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Mutagenicity of expectorate from workers in a coke plant
Mutation Research, 223 (1989) 213-219
213
Elsevier MTR 01407
Mutagenicity of expectorate from workers in a coke plant o
Ase Krokje Department of Botany, Section for Cell Biology, The University of Trondheim, Trondheim (Norway)
(Received2 February 1988) (Revision received2 January 1989) (Accepted 3 January 1989) Keywords: Expectorate; Coke plant workers
Summary Methanol extracts of hydrolyzed expectorate samples from workers in a coke plant were tested with the Salmonella reversion assay. The expectorates from exposed smokers (but also to a certain extent from exposed non-smokers) were mutagenic, however, the control samples from both smokers and non-smokers were not. The positive results produced by the expectorate samples from exposed smokers suggest a synergistic relationship between exposure to air pollution in the working atmosphere and smoking.
Expectorate is a mixture of sputum and mucus from the bronchi and alveoli. It contains both respirable and non-respirable particles on which mutagens have been adsorbed. The non-respirable particles are mostly swallowed. The mutagens exist in the expectorate either free, adsorbed onto particles, or phagocytized and subsequently metabolized in alveolar macrophages. Expectorate seems to be suitable to evaluate certain mutagens that are inhaled and are biologically available. A study of the Ames test on expectorate has previously been made in potrooms in an aluminum plant (Krokje et al., 1985). In the present study, results of mutagenicity studies on expectorate from workers on the top of the batteries in a coke plant are reported. The kind and degree of chemical pollution of the working atmosphere have already been relatively well investigated (Bruun et al.,
Correspondence: ,~se Krokje, Department of Botany, Section for Cell Biology,The University of Trondheim, 7055 Dragvoll (Norway).
1976; Bjorseth et al., 1977, 1978b). Polycyclic aromatic hydrocarbons (PAH) constitute a major part of the air pollution in the coke oven area. At the top of the ovens the amount of particulate PAH in the workers' breathing zone varied from 5 to 1044/~g/m 3 (Bjorseth et al., 1978a). 72% of the particulate PAH was found on particles smaller than 3 ~tm and only 1% was found on particles larger than 7/~m, which means that most of these compounds were within the respiratory size range. The ultimate aim of the present study is to determine the biological availability of the mutagens and to trace possible interactions between exposure to air pollution of the working atmosphere and smoking. The study is aimed at group exposure. The samples have been pooled into exposed smokers and exposed non-smokers respectively, since that, more than individual exposure, indicates the general effect of the working atmosphere. Individual factors (e.g., work hygiene, choice of food, hobbies) can more easily be overruled, while the effect from the common exposure at work will dominate the results.
0165-1218/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)
214 Material and methods
Collection of samples The workers gave their samples within half an hour of finishing the workshift. Samples were also given after a night's sleep: 'morning expectorate'. The samples were then frozen, and kept in polyethylene bags at - 20 ° C. Samples from a control group consisting of inhabitants of Trondheim were also investigated. The control group consisted of people of approximately the same age as the exposed workers. Each person was instructed to cough vigorously and the sputum was collected in a plastic cup. It was easy to obtain representative material containing cell types from the deep-seated areas of the lungs from both the control and the exposed persons. Coughing was repeated 3 times, and the persons gave samples on 3 consecutive days. All the people involved gave information about their age, smoking habits, place of work, previous exposure, use of medicines, etc., in their answers to a questionnaire. Our definition of a smoker was a person who had smoked more than 10 cigarettes per day for more than a year. A non-smoker was one who had either never smoked or had stopped smoking more than 2 years ago. Persons who fitted neither of these categories or who had used medicines the last 3 weeks were excluded from the study. The 4 groups studied were designated as follows: C-NS, control non-smokers; C-S, control smokers; B-NS, battery non-smokers; B-S, battery smokers. The numbers of individuals in the groups were different: 38 control non-smokers, 18 control smokers, 12 battery non-smokers and 15 battery smokers.
Chemicals The following chemicals were used in this study: cyclohexane p.a. (Merck); methanol p.a. (Merck); potassium hydroxide (KOH) p.a. (Merck); dimethyl sulfoxide (DMSO) spectrometric grade (Merck); benzo[a]pyrene, practical grade (Sigma); nutrient broth No. 2 (Oxoid Ltd.); Bacto-Agar (Difco); L-histidine HC1 (Sigma); d,biotin (Sigma); Vogel-Bonner Minimal 'E' Agar plates (Gibco Bio-cult); glucose-6-phosphate (Sigma); NADP (Sigma).
Extraction The extraction procedure has been described in detail previously (Krokje et al., 1985). The material from each group was hydrolyzed with alkaline methanol and extracted by cyclohexane. After the cyclohexane extract was evaporated to dryness, DMSO was added to dissolve the cyclohexane residue. The stock solutions were diluted with DMSO to obtain concentrations of 0.25, 0.50, 1.0, 2.0 and 3.0 g of expectorate per 100 /~1 of test solution.
Mutagenic analysis The Salmonella typhimurium strains TA98 and TA100 were kindly supplied by Dr. Bruce Ames, Berkeley, CA (U.S.A.). The mutagenic activity of extracts of expectorate from all groups was studied according to the Ames standard method (Ames et al., 1975; Maron and Ames, 1983). Tests were carried out in the presence and absence of $9 mix (50/~1 S9/plate).
Preparation of $9 mix Male Sprague-Dawley rats were treated with Aroclor 1254 (500 mg/kg) 5 days before they were killed. The 9000 X g liver supernatant ($9) was divided into 2-ml aliquots, frozen and stored at - 1 9 6 ° C . The protein content (25 mg/ml) was measured by the method of Lowry et al. (1951). The $9 mix ($9 with cofactors) was filtered through a Millipore filter (diameter 0.45/zm) before use.
Testing procedure The extracts from the different groups were tested at 5 different concentrations (triplicate plates) to establish the relationship between concentration and effect. The 'morning expectorate' was only tested with $9. Preliminary experiments without $9 did not show any effect of the expectorate samples, and therefore the samples were only tested with $9 in the further experiments. One concentration (1.0 g) in the linear area of the concentration/effect relationship was tested in the further experiments. The concentration was tested with both TA98 and TA100 in 5 parallel Petri plates. Each test was duplicated, in independent experiments, to ensure reproducibility.
215
Results
Control smokers The expectorate from control smokers does not contain mutagenic compounds at the investigated concentrations (Fig. 1, Table 1). The response of the samples from the control smokers did not differ significantly from that of the control nonsmokers.
Control non-smokers The test solution from the control group of non-smokers showed no indication of a concentration/effect relationship (Fig. 1) either for TA98 or TA100, and the C-NS expectorate does not suggest that any mutagenic compounds are present.
Exposed non-smokers The extract from the battery top non-smokers showed a positive relationship between concentration and effect on both strains of bacteria after $9 activation (Fig. 1). Ames' criterion that the response should be double that of the spontaneous
The statistical significance of all the results was determined with Student's t-test (Weinstein and Lewinson, 1978; Ehrenberg, 1979). Checks on the activating capacity of the $9 mix were made in each experiment using 1/~g benzo[a]pyrene.
aT 500.
TA
100 + S9
480.
we.-S
460440. 420. 400.~ TA
98 + $9
aSO360-
Rr 130 i B-S
340,
120-
320.
110.
300. 280-
100.
260-
90-
240-
80-
220200-
70-
180-
60-
160B-NS
50B-S
/"
30. 20..====
B-NS
~
120-
/I
40.
J
140.
.13B-NS
/
100
. . . .~.~ ~ -B ' ' "o
C-S
80.
~
C-NS
60 2~~'I
N
~
C-NS
:
• C-S
, 2
, .~ 3 g expectorate
" 1B- S ÷
40 100 0
I 1
I= 2
, = 3 g expectorate
0
! 1
Fig. 1. Concentrations and mutagenic effects of the extracts of expectorate from exposed workers (smokers (B-S) and non-smokers
(B-NS)) and from control groups (smokers (C-S) and non-smokers (C-NS)). The total responses of the bacteria (RT) after 48 h incubation are shown. For some of the concentrations each data symbol represents the mean of the R T values for 2 or more independent experiments. A solid line ( ) indicates samples given at the end of the workshift, and a broken line (. . . . . . ) indicates 'morning expectorate'. + indicates an observed reduction in the growth of the background.
216 TABLE 1 M U T A G E N I C EFFECTS OF EXTRACTS OF E X P E C T O R A T E (1.0 g) F R O M EXPOSED W O R K E R S (SMOKERS (B-S) A N D N O N - S M O K E R S (B-NS)), G I V E N AT T H E E N D OF T H E SHIFT, A N D F R O M T H E C O N T R O L S (S MO K ER S (C-S) A N D N O N - S M O K E R S (C-NS)) Test solution C-NS C-S B-NS B-S
TA98 + S9 R ~ 4-_SE 0.0+1.4 3.9_+1.4 -0.8_+3.1 0.5±2.1 13.1_+2.4 15.1-+2.5 48.1_+8.1
TA100 + $9 Ho: R T = R ~ ns + ns ns + + + +++ + + +
Ho: R N = R K
+ + +
R N _+SE
Ho: R-r = R K
7.5_+ 5.6 -12.1_+ 4.0 7.5_+ 6.9 -5.7_+ 5.3 34.6+11.4
ns
159.2_+15.6 52.9_+ 5.5
Ho: R N = R K
ns ns
+++ +++ +++
+++
The net responses_+ standard error (R s _+SE, after 48 h incubation) are tabulated. The net yield responses (R N) can be estimated from the difference between the number of revertants of the test plates (RT) and the control value of the solvent (RK). Results of Student's t-test for the hypotheses H0: R T = R K and H0: R N = R K (ns p > 0.05; R T > R K or R N > RK: + 0.01< p < 0.05; + + 0.001
control was not fulfilled at the concentration most often investigated (1.0 g). But this response together with the increased response at the higher concentration showed that the expectorate from exposed non-smokers contains mutagenic compounds. The response obtained was clearly greater than that of the control non-smokers (Fig. 1). The 'morning expectorate' also showed a weak relationship between concentration and effect. The response was weaker than from the expectorate samples given at the end of the shift.
Exposed smokers The expectorate from the workers on the top of the coke oven batteries produced mutagenic effects on both TA98 and TA100 (Fig. 1, Table 1) and the relationship between concentration and effect was clearly positive with $9 activation. The mutagenic test solution from the exposed smokers' expectorate gave a response which was significantly higher than the non-mutagenic test solution from the control smokers ( p < 0.001), and also higher than that for the control smokers plus exposed non-smokers. The 'morning expectorate' from the exposed smokers showed a mutagenic effect on TA98 while it had a toxic effect on TA100. With regard to
TA98 the effect was weaker than that of samples expectorated at the end of the shift. Discussion
This study has demonstrated that the Salmonella assay can reveal the presence of mutagenic activity in a methanol extract of 0.25-3.0 g hydrolyzed expectorate. The positive response could be correlated to exposure to the working atmosphere on the top of the batteries in a coke plant. The expectorates from the exposed smokers (B-S) (but also to a certain extent from non-smokers (B-NS)) had a positive effect on the bacteria; however, the control samples from both smokers (C-S) and non-smokers (C-NS) did not contain mutagens. The results are in agreement with the ones shown in the study from the potrooms in an aluminum plant (with PAH exposure) (Krokje et al., 1985). There is a synergistic relationship between exposure to occupational pollution and smoking, a weak effect from occupational exposure alone and none from smoking only. The mutagenicity in the expectorate extract indicates that the workers on the top of the batteries in a coke plant are exposed to potential mutagcns which are biologically available. The analysis of
217 the 'morning expectorate' shows that inhaled pollution with potential mutagens is not effectively removed from the respiratory system during the night. From the results of chemical analysis of the work atmosphere at the coke plant investigated (Bjorseth et al., 1978b) and the results from the Salmonella test on air filter samples (Krokje, unpublished) a mutagenic activity in the workers expectorate was expected. A clear effect of indirect mutagens is consistent with the PAH exposure at the battery top. PAH require metabolic activation before they have a mutagenic effect (Ames et al., 1973). Cigarette smoking and occupational exposure may interact through effects of smoking on the dose of genotoxic compounds that reaches the cell. Long-term exposure to cigarette smoke impairs mucociliary clearance (U.S. Department of Health and Human Services, 1982) and could alter the retained dose of an occupational agent. Benzo[a]pyrene and other mutagens may adsorb to particulates in smoke or to environmental dusts (Natusch et al., 1974; Mossman et al., 1983) resulting in a higher fractional retention or different distribution in the lung. The adsorption to dust may also facilitate or inhibit transport of carcinogens through the mucus layer. Cigarette smoke has been shown to increase the epithehal permeability in the tracheobronchial tree (Sirnani et al., 1974); the effect may increase the exposure of the underlying cell to an occupational agent. Another potential site of biologic interaction is the metabolic activation of a genotoxic compound. A number of constituents of cigarette smoke have by increased induction of microsomal enzymes been shown to affect the metabolic activation of other substances (U.S. Department of Health, Education, and Welfare, 1979). Cigarette smoking also alters the cellular composition of the lung, increasing the number of neutrophils and activated macrophages in the lung (U.S. Department of Health and Human Services, 1984); these cells also play a role in the metabolic transformation of occupational agents. Investigations of condensate of tobacco smoke have shown that it does contain mutagens (Kier et al., 1974; Sato et al., 1977). Moller and Dybing (1980) investigated the urine from workers at the
same coke plant. Mutagenic activity in the urine was not related to occupational exposure, but was strongly related to smoking. The negative results obtained for the group of control smokers are therefore surprising. One explanation for the present findings may be that the greatest part of the effect of smoking may be co-mutagenic, vide the increase in mutagenie effect recorded for the group of battery top smokers. In the same way the content of mutagens registered in the urine from smokers (Yahagi et al., 1977; van Doom et al., 1979; Falck et al., 1980; MoUer and Dybing, 1980; Aeschbacher and Chappuis, 1981; Dolara et al., 1981; Putzrath et al., 1981; Bos et al., 1982; Falck, 1982) need not be from the cigarette smoke itself. Hannan et al. (1981) found that cigarette smoke condensate and urine concentrate from smokers exhibit a co-mutagenic action on the mutagenicity of 2-aminoanthracene. Co-mutagens from the cigarette smoke may cooperate with mutagens which have entered the body via other absorption ways than the lungs. The expectorate analysis, in contrast to filter analysis, is corrected for local and time variations in intensity of exposure, the variable use of protective gear, and individual lifestyle factors. I suggest that the Salmonella typhimurium test used on expectorate samples may be effective in certain types of industries, viz. where the atmosphere contains particulates acting as carriers for mutagens apt to be eluated by the body fluids or cellular membranes. Such a test constitutes a tool by which one may monitor or test improvements in the occupational environment. PAH compounds will depend on the availability of carriers, mostly air particulates, to get into the lungs. It may therefore not be sufficient to measure the content of PAH in the working atmosphere, but the biological availability of these compounds should be evaluated.
Acknowledgements I wish to thank tlie Health Service Center and the laboratory personnel at Norsk Koksverk A / S for help during collection of samples. Thanks are also due to the participating workers. I would also like to thank cand.real. K. Zahlsen at the Department of Pharmacology and Toxicology, Faculty of
218
Medicine for preparing the $9. Financial support was obtained from the Norwegian Council for Scientific and Industrial Research, Norsk Koksverk A / S and the Norwegian Cancer Society. References Aeschbacher, H.U., and C. Chappuis (1981) Non-mutagenicity of urine from coffee drinkers compared with that from cigarette smokers, Mutation Res., 89, 161-177. Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. (U.S.A.), 70, 2281-2285. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-rnicrosome mutagenicity test, Mutation Res., 31, 347-364. Bjorseth, A., O. Bjorseth and P.E. Fjeldstad (1977) Kartlegging av PAH ved Norsk Koksverk A/S, Teknisk-hygienisk rapport nr. 2 (NTNF-rapport), Oslo, HD 742/770922, 23 pp. (in Norwegian). Bjorseth, A., O. Bjorseth and P.E. Fjeldstad (1978a) Polycyclic aromatic hydrocarbons in the work atmosphere. II. Determination in a coke plant, Scand. J. Work Environ. Health, 4, 224-236. Bjorseth, O., P.E. Fjeldstad, T. Frost and K. Halgard (1978b) Polysykliske aromatiske hydrokarboner. Eksponeringsmalinger ved Norsk Koksverk A/S, Mo i Rana, Yrkeshygienisk Institutt, Oslo, HD 782/781109, 30 pp. (in Norwegian). Bos, R.P., A.O. Lenaars, J.L.G. Theuws and P.T. Henderson (1982) Mutagenicity of urine from nurses handling cytostatic drugs, influence of smoking, Int. Arch. Occup. Environ. Health, 50, 359-369. Bruun, R., M. Gjolstad, T. Torgrimsen and B. Wermundsen (1976) Teknisk hygieniske undersokelser ved Norsk Koksverk A / S , Mo i Rana 17.-20. november 1975, Yrkeshygienisk Institutt, Oslo, HD 676/760302, 82 pp. (in Norwegian). Dolara, P., S. Mazzoli, D. Rosi, E. Buiatti, S. Baccetti, A. Turchi and V. Vannucci (1981) Exposure to carcinogenic chemicals and smoking increases urinary excretion of mutagens in humans, J. Toxicol. Environ. Health, 8, 95-103. van Doom, R., R.P. Bos, Ch.-M. Leijdekkers, M.A.P. Wagenaars-Zegers, J.L.G. Theuws and P.Th. Henderson (1979) Thioether concentration and mutagenicity of urine from cigarette smokers, Int. Arch. Occup. Environ. Health, 43, 159-166. Ehrenberg, L. (1979) Aspects of statistical inference in testing for genetic toxicity, in: B.J. Kilbey, M. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 419-461. Falck, K. (1982) Urinary mutagenicity caused by smoking, in: M. Sorsa and H. Vainio (Eds.), Mutagens in Our Environment, Proceedings of the Twelfth Annual Meeting of the European Environmental Mutagen Society, Espoo (Fin-
land) June 20-24 1982, Alan R. Liss, New York, pp. 387-400. Falck, K., M. Sorsa, H. Vainio and I. Kilpikari (1980) Mutagenicity in urine of workers in rubber industry, Mutation Res., 79, 45-52. Hannah, M.A., L. Recio, P.P. Deluca and H. Enoch (1981) Comutagenic effects of 2-aminoanthracene and cigarette smoke condensate on smoker's urine in the Ames Salmonella assay system, Cancer Lett., 13, 203-212. Kier, L.D., E. Yamasaki and B.N. Ames (1974) Detection of mutagenic activity in cigarette smoke condensates, Proc. Natl. Acad. Sci. (U.S.A.), 71, 4159-4163. Krokje, A. (n.d.) Mutagenicity of air samples in a coke plant (Unpublished). Krokje, A., A. Tiltnes, E. Mylius and B. Gullvhg (1985) Testing for mutagens in an aluminium plant. The results of Salmonella typhimurium tests on expectorates from exposed workers, Mutation Res., 156, 147-152. Lowry, O.H.,, N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the foliphenol reagent, J. Biol. Chem., 193, 265-275. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mossman, B., W. Light and E. Wei (1983) Asbestos: mechanisms of toxicity and carcinogenicity in the respiratory tract, Annu. Rev. Pharmacol. Toxicol., 23, 595-615. Moller, M., and E. Dybing (1980) Mutagenicity studies with urine concentrates from coke plant workers, Scand. J. Work Environ. Health, 6, 216-220. Natusch, D.F.S., J.R. Wallace and C.A. Evans Jr. (1974) Toxic trace elements: preferential concentration in respirable particles, Science, 183, 202-204. Putzrath, R.M., D. Langley and E. Elsenstadt (1981) Analysis of mutagelaic activity in cigarette smokers urine by high performance liquid chromatography, Mutation Res., 85, 97-108. Sato, S., Y. Seino, T. Ohka, T. Yahagi, N. Nagao, T. Matsushima and T. Sugimura (1977) Mutagenicity of smoke condensates from cigarettes, cigars and pipe tobacco, Cancer Lett., 3, 1-8. Simani, A.S., S. Inove and J.C. Hogg (1974) Penetration of the respiratory epithelium of guinea pigs following exposure to cigarette smoke, Lab. Invest., 31, 75-81. U.S. Department of Health, Education, and Welfare (1979) Smoking and Health: A Report of the Surgeon General, U.S. Department of Health, Education, and Welfare, Public Health Service, Office on Smoking and Health, DHEW Publ. No. (PHS)79-50066, 1, 136 pp. U.S. Department of Health and Human Services (1982) The Health Consequences of Smoking: Cancer. A Report of the Surgeon General, U.S. Department of Health and Human Services, Public Health Service, Office of the Assistant Secretary for Health, Office on Smoking and Health, DHHS Publ. No. (PHS)82-50179, 322 pp. U.S. Department of Health and Human Services (1984) The Health Consequences of Smoking: Chronic Obstructive Lung Disease, A Report of the Surgeon General, U.S. Department of Health and Human Services, Public Health Service, Office on Smoking and Health, DHEW Publ. No.
219 (PHS)8Zl-50205, 545 pp. Weinstein, D., and T.M. Lewinson (1978) A statistical treatment of the Ames mutagenicity assay, Mutation Res., 51, 433-434. Yahagi, T., M. Nagao, Y. Seino, T. Matsushima, T. Sugimura and M. Okada (1977) Mutagenicities of N-nitrosamines on
Salmonella, Mutation Res., 48, 121-130. Yamasaki, E., and B.N. Ames (1977) Concentration of mutagens from urine by adsorption with the nonpolar resin XAD-2: cigarette smokers have mutagenic urine, Proc. Natl. Acad. Sci. (U.S.A.), 74, 3555-3559.
213
Elsevier MTR 01407
Mutagenicity of expectorate from workers in a coke plant o
Ase Krokje Department of Botany, Section for Cell Biology, The University of Trondheim, Trondheim (Norway)
(Received2 February 1988) (Revision received2 January 1989) (Accepted 3 January 1989) Keywords: Expectorate; Coke plant workers
Summary Methanol extracts of hydrolyzed expectorate samples from workers in a coke plant were tested with the Salmonella reversion assay. The expectorates from exposed smokers (but also to a certain extent from exposed non-smokers) were mutagenic, however, the control samples from both smokers and non-smokers were not. The positive results produced by the expectorate samples from exposed smokers suggest a synergistic relationship between exposure to air pollution in the working atmosphere and smoking.
Expectorate is a mixture of sputum and mucus from the bronchi and alveoli. It contains both respirable and non-respirable particles on which mutagens have been adsorbed. The non-respirable particles are mostly swallowed. The mutagens exist in the expectorate either free, adsorbed onto particles, or phagocytized and subsequently metabolized in alveolar macrophages. Expectorate seems to be suitable to evaluate certain mutagens that are inhaled and are biologically available. A study of the Ames test on expectorate has previously been made in potrooms in an aluminum plant (Krokje et al., 1985). In the present study, results of mutagenicity studies on expectorate from workers on the top of the batteries in a coke plant are reported. The kind and degree of chemical pollution of the working atmosphere have already been relatively well investigated (Bruun et al.,
Correspondence: ,~se Krokje, Department of Botany, Section for Cell Biology,The University of Trondheim, 7055 Dragvoll (Norway).
1976; Bjorseth et al., 1977, 1978b). Polycyclic aromatic hydrocarbons (PAH) constitute a major part of the air pollution in the coke oven area. At the top of the ovens the amount of particulate PAH in the workers' breathing zone varied from 5 to 1044/~g/m 3 (Bjorseth et al., 1978a). 72% of the particulate PAH was found on particles smaller than 3 ~tm and only 1% was found on particles larger than 7/~m, which means that most of these compounds were within the respiratory size range. The ultimate aim of the present study is to determine the biological availability of the mutagens and to trace possible interactions between exposure to air pollution of the working atmosphere and smoking. The study is aimed at group exposure. The samples have been pooled into exposed smokers and exposed non-smokers respectively, since that, more than individual exposure, indicates the general effect of the working atmosphere. Individual factors (e.g., work hygiene, choice of food, hobbies) can more easily be overruled, while the effect from the common exposure at work will dominate the results.
0165-1218/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)
214 Material and methods
Collection of samples The workers gave their samples within half an hour of finishing the workshift. Samples were also given after a night's sleep: 'morning expectorate'. The samples were then frozen, and kept in polyethylene bags at - 20 ° C. Samples from a control group consisting of inhabitants of Trondheim were also investigated. The control group consisted of people of approximately the same age as the exposed workers. Each person was instructed to cough vigorously and the sputum was collected in a plastic cup. It was easy to obtain representative material containing cell types from the deep-seated areas of the lungs from both the control and the exposed persons. Coughing was repeated 3 times, and the persons gave samples on 3 consecutive days. All the people involved gave information about their age, smoking habits, place of work, previous exposure, use of medicines, etc., in their answers to a questionnaire. Our definition of a smoker was a person who had smoked more than 10 cigarettes per day for more than a year. A non-smoker was one who had either never smoked or had stopped smoking more than 2 years ago. Persons who fitted neither of these categories or who had used medicines the last 3 weeks were excluded from the study. The 4 groups studied were designated as follows: C-NS, control non-smokers; C-S, control smokers; B-NS, battery non-smokers; B-S, battery smokers. The numbers of individuals in the groups were different: 38 control non-smokers, 18 control smokers, 12 battery non-smokers and 15 battery smokers.
Chemicals The following chemicals were used in this study: cyclohexane p.a. (Merck); methanol p.a. (Merck); potassium hydroxide (KOH) p.a. (Merck); dimethyl sulfoxide (DMSO) spectrometric grade (Merck); benzo[a]pyrene, practical grade (Sigma); nutrient broth No. 2 (Oxoid Ltd.); Bacto-Agar (Difco); L-histidine HC1 (Sigma); d,biotin (Sigma); Vogel-Bonner Minimal 'E' Agar plates (Gibco Bio-cult); glucose-6-phosphate (Sigma); NADP (Sigma).
Extraction The extraction procedure has been described in detail previously (Krokje et al., 1985). The material from each group was hydrolyzed with alkaline methanol and extracted by cyclohexane. After the cyclohexane extract was evaporated to dryness, DMSO was added to dissolve the cyclohexane residue. The stock solutions were diluted with DMSO to obtain concentrations of 0.25, 0.50, 1.0, 2.0 and 3.0 g of expectorate per 100 /~1 of test solution.
Mutagenic analysis The Salmonella typhimurium strains TA98 and TA100 were kindly supplied by Dr. Bruce Ames, Berkeley, CA (U.S.A.). The mutagenic activity of extracts of expectorate from all groups was studied according to the Ames standard method (Ames et al., 1975; Maron and Ames, 1983). Tests were carried out in the presence and absence of $9 mix (50/~1 S9/plate).
Preparation of $9 mix Male Sprague-Dawley rats were treated with Aroclor 1254 (500 mg/kg) 5 days before they were killed. The 9000 X g liver supernatant ($9) was divided into 2-ml aliquots, frozen and stored at - 1 9 6 ° C . The protein content (25 mg/ml) was measured by the method of Lowry et al. (1951). The $9 mix ($9 with cofactors) was filtered through a Millipore filter (diameter 0.45/zm) before use.
Testing procedure The extracts from the different groups were tested at 5 different concentrations (triplicate plates) to establish the relationship between concentration and effect. The 'morning expectorate' was only tested with $9. Preliminary experiments without $9 did not show any effect of the expectorate samples, and therefore the samples were only tested with $9 in the further experiments. One concentration (1.0 g) in the linear area of the concentration/effect relationship was tested in the further experiments. The concentration was tested with both TA98 and TA100 in 5 parallel Petri plates. Each test was duplicated, in independent experiments, to ensure reproducibility.
215
Results
Control smokers The expectorate from control smokers does not contain mutagenic compounds at the investigated concentrations (Fig. 1, Table 1). The response of the samples from the control smokers did not differ significantly from that of the control nonsmokers.
Control non-smokers The test solution from the control group of non-smokers showed no indication of a concentration/effect relationship (Fig. 1) either for TA98 or TA100, and the C-NS expectorate does not suggest that any mutagenic compounds are present.
Exposed non-smokers The extract from the battery top non-smokers showed a positive relationship between concentration and effect on both strains of bacteria after $9 activation (Fig. 1). Ames' criterion that the response should be double that of the spontaneous
The statistical significance of all the results was determined with Student's t-test (Weinstein and Lewinson, 1978; Ehrenberg, 1979). Checks on the activating capacity of the $9 mix were made in each experiment using 1/~g benzo[a]pyrene.
aT 500.
TA
100 + S9
480.
we.-S
460440. 420. 400.~ TA
98 + $9
aSO360-
Rr 130 i B-S
340,
120-
320.
110.
300. 280-
100.
260-
90-
240-
80-
220200-
70-
180-
60-
160B-NS
50B-S
/"
30. 20..====
B-NS
~
120-
/I
40.
J
140.
.13B-NS
/
100
. . . .~.~ ~ -B ' ' "o
C-S
80.
~
C-NS
60 2~~'I
N
~
C-NS
:
• C-S
, 2
, .~ 3 g expectorate
" 1B- S ÷
40 100 0
I 1
I= 2
, = 3 g expectorate
0
! 1
Fig. 1. Concentrations and mutagenic effects of the extracts of expectorate from exposed workers (smokers (B-S) and non-smokers
(B-NS)) and from control groups (smokers (C-S) and non-smokers (C-NS)). The total responses of the bacteria (RT) after 48 h incubation are shown. For some of the concentrations each data symbol represents the mean of the R T values for 2 or more independent experiments. A solid line ( ) indicates samples given at the end of the workshift, and a broken line (. . . . . . ) indicates 'morning expectorate'. + indicates an observed reduction in the growth of the background.
216 TABLE 1 M U T A G E N I C EFFECTS OF EXTRACTS OF E X P E C T O R A T E (1.0 g) F R O M EXPOSED W O R K E R S (SMOKERS (B-S) A N D N O N - S M O K E R S (B-NS)), G I V E N AT T H E E N D OF T H E SHIFT, A N D F R O M T H E C O N T R O L S (S MO K ER S (C-S) A N D N O N - S M O K E R S (C-NS)) Test solution C-NS C-S B-NS B-S
TA98 + S9 R ~ 4-_SE 0.0+1.4 3.9_+1.4 -0.8_+3.1 0.5±2.1 13.1_+2.4 15.1-+2.5 48.1_+8.1
TA100 + $9 Ho: R T = R ~ ns + ns ns + + + +++ + + +
Ho: R N = R K
+ + +
R N _+SE
Ho: R-r = R K
7.5_+ 5.6 -12.1_+ 4.0 7.5_+ 6.9 -5.7_+ 5.3 34.6+11.4
ns
159.2_+15.6 52.9_+ 5.5
Ho: R N = R K
ns ns
+++ +++ +++
+++
The net responses_+ standard error (R s _+SE, after 48 h incubation) are tabulated. The net yield responses (R N) can be estimated from the difference between the number of revertants of the test plates (RT) and the control value of the solvent (RK). Results of Student's t-test for the hypotheses H0: R T = R K and H0: R N = R K (ns p > 0.05; R T > R K or R N > RK: + 0.01< p < 0.05; + + 0.001
control was not fulfilled at the concentration most often investigated (1.0 g). But this response together with the increased response at the higher concentration showed that the expectorate from exposed non-smokers contains mutagenic compounds. The response obtained was clearly greater than that of the control non-smokers (Fig. 1). The 'morning expectorate' also showed a weak relationship between concentration and effect. The response was weaker than from the expectorate samples given at the end of the shift.
Exposed smokers The expectorate from the workers on the top of the coke oven batteries produced mutagenic effects on both TA98 and TA100 (Fig. 1, Table 1) and the relationship between concentration and effect was clearly positive with $9 activation. The mutagenic test solution from the exposed smokers' expectorate gave a response which was significantly higher than the non-mutagenic test solution from the control smokers ( p < 0.001), and also higher than that for the control smokers plus exposed non-smokers. The 'morning expectorate' from the exposed smokers showed a mutagenic effect on TA98 while it had a toxic effect on TA100. With regard to
TA98 the effect was weaker than that of samples expectorated at the end of the shift. Discussion
This study has demonstrated that the Salmonella assay can reveal the presence of mutagenic activity in a methanol extract of 0.25-3.0 g hydrolyzed expectorate. The positive response could be correlated to exposure to the working atmosphere on the top of the batteries in a coke plant. The expectorates from the exposed smokers (B-S) (but also to a certain extent from non-smokers (B-NS)) had a positive effect on the bacteria; however, the control samples from both smokers (C-S) and non-smokers (C-NS) did not contain mutagens. The results are in agreement with the ones shown in the study from the potrooms in an aluminum plant (with PAH exposure) (Krokje et al., 1985). There is a synergistic relationship between exposure to occupational pollution and smoking, a weak effect from occupational exposure alone and none from smoking only. The mutagenicity in the expectorate extract indicates that the workers on the top of the batteries in a coke plant are exposed to potential mutagcns which are biologically available. The analysis of
217 the 'morning expectorate' shows that inhaled pollution with potential mutagens is not effectively removed from the respiratory system during the night. From the results of chemical analysis of the work atmosphere at the coke plant investigated (Bjorseth et al., 1978b) and the results from the Salmonella test on air filter samples (Krokje, unpublished) a mutagenic activity in the workers expectorate was expected. A clear effect of indirect mutagens is consistent with the PAH exposure at the battery top. PAH require metabolic activation before they have a mutagenic effect (Ames et al., 1973). Cigarette smoking and occupational exposure may interact through effects of smoking on the dose of genotoxic compounds that reaches the cell. Long-term exposure to cigarette smoke impairs mucociliary clearance (U.S. Department of Health and Human Services, 1982) and could alter the retained dose of an occupational agent. Benzo[a]pyrene and other mutagens may adsorb to particulates in smoke or to environmental dusts (Natusch et al., 1974; Mossman et al., 1983) resulting in a higher fractional retention or different distribution in the lung. The adsorption to dust may also facilitate or inhibit transport of carcinogens through the mucus layer. Cigarette smoke has been shown to increase the epithehal permeability in the tracheobronchial tree (Sirnani et al., 1974); the effect may increase the exposure of the underlying cell to an occupational agent. Another potential site of biologic interaction is the metabolic activation of a genotoxic compound. A number of constituents of cigarette smoke have by increased induction of microsomal enzymes been shown to affect the metabolic activation of other substances (U.S. Department of Health, Education, and Welfare, 1979). Cigarette smoking also alters the cellular composition of the lung, increasing the number of neutrophils and activated macrophages in the lung (U.S. Department of Health and Human Services, 1984); these cells also play a role in the metabolic transformation of occupational agents. Investigations of condensate of tobacco smoke have shown that it does contain mutagens (Kier et al., 1974; Sato et al., 1977). Moller and Dybing (1980) investigated the urine from workers at the
same coke plant. Mutagenic activity in the urine was not related to occupational exposure, but was strongly related to smoking. The negative results obtained for the group of control smokers are therefore surprising. One explanation for the present findings may be that the greatest part of the effect of smoking may be co-mutagenic, vide the increase in mutagenie effect recorded for the group of battery top smokers. In the same way the content of mutagens registered in the urine from smokers (Yahagi et al., 1977; van Doom et al., 1979; Falck et al., 1980; MoUer and Dybing, 1980; Aeschbacher and Chappuis, 1981; Dolara et al., 1981; Putzrath et al., 1981; Bos et al., 1982; Falck, 1982) need not be from the cigarette smoke itself. Hannan et al. (1981) found that cigarette smoke condensate and urine concentrate from smokers exhibit a co-mutagenic action on the mutagenicity of 2-aminoanthracene. Co-mutagens from the cigarette smoke may cooperate with mutagens which have entered the body via other absorption ways than the lungs. The expectorate analysis, in contrast to filter analysis, is corrected for local and time variations in intensity of exposure, the variable use of protective gear, and individual lifestyle factors. I suggest that the Salmonella typhimurium test used on expectorate samples may be effective in certain types of industries, viz. where the atmosphere contains particulates acting as carriers for mutagens apt to be eluated by the body fluids or cellular membranes. Such a test constitutes a tool by which one may monitor or test improvements in the occupational environment. PAH compounds will depend on the availability of carriers, mostly air particulates, to get into the lungs. It may therefore not be sufficient to measure the content of PAH in the working atmosphere, but the biological availability of these compounds should be evaluated.
Acknowledgements I wish to thank tlie Health Service Center and the laboratory personnel at Norsk Koksverk A / S for help during collection of samples. Thanks are also due to the participating workers. I would also like to thank cand.real. K. Zahlsen at the Department of Pharmacology and Toxicology, Faculty of
218
Medicine for preparing the $9. Financial support was obtained from the Norwegian Council for Scientific and Industrial Research, Norsk Koksverk A / S and the Norwegian Cancer Society. References Aeschbacher, H.U., and C. Chappuis (1981) Non-mutagenicity of urine from coffee drinkers compared with that from cigarette smokers, Mutation Res., 89, 161-177. Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. (U.S.A.), 70, 2281-2285. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-rnicrosome mutagenicity test, Mutation Res., 31, 347-364. Bjorseth, A., O. Bjorseth and P.E. Fjeldstad (1977) Kartlegging av PAH ved Norsk Koksverk A/S, Teknisk-hygienisk rapport nr. 2 (NTNF-rapport), Oslo, HD 742/770922, 23 pp. (in Norwegian). Bjorseth, A., O. Bjorseth and P.E. Fjeldstad (1978a) Polycyclic aromatic hydrocarbons in the work atmosphere. II. Determination in a coke plant, Scand. J. Work Environ. Health, 4, 224-236. Bjorseth, O., P.E. Fjeldstad, T. Frost and K. Halgard (1978b) Polysykliske aromatiske hydrokarboner. Eksponeringsmalinger ved Norsk Koksverk A/S, Mo i Rana, Yrkeshygienisk Institutt, Oslo, HD 782/781109, 30 pp. (in Norwegian). Bos, R.P., A.O. Lenaars, J.L.G. Theuws and P.T. Henderson (1982) Mutagenicity of urine from nurses handling cytostatic drugs, influence of smoking, Int. Arch. Occup. Environ. Health, 50, 359-369. Bruun, R., M. Gjolstad, T. Torgrimsen and B. Wermundsen (1976) Teknisk hygieniske undersokelser ved Norsk Koksverk A / S , Mo i Rana 17.-20. november 1975, Yrkeshygienisk Institutt, Oslo, HD 676/760302, 82 pp. (in Norwegian). Dolara, P., S. Mazzoli, D. Rosi, E. Buiatti, S. Baccetti, A. Turchi and V. Vannucci (1981) Exposure to carcinogenic chemicals and smoking increases urinary excretion of mutagens in humans, J. Toxicol. Environ. Health, 8, 95-103. van Doom, R., R.P. Bos, Ch.-M. Leijdekkers, M.A.P. Wagenaars-Zegers, J.L.G. Theuws and P.Th. Henderson (1979) Thioether concentration and mutagenicity of urine from cigarette smokers, Int. Arch. Occup. Environ. Health, 43, 159-166. Ehrenberg, L. (1979) Aspects of statistical inference in testing for genetic toxicity, in: B.J. Kilbey, M. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 419-461. Falck, K. (1982) Urinary mutagenicity caused by smoking, in: M. Sorsa and H. Vainio (Eds.), Mutagens in Our Environment, Proceedings of the Twelfth Annual Meeting of the European Environmental Mutagen Society, Espoo (Fin-
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