B[a]P-tetrol-I-1 standard (purchased from NCI Chemical Carcinogen Reference Standard Repository, Kansas City, MO), analysed prior to and immediately following the analysis of each set of samples. The minimum correlation coefficient was 0.9998 and the mean coefficient of variation for analyses repeated on different days was 10%. The detection threshold of B[a]P-tetrol-I-1 was 1 pg (signal/noise >3) so that, in the present study, with 100 µg DNA, this assay can measure 1 adduct/108 nt (1 fmol/µg DNA = 30 adducts/108 nt).

GSTM1 and NER gene polymorphisms
A multiplex polymerase chain reaction (PCR) method was used to detect the presence or absence of the GSTM1 gene, according to our previous study (27). This PCR method applied, in the same amplification mixture, the GSTM1-specific primer pair and a primer pair for ß-globin, as an internal positive PCR control. ß-Globin (285 bp) and GSTM1 (215 bp) amplification products were resolved in an ethidium bromide-stained 2% agarose gel. The absence of the specific GSTM1 fragment indicated the corresponding null genotype (*0/*0), and its presence corresponded to the *1/*1 and *0/*l genotypes; the ß-globin-specific fragment confirmed the presence of amplifiable DNA in the reaction mixture.

Polymorphisms of XPD-exon 23 A35931C Lys751Gln, XPD-exon 10 G23591A Asp312Asn and XPA 5' non-coding region-A23G were characterized by the PCR–RFLP technique. XPA-A23G genotypes were determined following the original method of Park et al. (29). PCR primers for the A23G polymorphism (GenBank accession no. U16815) were 10 pmol of 5'-TTAACTGCGCAGGCGCTCTCACTC-3' (bases 1689–1712 of XPA) and 5'-AAAGCCCCGTCGGCCGCCGCCAT-3' (bases 1846–1824 of XPA), which generate a 158-bp fragment. Overnight digestion with restriction enzyme MspI (New England BioLabs, Beverly, MA) was used to distinguish the A23G polymorphism: the wild-type (A) allele (23A) has a single band representing the entire 158-bp fragment, and the polymorphic (G) allele (23G) has two bands (132 and 26 bp).

XPD-312 and -751 polymorphisms were determined according to the method described by Lunn et al. (30), using 0.8 µM of primers 22872F (5'-CTGTTGGTGGGTGCCCGTATCTGTTGGTCT) and 23952R (5'-TAATATCGGGGCTCACCCTGCAGCACTTCCT) for 312 polymorphism analysis, and 35844F (5'-CCT CTCCCTTTCCTCTGTTC) and 36560R (5'-CAGGTGAGGGGGACATCT; Invitrogen Life Technologies, Milan, Italy) for 751 polymorphism analysis. Codon 312 was amplified using 5% DMSO as an additive. PCR amplifications of 312 and 751 XPD polymorphisms (757 and 734 bp) were digested overnight with discriminating restriction enzymes SytI and MboII, respectively (New England Bio-Labs, Milan, Italy). Digestion products were separated by 3% agarose gel. The wild-type (23591G) allele at codon 312 produced a fragment of 151 bp, and the variant A allele produced two bands (34 and 117 bp); the wild-type (35931A) allele at codon 751 produced two bands (98 and 505 bp), and the variant C allele produced a fragment of 603 bp.

Differently, the XPC-poly (AT) deletion>insertion of 83 bp in intron 9 (PAT–/+) was determined by PCR amplification using a forward primer at intron 9 (base pair 1258–1276), designated N1m (5'-TAGCACCCAGCAGTCAAAG-3'), and a reverse primer at intron 9 (base pair 1523–1504), designated N2n (5'-TGTGAATGTGCTTAATGCTG-3'), as described by Khan et al. (31). The XPC-PAT+ fragment was 344 bp and the XPC-PAT– fragment was 266 bp, a sequence of the variant PAT+ allele showing the insertion of 83 bases.

Statistical analysis
Statistical comparisons between various groups were made using the non-parametric Mann–Whitney U-test or the {chi}2 test. Multiple linear regression analysis was used to assess the influence of occupational exposure to PAHs (evaluated by urinary levels of 1-pyrenol µmol/mmol creatinine), GSTM1 and four polymorphisms of NER genes on anti-B[a]PDE–DNA adduct levels (ln value). The GSTM1 genotype was considered as a dichotomous variable, and values of 0 or 1 were attributed, referring to GSTM1 active or *0/*0. For each NER DNA repair genotype, values of 2, 1 or 0 were attributed, referring to the presence of two, one or no copies, of the low-DNA repair capacity XPC-PAT+, XPA-23A, XPD-312Asn and XPD–751Gln alleles, respectively, assuming the addictive affect of the presence of at least one allele at risk. All statistical tests were two-sided, and were performed with StatsDIRECT Statistical software (Ashwell, Herts, UK).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study evaluated the influence of four polymorphisms of NER genes [XPC-poly (AT) deletion>insertion of 83 bp in intron 9 (PAT–/+), XPA 5' non-coding region-A23G, XPD-exon 23 Lys 751Gln, XPD-exon 10 Asp312Asn) and GSTM1 (active or null) on anti-B[a]PDE–DNA adducts in LMF of 67 coke oven workers highly exposed to PAH [mean ± SD of urinary 1-pyrenol 9.33 ± 7.34 (range 2.31–31.4) µmol/mol creatinine]. The characteristics of the study population are shown in Table I.


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Table I. Characteristics of coke oven workers

 
Table II shows the frequencies of NER gene polymorphisms, in decreasing order of DNA repair capacity (XPC-PAT–/+, XPA-G23A, XPD-Lys 751Gln, XPD-Asp312Asn) and that of GSTM1 active (*1/*1 or *1/*0) and null (*0/*0) with the relative levels of 1-pyrenol and anti-B[a]PDE–DNA (range, mean ± SD). All subjects except one (66 out of 67) had detectable adduct levels (i.e. >1 adduct/108 nt). Coke oven workers with XPC PAT+/+ and XPA-A23A genotype with low DNA repair capacity showed significant increases in adduct levels with respect to those with none or only one copy of these at-risk polymorphisms (XPC PAT+/+ versus PAT–/–, 5.37 ± 3.86 versus 3.12 ± 2.00 adducts/108 bases, Mann–Whitney U-test, z = 2.24, P = 0.02; XPA-A23A versus G23G and versus A23G, 8.73 ± 5.98 versus 4.77 ± 5.24 and versus 3.29 ± 2.22 adducts/108 bases, Mann–Whitney U-test, z = 2.65, P = 0.01 and z = 2.15, P = 0.03): PAH exposure being quite similar in these subgroups. The other subgroups, with variants XPD-312Asn and XPD-751Gln, had higher anti-B[a]PDE–DNA adduct levels than those with wild-type alleles, but the difference was not statistically significant. Moreover, confirming our previous results (15,27), coke oven workers with a GSTM1-null genotype (30%) had significantly higher adduct levels than those with GSTM1-active (6.73 ± 6.61 versus 3.37 ± 2.20 adducts/108 bases, Mann–Whitney U-test, z = 2.25, P = 0.0246). No contribution of smoking to the levels of 1-pyrenol (mean ± SD, 46 smokers/21 non-smokers, 9.25 ± 8.00/9.53 ± 5.78 µmol/mol creatinine Mann–Whitney U-test, z = 0.87, P = 0.166) and those of anti-B[a]PDE–DNA was detected (mean ± SD, 46 smokers versus 21 non-smokers 4.28 ± 4.56 versus 4.57 ± 3.72 adduct/108 nt Mann–Whitney U-test, z = 0.68, P = 0.373).


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Table II. Anti-B[a]PDE–DNA adduct levels in LMF of PAH-exposed coke oven workers, according to GSTM1 and NER genotypes

 
Table III shows the frequencies of workers with DNA repair-unfavourable genotypes (XPC-PAT+/+, XPA-A23A, XPD-Asn312Asn and XPD-Gln751Gln), alone or in combination with GSTM1-null, according to tertile adduct levels: 1° tertile (≤2.27 adducts/108 nt), 2° tertile (>2.27 ≤4.11 adducts/108 nt) and 3° tertile (>4.11 adducts/108 nt). Subjects with unfavourable XPC-PAT+/+ and XPA-A23A genotypes significantly belonged to the 3° tertile with the highest adduct level (~65% of workers) (comparison between 1° and 3° tertile {chi}2 = 5.85, P = 0.0156 and {chi}2 = 5.40, P = 0.01 and P-value for trend P = 0.028 and 0.049). The few subjects with the unfavourable XPC-PAT+/+ and XPA-A23A genotype together with GSTM1-null deletion fell in the higher adduct tertiles, in particular, ~75% of workers fell in the highest one (P-value for trend = 0.024). No significant differences in the percentage of the low repair XPD polymorphisms were detected among tertile adduct levels.


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Table III. Frequencies of unfavourable NER genotypes alone or in combination with GSTM1-null according to anti-B[a]PDE–DNA adduct levels

 
Table IV shows the results of linear multiple regression analysis and the influence of occupational exposure of PAHs (i.e. individual post-shift urinary 1-pyrenol values), diet and smoking habits, NER genotypes and GSTM1 on anti-B[a]PDE–DNA adduct levels (ln value). The increase in anti-B[a]PDE–DNA adduct levels was significantly related to the intensity of occupational exposure to PAHs (B[a]P) of each subject (t = 2.661, P = 0.0115), lack of GSTM1 activity (t = 2.410, P = 0.0197) and lower DNA repair capacity of the XPC-PAT+/+ genotype (t = 2 .343, P = 0.0226). The other three NER polymorphisms (XPA-G23A, XPD-Asp312Asn, XPD-Lys751Gln) did not appear to modify anti-B[a]PDE–DNA adduct levels significantly in this statistical analysis. Neither PAH-rich diet nor smoking influenced the biomarker.


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Table IV. Influence of occupational exposure to PAHs, smoking, diet, GSTM1 and NER genotypes on anti-B[a]PDE–DNA adduct levels (ln value) in LMF of 67 coke oven workers: linear multiple regression analysis

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genetic variations and damaging exposure are two essential elements, which contribute to the risk of all diseases especially cancer. Many of the genetic variations associated with cancer seem to exert an influence only in the presence of exposure. The present study evaluated the influence of four polymorphisms of NER genes and that of GSTM1 (-active or -null) on anti-B[a]PDE–DNA adduct levels from LMF of highly PAH-exposed coke oven workers.

The frequencies of allele variants XPD-312Asn, XPD-751Gln and XPA-23A (0.40, 0.45 and 0.34, respectively) were similar to those found in two different studies on healthy Polish populations (32,33). The allele frequency of XPC-PAT+ (0.45) was similar to that found in the only study published until now on XPC-PAT polymorphism on differing American populations (34). The GSTM1-null frequency (30%) was lower than that found in larger Caucasian and Polish population studies (~50%) (35,36). The low number of subjects we examined was probably the main reason for this result, as there was no biological basis for this low proportion. In any case, the low number of GSTM1-null subjects in our sample population should not ‘force’ the results, in the sense of finding a difference between the two subgroups.

We found that coke oven workers with the unfavourable XPC-PAT+/+ and XPA-A23A genotypes at low DNA repair capacity have significantly higher anti-B[a]PDE–DNA adduct levels. No effect of XPD was detected. Until now, only a few studies have been published on the influence of NER gene polymorphisms on bulky polyaromatic–DNA adducts in LMF, with contrasting results. XPD-Gln751Gln, alone (19) or combined with the XPD-Asn312Asn genotype (16), is associated with increased DNA adduct levels in smokers (16) and low occupationally PAH-exposed people (19) (e.g. traffic workers). In contrast, one study indicated the influence of XPD-Gln751Gln in the adduct levels of non-smokers rather than smokers (18) while no effect was found by Duell et al. (17). In these studies, the PAH exposure was most likely low and not checked, and 32P post-labelling which, however, has the problem of quantifying polyaromatic–DNA adducts with unknown structure, was used for adduct analysis. In addition, the above studies examined some genotypes of base excision repair (BER) (XRCC1 Arg399Gln and XRCC3 Thr241Met), which are involved in the repair of minor base damage induced by alkylating and oxidizing agents (1719). Instead our results clearly show that genetic polymorphisms of key proteins (XPC, XPA) in the initial steps of the NER pathway influence anti-B[a]PDE–DNA adduct levels specifically detected by HPLC-fluorescence analysis. This emerges in our highly exposed workers, in whom PAH exposure was continually checked by 1-pyrenol analysis and who formed stable detectable DNA adducts, indicating that these two genetic polymorphisms may be considered susceptible factors in response to long-term genotoxic exposure to PAHs.

XPC plays an early role in the DNA repair reaction (37). It is the initial damage-recognizing step, which specifically recognizes helix deformation due to bulky adducts, and is essential for recruiting all subsequent NER proteins to the damaged DNA (38). In this context, XPA, which has many interactions with other NER components [e.g. single-strand binding complex replication protein A (RPA) (39), transcriptional repair factor complex (TFIIH) (40) and XPF endonuclease (41)], plans the repair machinery around the DNA lesion. Our results are consistent with decreased DNA repair capacity, due to these two polymorphisms, found to be associated to an increased risk of smoking-related cancers (34), whereas the low DNA repair capacity of XPD-751Gln and 312Asn allele was not clearly associated with cancer risk (20). Polymorphism of XPD, which is one of the six protein–TFIIH complexes involved in the subsequent lesion-demarcation step, most probably produces minor effects.

The clear effect of GSTM1 genotype on the levels of bulky anti-B[a]PDE–DNA adducts was also evident. Among all metabolic cancer susceptibility factors, GSTM1 deficiency is one of the most consistent risk factors: GSTM1 detoxifies reactive metabolites of B[a]P and other PAHs (10); GSTM1 active subjects have lower levels of DNA adducts in lung tissue (42); epidemiological studies consistently show that GSTM1 deficiency predisposes subjects to lung cancer, and this relationship becomes more evident in subjects with high tobacco exposure (43).

A number of molecular epidemiology studies on coke oven workers (for a review see ref. 14) showed that workers, with high occupational exposure to PAHs, have steadily increased adduct levels in LMF (for a review see ref. 2). However, the majority of studies found no association between genotypes and DNA adducts. Above all, the use of the post-labelling assay, which detects a broad-spectrum of PAH–DNA adducts (4446), the lowered-occupational exposure to PAHs of coke oven workers for the implementing of preventive measures (47), or else the biomarker aspecific for the exposure under investigation (e.g. 8-OH-deoxyguanosine on PAH exposure) (48) are the reasons for these negative results. Instead, in agreement with our present results, the clear-cut effect of GSTM1 alone (15) or in combination with phase I enzymes activating PAHs (greater activity of CYP1A1) (9) on BPDE–DNA adducts was found using the HPLC/fluorescence method. The higher PAH exposure and the use of a specific method for B[a]PDE–DNA adduct detection may account for the discrepancy between our results and the previously reported negative ones. No study was published until now on the influence of DNA repair genotypes and adducts in coke oven workers.

No influence of smoking or diet to urinary individual post-shift excretion of 1-pyrenol or anti-B[a]PDE–DNA levels was detected in our sample population. Above all, the overlap of high PAH exposure in coke oven workers may explain the negligible effect of PAH exposure to tobacco smoke and a charcoal-cooked meat diet. Coke oven workers are reported to have a significantly higher incidence of cancer, especially of the lung, estimated to be 1.3 (more than ~30%) exposed to the airborne threshold limit value (TLV) of coal-tar-pitch volatiles [i.e. 0.2 mg/m3 of ‘benzene soluble matter’, ACGIH (49)]. This TLV is probably too high for workers with these unfavourable genotypes of detoxification and DNA repair. Our findings suggest that elevated DNA adducts in LMF and the possible risk of lung cancer in coke oven workers, attributable to high and long-term occupational exposure to PAHs, are modulated by GSTM1-null and low DNA repair XPC+/+ genotypes. Actually, although nucleated blood cells are not considered the target cells for PAH-induced tumorigenesis, their DNA adduct levels have been found to be predictive of what occurs on the target tissues (47). Our results would suggest that workers with the highest DNA adduct levels in LMF for their GSTM1 and NER genotypes have the highest DNA adduct levels also in the target epithelial lung cell types.

In conclusion, the increase in anti-B[a]PDE–DNA adduct levels in LMF is significantly related to the intensity of occupational exposure to PAHs (B[a]P) of coke oven workers, together with some individual genetic factors such as lack of GSTM1 activity and impaired NER DNA repair capacity associated with the XPC-PAT+/+ and perhaps XPA-A23A genotypes. These genetic factors appear to be as important as occupational exposure to PAHs in modulating the levels of the biomarker. Although the additional information provided here requires further investigation regarding the influence of other NER polymorphisms on anti-B[a]PDE–DNA adduct formation in LMF, it does appear to be interesting for future environmental–occupational studies using this biomarker, in cases where PAH (B[a]P) exposure is a factor. Finally, it is very probable that these genetic polymorphisms contribute to coke oven workers' individual susceptibility to PAH-related cancers.


    Acknowledgments
 
Supported by grant B/40-2/DML/01 of National Institute of Occupational Safety and Prevention (ISPESL), Rome, Italy.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 6, 2004; revised September 1, 2004; accepted September 29, 2004.