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Carcinogenesis, Vol. 20, No. 3, 431-435, March 1999
© 1999 Oxford University Press

HPLC/fluorescence determination of anti-BPDE–DNA adducts in mononuclear white blood cells from PAH-exposed humans

Sofia Pavanello1,5, Donata Favretto2, Francesco Brugnone3, Giuseppe Mastrangelo1, Giorgio Dal Pra4 and Erminio Clonfero1

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


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to compare (±)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)–DNA adduct levels in groups of humans subjected to various levels of polycyclic aromatic hydrocarbon (PAH) (benzo[a]pyrene) exposure. An HPLC/fluorescence method was applied to detect specifically anti-BPDE–DNA adducts in mononuclear white blood cells [lymphocyte plus monocyte fraction (LMF)] from humans exposed to PAHs. A total of 130 subjects comprised the sample population: 26 psoriatic patients (3 days after clinical coal tar treatment of the skin), 15 coke oven workers, 19 chimney sweeps, 36 aluminium anode plant workers and 34 non-occupationally PAH-exposed subjects (controls). PAH exposure was assessed in each group by means of the urinary excretion of 1-pyrenol (mean group levels: 1.2, 0.7, 0.3, 65.0 and 0.1 µmol/mol creatinine in coke oven workers, chimney sweeps, aluminium plant anode workers, psoriatic patients and non-occupationally PAH-exposed subjects, respectively). HPLC/fluorescence analysis of BPDE–DNA adducts showed that 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 (46.7%) and chimney sweeps (21.0%) ({chi}2 test, P < 0.01 and P < 0.05, respectively) but not in aluminium plant workers (11.1%) and psoriatic patients (0%). The increase in BPDE–DNA adduct levels in LMF (Ln values) was significantly related to chronic inhalatory and high PAH exposure (linear multiple regression analysis, F = 6.37, P < 0.01; t = 4.2, P < 0.001). Skin acute (or short-term) and high PAH exposure, charcoal-grilled meat consumption and smoking habit did not seem to influence BPDE–DNA adduct formation in LMF.

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.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously distributed carcinogenic compounds (1,2). DNA adduct and urinary 1-pyrenol determination have been widely used to identify populations exposed to PAHs and thus at increased risk of developing cancer. The level of 1-pyrenol, a pyrene metabolite, in urine has been shown to be a reliable indicator of PAH exposure from occupational, environmental, dietary and therapeutic sources (3–5). Determination of the extent of PAH metabolites covalently bound to DNA (PAH–DNA adducts) has been suggested as a biomarker of exposure to genotoxic PAHs (6). Sensitive detection methods such as ELISA, 32P-post-labelling and SFS have all been optimized (7) to detect adduct formation in the DNA of blood cells as a more readily accessible DNA source than that of the target tissues (e.g. lung and bladder).

Elevated PAH–DNA 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 BPDE–DNA 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 BPDE–DNA 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-BPDE–DNA 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.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
Enzymes for DNA purification (Proteinase K and RNase A) and calf thymus DNA were obtained from Sigma (Milan, Italy). HPLC-grade ethanol and methanol were purchased from Prolabo (Fontenay, France). Water was purified by means of a Milli-Q purification system (Millipore, Milford, MA). HCl (0.5 N Titrisol) and NaOH (1 N Titrisol) were from Merck (Darmstadt, Germany). anti-BPDE was purchased from the NCI Chemical Carcinogen Reference Standard Repository (Kansas City, MO).

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 20–80% 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 0.4–2.4 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 (16–18). 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 (20–25 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 {chi}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 BPDE–DNA 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.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The presence of anti-BPDE tetrols (B[a]P tetrol I-1) released after acid hydrolysis from 130 LMF DNA samples were determined by means of reverse phase HPLC and fluorescence detection. The identity of the B[a]P tetrol I-1 peaks was also confirmed by GC/MS analysis of the collected fractions (~320 pg) from several HPLC runs of the positive samples. In these pooled samples, it was possible to detect the base peak (m/z 404+) of the tetramethylsilyl derivative of B[a]P tetrol I-1 at the same retention time (17.1 min) as that of an authentic B[a]P tetrol I-1 standard (data not shown). This ion is thought to derive from loss of (CH3)3-Si-O-CH=CH-O-Si-(CH3)3 from the molecular ion (m/z 608+). anti-BPDE–DNA adduct levels of the 15 coke oven workers, measured in the present work by the HPLC/fluorescence method, were compared with those corresponding to anti-BPDE–DNA spots detected by 32P-post-labelling analysis in our previous work (16). No significant relationship was found between the levels of anti-BPDE–DNA adducts measured by HPLC/fluorescence or by 32P-post-labelling (Spearman's correlation coefficient –0.2, z = –0.6, P = 0.5).

Table IGo lists smoking and dietary habits, mean (AM ± SD) urinary 1-pyrenol levels (in increasing order) and anti-BPDE–DNA 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-BPDE–DNA 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 IGo), and in eight of 34 (23%) control subjects (statistical comparison {chi}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%) ({chi}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-BPDE–DNA adducts.


View this table:
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  		Table I. anti-BPDE–DNA adduct levels in LMF and urinary 1-pyrenol excretion in PAH-exposed humans
 
Table IIGo shows the results of linear multiple regression analysis of the influence of occupational exposure to PAHs (psoriatic patients excluded), smoking and charcoal-grilled meat consumption on the presence of anti-BPDE–DNA adducts in the LMF of 104 subjects (aluminium anode plant workers, coke oven workers, chimney sweeps and controls). Dependent variables were DNA adduct levels (Ln values); PAH exposure, smoking and charcoal-grilled meat consumption were independent variables. The analysis shows that an increase in BPDE–DNA adduct levels (Ln values) in LMF was significantly related to occupational PAH exposure (F = 6.37, P < 0.01 and t = 4.2, P < 0.001) rather than to the two other confounding factors of PAH intake, charcoal-grilled meat consumption and smoking habit. No relationship was found between individual 1-pyrenol levels and individual adduct levels (data not shown).


View this table:
[in this window]
[in a new window]
  		Table II. Linear multiple regression analysis of influence of occupational PAH exposure, smoking habit and diet on anti-BPDE-DNA adduct levels (Ln value) (n = 104) in LMF (F = 6.37, P < 0.01)
 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the present study, the HPLC/fluorescence technique was applied to detect the in vivo formation of anti-BPDE–DNA adducts in LMF from five groups of PAH-exposed humans: psoriatic patients, coke oven workers, chimney sweeps, aluminium anode plant workers and non-occupationallly PAH-exposed subjects (controls). HPLC/fluorescence assays allowed the specific identification of B[a]P tetrol I-1 deriving from acid hydrolysis of anti-BPDE–DNA adducts and were found to be sufficiently sensitive (detection limit 2 adducts/108 nucleotides using 100 µg DNA). This analytical method is also rapid and simple: it does not require the specific precautions which are indispensable when handling high specific activity 32P, carrying out DNA adduct enrichment procedures or using specific polyclonal or monoclonal antibodies. This method may also be developed to detect other PAH–DNA adducts by exploiting the high fluorescence of their aromatic ring systems. The use of GC/MS confirmed the identity of the anti-BPDE tetrols from fractions collected in several HPLC runs.

High occupational PAH exposure in coke oven workers and chimney sweeps significantly increased levels of BPDE–DNA adducts, unlike aluminium workers and control subjects. No correlation was found between the levels of anti-BPDE–DNA 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 PAH–DNA 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 PAH–DNA 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 BPDE–DNA 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 BPDE–DNA 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 (28–31). 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-BPDE–DNA adducts in the five study groups. In other studies, no clear relationship has been shown between smoking habit and PAH–DNA adducts in non-target tissues, such as peripheral WBCs: negative correlations (16,32–34) 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 PAH–DNA adduct levels in LMF. In a few studies on occupational PAH exposure in which charcoal-grilled meat consumption was checked, a positive association between PAH–DNA 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, 10–50 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-BPDE–DNA adduct formation, as detected by HPLC/fluorescence. In our previous work (9), the levels of BPDE–DNA 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 PAH–DNA adducts, which enhances the responsiveness of the ELISA method versus structurally related PAH adducts (13,41). We also quantified PAH–DNA 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 BPDE–DNA 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 BPDE–DNA adducts in human lung and WBCs has been demonstrated (24). The study of the influence of these combined genotypes concerning BPDE–DNA 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.


   Acknowledgments
 
The English text was checked by Gabriel Walton. This research was supported by a grant from the Regione Veneto `Ricerche Sanitarie Finalizzate', Italy.


   Notes
 
5 To whom correspondence should be addressed Email: clonfero{at}uxl.unipd.it Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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