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Effects of formamide on the induction of chromosome structural changes by ethyleneimine in Vicia faba
249
Mutation Research, 41 (1976) 249--254 © Elsevier/North-Holland Biomedical Press
E F F E C T S OF F O R M A M I D E ON THE INDUCTION OF CHROMOSOME S T R U C T U R A L CHANGES BY ETHYLENEIMINE IN VICIA FABA
H. NICOLOFF Bulgarian Academy of Sciences, Institute of Genetics and Plant Breeding, Sofia 1113 (Bulgaria)
(Received February 18th, 1976) (Revision received July 12th, 1976) (Accepted August 2rid, 1976)
Summary Main roots of Vicia faba, k a r y o t y p e ACB, were treated with formamide (FA) alone, with ethyleneimine (EI) alone and simultaneously with formamide (FA). No increase in the spontaneous chromatid aberration rate in Vicia main r o o t meristems have been observed after treatment with FA alone. After treatment of primary roots with EI the maximum of aberrations (about 35.2%) was found after 21 h recovery time. After combined application of EI and FA the peak of aberration was found after 27 h i.e. a b o u t 6 h later, the frequency of aberrations approximately being doubled (50.3%) as compared with EI treatment alone.
Introduction Amides, members of the primary aliphatic amide series, at low concentrations cause large solubility increases of proteins without denaturation [6]. Nevo et al. [4] appealed to the fact that the amides modify the conformation of protein molecules (with denaturation as the extreme case) and serve in practice to oppose protein--protein interaction. In so far as the effects of formamide on chromosomes is concerned, according to the same authors, the action of formamide on the chromosomes at all stages of mitosis results in an unwinding of the condensed structure followed by a collapse of the threads into spherical masses, which give the impression of strongly refractile fluid drops [4]. Such phenomena, reported after application of amides, prompted us to make further studies with formamide concerning its effects in vivo on aberration induction b y chemical mutagens. In this paper we report some quantitative and qualitative modifications of aberration induction b y ethyleneimine in Vicia faba, connected with formamide application.
250
Material and methods Main roots (1--2 cm long) of Vicia faba var. Dornburger Ackerbohne karyotype ACB [3], were treated for 50 min at 24°C with formamide (FA) (5 - 10 -2 M) alone, with ethyleneimine (Ei) (7.7 • 10 -3 M) alone and simultaneously with 5 • 10 -2 M FA. The temperature during recovery in running tap-water was 24°C. Before fixation (3 parts ethanol, 1 part glacial acetic acid) the roots were immersed in 0.05% colchicine for 2 h. Fixation was made 15, 18, 21, 24, 27 and 30 h after treatment with the agents. Permanent Feulgen squashes were prepared by the dry-ice method. For intrachromosomal mapping of aberrations the metaphase chromosomes were subdivided into 28 segments as described previously [7,8]. Scoring of aberrations was done in the first metaphase after treatment with the agents. Results and conclusions No increase in the spontaneous chromatid aberration rate in Vicia main root meristems was observed after t r e a t m e n t with FA alone (5 • 10 -2 M, 50 min, 24°C). The percentage of metaphases with aberrations ranged during recovery times scored from 0.0 to 0.5 + 0.3. After treatment of primary roots with Ei, only chromatid aberrations of the " d e l a y e d " type [2] were observed; no aberrations occurred in metaphase for about 8 to 10 h after treatment of interphase. The m a x i m u m of aberrations (about 35.2%) was usually found after 21 h recovery time (Fig. 1). Combined application of Ei and FA indicated that simultaneous treatment with these agents exerted a significant delay on the appearance and an increase in frequency of aberrations induced. The peak of aberrations was found after 27 h, i.e. about 6 h later, the frequency of aberrations being approximately
cH2--c-u2",~ /
%
/ACB
NH
5O 40 .
5O 2O
10
0
t5
18
~t
24
~7
RECOVERY TiHE h Fig. 1. T h e f r e q u e n c y o f a b e r r a t i o n s i n d u c e d a f t e r t r e a t m e n t w i t h e t h y l e n e i m i n e .
251
C~'--~H2 + HC0.NH/ACB F.
NH
60
5O .
k,0 °
5O
2O 10
0 RECOVERY Tff'IE h Fig. 2. The f r e q u e n c y of a b e r r a t i o n s i n d u c e d a f t e r s i m u l t a n e o u s t r e a t m e n t w i t h e t h y l e n e i m i n e and f o r m amide.
22 7o EI/A.CB 20 18 16 14. 12 I0 8
o B"-538 I t'-206 D dd- 72 fl
eH2--CH2 \NFI/
d-82
0 ?
w LW
4
+
6 8 b ~0 12 t4
D 5"-609 t/- 178 I dd- 25
16
I
18 20 27
t/~I+FA~ACB
§ ct - 4~
c~--~Ht + HCO. NH z Nil
Fig. 3. The p e r c e n t a g e i n v o l v e m e n t in c h r o m a t i d a b e r r a t i o n s o f c h x o m o s o m e s e g m e n t s a f t e r t r e a t m e n t w i t h e t h y l e n e i m i n e alone (a), a n d a f t e r s i m u l t a n e o u s t z e a t m e n t w i t h e t h y l e n e i m i n e a n d f o r m a m i d e (b). B", isolocus b r e a k s ; d d , d e l e t i o n d u p l i c a t i o n s ; d, i n t e r c a l a r y deletions; t, lesions involved in c h r o m a t i d translocations.
252
doubled (50.3%) as compared with Ei treatment alone (Fig. 2). Intrachromosomal mapping of Ei-induced chromatid aberrations represented in Fig. 3a shows that intrachromosomal distribution of chromatid aberrations after Ei treatment was characterized by preferential involvement in aberrations of mainly two chromosome segments. Segment 19 of chromosome IV was the region preferentially involved in all types of El-induced aberrations and obviously represents a " h o t spot" of agent-specific aberration localization when alkylating agents are used [8,9]. Segment 23 (chromosome V) was preferentially involved in aberrations induced by Ei too, but represents a region of aberration clustering w i t h o u t agent specificity, having also been preferentially involved in aberrations induced by maleic hydrazide (MH), ethanol or hydroxylamine (HA) [5,7]. In segment 15 of chromosome I, segment 11 of chromosome II and segment 27 of chromosome VI the clustering of aberrations was comparatively less pronounced. A comparison of the intrachromosomal distribution patterns of chromatid aberrations after treatment with Ei alone and simultaneous treatment with Ei and FA reveals some differences. After the combined treatment with Ei and FA there was a preferential involvement in aberrations of some segments, e.g. segment 15 of chromosome I and segment 11 of chromosome II, which represents regions of highest aberration frequency (mainly breaks and translocations), whereas others, such as segment 23 (chromosome V), showed a decreased aberration frequency, a change in specificity which is difficult to explain (Fig. 3b). There is no d o u b t that a shift of segmental sensitivity and of inter- and intrachromosomal distribution patterns of Ei-induced chromosomal structural changes occurs as a result of application of FA. All segments preferentially involved in Ei-induced aberrations in k a r y t o t y p e ACB were regions known to be partly heterochromatic [1], but after treatment with Ei alone or simultaneously with Ei and FA the number of such segments was limited and did not exceed the number of major heterochromatic regions in Vicia faba. Segment 4, however, being also partly hererochromatic, showed preferential aberration clustering or high aberration frequency neither after Ei treatment alone nor after simultaneous treatment with Ei and FA. It is therefore tempting to suppose that the heterochromatic regions affected by application of FA exert a differential reaction in respect to sensitivity against Ei treatment. FA-dependent differential involvement in Ei-induced aberrations of the heterochromatic segments might be a reflection of differences and specificities in the molecular compositions or structure of heterochromatin as a functional state of chromosome regions of eukaryotic chromosomes. References 1 D S b e l , P., R. R i e g e r and A. Michaelis, T h e G i e m s a b a n d i n g p a t t e r n s o f the standard and f o u r r e c o n s t r u c t e d k a r y o t y p e s o f Vicia faba, C h r o m o s o m a (Berl.), 4 3 ( 1 9 7 3 ) 4 0 9 - - 4 2 2 . 2 K i h l m a n , B.A. A c t i o n o f C h e m i c a l s o n Dividing Ceils Prentice-Hall, E n g l e w o o d Cliffs N.J., ( 1 9 6 6 ) , p. 260. 3 Michaelis, A. a n d R. Rieger, O n t h e d i s t r i b u t i o n b e t w e e n c h r o m o s o m e s of c h e m i c a l l y i n d u c e d c h r o m arid aberrations: s t u d i e s w i t h a n e w k a r y o t y p e of Vicia faba, M u t a t i o n Res., 6 ( 1 9 6 8 ) 8 1 - - 9 2 .
253 4 Nevo, A.C., D. Mazia and P. Harris, Effects of amides on the structure of the m i t o t i c apparatus and on c h r o m o s o m e condensation, Exp. Cell. Res., 62 (1970) 173--183. 5 Nicoloff, H., R. Rieger and A. Michaelis, On the i n d u c t i o n of c h r o m o s o m e structural changes b y hyd r o x y l a m i n e in Vicia faba, M u t a t i o n Res., 13 (1971) 215---224. 6 Putman, F.W., in H. Neurath and K. Kai]ey (eds.), The Proteins, Vols. 1--8, Academic Press, New York, (1953) p. 807. 7 Rieger, R. and A. Michaells, I n t r a c h r o m o s o m a l clustering of c hroma t i d aberrations induced b y maleic hydrazide and e t h y l alkohol: studies with a new k a r y o t y p e of Vicia faba, Mut a t i on Res., 10 (1970) 162--164. 8 Rieger, R., A. Michaelis, I. Schubert, P. D~bel and H.-W. Jank, Non-random i n t r a c h r o m o s o m a l dist r i b u t i o n of chro matid aberrations induced by X-rays, alkylating agents and e t h a n o l in Vicia faba, M utation Res., 27 (1975) 69--79. 9 Rieger, R., H. Nicoloff and A. Michaelis, I n t r a c h r o m o s o m a l clustering of c hroma t i d aberrations induced b y N-methyl-N-nitroso-urethan in Vicia faba and Barley, Biol. Zentralblatt, 92,6 (1973) 681-689.
Mutation Research, 41 (1976) 249--254 © Elsevier/North-Holland Biomedical Press
E F F E C T S OF F O R M A M I D E ON THE INDUCTION OF CHROMOSOME S T R U C T U R A L CHANGES BY ETHYLENEIMINE IN VICIA FABA
H. NICOLOFF Bulgarian Academy of Sciences, Institute of Genetics and Plant Breeding, Sofia 1113 (Bulgaria)
(Received February 18th, 1976) (Revision received July 12th, 1976) (Accepted August 2rid, 1976)
Summary Main roots of Vicia faba, k a r y o t y p e ACB, were treated with formamide (FA) alone, with ethyleneimine (EI) alone and simultaneously with formamide (FA). No increase in the spontaneous chromatid aberration rate in Vicia main r o o t meristems have been observed after treatment with FA alone. After treatment of primary roots with EI the maximum of aberrations (about 35.2%) was found after 21 h recovery time. After combined application of EI and FA the peak of aberration was found after 27 h i.e. a b o u t 6 h later, the frequency of aberrations approximately being doubled (50.3%) as compared with EI treatment alone.
Introduction Amides, members of the primary aliphatic amide series, at low concentrations cause large solubility increases of proteins without denaturation [6]. Nevo et al. [4] appealed to the fact that the amides modify the conformation of protein molecules (with denaturation as the extreme case) and serve in practice to oppose protein--protein interaction. In so far as the effects of formamide on chromosomes is concerned, according to the same authors, the action of formamide on the chromosomes at all stages of mitosis results in an unwinding of the condensed structure followed by a collapse of the threads into spherical masses, which give the impression of strongly refractile fluid drops [4]. Such phenomena, reported after application of amides, prompted us to make further studies with formamide concerning its effects in vivo on aberration induction b y chemical mutagens. In this paper we report some quantitative and qualitative modifications of aberration induction b y ethyleneimine in Vicia faba, connected with formamide application.
250
Material and methods Main roots (1--2 cm long) of Vicia faba var. Dornburger Ackerbohne karyotype ACB [3], were treated for 50 min at 24°C with formamide (FA) (5 - 10 -2 M) alone, with ethyleneimine (Ei) (7.7 • 10 -3 M) alone and simultaneously with 5 • 10 -2 M FA. The temperature during recovery in running tap-water was 24°C. Before fixation (3 parts ethanol, 1 part glacial acetic acid) the roots were immersed in 0.05% colchicine for 2 h. Fixation was made 15, 18, 21, 24, 27 and 30 h after treatment with the agents. Permanent Feulgen squashes were prepared by the dry-ice method. For intrachromosomal mapping of aberrations the metaphase chromosomes were subdivided into 28 segments as described previously [7,8]. Scoring of aberrations was done in the first metaphase after treatment with the agents. Results and conclusions No increase in the spontaneous chromatid aberration rate in Vicia main root meristems was observed after t r e a t m e n t with FA alone (5 • 10 -2 M, 50 min, 24°C). The percentage of metaphases with aberrations ranged during recovery times scored from 0.0 to 0.5 + 0.3. After treatment of primary roots with Ei, only chromatid aberrations of the " d e l a y e d " type [2] were observed; no aberrations occurred in metaphase for about 8 to 10 h after treatment of interphase. The m a x i m u m of aberrations (about 35.2%) was usually found after 21 h recovery time (Fig. 1). Combined application of Ei and FA indicated that simultaneous treatment with these agents exerted a significant delay on the appearance and an increase in frequency of aberrations induced. The peak of aberrations was found after 27 h, i.e. about 6 h later, the frequency of aberrations being approximately
cH2--c-u2",~ /
%
/ACB
NH
5O 40 .
5O 2O
10
0
t5
18
~t
24
~7
RECOVERY TiHE h Fig. 1. T h e f r e q u e n c y o f a b e r r a t i o n s i n d u c e d a f t e r t r e a t m e n t w i t h e t h y l e n e i m i n e .
251
C~'--~H2 + HC0.NH/ACB F.
NH
60
5O .
k,0 °
5O
2O 10
0 RECOVERY Tff'IE h Fig. 2. The f r e q u e n c y of a b e r r a t i o n s i n d u c e d a f t e r s i m u l t a n e o u s t r e a t m e n t w i t h e t h y l e n e i m i n e and f o r m amide.
22 7o EI/A.CB 20 18 16 14. 12 I0 8
o B"-538 I t'-206 D dd- 72 fl
eH2--CH2 \NFI/
d-82
0 ?
w LW
4
+
6 8 b ~0 12 t4
D 5"-609 t/- 178 I dd- 25
16
I
18 20 27
t/~I+FA~ACB
§ ct - 4~
c~--~Ht + HCO. NH z Nil
Fig. 3. The p e r c e n t a g e i n v o l v e m e n t in c h r o m a t i d a b e r r a t i o n s o f c h x o m o s o m e s e g m e n t s a f t e r t r e a t m e n t w i t h e t h y l e n e i m i n e alone (a), a n d a f t e r s i m u l t a n e o u s t z e a t m e n t w i t h e t h y l e n e i m i n e a n d f o r m a m i d e (b). B", isolocus b r e a k s ; d d , d e l e t i o n d u p l i c a t i o n s ; d, i n t e r c a l a r y deletions; t, lesions involved in c h r o m a t i d translocations.
252
doubled (50.3%) as compared with Ei treatment alone (Fig. 2). Intrachromosomal mapping of Ei-induced chromatid aberrations represented in Fig. 3a shows that intrachromosomal distribution of chromatid aberrations after Ei treatment was characterized by preferential involvement in aberrations of mainly two chromosome segments. Segment 19 of chromosome IV was the region preferentially involved in all types of El-induced aberrations and obviously represents a " h o t spot" of agent-specific aberration localization when alkylating agents are used [8,9]. Segment 23 (chromosome V) was preferentially involved in aberrations induced by Ei too, but represents a region of aberration clustering w i t h o u t agent specificity, having also been preferentially involved in aberrations induced by maleic hydrazide (MH), ethanol or hydroxylamine (HA) [5,7]. In segment 15 of chromosome I, segment 11 of chromosome II and segment 27 of chromosome VI the clustering of aberrations was comparatively less pronounced. A comparison of the intrachromosomal distribution patterns of chromatid aberrations after treatment with Ei alone and simultaneous treatment with Ei and FA reveals some differences. After the combined treatment with Ei and FA there was a preferential involvement in aberrations of some segments, e.g. segment 15 of chromosome I and segment 11 of chromosome II, which represents regions of highest aberration frequency (mainly breaks and translocations), whereas others, such as segment 23 (chromosome V), showed a decreased aberration frequency, a change in specificity which is difficult to explain (Fig. 3b). There is no d o u b t that a shift of segmental sensitivity and of inter- and intrachromosomal distribution patterns of Ei-induced chromosomal structural changes occurs as a result of application of FA. All segments preferentially involved in Ei-induced aberrations in k a r y t o t y p e ACB were regions known to be partly heterochromatic [1], but after treatment with Ei alone or simultaneously with Ei and FA the number of such segments was limited and did not exceed the number of major heterochromatic regions in Vicia faba. Segment 4, however, being also partly hererochromatic, showed preferential aberration clustering or high aberration frequency neither after Ei treatment alone nor after simultaneous treatment with Ei and FA. It is therefore tempting to suppose that the heterochromatic regions affected by application of FA exert a differential reaction in respect to sensitivity against Ei treatment. FA-dependent differential involvement in Ei-induced aberrations of the heterochromatic segments might be a reflection of differences and specificities in the molecular compositions or structure of heterochromatin as a functional state of chromosome regions of eukaryotic chromosomes. References 1 D S b e l , P., R. R i e g e r and A. Michaelis, T h e G i e m s a b a n d i n g p a t t e r n s o f the standard and f o u r r e c o n s t r u c t e d k a r y o t y p e s o f Vicia faba, C h r o m o s o m a (Berl.), 4 3 ( 1 9 7 3 ) 4 0 9 - - 4 2 2 . 2 K i h l m a n , B.A. A c t i o n o f C h e m i c a l s o n Dividing Ceils Prentice-Hall, E n g l e w o o d Cliffs N.J., ( 1 9 6 6 ) , p. 260. 3 Michaelis, A. a n d R. Rieger, O n t h e d i s t r i b u t i o n b e t w e e n c h r o m o s o m e s of c h e m i c a l l y i n d u c e d c h r o m arid aberrations: s t u d i e s w i t h a n e w k a r y o t y p e of Vicia faba, M u t a t i o n Res., 6 ( 1 9 6 8 ) 8 1 - - 9 2 .
253 4 Nevo, A.C., D. Mazia and P. Harris, Effects of amides on the structure of the m i t o t i c apparatus and on c h r o m o s o m e condensation, Exp. Cell. Res., 62 (1970) 173--183. 5 Nicoloff, H., R. Rieger and A. Michaelis, On the i n d u c t i o n of c h r o m o s o m e structural changes b y hyd r o x y l a m i n e in Vicia faba, M u t a t i o n Res., 13 (1971) 215---224. 6 Putman, F.W., in H. Neurath and K. Kai]ey (eds.), The Proteins, Vols. 1--8, Academic Press, New York, (1953) p. 807. 7 Rieger, R. and A. Michaells, I n t r a c h r o m o s o m a l clustering of c hroma t i d aberrations induced b y maleic hydrazide and e t h y l alkohol: studies with a new k a r y o t y p e of Vicia faba, Mut a t i on Res., 10 (1970) 162--164. 8 Rieger, R., A. Michaelis, I. Schubert, P. D~bel and H.-W. Jank, Non-random i n t r a c h r o m o s o m a l dist r i b u t i o n of chro matid aberrations induced by X-rays, alkylating agents and e t h a n o l in Vicia faba, M utation Res., 27 (1975) 69--79. 9 Rieger, R., H. Nicoloff and A. Michaelis, I n t r a c h r o m o s o m a l clustering of c hroma t i d aberrations induced b y N-methyl-N-nitroso-urethan in Vicia faba and Barley, Biol. Zentralblatt, 92,6 (1973) 681-689.