Effects of ethidium bromide and nalidixic acid pretreatment on the induction of chromatid aberrations by TEM and maleic hydrazide in Vicia faba main root meristems

Mutation Research, 208 (1988) 83-86

83

Elsevier MTRL 0108

Effects of ethidium bromide and nalidixic acid pretreatment on the induction of chromatid aberrations by TEM and maleic hydrazide in Viciafaba main root meristems A. Michaelis, R. Rieger and H. Nicoloff Zentralinstitut fiir Genetic und Kulturpflanzenforschung der AdW der D.D.R., 4325 Gatersleben (G.D.R.)

(Accepted 27 January 1988)

Keywords." Clastogenic adaptation: Chromatid aberrations; Ethidium bromide; Maleic hydrazide; Nalidixic acid; TEM; Viciafaba.

Summary Pretreatment of Vicia f a b a main root meristems with ethidium bromide (EB) or nalidixic acid (NA) significantly reduced the yield of metaphases with chromatid aberrations induced by maleic hydrazide (MH), i.e., triggered clastogenic adaptation to M H . No such protection occurred when the alkylating agent triethylenemelamine (TEM) was used for challenge treatment. The differential response of pretreated cells to M H on the one hand (protection) and to T E M (no protection) on the other supports the conclusion that clastogenic adaptation is due to different inducible (repair?) functions, which eventually exert protection against clastogenic impacts.

Ethidium bromide (EB; 2,7-diamino- 10-ethyl-9phenyl-phenantridinium bromide) has been widely used as a probe of D N A structure inside chromatin. It intercalates between adjacent base pairs of the D N A double helix forming a molecular complex and inhibits D N A as well as RNA synthesis (for review see Newton, 1963). Its binding brings about unfolding of D N A inside chromatin or nucleosome core particles (Erard et al., 1979; Aubin et al., 1983) and resulted in significantly increased involvement of barley nucleolus organizer regions (NORs) in chromatid translocations Correspondence: Prof. Dr. R. Rieger, Zentralinstitut ffir Genetik und Kulturpflanzenforschung Gatersleben der AdW der D.D.R., 4325 Gatersleben (G.D.R.).

(Nicoloffet al., 1987). Nalidixic acid (NA; 1-ethyl-7methyl- 1,8-naphthyridine-4-on-3-carboxylic acid) is an antibiotic that primarily inhibits D N A replication by interaction with the A-component of bacterial D N A gyrase and affects various events of the cell cycle (Sugino et al., 1977). In the present paper we want to report on the effects of EB and NA pretreatments on the yield of rnetaphases with chromatid aberrations induced by either triethylene-melamine (TEM) or maleic hydrazide (MH; 1,2-dihydropyridazine-3,6-dione) in Vicia f a b a main root meristems. Various other pretreatments (low clastogen doses, heat shocks and at least some elicitors of heat shock proteins) resulted in a significant reduction of the yield of metaphases with chromatid aberrations (clastogenic adapta-

0165-7992/88/$ 03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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tion) in Vicia faba and the main conclusion from these results (for review see Heindorff et al., 1987a) was that the pretreatments are sensed as a cellular stress, which eventually induces protective (repair?) functions counteracting chromosome damage brought about by the challenge treatment. EB and NA were used to broaden the range of tested pretreatment procedures and to further substantiate this inference. Both EB and NA provoked clastogenic adaptation to MH but not to TEM.

treatment with only M H (0.5 h, curve 1) and after pretreatments with 6 x 10-5 M EB or 2.5 x 10-5 M EB (1 h) and, 2 h later, with 5 x 1 0 - 4 M MH (0.5 h, curves 2 and 3). It is evident that the pretreatments resulted in a significant reduction of the efficacy of M H challenge treatment. EB treatment alone (curve 4) did not increase the spontaneous yield of chromatid aberrations observed in

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Material and methods 50

Primary root tip meristems of 2-3 cm long seedlings of beans of the reconstructed Vicia faba karyotype ACB (cf. Michaelis and Rieger, 1971; D6bel et al., 1978) were treated at 24°C with maleic hydrazide (MH), triethylenemelamine (TEM), ethidium bromide (EB), and nalidixic acid (NA). For experimental details see legends to Figs. 1-3. Recovery times (RT) in running tap water (24°C) after challenge treatment were 12, 15, 18, 21 and 24 h. The roots were immersed in 0.05% colchicine solution for 2 h. 1 cm long cuts of the root tips were fixed in a mixture of absolute ethanol and glacial acetic acid (3:1); permanent slides were made according to the Feulgen method. At least 100 metaphases (50 per slide) of the first cell cycle after treatment were inspected for the presence of chromatid aberrations at each recovery time. Isochromatid breaks, chromatid breaks, intercalary deletions, duplication deletions, and chromatid translocations were scored. Each experiment was done at least twice and the data for each recovery time were pooled. Means of the frequencies of metaphases with chromatid aberrations (MwA) and the 95% confidence intervals were calculated for 4 root tips (50 metaphases each) for each recovery time. Results

Fig. 1 shows the yield of metaphases with chromatid aberrations obtained after challenge

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MwA %

40

30

20

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Fig. 1. R e d u c t i o n b y p r e t r e a t m e n t with d i f f e r e n t c o n c e n t r a t i o n s o f e t h i d i u m b r o m i d e (EB) o f the f r e q u e n c y o f m e t a p h a s e s with M H - i n d u c e d c h r o m a t i d a b e r r a t i o n s ( M w A % ) . RT - r e c o v e r y t i m e . C u r v e 1 : 0 . 5 h , 5 × 1 0 - 4 M M H ; c u r v e 2 : I h, 6 × 10 -s M EB-2h R T - 0 . 5 h 5 × 10 4 M M H ; c u r v e 3 : 1 h, 2.5 × 10 -5 M EB - 2 h R T - 0.5 h 5 x 10 `4 M M H ; c u r v e 4: [ h, 6 x 10-SMEB.

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main root meristem metaphases. Different results were obtained when, instead of MH, the alkylating agent TEM was used for challenging. Comparison of curves 1 and 2 shows (Fig. 2) that the yield of metaphases with TEM-induced chromatid aberrations (curve 1: challenge treatment with TEM alone; curve 2: pretreatment for 1 h with EB and, 2 h later, TEM challenging) remained uninfluenced

by EB pretreatment, as opposed to the results obtained when MH was used for induction of chromatid aberrations. A similar differential response o f cells to the 2 clastogens was obtained when N A (2 x 10 - 4 M , 1 h), instead of EB, was used for pretreatment before challenging with either MH or TEM (Fig. 3). Clastogenic adaptation after NA pretreatment

T

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Fig. 3. E f f e c t s o f p r e t r e a t m e n t with nalidixic a c i d ( N A ) o n the f r e q u e n c y o f m e t a p h a s e s with c h r o m a t i d a b e r r a t i o n s ( M w A % ) Fig. 2. N o r e d u c t i o n b y e t h i d i u m b r o m i d e (EB) p r e t r e a t m e n t o f the f r e q u e n c y o f m e t a p h a s e s with T E M - i n d u c e d c h r o m a t i d a b e r r a t i o n s ( M w A O7o). R T = r e c o v e r y time. C u r v e 1 : 0 . 5 h I0

4 M T E M ; c u r v e 2 : 1 h 6 x 10 -~ M E B - 2 M T E M ; c u r v e 3 : 1 h 6 x 10 -5 M EB.

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i n d u c e d b y M H a n d T E M , respectively. RT = r e c o v e r y time, C u r v e 1 : 0 . 5 h 5 x 10 - 4 M M H ; c u r v e 2 : 0 . 5 h 10 4 M T E M ; curve3:1 h2 x 10-4MNA-2hRT-0.5h5 x 10 4 M M H ; c u r v e 4 : 1 h 2 × 10 - 4 M N A - 2 h R T - 0.5 h 10 4 M T E M ; c u r v e 5 : 1 h 2 x 10 - 4 M N A .

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was observed in the case of M H challenge treatment (compare curves 1 and 3), no reduction (by NA pretreatment) of the yield of metaphases with induced chromatid aberrations occurred in the case of TEM challenging (compare curves 2 and 4).

Acknowledgement We are grateful to Dr. I. Schubert for a critical reading of the manuscript and helpful suggestions.

References Discussion No clastogenic cross-adaptation was observed when alkylating agents were used for conditioning pretreatments of V. faba main root meristems and MH for challenge treatment, or vice versa (Rieger et al., 1985). This result led to the conclusion that the conditioning pretreatment with M H on the one hand and alkylating agents on the other triggers different anticlastogenic (repair?) functions, which cannot substitute for each other and are possibly lesion-specific (Heindorff et al., 1987b). The differential response of cells to MH and TEM, respectively, on pretreatments with EB and NA provides additional evidence for agent-specific reactions of plant cells which eventually result in protective effects on clastogen impact: clastogenic adaptation occurred over the whole range of recovery times tested (and excludes the possibility that the phenomenon is due to influences on cell cycling) when both EB and NA were applied before M H challenging as is evident from the decreased yield of metaphases containing MH-induced chromatid aberrations. TEM challenge treatment remained uninfluenced by pretreatments with either EB or NA. This once more supports the idea that different routes may give rise to clastogenic adaptation and these are, in part at least, specific for the types of stress to which the cells try to respond (MH- and TEM-specific damage in the present case) in order to reduce the consequences of genotoxic impacts. We are well aware that the present data only describe the phenomenon of clastogenic adaptation and some of its parameters (see Heindorff et al., 1987b). They provide a basis for biochemical studies, which are expected to yield more detailed insights into the underlying molecular mechanisms belonging to the cell's repertoire for defence against stress conditions and the potential damage triggered by these.

Aubin, R., G. Frechette, G. de Murcia, F. Malouin, A. Lord, F. Mandel and G.G. Poirier (1983) Nucleosomal poly-(ADPribose) polymerase: properties and relaxation of the chromatin structure, in: Miwa et al. (Eds.), ADPRibosylation, DNA-Repair and Cancer, Jap. Sci. Soc. Press, Tokyo/VNU Science Press, Utrecht, pp. 83-91. D6bel, P., 1. Schubert and R. Rieger (1978) Distribution of heterochromatin in a reconstructed karyotype of Vicia faba as identified by banding- and DNA-late replication patterns, Chromosoma, 69, 193-209. Erard, M., G.C. Das, G. de Murcia, A. Mazen, I. Pouyet, M. Champagne and E. Daune (1979) Ethidium bromide binding to core particle: comparison with native chromatin, Nucleic Acids Res., 6, 3231-3253. Heindorff, K., R. Rieger, I. Schubert, A. Michaelis and O. Aurich (1987a) Clastogenic adaptation of plant ceils - reduction of the yield of clastogen-induced chromatid aberrations by various pretreatment procedures, Mutation Res., 181, 157-171. Heindorff, K., 1. Schubert, R. Rieger and A. Michaelis (1987b) Clastogenic adaptation of Vicia faba root tip meristem cells after consecutive treatments with S-phase dependent and Sphase independent agents, Biol. Zbl., 106, 439-448. Michaelis, A., and R. Rieger (1971) New karyotypes of Vicia faba, Chromosoma, 35, 1-8. Newton, B.A. (1963) Cations and anions: inhibitions and interactions in metabolism and in enzyme activity, in: R.M. Hochster and J.M. Quastel (Eds.), Metabolic lnhibitors, Vol. 2, Academic Press, New York, p. 285. Nicoloff, H., R. Rieger and G. Russev (1987) Mutations in rDNA. 5. Is translocation involvement of barley nucleolus organizer regions influenced by chromatin architecture?, Mutation Res., 181, 147-155. Rieger, R., A. Michaelis and S. Takehisa (1985) 'Clastogenic cross-adaptation' is dependent on the clastogens used for induction of chromatid aberrations in Vicia faba root tip meristems, Mutation Res., 144, 171-175. Sugino, A., C.L. Peebles, K.N. Kreuzer and N.R. Cozzarelli (1977) Mechanism of action of nalidixic acid: purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme, Proc. Natl. Acad. Sci. (U.S.A.), 74, 4767-4771. Communicated by J. Sch6neich