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Urinary and faecal mutagenicity in car mechanics exposed to diesel exhaust and in unexposed office workers
Mutation Research, 222 (1989) 375-391 Elsevier
375
MTR 01391
Urinary and faecal mutagenicity in car mechanics exposed to diesel exhaust and in unexposed office workers M.I. W i l l e m s 1, W . K . de R a a t 2 J.A. W e s s t r a 1 G.L. Bakker 2 G. D u b o i s 1 a n d W. v a n D o k k u m 1 I TNO-CIVO Toxicology and Nutrition Institute, Zeist (The Netherlands) and 2 TNO Division of Technology for Society, Delft (The Netherlands) (Received 4 August 1988) (Revision received 24 November 1988) (Accepted 25 November 1988)
Keywords: Urine; Faeces; Ames test; Office workers; Car mechanics
Summary The occurrence of mutagens in the urine and faeces of a group of car mechanics (n = 8) exposed to high concentrations of diesel exhaust in their working place and of a group of office workers (n = 9) not exposed to diesel exhaust during working hours was compared. The aim of the study was to investigate whether the specific diesel exposure a n d / o r other, more lifestyle-related, factors such as diet had any influence on the mutagenicity of excreta. Faeces were collected and pooled for a consecutive period of 48 h, urine was collected in the same period, but in 4 separate portions representing the urine produced during the day and at night on the 2 collection days. Information about food intake was collected by a 2-day dietary record method. Smoking habits and medicinal drug use were recorded as well. Air particulates were collected in and outside the garage during working hours. The mutagenicity of extracts of air particulates (methanol extracts), urine (XAD-2 and XAD-7 extracts) and faeces (acetone, ether and ether-NaOH extracts) was examined in the Ames test. The results did not suggest that exposure to diesel exhaust mutagens enhanced the incidence a n d / o r degree of either faecal or urinary mutagenicity. Urine of 2 mechanics appeared to contain rather high levels of XAD-7 mutagens, but in view of the uneven distribution over the different collection periods any relationship with the exposure to diesel exhaust mutagens seems improbable. Degree and frequency of faecal mutagenicity was higher in office workers than in mechanics. The pattern of faecal mutagenicity was characteristic of that of faecapentaenes. Statistical analysis did not reveal any consistent relationships between urinary and faecal mutagenicity and the various dietary variables.
Correspondence: Dr. M.I. Willems, TNO-CIVO Toxicology and Nutrition Institute, P.O. Box 360, 3700 AJ Zeist (The Netherlands).
Many studies have demonstrated that human excreta and other human biological materials can contain mutagenic compounds. As the presence of these components is often related to certain ex-
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
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posures or lifestyles, testing of human biological materials for mutagenicity has been suggested as a tool for the biological monitoring of exposure to genotoxic carcinogens. Well-known examples of exposures associated with excretion of urinary mutagens in man are tobacco smoking (Yamasaki and Ames, 1977; Dolara et al., 1981; Jaffe et al., 1983; Kriebel et al., 1985), handling of certain cytostatic drugs (Speck et al., 1976), application of coal tar containing polycyclic aromatic hydrocarbons (PAH) in the treatment of psoriasis (Clonfero et al., 1986), occupational exposures such as in coal liquefaction workers (Recio et al., 1984) and consumption of certain foodstuffs (Baker et al., 1982; Dolara et al., 1984; Oyama et al., 1987). Faecal mutagenicity has been widely investigated in studies on the relation between diet and colon cancer. It has been shown that the incidence and degree of mutagenic activity vary widely between groups differing in dietary pattern and lifestyle, and representing populations at varying risk of colon cancer (for references see Reddy, 1986). From these studies it has become clear that part of the human population excretes mutagens in their stools which are produced by bacteria belonging to the intestinal microflora. The chemical identity of these mutagens has been established by the groups of Bruce and co-workers and Wilkins and co-workers. (For a review on this subject, see Krepinsky et al. (1986) and Van Tassel et al. (1986).) The mutagenicity of faecal material has only incidentally been proposed as a possible indicator of occupational exposure to genotoxic carcinogens, although both faeces and urine are products of major pathways for excretion of potentially mutagenic metabolites via the enterohepatic circulation and the kidneys respectively. In studies with experimental animals in which we compared urinary and faecal mutagenicity after treatment of animals with representatives of various classes of chemical mutagens it clearly appeared that examination of urine and faeces side by side enlarged the possibility of detecting exposure to genotoxic carcinogens (Willems and de Raat, 1985; WiUems et al., 1987). In the present study the occurrence of mutagens in excreta of 2 different groups of workers
was compared. One group comprised car mechanics exposed to relatively high concentrations of diesel exhaust in their working place, the other office workers not exposed to diesel exhaust. Organic extracts of diesel exhaust particles contain both directly and indirectly acting bacterial mutagens (Huisingh et al., 1978; Clark and Vigil, 1980; Salmeen et al., 1984). Mutagenically active substances identified are polycyclic aromatic hydrocarbons (PAHs) and nitrated PAH derivatives (nitro-PAHs). Some of the nitro-PAHs occurring in diesel exhaust and also diesel exhaust as such have been shown to be carcinogenic in laboratory animals (Takayama et al., 1985; IARC, 1983; Heinrich et al., 1986). From studies in experimental animals it is known that the urine of rats treated with diesel exhaust particles by various routes showed a marked mutagenic activity in the Salmonella/microsome test. The highest response was observed with TA98 in the presence of metabolic activation (Belisario et al., 1984). The aim of the present study was to investigate whether the specific diesel exposure a n d / o r other, more lifestyle-related, factors such as diet had any influence on the mutagenicity of the excreta. Materials and methods
Subjects A group of 8 male car mechanics, who were exposed to diesel exhaust during their work in a service station for commercial diesel vehicles, and a group of 9 office workers, who were not exposed to diesel exhaust during working hours, participated in the study. Subjects were selected in such a way that both groups had a comparable age structure. As it was not possible to select only non-smokers, smokers were asked not to smoke during the urine and faeces collection period. If they did not succeed in abstaining from smoking, they had to make a note in their diaries on the kind and number of tobacco products smoked.
Recording of dietary food intake, medicine use and smoking habits Information about food intake was collected by using a 2-day record method (Bazarre and Meyers, 1979). Two trained interviewers (one dietician and one home economist) instructed the subjects in
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how to record food consumption in household measures an d how to describe the method of food preparation. Food intake was recorded on 2 consecutive days, i.e., on the first day of urine and faeces collection and on the preceding day, and records were checked afterwards. The use of medicinal drugs and smoking habits were inquired into by the interviewers and were recorded in the diaries. The food records were coded by the interviewers and analysed for nutrient composition using an extended computerised version of the Dutch food composition tables (Voorlichtingsbureau voor de Voeding, 1984).
Collection and processing of airborne particles The particulate fraction of the air was collected with Sartorius HV100 high-volume samplers on glass fibre filters (Sartorius SM13400; 25.7 cm, flow rate 80 m3/h). Sampling was done on 2 different occasions: in the period June-July 1984, and in October 1984 when the excreta were collected. In June-July 2 samples were collected simultaneously: one in the garage on a location where pollution by exhaust was thought to be most serious, and one at the upwind side outside the garage; in October samples were collected in the garage only. The air of the outside location can be regarded as representative of the air used to ventilate the garage. Sampling started at about 08.00 and lasted to about 17.00. The filters laden with particles were extracted with methanol (Rathburn, HPLC grade) in a Soxhlet apparatus for 8 h (about 20 cycles), in the dark; the solvent was evaporated with a rotatory evaporator under reduced pressure at 30°C. The residues were dissolved in dimethyl sulphoxide (DMSO, A.R. Merck, F.R.G.) and stored at - 3 0 ° C.
Collection and processing of urine Urine was collected during a consecutive period of 48 h in 4 separate portions representing the urine produced during the day and at night on both days and designated day-l, night-1 and day-2 and night-2 urine respectively. Day urine was collected from the start of the working day until sleeping time; night urine represented the urine produced during sleeping time until the start of the working day. Urine was collected in glass bottles and kept at 5 °C until completion of the
collection period when urine samples were pooled and stored at - 2 0 ° C until further processing. Storage time between collection and further processing was 2-3 months. A 2-step procedure was used for the extraction of mutagens from the urine samples. The procedure was developed from the one described by Yamasaki and Ames (1977), but besides the styrene-divinyl benzene copolymer XAD2, the methacrylate-based copolymer XAD7 was used (de Raat, 1984). The former resin is effective at adsorbing non-polar solutes, while the latter gives better recoveries for compounds of intermediate polarity. Amberlite XAD2 and XAD7 resins were kindly provided by Rohm and Haas Benelux N.V. (Belgium). Both resins were carefully purified by extraction with methanol and diethyl ether (24 h each) in a Soxhlet apparatus and stored, just covered with methanol, until use. Immediately before use, the resins were washed with MilliQ water and brought into glass-barrel columns (height 9 cm; internal diameter 1 cm). Subsequently, the resins were washed with 50 ml of acetone followed by about 50 ml of distilled water. Then 200 ml of urine was passed through the XAD2 and XAD7 resins (flow rate 4-5 ml/min). Before extraction the urine was warmed to 37 ° C, which resulted in a dissolution of the insoluble fractions. In a few samples residual insoluble substances remained, and these were removed by placing a quartz wool plug on top of the XAD2 column. The eluate of the XAD7 resin was nearly colourless. After passage of the urine both columns were eluted with 4-5 ml distilled water (flow rate 4-5 ml/min) to remove residual histidine. The free water was blown off with a nitrogen stream. Then the quartz wool plug was removed. The adsorbed organic fractions were desorbed with acetone (Rathburn, HPLC grade). Before elution (flow rate 4-5 ml/min) with acetone, the resin particles were submerged in acetone (in the column) and stirred to remove gas bubbles. The quartz wool plug was extracted with acetone by ultrasonic vibration and the extract was added to the XAD2 extract. The acetone was removed by evaporation (rotary evaporator, 30°C, reduced pressure). The 2 residues were dissolved in DMSO and stored at - 3 0 ° C . Within a few days the DMSO solutions were tested.
378
Collection and processing of faeces A standard method of stool collection and processing was followed throughout the study. Faeces were frozen by collecting them directly into a plastic bag fitting into a deep-freezing toilet, enabling freezing of the stool specimens immediately after defaecation. The faecal samples were coded and stored at - 3 0 ° C until further processing. Faeces were collected for a total period of 48 h. After thawing, just prior to extraction, the stool specimens were pooled per subject, and mixed thoroughly by kneading. The mixed faeces were weighed, and divided into portions. Ether extraction of faeces Portions of 60 g faeces were extracted by shaking vigorously with 100 ml of peroxide-free diethyl ether (A.R. Merck, F.R.G.) for 30 s, and supernatant and residue were separated by centrifugation for 2 min. Ether extraction of the residues was repeated twice with 80 ml ether each time. The ether fractions were pooled, and the volume adjusted with ether to 250 ml. Half of the extract was washed with 5 ml 1 N N a O H (p.a., Merck, F.R.G.) for 30 s and then with 3 or 4 portions of 10 ml distilled water until neutral again. The washed and unwashed extracts were dried by filtering over 75 g anhydrous Na2SO 4 ex (A.R. Merck, F.R.G.) and evaporated until almost dry in a rotary evaporator under reduced pressure at 40 ° C. The residues were dissolved in acetone, final concentrations being equivalent to 5 g faeces per ml acetone. This procedure provided the ether extract and the ether-NaOH extract. Acetone extraction The acetone extract was prepared as follows. Portions of 30 g faeces were mixed with 125 ml acetone (p.a., Merck) and then shaken vigorously for 15 min. Supernatant and residue were separated by centrifugation for 2 min. The supernatant was reduced as far as possible at 40 o C, and the last part at 60 o C to about 2 ml in a rotary evaporator. This still contained some water and was transferred with acetone to a calibrated tube and the volume adjusted to a concentration equivalent to 5 g faeces per ml of acetone. Blank
extracts were included in each series of extractions.
Mutagenicity testing The mutagenicity of the extracts of airborne particles, faeces and urine was examined in the S a l m o n e l l a / m i c r o s o m e plate incorporation assay according to procedures described by Ames et al. (1975) and Maron and Ames (1983). Briefly, the test was carried out as follows. To 2 ml molten soft agar, maintained at 46 ° C, were added in this sequence: 0.1 ml of a fully grown culture of the appropriate tester strain, 0.1 ml of the appropriate dilution of the extract, 0.5 ml $9 mix if indicated, and 0.1 ml of a fl-glucuronidase solution in water if indicated. The ingredients were thoroughly mixed and the mixture was immediately poured onto minimal-glucose agar plates. After incubation for 3 days at 37 ° C, the His + revertants were counted and the background growth of H i s bacteria in control and test plates was examined microscopically. Dilutions of the extracts, $9 mix and fl-glucuronidase solutions were prepared just before use. The $9 fractions and $9 mixes, containing 10% $9 ( v / v ) if not indicated otherwise, were prepared according to methods described by Ames et al. (1975). The liver homogenate fraction ($9) was prepared from livers of male rats ( C p b : W U ; Wistar random, 200 g) treated once interperitoneally with 500 mg Aroclor 1254 (Monsanto Company, U.S.A.) per kg body weight 5 days before sacrifice. The experiments were performed in duplicate or in triplicate as indicated in the captions of tables and figures. In each assay positive reference compounds and solvent controls were included. Extracts of the airborne particles were tested with strains TA98 and TA98NR. The latter strain is a mutant of the former which shows a markedly reduced sensitivity to m a n y nitro compounds because of deficient nitroreductase. Two tests were carried out: one for the first 2 days and one for the second 2 days. All doses were tested in duplicate. Urine extracts were tested with strain TA98. Combinations of the extracts of both days were tested in the presence of 1000 Sigma units of fl-glucuronidase (type IX from E. coli, Sigma). The activity of the enzyme was checked by testing
379
in its presence 8-hydroxyquinoline and its glucuronide as positive controls with strain TA100. The tested doses represented 0, 10, 30 and 60 ml of urine. The effects were summarised in one value by calculating straight lines with their standard deviations according to the maximum-likelihood method and assuming Poissonean sampling. The total number of revertants produced per day and per night was calculated by multiplying the slopes of the calculated lines (revertants per ml of urine) with the total volume of urine produced. In this way the excretion of mutagens could be corrected for individual differences in volume of urine. Faecal extracts were examined with tester strains TA98 and TA100. Doses applied represented 500, 167, 56, 19, and 6.5 mg faeces equivalents per plate except for the acetone extract of faeces sample L, which was tested at doses equivalent to 430, 143, 48, 16, and 5.3 mg faeces per plate, and the ether and ether-NaOH extracts of D, which were tested at doses representing 290, 97, 32, 10.7 and 3.6 mg faeces equivalents per plate. Because of shortage of material from subject D only the ether extracts could be made. The mutation index (slope of the dose-response curve) was determined from the part of the dose-effect curve that most approached linearity. The number of revertants per 100 mg faeces and per 48-h stool sample was calculated by multiplying the mutation index with the faecal weight. Results
Mutagenicity of airborne particles The results of the mutagenicity tests with the extracts of airborne particles are presented in Fig. 1. This Figure dearly reveals that the garage partides are much more strongly mutagenic than the outside air particles. There is also a qualitative difference: the effects of the garage particles clearly increased upon application of $9, while no influence was demonstrated on the outside particles. The effects obtained with the nitroreductase-deficient strain TA98NR were much less pronounced, indicating the contribution of compounds which exert their effect after reduction of a nitro group. The mutagenicity of the garage samples collected in October was comparable with that of the first collection period with respect to the extent of the
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Fig. 1. The mutagenicity of extracts from airborne particles with S. typhimurium TA98 and S. typhimurium TA98NR. The indicated values represent m e a n s of duplicates. The dose is expressed as the volume of air from which the tested extract originated. Top, strain TA98; bottom, strain TA98NR. o and e, inside air; [] and ,x outside air; o and n, with S9-rat; • and zx without S9-rat. The metabolic activation system contained 10% $9 from Aroclor-treated rats.
effect as well as the influence of the $9 and the NR mutation (results not shown). Exposure to mutagens is not likely to have been very different during the 2 periods. Mutagenicity of urine extracts Preliminary experiments with a smoker's urine First, 2 preliminary experiments were carried out with the urine of a subject smoking about 20
380 Revertants per plate 300-
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S. typhimuriurn TA98 in the presence of $9 mix. The points represent means of 2 plates. The test without fl-glucuronidase (zx, XAD2; 13, XADT) and the test with 1000 units flglucuronidase per plate (o, XAD2; o, XAD7) were carried out with extracts of samples collected at different dates. The metabolic activation system contained 10% $9 from Aroclortreated rats.
cigarettes p e r day. Fig. 2 gives the results o b t a i n e d with urine s a m p l e d on 2 d a y s in the presence o f $9 mix. N o clear m u t a g e n i c i t y was f o u n d in the a b sence of $9 mix (results n o t shown). B o t h tests d e m o n s t r a t e that indirect m u t a g e n s are present. T h e activity of b o t h the X A D 2 a n d the X A D 7 fractions seemed to b e stronger in the presence of fl-glucuronidase, a l t h o u g h it m u s t b e r e m a r k e d that the 2 tests were carried out with extracts of s a m p l e s collected o n different dates.
left b e h i n d b y X A D 2 . T h e clearest e x a m p l e s are shown in d e t a i l in Fig. 4. Fig. 3 shows t h a t m u t a g e n i c i t y is n e a r l y restricted to the tests c a r r i e d out in the presence of $9 fraction. W e a r b i t r a r i l y r e g a r d a s a m p l e to b e m u t a g e n i c if its effect exceeds twice the s t a n d a r d deviation. S o m e effects were negative, which is p r o b a b l y d u e to a t o x i c i t y - r e l a t e d decrease of the r e v e r t a n t s i n d u c e d d u r i n g the test. S o m e s a m p l e s were n o t tested b e c a u s e the a m o u n t of urine was t o o small; o t h e r s were too toxic for the bacteria. A t first sight, Fig. 3 reveals that, except for the results o b t a i n e d w i t h c a r m e c h a n i c s G a n d D, m u t a g e n o u t p u t was a b o u t evenly d i s t r i b u t e d over the 2 collection d a y s a n d h a d a b o u t the s a m e degree a n d f r e q u e n c y a m o n g c a r m e c h a n i c s a n d office workers. T h e relatively high m u t a g e n levels in the u r i n e o f c a r m e c h a n i c s G a n d D c o u l d largely b e a t t r i b u t e d to m u t a g e n s p i c k e d up b y the X A D 7 resin. S o m e o t h e r urine s a m p l e s of b o t h office w o r k e r s a n d c a r m e c h a n i c s a p p e a r e d to c o n t a i n i n c r e a s e d levels of X A D 7 m u t a g e n s as well, albeit in d i s t i n c t l y lower a m o u n t s t h a n the urine of c a r m e c h a n i c s G a n d D. W i t h o u t further o b s e r v a t i o n s , the i n c r e a s e in X A D 7 m u t a g e n s f o u n d in s o m e of the u r i n e samples of 2 of the car m e c h a n i c s is n o t clear. I n view of the u n e v e n d i s t r i b u t i o n over the collection periods, the increase in X A D 7 m u t a g e n s c a n n o t b e a s c r i b e d to e x p o s u r e to diesel exhaust mutagens. A d d i t i o n of f l - g l u c u r o n i d a s e d i d n o t l e a d to essentially different effects ( d a t a n o t given).
Mutagenicity of faeces extracts Car mechanics and office workers T h e results with urine s a m p l e s of car m e c h a n i c s a n d office workers are s u m m a r i s e d in Fig. 3. This F i g u r e shows that X A D 7 can p i c k up m u t a g e n s
A n u m b e r of subjects a p p e a r e d to p r o d u c e m u t a g e n i c a l l y active faeces. T h e m u t a g e n i c i t y o f the faecal extracts of the m u t a g e n p r o d u c e r s h a d the following c h a r a c t e r i s t i c s in c o m m o n .
Fig. 3. The mutagenicity of XAD2 and XAD7 urine concentrates from car mechanics and office workers, ranked according to total mutagenicity, excreted in the period indicated and given separately for day and night urine. The number 1-4 and 5-8 respectively, present the results obtained in the presence and the absence of $9 mix respectively. The number of revertants per ml urine (calculated from the dose-response relations) is multiplied by the volume of urine produced. The standard deviations (white bars) of the calculated effects (gradients of linear dose-response relations) are depicted after multiplication with the volume produced. The shaded bars represent the effects (light shading for office workers, dark shading for car mechanics). The metabolic activation system contained 10% $9 from Aroclor-treated rats. Legend: The letter is the subject code. The first number indicates the collection day: 1, urine collected during the first 24 h; 2, urine collected during the second 24 h. The second number denotes the collection period: 1, urine collected during the day; 2, urine collected at night. Example: the sample encoded H-2-2 represents night-2 urine of subject H.
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(1) Except for the faecal extracts of office worker S, the ether extracts induced clearly more revertants per mg faeces equivalent than the acetone extracts (Table 1). (2) The mutagenicity of the various extracts was most pronounced in the absence of the metabolic activation system. $9 mix seemed to inactivate or suppress the expression of mutagenicity of the extracts (data not shown).
(3) All extracts that were mutagenic with strain TA98 were also positive with strain TA100, but not vice versa. Strain TA100 was clearly more sensitive to the faecal mutagens than was strain TA98. Table 1 summarises the results obtained with ether (washed and not washed with N a O H ) and acetone extracts of faeces of mechanics and office workers with indicator strains TA98 and TA100 in
386 TABLE 1 MUTAGENICITY OF FAECAL EXTRACTS OF CAR MECHANICS AND OFFICE WORKERS IN THE AMES TEST EXPRESSED PER 100 mg FAECES EQUIVALENTS a Code
48-h stools (g)
Number of revertants per 100 mg faeces eq. with TA98 E-NaOH
Car mechanics A
544
B C D E F G H
78 469 44 305 200 259 127
TA100 E
A -
E-NaOH
E
A
95
-
131
NT
90
-
-
-
-
-
453
307
-
-
980 1739 264
1395 3726 -
130 285 -
Office workers K L M N O P Q R S
246 101 376 340 260 346 716 261 400
80 120 -
89 210 -
-
-
-
1 0 4
-
-
-
-
232
-
-
26 -
36 -
66 -
572 157
755 -
334 356
a Results obtained in the absence of $9 mix. The mutation index (slope of the dose-response curve) was determined from the part of the dose-response curve that most approached linearity. The conditions to qualify a response as positive were the presence of a clear dose-response effect with at least 1 dose level passing 3 times the control value. Abbreviations: E-NaOH, ether extract treated with NaOH; E, ether extract; A, acetone extract; NT, not tested; - extract not mutagenic. The number of spontaneous revertants for TA98 and TA100 varied between 22 and 44, and 110 and 178, respectively.
t h e a b s e n c e o f $9 m i x . Fig. 5 d e p i c t s t h e d o s e - e f fect c u r v e s o b t a i n e d w i t h the faecal e x t r a c t s o f t h e 6 most pronounced mutagen producers. T h e results d e m o n s t r a t e t h a t t h e i n c i d e n c e o f s t o o l s p e c i m e n s s h o w i n g m u t a g e n i c i t y was c l e a r l y h i g h e r in o f f i c e w o r k e r s t h a n in c a r m e c h a n i c s . Three out of 8 mechanics produced mutagenic f a e c e s ( s u b j e c t s A , B a n d F), w h i l e a m o n g t h e g r o u p of o f f i c e w o r k e r s 7 o u t o f 9 s h o w e d d i s t i n c t m u t a g e n i c i t y . I n a d d i t i o n , the d e g r e e o f m u t a g e n i c i t y was c l e a r l y h i g h e r in o f f i c e m u t a g e n p r o d u c e r s t h a n in g a r a g e m u t a g e n p r o d u c e r s .
Food intake The energy intake of car mechanics was slightly h i g h e r t h a n t h a t o f o f f i c e w o r k e r s , as c o u l d b e e x p e c t e d . T o t a l c a r b o h y d r a t e a n d t o t a l fat i n t a k e
were higher and total protein intake was lower among car mechanics than among office workers. N o n e o f t h e s e d i f f e r e n c e s w e r e s t a t i s t i c a l l y signific a n t (see T a b l e 2).
Relation between urinary and faecal mutagenicity and food intake figures To investigate the possible linear relationship between urinary and faecal mutagenicity and several dietary variables, Pearson's coefficients w e r e c a l c u l a t e d . T h e d i e t a r y v a r i a b l e s a n d the mutation indices for urine and faeces were calcul a t e d as i n d i c a t e d in M a t e r i a l s a n d m e t h o d s . Pearson's correlation coefficients did not reveal any consistent relations between urinary mutagenicity and the various dietary variables or between urinary and faecal mutagenicity.
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Fig. 5. Dose-effect curves obtained with faecal extracts of 'faecal mutagen producers' in the Ames test in the absence of $9 mix. Indicator strain: S. typhimurium TA100. t3, e t h e r - N a O H extract; o , ether extract; zx, acetone extract.
A number of rather high correlation coefficients, both positive and negative, were found between faecal mutagenicity and several dietary
variables. Within the group of car mechanics relatively high positive correlation coefficients were found between faecal mutagenicity and animal
388 TABLE2 MEAN DAILY INTAKE OF GARAGE AND O F ~ C E WORKERS a Car mechanics Office workers (n = 8) (n = 9)
Energy(kcal/day)
33305:600
2628+712
865:12 (10 en%) 39+ 8 47+ 12 138+ 33 (37 en%) 413 5 : 9 1 (50 en%) 36 5: 7 284 5:204
1005:22 (15 en%) 33+ 11 68+ 14 1045:37 (36 en%) 308 5 : 9 0 (47 en%) 36 5: 9 324 + 126
Food (solid and liquid, g) 3 291 5:458 Meat (g) 127 5 : 3 9 Potatoes/vegetables/fruit (g) 876 + 258
3 242 + 528 133 5 : 4 7 965 5:173
Nutrients Total protein (g) vegetable(g) animal(g) Total lipids (g) Total carbohydrates (g) Total dietary fibre (g) Cholesterol (mg)
Foodstuffs
a Mean 5: SEM; en%, percentage of the daily energy intake.
protein ( r = +0.71) and cholesterol ( r = +0.89) consumed, and a negative correlation coefficient with dietary fibre ( r = -0.55). Within the group of office workers most of the correlation coefficients calculated between faecal mutagenicity and the various dietary variables were negative. None of these values were statistically significant. Discussion
The results of the present study indicate that exposure to diesel exhaust mutagens has no enhancing effect on the incidence and degree of urinary and faecal mutagenicity. Belisario et al. (1984) have demonstrated that treatment of rats with diesel exhaust particulates, either intraperitoneally, orally or subcutaneously, leads to excretion of mutagens in urine. Urinary mutagenicity was apparent both after treatment with a suspension of diesel exhaust particulates and when diesel exhaust particulates were given as such in a gelatin capsule, indicating the biological availability of mutagens adsorbed onto diesel exhaust particulates in the absence of any suspend-
ing vehicle (Belisario et al., 1984). Although there are no data on the biological availability of mutagens from diesel exhaust particles after exposure by inhalation it seems reasonable to assume that upon exposition by inhalation mutagens adsorbed onto diesel exhaust particles become biologically available as well. About 90% of diesel exhaust particles is of respirable size, and inhaled particles are deposited and retained for a number of days in lung tissue. As pointed out in the Introduction mutagenically active substances identified in diesel exhaust particulates comprise PAHs and nitro-PAHs. Systemic uptake of PAHs has been demonstrated after intratracheal administration of hydrocarbons adsorbed onto particles of various sizes and chemical composition (IARC, 1983). Absorption may take place either via pulmonary tissues or via the enteral route after oral uptake through swallowing after mucociliary clearance of the particulate matter from the lungs (IARC, 1983). Once the PAHs are absorbed they are rapidly distributed. Part of PAHs or metabolites thereof are stored mainly in fat tissues and are slowly released. Hepatobiliary excretion and elimination through the faeces is the major route by which PAHs are removed from the body, regardless of the original route of administration (IARC, 1983). Data on absorption, distribution, kinetics, and excretion of nitro-PAHs are scarce. From studies with bacteria it is known that nitroPAHs are converted to their ultimate electrophilic intermediate by reduction of the nitro group. Anaerobic bacteria of the colonic flora are capable of activating the nitro-PAHs by nitroreduction (Rozenkranz and Mermelstein, 1983). For 1-nitropyrene it is proposed that the enterohepatic circulation is an important metabolic pathway in conventional rats, hydroxylation and conjugation taking place in the liver and nitroreduction and deconjugation in the intestinal bacteria (Tokiwa and Ohnishi, 1986). Various reasons, either alone or in combination, may explain why we did not find an increase in the incidence and degree of urinary and faecal mutagenicity in the present study. First it must be mentioned that the dose of particle-associated diesel exhaust mutagens to which car mechanics were exposed in the present study was only a fraction of the dose rats were exposed to in the study of
389 Belisario et al. (1984). To get an impression of the dose of particulate-associated mutagens car mechanics were exposed to, we examined the mutagenicity of air particles sampled during working hours (Fig. 1). Assuming that the average man inhales at most 8-10 m3 during an 8-h working day it becomes clear that the daily exposure was very low indeed. On the other hand, one should realise that the exposure takes place every working day and that particle-associated mutagens may accumulate in lung macrophages and be released gradually. A further point is the background noise of unidentified mutagenic activity in human excreta that makes an increase in incidence and degree of urinary and/or faecal mutagenicity hard to detect. Confounding factors in mutagenicity testing of human excreta are, among other things, various lifestyle-related factors such as smoking and diet. In fact 5 out of the 8 car mechanics had smoked one or a few cigarettes during the different collection periods, while none of the office workers had smoked. Our data do not, however, point to any relation between smoking pattern and the incidence and degree of urinary mutagens in either XAD2- or XAD7-treated urine samples. The number of cigarettes smoked was apparently too low to lead to detectable levels of mutagens in the urine. Another important point is the working-up procedure of the excreta. Recent work in which various extraction and working-up procedures of urine were compared has revealed that the use of 2 XAD resins of different polarity may considerably enlarge the amount a n d / o r spectrum of mutagens that are picked up (de Raat and van Ardenne, 1984; Belisario et al., 1984). Our results clearly confirmed this finding, both in the preliminary experiments in which a smoker's urine was used to evaluate the procedure and in the experiment with urine of office workers and car mechanics. We did not investigate whether the XAD7 mutagens were the result of an overloading of the XAD2 column, but our results merely point to the presence of specific mutagens of different polarity that are not retained on the XAD2 resin. The origin of the XAD7 mutagens is not clear: they were found both in smokers' and non-smokers' urine, and in urine of both office workers and car mechanics. In urine of rats treated with diesel exhaust par-
ticulates XAD2 and XAD7 mutagens were found as well, pointing to the presence of mutagens of different polarity (Belisario et al., 1984). An unexpected observation in the present study was the finding that the degree and incidence of faecal mutagenicity were distinctly higher among office workers than in car mechanics. In both groups faecal mutagenicity had a number of characteristics in common pointing to the presence of comparable mutagens in both groups of workers. The characteristics of faecal mutagenicity in the present study (TA100 being the most sensitive tester strain, mutagenicity being apparent without addition of $9 mix, an inhibitory fraction being present in the faecal extracts, which is eliminated at least partly by alkali washing) are consistent with reports on faecal mutagens in humans consuming a mixed western diet and are ascribed to the presence of faecapentaenes, mutagens originating from certain Bacteroides species in the intestinal tract (Wilkins and Van Tassel, 1983). However, the incidence of faecal mutagen producers among the group of office workers in the present study (78%) is not consistent with the reported studies. Although HPLC analysis reveals that faecapentaenes may occur in 50-75% of the individuals screened, the reported incidence of faecal mutagen producers is in general much lower and amounts to 10-20% in high-risk populations (Van Tassel, 1986). It is not clear to which factors the differences in faecal mutagenicity between office workers and car mechanics must be ascribed. Higher faecal mutagen levels may result from increases in mutagens but also from changes in the concentration of substances interfering with the expression of mutagenic activity (Hayatsu et al., 1981; Willems and de Raat, 1985). Differences in the incidence and degree of faecal mutagenicity have been found between populations differing in dietary habits and lifestyle (Kuhnlein et al., 1981; Ehrich et al., 1979; Reddy et al., 1980). It has been shown that the incidence and degree of faecal mutagenicity were higher in groups consuming a high-fat, high-meat, low-fibre diet than in groups consuming a high-fat, low-meat, high-fibre diet (Reddy et al., 1986). Two recently published diet intervention studies show that the production a n d / o r excretion of faecal mutagens becomes noticeably
390
lower when the diet of faecal mutagen producers is supplemented with fibre in the form of whole wheat and rye bread or of wheat b r a n (Reddy et al., 1987; Venitt et al., 1986). Furthermore, it appeared from observations in one faecal mutagen producer that addition of fat to the normal diet did not alter faecal mutagenicity (Venitt et al., 1986). In the present study total carbohydrate and fat intake figures tended to be slightly lower and total protein intake slightly higher in office workers than in car mechanics, without the differences reaching statistical significance. Analysis of the relationships between various dietary variables and faecal mutagenicity revealed a number of rather high correlation coefficients, negative as well as positive, but none of the correlation coefficients reached statistical significance. Statistical analysis did not reveal any relations between urinary mutagenicity and dietary variables or between urinary mutagenicity and faecal mutagenicity. Ingestion of food products such as fried pork, bacon and welldone ground beef has been reported to lead to an increased output of urinary and - in the case of well-done fried ground beef - also of faecal mutagens (Baker et al., 1982; Dolara et al., 1984; Hayatsu et al., 1985a,b). The present results did not show significant differences in consumption of fried meat between the 2 groups investigated. The higher incidence and degree of faecal mutagenicity among the office workers, therefore, cannot be ascribed to fried meat. In summary, our results did not indicate that exposure to diesel exhaust mutagens had any enhancing effect on degree and incidence of mutagen output in urine and faeces. Incidence and degree of faecal mutagen output was clearly higher among office workers than among car mechanics, while urinary mutagen output was about equal in both groups, except for 2 mechanics who did produce very mutagenic XAD7 extracts. The cause of the differences in faecal mutagen output between car mechanics and office workers is not clear, statistical analysis of possible links between various dietary variables and faecal mutagenicity being inconclusive and revealing other trends among car mechanics than among office workers. The present study did not reveal any relations between urine mutagen output and the various
dietary variables or between urinary and faecal mutagenicity.
Acknowledgements The study was carried out in close cooperation with the Regional Occupational Health Service of Groningen. Our special thanks are due to Drs. H.J. Logtenberg and R.P. Snorn.
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting c a r c i n o g e n s a n d m u t a g e n s with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Baker, R.D., A. Arlauskas, A. Bonin and D. Angus (1982) Detection of mutagenic activity in human urine following fried pork or bacon meals, Cancer Lett., 16, 81-89. Bazarre, T.L., and M.P. Meyers (1979) The collection of food intake data in cancer epidemiology studies, Nutr. Cancer, 1, 22-45. Befisario, M.A., V. Buonocore, E. De Marinis and F. De Lorenzo (1984) Biological availability of mutagenic compounds adsorbed onto diesel exhaust particulate, Mutation Res., 135, 1-9. Clark, C.R., and C.L. Vigil (1980) Influence of rat lung and liver homogenates on the mutagenicity of diesel exhaust particulate extracts, Toxicol. Appl. Pharmacol., 56,110-115. Clonfero, E., M. Zordan, D. Cottica, P. Venier, L. Pozzoli, E.L. Cardin, F. Sarto and A.G. Levis (1986) Mutagenic activity and polycychc aromatic hydrocarbon levels in urine of humans exposed to therapeutical coal tar, Carcinogenesis, 7, 819-823. 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. Dolara, P., G. Caderni, M. Salvadori, L. Tringale and M. Lodovici (1984) Urinary mutagens in humans after fried pork and bacon meals, Cancer Lett., 22, 275-280. Ehrich, M., J.E. Aswell, R.L. Van Tassel and T.D. Wiikins (1979) Mutagens in the feces of 3 South African populations at different levels of risk for colon cancer, Mutation Res., 64, 231-240. Hayatsu, H., S. Arimoto, K. Togawa and M. Makita (1981) Inhibitory effect of ether extract of human faeces on activity of mutagens: inhibition by oleic and linoleic acids, Mutation Res., 81, 287-293. Hayatsu, H., T. Hayatsu and Y. Ohara (1985a) Mutagenicity of human urine caused by ingestion of fried ground beef, Jpn. J. Cancer Res. (Gann), 76, 445-448. Hayatsu, H., T. Hayatsu, Y. Wataya and H.F. Mower (1985b) Fecal mutagenicity arising from ingestion of fried ground beef in the human, Mutation Res., 143, 207-211. Heinrich, U., H. Muhle, S. Takenaka, H. Ernst, R. Fuhst, U. Mohr, F. Pott and W. Stober (1986) Chronic effects on the
391 respiratory tract of hamsters, mice and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions, J. Appl. Toxicol., 6, 383-395. Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R. Zweidinger, S. Tejada et al. (1978) Application of bioassay to the characterization of diesel particle emissions, in: M.D. Waters, S. Nesnow, J.L. Huisingh et al. (Eds.), Application of Short-term Bioassays in the Fractionation and Analysis of Complex Environmental Mixtures. NTIS PB-299 429, pp. 381-418. IARC (1983) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Polynuclear aromatic compounds, Part I, Chemical, Environmental and Experimental Data, IARC, Lyon, 32 pp. Jaffe, R.L., W.J. Nicholson and A.J. Garro (1983) Urinary mutagen levels in smokers, Cancer Lett., 20, 37-42. Krepinsky, J.J., W.R. Bruce, I. Gupta, K. Suzuki, J.J. Rafter and P. Child (1986) Diet-related factors in the origin of cancer of the large bowel - A molecular scientist's point of view, in: I. Knudsen ('Ed.), Progress in Clinical and Biological Research, Genetic Toxicology of the Diet, Alan R. Liss, New York, pp. 183-197. Kriebel, D., J. Henry, J.C. Gold, A. Bronsdon and B. Commoner (1985) The mutagenicity of cigarette smokers' urine, J. Environ. Pathol. Toxicol., 6, 157-170. Kuhnlein, U., D. Bergstrom and H. Kuhnlein (1981) Mutagens in feces from vegetarians and non-vegetarians, Mutation Res., 85, 1-12. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Oyama, S., T. Inamasu, M. Ishisawa, N. Ishinishi and K. Matauura (1987) Mutagenicity of human urine after the consumption of fried salted salmon, Food Chem. Toxicol., 25, 147-153. Raat, W.K. de, and R.A.M. van Ardenne (1984) Sorption of organic compounds from urine in mutagenicity testing: choice of sorbent, J. Chromatogr., 310, 41-49. Recio, L., H.G. Enoch, M.A. Hannan and R.H. Hill (1984) Application of urine mutagenicity to monitor coal liquefaction workers, Mutation Res., 136, 201-207. Reddy, B.R., C.S. (1986) Fecal mutagens as a function of diet, in: I. Knudsen (Ed.), Progress in Clinical and Biological Research, Genetic Toxicology of the Diet, Alan R. Liss, New York, pp. 213-224. Reddy, B.R., C.S. Sharma, L. Darby, K. Laakso and E.L. Wynder (1980) Metabolic epidemiology of large bowel cancer; fecal mutagens in high and low-risk populations for colon cancer: a preliminary report, Mutation Res., 72, 511-519.
Reddy, B.R., C.S. Sharma, B. Simi, A. Engle, K. Laakso, P. Puska and R. Korpela (1987) Metabolic epidemioiogy of colon cancer: Effect of dietary fiber on fecal mutagens and bile acids in healthy subjects, Cancer Res., 47, 644-648. Rosenkranz, H.S., and R. Mermelstein (1983) Mutagenicity and genotoxicity of nitroarenes. All nitro-containing chemicals were not created equal, Mutation Res., 114, 217-267. Salmeen, I.T., A.M. Pero, R. Zator, D. Schuetzle and T.L. Riley (1984) Ames assay chromatograms and the identification of mutagens in diesel particle extracts, Environ. Sci. Technol., 18, 375-382. Speck, W.T., A.B. Stein and H.S. Rosenkranz (1976) Mutagenicity of metronidazole: presence of several active metabolites in human urine, J. Natl. Cancer Inst., 56, 283-284. Takayama, S., T. Ishikawa, H. Nakajima and S. Sato (1985) Lung carcinoma induction in Syrian golden hamsters by intratracheal instillation of 1,6-dinitropyrene, Jpn. J. Cancer Res. (Gann), 76, 457-461. Tassel, R.L. Van, R.M. Schram and T.D. Wilkins (1986) Microbial biosynthesis of fecapentaenes, in: I. Knudsen (Ed.), Progress in Clinical and Biological Research, Genetic Toxicology of the Diet, Alan R. Liss, New York, pp. 199-211. Tokiwa, H., and Y. Ohnishi (1986) Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment, CRC Crit. Rev. Toxicol., 23-60. Venitt, S., D. Bosworth and A.J. Alldrick (1986) Pilot study of the effect of diet on the mutagenicity of human faeces, Mutagenesis, 1, 353-358. Voorlichtingsbureau voor de Voeding (1984) Uitgebreide Voedingsmiddelen Tabel (UCV), The Hague. Wilkins, T.D., and R.L. Van Tassel (1983) Production of intestinal mutagens, in: D.J. Hentges (Ed.), Human Intestinal Microflora in Health and Disease, Academic Press, New York, pp. 265-288. Willems, M.I., and W.K. de Raat (1985) Mutagenicity of urine and faeces after treatment of rats with known genotoxins, Carcinogenesis, 6, 967-976. Willems, M.I., W.K. de Raat, R.A. Baan, J.W.G.M. Wilmer, M.J. Lansbergen and P.H.M. Lohman (1987) Monitoring the exposure of rats to 2-acetylaminofluorene by the estimation of mutagenic activity in excreta, sister-chromatid exchanges in peripheral blood cells and DNA adducts in peripheral blood, liver and spleen, Mutation Res., 176, 211-223. Yamasaki, E., and B.N. Ames (1977) Concentration of mutagens from urine by adsorption with the non-polar resin XAD-2: cigarette smokers have mutagenic urine, Proc. Natl. Acad. Sci. (U.S.A.), 74, 3555-3559.
375
MTR 01391
Urinary and faecal mutagenicity in car mechanics exposed to diesel exhaust and in unexposed office workers M.I. W i l l e m s 1, W . K . de R a a t 2 J.A. W e s s t r a 1 G.L. Bakker 2 G. D u b o i s 1 a n d W. v a n D o k k u m 1 I TNO-CIVO Toxicology and Nutrition Institute, Zeist (The Netherlands) and 2 TNO Division of Technology for Society, Delft (The Netherlands) (Received 4 August 1988) (Revision received 24 November 1988) (Accepted 25 November 1988)
Keywords: Urine; Faeces; Ames test; Office workers; Car mechanics
Summary The occurrence of mutagens in the urine and faeces of a group of car mechanics (n = 8) exposed to high concentrations of diesel exhaust in their working place and of a group of office workers (n = 9) not exposed to diesel exhaust during working hours was compared. The aim of the study was to investigate whether the specific diesel exposure a n d / o r other, more lifestyle-related, factors such as diet had any influence on the mutagenicity of excreta. Faeces were collected and pooled for a consecutive period of 48 h, urine was collected in the same period, but in 4 separate portions representing the urine produced during the day and at night on the 2 collection days. Information about food intake was collected by a 2-day dietary record method. Smoking habits and medicinal drug use were recorded as well. Air particulates were collected in and outside the garage during working hours. The mutagenicity of extracts of air particulates (methanol extracts), urine (XAD-2 and XAD-7 extracts) and faeces (acetone, ether and ether-NaOH extracts) was examined in the Ames test. The results did not suggest that exposure to diesel exhaust mutagens enhanced the incidence a n d / o r degree of either faecal or urinary mutagenicity. Urine of 2 mechanics appeared to contain rather high levels of XAD-7 mutagens, but in view of the uneven distribution over the different collection periods any relationship with the exposure to diesel exhaust mutagens seems improbable. Degree and frequency of faecal mutagenicity was higher in office workers than in mechanics. The pattern of faecal mutagenicity was characteristic of that of faecapentaenes. Statistical analysis did not reveal any consistent relationships between urinary and faecal mutagenicity and the various dietary variables.
Correspondence: Dr. M.I. Willems, TNO-CIVO Toxicology and Nutrition Institute, P.O. Box 360, 3700 AJ Zeist (The Netherlands).
Many studies have demonstrated that human excreta and other human biological materials can contain mutagenic compounds. As the presence of these components is often related to certain ex-
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
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posures or lifestyles, testing of human biological materials for mutagenicity has been suggested as a tool for the biological monitoring of exposure to genotoxic carcinogens. Well-known examples of exposures associated with excretion of urinary mutagens in man are tobacco smoking (Yamasaki and Ames, 1977; Dolara et al., 1981; Jaffe et al., 1983; Kriebel et al., 1985), handling of certain cytostatic drugs (Speck et al., 1976), application of coal tar containing polycyclic aromatic hydrocarbons (PAH) in the treatment of psoriasis (Clonfero et al., 1986), occupational exposures such as in coal liquefaction workers (Recio et al., 1984) and consumption of certain foodstuffs (Baker et al., 1982; Dolara et al., 1984; Oyama et al., 1987). Faecal mutagenicity has been widely investigated in studies on the relation between diet and colon cancer. It has been shown that the incidence and degree of mutagenic activity vary widely between groups differing in dietary pattern and lifestyle, and representing populations at varying risk of colon cancer (for references see Reddy, 1986). From these studies it has become clear that part of the human population excretes mutagens in their stools which are produced by bacteria belonging to the intestinal microflora. The chemical identity of these mutagens has been established by the groups of Bruce and co-workers and Wilkins and co-workers. (For a review on this subject, see Krepinsky et al. (1986) and Van Tassel et al. (1986).) The mutagenicity of faecal material has only incidentally been proposed as a possible indicator of occupational exposure to genotoxic carcinogens, although both faeces and urine are products of major pathways for excretion of potentially mutagenic metabolites via the enterohepatic circulation and the kidneys respectively. In studies with experimental animals in which we compared urinary and faecal mutagenicity after treatment of animals with representatives of various classes of chemical mutagens it clearly appeared that examination of urine and faeces side by side enlarged the possibility of detecting exposure to genotoxic carcinogens (Willems and de Raat, 1985; WiUems et al., 1987). In the present study the occurrence of mutagens in excreta of 2 different groups of workers
was compared. One group comprised car mechanics exposed to relatively high concentrations of diesel exhaust in their working place, the other office workers not exposed to diesel exhaust. Organic extracts of diesel exhaust particles contain both directly and indirectly acting bacterial mutagens (Huisingh et al., 1978; Clark and Vigil, 1980; Salmeen et al., 1984). Mutagenically active substances identified are polycyclic aromatic hydrocarbons (PAHs) and nitrated PAH derivatives (nitro-PAHs). Some of the nitro-PAHs occurring in diesel exhaust and also diesel exhaust as such have been shown to be carcinogenic in laboratory animals (Takayama et al., 1985; IARC, 1983; Heinrich et al., 1986). From studies in experimental animals it is known that the urine of rats treated with diesel exhaust particles by various routes showed a marked mutagenic activity in the Salmonella/microsome test. The highest response was observed with TA98 in the presence of metabolic activation (Belisario et al., 1984). The aim of the present study was to investigate whether the specific diesel exposure a n d / o r other, more lifestyle-related, factors such as diet had any influence on the mutagenicity of the excreta. Materials and methods
Subjects A group of 8 male car mechanics, who were exposed to diesel exhaust during their work in a service station for commercial diesel vehicles, and a group of 9 office workers, who were not exposed to diesel exhaust during working hours, participated in the study. Subjects were selected in such a way that both groups had a comparable age structure. As it was not possible to select only non-smokers, smokers were asked not to smoke during the urine and faeces collection period. If they did not succeed in abstaining from smoking, they had to make a note in their diaries on the kind and number of tobacco products smoked.
Recording of dietary food intake, medicine use and smoking habits Information about food intake was collected by using a 2-day record method (Bazarre and Meyers, 1979). Two trained interviewers (one dietician and one home economist) instructed the subjects in
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how to record food consumption in household measures an d how to describe the method of food preparation. Food intake was recorded on 2 consecutive days, i.e., on the first day of urine and faeces collection and on the preceding day, and records were checked afterwards. The use of medicinal drugs and smoking habits were inquired into by the interviewers and were recorded in the diaries. The food records were coded by the interviewers and analysed for nutrient composition using an extended computerised version of the Dutch food composition tables (Voorlichtingsbureau voor de Voeding, 1984).
Collection and processing of airborne particles The particulate fraction of the air was collected with Sartorius HV100 high-volume samplers on glass fibre filters (Sartorius SM13400; 25.7 cm, flow rate 80 m3/h). Sampling was done on 2 different occasions: in the period June-July 1984, and in October 1984 when the excreta were collected. In June-July 2 samples were collected simultaneously: one in the garage on a location where pollution by exhaust was thought to be most serious, and one at the upwind side outside the garage; in October samples were collected in the garage only. The air of the outside location can be regarded as representative of the air used to ventilate the garage. Sampling started at about 08.00 and lasted to about 17.00. The filters laden with particles were extracted with methanol (Rathburn, HPLC grade) in a Soxhlet apparatus for 8 h (about 20 cycles), in the dark; the solvent was evaporated with a rotatory evaporator under reduced pressure at 30°C. The residues were dissolved in dimethyl sulphoxide (DMSO, A.R. Merck, F.R.G.) and stored at - 3 0 ° C.
Collection and processing of urine Urine was collected during a consecutive period of 48 h in 4 separate portions representing the urine produced during the day and at night on both days and designated day-l, night-1 and day-2 and night-2 urine respectively. Day urine was collected from the start of the working day until sleeping time; night urine represented the urine produced during sleeping time until the start of the working day. Urine was collected in glass bottles and kept at 5 °C until completion of the
collection period when urine samples were pooled and stored at - 2 0 ° C until further processing. Storage time between collection and further processing was 2-3 months. A 2-step procedure was used for the extraction of mutagens from the urine samples. The procedure was developed from the one described by Yamasaki and Ames (1977), but besides the styrene-divinyl benzene copolymer XAD2, the methacrylate-based copolymer XAD7 was used (de Raat, 1984). The former resin is effective at adsorbing non-polar solutes, while the latter gives better recoveries for compounds of intermediate polarity. Amberlite XAD2 and XAD7 resins were kindly provided by Rohm and Haas Benelux N.V. (Belgium). Both resins were carefully purified by extraction with methanol and diethyl ether (24 h each) in a Soxhlet apparatus and stored, just covered with methanol, until use. Immediately before use, the resins were washed with MilliQ water and brought into glass-barrel columns (height 9 cm; internal diameter 1 cm). Subsequently, the resins were washed with 50 ml of acetone followed by about 50 ml of distilled water. Then 200 ml of urine was passed through the XAD2 and XAD7 resins (flow rate 4-5 ml/min). Before extraction the urine was warmed to 37 ° C, which resulted in a dissolution of the insoluble fractions. In a few samples residual insoluble substances remained, and these were removed by placing a quartz wool plug on top of the XAD2 column. The eluate of the XAD7 resin was nearly colourless. After passage of the urine both columns were eluted with 4-5 ml distilled water (flow rate 4-5 ml/min) to remove residual histidine. The free water was blown off with a nitrogen stream. Then the quartz wool plug was removed. The adsorbed organic fractions were desorbed with acetone (Rathburn, HPLC grade). Before elution (flow rate 4-5 ml/min) with acetone, the resin particles were submerged in acetone (in the column) and stirred to remove gas bubbles. The quartz wool plug was extracted with acetone by ultrasonic vibration and the extract was added to the XAD2 extract. The acetone was removed by evaporation (rotary evaporator, 30°C, reduced pressure). The 2 residues were dissolved in DMSO and stored at - 3 0 ° C . Within a few days the DMSO solutions were tested.
378
Collection and processing of faeces A standard method of stool collection and processing was followed throughout the study. Faeces were frozen by collecting them directly into a plastic bag fitting into a deep-freezing toilet, enabling freezing of the stool specimens immediately after defaecation. The faecal samples were coded and stored at - 3 0 ° C until further processing. Faeces were collected for a total period of 48 h. After thawing, just prior to extraction, the stool specimens were pooled per subject, and mixed thoroughly by kneading. The mixed faeces were weighed, and divided into portions. Ether extraction of faeces Portions of 60 g faeces were extracted by shaking vigorously with 100 ml of peroxide-free diethyl ether (A.R. Merck, F.R.G.) for 30 s, and supernatant and residue were separated by centrifugation for 2 min. Ether extraction of the residues was repeated twice with 80 ml ether each time. The ether fractions were pooled, and the volume adjusted with ether to 250 ml. Half of the extract was washed with 5 ml 1 N N a O H (p.a., Merck, F.R.G.) for 30 s and then with 3 or 4 portions of 10 ml distilled water until neutral again. The washed and unwashed extracts were dried by filtering over 75 g anhydrous Na2SO 4 ex (A.R. Merck, F.R.G.) and evaporated until almost dry in a rotary evaporator under reduced pressure at 40 ° C. The residues were dissolved in acetone, final concentrations being equivalent to 5 g faeces per ml acetone. This procedure provided the ether extract and the ether-NaOH extract. Acetone extraction The acetone extract was prepared as follows. Portions of 30 g faeces were mixed with 125 ml acetone (p.a., Merck) and then shaken vigorously for 15 min. Supernatant and residue were separated by centrifugation for 2 min. The supernatant was reduced as far as possible at 40 o C, and the last part at 60 o C to about 2 ml in a rotary evaporator. This still contained some water and was transferred with acetone to a calibrated tube and the volume adjusted to a concentration equivalent to 5 g faeces per ml of acetone. Blank
extracts were included in each series of extractions.
Mutagenicity testing The mutagenicity of the extracts of airborne particles, faeces and urine was examined in the S a l m o n e l l a / m i c r o s o m e plate incorporation assay according to procedures described by Ames et al. (1975) and Maron and Ames (1983). Briefly, the test was carried out as follows. To 2 ml molten soft agar, maintained at 46 ° C, were added in this sequence: 0.1 ml of a fully grown culture of the appropriate tester strain, 0.1 ml of the appropriate dilution of the extract, 0.5 ml $9 mix if indicated, and 0.1 ml of a fl-glucuronidase solution in water if indicated. The ingredients were thoroughly mixed and the mixture was immediately poured onto minimal-glucose agar plates. After incubation for 3 days at 37 ° C, the His + revertants were counted and the background growth of H i s bacteria in control and test plates was examined microscopically. Dilutions of the extracts, $9 mix and fl-glucuronidase solutions were prepared just before use. The $9 fractions and $9 mixes, containing 10% $9 ( v / v ) if not indicated otherwise, were prepared according to methods described by Ames et al. (1975). The liver homogenate fraction ($9) was prepared from livers of male rats ( C p b : W U ; Wistar random, 200 g) treated once interperitoneally with 500 mg Aroclor 1254 (Monsanto Company, U.S.A.) per kg body weight 5 days before sacrifice. The experiments were performed in duplicate or in triplicate as indicated in the captions of tables and figures. In each assay positive reference compounds and solvent controls were included. Extracts of the airborne particles were tested with strains TA98 and TA98NR. The latter strain is a mutant of the former which shows a markedly reduced sensitivity to m a n y nitro compounds because of deficient nitroreductase. Two tests were carried out: one for the first 2 days and one for the second 2 days. All doses were tested in duplicate. Urine extracts were tested with strain TA98. Combinations of the extracts of both days were tested in the presence of 1000 Sigma units of fl-glucuronidase (type IX from E. coli, Sigma). The activity of the enzyme was checked by testing
379
in its presence 8-hydroxyquinoline and its glucuronide as positive controls with strain TA100. The tested doses represented 0, 10, 30 and 60 ml of urine. The effects were summarised in one value by calculating straight lines with their standard deviations according to the maximum-likelihood method and assuming Poissonean sampling. The total number of revertants produced per day and per night was calculated by multiplying the slopes of the calculated lines (revertants per ml of urine) with the total volume of urine produced. In this way the excretion of mutagens could be corrected for individual differences in volume of urine. Faecal extracts were examined with tester strains TA98 and TA100. Doses applied represented 500, 167, 56, 19, and 6.5 mg faeces equivalents per plate except for the acetone extract of faeces sample L, which was tested at doses equivalent to 430, 143, 48, 16, and 5.3 mg faeces per plate, and the ether and ether-NaOH extracts of D, which were tested at doses representing 290, 97, 32, 10.7 and 3.6 mg faeces equivalents per plate. Because of shortage of material from subject D only the ether extracts could be made. The mutation index (slope of the dose-response curve) was determined from the part of the dose-effect curve that most approached linearity. The number of revertants per 100 mg faeces and per 48-h stool sample was calculated by multiplying the mutation index with the faecal weight. Results
Mutagenicity of airborne particles The results of the mutagenicity tests with the extracts of airborne particles are presented in Fig. 1. This Figure dearly reveals that the garage partides are much more strongly mutagenic than the outside air particles. There is also a qualitative difference: the effects of the garage particles clearly increased upon application of $9, while no influence was demonstrated on the outside particles. The effects obtained with the nitroreductase-deficient strain TA98NR were much less pronounced, indicating the contribution of compounds which exert their effect after reduction of a nitro group. The mutagenicity of the garage samples collected in October was comparable with that of the first collection period with respect to the extent of the
Revertants per plate 800 -
600 -
400 -
200 -
~
;
;
1'o
8
10
600
400
J
200 ~ 0
2
4
6
Cubic meter equivalent per plate
Fig. 1. The mutagenicity of extracts from airborne particles with S. typhimurium TA98 and S. typhimurium TA98NR. The indicated values represent m e a n s of duplicates. The dose is expressed as the volume of air from which the tested extract originated. Top, strain TA98; bottom, strain TA98NR. o and e, inside air; [] and ,x outside air; o and n, with S9-rat; • and zx without S9-rat. The metabolic activation system contained 10% $9 from Aroclor-treated rats.
effect as well as the influence of the $9 and the NR mutation (results not shown). Exposure to mutagens is not likely to have been very different during the 2 periods. Mutagenicity of urine extracts Preliminary experiments with a smoker's urine First, 2 preliminary experiments were carried out with the urine of a subject smoking about 20
380 Revertants per plate 300-
200
'°°IZ 50 10
20
30
-
-
40
50
60
ml urine per plate Fig. 2. The mutagenicity of urine extracts from a smoker with
S. typhimuriurn TA98 in the presence of $9 mix. The points represent means of 2 plates. The test without fl-glucuronidase (zx, XAD2; 13, XADT) and the test with 1000 units flglucuronidase per plate (o, XAD2; o, XAD7) were carried out with extracts of samples collected at different dates. The metabolic activation system contained 10% $9 from Aroclortreated rats.
cigarettes p e r day. Fig. 2 gives the results o b t a i n e d with urine s a m p l e d on 2 d a y s in the presence o f $9 mix. N o clear m u t a g e n i c i t y was f o u n d in the a b sence of $9 mix (results n o t shown). B o t h tests d e m o n s t r a t e that indirect m u t a g e n s are present. T h e activity of b o t h the X A D 2 a n d the X A D 7 fractions seemed to b e stronger in the presence of fl-glucuronidase, a l t h o u g h it m u s t b e r e m a r k e d that the 2 tests were carried out with extracts of s a m p l e s collected o n different dates.
left b e h i n d b y X A D 2 . T h e clearest e x a m p l e s are shown in d e t a i l in Fig. 4. Fig. 3 shows t h a t m u t a g e n i c i t y is n e a r l y restricted to the tests c a r r i e d out in the presence of $9 fraction. W e a r b i t r a r i l y r e g a r d a s a m p l e to b e m u t a g e n i c if its effect exceeds twice the s t a n d a r d deviation. S o m e effects were negative, which is p r o b a b l y d u e to a t o x i c i t y - r e l a t e d decrease of the r e v e r t a n t s i n d u c e d d u r i n g the test. S o m e s a m p l e s were n o t tested b e c a u s e the a m o u n t of urine was t o o small; o t h e r s were too toxic for the bacteria. A t first sight, Fig. 3 reveals that, except for the results o b t a i n e d w i t h c a r m e c h a n i c s G a n d D, m u t a g e n o u t p u t was a b o u t evenly d i s t r i b u t e d over the 2 collection d a y s a n d h a d a b o u t the s a m e degree a n d f r e q u e n c y a m o n g c a r m e c h a n i c s a n d office workers. T h e relatively high m u t a g e n levels in the u r i n e o f c a r m e c h a n i c s G a n d D c o u l d largely b e a t t r i b u t e d to m u t a g e n s p i c k e d up b y the X A D 7 resin. S o m e o t h e r urine s a m p l e s of b o t h office w o r k e r s a n d c a r m e c h a n i c s a p p e a r e d to c o n t a i n i n c r e a s e d levels of X A D 7 m u t a g e n s as well, albeit in d i s t i n c t l y lower a m o u n t s t h a n the urine of c a r m e c h a n i c s G a n d D. W i t h o u t further o b s e r v a t i o n s , the i n c r e a s e in X A D 7 m u t a g e n s f o u n d in s o m e of the u r i n e samples of 2 of the car m e c h a n i c s is n o t clear. I n view of the u n e v e n d i s t r i b u t i o n over the collection periods, the increase in X A D 7 m u t a g e n s c a n n o t b e a s c r i b e d to e x p o s u r e to diesel exhaust mutagens. A d d i t i o n of f l - g l u c u r o n i d a s e d i d n o t l e a d to essentially different effects ( d a t a n o t given).
Mutagenicity of faeces extracts Car mechanics and office workers T h e results with urine s a m p l e s of car m e c h a n i c s a n d office workers are s u m m a r i s e d in Fig. 3. This F i g u r e shows that X A D 7 can p i c k up m u t a g e n s
A n u m b e r of subjects a p p e a r e d to p r o d u c e m u t a g e n i c a l l y active faeces. T h e m u t a g e n i c i t y o f the faecal extracts of the m u t a g e n p r o d u c e r s h a d the following c h a r a c t e r i s t i c s in c o m m o n .
Fig. 3. The mutagenicity of XAD2 and XAD7 urine concentrates from car mechanics and office workers, ranked according to total mutagenicity, excreted in the period indicated and given separately for day and night urine. The number 1-4 and 5-8 respectively, present the results obtained in the presence and the absence of $9 mix respectively. The number of revertants per ml urine (calculated from the dose-response relations) is multiplied by the volume of urine produced. The standard deviations (white bars) of the calculated effects (gradients of linear dose-response relations) are depicted after multiplication with the volume produced. The shaded bars represent the effects (light shading for office workers, dark shading for car mechanics). The metabolic activation system contained 10% $9 from Aroclor-treated rats. Legend: The letter is the subject code. The first number indicates the collection day: 1, urine collected during the first 24 h; 2, urine collected during the second 24 h. The second number denotes the collection period: 1, urine collected during the day; 2, urine collected at night. Example: the sample encoded H-2-2 represents night-2 urine of subject H.
381 3000,
TA98 + $9 XAD2 day udne
® 2000,
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standard deviations A - H: car mechanics K - S: desk workers
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382
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Fig. 3 (continued).
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383 1000
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(~
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day urine
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standard deviations A - H: car mechanics K- S: desk workers
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standard deviations I A - H: car mechanics K- S: desk workers
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384 6oo,
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Fig. 3 (continued).
K- S: desk warkers
1
385 Revertants per plate
Revertants per plate 250
250-
sa
sample D-2-2 200 -
200
150-
150
100"
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50'
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~
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Revertants per plate
Revertants per plate
250
250 sample K-1-2
sample K-2-1
200-
200
150"
150
100"
100
J
J
---o 50'
0
6'0
ml urine per plate
o
50
lo
2'0
~o
4'o
50
60
o
1'o
io
3'0
io
5'0
6'0
ml urine per plate ml urine per plate Fig. 4. Dose-effect curves obtained with urine extracts from car mechanics D and G, and office worker K in the presence of a metabolic activation system, containing 10% S9 from Aroclor-treated rats. Indicator strain S. typhimurium TA98. Each point represents the mean of 2 plates. D, XAD2; o, XADT. For explanation of sample codes, see Fig. 3.
(1) Except for the faecal extracts of office worker S, the ether extracts induced clearly more revertants per mg faeces equivalent than the acetone extracts (Table 1). (2) The mutagenicity of the various extracts was most pronounced in the absence of the metabolic activation system. $9 mix seemed to inactivate or suppress the expression of mutagenicity of the extracts (data not shown).
(3) All extracts that were mutagenic with strain TA98 were also positive with strain TA100, but not vice versa. Strain TA100 was clearly more sensitive to the faecal mutagens than was strain TA98. Table 1 summarises the results obtained with ether (washed and not washed with N a O H ) and acetone extracts of faeces of mechanics and office workers with indicator strains TA98 and TA100 in
386 TABLE 1 MUTAGENICITY OF FAECAL EXTRACTS OF CAR MECHANICS AND OFFICE WORKERS IN THE AMES TEST EXPRESSED PER 100 mg FAECES EQUIVALENTS a Code
48-h stools (g)
Number of revertants per 100 mg faeces eq. with TA98 E-NaOH
Car mechanics A
544
B C D E F G H
78 469 44 305 200 259 127
TA100 E
A -
E-NaOH
E
A
95
-
131
NT
90
-
-
-
-
-
453
307
-
-
980 1739 264
1395 3726 -
130 285 -
Office workers K L M N O P Q R S
246 101 376 340 260 346 716 261 400
80 120 -
89 210 -
-
-
-
1 0 4
-
-
-
-
232
-
-
26 -
36 -
66 -
572 157
755 -
334 356
a Results obtained in the absence of $9 mix. The mutation index (slope of the dose-response curve) was determined from the part of the dose-response curve that most approached linearity. The conditions to qualify a response as positive were the presence of a clear dose-response effect with at least 1 dose level passing 3 times the control value. Abbreviations: E-NaOH, ether extract treated with NaOH; E, ether extract; A, acetone extract; NT, not tested; - extract not mutagenic. The number of spontaneous revertants for TA98 and TA100 varied between 22 and 44, and 110 and 178, respectively.
t h e a b s e n c e o f $9 m i x . Fig. 5 d e p i c t s t h e d o s e - e f fect c u r v e s o b t a i n e d w i t h the faecal e x t r a c t s o f t h e 6 most pronounced mutagen producers. T h e results d e m o n s t r a t e t h a t t h e i n c i d e n c e o f s t o o l s p e c i m e n s s h o w i n g m u t a g e n i c i t y was c l e a r l y h i g h e r in o f f i c e w o r k e r s t h a n in c a r m e c h a n i c s . Three out of 8 mechanics produced mutagenic f a e c e s ( s u b j e c t s A , B a n d F), w h i l e a m o n g t h e g r o u p of o f f i c e w o r k e r s 7 o u t o f 9 s h o w e d d i s t i n c t m u t a g e n i c i t y . I n a d d i t i o n , the d e g r e e o f m u t a g e n i c i t y was c l e a r l y h i g h e r in o f f i c e m u t a g e n p r o d u c e r s t h a n in g a r a g e m u t a g e n p r o d u c e r s .
Food intake The energy intake of car mechanics was slightly h i g h e r t h a n t h a t o f o f f i c e w o r k e r s , as c o u l d b e e x p e c t e d . T o t a l c a r b o h y d r a t e a n d t o t a l fat i n t a k e
were higher and total protein intake was lower among car mechanics than among office workers. N o n e o f t h e s e d i f f e r e n c e s w e r e s t a t i s t i c a l l y signific a n t (see T a b l e 2).
Relation between urinary and faecal mutagenicity and food intake figures To investigate the possible linear relationship between urinary and faecal mutagenicity and several dietary variables, Pearson's coefficients w e r e c a l c u l a t e d . T h e d i e t a r y v a r i a b l e s a n d the mutation indices for urine and faeces were calcul a t e d as i n d i c a t e d in M a t e r i a l s a n d m e t h o d s . Pearson's correlation coefficients did not reveal any consistent relations between urinary mutagenicity and the various dietary variables or between urinary and faecal mutagenicity.
387
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2000
F
¢0
1500
1500L
m
o ..---49
1000-
1000-
500-
500-
> t.
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160
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460
6
5130
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______._____-a
M
1500-
1500
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2000
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2000
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1500
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260
36o
460
56o
mg faeces equlva[en{s pap pta{e
O.
o
mg faecea equlvalen{s Oar" p|a~e
Fig. 5. Dose-effect curves obtained with faecal extracts of 'faecal mutagen producers' in the Ames test in the absence of $9 mix. Indicator strain: S. typhimurium TA100. t3, e t h e r - N a O H extract; o , ether extract; zx, acetone extract.
A number of rather high correlation coefficients, both positive and negative, were found between faecal mutagenicity and several dietary
variables. Within the group of car mechanics relatively high positive correlation coefficients were found between faecal mutagenicity and animal
388 TABLE2 MEAN DAILY INTAKE OF GARAGE AND O F ~ C E WORKERS a Car mechanics Office workers (n = 8) (n = 9)
Energy(kcal/day)
33305:600
2628+712
865:12 (10 en%) 39+ 8 47+ 12 138+ 33 (37 en%) 413 5 : 9 1 (50 en%) 36 5: 7 284 5:204
1005:22 (15 en%) 33+ 11 68+ 14 1045:37 (36 en%) 308 5 : 9 0 (47 en%) 36 5: 9 324 + 126
Food (solid and liquid, g) 3 291 5:458 Meat (g) 127 5 : 3 9 Potatoes/vegetables/fruit (g) 876 + 258
3 242 + 528 133 5 : 4 7 965 5:173
Nutrients Total protein (g) vegetable(g) animal(g) Total lipids (g) Total carbohydrates (g) Total dietary fibre (g) Cholesterol (mg)
Foodstuffs
a Mean 5: SEM; en%, percentage of the daily energy intake.
protein ( r = +0.71) and cholesterol ( r = +0.89) consumed, and a negative correlation coefficient with dietary fibre ( r = -0.55). Within the group of office workers most of the correlation coefficients calculated between faecal mutagenicity and the various dietary variables were negative. None of these values were statistically significant. Discussion
The results of the present study indicate that exposure to diesel exhaust mutagens has no enhancing effect on the incidence and degree of urinary and faecal mutagenicity. Belisario et al. (1984) have demonstrated that treatment of rats with diesel exhaust particulates, either intraperitoneally, orally or subcutaneously, leads to excretion of mutagens in urine. Urinary mutagenicity was apparent both after treatment with a suspension of diesel exhaust particulates and when diesel exhaust particulates were given as such in a gelatin capsule, indicating the biological availability of mutagens adsorbed onto diesel exhaust particulates in the absence of any suspend-
ing vehicle (Belisario et al., 1984). Although there are no data on the biological availability of mutagens from diesel exhaust particles after exposure by inhalation it seems reasonable to assume that upon exposition by inhalation mutagens adsorbed onto diesel exhaust particles become biologically available as well. About 90% of diesel exhaust particles is of respirable size, and inhaled particles are deposited and retained for a number of days in lung tissue. As pointed out in the Introduction mutagenically active substances identified in diesel exhaust particulates comprise PAHs and nitro-PAHs. Systemic uptake of PAHs has been demonstrated after intratracheal administration of hydrocarbons adsorbed onto particles of various sizes and chemical composition (IARC, 1983). Absorption may take place either via pulmonary tissues or via the enteral route after oral uptake through swallowing after mucociliary clearance of the particulate matter from the lungs (IARC, 1983). Once the PAHs are absorbed they are rapidly distributed. Part of PAHs or metabolites thereof are stored mainly in fat tissues and are slowly released. Hepatobiliary excretion and elimination through the faeces is the major route by which PAHs are removed from the body, regardless of the original route of administration (IARC, 1983). Data on absorption, distribution, kinetics, and excretion of nitro-PAHs are scarce. From studies with bacteria it is known that nitroPAHs are converted to their ultimate electrophilic intermediate by reduction of the nitro group. Anaerobic bacteria of the colonic flora are capable of activating the nitro-PAHs by nitroreduction (Rozenkranz and Mermelstein, 1983). For 1-nitropyrene it is proposed that the enterohepatic circulation is an important metabolic pathway in conventional rats, hydroxylation and conjugation taking place in the liver and nitroreduction and deconjugation in the intestinal bacteria (Tokiwa and Ohnishi, 1986). Various reasons, either alone or in combination, may explain why we did not find an increase in the incidence and degree of urinary and faecal mutagenicity in the present study. First it must be mentioned that the dose of particle-associated diesel exhaust mutagens to which car mechanics were exposed in the present study was only a fraction of the dose rats were exposed to in the study of
389 Belisario et al. (1984). To get an impression of the dose of particulate-associated mutagens car mechanics were exposed to, we examined the mutagenicity of air particles sampled during working hours (Fig. 1). Assuming that the average man inhales at most 8-10 m3 during an 8-h working day it becomes clear that the daily exposure was very low indeed. On the other hand, one should realise that the exposure takes place every working day and that particle-associated mutagens may accumulate in lung macrophages and be released gradually. A further point is the background noise of unidentified mutagenic activity in human excreta that makes an increase in incidence and degree of urinary and/or faecal mutagenicity hard to detect. Confounding factors in mutagenicity testing of human excreta are, among other things, various lifestyle-related factors such as smoking and diet. In fact 5 out of the 8 car mechanics had smoked one or a few cigarettes during the different collection periods, while none of the office workers had smoked. Our data do not, however, point to any relation between smoking pattern and the incidence and degree of urinary mutagens in either XAD2- or XAD7-treated urine samples. The number of cigarettes smoked was apparently too low to lead to detectable levels of mutagens in the urine. Another important point is the working-up procedure of the excreta. Recent work in which various extraction and working-up procedures of urine were compared has revealed that the use of 2 XAD resins of different polarity may considerably enlarge the amount a n d / o r spectrum of mutagens that are picked up (de Raat and van Ardenne, 1984; Belisario et al., 1984). Our results clearly confirmed this finding, both in the preliminary experiments in which a smoker's urine was used to evaluate the procedure and in the experiment with urine of office workers and car mechanics. We did not investigate whether the XAD7 mutagens were the result of an overloading of the XAD2 column, but our results merely point to the presence of specific mutagens of different polarity that are not retained on the XAD2 resin. The origin of the XAD7 mutagens is not clear: they were found both in smokers' and non-smokers' urine, and in urine of both office workers and car mechanics. In urine of rats treated with diesel exhaust par-
ticulates XAD2 and XAD7 mutagens were found as well, pointing to the presence of mutagens of different polarity (Belisario et al., 1984). An unexpected observation in the present study was the finding that the degree and incidence of faecal mutagenicity were distinctly higher among office workers than in car mechanics. In both groups faecal mutagenicity had a number of characteristics in common pointing to the presence of comparable mutagens in both groups of workers. The characteristics of faecal mutagenicity in the present study (TA100 being the most sensitive tester strain, mutagenicity being apparent without addition of $9 mix, an inhibitory fraction being present in the faecal extracts, which is eliminated at least partly by alkali washing) are consistent with reports on faecal mutagens in humans consuming a mixed western diet and are ascribed to the presence of faecapentaenes, mutagens originating from certain Bacteroides species in the intestinal tract (Wilkins and Van Tassel, 1983). However, the incidence of faecal mutagen producers among the group of office workers in the present study (78%) is not consistent with the reported studies. Although HPLC analysis reveals that faecapentaenes may occur in 50-75% of the individuals screened, the reported incidence of faecal mutagen producers is in general much lower and amounts to 10-20% in high-risk populations (Van Tassel, 1986). It is not clear to which factors the differences in faecal mutagenicity between office workers and car mechanics must be ascribed. Higher faecal mutagen levels may result from increases in mutagens but also from changes in the concentration of substances interfering with the expression of mutagenic activity (Hayatsu et al., 1981; Willems and de Raat, 1985). Differences in the incidence and degree of faecal mutagenicity have been found between populations differing in dietary habits and lifestyle (Kuhnlein et al., 1981; Ehrich et al., 1979; Reddy et al., 1980). It has been shown that the incidence and degree of faecal mutagenicity were higher in groups consuming a high-fat, high-meat, low-fibre diet than in groups consuming a high-fat, low-meat, high-fibre diet (Reddy et al., 1986). Two recently published diet intervention studies show that the production a n d / o r excretion of faecal mutagens becomes noticeably
390
lower when the diet of faecal mutagen producers is supplemented with fibre in the form of whole wheat and rye bread or of wheat b r a n (Reddy et al., 1987; Venitt et al., 1986). Furthermore, it appeared from observations in one faecal mutagen producer that addition of fat to the normal diet did not alter faecal mutagenicity (Venitt et al., 1986). In the present study total carbohydrate and fat intake figures tended to be slightly lower and total protein intake slightly higher in office workers than in car mechanics, without the differences reaching statistical significance. Analysis of the relationships between various dietary variables and faecal mutagenicity revealed a number of rather high correlation coefficients, negative as well as positive, but none of the correlation coefficients reached statistical significance. Statistical analysis did not reveal any relations between urinary mutagenicity and dietary variables or between urinary mutagenicity and faecal mutagenicity. Ingestion of food products such as fried pork, bacon and welldone ground beef has been reported to lead to an increased output of urinary and - in the case of well-done fried ground beef - also of faecal mutagens (Baker et al., 1982; Dolara et al., 1984; Hayatsu et al., 1985a,b). The present results did not show significant differences in consumption of fried meat between the 2 groups investigated. The higher incidence and degree of faecal mutagenicity among the office workers, therefore, cannot be ascribed to fried meat. In summary, our results did not indicate that exposure to diesel exhaust mutagens had any enhancing effect on degree and incidence of mutagen output in urine and faeces. Incidence and degree of faecal mutagen output was clearly higher among office workers than among car mechanics, while urinary mutagen output was about equal in both groups, except for 2 mechanics who did produce very mutagenic XAD7 extracts. The cause of the differences in faecal mutagen output between car mechanics and office workers is not clear, statistical analysis of possible links between various dietary variables and faecal mutagenicity being inconclusive and revealing other trends among car mechanics than among office workers. The present study did not reveal any relations between urine mutagen output and the various
dietary variables or between urinary and faecal mutagenicity.
Acknowledgements The study was carried out in close cooperation with the Regional Occupational Health Service of Groningen. Our special thanks are due to Drs. H.J. Logtenberg and R.P. Snorn.
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