- Email: [email protected]
The effect of the pesticides, dexon, captan and roundup, on sister-chromatid exchanges in human lymphocytes in vitro
53
Mutation Research, 79 (1980) 53--57
© Elsevier/North-Holland Biomedical Press
THE E F F E C T OF THE PESTICIDES, DEXON, CAPTAN AND ROUNDUP, ON SISTER-CHROMATID EXCHANGES IN HUMAN LYMPHOCYTES IN VITRO
N.V. VIGFUSSON a and E.R. VYSE b a Department o f Biology, Eastern Washington University, Cheney, WA, and Sacred Heart Medical Center, Spokane, WA, and b Department o f Biology, Montana State University, Bozeman, M T (U.S.A.)
(Received 27 August 1979) (Revision received 7 March 1980) (Accepted 26 March 1980)
Summary 3 pesticides at varying concentration were tested for the induction of SCE in human lymphocytes in vitro. The fungicide, Dexon, sodium (4-(dimethylamino)phenyldiazene sulfate, was found to cause the greatest increase in SCE frequency and the response is dose related. The herbicide, Roundup, isopropylamine salt of N-(phosphonomethyl)glycine, had the least effect on SCE requiring the use of much higher concentrations to produce an effect. Limited results were obtained with the fungicide Captan, c i s - N - ( ( t r i c h l o r o m e t h y l ) t h i o ) - 4 - c y c l o hexene-1, 2~licarboximide because of toxic levels of either the fungicide or solvent used.
The fungicide, Captan, c i s - N - ( ( t r i c h l o r o m e t h y l ) t h i o ) - 4 - c y c l o h e x e n e - l , 2 dicarboximide, is c o m m o n l y used in agriculture for control of fungal diseases and for prevention of spoilage of agricultural products in transit. It is notew o r t h y that Captan is allowed to remain on harvested crops in the U.S. at levels of 100 ppm, the highest of any organic pesticide [19]. The fungicide Dexon (also called Lesan or Fenaminosulf), sodium (4-(dimethylamino)phenyl)diazene sulfonate, is recommended for use on germinating seeds and seedlings of a variety of crops. The mutagenicity and carcinogenicity of these substances is therefore of concern. Dexon has been shown to be mutagenic in prokaryotic and eukaryotic test systems [9,14,18,20,24]. Captan has been shown to produce point mutations in bacteria [6,9,14,19] and in Chinese hamster cells [2] as well as to increase the frequency of chromosomal aberrations [13]. Other data on the mutagenicity of Captan shows conflicting results in mammalian systems [4] and in Drosophila [11,18]. The herbicide R o u n d u p , isopropyl-
54 amine salt o f N - ( p h o s p h o n o m e t h y l ) g l y c i n e , is relatively new on the market and, although no published data on its mutagenicity/carcinogenicity have been r e p o r ted , it is considered to be relatively innocuous. I n d u ctio n o f SCE in mammalian cells is a sensitive m e t h o d of measuring the potential mutagenicity of chemicals [ 1, 3, 5, 7, 10, 12, 15, 21--23]. SCEs have been f o u n d to increase with increasing concentrations of mutagens and carcinogens w i t h o u t necessarily increasing c h r o m o s o m e aberrations [17]. There is also a correlation between the induction of SCE and the induction of single-locus nlutations in mammalian cells [5]. In this study the SCE index was used as a measure of the potential mutagenicity/carcinogenicity of these pesticides by applying varying concentrations to h u m a n l y m p h o c y t e s and determining subsequent SCE frequencies. Ethylm e t h a n e sulfonate (EMS) was used as a positive control since it has been d e m o n s t r a t e d to be mutagenic and to cause a significant increase in SCE freq u e n c y [5,7,15]. Results are i m c o m p l e t e for Captan as a result of cellular t o x i c i t y experienced at the higher levels of concentration. Materials and m e t h o d s Human l y m p h o c y t e s were cultured in McCoys 5A m edi um (Gibco) with the addition of 10% fetal calf serum, 1% PHA (Gibco), 1% pen-strep solution ( 1 0 0 0 0 units pencillin and 1 0 0 0 0 pg s t r e p t o m y c i n / m l ) , and 30.7 pg/ml 5 - b r o m o d e o x y u r i d i n e (10 -4 M). Also, excepting controls, each 5-ml culture contained in addition either EMS, D e xon or Captan in concentrations ranging fr o m 10 -S to 1 0 - 3 M or R o u n d u p at 65X higher concentrations (Table 1). Captan was dissolved in 7% ethanol followed by a 1 : 4 dilution in water. All o t h e r chemicals were dissolved in distilled water.
TABLE 1 SISTER-CHROMATID EXCHANGE FREQUENCIES VARYING CONCENTRATIONS OF PESTICIDES
Treatment
Concentration mg/ml medium (M)
Control
IN
HUMAN
Subject No. 1 Mean SCE
LYMPHOCYTES
EXPOSED
TO
S u b j e c t No. 2 Sign.
Mean SCE
1 4 . 1 8 ± 2.9
Sign.
1 7 . 0 6 e 3.5
EMS EMS EMS
1 . 2 4 ( 1 0 -5 ) 12.4 (10 -4 ) 124.0 ( 1 0 -3 )
1 4 . 6 8 ± 3.5 16.86 ± 3.4 2 1 . 8 4 ± 4.0
0.5 0.001 0.001
1 6 . 5 0 ± 2.8 1 8 . 2 2 ~ 3.1 2 4 . 6 8 ~ 4.7
0.30 0.100 0.001
Dexon Dexon Dexon
2.5 25.0 250.0
( 1 0 -5 ) ( 1 0 -4 ) ( 1 0 -3 )
18.52 ± 3.4 1 9 . 8 4 ± 3.9 2 6 . 5 0 ± 4.1
0.001 0.001 0.001
1 7 . 7 5 * 3.2 19.22 ~ 3.4 2 6 . 4 2 ± 4.1
0.50 0.01 0.001
Captan Captan Captan
3.0 30.0 300.0
( 1 0 -5 ) ( 1 0 -4 ) ( 1 0 -3 )
1 8 . 9 0 ± 3.5 n.g.a n.g.a
0.001
18.44 ± 3.4 n.g.a n.g.a
0.05
1 6 . 4 8 ± 3.7 1 8 . 9 0 + 3.3 n.g.a
0.001 0.001
1 9 . 5 0 ~ 2.9 1 8 . 1 4 + 3.2 n.g.a
0.001 0.10
Roundup Roundup Roundup a No growth.
0 . 2 5 ( 6 5 X 1 0 -5 ) 2.5 ( 6 5 X 10 -4 ) 2 5 . 0 ( 6 5 X 10 -3 )
55 2 subjects were chosen to act as regular donors of blood cells. 5 ml culture tubes were inoculated with 0.2 ml of a gravity settled leukocyte suspension and incubated for 72 h. Harvesting was done by traditional methods and cells were fixed on slides with methanol : acetic acid (3 : 1). After overnight drying slides were stained by the FPG technique [16]. For each sample and concentration, 50 well spread and completely differentially stained metaphases were analyzed for SCE frequency from each subject. Results
Results for the 3 pesticides tested and the controls are shown in Table 1 with the mean SCE f r e q u e n c y of 2 experiments, standard devidations and significance levels as determined by Student's t-test. In addition, dose--response curves for EMS and Dexon are presented in Fig. 1. Similar data for Captan and R o u n d u p are n o t presented because the higher concentrations are toxic (Table 1). Paired Student's t-test were determined for all pairs of data in both subjects. EMS at the lowest concentration did n o t significantly increase SCE over the controls. The highest concentrations of EMS (10 -4 and 10 -3 M) did significantly increase the level of SCE over the controls and over the 10 -s M concentrations in both subjects (Table 1 and Fig. 1). In subject number one, Dexon increased SCE significantly at all concentrations tested. In subject number two, Dexon did not significantly increase the SCE at the lowest concentration but did produce a significant increase at the higher concentrations. At the 3 concentrations tested, Dexon produced a significantly higher level of SCE than EMS (P 0.05) at each of the concentrations tested in both subjects. Captan at the lowest concentration (10 -s M) significantly increased SCE
260
=
25,0
"--
24.0 ~J
= EMS Subject I EMS Subject 2 -'Dexon Subject I Dexon Subject 2 A C o n t r o l Subject I ZkControl Subject 2
23.0 i,i o
22.0
U_ 0
2 IO
I~:
20.0
oD
19.0 z
18.0
t~
17.0 ¢6.0 t5.0
14.0
I 10-5
I jO-4
I 10-3
MOLARITY
F i g . 1. S C E d o s e - - r e s p o n s e
for varying concentrations
of Dexon and EMS.
56 levels over controls and EMS at the same c o n c e n t r a t i o n (P 0.05). Direct comparisons of the R o u n d u p results to those of EMS and Dexon are n o t feasible because the concentrations used for R o u n d u p were 65X higher. The results at the concentrations for which data is available are significantly higher than the controls e x c e p t for the 65X 10 -4 M c o n c e n t r a t i o n in the second subject. The slightly reduced ef f ect of the higher c o n c e n t r a t i o n in the second subject is n o t easily explained unless there is a t o x i c i t y effect removing some affected cells f r o m the population.
Discussion Carrano and co-workers [5] r e c e nt l y r e p o r t e d a linear relationship between the induction of SCEs and the p r o d u c t i o n of mutations. The increased SCE f r e q u e n c y f o u n d in h u m a n l y m p h o c y t e s exposed to D exon corresponds to previous data on the mutagenicity of D e x o n [14,18--20,24] and lend furt her s u p p o r t to the use of the SCE index in mutagenicity testing. In our test, the SCE response is d o s e - dependent (Fig. 1) as r e p o r t e d for ot her mutagens [5,15]. The SCE induced in l y m p h o c y t e s exposed to D exon are significantly higher than those exposed to the same c o n c e n t r a t i o n of EMS, a well know n mutagen. D e x o n is a p o t e n t mutagen and inducer o f SCE but it has recently been r e p o r t e d n o t to be carcinogenic in a test involving 50 treated rats and mice (NCI Report). Based on the previous mutagenicity tests and the mammalian SCE results r e p o r t e d here the carcinogenicity tests should probabl y be repeated. Captan has been d e m o n s t r a t e d to be mutagenic in several test systems [2,4,6,9,13,14,19,20] and based on this data we would e x p e c t t h i s chemical to evoke a significant increase in the SCE index. U n f o r t u n a t e l y this could only be tested at the lowest c o n c e n t r a t i o n because bo t h higher concentrations inhibited growth of h u m a n l y m p h o c y t e s . The limited data shows that the SCEs are significantly higher when cells are exposed to Captan than to EMS at the same c o n c e n t r a t i o n . The United States Environmental Prot ect i on Agency has re c en tly triggered Captan for RPAR ( R ebut t abl e Presumption Against Registration) because o f its mutagenicity, possible oncogenicity (NCI Report ) and teratogenicity. Although n o t necessarily a consequence of presumption, triggering may result in restricted use, suspension, or ultimate cancellation of registrations of the pesticide for agricultural use. The presumption and ensuing rebuttal and evaluation of risks versus benefits is called RPAR [8]. To the best of our knowledge R o u n d u p has n o t previously been tested for its e ff e c t on DNA. The results r e p o r t e d here show significant increases in the SCE index upon exposure to R o u n d u p , but only when concentrations were increased 65X over those used for the ot her pesticides. Although our conclusions are tentative because of the apparent toxicity, we suggest t hat this chemical is at most weakly mutagenic based on the SCE test. These results suggest that R o u n d u p should be evaluated in ot her genetic tests t hat measure poi nt mutations and c h r o m o s o m e aberrations. F u r t h e r m o r e , this herbicide is probably n o t a biological hazard e x c e p t during application because it is inactivated on c o n t a c t with the soil.
57
Acknowledgement The technical assistance provided by Marilyn Lloyd and John Thomas is greatly appreciated. References 1 Allen, J.W., a n d S.A. L a t t , A n a l y s i s o f sister c h r o m a t i d e x c h a n g e f o r m a t i o n in vivo in m o u s e s p e r m a t o g o n i a as a n e w test s y s t e m f o r e n v i r o n m e n t a l m u t a g e n s , N a t u r e ( L o n d o n ) , 2 6 0 ( 1 9 7 6 ) 4 4 9 - - 4 5 1 . 2 A r l e t t , C.F., D. T u r n b u l l , S.A. H a r c o u r t , A.R. L e h m a n a n d C.M. Colella, A c o m p a r i s o n of t h e 8-azag u a n i n e a n d o u a b a i n - r e s i s t a n c e s y s t e m s for s e l e c t i o n of i n d u c e d m u t a n t Chinese h a m s t e r cells, Mutat i o n Res., 33 ( 1 9 7 5 ) 2 6 1 - - 2 7 8 . 3 Beck, B., a n d G. Obe, T h e h u m a n l e u c o c y t e t e s t s y s t e m s , VI. T h e use of sister c h r o m a t i d e x c h a n g e s as possible i n d i c a t o r s f o r m u t a g e n i c activities, H u m a n g e n e t i k , 29 ( 1 9 7 8 ) 5 5 1 - - 5 5 3 . 4 Bridges, B.A., T h e m u t a g e n i c i t y o f C a p t a n a n d r e l a t e d fungicides, M u t a t i o n Res., 32 ( 1 9 7 5 ) 3 - - 3 4 . 5 C a r r a n o , A.V., L . H . T h o m p s o n , P.A. L i n d l a n d J . L . Minkier, Sister c h r o m a t i d e x c h a n g e as an indicat o r of m u t a g e n e s i s , N a t u r e ( L o n d o n ) , 271 ( 1 9 7 8 ) 5 5 1 - - 5 5 3 . 6 Clarke, C.H., T h e m u t a g e n i c specificities o f p e n t a c h l o r o n i t r o b e n z e n e a n d C a p t a n , t w o e n v i r o n m e n t a l m u t a g e n s , M u t a t i o n Res., ( 1 9 7 1 ) 2 4 7 - - 2 4 8 . 7 C r a i g - H o l m e s , A.P., a n d M.W. S h a w , E f f e c t s of six c a r c i n o g e n s on SCE f r e q u e n c y a n d cell k i n e t i c s in c u l t u r e d h u m a n l y m p h o c y t e s , M u t a t i o n Res., 4 6 ( 1 9 7 7 ) 3 7 5 - - 3 8 4 . 8 F e d e r a l Register, 4 0 ( 1 9 7 5 ) 2 6 8 9 9 . 9 K a d a , T., M. M o r i y a a n d Y. Shirasu, S c r e e n i n g o f p e s t i c i d e s for D N A i n t e r a c t i o n s b y " R e - a s s a y " a n d m u t a g e n e s i s testing, a n d f r a m e s h i f t m u t a t i o n s d e t e c t e d , M u t a t i o n Res., 26 ( 1 9 7 4 ) 2 4 3 - - 2 4 8 . 10 K a t o , H., a n d H. S h i m a d a , Sister c h r o m a t i d e x c h a n g e s i n d u c e d b y M i t o m y c i n C: A n e w m e t h o d of d e t e c t i n g D N A d a m a g e at the c h r o m o s o m a l level, M u t a t i o n Res., 28 ( 1 9 7 5 ) 4 5 9 - - 4 6 4 . 11 K r a m e r s , P . G . N . , a n d A.G. K n a a p , M u t a g e n i c i t y tests w i t h C a p t a n a n d F o l p e t in D r o s o p h i l a m e l a n o gaster, M u t a t i o n Res., 21 ( 1 9 7 3 ) 1 4 9 - - 1 5 4 . 12 L a t t , S.A., Sister c h r o m a t i d e x c h a n g e s , indices o f h u m a n c h r o m o s o m e d a m a g e a n d r e p a i r : D e t e c t i o n b y f l u o r e s c e n c e a n d i n d u c t i o n b y M i t o m y c i n C, Proc. Natl. A c a d . Sci. ( U . S . A . ) , 71 ( 1 9 7 4 ) 3 1 6 2 - 3166. 13 L e g a t o r , M.S., F.J. K e l l y , S. G r e e n a n d E.J. O s w a l d , M u t a g e n i c e f f e c t s of C a p t a n , A n n . N.Y. A c a d . Sci., 1 6 0 ( 1 9 6 9 ) 3 4 4 - - 3 5 1 . 14 M c C a n n , J., E. Choi, E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s in the Salm o n e l l a / m i c r o s o m e test: A s s a y o f 3 0 0 c h e m i c a l s , Proc. Natl. A c a d . Sci. ( U . S . A . ) , 72 ( 1 9 7 5 ) 5 1 3 5 - 5139. 15 P e r r y , P., a n d H.J. E v a n s , C y t o l o g i c a l d e t e c t i o n o f m u t a g e n - - c a r c i n o g e n e x p o s u r e b y sister c h r o m a t i d exchanges, Nature ( L o n d o n ) , 258 (1975) 121--125. 16 P e r r y , P., a n d S. Wolff, N e w G i e m s a m e t h o d f o r the d i f f e r e n t i a l s t a i n i n g of c h r o m o s o m e s , N a t u r e ( L o n d o n ) , 251 ( 1 9 7 4 ) 1 5 6 - - 1 5 8 . 17 P o p e s c u , N.C., D. T u r n b u l l a n d J . A . D i P a u l o , Sister c h r o m a t i d e x c h a n g e a n d c h r o m o s o m e a b e r r a t i o n analysis w i t h t h e use o f several c a r c i n o g e n s a n d n o n c a r c i n o g e n s : Brief c o m m u n i c a t i o n , J. Natl. C a n c e r Inst., 59 ( 1 9 7 7 ) 2 8 9 - - 2 9 3 . 18 R o g e r s , S.J., P.D. S k a a r , K . R . Tindall, E.R. Vyse a n d G. W a r r e n , M u t a g e n i c i t y of t h e f u n g i c i d e D e x o n , M u t a t i o n Res., 53 ( 1 9 7 8 ) 91. 19 Shea, K.P., C a p t a n a n d f o l p e t . E n v i r o n m e n t , 14 ( 1 9 7 2 ) 2 2 - - 3 3 . 20 Shirasu, Y., M. M o r i y a , K. K a t o , A. F u r u h a s h i a n d T. K a d a , M u t a g e n i c i t y s c r e e n i n g of p e s t i c i d e s in t h e m i c r o b i a l s y s t e m , M u t a t i o n Res., 4 0 ( 1 9 7 6 ) 1 9 - - 3 0 . 21 S o l o m o n , E., a n d M. B o b r o w , Sister c h r o m a t i d e x c h a n g e - - a sensitive assay of a g e n t s d a m a g i n g h u m a n c h r o m o s o m e s , M u t a t i o n Res., 30 ( 1 9 7 5 ) 2 7 3 - - 2 7 8 . 22 S t e t k a , D . G . , a n d S. Wolff, Sister c h r o m a t i d e x c h a n g e s as an assay for g e n e t i c d a m a g e i n d u c e d b y m u t a g e n ~ a r c i n o g e n s , II. In v i t r o test for c o m p o u n d s r e q u i r i n g 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 Res., 41 ( 1 9 7 6 ) 3 4 3 - - 3 5 0 . 23 Vogel, W., a n d T. B a u k n e c h t , D i f f e r e n t i a l c h r o m a t i d staining in vivo t r e a t m e n t as a m u t a g e n i c i t y test system, Nature (London), 260 (1976) 448--449. 24 Zutshi, U., a n d B.L. K a u l , S t u d i e s on t h e c y t o g e n e t i c a c t i v i t y of s o m e c o m m o n f u n g i c i d e s in h i g h e r p l a n t s , C y t o b i o s , 12 ( 1 9 7 5 ) 6 1 - - 6 7 .
Mutation Research, 79 (1980) 53--57
© Elsevier/North-Holland Biomedical Press
THE E F F E C T OF THE PESTICIDES, DEXON, CAPTAN AND ROUNDUP, ON SISTER-CHROMATID EXCHANGES IN HUMAN LYMPHOCYTES IN VITRO
N.V. VIGFUSSON a and E.R. VYSE b a Department o f Biology, Eastern Washington University, Cheney, WA, and Sacred Heart Medical Center, Spokane, WA, and b Department o f Biology, Montana State University, Bozeman, M T (U.S.A.)
(Received 27 August 1979) (Revision received 7 March 1980) (Accepted 26 March 1980)
Summary 3 pesticides at varying concentration were tested for the induction of SCE in human lymphocytes in vitro. The fungicide, Dexon, sodium (4-(dimethylamino)phenyldiazene sulfate, was found to cause the greatest increase in SCE frequency and the response is dose related. The herbicide, Roundup, isopropylamine salt of N-(phosphonomethyl)glycine, had the least effect on SCE requiring the use of much higher concentrations to produce an effect. Limited results were obtained with the fungicide Captan, c i s - N - ( ( t r i c h l o r o m e t h y l ) t h i o ) - 4 - c y c l o hexene-1, 2~licarboximide because of toxic levels of either the fungicide or solvent used.
The fungicide, Captan, c i s - N - ( ( t r i c h l o r o m e t h y l ) t h i o ) - 4 - c y c l o h e x e n e - l , 2 dicarboximide, is c o m m o n l y used in agriculture for control of fungal diseases and for prevention of spoilage of agricultural products in transit. It is notew o r t h y that Captan is allowed to remain on harvested crops in the U.S. at levels of 100 ppm, the highest of any organic pesticide [19]. The fungicide Dexon (also called Lesan or Fenaminosulf), sodium (4-(dimethylamino)phenyl)diazene sulfonate, is recommended for use on germinating seeds and seedlings of a variety of crops. The mutagenicity and carcinogenicity of these substances is therefore of concern. Dexon has been shown to be mutagenic in prokaryotic and eukaryotic test systems [9,14,18,20,24]. Captan has been shown to produce point mutations in bacteria [6,9,14,19] and in Chinese hamster cells [2] as well as to increase the frequency of chromosomal aberrations [13]. Other data on the mutagenicity of Captan shows conflicting results in mammalian systems [4] and in Drosophila [11,18]. The herbicide R o u n d u p , isopropyl-
54 amine salt o f N - ( p h o s p h o n o m e t h y l ) g l y c i n e , is relatively new on the market and, although no published data on its mutagenicity/carcinogenicity have been r e p o r ted , it is considered to be relatively innocuous. I n d u ctio n o f SCE in mammalian cells is a sensitive m e t h o d of measuring the potential mutagenicity of chemicals [ 1, 3, 5, 7, 10, 12, 15, 21--23]. SCEs have been f o u n d to increase with increasing concentrations of mutagens and carcinogens w i t h o u t necessarily increasing c h r o m o s o m e aberrations [17]. There is also a correlation between the induction of SCE and the induction of single-locus nlutations in mammalian cells [5]. In this study the SCE index was used as a measure of the potential mutagenicity/carcinogenicity of these pesticides by applying varying concentrations to h u m a n l y m p h o c y t e s and determining subsequent SCE frequencies. Ethylm e t h a n e sulfonate (EMS) was used as a positive control since it has been d e m o n s t r a t e d to be mutagenic and to cause a significant increase in SCE freq u e n c y [5,7,15]. Results are i m c o m p l e t e for Captan as a result of cellular t o x i c i t y experienced at the higher levels of concentration. Materials and m e t h o d s Human l y m p h o c y t e s were cultured in McCoys 5A m edi um (Gibco) with the addition of 10% fetal calf serum, 1% PHA (Gibco), 1% pen-strep solution ( 1 0 0 0 0 units pencillin and 1 0 0 0 0 pg s t r e p t o m y c i n / m l ) , and 30.7 pg/ml 5 - b r o m o d e o x y u r i d i n e (10 -4 M). Also, excepting controls, each 5-ml culture contained in addition either EMS, D e xon or Captan in concentrations ranging fr o m 10 -S to 1 0 - 3 M or R o u n d u p at 65X higher concentrations (Table 1). Captan was dissolved in 7% ethanol followed by a 1 : 4 dilution in water. All o t h e r chemicals were dissolved in distilled water.
TABLE 1 SISTER-CHROMATID EXCHANGE FREQUENCIES VARYING CONCENTRATIONS OF PESTICIDES
Treatment
Concentration mg/ml medium (M)
Control
IN
HUMAN
Subject No. 1 Mean SCE
LYMPHOCYTES
EXPOSED
TO
S u b j e c t No. 2 Sign.
Mean SCE
1 4 . 1 8 ± 2.9
Sign.
1 7 . 0 6 e 3.5
EMS EMS EMS
1 . 2 4 ( 1 0 -5 ) 12.4 (10 -4 ) 124.0 ( 1 0 -3 )
1 4 . 6 8 ± 3.5 16.86 ± 3.4 2 1 . 8 4 ± 4.0
0.5 0.001 0.001
1 6 . 5 0 ± 2.8 1 8 . 2 2 ~ 3.1 2 4 . 6 8 ~ 4.7
0.30 0.100 0.001
Dexon Dexon Dexon
2.5 25.0 250.0
( 1 0 -5 ) ( 1 0 -4 ) ( 1 0 -3 )
18.52 ± 3.4 1 9 . 8 4 ± 3.9 2 6 . 5 0 ± 4.1
0.001 0.001 0.001
1 7 . 7 5 * 3.2 19.22 ~ 3.4 2 6 . 4 2 ± 4.1
0.50 0.01 0.001
Captan Captan Captan
3.0 30.0 300.0
( 1 0 -5 ) ( 1 0 -4 ) ( 1 0 -3 )
1 8 . 9 0 ± 3.5 n.g.a n.g.a
0.001
18.44 ± 3.4 n.g.a n.g.a
0.05
1 6 . 4 8 ± 3.7 1 8 . 9 0 + 3.3 n.g.a
0.001 0.001
1 9 . 5 0 ~ 2.9 1 8 . 1 4 + 3.2 n.g.a
0.001 0.10
Roundup Roundup Roundup a No growth.
0 . 2 5 ( 6 5 X 1 0 -5 ) 2.5 ( 6 5 X 10 -4 ) 2 5 . 0 ( 6 5 X 10 -3 )
55 2 subjects were chosen to act as regular donors of blood cells. 5 ml culture tubes were inoculated with 0.2 ml of a gravity settled leukocyte suspension and incubated for 72 h. Harvesting was done by traditional methods and cells were fixed on slides with methanol : acetic acid (3 : 1). After overnight drying slides were stained by the FPG technique [16]. For each sample and concentration, 50 well spread and completely differentially stained metaphases were analyzed for SCE frequency from each subject. Results
Results for the 3 pesticides tested and the controls are shown in Table 1 with the mean SCE f r e q u e n c y of 2 experiments, standard devidations and significance levels as determined by Student's t-test. In addition, dose--response curves for EMS and Dexon are presented in Fig. 1. Similar data for Captan and R o u n d u p are n o t presented because the higher concentrations are toxic (Table 1). Paired Student's t-test were determined for all pairs of data in both subjects. EMS at the lowest concentration did n o t significantly increase SCE over the controls. The highest concentrations of EMS (10 -4 and 10 -3 M) did significantly increase the level of SCE over the controls and over the 10 -s M concentrations in both subjects (Table 1 and Fig. 1). In subject number one, Dexon increased SCE significantly at all concentrations tested. In subject number two, Dexon did not significantly increase the SCE at the lowest concentration but did produce a significant increase at the higher concentrations. At the 3 concentrations tested, Dexon produced a significantly higher level of SCE than EMS (P 0.05) at each of the concentrations tested in both subjects. Captan at the lowest concentration (10 -s M) significantly increased SCE
260
=
25,0
"--
24.0 ~J
= EMS Subject I EMS Subject 2 -'Dexon Subject I Dexon Subject 2 A C o n t r o l Subject I ZkControl Subject 2
23.0 i,i o
22.0
U_ 0
2 IO
I~:
20.0
oD
19.0 z
18.0
t~
17.0 ¢6.0 t5.0
14.0
I 10-5
I jO-4
I 10-3
MOLARITY
F i g . 1. S C E d o s e - - r e s p o n s e
for varying concentrations
of Dexon and EMS.
56 levels over controls and EMS at the same c o n c e n t r a t i o n (P 0.05). Direct comparisons of the R o u n d u p results to those of EMS and Dexon are n o t feasible because the concentrations used for R o u n d u p were 65X higher. The results at the concentrations for which data is available are significantly higher than the controls e x c e p t for the 65X 10 -4 M c o n c e n t r a t i o n in the second subject. The slightly reduced ef f ect of the higher c o n c e n t r a t i o n in the second subject is n o t easily explained unless there is a t o x i c i t y effect removing some affected cells f r o m the population.
Discussion Carrano and co-workers [5] r e c e nt l y r e p o r t e d a linear relationship between the induction of SCEs and the p r o d u c t i o n of mutations. The increased SCE f r e q u e n c y f o u n d in h u m a n l y m p h o c y t e s exposed to D exon corresponds to previous data on the mutagenicity of D e x o n [14,18--20,24] and lend furt her s u p p o r t to the use of the SCE index in mutagenicity testing. In our test, the SCE response is d o s e - dependent (Fig. 1) as r e p o r t e d for ot her mutagens [5,15]. The SCE induced in l y m p h o c y t e s exposed to D exon are significantly higher than those exposed to the same c o n c e n t r a t i o n of EMS, a well know n mutagen. D e x o n is a p o t e n t mutagen and inducer o f SCE but it has recently been r e p o r t e d n o t to be carcinogenic in a test involving 50 treated rats and mice (NCI Report). Based on the previous mutagenicity tests and the mammalian SCE results r e p o r t e d here the carcinogenicity tests should probabl y be repeated. Captan has been d e m o n s t r a t e d to be mutagenic in several test systems [2,4,6,9,13,14,19,20] and based on this data we would e x p e c t t h i s chemical to evoke a significant increase in the SCE index. U n f o r t u n a t e l y this could only be tested at the lowest c o n c e n t r a t i o n because bo t h higher concentrations inhibited growth of h u m a n l y m p h o c y t e s . The limited data shows that the SCEs are significantly higher when cells are exposed to Captan than to EMS at the same c o n c e n t r a t i o n . The United States Environmental Prot ect i on Agency has re c en tly triggered Captan for RPAR ( R ebut t abl e Presumption Against Registration) because o f its mutagenicity, possible oncogenicity (NCI Report ) and teratogenicity. Although n o t necessarily a consequence of presumption, triggering may result in restricted use, suspension, or ultimate cancellation of registrations of the pesticide for agricultural use. The presumption and ensuing rebuttal and evaluation of risks versus benefits is called RPAR [8]. To the best of our knowledge R o u n d u p has n o t previously been tested for its e ff e c t on DNA. The results r e p o r t e d here show significant increases in the SCE index upon exposure to R o u n d u p , but only when concentrations were increased 65X over those used for the ot her pesticides. Although our conclusions are tentative because of the apparent toxicity, we suggest t hat this chemical is at most weakly mutagenic based on the SCE test. These results suggest that R o u n d u p should be evaluated in ot her genetic tests t hat measure poi nt mutations and c h r o m o s o m e aberrations. F u r t h e r m o r e , this herbicide is probably n o t a biological hazard e x c e p t during application because it is inactivated on c o n t a c t with the soil.
57
Acknowledgement The technical assistance provided by Marilyn Lloyd and John Thomas is greatly appreciated. References 1 Allen, J.W., a n d S.A. L a t t , A n a l y s i s o f sister c h r o m a t i d e x c h a n g e f o r m a t i o n in vivo in m o u s e s p e r m a t o g o n i a as a n e w test s y s t e m f o r e n v i r o n m e n t a l m u t a g e n s , N a t u r e ( L o n d o n ) , 2 6 0 ( 1 9 7 6 ) 4 4 9 - - 4 5 1 . 2 A r l e t t , C.F., D. T u r n b u l l , S.A. H a r c o u r t , A.R. L e h m a n a n d C.M. Colella, A c o m p a r i s o n of t h e 8-azag u a n i n e a n d o u a b a i n - r e s i s t a n c e s y s t e m s for s e l e c t i o n of i n d u c e d m u t a n t Chinese h a m s t e r cells, Mutat i o n Res., 33 ( 1 9 7 5 ) 2 6 1 - - 2 7 8 . 3 Beck, B., a n d G. Obe, T h e h u m a n l e u c o c y t e t e s t s y s t e m s , VI. T h e use of sister c h r o m a t i d e x c h a n g e s as possible i n d i c a t o r s f o r m u t a g e n i c activities, H u m a n g e n e t i k , 29 ( 1 9 7 8 ) 5 5 1 - - 5 5 3 . 4 Bridges, B.A., T h e m u t a g e n i c i t y o f C a p t a n a n d r e l a t e d fungicides, M u t a t i o n Res., 32 ( 1 9 7 5 ) 3 - - 3 4 . 5 C a r r a n o , A.V., L . H . T h o m p s o n , P.A. L i n d l a n d J . L . Minkier, Sister c h r o m a t i d e x c h a n g e as an indicat o r of m u t a g e n e s i s , N a t u r e ( L o n d o n ) , 271 ( 1 9 7 8 ) 5 5 1 - - 5 5 3 . 6 Clarke, C.H., T h e m u t a g e n i c specificities o f p e n t a c h l o r o n i t r o b e n z e n e a n d C a p t a n , t w o e n v i r o n m e n t a l m u t a g e n s , M u t a t i o n Res., ( 1 9 7 1 ) 2 4 7 - - 2 4 8 . 7 C r a i g - H o l m e s , A.P., a n d M.W. S h a w , E f f e c t s of six c a r c i n o g e n s on SCE f r e q u e n c y a n d cell k i n e t i c s in c u l t u r e d h u m a n l y m p h o c y t e s , M u t a t i o n Res., 4 6 ( 1 9 7 7 ) 3 7 5 - - 3 8 4 . 8 F e d e r a l Register, 4 0 ( 1 9 7 5 ) 2 6 8 9 9 . 9 K a d a , T., M. M o r i y a a n d Y. Shirasu, S c r e e n i n g o f p e s t i c i d e s for D N A i n t e r a c t i o n s b y " R e - a s s a y " a n d m u t a g e n e s i s testing, a n d f r a m e s h i f t m u t a t i o n s d e t e c t e d , M u t a t i o n Res., 26 ( 1 9 7 4 ) 2 4 3 - - 2 4 8 . 10 K a t o , H., a n d H. S h i m a d a , Sister c h r o m a t i d e x c h a n g e s i n d u c e d b y M i t o m y c i n C: A n e w m e t h o d of d e t e c t i n g D N A d a m a g e at the c h r o m o s o m a l level, M u t a t i o n Res., 28 ( 1 9 7 5 ) 4 5 9 - - 4 6 4 . 11 K r a m e r s , P . G . N . , a n d A.G. K n a a p , M u t a g e n i c i t y tests w i t h C a p t a n a n d F o l p e t in D r o s o p h i l a m e l a n o gaster, M u t a t i o n Res., 21 ( 1 9 7 3 ) 1 4 9 - - 1 5 4 . 12 L a t t , S.A., Sister c h r o m a t i d e x c h a n g e s , indices o f h u m a n c h r o m o s o m e d a m a g e a n d r e p a i r : D e t e c t i o n b y f l u o r e s c e n c e a n d i n d u c t i o n b y M i t o m y c i n C, Proc. Natl. A c a d . Sci. ( U . S . A . ) , 71 ( 1 9 7 4 ) 3 1 6 2 - 3166. 13 L e g a t o r , M.S., F.J. K e l l y , S. G r e e n a n d E.J. O s w a l d , M u t a g e n i c e f f e c t s of C a p t a n , A n n . N.Y. A c a d . Sci., 1 6 0 ( 1 9 6 9 ) 3 4 4 - - 3 5 1 . 14 M c C a n n , J., E. Choi, E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s in the Salm o n e l l a / m i c r o s o m e test: A s s a y o f 3 0 0 c h e m i c a l s , Proc. Natl. A c a d . Sci. ( U . S . A . ) , 72 ( 1 9 7 5 ) 5 1 3 5 - 5139. 15 P e r r y , P., a n d H.J. E v a n s , C y t o l o g i c a l d e t e c t i o n o f m u t a g e n - - c a r c i n o g e n e x p o s u r e b y sister c h r o m a t i d exchanges, Nature ( L o n d o n ) , 258 (1975) 121--125. 16 P e r r y , P., a n d S. Wolff, N e w G i e m s a m e t h o d f o r the d i f f e r e n t i a l s t a i n i n g of c h r o m o s o m e s , N a t u r e ( L o n d o n ) , 251 ( 1 9 7 4 ) 1 5 6 - - 1 5 8 . 17 P o p e s c u , N.C., D. T u r n b u l l a n d J . A . D i P a u l o , Sister c h r o m a t i d e x c h a n g e a n d c h r o m o s o m e a b e r r a t i o n analysis w i t h t h e use o f several c a r c i n o g e n s a n d n o n c a r c i n o g e n s : Brief c o m m u n i c a t i o n , J. Natl. C a n c e r Inst., 59 ( 1 9 7 7 ) 2 8 9 - - 2 9 3 . 18 R o g e r s , S.J., P.D. S k a a r , K . R . Tindall, E.R. Vyse a n d G. W a r r e n , M u t a g e n i c i t y of t h e f u n g i c i d e D e x o n , M u t a t i o n Res., 53 ( 1 9 7 8 ) 91. 19 Shea, K.P., C a p t a n a n d f o l p e t . E n v i r o n m e n t , 14 ( 1 9 7 2 ) 2 2 - - 3 3 . 20 Shirasu, Y., M. M o r i y a , K. K a t o , A. F u r u h a s h i a n d T. K a d a , M u t a g e n i c i t y s c r e e n i n g of p e s t i c i d e s in t h e m i c r o b i a l s y s t e m , M u t a t i o n Res., 4 0 ( 1 9 7 6 ) 1 9 - - 3 0 . 21 S o l o m o n , E., a n d M. B o b r o w , Sister c h r o m a t i d e x c h a n g e - - a sensitive assay of a g e n t s d a m a g i n g h u m a n c h r o m o s o m e s , M u t a t i o n Res., 30 ( 1 9 7 5 ) 2 7 3 - - 2 7 8 . 22 S t e t k a , D . G . , a n d S. Wolff, Sister c h r o m a t i d e x c h a n g e s as an assay for g e n e t i c d a m a g e i n d u c e d b y m u t a g e n ~ a r c i n o g e n s , II. In v i t r o test for c o m p o u n d s r e q u i r i n g 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 Res., 41 ( 1 9 7 6 ) 3 4 3 - - 3 5 0 . 23 Vogel, W., a n d T. B a u k n e c h t , D i f f e r e n t i a l c h r o m a t i d staining in vivo t r e a t m e n t as a m u t a g e n i c i t y test system, Nature (London), 260 (1976) 448--449. 24 Zutshi, U., a n d B.L. K a u l , S t u d i e s on t h e c y t o g e n e t i c a c t i v i t y of s o m e c o m m o n f u n g i c i d e s in h i g h e r p l a n t s , C y t o b i o s , 12 ( 1 9 7 5 ) 6 1 - - 6 7 .