Reduction by heat shock of maleic hydrazide-induced aberration yield is dependent on temperature and duration of heat pretreatment

Mutation Research, 174 (1986) 199-204 Elsevier

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MRLett. 0865

Reduction by heat shock of maleic hydrazide-induced aberration yield is dependent on temperature and duration of heat pretreatment R. Rieger, A. Michaelis and I. Schubert Zentralinstitut ffir Genetik und Kulturpflanzenforschung Gatersleben der A d W der DDR, DDR-4325 Gatersleben (German Democratic Republic) (Accepted 18 February 1986)

Summary Heat shocks (HS) applied prior to challenge treatment with maleic hydrazide (MH) significantly reduced the yield of metaphases with chromatid structural changes in Viciafaba root tip meristems. The protective effect was found to be dependent both on temperature and duration of HS pretreatment. With HS at 40°C a 1-min pretreatment was sufficient to trigger the full protective effect, with HS at 38°C this was true for only part of the recovery times tested. Heat pretreatment at 36°C for 10 min was without protective effect.

Heat shocks (10 and 30 min at 40°C) prior to treatment of Vicia faba root tip meristems with MH or TEM have previously been observed to reduce significantly the yield of metaphases with chromatid aberrations (Rieger et al., 1985a). The experimental results reported here show that the protective effect of heat shocks (HS), given 1 h before treatment with MH, is dependent on temperature and duration of heat pretreatment. HS protection of root meristem cells against aberration induction by MH or TEM is reminiscent of what has been called 'clastogenic adaptation', i.e., reduction of the yield of chromatid aberrations induced by a high (challenge) clastogen dose after pretreatment with a low (conditioning) dose of the same or certain other clastogens Abbreviations: HS, heat shock; MH, maleic hydrazide; MwA, metaphases with chromatid aberrations; RT, recovery time; TEM, triethylenemelamine.

(Rieger et al., 1982, 1984, 1985b, 1986; Heindorff et al., 1985a,b; Michaelis et al., 1986). Our preliminary conclusion from the presently available data is that all the pretreatments used exert stress on the root meristem ceils and the stressors trigger inducible functions. Such functions are assumed to counteract at least a fraction of the genotoxic effects of the challenge treatments with clastogens and thereby reduce the yield of cells containing chromatid aberrations. The data on the biochemical effects of HS in prokaryotic and eukaryotic cells and the reduction of heat damage by the 'heat shock response' (for review see Nover et al., 1984; Neidhardt et al., 1984) stimulated our present experiments. The intention underlying a more detailed investigation of HS pretreatments on clastogen activity was to provide a basis for searching for correlations between HS protection against aberration formation and induction of HS proteins. Biochemical studies on the induction by

0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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heat o f such proteins in V. faba are presently being done. Material and methods Primary roots of about 30 mm length of the reconstructed karyotype ACB of Vicia faba (Michaelis and Rieger, 1971; Rieger and Michaelis, 1972) were used for the experiments. Maleic hydrazide (l,2-dihydropyridazine-3,6-dione, MH) was applied for 0.5 h at a concentration of 5 × 10 -4 M. For treatment conditions (duration of HS, temperature, time intervals between HS and MH treatment see legends to Figs. 1-4). After 12-24 h recovery times in running tap water (24°C) following the challenge treatment with MH, the roots were immersed in 0.05°7o colchicine for 2 h, fixed in absolute ethanol and glacial acetic acid (3:1) and Feulgen-stained. For each recovery time 200 metaphases of the first cell-cycle after treatment were scored from 4 root tips, and the percentage of metaphases with isochromatid breaks, chromatid translocations, duplication deletions, and intercalary deletions was established. At least two repetitions of each experiment were made and the data were pooled. As to the statistics used, see legend to Fig. 1.

Results

(1) A HS o f 1 min (40 °C) is sufficient to exert protective effects on aberration induction by M H Main roots o f V. faba were exposed to a temperature of 40°C for 5, 2.5 or 1 min and 1 h later treated with M H (0.5 h, 5 x 10 - 4 M ) . The yield of metaphases with chromatid aberrations was established after recovery times of 12, 15, 18, 21 and 24 h. It is evident from Fig. 1 that all three time spans of a 40°C HS pretreatment reduce significantly the yield of metaphases with MHinduced chromatid aberrations as compared to the aberration yield obtained after treatment with M H alone (curve 1 as compared to curves 2, 3 and 4). The reduction of aberration yield was the same after HS for 5, 2.5 or 1 min. A 1-min HS at 40°C

is thus sufficient to trigger protection against MH clastogenicity.

(2) The temperature used for HS is decisive for the protective effect When temperatures of 36°C, 38°C and 40°C were used for a 10-min heat treatment 1 h before

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M H (0.5 h, 5 × 10 - 4 M), reduced aberration yields were observed for all recovery times after HS of 40°C and 38°C (Fig. 2). The protective effect was quantitatively the same for these two temperatures (curves 3 and 4). No protection occurred when the pretreatment temperature was 36°C. A certain temperature level is therefore obligatory for triggering protective effects. For 10min heat pretreatment the critical temperature is 38°C or near to that value. It was shown in Fig. 1 that heat pretreatment at 40°C for 1 min is suffi-

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cient to trigger as much protection against MH as does a 10-min heat pretreatment. Although 10-min heat pretreatments at 40°C and 38°C proved to be equally effective with respect to protection against aberration induction by MH (Fig. 2), the situation was different when the heat pretreatments at 40°C and 38°C were for only 1 min. Under these circumstances (Fig. 3), no reduction of aberration yield occurred for the early recovery times (12 and 15 h) when the heat pretreatment for 1 min was at 38°C, as opposed to the situation for a 1 min HS at 40°C (Fig. 3, curve 3). During later recovery times, however, 1-min pretreatments at 38°C also significantly reduced MH-induced aberration yields (Fig. 3, curve 2), as compared to the yield of aberrations scored after MH treatment without HS (Fig. 3, curve 1). Metaphases observed during early recovery time had a later cell cycle position when they came under clastogen influence, and vice versa. From this it may be inferred, that the heat-induced protective functions, which eventually reduce clastogen responses, are triggered incompletely under these pretreatment conditions so that their activity is already 'used up' by the earlier cycling cells which are scored during the later recovery times and are reduced in chromosome damage, similar to those observed after pretreatment at 38°C for 10 instead of 1 min.

(3) HS prior to heat challenge reduce root lethality in Vicia faba The HS response confers increased resistance to the deleterious effects of high temperature and other stressors, but the underlying mechanisms are not yet understood (Neidhardt et al., 1984). In addition to our experiments on the effects of HS on the induction of chromatid structural changes (Rieger et al., 1985 and this paper), we looked for influences of HS on the survival of V. faba primary roots after subsequent heat challenge. The data obtained are shown in Fig. 4 and provide clear evidence that conditioning pretreatments with heat increase thermoresistance of the roots as is obvious from their reduced lethality after temperature increases.

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Fig. 3. The yield of metaphases with chromatid aberrations (% MwA) as affected by HS treatments for 1 min at different temperatures followed by a recovery time o f 1 h and a subsequent treatment with 5 x 10 -4 M M H (0.5 h). Curve h 0.5 h 5 x 10 -4 M MH; curve 2 : 1 rain HS at 38°C prior to MH; curve 3 : 1 rain HS at 40°C prior to MH.

Fig. 4. The influence o f a 0.5 h HS treatment at different temperatures from 40°C up to 44°C (curve l) on the lethality of main roots of Viciafaba and the reduction of lethality after pretreatment of 0.5 h HS at 40°C followed by a recovery time o f 2 h at 24°C and a second HS with temperatures ranging from 40°C up to 44°C (curve 2). The values observed after challenge treatment at 42°C are significant at ct = 2.5% and those at 43°C and 44°C at c~ = 0.5% according to the x2-test (v.d. Waerden and Nievergelt, 1956).

Discussion

al., 1982). There is evidence that the HS response protects cells from stress but the mechanisms by which an accumulation of stress proteins within cells makes these cells more resistant to further stress have not yet been elucidated. It is also not known whether or not the production of stress proteins in heat-treated cells is correlated with the heat reduction of aberration formation in V. faba. The data presented here will

The general pattern and time course of the HS response is similar in prokaryotic and eukaryotic cells: A family o f HS genes can be activated by diverse physical, chemical and biophysical stresses; the HS genes produce stress proteins, and are regulated at the transcriptional and translational level (Ashburner and Bonnet, 1979; Schlesinger et

203

serve for appropriate biochemical studies on the production of HS proteins in II. faba and possible correlations of these with HS effects on clastogenicity. Although this question is open, there is no doubt that HS and other stressors are able to modulate quantitatively aberration production in root tip meristem cells. The conclusions from the results presented in this paper are: (1) In addition to increased thermotolerance of primary root tips (Fig. 4), HS (and other stressors) prior to MH (and other clastogens) trigger cellular functions that eventually reduce the clastogenicity of consecutive challenge treatments with the clastogens as evidenced by significantly decreased frequencies of induced chromatid aberrations. (2) With the appropriate temperature (40°C) used for conditioning, a very short duration of heat treatment (1 min) is sufficient for triggering these inducible functions (Fig. 1). (3) Conditioning temperature treatment for 10 rain at 36°C remains without effect on MHinduced aberration yield (Fig. 2). (4) Conditioning heat pretreatment at 38°C for 1 min reduces the MH-induced yield of chromatid aberrations in cells observed at later recovery times (18, 21 and 24 h), not in those scored during early recovery times (12 and 15 h; Fig. 3). Contrary to this, conditioning by temperatures of 40°C results in reduced aberration frequencies in all recovery times. Since metaphases scored during early recovery stem from cells that were later in cell cycle at the time of MH treatment than those observed at later recovery times, we presume that with 38°C heat treatment for 1 min the inducible functions resulting in protective effects are incompletely triggered in quantity under such heat treatment conditions. The data presented in this and earlier papers (summarized in Rieger et al., 1986) invite the speculation that specific functions are activated when a cell responds to stress such as an increase in temperature or exposure to other stressors and that these functions ultimately alleviate the damaging effects of stressors, including genotoxic ones. HS induction of resistance to the lethal effects of ionizing radiation and ultraviolet light has been

observed by Mitchel and Morrison (1982, 1983) in Saccharomyces cerevisiae. HS was found by Yager et al. (1985) to induce, in African green monkey kidney epithelial cells, the capacity to carry out enhanced reactivation of UV-irradiated herpes virus, and Piperakis and McLennan (1984) reported that hyperthermia resulted in enhanced reactivation of UV-irradiated adenovirus in HeLa cells. These observations and our results reported here indicate that thermotolerance induced by HS is only one of several endpoints triggered by heat stress. References Ashburner, M., and J.J. Bonner (1979) The induction of gene activity in Drosophila by heat shock, Cell, 17, 241-254. Heindorff, K., O. Aurich, R. Rieger and A. Michaelis (1985a) Pretreatment of Vicia faba root tip meristems with hydrazines results in 'clastogenic adaptation' to maleic hydrazide, Mutation Res., 142, 183-186. Heindorff, K., A. Michaelis, O. Aurich and R. Rieger (1985b) Peroxide treatment of Viciafaba root tip meristems results in 'clastogenic adaptation' to maleic hydrazide but not to TEM, Mutation Res., 142, 23-27. Michaelis, A., and R. Rieger (1971) New karyotypes of Vicia faba, Chromosoma, 35, 1-8. Michaelis, A., S. Takehisa, R. Rieger and O. Aurich (1986) Ammonium chloride and zinc sulfate pretreatments reduce the yield of chromatid aberrations induced by TEM and maleic hydrazide in Viciafaba, Mutation Res., 173, 187-191. Mitchel, R.E.J., and D.P. Morrison (1982) Heat-shock induction of ionizing radiation resistance in Saccharomyces cerevisiae, Transient changes in growth cycle distribution and recombinational ability, Radiation Res., 92, 182-187. Mitchel, R.E.J., and D.P. Morrison (1983) Heat-shock induction of ultraviolet light resistance in Saccharomyces cerevisiae, Radiation Res., 96, 95-99. Neidhardt, F.C., R.A. van Bogelen and V. Vaugh (1984) The genetics and regulation of heat-shock proteins, Annu. Rev. Genet., 18, 295-329. Nover, L., D. Hellmund, D. Neumann, K.-D. Scharf and E. Serfling (1984) The heat shock response of eukaryotic cells, Biol. Zbl., 103, 357-435. Piperakis, S.M., and A.G. McLennan (1984) Hyperthermia enhances the reactivation of irradiated adenovirus in HeLa cells, Br. J. Cancer, 49, 199-205. Rieger, R., and A. Michaelis (1972) Effects of chromosome repatterning in Vicia faba L., I. Aberration distribution, aberration spectrum and karyotype sensitivity after treatment with ethanol of differently reconstructed chromosome complements, Biol. Zbl., 91, 151-169.

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cross-adaptation' is dependent on the clastogens used for induction of chromatid aberrations in Vicia faba root tip meristems, Mutation Res., 144, 171-175. Rieger, R., A. Michaelis and H. Nicoloff 0986) Effects of stress factors on the clastogen response of Viciafaba root tip meristems - - 'Clastogenic adaptation', Biol. Zbl., 105, 18-28. Schlesinger, M.J., M. Ashburner and A. Tissibres (Eds.) (1981) Heat Shock from Bacteria to Man, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. v.d. Waerden, B.L., and E. Nievergelt (1956) Tafeln zum Vergleich zweier Stichproben mittels Test und Zeichentest, Springer, Berlin. Yager, J.D., J. Zurlo and A.L. Penn 0985) Heat-shockinduced enhanced reactivation of UV-irradiated herpes virus, Mutation Res., 146, 121-128. Communicated by J. Sch6neich