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Mutagenic activity of thiram in ames tester strains of Salmonella typhimurium
Mutation Research, 68 (1979) 9--13
© Elsevier/North-HollandBiomedicalPress
MUTAGENIC ACTIVITY OF THIRAM IN AMES TESTER STRAINS OF SALMONELLA
MALGORZATA SZYMCZYK
TYPHIMURIUM
ZDZIENICKA, M A R I A ZIELEI~SKA, B A R B A R A T U D E K and T E R E S A
Department of Biochemistry, Warsaw Medical School, Banacha 1, 02-097 Warsaw (Poland)
(Received 16 January 1979) (Revision received 25 April 1979) (Accepted 1 May 1979)
Summary The mutagenic activity of thiram was investigated in 4 histidine-requiring strains of S a l m o n e l l a t y p h i m u r i u m (TA1535, TA100, TA1538, TA98) with and without activation by liver microsomes. In strains TA1535 and TA100, thiram induces mutations without metabolic activation. The presence of ratliver microsome fraction, cysteine or glutathione abolish its mutagenic activity in these strains. In contrast, thiram requires metabolic activation for the expression of its mutagenic activity in TA1538 and TA98 strains. The compounds containing the sulphydryl group abolish mutagenic activity of thiram in these strains, too.
Many pesticides have been evaluated for possible mutagenicity [3,4,10]. Thiram, a fungicide of the dimethyldithiocarbamate class, is widely used in agriculture. Thus, man may be exposed to thiram both through direct occupational contact and by consuming food containing residues of that fungicide. Since most carcinogens axe also mutagens [8,9] it seemed of interest to determine the ability of thiram to induce mutations in the Salmonella test system of Ames [1,2]. Materials and methods Chemicals
Thiram (purity 98%) was a product of "Organica-Azot" (Jaworzno-Poland); biotin, L-histidine, L-cysteine, glutathione and MNNG were from Koch-Light; DMS0, dimethylsulphoxide; MMS, methyl methanesulphonate; MNNO, N'-methylN'-nitrosoguanldlne; NADP, n i c o t i n a m i d e adenine d i n u c l e o t i d e p h o s p h a t e . Abbreviations:
10 glucose 6-phosphate and NADP from Sigma Chemicals Ltd.; ampicillin (binotal) was a p r o d u c t of Bayer Leverkusen; MMS of K and K Lab.; 2-aminofluorene of Aldrich Co.; Aroclor 1254 was a gift from W.B. Papageorge, Monsanto Chemical Co. (St. Louis, U.S.A.). Bacterial strains Salmonella typhimurium strains were generously provided b y Dr. B.N. Ames (Biochemistry Department, University of California, Berkeley, U.S.A.). All strains were routinely checked for efficiency as recommended by Ames et al. [1,2]. Positive mutagenesis controls, with MNNG (TA1535), MMS (TA100) and 2-aminofluorene (TA1538 and TA98), were also included. The average numbers of spontaneous mutants per plate for TA1535, TA100, T A 1 5 3 8 and TA98 without $9 mix were 21 + 2, 165 + 8, 23 + 1, and 39 + 3, and with $9 mix 19 + 2, 171 + 6, 22 + 3 and 41 + 2, respectively. Induction o f rat-liver e n z y m e system Liver microsomal fraction $9 was prepared from rats treated with Aroclor 1254, as described by Ames et al. [1]. Male Wistar rats (200 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw) 5 days before they were killed b y decapitation. Livers were collected and homogenized in 0.15 M KC1 (3 ml/g wet tissue). The homogenate was centrifuged for 10 min at 9000 g; the supernatant was decanted and served as the source of soluble microsomal enzymes. The protein content in the $9 fraction, determined according to Lowry et al. [5], usually ranged from 36 to 42 mg/ml. Mutagenesis assay Mutagenicity test using plate-incorporation assays and S. typhimurium strains TA1535, TA100, T A 1 5 3 8 and TA98 were carried out as described by Ames et al. [1,2]. Thiram dissolved in DMSO was added to 2.0 ml of molten t o p agar (0.6% agar, 0.5% NaC1, 0.05 mM L-histidine and 0.05 mM biotin), 0.1 ml of tester strain grown overnight in Difco nutrient broth and (or omitted) 0.5 ml of the $9 mix composed of 100 pl of rat-liver microsomal fraction, 4 pmol MgC12, 16.5 #mol KC1, 2.5 pmol glucose 6-phosphate, 2 pmol NADP and 50 pmol sodium phosphate buffer (pH 7.4). In experiments with pre-incubation, procedures were based on those of Ames et al. [1,2] with a slight modification described b y Moriya et al. [10]. Thiram dissolved in DMSO (20 pl) was added to a test-tube containing 0.5 ml of one of the following solutions: $9 mix containing 0.3 ml S9 fraction per ml; $9 mix and 20 mM L-cysteine; $9 mix and 20 mM glutathione; 20 mM L-cysteine; 20 mM glutathione; 0.5 ml H20 (control). The test-tube was incubated in a water-bath with gentle shaking at 37°C for 20 min. After the incubation, 0.1 ml of an overnight broth culture of bacterial tester strain and 2 ml of molten t o p agar containing 0.05 mM L-histidine and 0.05 mM biotin were added to the test-tube. The mixture was stirred vigorously, poured immediately onto plates of minimal agar, and incubated at 37°C. Mutagenicity assays were carried o u t in triplicate, and the number o f his + revertant colonies were scored after incubation for 3 days. The number of
11
300
25( LU
~. 200 p4o UJ u.J lOO wr
2o
50
50
%00 /ug
50
200
150
THIRAM/PLATE
I00
150
200
JJg T H I R A M / P L A T E
Fig. 1. D o s e - - r a s p o n s e c u r v e s o f m u t a t i o n in Salmonella t y p h i m u r i u m w i t h v a r i o u s a m o u n t s o f t h i r a m in t h e a b s e n c e o f r a t - l i v e r $ 9 m i x . S t r a i n s : T A 1 5 3 5 (o); T A 1 0 0 ( e ) ; T A 1 5 3 8 (4); T A 9 8 (A). T h e n u m b e r o f s p o n t a n e o u s r e v e r t a n t s h a s b e e n s u b t r a c t e d f r o m t h e r e v e r t a n t v a l u e s p l o t t e d against t h i r a m c o n c e n t r a tion. Fig. 2. D o s e - - r e s p o n s e c u r v e s o f m u t a t i o n s in S. t y p h i m u r i u m T A 1 5 3 8 (o) a n d T A 9 8 ( e ) w i t h v a r i o u s a m o u n t s o f t h i r a m i n t h e p r e s e n c e o f rat-Hver $ 9 m i x . T h e n u m b e r o f s p o n t a n e o u s r e v e r t a n t s h a s b e e n s u b t r a c t e d f r o m t h e r e v e r t a n t v a l u e s p l o t t e d against t h i r a m c o n c e n t r a t i o n .
revertants per plate is an average number from at least 3 separate experiments. Mean values + S.E. of 3--5 expts, are listed Results and discussion The dose--response curves (Fig. 1) show the effect of thiram on induction o f h i s + mutations in T A 1 5 3 5 , TA100, T A 1 5 3 8 and TA98 strains of S. t y p h i r n u r i urn. Without metabolic activation thirarn was n o t mutagenic towards T A 1 5 3 8
or T A 9 8 but induced mutation in T A 1 5 3 5 and TA100. Thiram in the amount of 100 #g/plate increased the mutation frequency about 3-fold in T A 1 5 3 5 and 2.5-fold in TA100. In the presence of the microsomal fraction of rat liver, the mutagenic activity of thiram was decreased in these strains (Tables 1 and 2),
TABLE 1 E F F E C T O F T I M E O F P R E - I N C U B A T I O N O F T H I R A M ( I 0 0 # g / P L A T E ) W I T H $ 9 M I X O N ITS MUT A G E N I C A C T I V I T Y I N S T R A I N S T A 1 5 3 5 , T A 1 0 0 , T A 1 5 3 8 A N D T A 9 8 O F S. T Y P H I M U R I U M Time of pre-incubation (min)
0 5 10 15 20
Number of revertants/plate TA1535
TA100
TA1538
TA98
49±2 37±1 29±3 26 ± 1 22±2
280± 9 204± 6 186±13 172 ± 10 160± 7
54±3 55±2 53±1 55 ± 4 54±2
88±1 87±3 83±2 85 ± 3 87 ±3
12 TABLE 2 E F F E C T O F C Y S T E I N E O R G L U T A T H I O N E ON M U T A G E N I C A C T I V I T Y O F T H I R A M ( 1 0 0 # g / P L A T E ) I N S T R A I N S T A 1 5 3 5 , T A 1 0 0 , T A 1 5 3 8 A N D T A 9 8 O F S. T Y P H I M U R I U M Strain
Number of revertants/Plate Without thiram
TA1535 TA100 TA1538 TA98
With t h i r a m
H20
+cys.
+glut.
H 20
+cys.
+glut.
20+2 165 ± 6 22 ± 3 41± 2
23±1 161 ± 7 25 ± 2 66 ± 4
24±2 156 ± 9 28 ± 1 57 ± 3
72± 4 4 4 4 ± 11 54 ± 2 88 • 3
20± 1 154 ± 10 24 ± 1 65 ± 4
18±3 142 ± 9 27 ± 3 58 ± 3
$ 9 m i x w a s a d d e d t o t h e p l a t e s o n l y w h e n T A 1 5 3 8 a n d T A 9 8 strains w e r e u s e d .
which indicates that thiram was not metabolically activated by this fraction. The mutagenic activity of thiram disappeared after 20 min pre-incubation with $9 mix both in TA1535 and TA100 strains (Table 1). L-Cysteine and glutathione had a similar effect (Table 2). The frequency of the thiram-induced mutations in TA1538 and TA98 strains indicated that thiram is mutagenic for these strains only in the presence of microsomal enzymes of rat-liver (Figs. 1 and 2). The number of revertants was raised by thiram about 2.5-fold in TA1538 strain (at doses of 50--150 pg/plate) and TA98 strain (50 #g/plate). The mutagenic activity in strains TA1538 and TA98 that appeared only in the presence of microsomal fraction of rat liver was not increased by 20 rain pre-incubation of thiram with $9 mix (Table 1). On the other hand, 20 min pre-incubation of thiram with $9 mix and L-cysteine or glutathione completely destroyed its mutagenic activity in these strains (Table 2). The results suggest that mutagenic activity of thiram in all tester strains is rapidly destroyed by compounds that contain the sulphydryl group. This is in agreement with the results obtained by Matthiaschk [7], who reported that simultaneous administration of thiram and L-cysteine to pregnant mice resulted in a significant decrease both in the severity and the number of embryopathies. Assuming that carcinogenesis and teratogenesis involve similar molecular mechanisms and that these mechanisms are similar to those in mutagenesis [6], the potential hazard of thiram for man should be taken into consideration. References 1 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 , 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 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., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 A m e s , B.N., W.E. D u r t s o n , E. Y a m a s a k i a n d F.D. L e e , C a r c i n o g e n s are mutagens: a s i m p l e t e s t s y s t e m c o m b i n i n g liver h o m o g e n a t e s for activation a n d b a c t e r i a for d e t e c t i o n , P r o c . Natl. A c a d . Sci. ( U . S . A . ) , 70 ( 1 9 7 3 ) 2 2 8 1 - - 2 2 8 5 . 3 D u r h a m , W.F., a n d C.H. Williams, M u t a g e n l c , t e r a t o g e n i c , a n d c a r c i n o g e n i c p r o p e r t i e s o f pesticides, A n n u . R e v . E n t o m . , 17 ( 1 9 7 2 ) 1 2 3 - - 1 4 8 . 4 F a h r i g , R., C o m p a r a t i v e M u t a g e n i c i t y S t u d i e s w i t h Pesticides, I A R C Scient. P u b l . N o . 10, I n t . A g e n c y Res. Cancer, L y o n , 1 9 7 4 .
13
5 Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein m e a s u r c m e n t w i t h t he Folin p h e n o l reagent, J. Biol. Chem., 193 (1951) 265--275. 6 Magee, P.N., The relationship b e t w e e n mutagenesis, carcinogenesis and teratogenesis, in: D. Scott, B.A. Bridges and F.H. Sobeis (Eds.), Progress in Genetic Toxicology, Elsevier/North-Holland, Amsterdam , 1977, pp. 15--27, 7 Matthiaschk, G., Uber den Einfluss von L-cystein auf die Teratogenese d t u c h Thiram (TMTD) bei NMRI-MKusen, Arch. Toxikol., 30 (1973) 251--262. 8 McCann, J., and B.N. Ames, Detection of carcinogens as mutagens in the Salmonena/microaome test: Assay of 300 chemicals: Discussion, Proc. Natl. Acad. Sci. (U.S.A.), 73 (1976) 950--954. 9 McCann, J., E. Choi, E. Yamasaki and B.N. Ames, D e t e c t i o n of carcinogens as mutagens in t he Salm o n e l l a / m i c r o s o m e test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 5135-5139. 10 Moriya, M., K. Kato and Y. Shirasu, Effects Of cysteine and a liver metabolic activation s ys t e m on the activities of mutagenic pesticides, Mutation Res., 57 (1978) 259--263.
© Elsevier/North-HollandBiomedicalPress
MUTAGENIC ACTIVITY OF THIRAM IN AMES TESTER STRAINS OF SALMONELLA
MALGORZATA SZYMCZYK
TYPHIMURIUM
ZDZIENICKA, M A R I A ZIELEI~SKA, B A R B A R A T U D E K and T E R E S A
Department of Biochemistry, Warsaw Medical School, Banacha 1, 02-097 Warsaw (Poland)
(Received 16 January 1979) (Revision received 25 April 1979) (Accepted 1 May 1979)
Summary The mutagenic activity of thiram was investigated in 4 histidine-requiring strains of S a l m o n e l l a t y p h i m u r i u m (TA1535, TA100, TA1538, TA98) with and without activation by liver microsomes. In strains TA1535 and TA100, thiram induces mutations without metabolic activation. The presence of ratliver microsome fraction, cysteine or glutathione abolish its mutagenic activity in these strains. In contrast, thiram requires metabolic activation for the expression of its mutagenic activity in TA1538 and TA98 strains. The compounds containing the sulphydryl group abolish mutagenic activity of thiram in these strains, too.
Many pesticides have been evaluated for possible mutagenicity [3,4,10]. Thiram, a fungicide of the dimethyldithiocarbamate class, is widely used in agriculture. Thus, man may be exposed to thiram both through direct occupational contact and by consuming food containing residues of that fungicide. Since most carcinogens axe also mutagens [8,9] it seemed of interest to determine the ability of thiram to induce mutations in the Salmonella test system of Ames [1,2]. Materials and methods Chemicals
Thiram (purity 98%) was a product of "Organica-Azot" (Jaworzno-Poland); biotin, L-histidine, L-cysteine, glutathione and MNNG were from Koch-Light; DMS0, dimethylsulphoxide; MMS, methyl methanesulphonate; MNNO, N'-methylN'-nitrosoguanldlne; NADP, n i c o t i n a m i d e adenine d i n u c l e o t i d e p h o s p h a t e . Abbreviations:
10 glucose 6-phosphate and NADP from Sigma Chemicals Ltd.; ampicillin (binotal) was a p r o d u c t of Bayer Leverkusen; MMS of K and K Lab.; 2-aminofluorene of Aldrich Co.; Aroclor 1254 was a gift from W.B. Papageorge, Monsanto Chemical Co. (St. Louis, U.S.A.). Bacterial strains Salmonella typhimurium strains were generously provided b y Dr. B.N. Ames (Biochemistry Department, University of California, Berkeley, U.S.A.). All strains were routinely checked for efficiency as recommended by Ames et al. [1,2]. Positive mutagenesis controls, with MNNG (TA1535), MMS (TA100) and 2-aminofluorene (TA1538 and TA98), were also included. The average numbers of spontaneous mutants per plate for TA1535, TA100, T A 1 5 3 8 and TA98 without $9 mix were 21 + 2, 165 + 8, 23 + 1, and 39 + 3, and with $9 mix 19 + 2, 171 + 6, 22 + 3 and 41 + 2, respectively. Induction o f rat-liver e n z y m e system Liver microsomal fraction $9 was prepared from rats treated with Aroclor 1254, as described by Ames et al. [1]. Male Wistar rats (200 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw) 5 days before they were killed b y decapitation. Livers were collected and homogenized in 0.15 M KC1 (3 ml/g wet tissue). The homogenate was centrifuged for 10 min at 9000 g; the supernatant was decanted and served as the source of soluble microsomal enzymes. The protein content in the $9 fraction, determined according to Lowry et al. [5], usually ranged from 36 to 42 mg/ml. Mutagenesis assay Mutagenicity test using plate-incorporation assays and S. typhimurium strains TA1535, TA100, T A 1 5 3 8 and TA98 were carried out as described by Ames et al. [1,2]. Thiram dissolved in DMSO was added to 2.0 ml of molten t o p agar (0.6% agar, 0.5% NaC1, 0.05 mM L-histidine and 0.05 mM biotin), 0.1 ml of tester strain grown overnight in Difco nutrient broth and (or omitted) 0.5 ml of the $9 mix composed of 100 pl of rat-liver microsomal fraction, 4 pmol MgC12, 16.5 #mol KC1, 2.5 pmol glucose 6-phosphate, 2 pmol NADP and 50 pmol sodium phosphate buffer (pH 7.4). In experiments with pre-incubation, procedures were based on those of Ames et al. [1,2] with a slight modification described b y Moriya et al. [10]. Thiram dissolved in DMSO (20 pl) was added to a test-tube containing 0.5 ml of one of the following solutions: $9 mix containing 0.3 ml S9 fraction per ml; $9 mix and 20 mM L-cysteine; $9 mix and 20 mM glutathione; 20 mM L-cysteine; 20 mM glutathione; 0.5 ml H20 (control). The test-tube was incubated in a water-bath with gentle shaking at 37°C for 20 min. After the incubation, 0.1 ml of an overnight broth culture of bacterial tester strain and 2 ml of molten t o p agar containing 0.05 mM L-histidine and 0.05 mM biotin were added to the test-tube. The mixture was stirred vigorously, poured immediately onto plates of minimal agar, and incubated at 37°C. Mutagenicity assays were carried o u t in triplicate, and the number o f his + revertant colonies were scored after incubation for 3 days. The number of
11
300
25( LU
~. 200 p4o UJ u.J lOO wr
2o
50
50
%00 /ug
50
200
150
THIRAM/PLATE
I00
150
200
JJg T H I R A M / P L A T E
Fig. 1. D o s e - - r a s p o n s e c u r v e s o f m u t a t i o n in Salmonella t y p h i m u r i u m w i t h v a r i o u s a m o u n t s o f t h i r a m in t h e a b s e n c e o f r a t - l i v e r $ 9 m i x . S t r a i n s : T A 1 5 3 5 (o); T A 1 0 0 ( e ) ; T A 1 5 3 8 (4); T A 9 8 (A). T h e n u m b e r o f s p o n t a n e o u s r e v e r t a n t s h a s b e e n s u b t r a c t e d f r o m t h e r e v e r t a n t v a l u e s p l o t t e d against t h i r a m c o n c e n t r a tion. Fig. 2. D o s e - - r e s p o n s e c u r v e s o f m u t a t i o n s in S. t y p h i m u r i u m T A 1 5 3 8 (o) a n d T A 9 8 ( e ) w i t h v a r i o u s a m o u n t s o f t h i r a m i n t h e p r e s e n c e o f rat-Hver $ 9 m i x . T h e n u m b e r o f s p o n t a n e o u s r e v e r t a n t s h a s b e e n s u b t r a c t e d f r o m t h e r e v e r t a n t v a l u e s p l o t t e d against t h i r a m c o n c e n t r a t i o n .
revertants per plate is an average number from at least 3 separate experiments. Mean values + S.E. of 3--5 expts, are listed Results and discussion The dose--response curves (Fig. 1) show the effect of thiram on induction o f h i s + mutations in T A 1 5 3 5 , TA100, T A 1 5 3 8 and TA98 strains of S. t y p h i r n u r i urn. Without metabolic activation thirarn was n o t mutagenic towards T A 1 5 3 8
or T A 9 8 but induced mutation in T A 1 5 3 5 and TA100. Thiram in the amount of 100 #g/plate increased the mutation frequency about 3-fold in T A 1 5 3 5 and 2.5-fold in TA100. In the presence of the microsomal fraction of rat liver, the mutagenic activity of thiram was decreased in these strains (Tables 1 and 2),
TABLE 1 E F F E C T O F T I M E O F P R E - I N C U B A T I O N O F T H I R A M ( I 0 0 # g / P L A T E ) W I T H $ 9 M I X O N ITS MUT A G E N I C A C T I V I T Y I N S T R A I N S T A 1 5 3 5 , T A 1 0 0 , T A 1 5 3 8 A N D T A 9 8 O F S. T Y P H I M U R I U M Time of pre-incubation (min)
0 5 10 15 20
Number of revertants/plate TA1535
TA100
TA1538
TA98
49±2 37±1 29±3 26 ± 1 22±2
280± 9 204± 6 186±13 172 ± 10 160± 7
54±3 55±2 53±1 55 ± 4 54±2
88±1 87±3 83±2 85 ± 3 87 ±3
12 TABLE 2 E F F E C T O F C Y S T E I N E O R G L U T A T H I O N E ON M U T A G E N I C A C T I V I T Y O F T H I R A M ( 1 0 0 # g / P L A T E ) I N S T R A I N S T A 1 5 3 5 , T A 1 0 0 , T A 1 5 3 8 A N D T A 9 8 O F S. T Y P H I M U R I U M Strain
Number of revertants/Plate Without thiram
TA1535 TA100 TA1538 TA98
With t h i r a m
H20
+cys.
+glut.
H 20
+cys.
+glut.
20+2 165 ± 6 22 ± 3 41± 2
23±1 161 ± 7 25 ± 2 66 ± 4
24±2 156 ± 9 28 ± 1 57 ± 3
72± 4 4 4 4 ± 11 54 ± 2 88 • 3
20± 1 154 ± 10 24 ± 1 65 ± 4
18±3 142 ± 9 27 ± 3 58 ± 3
$ 9 m i x w a s a d d e d t o t h e p l a t e s o n l y w h e n T A 1 5 3 8 a n d T A 9 8 strains w e r e u s e d .
which indicates that thiram was not metabolically activated by this fraction. The mutagenic activity of thiram disappeared after 20 min pre-incubation with $9 mix both in TA1535 and TA100 strains (Table 1). L-Cysteine and glutathione had a similar effect (Table 2). The frequency of the thiram-induced mutations in TA1538 and TA98 strains indicated that thiram is mutagenic for these strains only in the presence of microsomal enzymes of rat-liver (Figs. 1 and 2). The number of revertants was raised by thiram about 2.5-fold in TA1538 strain (at doses of 50--150 pg/plate) and TA98 strain (50 #g/plate). The mutagenic activity in strains TA1538 and TA98 that appeared only in the presence of microsomal fraction of rat liver was not increased by 20 rain pre-incubation of thiram with $9 mix (Table 1). On the other hand, 20 min pre-incubation of thiram with $9 mix and L-cysteine or glutathione completely destroyed its mutagenic activity in these strains (Table 2). The results suggest that mutagenic activity of thiram in all tester strains is rapidly destroyed by compounds that contain the sulphydryl group. This is in agreement with the results obtained by Matthiaschk [7], who reported that simultaneous administration of thiram and L-cysteine to pregnant mice resulted in a significant decrease both in the severity and the number of embryopathies. Assuming that carcinogenesis and teratogenesis involve similar molecular mechanisms and that these mechanisms are similar to those in mutagenesis [6], the potential hazard of thiram for man should be taken into consideration. References 1 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 , 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 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., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 A m e s , B.N., W.E. D u r t s o n , E. Y a m a s a k i a n d F.D. L e e , C a r c i n o g e n s are mutagens: a s i m p l e t e s t s y s t e m c o m b i n i n g liver h o m o g e n a t e s for activation a n d b a c t e r i a for d e t e c t i o n , P r o c . Natl. A c a d . Sci. ( U . S . A . ) , 70 ( 1 9 7 3 ) 2 2 8 1 - - 2 2 8 5 . 3 D u r h a m , W.F., a n d C.H. Williams, M u t a g e n l c , t e r a t o g e n i c , a n d c a r c i n o g e n i c p r o p e r t i e s o f pesticides, A n n u . R e v . E n t o m . , 17 ( 1 9 7 2 ) 1 2 3 - - 1 4 8 . 4 F a h r i g , R., C o m p a r a t i v e M u t a g e n i c i t y S t u d i e s w i t h Pesticides, I A R C Scient. P u b l . N o . 10, I n t . A g e n c y Res. Cancer, L y o n , 1 9 7 4 .
13
5 Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein m e a s u r c m e n t w i t h t he Folin p h e n o l reagent, J. Biol. Chem., 193 (1951) 265--275. 6 Magee, P.N., The relationship b e t w e e n mutagenesis, carcinogenesis and teratogenesis, in: D. Scott, B.A. Bridges and F.H. Sobeis (Eds.), Progress in Genetic Toxicology, Elsevier/North-Holland, Amsterdam , 1977, pp. 15--27, 7 Matthiaschk, G., Uber den Einfluss von L-cystein auf die Teratogenese d t u c h Thiram (TMTD) bei NMRI-MKusen, Arch. Toxikol., 30 (1973) 251--262. 8 McCann, J., and B.N. Ames, Detection of carcinogens as mutagens in the Salmonena/microaome test: Assay of 300 chemicals: Discussion, Proc. Natl. Acad. Sci. (U.S.A.), 73 (1976) 950--954. 9 McCann, J., E. Choi, E. Yamasaki and B.N. Ames, D e t e c t i o n of carcinogens as mutagens in t he Salm o n e l l a / m i c r o s o m e test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 5135-5139. 10 Moriya, M., K. Kato and Y. Shirasu, Effects Of cysteine and a liver metabolic activation s ys t e m on the activities of mutagenic pesticides, Mutation Res., 57 (1978) 259--263.