Suppressive activity by fluoride on the induction of dominant lethals with trenimon in spermatozoa of Phryne cincta (Nematocera, Diptera)

287 Mutation Research, 44 (1977) 287--290 © Elsevier/North-Holland Biomedical Press

Short communication SUPPRESSIVE ACTIVITY BY F L U O R I D E ON THE INDUCTION OF DOMINANT LETHALS WITH TRENIMON IN S P E R M A T O Z O A OF PHR YNE CINCTA (NEMATOCERA, DIPTERA)

NORBERT ISRAELEWSKI and GUNTER OBE Institut fi~r Genetik, Freie Universitdt Berlin, Arnimallee 5--7, D-I O00 Berlin 33 (BRD)

(Received January 26th, 1977) (Accepted March 10th, 1977)

Sodium fluoride (NaF) has a suppressive effect on the yield of chromosomal aberrations induced by chemicals in Drosophila melanogaster [5,11] and human l y m p h o c y t e chromosomes in vitro [8,9]. With human lymphocytes in vitro, Slacik-Erben and Obe [9] have shown that, in the presence of trenimon, NaF has an enhancing effect on the DNA synthesis, b u t n o t on the yield of mitosis, indicating that a rate-limiting process that leads to suppression and later occurrence of mitoses under the influence of trenimon as compared with untreated cells is n o t influenced by NaF. In this communication we show that the yield of dominant lethals induced by trenimon in spermatozoa of Phryne cincta (Nematocera, Diptera) is suppressed by NaF, again indicating a suppressive influence of this agent on chemically induced chromosomal alterations. Phryne cincta was reared in our laboratory as described by Israelewski [6] and Wolf [12]. Males 3--5 days old were fed with trenimon, NaF and with a combination of both substances. The chemicals were dissolved in 5% sucrose solution. The feeding experiments were performed at 15°C in glass tube. To ensure that the animals t o o k up the substances, they were deprived of drinking possibilities for 48 h before they were given small filter papers saturated with solutions of the test substances. Under our experimental conditions the animals immediately began to suck on the filter papers. The concentrations tested were, for trenimon 10 -~, 5 • 10 -6, 10 -s, 5 • 10 -s, 10 -4, 5 • 10 -4, 10 -3 M, and for NaF, 10 -2 M in the experimental series, 5 • 10 -1, 10 -2, 10 -3 M in the control series. The animals were allowed to suck on the filter papers for 12 h in a moist chamber. Then each treated male was mated with an untreated 3--5-day-old female for 6 days at 15°C in a small (50 cc) glass t u b e whose b o t t o m was covered with agar (1.5% agar in tap water). The animals were transferred to fresh agar every 48 h. Each female deposits 3--10 times a b o u t 150 eggs encapsulated in a gelatinous substance in a period of 4--6 days after encountering the males. The experimental schedule used ensured that only treated spermatozoa were

288 tested [6]. Four days after deposition the eggs were freed from the gelatinous material encapsulating them into a 0.9% NaC1 solution and analyzed under a binocular microscope with low magnification (32 times) for dead eggs or embryos. Dead eggs or embryos are whitish and opaque, the dead embryos showing a clearly disorganized morphology. The living embryos are short before hatching after 4 days of embryogenesis and exhibit movements inside the egg capsule; hatched larvae cannot be seen 4 days after oviposition (Fig. 1). For each concentration of substance and for the controls 500 to 2000 eggs per female were analyzed, i.e. 3 to 14 males (Fig. 2). All experiments were carried out with two stocks of P h r y n e cincta that were different in their alpha-heterochromatin content in the X-chromosomes, the as stock having 3 blocks of alpha-heterochromatin in the X, the bs stock only 1 [12,13]. The results of our analyses can be seen in Fig. 2. Trenimon treatment of spermatozoa led to a clear d o s e ~ f f e c t relationship of embryonic dominant lethals, with the as stock being significantly more sensitive than the bs stock (P < 0.05, t test, except at 10 -6 and 10 -3 M, where the values were the same for as and bs). A similar difference in the sensitivity of as and bs was found for X-ray-induced d o m i n a n t lethals as well as chromosomal aberrations in the polytene salivary gland chromosomes of the F~ generation of X-irradiated males (ref. [6] and unpublished). The results with trenimon treatment alone show that P h r y n e cincta is more sensitive than D. melanogaster. Vogel [11], using 1 0 - S M trenimon, found

Fig. 1. Eggs o f the g n a t P h r y n e cincta f o u r d a y s a f t e r o v i p o s i t i o n a f t e r t r e a t m e n t o f s p e r m a t o z o a w i t h 10 -5 M t r e n i m o n . ( a ) D e a d eggs; ( h - - d ) d e a d e m b r y o s in early (b), m i d d l e (c) a n d late (d) s t a g e o f d e v e l o p r n e n t ; (e) l i v i n g e m b r y o s s h o r t l y b e f o r e h a t c h i n g . T h e l e n g t h o f o n e e g g is a b o u t 0.3 turn.

289

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Fig. 2. D o s e - e f f e c t r e l a t i o n s h i p s o f d o m i n a n t l e t h a l i t y a f t e r t r e a t m e n t o f males o f the g n a t Phryne cincta w i t h 10 -6 to 10 -3 M t r e n i m o n ( o p e n circles, as s t o c k ; c l o s e d circles, bs s t o c k ) a n d w i t h 10 -6 t o 10- 3 M txen i m o n in c o m b i n a t i o n w i t h 10- 2 M N a F ( o p e n s q u a r e s , a s s t o c k ; c l o s e d s q u a r e s , b s s t o c k ) . F o u r e x p e r i m e n t a l s e r i e s w e r e p e r f o r m e d . T h e r e s u l t s a f t e r c h e m i c a l t r e a t m e n t are c o r r e c t e d w i t h t h e r e s p e c t i v e c o n trol levels o f d o m i n a n t l e t h a l s ( a v e r a g e c o n t r o l levels i n f o u r e x p e r i m e n t s : a s s t o c k , 5.3%; b s s t o c k , 4 . 6 % ) . I n e a c h e x p e r i m e n t , m a l e s t r e a t e d w i t h 10- 5 M t r e n i m o n w e r e r u n as p o s i t i v e c o n t r o l s w i t h t h e r e s p e c t i v e d a t a s h o w i n g n o s i g n i f i c a n t d i f f e r e n c e s . A f t e r m a l e s h a d b e e n t r e a t e d w i t h 10- 2 M N a F , 6 . 1 % d o m i n a n t l e t h a l s in t h e as a n d 5.9% in t h e b s s t o c k w e r e f o u n d , t h e s e v a l u e s b e i n g n o t s i g n i f i c a n t l y d i f f e r e n t f r o m the c o n t r o l v a l u e s . T h e n u m b e r s w r i t t e n to t h e l e f t o f e a c h p o i n t in t h e c u r v e s i n d i c a t e t h e n u m b e r o f m a l e s t e s t e d . T h e v a l u e s are g i v e n w i t h t h e i r s t a n d a r d e r r o r s .

a b o u t 30% d o m i n a n t lethals in D. melanogaster, in Phryne the respective values were a b o u t 80% in as and 65% in bs. These differences c a n n o t result from different stages o f the spermatogenesis being treated with the mutagen, because in b o th , s p e r m a t o z o a were treated. With X-rays, too, Phryne is clearly more sensitive than D. melanogaster [2], and this again with respect to d o m i n a n t lethals as well as aberrations in the giant chromosomes [6]. The parallelisms o f the results with t r e n i m o n and d o m i n a n t lethals and with X-rays and dominant lethals as well as aberrations in the giant chrom osom es are strong indications th at the d o m i n a n t lethals in Phryne after t r e a t m e n t with t reni m on may result from ch r o m os om al aberrations and n o t from unspecific toxic effects. T h a t d o m i n a n t lethals may result mainly from chrom osom al aberrations has also been shown in mammals [1,4,7] and in Drosophila [3,10]. Feeding the animals with a com bi na t i on of t r eni m on and NaF led to a suppression o f the frequencies o f d o m i n a n t lethals obtained after t r e a t m e n t with trenimon alone. NaF t r e a t m e n t alone had no effect on the induction of d o m i n a n t lethals in a c o n c e n t r a t i o n range from 5 • 10 -1 to 10 -3 M. A ft er a combinational t r e a t m e n t with t r e n i m o n and NaF the d o s e ~ f f e c t curve of the d o m i n a n t lethals begins at a t r e n i m o n c o n c e n t r a t i o n of 10 -s M instead o f at 10 -6 M with t r e n i m o n alone. The curve after combinational t r e a t m e n t runs parallel to t hat after trenim o n alone reaching 100% at around 10 -3 M, i.e. at the same c o n c e n t r a t i o n as with t r e n i m o n alone. In the combinational series also as was significantly more sensitive than bs (P < 0.05, t test). The difference between the two strains seems to be the same as with t r eni m on alone. These results with NaF again indicate th at this substance has a suppressive activity on the induction of chro-

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mosomal aberrations by polyfunctional alkylating agents. Our results show further that the additional heterochromatin in the as strain had no effect on the anti-mutagenic activity of NaF in Phryne. The parallelism of the dose-effect curves of dominant lethals with trenimon as compared with that with trenimon and NaF in combination could simply result from a pure chemical reaction between NaF and trenimon, leading to an inactive product. From chemical analyses [11] and from experiments with human lymphocytes in vitro showing that post-treatment with NaF also leads to a suppressive effect [9] a pure chemical reaction between NaF and trenimon is not very probable. Trenimon-induced chromosomal damage in spermatozoa of D. melanogaster is not influenced by treatment of females with NaF [5], indicating that the repair capacities of the egg are not influenced by NaF. The NaF activity is possibly caused by a chemical reaction with the chromosome-breaking agent in conjunction with the organized structure of the chromosome. References 1 Basler, A., B. B u s e l m a l e r a n d G. R 6 h r b o r n , E l i m i n a t i o n of s p o n t a n e o u s a n d c h e m i c a l l y i n d u c e d c h r o m o s o m e a b e r r a t i o n s in m i c e d u r i n g e a r l y e m b r y o g e n e s i s , H u m a n G e n e t . , 33 ( 1 9 7 6 ) 1 2 1 - - 1 3 0 . 2 Bauer, H., R 6 n t g e n a u s l S s u n g y o n C h r o m o s o m e n m u t a t i o n e n bei Drosophila melanogaster. I. Bruchh~/ufigkeit, -verteilung u n d - r e k o m b i n a t i o n n a e h S p e i c h e l d r i i s e n u n t e r s u c h u n g , C h r o m o s o m a (BEE.), 1 (1939) 343--390. 3 Borstel, R.C. y o n a n d M.L. R e k e m e y e r , R a d i a t i o n - i n d u c e d a n d g e n e t i c a l l y c o n t r i v e d d o m i n a n t lethality in H a b r o b r a c o n a n d D r o s o p h i l a , G e n e t i c s , 44 ( 1 9 5 9 ) 1 0 5 3 - - 1 0 7 4 . 4 B r e w e n , J . G . , H.S. P a y n e , K.P. J o n e s and R.J. P r e s t o n , S t u d i e s on c h e m i c a l l y i n d u c e d d o m i n a n t l e t h a l i t y . I. T h e c y t o g e n e t i c basis of M M S - i n d u e e d d o m i n a n t l e t h a l i t y in p o s t - m e i o t i c m a l e g e r m cells, M u t a t i o n Res., 33 ( 1 9 7 5 ) 2 3 9 - - 2 5 0 . 5 B~chi, R. a n d K. Bfirki, T h e origin o f c h r o m o s o m e a b e r r a t i o n s in m a t u r e s p e r m of D r o s o p h i l a : I n f l u e n c e o f s o d i u m f l u o r i d e on t r e a t m e n t s w i t h t r e n i m o n a n d 1 - p h e n y l - 3 , 3 - d i m e t h y l t r i a z e n e , A r c h . G e n e t . , 48 ( 1 9 7 5 ) 5 9 - - 6 7 . 6 Israclewski, N., X-ray i n d u c e d visible a l t e r a t i o n s in the giant c h r o m o s o m e s of Phryne cincta ( N e m a t o cera, D i p t e r a ) . R e l a t i o n o f r a d i a t i o n sensitivity to p r o n u c l e a r c h r o m o s o m e s t r u c t u r e , C h r o m o s o m a (Berl.) 53 ( 1 9 7 5 ) 2 4 3 - - 2 6 3 . 7 M a t t e r , B.E. and I. J a e g e r , P r e m a t u r e c h r o m o s o m e c o n d e n s a t i o n , s t r u c t u r a l c h r o m o s o m e a b e r r a t i o n s , a n d m i c r o n u c l e i in early m o u s e e m b r y o s a f t e r t r e a t m e n t of p a t e r n a l p o s t m e i o t i c g e r m cells w i t h t r i e t h y l e n c m e l a m i n e : possible m e c h a n i s m s for c h e m i c a l l y i n d u c e d d o m i n a n t - l e t h a l m u t a t i o n s , Mutation Res., 33 ( 1 9 7 5 ) 2 5 1 - - 2 6 0 . 8 Obc, G. a n d R. S l a e i k - E r b e n , Suppressive a c t i v i t y by fluoride on the i n d u c t i o n of c h r o m o s o m e aberrations in h u m a n ceils w i t h a l k y l a t i n g a g e n t s in v i t r o , M u t a t i o n Res., 19 ( 1 9 7 3 ) 3 6 9 - - 3 7 1 . 9 S l a c i k - E r b c n , R. a n d G. Obe, T h e e f f e c t of s o d i u m f l u o r i d e on D N A s y n t h e s i s , m i t o t i c indices a n d c h r o m o s o m a l a b e r r a t i o n s in h u m a n l e u k o c y t e s t r e a t e d w i t h t r e n i m o n in v i t r o , M u t a t i o n Res., 37 (1976) 253--266. 10 T r a u t , H., E x p e r i m e n t s on the m e c h a n i s m of X-ray i n d u c e d c h r o m o s o m e loss, M u t a t i o n Res., 6 (1968) 109--115. 11 Vogel, E., S t r o n g a n t i m u t a g e n i c e f f e c t s of fluoride on m u t a t i o n i n d u c t i o n by t r e n i m o n and l - p h c n y l 3 , 3 - d i m e t h y l t r i a z e n e in Drosophila melanogaster. M u t a t i o n Res., 20 ( 1 9 7 3 ) 3 3 9 - - 3 5 2 . 12 Wolf, B.E., T e m p e r a t u r a b h ~ ' n g i g e A l l o z y k l i e des p o l y t ~ n e n X - C h r o m o s o m s in d e n K e r n e n d e r S o m a zellen y o n Phryne cincta, C h r o m o s o m a (Berl.), 8 ( 1 9 5 7 ) 3 9 6 - - 4 3 5 . 13 Wolf, B.E. a n d S. S o k o l o f f , M i g r a t i o n of ~ - h e t e r o c h r o m a t i n f r o m the giant X c h r o m o s o m e of the g n a t Phryne eincta, in: J. W a h r m a n a n d K . R . L e w i s (eds.), C h r o m o s o m e s T o d a y , Vol. 4, J o h n Wiley & Sons, N e w Y o r k a n d T o r o n t o a n d Israel U n i v e r s i t y Press, J e r u s a l e m , 1 9 7 3 , Pp. 1 2 9 - - 1 4 2 .