Mutation Research, 282 (1992) 69-72 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
69
MUTLET 0660
Low temperature between conditioning and challenge treatment prevents the 'adaptive response' of Viciafaba root tip meristem cells R. Rieger a, A. Michaelis a and S. Takehisa
~'
a Institut fiir Pflanzengenetik und Kulturpflanzenforschung Gatersleben, 0-4325 Gatersleben (Germany) and b Department of Biology, Keio University, Yokohama (Japan) (Received 20 January 1992) (Accepted 27 January 1992)
Keywords: Adaptive response; Chromatid aberrations; Maleic hydrazide; Triethylenemelamine; l/icia faba
Summary When the temperature during intertreatment time (2 h) between conditioning and challenge treatment of l/icia faba root tip meristems with either triethylenemelamine or maleic hydrazide was reduced from 24°C to 12°C no adaptive response occurred any more. The yield of metaphases with chromatid aberrations under these circumstances was similar to that observed after challenge _treatment alone, i.e., no reduction occurred. This indicates that the metabolic state of the cells is of critical importance for the presence or absence of adaptive responses.
Bacteria (Samson and Cairns, 1977; Demple and Halbrook, 1983) and eukaryotic cells (Samson and Schwartz, 1980; Frosina and Abbondandolo, 1985; Rieger et al., 1982, 1990) confronted with stressors of various kinds may react by 'adaptive responses' which eventually enhance their resistance to consecutive damaging impacts. In some cases at least, the adaptive response is due to stress-induced repair processes acting on DNA lesions (Karran et al., 1979; Olsson and Lindahl, 1980; Wolff et al., 1989, 1990). When root tip meristem ceils (l/icia faba, Hordeum vulgare) are pretreated with low clastogen doses, heat shock, or heavy metal salts they
Correspondence: Prof. R. Rieger, Institut fiir Pflanzengenetik und Kulturpflanzenforschung, Correnstrasse 3, 0-4325 Gatersleben (Germany).
may become more resistant to a consecutive challenge treatment with a high clastogen dose (challenge treatment) as evident from a yield of chromatid aberrations lower than that induced by challenge treatment alone (for review see Rieger et al., 1990). The data at hand allow to conclude that conditioning treatments trigger protective functions in the pretreated cells and eventually reduce the effects of challenge treatment. The underlying mechanisms are obscure but unimpaired protein synthesis is a prerequisite for the occurrence of the adaptive response and protection against aberration induction by challenge treatment. In our previous experiments in which small clastogen doses were used for conditioning and higher ones for challenging, the temperature during the intertreatment period between the two clastogen treatments was 24°C, i.e., a tempera-
70
ture at which cellular metabolism is expected to proceed normally. In this paper we wish to report on the effect of reduced temperature (from 24°C down to 12°C) during the intertreatment period before challenging. Under these circumstances no adaptive response occurred; this indicates that induction of protective functions is dependent on unimpaired cellular metabolism (in conformity with the effects of cycloheximide on the adaptive response).
50MwA % 40
30
Material and methods
Primary roots (2-3 cm long) of broad bean seedlings of the reconstructed karyotype ACB (see Michaelis and Rieger, 1971; D6bel et al., 1973, 1978) were treated at 24°C with the clastogens triethylenemelamine (triethyleniminotriazine; TEM), or maleic hydrazide (1,2-dihydropyridazine-3,6-dione; MH). For details of treatment see the legends of the figures. The percentage of metaphases containing chromatid aberrations (chromatid breaks, isochromatid breaks, intercalary deletions, duplication deletions, and chromatid translocations) was established after the different treatment regimes. Intertreatment time (IT) between conditioning and challenging was in tap water at either 24 or 12°C. Recovery times in running tap water (24°C) after the last clastogen treatments were 12, 18, 24 and 36 h. The roots were immersed in 0.05% colchicine solution for 2 h, fixed in a mixture of absolute ethanol and glacial acetic acid (3:1), and Feulgen-stained. 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. Each experiment was done at least twice and the data obtained 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 roots (50 metaphases each) for each recovery time. Results and conclusions
Figs. 1 and 2 summarize the data obtained when Vicia faba root tip meristems were treated with triethylenemelamine (Fig. 1) or maleic hy-
20-
T ±
x!
10-
~-~-
T--
I
I
I
12
18
24
l -~ 36 hRT
Fig. 1. No reduction of the yield of metaphases with TEM-induced chromatid aberrations when the temperature during intertreatment (2 h) between TEM conditioning and TEM challenge treatment was 12°C (curve 4) instead of 24°C (curve 3). Curve 1:0.5 h, l0 -5 mole/l TEM; curve 2:0.5 h, 10 4 mole/1 TEM; curve 3:0.5 h, 10 -5 mole/l TEM ~ 2 h 24°C IT-}0.5 h, 10 -4 mole/l TEM; curve 4:0.5 h, 10 -5 mole/l TEM-~ 2 h 12°C IT ~ 0.5 h, 10 -4 mole/l TEM. MwA: % metaphases containing chromatid aberrations; hRT: hours recovery time.
drazide (Fig. 2). When the root tips were consecutively treated first with a small clastogen dose (conditioning) and, 2 h later, with a higher challenge dose, the yield of TEM- or MH-induced chromatid aberrations was significantly lower than after treatment with only the challenge doses of TEM or MH (curves 2 and 3 in Figs. 1 and 2), i.e., the conditioning treatments resulted in an adaptive response of the cells. Temperature between conditioning and challenge (intertreatment time) was 24°C in these cases. When IT temperature was reduced to 12°C no adaptive response was triggered (curves 4 in Figs. 1 and 2). Basically the same result was obtained when conditioning
71 MwA
the adaptive r e s p o n s e ( R i e g e r et al., 1984, 1986). No such effect of I T t e m p e r a t u r e r e d u c t i o n occ u r r e d when, i n s t e a d of low clastogen doses a n d heat shock, C u S O 4 or P b ( N O 3 ) 2 were used for c o n d i t i o n i n g t r e a t m e n t prior to T E M or M H c h a l l e n g i n g ( u n p u b l i s h e d results); this once m o r e indicates that various m e c h a n i s m s u n d e r l i e adaptive responses of p l a n t cells a n d that the m e t a b o l i c state of cells d u r i n g a n d after c o n d i t i o n i n g is of i m p o r t a n c e for the inducibility of adaptive responses (see also Olivieri a n d Bosi, 1990).
%
40-
30-
References
20-
± 103
3-_...._.__ 1 ~ - _ _ _ _ _ _ _ _ _ ~
~
I 12
f 36 hRT
I 18
I 24
Fig. 2. No reduction of the yield of metaphases with MH-induced chromatid aberrations when the temperature during intertreatment (2 h) between MH conditioning and MH challenge treatment was 12°C (curve 4) instead of 24°C (curve 3). Curve 1:0.5 h, 5 × 10 -5 mole/l MH; curve 2:0.5 h, 5 × 10 - 4 mole/1 MH; curve 3:0.5 h, 5 × 10-5 mole/1 MH-~ 2 h 24°C I T s 0 . 5 h, 5×10 -4 mole/I MH; curve 4:0.5 h, 5×10 -5 mole/l MH -, 2 h 12°C IT -~ 0.5 h, 5 × 10 - 4 mole/l MH.
a n d c h a l l e n g e t r e a t m e n t were d o n e with E N U or w h e n a heat shock (40°C for 10 m i n ) was used for c o n d i t i o n i n g t r e a t m e n t p r i o r to T E M or M H (data n o t shown). T h e s e o b s e r v a t i o n s allow the c o n c l u s i o n that u n i m p a i r e d cellular m e t a b o l i s m d u r i n g i n t e r t r e a t m e n t time is a p r e r e q u i s i t e for the o c c u r r e n c e of the adaptive r e s p o n s e in l/icia faba. T e m p e r a t u r e r e d u c t i o n d u r i n g I T from 24°C down to 12°C p r e v e n t s the r e d u c t i o n of the yield of c h a l l e n g e t r e a t m e n t - 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 characteristic of the adaptive response. This o b s e r v a t i o n agrees with e a r l i e r results which showed that i n h i b i t i o n of p r o t e i n synthesis by cycloheximide prior to c o n d i t i o n i n g t r e a t m e n t also p r e v e n t e d
Demple, B., and J. Halbrook (1983) Inducible repair of oxidative DNA damage in Escherichia coli, Nature (London), 304, 466-468. D6bel, P., R. Rieger and A. Michaelis (1973) The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba, Chromosoma, 43, 409-422. D6bel, P., I. 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. Frosina, G., and A. Abbondandolo (1985) The current evidence for an adaptive response to alkylating agents in mammalian cells with special reference to experiments with in vitro cultures, Mutation Res., 154, 85-100. Karran, P., T. Lindahl and B. Griffin (1979) Adaptive reponse to alkylating agents involves alteration in situ of O6omethylguanine residues in DNA, Nature (London), 280, 76-77. Olivieri, G., and A. Bosi (1990) Possible causes of variability of the adaptive response in human lymphocytes, in: G. Obe and A.T. Natarajan (Eds.), Chromosomal Aberrations, Springer, Berlin, pp. 130-139. Olsson, M., and T. Lindahl (1980) Repair of alkylated DNA in Escherichia coli: methyl group transfer from O6-methylguanine to a protein cysteine residue, J. Biol. Chem., 255, 10569-10571. Rieger, R., A. Michaelis and H. Nicoloff (1982) Inducible repair processes in plant root tip meristems? 'Below-additivity effects' of unequally fractionated clastogen concentrations, Biol. Zbl., 101, 125-138. Rieger, R., A. Michaelis and H. Nicoloff (1984) 'Clastogenic adaptation' of the I/icia faba root tip meristem as affected by various treatment parameters, Mutation Res., 140, 99102. Rieger, R., A. Michaelis and H. Nicoloff (1986) Effects of stress factors on the clastogen response of Vicia faba root tip meristems: 'Clastogenic adaptation', Biol. Zbl., 105, 19-28. Rieger, R., A. Michaelis and S. Takehisa (1990) On adaptive responses of plant meristem cells in vivo - protection against induction of chromatid aberrations, in: G. Obe and A.T. Natarajan (Eds.), Chromosomal Aberrations, Springer, Berlin, pp. 163-179.
72 Samson, L., and J. Cairns (1977) A new pathway for DNA repair in Escherichia coli, Nature (London), 267, 281-283. Samson, L., and J.L. Schwartz (1980) Evidence for an adaptive DNA repair pathway in CHO and human skin fibroblast cell lines, Nature (London), 287, 861-863. Wolff, S., G. Olivieri and V. Afzal (1990) Adaptation of human lymphocytes to radiation or chemical mutagens:
differences in cytogenetic repair, in: G. Obe and A.T. Natarajan (Eds.), Chromosomal Aberrations, Springer, Berlin, pp. 140-150.
Communicated by J. Velemlnsl~
69
MUTLET 0660
Low temperature between conditioning and challenge treatment prevents the 'adaptive response' of Viciafaba root tip meristem cells R. Rieger a, A. Michaelis a and S. Takehisa
~'
a Institut fiir Pflanzengenetik und Kulturpflanzenforschung Gatersleben, 0-4325 Gatersleben (Germany) and b Department of Biology, Keio University, Yokohama (Japan) (Received 20 January 1992) (Accepted 27 January 1992)
Keywords: Adaptive response; Chromatid aberrations; Maleic hydrazide; Triethylenemelamine; l/icia faba
Summary When the temperature during intertreatment time (2 h) between conditioning and challenge treatment of l/icia faba root tip meristems with either triethylenemelamine or maleic hydrazide was reduced from 24°C to 12°C no adaptive response occurred any more. The yield of metaphases with chromatid aberrations under these circumstances was similar to that observed after challenge _treatment alone, i.e., no reduction occurred. This indicates that the metabolic state of the cells is of critical importance for the presence or absence of adaptive responses.
Bacteria (Samson and Cairns, 1977; Demple and Halbrook, 1983) and eukaryotic cells (Samson and Schwartz, 1980; Frosina and Abbondandolo, 1985; Rieger et al., 1982, 1990) confronted with stressors of various kinds may react by 'adaptive responses' which eventually enhance their resistance to consecutive damaging impacts. In some cases at least, the adaptive response is due to stress-induced repair processes acting on DNA lesions (Karran et al., 1979; Olsson and Lindahl, 1980; Wolff et al., 1989, 1990). When root tip meristem ceils (l/icia faba, Hordeum vulgare) are pretreated with low clastogen doses, heat shock, or heavy metal salts they
Correspondence: Prof. R. Rieger, Institut fiir Pflanzengenetik und Kulturpflanzenforschung, Correnstrasse 3, 0-4325 Gatersleben (Germany).
may become more resistant to a consecutive challenge treatment with a high clastogen dose (challenge treatment) as evident from a yield of chromatid aberrations lower than that induced by challenge treatment alone (for review see Rieger et al., 1990). The data at hand allow to conclude that conditioning treatments trigger protective functions in the pretreated cells and eventually reduce the effects of challenge treatment. The underlying mechanisms are obscure but unimpaired protein synthesis is a prerequisite for the occurrence of the adaptive response and protection against aberration induction by challenge treatment. In our previous experiments in which small clastogen doses were used for conditioning and higher ones for challenging, the temperature during the intertreatment period between the two clastogen treatments was 24°C, i.e., a tempera-
70
ture at which cellular metabolism is expected to proceed normally. In this paper we wish to report on the effect of reduced temperature (from 24°C down to 12°C) during the intertreatment period before challenging. Under these circumstances no adaptive response occurred; this indicates that induction of protective functions is dependent on unimpaired cellular metabolism (in conformity with the effects of cycloheximide on the adaptive response).
50MwA % 40
30
Material and methods
Primary roots (2-3 cm long) of broad bean seedlings of the reconstructed karyotype ACB (see Michaelis and Rieger, 1971; D6bel et al., 1973, 1978) were treated at 24°C with the clastogens triethylenemelamine (triethyleniminotriazine; TEM), or maleic hydrazide (1,2-dihydropyridazine-3,6-dione; MH). For details of treatment see the legends of the figures. The percentage of metaphases containing chromatid aberrations (chromatid breaks, isochromatid breaks, intercalary deletions, duplication deletions, and chromatid translocations) was established after the different treatment regimes. Intertreatment time (IT) between conditioning and challenging was in tap water at either 24 or 12°C. Recovery times in running tap water (24°C) after the last clastogen treatments were 12, 18, 24 and 36 h. The roots were immersed in 0.05% colchicine solution for 2 h, fixed in a mixture of absolute ethanol and glacial acetic acid (3:1), and Feulgen-stained. 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. Each experiment was done at least twice and the data obtained 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 roots (50 metaphases each) for each recovery time. Results and conclusions
Figs. 1 and 2 summarize the data obtained when Vicia faba root tip meristems were treated with triethylenemelamine (Fig. 1) or maleic hy-
20-
T ±
x!
10-
~-~-
T--
I
I
I
12
18
24
l -~ 36 hRT
Fig. 1. No reduction of the yield of metaphases with TEM-induced chromatid aberrations when the temperature during intertreatment (2 h) between TEM conditioning and TEM challenge treatment was 12°C (curve 4) instead of 24°C (curve 3). Curve 1:0.5 h, l0 -5 mole/l TEM; curve 2:0.5 h, 10 4 mole/1 TEM; curve 3:0.5 h, 10 -5 mole/l TEM ~ 2 h 24°C IT-}0.5 h, 10 -4 mole/l TEM; curve 4:0.5 h, 10 -5 mole/l TEM-~ 2 h 12°C IT ~ 0.5 h, 10 -4 mole/l TEM. MwA: % metaphases containing chromatid aberrations; hRT: hours recovery time.
drazide (Fig. 2). When the root tips were consecutively treated first with a small clastogen dose (conditioning) and, 2 h later, with a higher challenge dose, the yield of TEM- or MH-induced chromatid aberrations was significantly lower than after treatment with only the challenge doses of TEM or MH (curves 2 and 3 in Figs. 1 and 2), i.e., the conditioning treatments resulted in an adaptive response of the cells. Temperature between conditioning and challenge (intertreatment time) was 24°C in these cases. When IT temperature was reduced to 12°C no adaptive response was triggered (curves 4 in Figs. 1 and 2). Basically the same result was obtained when conditioning
71 MwA
the adaptive r e s p o n s e ( R i e g e r et al., 1984, 1986). No such effect of I T t e m p e r a t u r e r e d u c t i o n occ u r r e d when, i n s t e a d of low clastogen doses a n d heat shock, C u S O 4 or P b ( N O 3 ) 2 were used for c o n d i t i o n i n g t r e a t m e n t prior to T E M or M H c h a l l e n g i n g ( u n p u b l i s h e d results); this once m o r e indicates that various m e c h a n i s m s u n d e r l i e adaptive responses of p l a n t cells a n d that the m e t a b o l i c state of cells d u r i n g a n d after c o n d i t i o n i n g is of i m p o r t a n c e for the inducibility of adaptive responses (see also Olivieri a n d Bosi, 1990).
%
40-
30-
References
20-
± 103
3-_...._.__ 1 ~ - _ _ _ _ _ _ _ _ _ ~
~
I 12
f 36 hRT
I 18
I 24
Fig. 2. No reduction of the yield of metaphases with MH-induced chromatid aberrations when the temperature during intertreatment (2 h) between MH conditioning and MH challenge treatment was 12°C (curve 4) instead of 24°C (curve 3). Curve 1:0.5 h, 5 × 10 -5 mole/l MH; curve 2:0.5 h, 5 × 10 - 4 mole/1 MH; curve 3:0.5 h, 5 × 10-5 mole/1 MH-~ 2 h 24°C I T s 0 . 5 h, 5×10 -4 mole/I MH; curve 4:0.5 h, 5×10 -5 mole/l MH -, 2 h 12°C IT -~ 0.5 h, 5 × 10 - 4 mole/l MH.
a n d c h a l l e n g e t r e a t m e n t were d o n e with E N U or w h e n a heat shock (40°C for 10 m i n ) was used for c o n d i t i o n i n g t r e a t m e n t p r i o r to T E M or M H (data n o t shown). T h e s e o b s e r v a t i o n s allow the c o n c l u s i o n that u n i m p a i r e d cellular m e t a b o l i s m d u r i n g i n t e r t r e a t m e n t time is a p r e r e q u i s i t e for the o c c u r r e n c e of the adaptive r e s p o n s e in l/icia faba. T e m p e r a t u r e r e d u c t i o n d u r i n g I T from 24°C down to 12°C p r e v e n t s the r e d u c t i o n of the yield of c h a l l e n g e t r e a t m e n t - 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 characteristic of the adaptive response. This o b s e r v a t i o n agrees with e a r l i e r results which showed that i n h i b i t i o n of p r o t e i n synthesis by cycloheximide prior to c o n d i t i o n i n g t r e a t m e n t also p r e v e n t e d
Demple, B., and J. Halbrook (1983) Inducible repair of oxidative DNA damage in Escherichia coli, Nature (London), 304, 466-468. D6bel, P., R. Rieger and A. Michaelis (1973) The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba, Chromosoma, 43, 409-422. D6bel, P., I. 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. Frosina, G., and A. Abbondandolo (1985) The current evidence for an adaptive response to alkylating agents in mammalian cells with special reference to experiments with in vitro cultures, Mutation Res., 154, 85-100. Karran, P., T. Lindahl and B. Griffin (1979) Adaptive reponse to alkylating agents involves alteration in situ of O6omethylguanine residues in DNA, Nature (London), 280, 76-77. Olivieri, G., and A. Bosi (1990) Possible causes of variability of the adaptive response in human lymphocytes, in: G. Obe and A.T. Natarajan (Eds.), Chromosomal Aberrations, Springer, Berlin, pp. 130-139. Olsson, M., and T. Lindahl (1980) Repair of alkylated DNA in Escherichia coli: methyl group transfer from O6-methylguanine to a protein cysteine residue, J. Biol. Chem., 255, 10569-10571. Rieger, R., A. Michaelis and H. Nicoloff (1982) Inducible repair processes in plant root tip meristems? 'Below-additivity effects' of unequally fractionated clastogen concentrations, Biol. Zbl., 101, 125-138. Rieger, R., A. Michaelis and H. Nicoloff (1984) 'Clastogenic adaptation' of the I/icia faba root tip meristem as affected by various treatment parameters, Mutation Res., 140, 99102. Rieger, R., A. Michaelis and H. Nicoloff (1986) Effects of stress factors on the clastogen response of Vicia faba root tip meristems: 'Clastogenic adaptation', Biol. Zbl., 105, 19-28. Rieger, R., A. Michaelis and S. Takehisa (1990) On adaptive responses of plant meristem cells in vivo - protection against induction of chromatid aberrations, in: G. Obe and A.T. Natarajan (Eds.), Chromosomal Aberrations, Springer, Berlin, pp. 163-179.
72 Samson, L., and J. Cairns (1977) A new pathway for DNA repair in Escherichia coli, Nature (London), 267, 281-283. Samson, L., and J.L. Schwartz (1980) Evidence for an adaptive DNA repair pathway in CHO and human skin fibroblast cell lines, Nature (London), 287, 861-863. Wolff, S., G. Olivieri and V. Afzal (1990) Adaptation of human lymphocytes to radiation or chemical mutagens:
differences in cytogenetic repair, in: G. Obe and A.T. Natarajan (Eds.), Chromosomal Aberrations, Springer, Berlin, pp. 140-150.
Communicated by J. Velemlnsl~
