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Non-smoking coke oven workers show an occupational PAH exposure-related increase in urinary mutagens
Mutation Research 562 (2004) 103–110
Non-smoking coke oven workers show an occupational PAH exposure-related increase in urinary mutagens P. Simioli a , S. Lupi a , P. Gregorio a , E. Siwinska b , D. Mielzynska b , E. Clonfero c , S. Pavanello c,∗ a
Section of Hygiene and Occupational Medicine, Department of Clinical and Experimental Medicine, University of Ferrara, Italy b Institute of Occupational Health, Sosnowiec, Poland c Occupational Health Section, Department of Environmental Medicine and Public Health, University of Padova, Italy Received 15 March 2004; received in revised form 5 May 2004; accepted 20 May 2004
Abstract We examined the urinary mutagenicity in the YG1024 Salmonella typhimurium strain in the presence of S9 mix, of 31 male non-smoking coke oven workers and an equal number of controls matched for gender and dietary habits. Occupational PAH exposure to the workers was assessed by means of the individual urinary post-shift excretion of 1-pyrenol (mean ± S.D.: 5.41 ± 6.06 mole/mol creatinine). Eleven urinary extracts of workers (35.5%) were clearly mutagenic (with at least a doubling of the number of spontaneous revertants), against only two samples in the control group (6.5%) (χ2 -test; χ2 = 7.883; P < 0.01). Moreover, the mean mutagenic activity level corrected for dilution/concentration of the urine was about three times higher in coke oven workers than in matched controls (mean ± S.D. (range) 495 ± 407 (89.7–1603) versus 186 ± 113 (14.2–524) net revertants/mmol creatinine; Mann–Whitney U-test, z = 3.86, P < 0.001). Simple linear regression analysis showed that the coke workers’ urinary mutagenic activity is associated with the PAH occupation-related urinary excretion of 1-pyrenol (r = 0.41, P = 0.0215). This study definitely demonstrates an occupation-related exposure of coke oven workers’ bladder epithelium to mutagenic PAH metabolites. This factor, mainly in the case of high exposure studied here, may account for a higher bladder cancer risk in coke oven workers. © 2004 Elsevier B.V. All rights reserved. Keywords: Coke oven workers; Urinary 1-pyrenol; Urinary mutagenicity
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are generated from incomplete combustion of organic ∗ Corresponding author. Tel.: +39 049 8216600; fax: +39 049 8212542. E-mail address: [email protected] (S. Pavanello).
materials and are widely distributed in the environment. Many PAHs have been identified as cancer-inducing chemicals for animals and/or humans [1]. Human exposure to PAHs may occur through smoking, occupational factors, polluted air, food consumption [2] and in medications [3,4]. Occupationally PAH-exposed groups include coke oven workers, chimney sweeps, aluminium workers, etc. There is
1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.05.009
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strong epidemiological evidence that these exposed groups have an increased risk for developing lung, bladder and skin cancer [5,6]. Many biomarkers have been developed to estimate PAH exposure levels and to assess the risk of adverse health effects in coke oven workers. Urinary 1-pyrenol is a well-validated and commonly used indicator of absorbed dose of aromatic hydrocarbons [7]. Urinary mutagenicity has long been used as a marker of exposure to environmental genotoxins of variable nature. Increased urinary mutagenicity has been demonstrated among people who smoke cigarettes [8], are exposed to mutagenic drugs [9], eat fried meats [10], and workers exposed to aromatic amines as in the rubber [11], textiles [12], ink and pharmaceutical [13] industries. Coke oven workers have been extensively studied over the last decade by means of this biologically relevant marker of genotoxic exposure. Both positive and negative findings on mutagenic activity in urine samples of PAH-exposed workers have been found by several authors. Some studies report increased urinary mutagenicity levels, but these results may be ascribed to insufficient control of confounding factors like smoking habits [14] and diet [15–17]. However, most studies suggest that coke oven urine samples are not mutagenic [18–20] because of the low sensitivity of most of the urinary mutagenicity assays in detecting occupation-related increases in mutagen excretion [21]. Our previous study [21], on low-exposed coke oven workers, reported clear mutagenic activity, detectable only in smokers’ urine samples and in a single positive non-smoking subject who was highly exposed. This finding suggests that smoke is able to mask urinary mutagenicity due to PAHs and the technique (assay or strain) used may not be sensitive enough. Until now, the presence of mutagens in PAH-exposed workers’ urine has been taken for granted, often including smokers, but no study was able to show it clearly. The aim of the present study was to clarify definitely if coke oven workers have a demonstrable occupation-related presence of mutagens in their urine. Only non-smoking subjects were enrolled, and diet-caused urinary mutagenicity was carefully controlled for. Biological monitoring of occupational PAH exposure by means of urinary 1-pyrenol analysis was assessed in coke oven workers. Urinary mutagenic activity was evaluated with the plate incorporation preincubation technique with S9, using the YG1024
Salmonella typhimurium strain over-sensitive to aromatic, heterocyclic amine and also PAH metabolites [22,23].
2. Materials and methods 2.1. Subjects Workers occupationally exposed to PAHs in a coke oven plant (n = 31) were studied and compared with healthy control subjects not occupationally exposed to PAHs (n = 31). All exposed workers (mean age 37, range 24–53) and all control subjects (clerks involved in a check-up sanitary program) were non-smoking males (mean age: 45; range 28–58). At the moment of urine collection participants were asked if they had consumed in the previous 24 h food with increasing urinary mutagenic capacity (i.e, high-temperature cooked meats). Controls’ urine sample collection was continued to obtain an equal number of subjects as that of coke oven workers matched for diet. All participants gave their informed consent. The occupation-exposed subjects were members of a group of 95 from a coke oven industry examined in our previous work with respect to the GSTM1 active and null genotype [24]. 2.2. Sample collection Urine samples from coke oven workers (at least 50 ml) were collected in polyethylene containers at the end of the work-shift, after at least three consecutive days of exposure to PAHs. Urine samples from controls were also collected in the late afternoon. All samples were stored in the dark at −20 ◦ C until analysis. 2.3. Evaluation of occupational PAH exposure 1-Pyrenol in urine samples was determined as previously described [21], following the original method of [25]. 2.4. Correction of measured values with respect to dilution/concentration of urine samples Concentrations of 1-pyrenol and urinary mutagenicity were adjusted for urinary creatinine, determined
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spectrophotometrically at 520 nm using a commercial kit (Boehringer, Mannheim, Germany) based on the reaction of creatinine with picrate in alkaline medium. 2.5. Urinary mutagenicity After being thawed and filtered, urine samples were concentrated in glass columns (1.5 cm × 10 cm) packed with washed XAD-2 resin (4 g/100 ml urine) [8] and eluted with 15 ml of acetone. Extracts were dried under a nitrogen stream, resuspended in dimethylsulfoxide (DMSO) (250 ml urine/ml) and placed in the dark at −20 ◦ C. Mutagenic activity was determined using the plate-incorporation preincubation technique on the YG1024 Salmonella typhimurium strain in the presence of Aroclor 1254-induced rat liver S9 (50 l per plate) (Moltox, Boone, NC, USA) [26]. The mean ± S.D. of spontaneous revertants in all experiments (n = 17) was 65.0 ± 7.2. 2-Aminofluorene (0.2 g per plate) was used as a positive control. Mutagenic activity was taken as positive when at least one of the tested doses was able to double the number of revertants with respect to spontaneous ones, and expressed as the slope of the linear portion of the dose–response curve calculated by the linear regression method, from at least two urine extract doses different from zero, as number of revertants/ml urine and number of revertants/mmole of creatinine. 2.6. Statistical analysis Frequencies of positive mutagenic urine samples of PAH-exposed workers and controls were compared by the χ2 -test. Comparison of urinary mutagenicity levels of workers and the control group was carried out using the Mann–Whitney U-test. Simple linear regression analysis was used to assess the influence of PAH exposure, evaluated by 1-pyrenol urinary excretion, on urinary mutagenic activity. All statistical tests were two-tailed and were performed with Statsdirect Statistical software (Ashwell, Herts, UK).
3. Results Thirty-one non-smoking male coke oven workers and controls, matched for dietary habits during the
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24 h before urine collection, comprised the analysed population. Eleven out of 31 workers and controls (35.5%) declared that they had consumed in the previous 24 h food with increasing urinary mutagenic capacity (i.e, high-temperature cooked meats). All data of urinary mutagenicity (expressed as number (N) of positive samples (at least a doubling of the number of spontaneous revertants at one assayed dose), Net rev/ml and Net rev/mmole creatinine), exposure to passive smoke and diet, for every coke-oven worker and control are shown in Tables 1 and 2. Occupational PAH-exposure was present in coke oven workers, as detected by urinary 1-pyrenol, a marker of occupationally PAH-absorbed dose, but not in controls. The sensitivity of our 1-pyrenol assay (LOQ) was 0.01 g/L. No subject was below the LOQ-value since all our subjects were highly PAH-exposed workers. The mean urinary 1-pyrenol level was 5.41 ± 6.06 (range 0.25–23.6) moles/mole creatinine and sixteen subjects (51%) exceeded the suggested biological exposure index of 2.3 moles/mole creatinine [27]. In our control subjects 1-pyrenol determination was not performed. Data from our laboratory showed very low urinary 1-pyrenol levels in a non-smoking control population: more than 80% subjects did not exceed the 0.10 mole/mole creatinine level for 1-pyrenol (mean ± S.D., 0.07 ± 0.1 mole/mole creatinine) and no relationship with mutagenic activity has been found since its increase is mainly associated to the dietary intake of food with increasing urinary mutagenic capacity [22,28]. Urinary mutagenicity values (the frequency of positive samples and mean ± S.D. urinary mutagenicity ) of non-smoking coke oven workers and controls are shown in Table 3. The urine extracts of 11 workers (35.5%) were clearly mutagenic (capable of at least doubling the number of spontaneous revertants at one assayed dose) whereas only 2 controls (6.5%) showed mutagenic urine (χ2 test, χ2 = 7.883; P < 0.01). As seen in Table 1, the frequency of coke oven workers’ positive samples for urinary mutagenicity with 1-pyrenol less than 1.50 mole/mole creatinine becomes quite similar to that found in non-exposed controls (1 out of 9 workers (11%) versus 2 out of 31 controls (6.5%)). Overall, coke oven workers showed twice as much urinary mutagenicity than the controls (4.6 ± 3.0
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Table 1 Diet, passive smoke, urinary 1-pyrenol and mutagenic activity of coke oven workers (in order of 1-pyrenol content) Coke oven workers
Dieta
Passive smokea
1-Pyrenol (moles/mole creatinine)
Positive sampleb
Net revertants/ml
Net revertants/ mmole creatinine
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 0 0 0 0 1 0 0 1 1 1 0 0 0 0 1
1 0 0 0 0 1 1 1 0 1 0 1 0 0 1 1 1 1 0 1 0 0 1 0 0 0 0 0 0 0 1
23.63 18.97 17.00 15.53 10.96 8.77 8.26 8.05 7.87 5.55 5.37 5.22 4.68 3.78 3.56 2.31 1.90 1.76 1.75 1.70 1.63 1.58 1.46 1.26 1.06 1.03 0.92 0.84 0.50 0.46 0.25
1 1 1 1 0 0 0 0 0 0 1 0 0 0 1 1 0 1 1 0 0 1 0 0 0 0 0 0 1 0 0
4.74 5.46 6.14 5.36 13.64 2.03 1.29 4.32 3.17 6.55 4.44 2.1 1.02 1.63 4.96 5.24 3.14 5.12 4.49 1.98 4.45 4.8 4.07 11.81 4.89 1.27 1.92 2.02 3.92 4.42 11.02
1094 532 1579 583 952 90 99 489 221 242 461 159 769 293 1603 498 740 240 430 222 1049 177 196 846 390 158 153 174 363 250 282
a 1 or 0, Consumption or not of food with increasing urinary mutagenic capacity and exposure or not to passive smoke 24 h before urine collection. b Mutagenic activity was taken as positive or not (1 or 0), when at least one of the tested doses was able to double the number of revertants with respect to the spontaneous level.
versus 2.4 ± 1.4 net revertants/ml, Mann–Whitney U-test, z = 3.36, P < 0.001). After adjustment of mutagenic activity for dilution/concentration of urine, PAH-exposed workers had about three times higher mutagen excretion than controls (mean ± S.D. 495 ± 407 versus 186 ± 113 net revertants/mmole creatinine, Mann–Whitney U-test, z = 3.86, P < 0.001). Simple regression analysis between mutagenic activity and 1-pyrenol levels in coke oven workers (Fig. 1) indicated that urinary mutagenicity was fairly low, but significantly related to occupational PAH exposure (r = 0.41, P = 0.0215).
4. Discussion We investigated the influence of occupational PAH exposure on urinary mutagenicity in non-smoking coke oven workers, detected with the sensitive YG1024 Salmonella typhimurium strain in the presence of an S9 metabolizing system. Since it is well known that cigarette smoking and diet influence urinary mutagenic activity [2], we enrolled only non-smoking people. Coke oven workers and controls were also matched for dietary habits in order to exclude the most important non-occupational source
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Table 2 Diet, passive smoke and mutagenic activity of controls Control-codex number
Dieta
Passive-smokea
Positive sampleb
Net revertants/ml
Net revertants/mmole creatinine
36 46 50 53 58 61 62 68 71 76 77 79 80 86 88 89 91 93 95 96 97 98 101 103 105 109 118 123 132 133 135
0 1 1 1 0 1 1 1 0 1 0 0 1 1 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3.4 4.1 4.1 0.2 3.4 2.3 1.3 2.9 1.6 0.6 2.5 1.4 7.0 1.0 5.2 2.8 2.6 1.9 1.7 2.2 1.7 2.1 2.4 1.5 1.9 2.6 2.4 1.3 2.3 3.3 1.4
195 229 257 14 125 172 309 117 85 75 227 524 393 85 390 301 238 144 222 178 150 82 112 114 277 211 112 58 134 254 88
a 1 or 0, Consumption or not of food with increasing urinary mutagenic capacity and exposure or not to passive smoke 24 h before urine collection. b Mutagenic activity was taken as positive or not (1 or 0), when at least one of the tested doses was able to double the number of revertants with respect to spontaneous ones.
Table 3 Mutagenic activity assayed on YG 1024 Salmonella typhimurium strain with metabolizing system (S9) in the urine of coke oven workers and controls Groups
Coke oven workers Controls
Urinary mutagenicity Positive samplesa N (%)
Net revertants/ml Mean ± S.D. (range)
Net revertants/mmole creatinine
11∗ (35.5) 2 (6.5)
4.6 ± 3.0 (1.0–13.6)∗∗ 2.4 ± 1.4 (0.2–7.0)
495 ± 407 (89.7–1603)∗∗ 186 ± 113 (14.2–524)
Statistical comparison: ∗ χ2 =7.883, P < 0.01; ∗∗ Mann–Whitney U-test, z = 3.36 and 3.86, P < 0.001. a At least doubling the number of spontaneous revertants.
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1600
1200
Urinary mutagenicity Net rev/mmole creatinine 800
400
0 10
0
20
1-pyrenol µmoles/mole creatinine Fig. 1. Simple regression analysis between mutagenic activity and 1-pyrenol concentration in urine sample from non-smoking coke oven workers. Urinary mutagenic activity (net revertants/mmole creatinine) = 345 + 27.6 × 1-pyrenol moles/mole creatinine. r = 0.41, P = 0.0215.
of urinary mutagens in non-smokers [29,30]. Even if passive smoke may have some influence in the controls, its effect is masked by the high occupational exposure to PAHs in coke oven workers. The mean value of urinary 1-pyrenol in the urine of exposed workers in this study (5.41 mole/mole creatinine) was about 5 times higher than the level found in coke oven workers in our previous work (mean ± S.D., 1.28 ± 1.11 mol/mol creatinine) [21], and the levels reported by [31] comparing the 1-pyrenol urinary levels found by several researchers between 1990 and 2000, in different coke oven plants. Although these studies also included old and not technologically updated plants, the average 1-pyrenol excretion was lower than 5.0 mole/mole creatinine. In the present study, urinary mutagenicity was significantly higher in coke oven workers than in controls. However, the available literature on coke production workers and urinary mutagenicity does not give a clear picture. [19] found no difference in this respect between non-smoking coke oven workers and non-smoking control workers. The only element
influencing mutagenicity was smoke. Instead, [16] reported, without controlling for diet as a confounding factor and without using a very sensitive mutant strain (TA98 and TA100), that non-smoking steel workers exposed to coke oven emissions had significantly higher urinary mutagenic activity than non-smoking controls, besides the strong influence of smoke. A Swedish study, using the same TA98 strain with S9 and providing a mutagen-free controlled diet, detected lower levels of urinary mutagens in non-smoking coke oven workers than in non-smoking control [20] confirming indirectly the importance of other unknown sources of variability of the assay. Ferreira et al. [32] and Mielzynska et al. [33], using TA98 with S9, reported higher mutagenic activity in exposed coke oven workers, but were unable to demonstrate a statistically significant difference attributable to occupational exposure, although in the second study 1-pyrenol levels were similar to those found in the present work. The latter result can be attributed to the low sensitivity of the Salmonella typhimurium strains employed. Both studies confirmed the significant influence of smoking habits on urinary mutagenicity.
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More recently [34], experiments using the oversensitive YG1024 Salmonella typhimurium strain [23] with metabolic S9 activation showed that urinary mutagenicity was higher in coke oven workers than in controls, but the difference was not statistically significant. Differences were found only between smoking (more mutagenic) and non-smoking coke oven workers. Again, the non-exclusion of smokers (most of both workers and controls) on occupational factors and the low number of controls (n = 10) may explain these findings. Significantly higher urinary mutagenicity was in fact observed in smoking workers, and the authors concluded that the only factor having a statistically significant effect on urinary mutagenic activity was smoke. Instead, our study shows that non-smoking coke oven workers’ urine, assayed by the plate-incorporation test with YG1024 Salmonella typhimurium strain and S9, has significantly increased mutagen levels in comparison with urine from non-exposed diet-matched controls. This mutagenic activity is related to urinary 1-pyrenol excretion which, at the high PAH-exposure levels of non-smoking coke oven workers, depends only on occupational factors [27]. Moreover, as already reported for non-smoking psoriatic patients treated with coal tar-based ointment rich in PAHs [23], it seems that coke oven workers without GSTM1 activity (GSTM1-null) have higher excretion of mutagens, detected by YG1024 strain, than those with active GSTM1. NAT2 can also influence urinary mutagenicity of coke oven workers, since in our previous work we demonstrated that NAT2 slow combined with GSTM1-null genotypes significantly increase mutagenic activity detected by normal-sensitive Salmonella TA 98 strain, but only in smoking coke oven workers [35]. This study suggests that a high-sensitivity urinary mutagenicity assay can be used for monitoring genotoxic PAH exposure of workers. However, special attention should be paid to the control of confounding factors such as smoking and diet habits. A PAH exposure-related level of 1.50 mole 1-pyrenol/mole creatinine, which is easily found in the post-shift coke oven workers’ urine and in other PAH-exposed workers particularly in cases of extensive contamination of the skin [36,37], has been suggested as benchmark for the presence of increased levels of urinary mutagens. The latter implies genotoxic exposure of coke oven
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workers’ bladder epithelium, which may account for the increased risk of bladder cancer in this population.
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Non-smoking coke oven workers show an occupational PAH exposure-related increase in urinary mutagens P. Simioli a , S. Lupi a , P. Gregorio a , E. Siwinska b , D. Mielzynska b , E. Clonfero c , S. Pavanello c,∗ a
Section of Hygiene and Occupational Medicine, Department of Clinical and Experimental Medicine, University of Ferrara, Italy b Institute of Occupational Health, Sosnowiec, Poland c Occupational Health Section, Department of Environmental Medicine and Public Health, University of Padova, Italy Received 15 March 2004; received in revised form 5 May 2004; accepted 20 May 2004
Abstract We examined the urinary mutagenicity in the YG1024 Salmonella typhimurium strain in the presence of S9 mix, of 31 male non-smoking coke oven workers and an equal number of controls matched for gender and dietary habits. Occupational PAH exposure to the workers was assessed by means of the individual urinary post-shift excretion of 1-pyrenol (mean ± S.D.: 5.41 ± 6.06 mole/mol creatinine). Eleven urinary extracts of workers (35.5%) were clearly mutagenic (with at least a doubling of the number of spontaneous revertants), against only two samples in the control group (6.5%) (χ2 -test; χ2 = 7.883; P < 0.01). Moreover, the mean mutagenic activity level corrected for dilution/concentration of the urine was about three times higher in coke oven workers than in matched controls (mean ± S.D. (range) 495 ± 407 (89.7–1603) versus 186 ± 113 (14.2–524) net revertants/mmol creatinine; Mann–Whitney U-test, z = 3.86, P < 0.001). Simple linear regression analysis showed that the coke workers’ urinary mutagenic activity is associated with the PAH occupation-related urinary excretion of 1-pyrenol (r = 0.41, P = 0.0215). This study definitely demonstrates an occupation-related exposure of coke oven workers’ bladder epithelium to mutagenic PAH metabolites. This factor, mainly in the case of high exposure studied here, may account for a higher bladder cancer risk in coke oven workers. © 2004 Elsevier B.V. All rights reserved. Keywords: Coke oven workers; Urinary 1-pyrenol; Urinary mutagenicity
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are generated from incomplete combustion of organic ∗ Corresponding author. Tel.: +39 049 8216600; fax: +39 049 8212542. E-mail address: [email protected] (S. Pavanello).
materials and are widely distributed in the environment. Many PAHs have been identified as cancer-inducing chemicals for animals and/or humans [1]. Human exposure to PAHs may occur through smoking, occupational factors, polluted air, food consumption [2] and in medications [3,4]. Occupationally PAH-exposed groups include coke oven workers, chimney sweeps, aluminium workers, etc. There is
1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.05.009
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strong epidemiological evidence that these exposed groups have an increased risk for developing lung, bladder and skin cancer [5,6]. Many biomarkers have been developed to estimate PAH exposure levels and to assess the risk of adverse health effects in coke oven workers. Urinary 1-pyrenol is a well-validated and commonly used indicator of absorbed dose of aromatic hydrocarbons [7]. Urinary mutagenicity has long been used as a marker of exposure to environmental genotoxins of variable nature. Increased urinary mutagenicity has been demonstrated among people who smoke cigarettes [8], are exposed to mutagenic drugs [9], eat fried meats [10], and workers exposed to aromatic amines as in the rubber [11], textiles [12], ink and pharmaceutical [13] industries. Coke oven workers have been extensively studied over the last decade by means of this biologically relevant marker of genotoxic exposure. Both positive and negative findings on mutagenic activity in urine samples of PAH-exposed workers have been found by several authors. Some studies report increased urinary mutagenicity levels, but these results may be ascribed to insufficient control of confounding factors like smoking habits [14] and diet [15–17]. However, most studies suggest that coke oven urine samples are not mutagenic [18–20] because of the low sensitivity of most of the urinary mutagenicity assays in detecting occupation-related increases in mutagen excretion [21]. Our previous study [21], on low-exposed coke oven workers, reported clear mutagenic activity, detectable only in smokers’ urine samples and in a single positive non-smoking subject who was highly exposed. This finding suggests that smoke is able to mask urinary mutagenicity due to PAHs and the technique (assay or strain) used may not be sensitive enough. Until now, the presence of mutagens in PAH-exposed workers’ urine has been taken for granted, often including smokers, but no study was able to show it clearly. The aim of the present study was to clarify definitely if coke oven workers have a demonstrable occupation-related presence of mutagens in their urine. Only non-smoking subjects were enrolled, and diet-caused urinary mutagenicity was carefully controlled for. Biological monitoring of occupational PAH exposure by means of urinary 1-pyrenol analysis was assessed in coke oven workers. Urinary mutagenic activity was evaluated with the plate incorporation preincubation technique with S9, using the YG1024
Salmonella typhimurium strain over-sensitive to aromatic, heterocyclic amine and also PAH metabolites [22,23].
2. Materials and methods 2.1. Subjects Workers occupationally exposed to PAHs in a coke oven plant (n = 31) were studied and compared with healthy control subjects not occupationally exposed to PAHs (n = 31). All exposed workers (mean age 37, range 24–53) and all control subjects (clerks involved in a check-up sanitary program) were non-smoking males (mean age: 45; range 28–58). At the moment of urine collection participants were asked if they had consumed in the previous 24 h food with increasing urinary mutagenic capacity (i.e, high-temperature cooked meats). Controls’ urine sample collection was continued to obtain an equal number of subjects as that of coke oven workers matched for diet. All participants gave their informed consent. The occupation-exposed subjects were members of a group of 95 from a coke oven industry examined in our previous work with respect to the GSTM1 active and null genotype [24]. 2.2. Sample collection Urine samples from coke oven workers (at least 50 ml) were collected in polyethylene containers at the end of the work-shift, after at least three consecutive days of exposure to PAHs. Urine samples from controls were also collected in the late afternoon. All samples were stored in the dark at −20 ◦ C until analysis. 2.3. Evaluation of occupational PAH exposure 1-Pyrenol in urine samples was determined as previously described [21], following the original method of [25]. 2.4. Correction of measured values with respect to dilution/concentration of urine samples Concentrations of 1-pyrenol and urinary mutagenicity were adjusted for urinary creatinine, determined
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spectrophotometrically at 520 nm using a commercial kit (Boehringer, Mannheim, Germany) based on the reaction of creatinine with picrate in alkaline medium. 2.5. Urinary mutagenicity After being thawed and filtered, urine samples were concentrated in glass columns (1.5 cm × 10 cm) packed with washed XAD-2 resin (4 g/100 ml urine) [8] and eluted with 15 ml of acetone. Extracts were dried under a nitrogen stream, resuspended in dimethylsulfoxide (DMSO) (250 ml urine/ml) and placed in the dark at −20 ◦ C. Mutagenic activity was determined using the plate-incorporation preincubation technique on the YG1024 Salmonella typhimurium strain in the presence of Aroclor 1254-induced rat liver S9 (50 l per plate) (Moltox, Boone, NC, USA) [26]. The mean ± S.D. of spontaneous revertants in all experiments (n = 17) was 65.0 ± 7.2. 2-Aminofluorene (0.2 g per plate) was used as a positive control. Mutagenic activity was taken as positive when at least one of the tested doses was able to double the number of revertants with respect to spontaneous ones, and expressed as the slope of the linear portion of the dose–response curve calculated by the linear regression method, from at least two urine extract doses different from zero, as number of revertants/ml urine and number of revertants/mmole of creatinine. 2.6. Statistical analysis Frequencies of positive mutagenic urine samples of PAH-exposed workers and controls were compared by the χ2 -test. Comparison of urinary mutagenicity levels of workers and the control group was carried out using the Mann–Whitney U-test. Simple linear regression analysis was used to assess the influence of PAH exposure, evaluated by 1-pyrenol urinary excretion, on urinary mutagenic activity. All statistical tests were two-tailed and were performed with Statsdirect Statistical software (Ashwell, Herts, UK).
3. Results Thirty-one non-smoking male coke oven workers and controls, matched for dietary habits during the
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24 h before urine collection, comprised the analysed population. Eleven out of 31 workers and controls (35.5%) declared that they had consumed in the previous 24 h food with increasing urinary mutagenic capacity (i.e, high-temperature cooked meats). All data of urinary mutagenicity (expressed as number (N) of positive samples (at least a doubling of the number of spontaneous revertants at one assayed dose), Net rev/ml and Net rev/mmole creatinine), exposure to passive smoke and diet, for every coke-oven worker and control are shown in Tables 1 and 2. Occupational PAH-exposure was present in coke oven workers, as detected by urinary 1-pyrenol, a marker of occupationally PAH-absorbed dose, but not in controls. The sensitivity of our 1-pyrenol assay (LOQ) was 0.01 g/L. No subject was below the LOQ-value since all our subjects were highly PAH-exposed workers. The mean urinary 1-pyrenol level was 5.41 ± 6.06 (range 0.25–23.6) moles/mole creatinine and sixteen subjects (51%) exceeded the suggested biological exposure index of 2.3 moles/mole creatinine [27]. In our control subjects 1-pyrenol determination was not performed. Data from our laboratory showed very low urinary 1-pyrenol levels in a non-smoking control population: more than 80% subjects did not exceed the 0.10 mole/mole creatinine level for 1-pyrenol (mean ± S.D., 0.07 ± 0.1 mole/mole creatinine) and no relationship with mutagenic activity has been found since its increase is mainly associated to the dietary intake of food with increasing urinary mutagenic capacity [22,28]. Urinary mutagenicity values (the frequency of positive samples and mean ± S.D. urinary mutagenicity ) of non-smoking coke oven workers and controls are shown in Table 3. The urine extracts of 11 workers (35.5%) were clearly mutagenic (capable of at least doubling the number of spontaneous revertants at one assayed dose) whereas only 2 controls (6.5%) showed mutagenic urine (χ2 test, χ2 = 7.883; P < 0.01). As seen in Table 1, the frequency of coke oven workers’ positive samples for urinary mutagenicity with 1-pyrenol less than 1.50 mole/mole creatinine becomes quite similar to that found in non-exposed controls (1 out of 9 workers (11%) versus 2 out of 31 controls (6.5%)). Overall, coke oven workers showed twice as much urinary mutagenicity than the controls (4.6 ± 3.0
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Table 1 Diet, passive smoke, urinary 1-pyrenol and mutagenic activity of coke oven workers (in order of 1-pyrenol content) Coke oven workers
Dieta
Passive smokea
1-Pyrenol (moles/mole creatinine)
Positive sampleb
Net revertants/ml
Net revertants/ mmole creatinine
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 0 0 0 0 1 0 0 1 1 1 0 0 0 0 1
1 0 0 0 0 1 1 1 0 1 0 1 0 0 1 1 1 1 0 1 0 0 1 0 0 0 0 0 0 0 1
23.63 18.97 17.00 15.53 10.96 8.77 8.26 8.05 7.87 5.55 5.37 5.22 4.68 3.78 3.56 2.31 1.90 1.76 1.75 1.70 1.63 1.58 1.46 1.26 1.06 1.03 0.92 0.84 0.50 0.46 0.25
1 1 1 1 0 0 0 0 0 0 1 0 0 0 1 1 0 1 1 0 0 1 0 0 0 0 0 0 1 0 0
4.74 5.46 6.14 5.36 13.64 2.03 1.29 4.32 3.17 6.55 4.44 2.1 1.02 1.63 4.96 5.24 3.14 5.12 4.49 1.98 4.45 4.8 4.07 11.81 4.89 1.27 1.92 2.02 3.92 4.42 11.02
1094 532 1579 583 952 90 99 489 221 242 461 159 769 293 1603 498 740 240 430 222 1049 177 196 846 390 158 153 174 363 250 282
a 1 or 0, Consumption or not of food with increasing urinary mutagenic capacity and exposure or not to passive smoke 24 h before urine collection. b Mutagenic activity was taken as positive or not (1 or 0), when at least one of the tested doses was able to double the number of revertants with respect to the spontaneous level.
versus 2.4 ± 1.4 net revertants/ml, Mann–Whitney U-test, z = 3.36, P < 0.001). After adjustment of mutagenic activity for dilution/concentration of urine, PAH-exposed workers had about three times higher mutagen excretion than controls (mean ± S.D. 495 ± 407 versus 186 ± 113 net revertants/mmole creatinine, Mann–Whitney U-test, z = 3.86, P < 0.001). Simple regression analysis between mutagenic activity and 1-pyrenol levels in coke oven workers (Fig. 1) indicated that urinary mutagenicity was fairly low, but significantly related to occupational PAH exposure (r = 0.41, P = 0.0215).
4. Discussion We investigated the influence of occupational PAH exposure on urinary mutagenicity in non-smoking coke oven workers, detected with the sensitive YG1024 Salmonella typhimurium strain in the presence of an S9 metabolizing system. Since it is well known that cigarette smoking and diet influence urinary mutagenic activity [2], we enrolled only non-smoking people. Coke oven workers and controls were also matched for dietary habits in order to exclude the most important non-occupational source
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Table 2 Diet, passive smoke and mutagenic activity of controls Control-codex number
Dieta
Passive-smokea
Positive sampleb
Net revertants/ml
Net revertants/mmole creatinine
36 46 50 53 58 61 62 68 71 76 77 79 80 86 88 89 91 93 95 96 97 98 101 103 105 109 118 123 132 133 135
0 1 1 1 0 1 1 1 0 1 0 0 1 1 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3.4 4.1 4.1 0.2 3.4 2.3 1.3 2.9 1.6 0.6 2.5 1.4 7.0 1.0 5.2 2.8 2.6 1.9 1.7 2.2 1.7 2.1 2.4 1.5 1.9 2.6 2.4 1.3 2.3 3.3 1.4
195 229 257 14 125 172 309 117 85 75 227 524 393 85 390 301 238 144 222 178 150 82 112 114 277 211 112 58 134 254 88
a 1 or 0, Consumption or not of food with increasing urinary mutagenic capacity and exposure or not to passive smoke 24 h before urine collection. b Mutagenic activity was taken as positive or not (1 or 0), when at least one of the tested doses was able to double the number of revertants with respect to spontaneous ones.
Table 3 Mutagenic activity assayed on YG 1024 Salmonella typhimurium strain with metabolizing system (S9) in the urine of coke oven workers and controls Groups
Coke oven workers Controls
Urinary mutagenicity Positive samplesa N (%)
Net revertants/ml Mean ± S.D. (range)
Net revertants/mmole creatinine
11∗ (35.5) 2 (6.5)
4.6 ± 3.0 (1.0–13.6)∗∗ 2.4 ± 1.4 (0.2–7.0)
495 ± 407 (89.7–1603)∗∗ 186 ± 113 (14.2–524)
Statistical comparison: ∗ χ2 =7.883, P < 0.01; ∗∗ Mann–Whitney U-test, z = 3.36 and 3.86, P < 0.001. a At least doubling the number of spontaneous revertants.
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1600
1200
Urinary mutagenicity Net rev/mmole creatinine 800
400
0 10
0
20
1-pyrenol µmoles/mole creatinine Fig. 1. Simple regression analysis between mutagenic activity and 1-pyrenol concentration in urine sample from non-smoking coke oven workers. Urinary mutagenic activity (net revertants/mmole creatinine) = 345 + 27.6 × 1-pyrenol moles/mole creatinine. r = 0.41, P = 0.0215.
of urinary mutagens in non-smokers [29,30]. Even if passive smoke may have some influence in the controls, its effect is masked by the high occupational exposure to PAHs in coke oven workers. The mean value of urinary 1-pyrenol in the urine of exposed workers in this study (5.41 mole/mole creatinine) was about 5 times higher than the level found in coke oven workers in our previous work (mean ± S.D., 1.28 ± 1.11 mol/mol creatinine) [21], and the levels reported by [31] comparing the 1-pyrenol urinary levels found by several researchers between 1990 and 2000, in different coke oven plants. Although these studies also included old and not technologically updated plants, the average 1-pyrenol excretion was lower than 5.0 mole/mole creatinine. In the present study, urinary mutagenicity was significantly higher in coke oven workers than in controls. However, the available literature on coke production workers and urinary mutagenicity does not give a clear picture. [19] found no difference in this respect between non-smoking coke oven workers and non-smoking control workers. The only element
influencing mutagenicity was smoke. Instead, [16] reported, without controlling for diet as a confounding factor and without using a very sensitive mutant strain (TA98 and TA100), that non-smoking steel workers exposed to coke oven emissions had significantly higher urinary mutagenic activity than non-smoking controls, besides the strong influence of smoke. A Swedish study, using the same TA98 strain with S9 and providing a mutagen-free controlled diet, detected lower levels of urinary mutagens in non-smoking coke oven workers than in non-smoking control [20] confirming indirectly the importance of other unknown sources of variability of the assay. Ferreira et al. [32] and Mielzynska et al. [33], using TA98 with S9, reported higher mutagenic activity in exposed coke oven workers, but were unable to demonstrate a statistically significant difference attributable to occupational exposure, although in the second study 1-pyrenol levels were similar to those found in the present work. The latter result can be attributed to the low sensitivity of the Salmonella typhimurium strains employed. Both studies confirmed the significant influence of smoking habits on urinary mutagenicity.
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More recently [34], experiments using the oversensitive YG1024 Salmonella typhimurium strain [23] with metabolic S9 activation showed that urinary mutagenicity was higher in coke oven workers than in controls, but the difference was not statistically significant. Differences were found only between smoking (more mutagenic) and non-smoking coke oven workers. Again, the non-exclusion of smokers (most of both workers and controls) on occupational factors and the low number of controls (n = 10) may explain these findings. Significantly higher urinary mutagenicity was in fact observed in smoking workers, and the authors concluded that the only factor having a statistically significant effect on urinary mutagenic activity was smoke. Instead, our study shows that non-smoking coke oven workers’ urine, assayed by the plate-incorporation test with YG1024 Salmonella typhimurium strain and S9, has significantly increased mutagen levels in comparison with urine from non-exposed diet-matched controls. This mutagenic activity is related to urinary 1-pyrenol excretion which, at the high PAH-exposure levels of non-smoking coke oven workers, depends only on occupational factors [27]. Moreover, as already reported for non-smoking psoriatic patients treated with coal tar-based ointment rich in PAHs [23], it seems that coke oven workers without GSTM1 activity (GSTM1-null) have higher excretion of mutagens, detected by YG1024 strain, than those with active GSTM1. NAT2 can also influence urinary mutagenicity of coke oven workers, since in our previous work we demonstrated that NAT2 slow combined with GSTM1-null genotypes significantly increase mutagenic activity detected by normal-sensitive Salmonella TA 98 strain, but only in smoking coke oven workers [35]. This study suggests that a high-sensitivity urinary mutagenicity assay can be used for monitoring genotoxic PAH exposure of workers. However, special attention should be paid to the control of confounding factors such as smoking and diet habits. A PAH exposure-related level of 1.50 mole 1-pyrenol/mole creatinine, which is easily found in the post-shift coke oven workers’ urine and in other PAH-exposed workers particularly in cases of extensive contamination of the skin [36,37], has been suggested as benchmark for the presence of increased levels of urinary mutagens. The latter implies genotoxic exposure of coke oven
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workers’ bladder epithelium, which may account for the increased risk of bladder cancer in this population.
Acknowledgements Supported by grant B/40-2/DML/01 of National Institute of Occupational Safety and Prevention (ISPESL), Rome, Italy. References [1] IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Polycyclyc Aromatic Hydrocarbons. Part 1. Chemical, Environmental and Experimental Data, vol. 32, International Agency for Research on Cancer, Lyon, France, 1983. [2] J.G.M. Van Rooij, M.M.S. Veeger, M.M. Bodelier-Bade, P.T. Scheepers, F.J. Jongeneelen, Smoking and dietary intake of polycyclic aromatic hydrocarbons as source of interindividual variability in the baseline excretion of 1-hydroxypyrene in urine, Int. Arch. Occup. Environ. Health 66 (1994a) 55–65. [3] E. Clonfero, M. Zordan, P. Venier, M. Paleologo, A.G. Levis, D. Cottica, L. Pozzoli, F.J. Jongeneelen, R.P. Bos, R.B. Anzion, Biological monitoring of human exposure to coal tar. Urinary excretion of total polycyclic aromatic hydrocarbons, 1-hydroxypyrene and mutagens in psoriatic patients, Int. Arch. Occup. Environ. Health 61 (1989) 363–368. [4] R.M. Santella, M.G. Nunes, R. Blaskovic, F.P. Perera, D. Tang, A. Beachman, J.H. Lin, V.A. DeLeo, Quantitation of polycyclic aromatic hydrocarbons, 1-hydroxypyrene and mutagenicity in urine of coal tar-treated psoriasis patients and untreated volunteers, Cancer Epidemiol. Biomarkers Prev. 3 (1994) 137–140. [5] IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Polycyclic Aromatic Hydrocarbons. Part 3. Industrial Exposures in Aluminium Production, Coal Gasification, Coke Production, and Iron and Steel Founding, vol. 34, International Agency for Research on Cancer, Lyon, France, 1984. [6] E.S. Hansen, Cancer mortality in asphalt industry: a 10 year follow up of an occupational cohort, Br. J. Ind. Med. 46 (1989) 582–585. [7] F.J. Jongeneelen, F.E. van Leeuwen, S. Oosterink, R.B. Anzion, F. van der Loop, R.P. Bos, H.G. van Veen, Ambient and biological monitoring of the coke oven workers: determinants of the internal dose of polycyclic aromatic hydrocarbons, Br. J. Int. Med. 47 (1990) 454–461. [8] E. Yamasaki, B.N. Ames, Concentration of mutagens from urine by adsorption with the non-polar resin XAD-2: cigarette smokers have mutagenic urine, Proc. Natl. Acad. Sci. USA 74 (1977) 3555–3559. [9] K. Falck, P. Gröhn, M. Sorsa, H. Vainio, E. Heinonen, L.R. Holsti, Mutagenicity in urine of nurses handling cytostatic drugs, Lancet 1 (1979) 1250–1251.
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