Tnt A-Iti J~11l 1z-t I tl VCCU~,l,
trn S JiI Tla 1781 i Qi)fl ) U J-~tl Vlt UU 11 ~tlllt 177/~) UJ J 71
't~J
International e Archives of
Environmental Health © Springer-Verlag 1992
Evaluation of urinary excretion of 1-hydroxypyrene and thioethers in workers exposed to bitumen fumes Sema Burgazl , Paul J A Borm 2 , and Frans J Jongeneelen 3 1Department of Toxicology, Faculty of Pharmacy, Gazi University, 06330, Hipodrom, Ankara, Turkey Department of Occupational and Environmental Health & Toxicology, University of Limburg, PO Box 616, 6200 MD Maastricht, The Netherlands 3 Department of Toxicology, University of Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands 2
Received October 24, 1990 / Accepted October 21, 1991
Summary Biological monitoring of exposure to bitumen fumes during road-paving operations was carried out In order to evaluate the biological uptake of the workers, the nonselective urinary thioether assay and a selective method for the determination of urinary 1-hydroxypyrene were used Urinary thioether data of exposed workers were higher than those of nonexposed subjects The effect of smoking, however, was stronger than the effect of occupational exposure Levels of 1-hydroxypyrene in road-paving workers were significantly higher than those in control subjects The 1-hydroxypyrene level was also influenced by smoking habits, but the effect of occupational exposure was stronger Our present data suggest that enhanced urine levels of both thioethers and 1-hydroxypyrene in bitumen workers may indicate an increased genotoxic risk Furthermore, our results demonstrate the applicability of the 1-hydroxypyrene assay after occupational exposure to petroleum-based products. Key words: Urinary thioether 1-Hydroxypyrene excretion Bitumen fumes Occupational exposure
Introduction It is estimated that the current annual use of bitumen in Turkey is approximately 0 35 million tons As bitumens are used in many different forms of road construction and maintenance, no data are available on the number of workers exposed to bitumens and bitumen fumes. There is also no information on the extent of their exposure and little on their health risks Thus, it is important to assess the exposure of workers to polycyclic aromatic hydrocarbons (PA Hs) during road-paving operations with bitumens. Bitumen samples and fumes consist essentially of hydrocarbons and their derivatives, some of which are known to be carcinogenic or cocarcinogenic The levels Offprint requests to: S Burgaz
of total PA Hs are moderate when compared with coal-tar products l4, 12, 24, 37 l There are only a few data on the potential carcinogenicity and/or toxic effects of bitumens on humans l9, 12, 27 l. In the previous study, biological monitoring of exposure to bitumen fumes was carried out using the nonselective thioether assay l3 l In that study, no significant increase in thioether concentration was detected that could be related to work exposure Recent studies have shown that 1-hydroxypyrene in urine can be considered an useful biological marker for occupational exposure to PA Hs Increased urinary levels of 1-hydroxypyrene have been found in patients cutaneously treated with coal tar, in workers exposed to the wood preservative creosote oil, in coal tar distillary workers, and in road-paving workers using coal tar blended binder l6, 16-19l. The present study was undertaken to investigate the bitumen exposure of paving workers with the aim of evaluating (a) the urinary thioethers and 1-hydroxypyrene excretion data and (b) the utility of the above two parameters for biological monitoring of exposure to petroleum-based products.
Materials and methods Road-paving operations Typical paving operations with bitumens are as follows: A hot mixture of stone chips and bitumen is delivered to a finishing machine, where it is discharged into a hopper. Conveyors in the machine then spread the mixture to a fixed width, and it is levelled and compacted to the thickness required. Further compaction is conducted using steel-tyred rollers by roller drivers The finishing-machine operator sits about 2 m above road level; the rakermen correct imperfections and complete edges on the newly laid asphalt layer immediately behind the finisher and/ or, depending on the nature of application, hot bitumen mixture is shovelled into a wheelbarrow, and throwers apply it to the surface by shovel The rakermen spread the mixture immediately behind the steel-tyred rollers A foreman controls all these operations. The exposure to bitumen fumes is evident during these road-paving processes. Subjects The exposed workers were 39 men employed in road bituminization Some 29 men selected from university staff and
398 Table 1 Characteristics of exposed group and controls with respect to age, smoking, use of alcohol, and medication Exposed group (n = 39)
Controls (n = 29)
Mean age (years)
35 5 (22-55)
36 3 (21-62)
Cigarettes/day (%) 0 1-10 11-20 > 20 Alcohol (yes) Medication (yes)
46 2 51 38 5 10 2 5 13
51 7 20 7 24 1 35 1 9
students served as a control group None of the referents had been occupationally exposed to potential genotoxic agents, and no subject (exposed or control) was using genotoxic agents in connection with leisure-time activities All subjects were asked to complete a questionnaire which inquired about age, smoking habits, recent use of medications, and alcohol intake The characteristics of the exposed and control group are given in Table 1. The majority of the exposed workers had worked on road-paving operations for at least 2 weeks prior to the study In order to evaluate the job exposure differences, the workers were classified as follows; roller driver (n = 10), thrower (n = 12), rakerman (n = 14), finishing machine operator (n = 2), and foreman (n = 1). All subjects ate a normal diet Most of the bitumens used in these applications were steam-refined and had 75-100 penetration values (class 1) Small amounts of cutback bitumens (class 3) were used in minor applications. Urine samples Urine samples from exposed subjects were collected at the end of the (8-h) working day on Thursday or Friday. Urine samples from control subjects were collected at different times of the day, whenever convenient After collection, the urine samples were divided into two aliquots to be used for the thioether assay and the analysis of 1-hydroxypyrene content A few milliliters were stored separately for the creatinine assay All urine samples were stored at -20 °C without any preservatives until analysis. Urinary thioethers were determined by the method described by Seutter-Berlage et al l29 l Urinary levels of thioether compounds were expressed as mmol SH-/mol creatinine Coefficient of variation (CV) of repeated analysis on different days was 2 9 %. The method for the determination of urinary levels of 1-hydroxypyrene was developed by Jongeneelen et al l 15 l The determination is based on the enzymatic hydrolysis of the conjugated metabolites, followed by their solid phase extraction The retained metabolites are then eluted and 1-hydroxypyrene in the eluate is determined by high-performance liquid chromatography (HPLC). HPLC analysis allowed the quantification of the sum of free and conjugated 1-hydroxypyrene in urine at the nmol/l level Urinary 1-hydroxypyrene concentrations were corrected for creatinine. Coefficient of variation (CV) repeated analysis on different days was 12 6 %. The creatinine content of the urine samples was measured by the method based on the reaction of creatinine with alkaline picrate l 8l Urine samples with creatinine values below 5 mmol/l were regarded as not reliable and excluded from the analysis of data. Statistical analysis The different sets of data were examined for normal distribution by the Shapiro and Wilk test Since observed distributions of biological factors data, thioether and 1-hydroxypyrene excretion, deviated from normality, the values were transformed into logarithms However, means and standard deviations are presented as untransformed values for ease of interpretation. To evaluate the several types of possible influences on the biologi-
cal parameters expressing PA Hs exposure, five-way analysis of variance (ANOVA) was used This analysis includes exposure (yes/no), age class, smoking (yes/no), drug intake (yes/no), and recent alcohol intake (yes/no). The effect of job category of the exposed workers and smoking on thioether and 1-hydroxypyrene excretion was also tested with two-way ANOVA. Correlation was qualified with the linear regression analysis. All tests were two-sided.
Results The concentrations of thioethers and 1-hydroxypyrene in urine samples of exposed and nonexposed groups are given in Table 2 Table 3 indicates the results of the ANOVA from which we can observe that exposure and smoking significantly (P < 0 01) contribute to the sum of squares for each of the two biological parameters, thio-
ethers and 1-hydroxypyrene From Tables 2 and 3, it is evident that the bitumen fumes during road-surfacing operations are absorbed by exposed workers Increases in the thioether excretion may be related to exposure (P = 0 011) Thioether excretion among nonsmokers
Table 2 Mean values (+ SD) of urinary thioether and 1-hydroxypyrene excretions of exposed and nonexposed groups, according to smoking habits Groups
Urinary thio ether excretion (mmol SH-/mol creatinine)
Urinary 1-hydroxypyrene excretion (gmol/mol creatinine)
Total exposed (n = 39)
7 76 + 4 70
0 61 + 0 38
Total nonexposed (n = 29)
4 61 ±+2 59
0 28 ± 0 17
Exposed Nonsmokers (n = 18) Smokers (n = 21)
6 93 + 3 68 8 47 + 5 42
0 53 ± 0 45 0 67 + 0 30
Nonexposed Nonsmokers (n = 15) Smokers (n = 14)
3 20 ± 2 40 6 12 + 1 88
0 24 + 0 13 0 33 ± 0 19
Table 3 Analyses of variance for the dependent variables 1-hydroxypyrene and thioethers Source of variance
df
Exposureb Smokingb Medicationb Alcoholb Age class
1 1 1 1 3
Log (hydroxypyrene)
Log (thioethers)
F value
P value
F value
P value
25 36 6 86 0 10 0 48 0 63
0 0001 a 0 0111 a 0 748 0 491 0 600
6 9 3 0 0
0 0 0 0 0
df, degrees of freedom a F value significant at 1 % level b Yes = 1, no= O c Class 1, < 30 years; 2, 30-39 ; 3, 40-49 ; 4, > 49
93 14 76 04 24
0107 a 0037 a 057 839 870
399 NS
S
NS
S
S
NS -
o
20.
.2.0
.
0 e 16.
.1.6
0
:'
0
0
0
12.
0
·
1.2 O
.
E I I
S
8 E E
o o
0
0
4.
8
0
0o
_
-0
_
r
o
a
0
0
0 o
o S
0
o
0
.Os 0.8
0
·
0
s.
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·
.0.4
o ___T
Roller driver
S.
0
0
.
·
Thrower
S
0o
Fig 1 Urinary thioether (0) (mmol SH-/mol creatinine) and 1-hydroxypyrene levels () (gmol/mol creatinine) of workers exposed to bitumen fumes, according to job classification and smoking habits S, smoking workers; NS, nonsmoking workers Straight line is the mean control level of thioethers for each group
Rakerman
was statistically higher in exposed that in nonexposed subjects On the other hand, the effect of smoking (P = 0.004) was found to be stronger than that of occupational exposure, and medication seems to influence the urinary thioethers level (P = 0 057) The most frequently taken medicines by subjects were analgesics, stomach pills, and vitamins. Table 3 also indicates that the effect of occupational exposure on 1-hydroxypyrene levels (P = 0 0001) is stronger that that of smoking (P = 0 011) Tables 2 and 3 show that smoking increases the excretion of 1-hydroxypyrene, but it is not a strong determinant Medication did not influence the 1-hydroxypyrene level (P = 0 748) Age and alcohol intake have no significant effect on both biological parameters (Table 3). The correlation between the 1-hydroxypyrene excretion and urinary thioethers was tested Both biological factors do not show any relationship (r = 0 09, P > 0 05, n = 68). Most of the workers in the exposed group were rakermen and throwers (50 %) None of the workers wore gloves or masks The relationship of job category of the exposed workers and thioether and 1-hydroxypyrene levels was studied Since smoking seems to enhance the urinary thioether and 1-hydroxypyrene excretions, the data were analyzed after dividing all these workers in different job categories into two groups (smoking and nonsmoking) (Fig 1) Two-way ANOVA indicates no effect of smoking or job on urinary thioethers (P = 0 99; P = 0.47, respectively) and 1-hydroxypyrene excretion (P = 0.10 ; P = 0 93, respectively) Owing to the small number of subjects involved in two job categories foreman and finishing machine operator, they were excluded in the statistical analysis.
Discussion In the earlier studies, it was shown that potential exposure to bitumen fumes involved in road-paving operations had been rather low l4, 24 l A potential hazard, however, was still present to an appreciable extent in all the user applications l4, 7 l It is therefore appropriate to characterize better the genotoxic hazards associated with occupational exposure to bitumen fumes, especially using biological monitoring methods. In this study, we have used two biological monitoring methods: nonselective urinary thioether assay l3l and a selective method for the detection of 1-hydroxypyrene in urine l 15l Previous studies on the urinary thioether excretions of road-paving workers have found either occupational asphalt exposure l23l or a synergistic effect of bitumen exposure and smoking l26 l, while others l3, 32 l have not found any occupational effect In all exposed subjects as well as in nonsmokers, excretion of thioethers was significantly higher than in nonexposed subjects in our study Occupational exposure to bitumen fumes seemed to influence this parameter On the other hand, in agreement with data previously published l1, 3, 13, 21, 23, 25, 26, 30 l, the effect of smoking on thioether excretion was substantial At present, there is not much known about the conversion of some drugs to urinary thioethers in humans However, it is known that sulfur-containing drugs may interfere with the thioether assay l35 l Vainio and coworkers l33l found higher values in subjects using medicaments such as blood pressure drugs, diuretics, and heart drugs In our study, it also seems that medication contributes to the urinary thioether excretion Compared with the effect of smoking, however, that of medication is slight.
400
Despite these confounding variables, the present data suggest an exposure to electrophilic agents in bitumen fumes The urinary thioether assay is considered a signal of exposure and absorption of one or more electrophilic substances l11, 32, 35 l, although it should be pointed out that the significance of this parameter is still not clear l31, 36l The presence of 1-hydroxypyrene in urine in fact reflects exposure to pyrene and leads to a more detailed evaluation of exposure to genotoxic PA Hs in bitumen fumes The enhanced level of 1-hydroxypyrene in road-paving workers' urine compared with that of controls is obvious. Our results indicate that smoking is not a strong determinant in urinary 1-hydroxypyrene excretion The 1hydroxypyrene concentrations in smoking and nonsmoking controls in this study are of the same magnitude as reported in previous studies l17, 19l In the present paper we observed that occupational exposure to PA Hs from bitumen fumes seemed to influence urinary 1-hydroxypyrene levels to a lesser extent when compared with refined coal-tar products l 6, 17, 18l Since it is known that the concentration of PA Hs is moderate when petroleum bitumens (0 1-10 mg/kg) are used l4, 24l, our observation seems to be logical In this study and also in previous studies l16, 19 l, 1-hydroxypyrene has been shown to be a sensitive biological indicator of exposure to PA Hs Moreover, our results demonstrate the applicability of the 1-hydroxypyrene assay after occupational exposure to petroleum-based products As previously indicated, PAHs have a rather complex metabolism, and after exposure to electrophilic PAHs, mutagens, thioether, and some identified metabolites could appear in urine l2, 11, 35 l Some studies have shown that smokers show higher values of urinary mutagenicity and thioethers than nonsmokers l 10, 34, 35 l Recent studies also reveal that there is a good correlation between the urinary levels of 1-hydroxypyrene and urinary mutagenicity in both animal and human experiments following exposure to coal tar l6, 16l We did not observe a relationship between the urinary thioethers and 1-hydroxypyrene levels in our study There may be several explanations for this finding (a) Thioethers and 1-hydroxypyrene have different parent compounds in bitumen fume l5, 11, 14 l, and the ratio of the parent compounds might vary (b) The enzyme systems responsible for the biotransformation pathways of electrophilic compounds in bitumen fumes to thioethers and pyrene to 1-hydroxypyrene are different l5, 14 l (c) Large interindividual differences in the metabolic systems may result in a large variance of thioether and 1-hydroxypyrene excretion. Interindividual differences of the two different enzyme systems are probably not correlated. In this study, significant increases of biological exposure indicators in road-paving workers indicate that intake of PAH by inhalation and/or by skin contact are common to all jobs, as also found by other researchers l4, 7, 18l It is known that during field studies of bitumen exposure, factors such as the nature of the application, the type of job performed, daily changing paving temperatures of the bitumen, and external conditions such as the weather can affect emission and exposure l4, 9,
22l Brandt et al l4l reported that rakermen ( 1 2 mg/m3 8-h time-weighted average) were significantly more exposed to bitumen fumes than roller drivers ( 0 3 mg/m3 8-h timeweighted average) during road-paving operations, based on standardized conditions Our results showed no effect of the job category at the road-paving operations on urinary thioether and 1-hydroxypyrene excretion The reason for this could be that there is no constancy of the position of the workers and duration of the task So, in this case, we feel that tasks are shared according to need, and jobs are not rigidly definable For further clarification, it may be necessary to measure the skin concentration of PA Hs as well as PAH concentration and PAH profile in airborne particulates at the work site l17, 18, 20l. Conclusion There is great concern about health problems associated with occupational exposure to bitumen fumes, and among the suggested hazards is an increased risk of cancer l9, 28l In fact, only a few researchers have performed biological monitoring of workers after occupational exposure to bitumen fumes l3, 26, 31 l Our results reveal that the increase of both thioethers and 1-hydroxypyrene levels in road-paving workers may not only be a sign of exposure and biological uptake but also an indication of an increased genotoxic risk We do believe that this type of combined nonselective and selective methods ensures a better evaluation of exposure to bitumens Attention needs to be given to minimizing exposure, either by improved working procedures or by protective control measures, in the application of the bituminous material. References 1 Bayhan A, Burgaz S, Karakaya AE (1987) Urinary thioether excretion in nurses at an oncologic department J Clin Pharmacol Ther 12:303-306 2 Bos RP, Jongeneelen FJ (1988) Nonselective and selective methods for biological monitoring of exposure to coal-tar products In: Bartsch H, Hemminki K, O'Neill IK (eds) Methods for detecting DNA-damaging agents in humans; applications in cancer epidemiology and prevention (IARC Scientific Publications no 89) International Agency for Research on Cancer, Lyon, pp 389-395 3 Burgaz S, Bayhan A, Karakaya AE (1988) Thioether excretion of workers exposed to bitumen fumes Int Arch Occup Environ Health 60:347-349 4 Brandt HCA, De Groot PC, Molyneux MKB, Tindlet PE (1985) Sampling and analysis of bitumen fumes Ann Occup Hyg 29:27-80 5 Chasseaud LF (1979) The role of glutathione and glutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents Adv Cancer Res 29: 175-274 6 Clonfero E, Zordan M, Venier P, Paleologo M, Levis AG, Cottica D, Pozzoli L, Jongeneelen FJ, Bos RP, Anzion RBM (1989) Biological monitoring of human exposure to coal tar. Int Arch Occup Environ Health 61: 363-368 7 Darby FW, Willis AF, Winchester RV (1986) Occupational health hazards from road construction and sealing work Ann Occup Hyg 30:445-454 8 Donald M, Zimmer A (1967) Atlas of clinical laboratory procedures, vol 1 McGraw-Hill, New York
401 9 Hansen ES (1989) Cancer mortality in the asphalt industry: a ten year follow up of an occupational cohort Br J Ind Med 46:582-585 10 Heinonen T, Kytoniemi V, Sorsa M, Vainio H (1983) Correlation of urinary thioethers and mutagenicity among volunteer smokers of low-tar and medium-tar cigarettes Int Arch Occup Environ Health 52:11-16 11 Henderson PT, Doorn R van, Leijdekkers C-M, Bos RP (1984) Excretion of thioethers in urine after exposure to electrophilic chemicals In: Berlin A, Draper M, Hemminki K, Vainio H (eds) Monitoring human exposure to carcinogenic and mutagenic agents (IARC Scientific Publications no 59). International Agency for Research on Cancer, Lyon, pp 173187 12 International Agency for Research on Cancer (1985) IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans Polynuclear compounds, bitumens, coaltars and derived products, shale oils and soots, part 4, vol 35. IARC, Lyon 13 Jagun O, Ryan M, Waldron HA (1982) Urinary thioether excretion in nurses handling cytotoxic drugs Lancet I 1:443444 14 Jongeneelen FJ (1987) Biological monitoring of occupational exposure to PAH (PhD thesis) Scientific Publishers, Nijmegen, pp 31-48 15 Jongeneelen FJ, Anzion RBM, Henderson PT (1987) Determination of hydroxylated metabolites of polycyclic aromatic hydrocarbons in urine J Chromatogr 413:227-232 16 Jongeneelen FK, Akker W van der, Bos RP, Anzion RBM, Theuws JLG, Roelofs HMJ, Henderson PT (1988) 1-Hydroxypyrene as an indicator of the mutagenicity of coal tar after activation with human liver preparations Mutat Res 204: 195-201 17 Jongeneelen FJ, Anzion RBM, Scheepers PTJ, Bos RP, Henderson PT, Nijenhuis EH, Veenstra SJ, Brouns RME, Winkes A (1988) 1-Hydroxypyrene in urine as an biological indicator of exposure to polycyclic aromatic hydrocarbons in several work environments Ann Occup Hyg 32:35-43 18 Jongeneelen FJ, Scheepers PTJ, Groenendijk A, Van Aerts LAJM, Anzion RBM, Bos RP, Veenstra SJ (1988) Airborne concentrations, skin contamination, and urinary metabolite excretion of polycyclic aromatic hydrocarbons among paving workers exposed to coal tar derived road tars Am Ind Hyg Assoc J 49:600-607 19 Jongeneelen FK, Anzion RBM, Theuws JLG, Bos RP (1989) Urinary 1-hydroxypyrene levels in workers handling petroleum coke J Toxicol Environ Health 26:133-136 20 Jongeneelen FK, Leeuwen FE van, Oosterink S, Anzion RBM, Loop F van der, Bos RP, Veen HG van (1990) Ambient and biological monitoring of cokeoven workers: determinants of the internal dose of polycyclic aromatic hydrocarbons Br J Ind Med 47:454-461 21 Kilpikari I, Savolainen H (1982) Increased urinary excretion of thioether in new rubber workers Br J Ind Med 39:401-403 22 Knecht U, Woitowitz HJ (1989) Risk of cancer from the use of tar bitumen in road works Br J Ind Med 46:24-30
23 Lafuente A, Mallol J (1987) Urinary thioethers in workers exposed to asphalt: an impairment of glutathione-S-transferase activity J Toxicol Environ Health 21:533-534 24 Monarca S, Pasquini R, Scassellati Sforzolini G, Savino A, Bauleo FA, Angeli G (1987) Environmental monitoring of mutagenic/carcinogenic hazards during road paving operations with bitumens Int Arch Occup Environ Health 59:393-402 25 Pasquini R, Monarca S, Scassellati Sforzolini G, Bauleo FA, Angeli D, Cerami F (1989) Thioethers, mutagens and o-glucaric acid in urine of operating room personnel exposed to anesthetics Teratogenesis Carcinog Mutagen 9:359-368 26 Pasquini R, Monarca S, Scassellati Sforzolini G, Savino A, Bauleo FA, Angeli D (1989) Urinary excretion of mutagens, thioether and D-glucaric acid in workers exposed to bitumen fumes Int Arch Occup Environ Health 61:335-340 27 Robinson M, Bull RJ, Munch J, Meier J (1984) Comparative carcinogenic and mutagenic activity of coal tar and petroleum asphalt paints used in potable water supply systems J Appl Toxicol 4:49-56 28 Schoket B, Hewer A, Grover PL, Phillips DH (1988) Formation of DNA adducts in human skin maintained in short-term organ culture and treated with coal-tar, creosote or bitumen. Int J Cancer 42: 622-626 29 Seutter-Berlage F, Selten GCM, Oostendorp SGML, Hoog Antink JMT (1979) The modified thioether test In: Strik JJTWA, Koeman JH (eds) Chemical porphyria in man Elsevier/North-Holland Biomedical, Amsterdam 30 Stock JK, Priestly BG (1986) Urinary thioether output as a index of occupational chemical exposure in petroleum retailers Br J Ind Med 43: 718-720 31 Thiringer G, Granung G, Holmen A, Hogstedt B, Jarvholm B, Jonsson D, Persson L, Wahlstr 6m J, Westin K (1991) Comparison of methods for the biomonitoring of nurses handling antitumor drugs Scand J Work Environ Health 17:133-138 32 Triebig G, Moser S, Schaller KH (1986) Pilot study for the diagnostic validity of urinary thioether-excretion in occupational medicine Zbl Arbeitsmed 36:301-307 33 Vainio H, Savolainen H, Kilpikari I (1978) Urinary thioether of employees of a chemical plant Br J Ind Med 35:232-234 34 Van Doorn R, Bos RP, Leijdekkers CM, Wagennaszegers MAP, Theuws JLG, Henderson PT (1979) Thioether concentration and mutagenicity of urine from cigarette smokers Int Arch Occup Environ Health 43:159-166 35 Van Doom R, Leijdekkers CM, Bos RP, Brouns RME, Henderson PT (1981) Detection of human exposure to electrophilic compounds by assay of thioether detoxication products in urine Ann Occup Hyg 24:77-92 36 Van Welie RTH, Marrewijk CM van, Wolff FA, Vermeulen NPE (1991) Thioether excretion in urine of applicators exposed to 1,3-dichloropropene: a comparison with urinary mercapturic acid excretion Br J Ind Med 48:492-498 37 Wallcave L, Garcia H, Feldman R, Lijinsky W, Shubik P (1971) Skin tumorigenesis in mice by petroleum asphalts and coal-tar pitches of known polynuclear aromatic hydrocarbon content Toxicol Appl Pharmacol 18:41-52
trn S JiI Tla 1781 i Qi)fl ) U J-~tl Vlt UU 11 ~tlllt 177/~) UJ J 71
't~J
International e Archives of
Environmental Health © Springer-Verlag 1992
Evaluation of urinary excretion of 1-hydroxypyrene and thioethers in workers exposed to bitumen fumes Sema Burgazl , Paul J A Borm 2 , and Frans J Jongeneelen 3 1Department of Toxicology, Faculty of Pharmacy, Gazi University, 06330, Hipodrom, Ankara, Turkey Department of Occupational and Environmental Health & Toxicology, University of Limburg, PO Box 616, 6200 MD Maastricht, The Netherlands 3 Department of Toxicology, University of Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands 2
Received October 24, 1990 / Accepted October 21, 1991
Summary Biological monitoring of exposure to bitumen fumes during road-paving operations was carried out In order to evaluate the biological uptake of the workers, the nonselective urinary thioether assay and a selective method for the determination of urinary 1-hydroxypyrene were used Urinary thioether data of exposed workers were higher than those of nonexposed subjects The effect of smoking, however, was stronger than the effect of occupational exposure Levels of 1-hydroxypyrene in road-paving workers were significantly higher than those in control subjects The 1-hydroxypyrene level was also influenced by smoking habits, but the effect of occupational exposure was stronger Our present data suggest that enhanced urine levels of both thioethers and 1-hydroxypyrene in bitumen workers may indicate an increased genotoxic risk Furthermore, our results demonstrate the applicability of the 1-hydroxypyrene assay after occupational exposure to petroleum-based products. Key words: Urinary thioether 1-Hydroxypyrene excretion Bitumen fumes Occupational exposure
Introduction It is estimated that the current annual use of bitumen in Turkey is approximately 0 35 million tons As bitumens are used in many different forms of road construction and maintenance, no data are available on the number of workers exposed to bitumens and bitumen fumes. There is also no information on the extent of their exposure and little on their health risks Thus, it is important to assess the exposure of workers to polycyclic aromatic hydrocarbons (PA Hs) during road-paving operations with bitumens. Bitumen samples and fumes consist essentially of hydrocarbons and their derivatives, some of which are known to be carcinogenic or cocarcinogenic The levels Offprint requests to: S Burgaz
of total PA Hs are moderate when compared with coal-tar products l4, 12, 24, 37 l There are only a few data on the potential carcinogenicity and/or toxic effects of bitumens on humans l9, 12, 27 l. In the previous study, biological monitoring of exposure to bitumen fumes was carried out using the nonselective thioether assay l3 l In that study, no significant increase in thioether concentration was detected that could be related to work exposure Recent studies have shown that 1-hydroxypyrene in urine can be considered an useful biological marker for occupational exposure to PA Hs Increased urinary levels of 1-hydroxypyrene have been found in patients cutaneously treated with coal tar, in workers exposed to the wood preservative creosote oil, in coal tar distillary workers, and in road-paving workers using coal tar blended binder l6, 16-19l. The present study was undertaken to investigate the bitumen exposure of paving workers with the aim of evaluating (a) the urinary thioethers and 1-hydroxypyrene excretion data and (b) the utility of the above two parameters for biological monitoring of exposure to petroleum-based products.
Materials and methods Road-paving operations Typical paving operations with bitumens are as follows: A hot mixture of stone chips and bitumen is delivered to a finishing machine, where it is discharged into a hopper. Conveyors in the machine then spread the mixture to a fixed width, and it is levelled and compacted to the thickness required. Further compaction is conducted using steel-tyred rollers by roller drivers The finishing-machine operator sits about 2 m above road level; the rakermen correct imperfections and complete edges on the newly laid asphalt layer immediately behind the finisher and/ or, depending on the nature of application, hot bitumen mixture is shovelled into a wheelbarrow, and throwers apply it to the surface by shovel The rakermen spread the mixture immediately behind the steel-tyred rollers A foreman controls all these operations. The exposure to bitumen fumes is evident during these road-paving processes. Subjects The exposed workers were 39 men employed in road bituminization Some 29 men selected from university staff and
398 Table 1 Characteristics of exposed group and controls with respect to age, smoking, use of alcohol, and medication Exposed group (n = 39)
Controls (n = 29)
Mean age (years)
35 5 (22-55)
36 3 (21-62)
Cigarettes/day (%) 0 1-10 11-20 > 20 Alcohol (yes) Medication (yes)
46 2 51 38 5 10 2 5 13
51 7 20 7 24 1 35 1 9
students served as a control group None of the referents had been occupationally exposed to potential genotoxic agents, and no subject (exposed or control) was using genotoxic agents in connection with leisure-time activities All subjects were asked to complete a questionnaire which inquired about age, smoking habits, recent use of medications, and alcohol intake The characteristics of the exposed and control group are given in Table 1. The majority of the exposed workers had worked on road-paving operations for at least 2 weeks prior to the study In order to evaluate the job exposure differences, the workers were classified as follows; roller driver (n = 10), thrower (n = 12), rakerman (n = 14), finishing machine operator (n = 2), and foreman (n = 1). All subjects ate a normal diet Most of the bitumens used in these applications were steam-refined and had 75-100 penetration values (class 1) Small amounts of cutback bitumens (class 3) were used in minor applications. Urine samples Urine samples from exposed subjects were collected at the end of the (8-h) working day on Thursday or Friday. Urine samples from control subjects were collected at different times of the day, whenever convenient After collection, the urine samples were divided into two aliquots to be used for the thioether assay and the analysis of 1-hydroxypyrene content A few milliliters were stored separately for the creatinine assay All urine samples were stored at -20 °C without any preservatives until analysis. Urinary thioethers were determined by the method described by Seutter-Berlage et al l29 l Urinary levels of thioether compounds were expressed as mmol SH-/mol creatinine Coefficient of variation (CV) of repeated analysis on different days was 2 9 %. The method for the determination of urinary levels of 1-hydroxypyrene was developed by Jongeneelen et al l 15 l The determination is based on the enzymatic hydrolysis of the conjugated metabolites, followed by their solid phase extraction The retained metabolites are then eluted and 1-hydroxypyrene in the eluate is determined by high-performance liquid chromatography (HPLC). HPLC analysis allowed the quantification of the sum of free and conjugated 1-hydroxypyrene in urine at the nmol/l level Urinary 1-hydroxypyrene concentrations were corrected for creatinine. Coefficient of variation (CV) repeated analysis on different days was 12 6 %. The creatinine content of the urine samples was measured by the method based on the reaction of creatinine with alkaline picrate l 8l Urine samples with creatinine values below 5 mmol/l were regarded as not reliable and excluded from the analysis of data. Statistical analysis The different sets of data were examined for normal distribution by the Shapiro and Wilk test Since observed distributions of biological factors data, thioether and 1-hydroxypyrene excretion, deviated from normality, the values were transformed into logarithms However, means and standard deviations are presented as untransformed values for ease of interpretation. To evaluate the several types of possible influences on the biologi-
cal parameters expressing PA Hs exposure, five-way analysis of variance (ANOVA) was used This analysis includes exposure (yes/no), age class, smoking (yes/no), drug intake (yes/no), and recent alcohol intake (yes/no). The effect of job category of the exposed workers and smoking on thioether and 1-hydroxypyrene excretion was also tested with two-way ANOVA. Correlation was qualified with the linear regression analysis. All tests were two-sided.
Results The concentrations of thioethers and 1-hydroxypyrene in urine samples of exposed and nonexposed groups are given in Table 2 Table 3 indicates the results of the ANOVA from which we can observe that exposure and smoking significantly (P < 0 01) contribute to the sum of squares for each of the two biological parameters, thio-
ethers and 1-hydroxypyrene From Tables 2 and 3, it is evident that the bitumen fumes during road-surfacing operations are absorbed by exposed workers Increases in the thioether excretion may be related to exposure (P = 0 011) Thioether excretion among nonsmokers
Table 2 Mean values (+ SD) of urinary thioether and 1-hydroxypyrene excretions of exposed and nonexposed groups, according to smoking habits Groups
Urinary thio ether excretion (mmol SH-/mol creatinine)
Urinary 1-hydroxypyrene excretion (gmol/mol creatinine)
Total exposed (n = 39)
7 76 + 4 70
0 61 + 0 38
Total nonexposed (n = 29)
4 61 ±+2 59
0 28 ± 0 17
Exposed Nonsmokers (n = 18) Smokers (n = 21)
6 93 + 3 68 8 47 + 5 42
0 53 ± 0 45 0 67 + 0 30
Nonexposed Nonsmokers (n = 15) Smokers (n = 14)
3 20 ± 2 40 6 12 + 1 88
0 24 + 0 13 0 33 ± 0 19
Table 3 Analyses of variance for the dependent variables 1-hydroxypyrene and thioethers Source of variance
df
Exposureb Smokingb Medicationb Alcoholb Age class
1 1 1 1 3
Log (hydroxypyrene)
Log (thioethers)
F value
P value
F value
P value
25 36 6 86 0 10 0 48 0 63
0 0001 a 0 0111 a 0 748 0 491 0 600
6 9 3 0 0
0 0 0 0 0
df, degrees of freedom a F value significant at 1 % level b Yes = 1, no= O c Class 1, < 30 years; 2, 30-39 ; 3, 40-49 ; 4, > 49
93 14 76 04 24
0107 a 0037 a 057 839 870
399 NS
S
NS
S
S
NS -
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20.
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.1.6
0
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0
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8
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Roller driver
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Fig 1 Urinary thioether (0) (mmol SH-/mol creatinine) and 1-hydroxypyrene levels () (gmol/mol creatinine) of workers exposed to bitumen fumes, according to job classification and smoking habits S, smoking workers; NS, nonsmoking workers Straight line is the mean control level of thioethers for each group
Rakerman
was statistically higher in exposed that in nonexposed subjects On the other hand, the effect of smoking (P = 0.004) was found to be stronger than that of occupational exposure, and medication seems to influence the urinary thioethers level (P = 0 057) The most frequently taken medicines by subjects were analgesics, stomach pills, and vitamins. Table 3 also indicates that the effect of occupational exposure on 1-hydroxypyrene levels (P = 0 0001) is stronger that that of smoking (P = 0 011) Tables 2 and 3 show that smoking increases the excretion of 1-hydroxypyrene, but it is not a strong determinant Medication did not influence the 1-hydroxypyrene level (P = 0 748) Age and alcohol intake have no significant effect on both biological parameters (Table 3). The correlation between the 1-hydroxypyrene excretion and urinary thioethers was tested Both biological factors do not show any relationship (r = 0 09, P > 0 05, n = 68). Most of the workers in the exposed group were rakermen and throwers (50 %) None of the workers wore gloves or masks The relationship of job category of the exposed workers and thioether and 1-hydroxypyrene levels was studied Since smoking seems to enhance the urinary thioether and 1-hydroxypyrene excretions, the data were analyzed after dividing all these workers in different job categories into two groups (smoking and nonsmoking) (Fig 1) Two-way ANOVA indicates no effect of smoking or job on urinary thioethers (P = 0 99; P = 0.47, respectively) and 1-hydroxypyrene excretion (P = 0.10 ; P = 0 93, respectively) Owing to the small number of subjects involved in two job categories foreman and finishing machine operator, they were excluded in the statistical analysis.
Discussion In the earlier studies, it was shown that potential exposure to bitumen fumes involved in road-paving operations had been rather low l4, 24 l A potential hazard, however, was still present to an appreciable extent in all the user applications l4, 7 l It is therefore appropriate to characterize better the genotoxic hazards associated with occupational exposure to bitumen fumes, especially using biological monitoring methods. In this study, we have used two biological monitoring methods: nonselective urinary thioether assay l3l and a selective method for the detection of 1-hydroxypyrene in urine l 15l Previous studies on the urinary thioether excretions of road-paving workers have found either occupational asphalt exposure l23l or a synergistic effect of bitumen exposure and smoking l26 l, while others l3, 32 l have not found any occupational effect In all exposed subjects as well as in nonsmokers, excretion of thioethers was significantly higher than in nonexposed subjects in our study Occupational exposure to bitumen fumes seemed to influence this parameter On the other hand, in agreement with data previously published l1, 3, 13, 21, 23, 25, 26, 30 l, the effect of smoking on thioether excretion was substantial At present, there is not much known about the conversion of some drugs to urinary thioethers in humans However, it is known that sulfur-containing drugs may interfere with the thioether assay l35 l Vainio and coworkers l33l found higher values in subjects using medicaments such as blood pressure drugs, diuretics, and heart drugs In our study, it also seems that medication contributes to the urinary thioether excretion Compared with the effect of smoking, however, that of medication is slight.
400
Despite these confounding variables, the present data suggest an exposure to electrophilic agents in bitumen fumes The urinary thioether assay is considered a signal of exposure and absorption of one or more electrophilic substances l11, 32, 35 l, although it should be pointed out that the significance of this parameter is still not clear l31, 36l The presence of 1-hydroxypyrene in urine in fact reflects exposure to pyrene and leads to a more detailed evaluation of exposure to genotoxic PA Hs in bitumen fumes The enhanced level of 1-hydroxypyrene in road-paving workers' urine compared with that of controls is obvious. Our results indicate that smoking is not a strong determinant in urinary 1-hydroxypyrene excretion The 1hydroxypyrene concentrations in smoking and nonsmoking controls in this study are of the same magnitude as reported in previous studies l17, 19l In the present paper we observed that occupational exposure to PA Hs from bitumen fumes seemed to influence urinary 1-hydroxypyrene levels to a lesser extent when compared with refined coal-tar products l 6, 17, 18l Since it is known that the concentration of PA Hs is moderate when petroleum bitumens (0 1-10 mg/kg) are used l4, 24l, our observation seems to be logical In this study and also in previous studies l16, 19 l, 1-hydroxypyrene has been shown to be a sensitive biological indicator of exposure to PA Hs Moreover, our results demonstrate the applicability of the 1-hydroxypyrene assay after occupational exposure to petroleum-based products As previously indicated, PAHs have a rather complex metabolism, and after exposure to electrophilic PAHs, mutagens, thioether, and some identified metabolites could appear in urine l2, 11, 35 l Some studies have shown that smokers show higher values of urinary mutagenicity and thioethers than nonsmokers l 10, 34, 35 l Recent studies also reveal that there is a good correlation between the urinary levels of 1-hydroxypyrene and urinary mutagenicity in both animal and human experiments following exposure to coal tar l6, 16l We did not observe a relationship between the urinary thioethers and 1-hydroxypyrene levels in our study There may be several explanations for this finding (a) Thioethers and 1-hydroxypyrene have different parent compounds in bitumen fume l5, 11, 14 l, and the ratio of the parent compounds might vary (b) The enzyme systems responsible for the biotransformation pathways of electrophilic compounds in bitumen fumes to thioethers and pyrene to 1-hydroxypyrene are different l5, 14 l (c) Large interindividual differences in the metabolic systems may result in a large variance of thioether and 1-hydroxypyrene excretion. Interindividual differences of the two different enzyme systems are probably not correlated. In this study, significant increases of biological exposure indicators in road-paving workers indicate that intake of PAH by inhalation and/or by skin contact are common to all jobs, as also found by other researchers l4, 7, 18l It is known that during field studies of bitumen exposure, factors such as the nature of the application, the type of job performed, daily changing paving temperatures of the bitumen, and external conditions such as the weather can affect emission and exposure l4, 9,
22l Brandt et al l4l reported that rakermen ( 1 2 mg/m3 8-h time-weighted average) were significantly more exposed to bitumen fumes than roller drivers ( 0 3 mg/m3 8-h timeweighted average) during road-paving operations, based on standardized conditions Our results showed no effect of the job category at the road-paving operations on urinary thioether and 1-hydroxypyrene excretion The reason for this could be that there is no constancy of the position of the workers and duration of the task So, in this case, we feel that tasks are shared according to need, and jobs are not rigidly definable For further clarification, it may be necessary to measure the skin concentration of PA Hs as well as PAH concentration and PAH profile in airborne particulates at the work site l17, 18, 20l. Conclusion There is great concern about health problems associated with occupational exposure to bitumen fumes, and among the suggested hazards is an increased risk of cancer l9, 28l In fact, only a few researchers have performed biological monitoring of workers after occupational exposure to bitumen fumes l3, 26, 31 l Our results reveal that the increase of both thioethers and 1-hydroxypyrene levels in road-paving workers may not only be a sign of exposure and biological uptake but also an indication of an increased genotoxic risk We do believe that this type of combined nonselective and selective methods ensures a better evaluation of exposure to bitumens Attention needs to be given to minimizing exposure, either by improved working procedures or by protective control measures, in the application of the bituminous material. References 1 Bayhan A, Burgaz S, Karakaya AE (1987) Urinary thioether excretion in nurses at an oncologic department J Clin Pharmacol Ther 12:303-306 2 Bos RP, Jongeneelen FJ (1988) Nonselective and selective methods for biological monitoring of exposure to coal-tar products In: Bartsch H, Hemminki K, O'Neill IK (eds) Methods for detecting DNA-damaging agents in humans; applications in cancer epidemiology and prevention (IARC Scientific Publications no 89) International Agency for Research on Cancer, Lyon, pp 389-395 3 Burgaz S, Bayhan A, Karakaya AE (1988) Thioether excretion of workers exposed to bitumen fumes Int Arch Occup Environ Health 60:347-349 4 Brandt HCA, De Groot PC, Molyneux MKB, Tindlet PE (1985) Sampling and analysis of bitumen fumes Ann Occup Hyg 29:27-80 5 Chasseaud LF (1979) The role of glutathione and glutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents Adv Cancer Res 29: 175-274 6 Clonfero E, Zordan M, Venier P, Paleologo M, Levis AG, Cottica D, Pozzoli L, Jongeneelen FJ, Bos RP, Anzion RBM (1989) Biological monitoring of human exposure to coal tar. Int Arch Occup Environ Health 61: 363-368 7 Darby FW, Willis AF, Winchester RV (1986) Occupational health hazards from road construction and sealing work Ann Occup Hyg 30:445-454 8 Donald M, Zimmer A (1967) Atlas of clinical laboratory procedures, vol 1 McGraw-Hill, New York
401 9 Hansen ES (1989) Cancer mortality in the asphalt industry: a ten year follow up of an occupational cohort Br J Ind Med 46:582-585 10 Heinonen T, Kytoniemi V, Sorsa M, Vainio H (1983) Correlation of urinary thioethers and mutagenicity among volunteer smokers of low-tar and medium-tar cigarettes Int Arch Occup Environ Health 52:11-16 11 Henderson PT, Doorn R van, Leijdekkers C-M, Bos RP (1984) Excretion of thioethers in urine after exposure to electrophilic chemicals In: Berlin A, Draper M, Hemminki K, Vainio H (eds) Monitoring human exposure to carcinogenic and mutagenic agents (IARC Scientific Publications no 59). International Agency for Research on Cancer, Lyon, pp 173187 12 International Agency for Research on Cancer (1985) IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans Polynuclear compounds, bitumens, coaltars and derived products, shale oils and soots, part 4, vol 35. IARC, Lyon 13 Jagun O, Ryan M, Waldron HA (1982) Urinary thioether excretion in nurses handling cytotoxic drugs Lancet I 1:443444 14 Jongeneelen FJ (1987) Biological monitoring of occupational exposure to PAH (PhD thesis) Scientific Publishers, Nijmegen, pp 31-48 15 Jongeneelen FJ, Anzion RBM, Henderson PT (1987) Determination of hydroxylated metabolites of polycyclic aromatic hydrocarbons in urine J Chromatogr 413:227-232 16 Jongeneelen FK, Akker W van der, Bos RP, Anzion RBM, Theuws JLG, Roelofs HMJ, Henderson PT (1988) 1-Hydroxypyrene as an indicator of the mutagenicity of coal tar after activation with human liver preparations Mutat Res 204: 195-201 17 Jongeneelen FJ, Anzion RBM, Scheepers PTJ, Bos RP, Henderson PT, Nijenhuis EH, Veenstra SJ, Brouns RME, Winkes A (1988) 1-Hydroxypyrene in urine as an biological indicator of exposure to polycyclic aromatic hydrocarbons in several work environments Ann Occup Hyg 32:35-43 18 Jongeneelen FJ, Scheepers PTJ, Groenendijk A, Van Aerts LAJM, Anzion RBM, Bos RP, Veenstra SJ (1988) Airborne concentrations, skin contamination, and urinary metabolite excretion of polycyclic aromatic hydrocarbons among paving workers exposed to coal tar derived road tars Am Ind Hyg Assoc J 49:600-607 19 Jongeneelen FK, Anzion RBM, Theuws JLG, Bos RP (1989) Urinary 1-hydroxypyrene levels in workers handling petroleum coke J Toxicol Environ Health 26:133-136 20 Jongeneelen FK, Leeuwen FE van, Oosterink S, Anzion RBM, Loop F van der, Bos RP, Veen HG van (1990) Ambient and biological monitoring of cokeoven workers: determinants of the internal dose of polycyclic aromatic hydrocarbons Br J Ind Med 47:454-461 21 Kilpikari I, Savolainen H (1982) Increased urinary excretion of thioether in new rubber workers Br J Ind Med 39:401-403 22 Knecht U, Woitowitz HJ (1989) Risk of cancer from the use of tar bitumen in road works Br J Ind Med 46:24-30
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