1 Institute of Occupational Health, University of Padova, Via Giustiniani 2, 35128 Padova,
2 CNR, Area di Ricerca, Corso Stati Uniti 4, Padova,
3 Institute of Occupational Health, University of Verona, Verona and
4 Occupational Health Service, Provincia di Bolzano, Boleano, Italy
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Abstract |
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Abbreviations: anti-BPDE, (±)-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-tetrahydro-benzo[a]pyrene; CT, coal tar; CYP, cytochrome P450; GST, glutathione S-transferase; LMF, lymphocyte plus monocyte fraction; PAH, polycyclic aromatic hydrocarbon; TLC, thin-layer chromatography; WBC, white blood cell.
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Introduction |
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Elevated PAHDNA adduct levels have been found in some groups of workers, mainly coke oven and foundry workers (for a review see ref. 8), in populations living in urban polluted areas compared with rural ones (8), in fire fighters who are accustomed to eating barbecued and grilled meats (8) and in psoriatic patients whose skin has been treated with coal tar (CT)-based ointments (9,10).
The formation of (±)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)DNA adducts is considered to be critical in the carcinogenic process of benzo[a]pyrene (B[a]P) (11). The new HPLC/fluorescence assay, which quantifies absolute levels of BPDEDNA adducts in human tissues (12), has recently been validated using white blood cells (WBCs) (13) and tissues from rats exposed to B[a]P (14). Good correlations and proportionality were found between the levels of BPDEDNA adducts measured by fluorometry and 32P-post-labelling. The detection limit of the HPLC/fluorescence assay is 0.5 adducts/108 nucleotides, while the detection limit of the 32P-post-labelling assay is ~1 adduct/109 nucleotides (14,15). The 32P-post-labelling assay is sensitive and capable of detecting exposure to complex mixtures, whereas HPLC/fluorescence can be used to identify BPDE isomers and may therefore be of value in risk assessment of individuals exposed to PAHs.
The aim of the present study was to compare anti-BPDEDNA adduct levels, determined by HPLC/fluorescence analysis (12), in groups of humans exposed to various levels of PAHs (B[a]P): 26 psoriatic patients (3 days after clinical CT treatment of the skin), 15 coke oven workers, 19 chimney sweeps, 36 aluminium anode plant workers and 34 non-occupationally exposed subjects (controls). PAH exposure was assessed in each group by means of the urinary excretion of 1-pyrenol.
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Materials and methods |
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Subjects
A total of 130 Caucasian subjects were involved in the study, as follows.
Twenty-six psoriatic patients (10 female and 16 male), hospitalized in the Dermatology Clinic of the University of Padova, were examined. Their ages ranged between 26 and 80 (mean ± SD 51 ± 14) years. Nine of them were smokers (14 ± 12 cigarettes/day). All patients were treated, in the skin lesions involving 2080% of the body surface, with a CT-based paste containing 2% CT, made up by the hospital pharmacy. The quantity applied varied between 20 and 120 g/day, i.e. CT applications of 0.42.4 g/day. The total PAH content of the CT was 3% (w/w).
The 15 male coke oven workers were members of a group of 98 from a coke oven factory examined in our previous works (1618). Their ages ranged between 37 and 50 (40 ± 15) years. Nine of them were smokers (17 ± 3 cigarettes/day). Subjects from whom sufficient quantities of DNA remained available (at least 100 µg) were analysed for DNA adduct detection. We sampled 19 male chimney sweeps, ages ranging between 15 and 46 (26 ± 9) years, four of whom were smokers (18 ± 8 cigarettes/day). There were 36 male aluminium anode plant workers, ages ranging between 28 and 51 (41 ± 6) years, 14 of whom were smokers (19 ± 6 cigarettes/day). Non-occupationally PAH-exposed subjects (controls), recruited from students and personnel of our Institute, were 34 healthy volunteers (20 female, 14 male), free from chronic illness, not subjected to pharmaceutical or hobby-related CT exposure. Their ages ranged between 26 and 51 (32 ± 6) years. Seventeen of them were smokers (19 ± 7 cigarettes/day).
For each subject, data regarding age, smoking and charcoal-grilled meat consumption, occupational or consistent environmental exposure to PAHs, medical history, domiciliary treatment for psoriasis and drugs taken during CT therapy were collected by means of a questionnaire, administered by trained interviewers. Recruitment was approved by the Ethics Committee of the University of Padova and all participants gave their informed consent. All information regarding participants was rendered anonymous after collection of data, urine and blood samples. Non-smokers were selected as individuals who had never smoked and smokers as those who smoked >10 cigarettes/day.
Sample collection
Urine (50 ml) and blood (2025 ml) samples were collected from 130 PAH-exposed subjects. Samples from psoriatic patients were collected 3 days after clinical CT treatment. Samples from workers were collected at the end of the work shift, after at least 3 consecutive days of work. Control urine samples were collected in the late afternoon.
Determination of urinary 1-pyrenol
1-Pyrenol in urine samples was determined as previously described (17), following the original method of Jongeneelen et al. (3). 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 alkaline medium.
DNA isolation from lymphocyte plus monocyte fraction (LMF)
LMF was isolated in Ficoll separating solution (Seromed, Berlin, Germany), as previously described (19). DNA from cells was isolated as previously described (20), following the original procedure described by Johns and Paulus-Thomas (21). DNA samples were precipitated with cold ethanol and washed several times with 70% ethanol. Ethanol washing was checked for the presence of peaks in the HPLC chromatograms at the retention time of anti-BPDE tetrols. The procedure provided DNA free of RNA and protein contamination, as checked by the 260:230 nm and 260:280 nm absorbance ratios of DNA, which were always ~2.3 and 1.7, respectively.
HPLC/fluorescence analysis of anti-BPDE tetrols
The method used in this study is identical to that reported by Alexandrov et al. (12) and validated by Rojas et al. (13). Briefly, a total amount of at least 100 µg DNA was used for each analysis. Many precautions were taken to avoid the presence of fluorescent contaminants: the absence of any fluorescent material in the purified HCl was checked by HPLC; tubes, HPLC syringes and other equipment were washed many times with HPLC-grade methanol and a blank injection was perfomed before each sample was subjected to HPLC analysis. DNA samples were dissolved in 0.1 N HCl and acid hydrolysis carried out at 90°C for 6 h. The resulting solution was analysed as described (12,13). The amounts of tetrols were calculated by comparing the areas of samples to be analysed with an external calibration curve, generated from the fluorescence peak of an authentic BPDE tetrol standard, every time a set of samples was analysed. Calibration was carried out with DNA from calf thymus, alone (background) and spiked with 2, 4, 10, 20, 40 and 100 pg anti-BPDE tetrol. These standard solutions were then treated in the same way as the tested samples (hydrolysed in 0.1 N HCl at 90°C for 6 h).The minimum correlation coefficient was 0.98 and the mean coefficient of variation for analyses repeated on different days was 16%. The detection threshold of anti-BPDE tetrols [r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P tetrol I-1) and r-7,t-9,t-10,t-8-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P tetrol I-2)] was 2 pg (signal-to-noise ratio >3) so that, in the present study, with 100 µg DNA, this assay can measure 2 adducts/108 nucleotides (1 fmol/µg DNA = 30 adducts/108 nucleotides).
Analysis of anti-BPDE tetrols by gas chromatography/mass spectroscopy (GC/MS)
The peak corresponding to B[a]P tetrol-I-1 collected from positive samples by different HPLC runs was analysed by means of GC/MS measurements after derivatization. Approximately 340 pg, calculated by fluorescence intensity, were treated with 50 µl bis(trimethylsilyl)acetamide in dimethylformamide (1:1) at 80°C for 1 h. Mass spectrometric measurements were performed with a Fisons (Altrincham, UK) MD 800 GC/MS instrument operating under electron ionization conditions (70 eV, 1800 µA, ion source temperature 180°C) and in the selected ion monitoring mode (m/z 389+ and 404+). GC conditions were as follows: column DB-1 (JZW, Folsom, CA) (300x0.25 mm, 0.25 µm film thickness); temperature program from 100 to 300°C at 15°C/min, then held for 10 min. Splitless injection was used with a sample size of 2 µl of the derivatized solution.
Statistic analysis
The 2 test was used in the proportion analysis of subjects with adduct levels exceeding the 95 percentile control subject value (8.9 adducts/108 nucleotides). P < 0.05 was considered to be significant. Linear multiple regression analysis was used to assess the influence on BPDEDNA adduct levels (Ln values) of three independent variables: occupational PAH exposure, smoking and dietary habit. Due to the exclusively skin PAH exposure of psoriatic patients, their data were not used in the regression analysis. PAH exposure was evaluated according to the mean urinary excretion of 1-pyrenol in each group. Subjects were divided into four exposure groups to which an arbitrary score was given according to the mean urinary excretion of 1-pyrenol of each group, as follows: 4, coke oven workers (mean ± SD urinary 1-pyrenol 1.2 ± 0.8 µmol/mol creatinine); 2, chimney sweeps (0.7 ± 0.7 µmol/mol creatinine); 1, aluminium anode plant workers (0.3 ± 0.3 µmol/mol creatinine); 0, non-occupationally PAH-exposed subjects (controls) (0.1 ± 0.1 µmol/mol creatinine). Smoking habit was attributed a value 1 or 0, referring to smokers and non-smokers, respectively. Diet was attributed a value 1 or 0 if charcoal-grilled meat consumption was more or less than once a week, respectively.
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Results |
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Table I lists smoking and dietary habits, mean (AM ± SD) urinary 1-pyrenol levels (in increasing order) and anti-BPDEDNA levels for each of the five groups examined (psoriatic patients, coke oven workers, chimney sweeps, aluminium anode plant workers and controls). For subjects with non-detectable adducts, a value of 2 adducts/108 nucleotides was assigned. Those with >2 adducts/108 nucleotides were considered positive. The presence of anti-BPDEDNA was detected in 20 of 70 (29%) samples from occupationally PAH-exposed subjects, most of them from coke oven workers (12 out of 22 positive samples; Table I
), and in eight of 34 (23%) control subjects (statistical comparison
2 test, coke oven workers versus controls, P < 0.01). The percentage of subjects with adduct levels exceeding the 95 percentile control subject value (8.9 adducts/108 nucleotides) was significantly high in coke oven workers (47%) and chimney sweeps (21%) (
2 test, P < 0.01 and P < 0.05, respectively) but not in aluminium plant workers (11%) or psoriatic patients (0%), compared with that of control subjects (3%). In particular, all 26 DNA samples from psoriatic patients, with high exposure to PAHs, as demonstrated by the exceptionally high levels of urinary 1-pyrenol (65.0 ± 57.9 µmol/mol creatinine), did not show markedly increased levels of anti-BPDEDNA adducts.
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Discussion |
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High occupational PAH exposure in coke oven workers and chimney sweeps significantly increased levels of BPDEDNA adducts, unlike aluminium workers and control subjects. No correlation was found between the levels of anti-BPDEDNA adducts measured in the 15 samples from coke oven workers by fluorescence in the present work and by 32P-post-labelling in the previous one (16). In the past 10 years, several studies have been reported on PAHDNA adduct levels in WBCs from occupationally exposed workers in coke oven plants, foundries and aluminium plants (see ref. 8 and references therein). Among the occupationally exposed groups, coke oven workers always had higher levels of aromatic adducts, as detected by the ELISA and 32P-post-labelling techniques. Direct comparison of DNA adduct values between different studies should be made with caution, because many variables may influence the results. The apparent levels of PAHDNA adducts in WBCs differ extensively depending on the method used. For example, the 32P-post-labelling assay applying thin-layer chromatography (TLC) often yields total PAH adduct levels lower than the observed BPDEDNA adduct levels alone by the HPLC/fluorescence technique (13,15,22,23). The cause is believed to be loss of adducts in the TLC analysis (23), while with the HPLC method such losses cannot take place (12,13,15,24). Thus, the previous results from 32P-post-labelling or ELISA, and hence the absolute quantification of total bulky adducts of unknown structure, must be regarded with some caution. One study has been described for BPDEDNA adduct levels in WBCs from coke oven workers using HPLC/fluorescence (15). The mean adduct levels and range match those of our present data. In the only study published on DNA adduct levels in chimney sweeps, a moderate but statistically insignificant increase in the level of WBC DNA adducts was detected by the 32P-post-labelling method. The authors concluded that a greater difference between chimney sweeps and controls could have been shown if purified lymphocytes had been used as a DNA source (25). The six studies evaluating PAH exposure in aluminium plant workers, determining DNA adducts using the 32P-post-labelling, ELISA and SFS methods, showed contrasting results: two positive (26,27) (i.e. with a significantly higher level of DNA adducts) and four negative (2831). This difference was attributed to varying PAH profiles and levels of exposure among the plants, together with different experimental procedures (DNA source and detection methods used).
In the present study, smoking habits did not enhance the levels of anti-BPDEDNA adducts in the five study groups. In other studies, no clear relationship has been shown between smoking habit and PAHDNA adducts in non-target tissues, such as peripheral WBCs: negative correlations (16,3234) and a synergistic effect between smoking and occupational PAH exposure (15,27) have been shown in the formation of DNA adducts in WBCs.
In the present study, charcoal-grilled meat consumption did not contribute to PAHDNA adduct levels in LMF. In a few studies on occupational PAH exposure in which charcoal-grilled meat consumption was checked, a positive association between PAHDNA adducts and diet was observed (35,36), but a negative association was shown if consumption had occurred >1 week prior to sampling (36,37). In two controlled feeding (grilled meat) studies with human volunteers, an overall response of increasing DNA adduct formation was not evident (38,39).
The acute skin PAH exposure of our psoriatic patients, estimated to be up to ~2 mg B[a]P/day, 1050 times higher than that of coke oven workers, as previously demonstrated not only by the exceptionally high levels of PAH metabolites and/or mutagenic compounds excreted in their urine, but also by the significant chromosomal damage detected in lymphocytes (40), surprisingly did not lead to anti-BPDEDNA adduct formation, as detected by HPLC/fluorescence. In our previous work (9), the levels of BPDEDNA adducts were determined by ELISA in the WBCs of 23 psoriatic patients during CT treatment. The mean adduct level was 7.7 ± 4.9 adducts/108 nucleotides, comparable with that reported more recently by Santella et al. (10) using ELISA. This discrepancy with the results of the present study may be explained by the cross-reactivity of the antiserum against various PAHDNA adducts, which enhances the responsiveness of the ELISA method versus structurally related PAH adducts (13,41). We also quantified PAHDNA adducts in LMF from psoriatic patients by the 32P-post-labelling technique, using both nuclease P1 enrichment and butanol extraction procedures. The mean total DNA adduct level after 8 days of continuous CT application was 0.54 ± 0.36 adducts/108 nucleotides, i.e. under the detection limit of our HPLC/fluorescence technique (42).
At present, the relative contribution of dermal and respiratory absorption of PAHs to the formation of DNA adducts is unknown. In two different studies on animal models, skin application of B[a]P in combination with various complex mixtures has been shown to decrease BPDEDNA adduct levels (43,44), indicating the presence of substances preventing B[a]P metabolites from binding to DNA (43). It should be noted that, although repeated topical treatment of psoriatic patients with CT gives rise to one of the highest PAH exposures, until now no clear evidence has been provided that CT therapy increases the incidence of skin cancer in such patients (45,46).
B[a]P metabolism is mediated by polymorphic enzymes: cytochrome P450 (CYP) 1A1 in the initial oxidation step and glutathione S-transferase (GST) in the conjugation step. The clear effect of the combination of CYP1A1 and GSTM1 genotypes on the formation of specific BPDEDNA adducts in human lung and WBCs has been demonstrated (24). The study of the influence of these combined genotypes concerning BPDEDNA adduct formation may help to identify high risk individuals among people exposed to PAHs.
In conclusion, our results show that the HPLC/fluorescence method may be applied to DNA adduct measurements to assess high chronic PAH exposure and that it is thus suitable for industrial health purposes. A large quantity of DNA is needed, ~100 µg DNA for each sample, to detect low occupational or environmental PAH exposure. In this study, in which no increased DNA adduct levels were seen in the LMF of psoriatic patients after skin CT treatment, the inhalatory pathway of PAH absorption probably contributes substantially to DNA adduct formation in the LMF of coke oven workers and chimney sweeps.
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Acknowledgments |
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Notes |
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References |
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