Mutagenicity in urine of workers in rubber industry

45

Mutation Research, 79 (1980) 45--52

© Elsevier/North-Holland Biomedical Press

M U T A G E N I C I T Y IN URINE OF WORKERS IN RUBBER I N D U S T R Y

KAI FALCK, MARJA SORSA, HARRI VAINIO Institute of Occupational Health, Department of Industrial Hygiene and Toxicology, SF-00290 Helsinki 29 (Finland)

and IIRO KILPIKARI Nokia Corporation Inc., Medical Department, SF-37100 Nokia (Finland)

(Received 11 March 1980) (Accepted 1 April 1980)

Summary Epidemiological studies have shown t hat those who w ork in rubber industry have an increased risk o f cancer. In the working e n v i r o n m e n t t h e y are exposed, p ro bab ly , to several h u n d r e d di f f er e nt chemicals some of t hem being k n o w n or suspected carcinogens and mutagens. The bacterial fluctuation test was used to det ect the mutagenicity in the urines o f exposed workers. A group of u n e x p o s e d office clerks served as controls. Both groups consisted of smokers and non-smokers, and t hat was taken into consideration in the results. R u b b e r workers, either smokers or non-smokers, exhibited significantly higher mutagenic activity in their urine than the occupationally u n e x p o s e d controls when th e base-pair substitution strain E. coli WP2 uvrA was used as indicator organism. Use of t he frameshift strain S. t y p h i m u r i u m T A 9 8 revealed increased mutagenicity in the urine of occupationally exposed smokers, nonsmokers and u n e x p o s e d smokers. The e x t e n t of mutagenicity in the urine of workers who smoked suggested a synergistic effect of smoking and occupational exposure. The bacterial f l uct uat i on test with urine samples as sources of mutagenicity is able to d etect chemical exposure if t he excret ed c o m p o u n d s are still in active f o rm or can be activated. T he m e t h o d can be used to identify hazardous working conditions long before the manifestation of possible pathological changes in exposed individuals.

Several epidemiological studies have shown t h a t workers in the rubber industry have an increased risk o f cancer (Monson and Fine, 1978; Hakama and

46 Kilpikari, 1980). The chemical exposure range in the rubber industry is especially wide and mixed, as several hundred chemical entities are used (Holmberg et al., 1977). The exposure pattern is further complicated by the interaction of chemicals and thermodegradation products. The ambient exposure may be monitored by bioassay systems able to detect mutagenic activity in body fluids of the exposed workers. This is conveniently performed with urine, which contains mixtures of activated or conjugated compounds (Commoner et al., 1974; Yamasaki and Ames, 1977; Seibert and Simon, 1978). The urine of workers in rubber factories has been reported to contain increased amounts of thioethers such as mercapturic acids (Vainio et al., 1978). Materials and methods Mutagenicity in the urine of male workers in the tire-manufacturing departm e n t of a rubber plant was investigated by the bacterial fluctuation test. An occupationally unexposed group of male office clerks served as controls. Both groups contained smokers (about 20 cigarettes per day) and non-smokers.

Urine samples Samples of urine were collected on Thursday afternoons after four 8-h workdays. The samples were frozen at --70°C and stored in a deep-freeze until used. The m e t h o d for concentrating urine has been described by Yamasaki and Ames (1977). A 100-ml volume of urine was loaded onto 2 ml of XAD-2 resin. The adsorbed components were eluted with 10 ml of freshly distilled acetone, and the eluant was evaporated to dryness at +60°C under a continuous flow of nitrogen. The residue was dissolved with dimethylsulfoxide (DMSO). Six 100pl concentrates, dilutions 1/2 (corresponding to 50 ml of urine), 1/4 (25 ml), 1/8 (12.5 ml), 1/16 (6.25 ml), 1/32 (3.125 ml) and 1/64 (1.56 ml) were used for the tests in order to avoid the bactericidal effect of urine. Bacterial strains The strains used were Escherichia coli WP2 uvrA (base-pair m u t a n t , kindly provided by Prof. B.A. Bridges) and Salmonella typhimurium TA98 (frameshift, kindly provided by Prof. B.N. Ames). Preparation of $9 mix Rats were treated with Aroclor 1254 (500 mg/kg b.wt) 5 days before they were killed. The 9O00-g liver supernatant ($9) was divided into 2-ml aliquots, frozen and stored at --70°C. The protein c o n t e n t (24.4 mg/ml) was measured by the m e t h o d of Lowry et al. (1951). The $9 mix (20%, v/v) was prepared according to Ames et al. (1975), and it was filtered through a Millipore filter (diam. 0.45 pm) before use. Determination of mutagenicity The mutagenicity in urine was determined with the bacterial fluctuation test according to Green et al. (1977), $9 mix being used for metabolic activation.

47 The urine concentrate in 100 pl of DMSO, together with 100 pl of bacterial suspension (log-phase, l 0 s cells/ml) and 100 pl of $9 mix, were added to 4.7 ml of Davis--Mingioli growth medium supplemented with glucose (4 mg/ml), biotin (160 ng/ml) and t r y p t o p h a n (2 pg/ml) or histidine (2 pg/ml). The solution was dispensed into 50 tubes and incubated at +37°C for 18--20 h. On the following day, 2 ml of histidine- or tryptophan-free growth medium containing bromcresol purple (5 pg/ml) was added to each tube. The selection phase t o o k 3 days at +37°C, after which the number of revertant tubes was counted. The creatinine concentrations in urine samples were determined by the m e t h o d of Jaffe (1879). The mutagenic activity was calculated as follows: Mutagenic activity -

revertants in sample (Rs) --revertants in solvent control (Re) creatinine (mmoles) in urine sample

The mutagenic activity was calculated separately for each of the 6 dilutions. From each sample, the dilution giving the highest mutagenic activity was included in the results. Results and discussion The results with individual urine samples are given in Tables 1 and 2. The highest numbers of induced revertants (Rs--Rc) were achieved when concentrates having the original volume of urine 12.5, 6.25 or 3.125 ml were tested (Fig. 1). With smaller dilutions (25 and 50 ml), the urinary toxicity reduced the number of viable revertants. The XAD-2 adsorption reduced histidine and t r y p t o p h a n in the urine concentrate to concentrations that were unable to support prolonged auxotrophic growth. The a m o u n t of histidine in the concentrate corresponding to 100 ml of urine was 0.2 pg; tryptophan could n o t be detected. In agreement with the data published earlier (Yamasaki and Ames, 1977; van D o o m et al., 1979) smokers showed mutagenicity in their urine, detectable by the frameshift strain S. typhimurium TA98. The group revealing the highest urinary mutagenicity was that of smoking workers. When the extent of mutagenicity in the groups of exposed non-smokers and unexposed smokers was ~ompared with the group of smoking workers, the results suggested that smoking acts synergistically with exposure to rubber chemicals in producing mutagenicity in urine (Fig. 2). The unexposed groups (when tested with E. coli strain WP2 uvrA) and the occupationally unexposed non-smokers (with S. typhimurium TA98) revealed no or only minimal mutagenicity. Both the smoking and non-smoking groups of workers showed significantly higher levels of mutagenicity in their urine as compared with their unexposed controls. The frameshift strain S. typhimurium TA98 was able to detect mutagenic activity caused both by smoking and by occupational exposure to chemicals (Fig. 2A). The base-pair substitution strain E. coli WP2 uvrA detected only the effect of occupational exposure (Fig. 2B). A practical application of this test system may be the evaluation of exposure to mutagens both in the occupational environment and in life-style, e.g. smoking. Epidemiological studies have led to the identification of a n u m b e r of

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"-~"-: Mea n e S D .

F i g . 2. M u t a g e n i c a c t i v i t y d e t e c t e d i n u r i n e o f r u b b e r w o r k e r s ( e x p o s e d ) a n d c o n t r o l s ( u n e x p o s e d ) w i t h S. t y p h i m u r i u m T A 9 8 ( A ) a n d E. coli W P 2 u v r A ( B ) . T h e s t a t i s t i c a l s i g n i f i c a n c e o f t h e d i f f e r e n c e s b e t w e e n r u b b e r w o r k e r s a n d t h e i r u n e x p o s e d c o n t r o l s m e a s u r e d w i t h S t u d e n t ' s t t e s t a r e a s f o l l o w s : e x p o s e d s m o k e r s vs. unexposed smokers (TA98) t = 3.66** P < 0.01; (WP2 uvrA) t = 5.365** P < 0.01; exposed non-smokers vs. unexposed non-smokers (TA98) t = 4.34** P < 0.01; ( W P 2 u v r A ) t = 8 . 6 3 * * * P < 0 . 0 0 1 . U n e x p o s e d s m o k e r s v s . e x p o s e d n o n - s m o k e r s ( T A 9 8 ) t = 1 . 7 3 N S P > 0 . 0 5 ; e x p o s e d s m o k e r s vs. e x p o s e d n o n - s m o k e r s ( W P 2 uvrA) t = 1.75 NS P > 0.05.

smokers

100

100

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500

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5O TABLE 1 MUTAGENICITY DETECTED WITH Salmonella typhirnurium TA98 EXPOSED TO RUBBER CHEMICALS AND OF UNEXPOSED PERSONS Person

A1 A2 A3 A4 A5 A6 A7 AS A9

Type of exposure

Exposed smoker

Mean ± S.D. B1 B2 B3 B4 B5 B6 B7

Exposed non-smoker

Mean ± S.D. D1 D2 D3 D4 D5 D6 D7 D8 D9 Mean ± S.D.

Unexposed non-smoker

smoker

Creatinine (mmoles/1)

3.125 6.25 1.56 3.125 3.125 1.56 3.125 12.5 12.5

6.0 6.3 6.3 10.4 S.0 10.2 6.4 5.5 3.3

7.6 4.2 17.8 11.5 22.8 4.0 18,6

10.3 ± 3.9

12.4 + 7.5

50.0 12.5 6.25 12.5 12.5 12.5 12.5 12.5 6.25 12.5 12.5

7.5 5.3 3.4 9.9 5.9 5.8 5.9 4.1 7.6 8.1 5.9

14.8 ± 12.0

6 . 3 + 1.9

15.3 ± 5.5

In sample

In control

42 30 31 40 37 38 32 36 43

19 15 23 19 19 23 19 18 18

23 30 48 50 49 47 50

17 14 17 17 17 17 17

49 19 24 29 24 29 28 29 22 28 30

22 16 15 18 18 18 18 18 14 18 18

19 21 17 17 17 27 23 20 22

17 17 17 17 17 17 17 17 17

6.9 * 2.3

3.125 6.25 12.5 12.5 12.5 12.5 12.5

25.0 25.0 12.5 12.5 12.5 12.5 12.5 12.5 12.5

URINE

Number of revertant tubes

Volume (ml)

5.2 ± 4.4 Unexposed

Mean ± S.D. C1 C2 C3 C4 C5 C6 C7 CS C9 C10 Cll

Urine sample

IN

5.5 6.1 6.3 3.7 2.1 9.3 18.6 5.5 19.9 8 . 6 _+ 6 . 4

OF

WORKERS

Mutagenic activity

1225 383 8:16 644 724 945 654 263 612 717.1 ÷ 235.6 253 604 140 232 113 603 143 298.2 ± 214.6 72 45 429 89 81 153 135 216 168 98 162 I49.8

÷ 90.4

17 33 0 0 0 86 26 44 20 25.1 * 27.7

S o l v e n t c o n t r o l ( 2 0 # l / m l D M S O ) 1 5 . 9 + 4 . 0 ( m e a n n u m b e r o f r e v e r t a n t s -+ S.D. i n 2 0 r e p e a t s ) . P o s i t i v e c o n t r o l ( 0 . 1 p g ] m l B [ a ] P ) 1 2 . 8 + 3.1 ( m e a n n u m b e r o f i n d u c e d r e v e r t a n t s e x c e e d i n g t h e s o l v e n t c o n t r o l + S.D. i n 1 0 r e p e a t s ) .

h a z a r d o u s w o r k i n g c o n d i t i o n s , b u t s u c h studies h a v e n o t c o n s i d e r e d individual variations in e x p o s u r e or e x t e n t o f r e s p o n s e . Our results indicate that t h e w o r k e r s in rubber-tire m a n u f a c t u r i n g are e x p o s e d t o m u t a g e n i c c h e m i c a l s .

51

TABLE 2 MUTAGENICITY DETECTED WITH Eschcrichia coli WP2 uvrA IN URINE TO RUBBER CHEMICALS AND OF UNEXPOSED PERSONS

Person

Type of exposure

Urine sample Volume (ml)

A1

Exposed smoker

1.56

A5 A6 A7 A8 A9

3.125 3.125 1.56 3.125 6.25

Mean ± S.D.

3.1 • 1.7

B3 B4 B5 B6 B7

Unexposed

Mean + S.D. C2 C4 C5 C6 C7 C8 C9 C10

Exposed non-smoker

Mean + S.D. D4 D5 D6 D7 D8 D9

Mean ± S.D.

Unexposed no n-smoker

smoker

Number of revertant tubes Creatinine (mmoles/l)

In control

6.0

35

24

1172

8.0 10.2 6.4 5.5 3.3

34 36 18 38 36

24 24 S 24 24

402 378 1006 818 587

6.6 ± 2.3 17.8 11.5 22.8 4.0 18.6

12.5 ± 0.0

14.9 ± 7.3

3.125 3.125 3.125 3.125 6.25 3.125 6.25 3.125

5.3 9.9 5.9 5.8 5.9 4.1 7.6 8.1

3.9 ± 1 . 5

6.6 ± 1.8

1 2 . 5 _+ 0 . 0

3.7 2.1 9.3 18.6 5.5 19.9 9 . 9 + 7.7

EXPOSED

Mutagenic activity

In sample

12.5 12.5 12.5 12.5 12.5

12.5 12.5 12.5 12.5 12.5 12.5

OF WORKERS

7 2 7 . 1 +_ 3 2 5 . 7 19 16 18 15 22

15 15 15 15 15

18 7 11 0 30

31 36 30 35 38 32 48 43

24 24 24 24 24 24 24 24

422 388 324 613 378 630 505 748

18 16 15 16 20 21

15 15 15 15 15 15

1 3 . 2 _+ 1 1 . 6

5 0 1 . 1 _+ 1 4 8 . 9 65 38 0 9 73 24 34.7 ± 29.5

S o l v e n t c o n t r o l ( 2 0 p l ] m l D M S O ) 2 0 . 9 -+ 2 . 2 ( m e a n n u m b e r o f r e v e r t a n t s -+ S . D . i n 1 6 r e p e a t s ) . P o s i t i v e control (cyclophosphamide, 1 0 0 P g ] m l ) 1 6 . 6 ± 2.1 ( m e a n n u m b e r o f i n d u c e d r e v e r t a n t s e x c e e d i n g t h e solvent control + S.D. in 12 repeats).

Because of the high experimental correlation between carcinogenicity and mutagenicity (McCann et al., 1 9 7 5 ) it is probable that this kind of exposure constitutes one of the etiological factors for the increased risk of cancer among workers in rubber factories. We feel that the fluctuation test of urine concentrates offers a sensitive m e t h o d for the monitoring of urinary mutagens among occupational groups exposed to mutagenic chemicals. This is possible in cases where the excreted c o m p o u n d s are still in active form or can be activated. Such information can help one to identify previously u n k n o w n chemical hazards (Falck et al., 1 9 7 9 ) years before possible manifestation as irreversible pathological changes.

52

Acknowledgements The authors thank Ms. Leena Tikkanen, State Technical Research Centre, for advice with determination of urinary amino acids, and Ms. Helena KivistS, Institute of Occupational Health, for performing the creatinine analyses. This work was supported by grants from the National Research Council for Sciences, Academy of Finland (No. 013) and the Swedish Work Environment Fund (No. 79/189). References A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i ( 1 9 7 5 ) M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y t e s t , M u t a t i o n Res., 3 1 , 3 4 7 - - 3 6 4 . C o m m o n e r , B., A . J . V i t h a y a t h i l a n d J.I. H e n r y ( 1 9 7 4 ) D e t e c t i o n o f m e t a b o l i c c a r c i n o g e n i n t e r m e d i a t e s in u r i n e o f c a r c i n o g e n - f e d r a t s b y m e a n s o f b a c t e r i a l m u t a g e n e s i s , N a t u r e ( L o n d o n ) , 2 4 9 , 8 5 0 - - 8 5 2 . v a n D o o r n , R . , R . P . B o s , C.-M. L e i j d e k k e r s , M . A . P . W a g e n a a s - Z e g e r s , J . L . G . T h e u w s a n d P.T. H e n d e r s o n (1979) Thioether concentration and mutagenicity of urine from cigarette smokers, Int. Arch. Occup. Environ. Health, 43, 159--166. F a l c k , K . , P. G r 6 h n , M. S o r s a , H. V a l n i o , E. H e i n o n e n a n d L . R . H o l s t i ( 1 9 7 9 ) M u t a g e n i c i t y in u r i n e o f n u r s e s h a n d l i n g c y t o s t a t i c d r u g s , L a n c e t , i, 1 2 5 0 - - 1 2 5 1 . G r e e n , M . H . L . , B . A . B r i d g e s , A.M. R o g e r s , G. H o r s p o o l , W.J. M u r i e l , J.W. B r i d g e s a n d J . R . F r y ( 1 9 7 7 ) M u t a g e n s c r e e n i n g b y a s i m p l i f i e d b a c t e r i a l f l u c t u a t i o n test: use o f m i c r o s o m a l p r e p a r a t i o n s a n d w h o l e liver cells f o r m e t a b o l i c a c t i v a t i o n , M u t a t i o n R e s . , 4 8 , 2 8 7 - - 2 9 4 . H a k a m a , M., a n d I. K i l p i k a r i ( 1 9 8 0 ) C a n c e r r i s k a m o n g r u b b e r w o r k e r s , J. T o x i c o l . E n v i r o n . H e a l t h , in press. H o l m b e r g , B., B. S j ~ s t r 6 m a n d S. O l s s o n ( 1 9 7 7 ) A t o x i c o l o g i c a l s u r v e y o f c h e m i c a l s u s e d in t h e S w e d i s h rubber industry and proposals for the elimination of chemical hazards in the rubber industry, Investigation Report No. 19, National Board of Occupational Safety and Health, Stockholm, pp. 1--127. J a f f e , M. ( 1 8 7 9 ) U~ber die n a c h E i n f f i h r n n g v o n B r o m b e n z o l , u n d C h l o r b e n z o l im O r g a n i s m u s e n t s t e h e n d e n s c h w e f e l h a l t i g e n S~'uren, Bet. D e u t s c h . C h e m . Ges., 1 2 , 1 0 9 2 - - 1 0 9 8 . L o w r y , O . H . . N . J . R o s e b r o u g h , A . L . F a r r a n d R . J . R a n d a l l ( 1 9 5 1 ) P r o t e i n m e a s u r e m e n t w i t h t h e folinp h e n o l r e a g e n t , J. Biol. C h e m . , 1 9 3 , 2 6 5 - - 2 7 5 . M c C a n n , J., E. C h o i , E. Y a m a s a k i a n d B . N . A m e s ( 1 9 7 5 ) D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t h e S a l m o n e l l a / m i c r o s o m e t e s t : A s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 , 5 1 3 5 - - - 5 1 3 9 . M o n s o n , R . R . , a n d L.J. F i n e ( 1 9 7 8 ) C a n c e r m o r t a l i t y a n d m o r b i d i t y a m o n g r u b b e r w o r k e r s , J. N a t l . Cancer Inst., 61, 1047--1053, S e i b e r t , P., a n d U. S i m o n ( 1 9 7 3 ) C y c l o p h o s p h a m i d e : P i l o t s t u d y o f g e n e t i c a l l y active m e t a b o l i t e s in u r i n e of treated human patient, Mutation Res., 19, 65--72. V a i n i o , H., H. S a v o l a i n e n a n d I. K i l p i k a r i ( 1 9 7 8 ) U r i n a r y t h i o e t h e r o f e m p l o y e e s o f a c h e m i c a l p l a n t , Br. J. I n d . M c d . , 3 5 , 2 3 2 - - 2 3 4 . Y a m a s a k i , E., a n d B. A m e s ( 1 9 7 7 ) C o n c e n t r a t i o n o f m u t a g e n s f r o m u r i n e w i t h t h e n o n p o l a r r e s i n X A D - 2 : C i g a r e t t e s m o k e r s h a v e m u t a g e n i c u r i n e , P r o e . N a t l . A c a d . Sci. ( U . S . A . ) , 7 4 , 3 5 5 5 - - 3 5 5 9 .