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Induction of somatic mutations by the promutagen dimethylnitrosoamine in hairs of tradescantia stamen
381
Mutation Research, 78 (1980) 381--384
© Elsevier/North-Holland Biomedical Press
Short Communication INDUCTION OF SOMATIC MUTATIONS BY THE P R O M U T A G E N DIMETHYLNITROSOAMINE IN HAIRS OF T R A D E S C A N T I A STAMEN
T. GICHNER, J. VELEMfNSK~"and A.G. UNDERBRINK a Institute of Experimental Botany, Flemingovo 2, Praha 2 (Czechoslovakia). and a Institute of Nuclear Physics, Krak6w (Poland)
(Received 19 November 1979) (Revision received 29 February 1980) (Accepted 14 March 1980)
Dimethylnitrosoamine (DMN) is widely acknowledged to be among the most potent, environmentally significant, carcinogens. DMN is a so-called promutagen, i.e., it is not mutagenic in itself, but it requires metabolic activation for conversion into a mutagen. In the absence of metabolic activation, DMN is nonmutagenic in bacteria, yeasts, molds and cultures of animal tissues (Geissler, 1962; Marquardt et al., 1963, 1964; Natarajan et al., 1976). However, the mutagenicity of DMN in these cells has been achieved by: (1) a host-mediated assay, {2) by adding hepatic microsomes, or (3) by adding an Udenfriend enzyme-like mixture to the DMN treatment solution (Gabridge and Legator, 1969; Malling, 1966, 1971; Marquardt et al., 1964; Mayer, 1971; Natarajan et al., 1976). In contrast with a lack of mutagenicity in microorganisms and animal tissue cells, DMN can induce mutations in Drosophila (Pasternak, 1964). The first plant species in which DMN proved to be mutagenic was Arabidopsis thaliana (Veleminsk:~ and Gichner, 1968, 1971). Seeds of Arabidopsis were postulated to contain enzymes or an enzyme-like system capable of converting DMN into a mutagenic intermediate and that the decline in the frequency of mutations and sterility following large DMN doses might be caused by the inhibition of the activation system. This communication concerns the mutagenic activity of DMN in 2 clones of Tradescantia that differ in their sensitivity to chemical mutagens. Clone 4430 (a hybrid of T. subacaulis × T. hirsutiflora) is 6--9 times more sensitive to 1,2dibromoethane {DBE) and ethyl methanesulfonate (EMS} (Nauman et al., 1976; Sparrow et al., 1974) than is clone 02 (a putative hybrid of T. occio dentalis × T. ohiensis).
DMN, dissolved in distilled water, was applied in two ways. (1) The stems of cuttings were immersed in the solution for 2 4 h at 25°C. (2) The entire inflorescenses were immersed in the solution for 24 h at 25 ° C. After treatment, the cuttings were washed for 1 h and placed in beakers with Hoagland's nutrient solution. The cultivation took place in a controlled-environment chamber with
382 a 16-h day and a p h o t o n fluence rate of 400--700 nm, 300 pmoles m -~s-' and 25/18°C day/night temperatures. 30 cuttings were used for each treatment; and each day about 10 flowers (i.e. about 3000 stamen hairs) were scored depending on how many flowers were available. The pink somatic mutations in the stamen hairs were scored according to methods described by Underbrink et al. (1973). One or more contiguous pink cells were considered to have resulted from one m u t a n t event. The frequency of spontaneous m u t a n t events in clone 4430 was 0.185% {131 200 hairs were analyzed) and in clone 02 it was 0.118% (67 700 hairs were analyzed). The peak of pink somatic mutations in clone 4430 after immersion of the stems of cuttings in solutions of 150 and 200 mM DMN for 24 h occurred 9 days after the onset of t r e a t m e n t (Fig. 1). Treatment with 250 and 300 mM DMN strongly inhibited flowering. In clone 02, treatment with 200 mM DMN resulted in a very low frequency of pink-mutant events which was only slightly higher than the spontaneous level. The application of 250 mM DMN partly inhibited flowering, and 300 mM DMN caused complete inhibition. The application of 150 mM DMN directly to the inflorescences of both clones caused the flowers to be badly damaged for the first 4--7 days after treatment. The stamen hairs were bleached and deformed, and scoring was difficult. The maximal frequency of pink events in clone 4430 was reached 7--8 days after the onset of treatment (Fig. 2). The frequency of pink mutations in clone 02 was again considerably lower, even after treatment with 200 mM DMN which, in clone 4430, caused complete inhibition of flowering. A comparison of the 2 methods used to apply the DMN solutions shows that a direct treatment to the inflorescences (Fig. 2) yielded a considerably higher frequency of pink mutations at equal mutagenic concentrations than treatment to the stems of cuttings (Fig. 1). The disadvantage in directly treating the inflorescences is that the flowers are badly damaged for the first few days after
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.....
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....
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~
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~-o-~ clone 02
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..z. ~o Z
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6 8 10 12 DAYS AFTER TREATMENT
14
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Fig. 2. T h e f r e q u e n c y o f p i n k - m u t a n t e v e n t s a f t e r i m m e r s i o n o f t h e i n f l o r e s c e n c e s o f T r a d e s c a n t i a c u t t i n g s in s o l u t i o n s o f 1 0 0 - - 2 0 0 m M d i m e t h y l n i t r o s o a m i n e f o r 24 h a t 2 5 ° C .
treatment. However, at the time when the mutation frequency peaks, the flowers are suitable for scoring. The results given in Figs. 1 and 2 support previous reports (Nauman et at., 1976; Sparrow et al., 1974) that clone 4430 is more sensitive to chemical mutagens than is clone 02. The difference in sensitivity towards DMN for the 2 clones was even greater than that reported for DBE and EMS (Sparrow et al., 1974). Because clones 4430 and 02 are almost equally sensitive to X-rays (Nauman et al., 1976} it might be postulated that the differences in sensitivity to chemical mutagens are due to a difference in the uptake of mutagens by the 2 clones. However, this is n o t true because the uptake of tritium-labeled DBE proved to be the same in flower tissues of both clones (Nauman et al., 1979). Possibly, the difference in mutation rates between the 2 clones may be linked with a larger amount of heterochromatin and a greater number of SAT chromosomes found in clone 4430 (Sparrow et al., 1974). The differences in the activity of repair enzymes in the two clones should also be considered. These results demonstrate that some plant species, in contrast with microorganisms, may have enzymes or enzyme-like systems capable of converting the promutagen, DMN, into a mutagenic compound. This is true for Arabidopsis, Tradescantia and also for soybeans (Arenaz and Vig, 1978). An activation system may be lacking in barley, because DMN induced a negligible amount of chlorophyll mutations (Ehrenberg, private communication; Gichner and Veleminsk:~, unpublished results).
384
References A r e n a z , P., a n d B.K. Vig ( 1 9 7 8 ) S o m a t i c c r o s s i n g - o v e r in G l y c i n e m a x (L.) merill: A c t i v a t i o n o f d i m e t h y l r t i t r o s o a m i n e b y p l a n t s e e d s a n d c o m p a r i s o n w i t h m e t h y l n i t r o s o u r e a in i n d u c i n g s o m a t i c m o s a i c i s m , M u t a t i o n Res., 5 2 , 3 6 7 - - 3 8 0 . G a b r i d g e , M.G., a n d M.S. L e g a t o r ( 1 9 6 9 ) A h o s t - m e d i a t e d m i c r o b i a l a s s a y f o r t h e d e t e c t i o n o f m u t a g e n i c c o m p o u n d s , P r o c . S o c . E x p . Biol. M e d . , 1 3 0 . 8 3 1 - - 8 3 4 . Geissler, E. ( 1 9 6 2 ) ~ b e r die Wi~'kung y o n N i t r o s o a m i n e n a u f M i k o r o r g a n i s m e n , N a t u r w i s s e n s c h a f t e n , 4 9 , 380--381. Mailing, H . V . ( 1 9 6 6 ) M u t a g e n i c i t y o f t w o p o t e n t c a r c i n o g e n s , d i m e t h y l n i t r o s o a m i n e a n d d i e t h y l n i t r o s o a m i n e in N e u r o s p o r a crassa, M u t a t i o n Res., 3, 5 3 7 - - 5 4 0 . Mailing, H . V . ( 1 9 7 1 ) D i m e t h y l n i t r o s o a m i n e : F o r m a t i o n o f m u t a g e n i c c o m p o u n d s b y i n t e r a c t i o n w i t h m o u s e - l i v e r m i c r o s o m e s , M u t a t i o n Res., 13, 4 2 5 - - 4 2 9 . M a r q u a r d t , H . L . , R. S c h w a l e r a n d F . K . Z i m m e r m a n n , ( 1 9 6 3 ) N i c h t - M u t a g e n i t ~ t y o n N i t r o s o a z n i n e n bei N e u r o s p o r a crassa, N a t u r w i s s e n s c h a f t e n , 5 0 , 1 3 5 - - 1 3 6 . M a r q u a r d t , If., F . K . Z i m m e r m a n n a n d R. S c h w a i e r ( 1 9 6 4 ) Die W t r k u n g k r e b s a u s i S s e n d e r N i t r o s a m i n e u n d N i t r o s a m i d e a u f d a s Aden,in 6 - 4 5 R i i c k m u t a t i o n s s y s t e m y o n S a c c h a r o m y c e s cerevisiae, Z. Vererbungsl., 95, 82--96. M a y e r , V.M. ( 1 9 7 1 ) M u t a g e n i c i t y o f d i m e t h y l m t r o s a m i n e a n d d i e t h y l n i t r o s a m i n e f o r S a e c h a x o m y c e s in a n in v i t r o h y d r o x y l a t i n g s y s t e m , Mol. G e m G e n e t . , 1 1 2 , 2 8 9 - - 2 9 4 . N a t a r a j a n , A . T . , A . D . T a t e s , P.P.W. v a n B u u l , M. Meijers a n d N. d e V o g e l ( 1 9 7 6 ) C y t o g e n e t i c e f f e c t s o f m u t a g e n s ] c a r c i n o g e n s a f t e r a c t i v a t i o n in a n in v i t r o m i c r o s o m a l s y s t e m , I. I n d u c t i o n o f c h r o m o s o m e aber~rations a n d sister c h r o m a t i d e x c h a n g e b y d i e t h y l n i t r o s o a m i n e ( D E N ) a n d d i m e t h y l r t i t r o s o a m i n e ( D M N ) in C H O cells in t h e p r e s e n c e o f r a t - l i v e r m i c r o s o m e s , M u t a t i o n R e s . , 3 7 , 8 3 - - 9 0 . Nauman, C.H., A.H. Sparrow and L.A. Schairer (1976) Comparative effects of ionizing radiation and two g a s e o u s c h e m i c a l m u t a g e n s o n s o m a t i c m u t a t i o n i n d u c t i o n in o n e m u t a b l e a n d t w o n o n - m u t a b l e c l o n e s o f T r a d e s c a n t i a , M u t a t i o n Res., 3 8 , 5 3 - - 7 0 . N a u m a n , C.II., P.J. K l o t z a n d L . A . S c h a i r e r ( 1 9 7 9 ) U p t a k e o f t r i t i a t e d 1 , 2 - d i b r o m o m e t h a n e b y T r a d e s c a n t i a floral tissues: r e l a t i o n t o i n d u c e d m u t a t i o n f r e q u e n c y in s t a m e n h a i r cells, E n v i r o n . E x p . Botany, 19, 201--215. P a s t e r n a k , L. ( 1 9 6 4 ) U n t e r ~ u c h u g e n ~iber die m u t a g e n e W i r k u n g v e r s c h i e d e n e r N i t r o s a m i n - u n d N i t r o s amid-Verbindungen, Arzneim.-Forsch., 14, 802--804. S p a r r o w , A . H . , L . A . S c h a i r e r a n d R. V i l l a i o b o s - P i e t r l n i ( 1 9 7 4 ) C o m p a r i s o n o f s o m a t i c m u t a t i o n r a t e s i n d u c e d in T r a d e s c a n t i a b y c h e m i c a l a n d p h y s i c a l m u t a g e n s , M u t a t i o n Res., 2 6 , 2 6 5 - - 2 7 6 . U n d e r b r i n k , A . G . , L . A . Schai~er mad A . H . S p a r r o w ( 1 9 7 3 ) T r a d e s c a n t i a s t a m e n h a i r s : A r a d i o b i o l o g i c a l t e s t s y s t e m a p p l i c a b l e to c h e m i c a l m u t a g e n e s i s , in: A. H o l l a e n d e r ( E d . ) , C h e m i c a l M u t a g e n s , Principles a n d M e t h o d s f o r t h e i r D e t e c t i o n , Vol. 3, P l e n u m , N e w Y o r k , p p . 1 7 1 - - 2 0 7 . V e l e m i n s k g , J., a n d T. G i c h n e r ( 1 9 6 8 ) T h e m u t a g e n i c a c t i v i t y o f n i t r o s a m i n e s in A r a b i d o p s i s thaliana. Mutation Res,, 5,429--431. V e l e m i n s k g , J., a n d T. G i c h n e r ( 1 9 7 1 ) T w o t y p e s o f d o s e - - r e s p o n s e c u r v e s in A r a b i d o p s i s thaliana a f t e r t h e a c t i o n o f n i t r o s a m i n e s , M u t a t i o n Res., 1 2 , 6 5 - - 7 0 .
Mutation Research, 78 (1980) 381--384
© Elsevier/North-Holland Biomedical Press
Short Communication INDUCTION OF SOMATIC MUTATIONS BY THE P R O M U T A G E N DIMETHYLNITROSOAMINE IN HAIRS OF T R A D E S C A N T I A STAMEN
T. GICHNER, J. VELEMfNSK~"and A.G. UNDERBRINK a Institute of Experimental Botany, Flemingovo 2, Praha 2 (Czechoslovakia). and a Institute of Nuclear Physics, Krak6w (Poland)
(Received 19 November 1979) (Revision received 29 February 1980) (Accepted 14 March 1980)
Dimethylnitrosoamine (DMN) is widely acknowledged to be among the most potent, environmentally significant, carcinogens. DMN is a so-called promutagen, i.e., it is not mutagenic in itself, but it requires metabolic activation for conversion into a mutagen. In the absence of metabolic activation, DMN is nonmutagenic in bacteria, yeasts, molds and cultures of animal tissues (Geissler, 1962; Marquardt et al., 1963, 1964; Natarajan et al., 1976). However, the mutagenicity of DMN in these cells has been achieved by: (1) a host-mediated assay, {2) by adding hepatic microsomes, or (3) by adding an Udenfriend enzyme-like mixture to the DMN treatment solution (Gabridge and Legator, 1969; Malling, 1966, 1971; Marquardt et al., 1964; Mayer, 1971; Natarajan et al., 1976). In contrast with a lack of mutagenicity in microorganisms and animal tissue cells, DMN can induce mutations in Drosophila (Pasternak, 1964). The first plant species in which DMN proved to be mutagenic was Arabidopsis thaliana (Veleminsk:~ and Gichner, 1968, 1971). Seeds of Arabidopsis were postulated to contain enzymes or an enzyme-like system capable of converting DMN into a mutagenic intermediate and that the decline in the frequency of mutations and sterility following large DMN doses might be caused by the inhibition of the activation system. This communication concerns the mutagenic activity of DMN in 2 clones of Tradescantia that differ in their sensitivity to chemical mutagens. Clone 4430 (a hybrid of T. subacaulis × T. hirsutiflora) is 6--9 times more sensitive to 1,2dibromoethane {DBE) and ethyl methanesulfonate (EMS} (Nauman et al., 1976; Sparrow et al., 1974) than is clone 02 (a putative hybrid of T. occio dentalis × T. ohiensis).
DMN, dissolved in distilled water, was applied in two ways. (1) The stems of cuttings were immersed in the solution for 2 4 h at 25°C. (2) The entire inflorescenses were immersed in the solution for 24 h at 25 ° C. After treatment, the cuttings were washed for 1 h and placed in beakers with Hoagland's nutrient solution. The cultivation took place in a controlled-environment chamber with
382 a 16-h day and a p h o t o n fluence rate of 400--700 nm, 300 pmoles m -~s-' and 25/18°C day/night temperatures. 30 cuttings were used for each treatment; and each day about 10 flowers (i.e. about 3000 stamen hairs) were scored depending on how many flowers were available. The pink somatic mutations in the stamen hairs were scored according to methods described by Underbrink et al. (1973). One or more contiguous pink cells were considered to have resulted from one m u t a n t event. The frequency of spontaneous m u t a n t events in clone 4430 was 0.185% {131 200 hairs were analyzed) and in clone 02 it was 0.118% (67 700 hairs were analyzed). The peak of pink somatic mutations in clone 4430 after immersion of the stems of cuttings in solutions of 150 and 200 mM DMN for 24 h occurred 9 days after the onset of t r e a t m e n t (Fig. 1). Treatment with 250 and 300 mM DMN strongly inhibited flowering. In clone 02, treatment with 200 mM DMN resulted in a very low frequency of pink-mutant events which was only slightly higher than the spontaneous level. The application of 250 mM DMN partly inhibited flowering, and 300 mM DMN caused complete inhibition. The application of 150 mM DMN directly to the inflorescences of both clones caused the flowers to be badly damaged for the first 4--7 days after treatment. The stamen hairs were bleached and deformed, and scoring was difficult. The maximal frequency of pink events in clone 4430 was reached 7--8 days after the onset of treatment (Fig. 2). The frequency of pink mutations in clone 02 was again considerably lower, even after treatment with 200 mM DMN which, in clone 4430, caused complete inhibition of flowering. A comparison of the 2 methods used to apply the DMN solutions shows that a direct treatment to the inflorescences (Fig. 2) yielded a considerably higher frequency of pink mutations at equal mutagenic concentrations than treatment to the stems of cuttings (Fig. 1). The disadvantage in directly treating the inflorescences is that the flowers are badly damaged for the first few days after
.'I
=:--="
ll"" ~
.
~ ~
.........
~
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~-M--~--*-*-~--~--~,
6 8 ~ AFTER TRE~TI~4T
~ K . ~. ~ ~ e ~ solutio~ of 15~200
.....
~
o.-~-
~
....
•
~
o~ p ~ k - ~ t e~e~s ~ t ~ " ~mm~z~o~ o[ ~ mM dimethy~itrosoamine f o r 2 4 h a t 2 5 ° C.
s~s
~
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383
~
clone 4430
~-o-~ clone 02
< -r
1SOmM
..z. ~o Z
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-~ •
6 8 10 12 DAYS AFTER TREATMENT
14
~6
Fig. 2. T h e f r e q u e n c y o f p i n k - m u t a n t e v e n t s a f t e r i m m e r s i o n o f t h e i n f l o r e s c e n c e s o f T r a d e s c a n t i a c u t t i n g s in s o l u t i o n s o f 1 0 0 - - 2 0 0 m M d i m e t h y l n i t r o s o a m i n e f o r 24 h a t 2 5 ° C .
treatment. However, at the time when the mutation frequency peaks, the flowers are suitable for scoring. The results given in Figs. 1 and 2 support previous reports (Nauman et at., 1976; Sparrow et al., 1974) that clone 4430 is more sensitive to chemical mutagens than is clone 02. The difference in sensitivity towards DMN for the 2 clones was even greater than that reported for DBE and EMS (Sparrow et al., 1974). Because clones 4430 and 02 are almost equally sensitive to X-rays (Nauman et al., 1976} it might be postulated that the differences in sensitivity to chemical mutagens are due to a difference in the uptake of mutagens by the 2 clones. However, this is n o t true because the uptake of tritium-labeled DBE proved to be the same in flower tissues of both clones (Nauman et al., 1979). Possibly, the difference in mutation rates between the 2 clones may be linked with a larger amount of heterochromatin and a greater number of SAT chromosomes found in clone 4430 (Sparrow et al., 1974). The differences in the activity of repair enzymes in the two clones should also be considered. These results demonstrate that some plant species, in contrast with microorganisms, may have enzymes or enzyme-like systems capable of converting the promutagen, DMN, into a mutagenic compound. This is true for Arabidopsis, Tradescantia and also for soybeans (Arenaz and Vig, 1978). An activation system may be lacking in barley, because DMN induced a negligible amount of chlorophyll mutations (Ehrenberg, private communication; Gichner and Veleminsk:~, unpublished results).
384
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