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Induction of SCE by maleic hydrazide throughout the cell cycle
Mutation Research, 163 (1986) 57-61
57
Elsevier MTR 04240
Induction of SCE by maleic hydrazide throughout the cell cycle Gardenia Gonz dez-Gil * and Matilde H. Navarrete Instituto de Biolog~a Celular, C.S.L C., Vel~zquez 144, 28006 Madrid (Spain)
(Received20 July 1984) (Revisionreceived2 May 1986) (Accepted20 May 1986)
Summary In this paper we studied the effectiveness of a treatment with maleic hydrazide to increase the sister-chromatid exchange yield in root-meristem cells of Allium cepa, in relation to the stage of the cell cycle in which the treatment is performed. Roots were grown in 5-bromodeoxyuridine for two cell cycles and pulse-treatments with maleic hydrazide were carried out at different times throughout both cycles. By analyzing the [3H]thymidine-labeled metaphases at the second mitosis, we established the position of ceils along the cycle when treated with maleic hydrazide. The results demonstrate that (1) there is a differential sensitivity of plant cells to maleic hydrazide throughout the cell cycle, being maximum during the period of DNA replication, (2) the effectiveness of maleic hydrazide in increasing the sister-chromatid exchange yield could be related to the time available for repair before the lesions are reached by the replicating forks, and (3) maleic hydrazide induces sister-chromatid exchange formation in a similar way to that of alkylating agents, even though it is not alkylating per se.
Sister-chromatid exchange is an interchange of genetic material between replication products in a chromosome at apparently homologous loci. Although their biological significance is as yet unclear, the SCE test has been proved to be a sensitive cytological indicator of DNA damage caused by S-dependent mutagens and carcinogens. Attempts to correlate SCE with any known DNA-repair processes have been unsuccessful, but the evidence found so far indicates that SCE might be a cytological endpoint of a damage-tolerating mechanism still unidentified in molecular terms, but operating when the damage is carded out during DNA replication (Sasaki, 1982). The herbicide MH (1,2-dihydro-3,6-pyridazinedione) is reported to be an effective clastogen
* To whomcorrespondenceshould be addressed.
in higher plants (Swietlinska and Zuk, 1978) and, together with its salts, proves to be highly mutagenie in the Tradescantia stamen hairs mutagenicity assay (Gichner et al., 1982). Studies performed by Evans and Scott (1964) in root-tip cells of Ficia faba indicated that MH induces chromosomal aberrations by an S-dependent mechafiism, as UV and alkylating agents do. Apparently, MH does not damage DNA per se, but only after transformation by the plant metabolism to some damaging derivatives (Plewa and Gentile, 1982). Among them, hydrazine is a known mutagen (Kimball, 1977) which could be directly implicated in this type of response of plant cells to MH. In this paper we have evaluated the effectiveness of a treatment with MH in increasing the SCE yield in Allium cepa root-meristem ceils in relation to the concentration of the chemical and
0027-5107/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)
58 the stage of the cell cycle in which the treatment is performed. Material and methods
Culture conditions Root meristem cells of Allium cepa L. were used. The onion bulbs were grown in 80-ml cylindrical receptacles containing filtered tap water, at a constant temperature of 25 + 0.5 °C, in the dark. The water was renewed every 24 h and continuously aerated by bubbling air at a constant rate of 15-20 ml/min. The bulbs were placed in such a way that only their bases remained submerged in the water and treatments began when the roots had reached steady-state kinetics (15-20 mm in length). DNA substitution with BrdUrd was carried out by exposing the growing roots to 0.1 mM BrdUrd (Sigma) in tap water for 36 h. Treatments with MH (Sigma) for 1 h were performed at the concentrations and times specified in each experiment. Since the chromosome-damaging effect of MH varies with the pH of the final treatment solution (Kihlman, 1956), the herbicide was always dissolved in 7 mM phosphate buffer, pH 6.0. For autoradiographic studies, 3H-Thd (Amersham) was present in the culture medium at 12 #Ci m1-1 (sp. act. 25 Ci mmole -1) for 30 rain at the times specified in each experiment. Metaphase accumulation was achieved by exposing the roots, still attached to the bulbs, to 1 mM colchicine (Sigma) for 2.5 h and fixations were made in ethanol-acetic acid (3 : 1) at 4°C) overnight. In order to minimize photolysis of BrdUrd and background SCE, the culture receptacles and the bulbs were kept away from light by wrapping them in aluminum foil. SCE analysis Squashes were stained with Hoechst 33258 (1 mg Hoechst in 1 ml ethanol and 0.2 ml of this solution in 100 ml of 0.5 × SSC) at room temperature for 20 rain. They were then mounted with a drop of 0.5 × SSC and exposed to a 313-nm light (Westinghouse FS 20) at 10 cm distance for 1 h. After removing the coverslips, slides were incubated in 0.5 x SSC at 50°C for 1 h, treated with 5 N HC1 at 20°C for 15 min and thoroughly washed with distilled water. Finally, preparations
were stained in 2% Giemsa (Merck) dissolved in 0.17 M phosphate buffer pH 6.8 for 7 min, airdried and mounted with Sandeural (Gonzhlez-Gil and Navarrete, 1982). Since the 16 chromosomes which make up the Allium cepa karyotype are similar in length and shape, SCE frequencies were calculated per chromosome after scoring at least 300 second-division chromosomes. Control data were only taken into account when the observed chromosome distribution fitted the theoretical Poisson distribution according to the X2 test. The theoretical distribution was calculated from the experimental mean value. In these cases, 95% confidence limits of the mean were also calculated.
Analysis of second-division metaphases In those studies in which sister-chromatid differentiation and autoradiography were combined, a modification of the "reverse" sister-chromatid differentiation method of Takayama and Sakanishi (1977) was used: fixed whole meristems were hydrated in distilled water for 30 min. They were then hydrolyzed in 6 N HCI at 20°C for 20 min, stained with the Schiff reagent at 20 °C for 30 min and squashed onto gelatinized slides. For autoradiography, slides were rehydrated in distilled water for 30 min, dipped in Kodak NTB2 emulsion (di: luted 1:1 in distilled water), dried and exposed for 6 days at - 7 0 ° C . Development was with Kodak D19 at 2°C for 5 min. 100 second-division metaphases were scored in each experiment and the proportion of labeled and unlabeled metaphases was calculated. Results and discussion
Allium cepa roots growing under fixed environmental conditions develop a constant growth rate and show a constant number of cells a t any given stage of the cell cycle over a long period (Navarrete et al., 1983). Under continuous treatment with 100 /~M BrdUrd, we measured the percentage of seconddivision chromosomes and found a maximum 36 h after the beginning of the treatment. Cell proliferation after a pulse-treatment with M H The toxicity of any clastogenic agent may be-
59 MH 25-
-1 0
3
6
9 Time (h}
12
15
18
Fig. 1. Mitotic index after treating the meristem cell population with different concentrations of MH for 1 h (shaded area): 1 mM ([3), 100/aM (O) and 10 jaM (©). The control value range observed was 12-145g.
come evident either in chromosome breakage or in alteration of specific cell kinetics parameters. Fig. 1 shows mitotic index after treating the meristems with MH at different concentrations for 1 h. The lowest concentration used (10/~M) apparently did not modify mitotic index during recovery. On the other hand, when roots were exposed to 1 mM MH the mitotic index dropped sharply just after treatment and did not reach the control values for the next 24 h (data not shown). After 100 #M MH, the mitotic index was reduced, but this alteration was easily overcome after a recovery period of 9 h and resulted in a small wave of mitotic synchrony by the 12th hour. Studies performed by other authors indicated that MH is an effective clastogen in higher plants (Evans and Scott, 1964; Kihlman and Sturelid, 1978). By contrast, in the experiment shown in Fig. 1, we did not find any chromosomal aberrations (up to 24 h after treatment), probably because 10 #M and 100 #M for 1 h are mild treatments and 1 mM produces a strong depression in cell cycle transit. When DNA was substituted with BrdUrd, some micronuclei and chromatin bridges were observed after treatment with 100 #M MH. These aberrations were more evident after a recovery period of at least 18 h. Control cells treated with BrdUrd for 36 h but without MH showed a slight proportion of micronuclei. Together these results confirm the greater sensitivity of BrdUrd-substituted DNA than native DNA to damaging agents (Dewey and Humphrey, 1965).
Second S-phase time in the presence of BrdUrd Fig. 2 presents the experimental protocol schematically. BrdUrd was always present in the culture medium for two complete cell cycles and pulse-treatment with 3H-Thd for 30 min (a) or with 3H-Thd for 30 min followed by 100 #M MH for 1 h (b) were performed at different times throughout both cycles. In every case the proportion of labeled and unlabeled second-division metaphases was calculated. When cells were pulsed with 3H-Thd 18 h before the end of culture (Fig. 3, open circles), only 13% of the metaphases appeared labeled, indicating that most of the cells were in a non-replicative period of the division cycle. 3 h later (h 15), 61% of the cells were located inside the S phase. 10 h before the end of culture, the majority of them was replicating (96%), and finally at h 5, only a slight proportion (4%) was in the S phase. Therefore, the second S phase started sometime between h 18 and h 15 and concluded near h 5. When treatments with 100 #M MH were carried out just after the 3H-Thd pulse (closed circles in Fig. 3), the maximum frequency of labeled metaphases was displaced to h 15. When MH was given at h 10 only 36% of the metaphases were labeled, suggesting a delay of the end of S and last G 2 periods. Induction of SCE by M H at various stages of the cell cycle Treatments with MH for 1 h were performed at the end of the first cycle or at different times during the second interphase (Fig. 4). The results, summarized in Fig. 5, show that the effectiveness of MH in inducing SCE increases with increasing
a
Fig. 2. Experimental protocol for monitoring the second S-phase time when cells were kept in BrdUrd for two cell cycles (a) and when additional treatments with MH were performed at different times throughout both cycles (b). Asterisks show the times of the pulse-treatment with 3H-Thd and horizontal bars represent the times of the MH treatment.
60
,00t
/°\
90t
¢,\
801
70 . 60
o
o
141312 11
/
9 8 o. to
6
$
24
18
15
10
5
Time before the end of the culture (h)
Fig. 3. Percentage of labeled metaphases obtained at the second mitosis after cells replicated in BrdUrd for two cell cycles and were pulsed with 3H-Thd alone (O) or with 3H-Thd followed by 100 btM MH ($) at different times throughout both cycles.
concentrations of MH and that within each concentration this efficiency varies according to the phase of the cell cycle in which the treatment is performed. Since MH interferes with normal DNA replication (Evans and Scott, 1964) it is to be expected that cells in S phase were the most affected by this herbicide. This is what we found with 3/~M MH for which the maximum SCE yield was obtained when the treatment was carried out upon replicating cells (see Fig. 3). When the treatment was carried out before the S phase (h 18), no increase of SCE compared to the control values was observed at the second mitosis, probably because the induced lesions could be efficiently repaired before replication. These results are similar to the ones obtained by Latt and Loveday (1978)
Fig. 4. Experimental protocol for studying SCE induction by MH at different times of the cell cycle. In every case, BrdUrd was present in the culture medium for two complete cell cycles and treatments with MH for 1 h (horizontal bars) were performed at different times throughout both cycles.
2/,,
18
t5
10
5
2
Time before the end of the culture (h)
Fig. 5. Effectiveness of a treatment with varying concentrations of MH in increasing the SCE yield at different times of the cell cycleL The abscissa represents the time (h) before the end of culture (h 0) at which treatments with MH were performed, and we considered h 0 to be the end of the BrdUrd treatment prior to metaphase accumulation with colchicine. The concentrations of MH used were 100 ~M (O), 10 /~M ([3) and 3 #M (zx). The shaded area shows the range of SCE control values. Range bars represent 95% confidence limits of the mean.
using 8-methoxypsoralen plus near-UV light in CHO cells and by Schvartzman and Guti6rrez (1980) exposing BrdUrd-substituted root-tip cells of A Ilium cepa to visible light, and it appears to be a general response of the cells to moderate DNA damage. As the concentration of MH increased, the SCE values gradually increased as well, suggesting a higher frequency of lesions in the DNA molecule. But the most striking fact was that the summit of the curve was also displaced to the left, and with 100 ttM MH it appeared inside the non-replicative periods before the last S phase (h 18). In this case, when more time for repair was available (h 24) the SCE value obtained was lower. At this time of the treatment (h 24) the level of DNA damage should be the same as at any other time, but the number of the non-repaired lesions which reached the replicating fork was apparently lower, thereby giving a lower number of SCE.
61
her skillful technical assistance. This research was supported by a fellowship from the "Caja de Ahorros y Monte de Piedad de Madrid" and by a grant from the "Comisi6n Asesora para la Investigaci6n Cientifica y T6cnica" and by the "Acuerdo de Cooperaci6n Cientifica CSIC (Spain)-Universidad de Chile (Chile)".
~3
3
10 30 1,48 concentration()JM)
100
Fig. 6. Dose-respon~ of SCE in meristem cells treated with
MH. SCE values at the time of highest sensitivity in the cycle (h 18 for 100/~M and h 10 for 10 #M, 3/~M and 1/tM) are represented as increments over the control SCE yield per chromosome (3.5).
Dose-response of SCE Sasaki (1982) demonstrated a linear dose-response for SCE produced by several alkylating agents, suggesting a single-hit mechanism for this event. In Fig. 6 we have plotted the highest SCE values obtained at every concentration of MH. Our data conform to a linear function of dose, indicating that most of the SCE produced by MH are single-hit events, originating from single lesions. This fact indicates that MH induces SCE in a similar way to that of alkylating agents.
Conclusions DNA damage caused by MH in plant cells can be accurately measured by SCE induction. By means of this test we have demonstrated a differential sensitivity of plant cells to MH throughout their division cycle. The maximum sensitivity is obtained during DNA replication, as it was clearly seen using short-time incubations in MH at low concentrations. Higher doses of MH suggest that SCE induction might be dependent on the time available for repair before the induced lesions are reached by the replicating forks. Lastly, SCE produced by MH are single-hit events, indicating that MH behaves as an alkylating agent.
Acknowledgements The authors are indebted to Prof. J.F. L6pezShez for his advice and constructive comments on the manuscript. We also thank M.L. Martinez for
References Dewey, W.C., and R.M. Humphrey (1965) Increase in radiosensitivity to ionizing radiation related to replacement of thymidine in mammalian cells with 5-bromodeoxyuridine, Radiation Res., 26, 538-553. Evans, H.J., and D. Scott (1964) Influence of DNA synthesis on the production of chromatid aberrations by X-rays and maleic hydrazide in Vicia faba, Genetics, 49, 17-38. Gichner, T., J. Veleminsk.~ and V. Pokorn~ (1982) Somatic mutations induced by maleic hydrazide and its potassium and diethanolamine salts in the Tradescantia mutation assay, Mutation Res., 103, 289-293. Gonz,~lez-Gil, G., and M.H. Navarrete (1982) On the mechanism of differential Cfiemsa staining of BrdU-substituted chromatids, Chromosoma, 86, 375-382. Kihlman, B.A. (1956) Factors affecting the production of chromosome aberrations by chemicals, J. Biophys. Biochem. Cytol., 2, 543-555. Kihlman, B.A., and S. Sturelid (1978) Effects of caffeine on the frequencies of chromosomal aberrations and sister-chromatid exchanges induced by chemical mutagens in root tips of Vicia faba, Hereditas, 88, 35-41. Kimball, R.F. (1977) The mutagenicity of hydrazine and some of its derivatives, Mutation Res., 39, 111-126. Latt, S.A., and K.S. Loveday (1978) Characterization of sister chromatid exchange induction by 8-methoxypsoralen plus near UV light, Cytogenet. Cell Genet., 21,184-200. Navarrete, M.H., A. Cuadrado and J.L. Chnovas (1983) Partial elimination of G 1 and G 2 periods in higher plant cells by increasing the S period, Exp. Cell Res., 148, 273-280. Plewa, M.J., and J.M. Gentile (1982) The activation of chemicals into mutagens by green plants, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 7, Plenum, New York, pp. 401-420. Sasaki, M.S. (1982) Sister chromatid exchange as a reflection of cellular DNA repair, in: A.A. Sandberg (Ed.), Sister Chromatid Exchange, pp. 135-161. Schvartzman, J.B., and C. Guti6rrez (1980) The relationship between the cell time available for repair and the effectiveness of a damaging treatment in provoking the formation of sister chromatid exchanges, Mutation Res., 72, 483-489. Swietlinska, Z., and J. Zuk (1978) Cytotoxic effects of maleic hydrazide, Mutation Res., 55, 15-30. Takayama, S., and S. Sakanishi (1977) Differential staining of sister chromatids after extraction with acids, Chromosoma, 64, 109-115.
57
Elsevier MTR 04240
Induction of SCE by maleic hydrazide throughout the cell cycle Gardenia Gonz dez-Gil * and Matilde H. Navarrete Instituto de Biolog~a Celular, C.S.L C., Vel~zquez 144, 28006 Madrid (Spain)
(Received20 July 1984) (Revisionreceived2 May 1986) (Accepted20 May 1986)
Summary In this paper we studied the effectiveness of a treatment with maleic hydrazide to increase the sister-chromatid exchange yield in root-meristem cells of Allium cepa, in relation to the stage of the cell cycle in which the treatment is performed. Roots were grown in 5-bromodeoxyuridine for two cell cycles and pulse-treatments with maleic hydrazide were carried out at different times throughout both cycles. By analyzing the [3H]thymidine-labeled metaphases at the second mitosis, we established the position of ceils along the cycle when treated with maleic hydrazide. The results demonstrate that (1) there is a differential sensitivity of plant cells to maleic hydrazide throughout the cell cycle, being maximum during the period of DNA replication, (2) the effectiveness of maleic hydrazide in increasing the sister-chromatid exchange yield could be related to the time available for repair before the lesions are reached by the replicating forks, and (3) maleic hydrazide induces sister-chromatid exchange formation in a similar way to that of alkylating agents, even though it is not alkylating per se.
Sister-chromatid exchange is an interchange of genetic material between replication products in a chromosome at apparently homologous loci. Although their biological significance is as yet unclear, the SCE test has been proved to be a sensitive cytological indicator of DNA damage caused by S-dependent mutagens and carcinogens. Attempts to correlate SCE with any known DNA-repair processes have been unsuccessful, but the evidence found so far indicates that SCE might be a cytological endpoint of a damage-tolerating mechanism still unidentified in molecular terms, but operating when the damage is carded out during DNA replication (Sasaki, 1982). The herbicide MH (1,2-dihydro-3,6-pyridazinedione) is reported to be an effective clastogen
* To whomcorrespondenceshould be addressed.
in higher plants (Swietlinska and Zuk, 1978) and, together with its salts, proves to be highly mutagenie in the Tradescantia stamen hairs mutagenicity assay (Gichner et al., 1982). Studies performed by Evans and Scott (1964) in root-tip cells of Ficia faba indicated that MH induces chromosomal aberrations by an S-dependent mechafiism, as UV and alkylating agents do. Apparently, MH does not damage DNA per se, but only after transformation by the plant metabolism to some damaging derivatives (Plewa and Gentile, 1982). Among them, hydrazine is a known mutagen (Kimball, 1977) which could be directly implicated in this type of response of plant cells to MH. In this paper we have evaluated the effectiveness of a treatment with MH in increasing the SCE yield in Allium cepa root-meristem ceils in relation to the concentration of the chemical and
0027-5107/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)
58 the stage of the cell cycle in which the treatment is performed. Material and methods
Culture conditions Root meristem cells of Allium cepa L. were used. The onion bulbs were grown in 80-ml cylindrical receptacles containing filtered tap water, at a constant temperature of 25 + 0.5 °C, in the dark. The water was renewed every 24 h and continuously aerated by bubbling air at a constant rate of 15-20 ml/min. The bulbs were placed in such a way that only their bases remained submerged in the water and treatments began when the roots had reached steady-state kinetics (15-20 mm in length). DNA substitution with BrdUrd was carried out by exposing the growing roots to 0.1 mM BrdUrd (Sigma) in tap water for 36 h. Treatments with MH (Sigma) for 1 h were performed at the concentrations and times specified in each experiment. Since the chromosome-damaging effect of MH varies with the pH of the final treatment solution (Kihlman, 1956), the herbicide was always dissolved in 7 mM phosphate buffer, pH 6.0. For autoradiographic studies, 3H-Thd (Amersham) was present in the culture medium at 12 #Ci m1-1 (sp. act. 25 Ci mmole -1) for 30 rain at the times specified in each experiment. Metaphase accumulation was achieved by exposing the roots, still attached to the bulbs, to 1 mM colchicine (Sigma) for 2.5 h and fixations were made in ethanol-acetic acid (3 : 1) at 4°C) overnight. In order to minimize photolysis of BrdUrd and background SCE, the culture receptacles and the bulbs were kept away from light by wrapping them in aluminum foil. SCE analysis Squashes were stained with Hoechst 33258 (1 mg Hoechst in 1 ml ethanol and 0.2 ml of this solution in 100 ml of 0.5 × SSC) at room temperature for 20 rain. They were then mounted with a drop of 0.5 × SSC and exposed to a 313-nm light (Westinghouse FS 20) at 10 cm distance for 1 h. After removing the coverslips, slides were incubated in 0.5 x SSC at 50°C for 1 h, treated with 5 N HC1 at 20°C for 15 min and thoroughly washed with distilled water. Finally, preparations
were stained in 2% Giemsa (Merck) dissolved in 0.17 M phosphate buffer pH 6.8 for 7 min, airdried and mounted with Sandeural (Gonzhlez-Gil and Navarrete, 1982). Since the 16 chromosomes which make up the Allium cepa karyotype are similar in length and shape, SCE frequencies were calculated per chromosome after scoring at least 300 second-division chromosomes. Control data were only taken into account when the observed chromosome distribution fitted the theoretical Poisson distribution according to the X2 test. The theoretical distribution was calculated from the experimental mean value. In these cases, 95% confidence limits of the mean were also calculated.
Analysis of second-division metaphases In those studies in which sister-chromatid differentiation and autoradiography were combined, a modification of the "reverse" sister-chromatid differentiation method of Takayama and Sakanishi (1977) was used: fixed whole meristems were hydrated in distilled water for 30 min. They were then hydrolyzed in 6 N HCI at 20°C for 20 min, stained with the Schiff reagent at 20 °C for 30 min and squashed onto gelatinized slides. For autoradiography, slides were rehydrated in distilled water for 30 min, dipped in Kodak NTB2 emulsion (di: luted 1:1 in distilled water), dried and exposed for 6 days at - 7 0 ° C . Development was with Kodak D19 at 2°C for 5 min. 100 second-division metaphases were scored in each experiment and the proportion of labeled and unlabeled metaphases was calculated. Results and discussion
Allium cepa roots growing under fixed environmental conditions develop a constant growth rate and show a constant number of cells a t any given stage of the cell cycle over a long period (Navarrete et al., 1983). Under continuous treatment with 100 /~M BrdUrd, we measured the percentage of seconddivision chromosomes and found a maximum 36 h after the beginning of the treatment. Cell proliferation after a pulse-treatment with M H The toxicity of any clastogenic agent may be-
59 MH 25-
-1 0
3
6
9 Time (h}
12
15
18
Fig. 1. Mitotic index after treating the meristem cell population with different concentrations of MH for 1 h (shaded area): 1 mM ([3), 100/aM (O) and 10 jaM (©). The control value range observed was 12-145g.
come evident either in chromosome breakage or in alteration of specific cell kinetics parameters. Fig. 1 shows mitotic index after treating the meristems with MH at different concentrations for 1 h. The lowest concentration used (10/~M) apparently did not modify mitotic index during recovery. On the other hand, when roots were exposed to 1 mM MH the mitotic index dropped sharply just after treatment and did not reach the control values for the next 24 h (data not shown). After 100 #M MH, the mitotic index was reduced, but this alteration was easily overcome after a recovery period of 9 h and resulted in a small wave of mitotic synchrony by the 12th hour. Studies performed by other authors indicated that MH is an effective clastogen in higher plants (Evans and Scott, 1964; Kihlman and Sturelid, 1978). By contrast, in the experiment shown in Fig. 1, we did not find any chromosomal aberrations (up to 24 h after treatment), probably because 10 #M and 100 #M for 1 h are mild treatments and 1 mM produces a strong depression in cell cycle transit. When DNA was substituted with BrdUrd, some micronuclei and chromatin bridges were observed after treatment with 100 #M MH. These aberrations were more evident after a recovery period of at least 18 h. Control cells treated with BrdUrd for 36 h but without MH showed a slight proportion of micronuclei. Together these results confirm the greater sensitivity of BrdUrd-substituted DNA than native DNA to damaging agents (Dewey and Humphrey, 1965).
Second S-phase time in the presence of BrdUrd Fig. 2 presents the experimental protocol schematically. BrdUrd was always present in the culture medium for two complete cell cycles and pulse-treatment with 3H-Thd for 30 min (a) or with 3H-Thd for 30 min followed by 100 #M MH for 1 h (b) were performed at different times throughout both cycles. In every case the proportion of labeled and unlabeled second-division metaphases was calculated. When cells were pulsed with 3H-Thd 18 h before the end of culture (Fig. 3, open circles), only 13% of the metaphases appeared labeled, indicating that most of the cells were in a non-replicative period of the division cycle. 3 h later (h 15), 61% of the cells were located inside the S phase. 10 h before the end of culture, the majority of them was replicating (96%), and finally at h 5, only a slight proportion (4%) was in the S phase. Therefore, the second S phase started sometime between h 18 and h 15 and concluded near h 5. When treatments with 100 #M MH were carried out just after the 3H-Thd pulse (closed circles in Fig. 3), the maximum frequency of labeled metaphases was displaced to h 15. When MH was given at h 10 only 36% of the metaphases were labeled, suggesting a delay of the end of S and last G 2 periods. Induction of SCE by M H at various stages of the cell cycle Treatments with MH for 1 h were performed at the end of the first cycle or at different times during the second interphase (Fig. 4). The results, summarized in Fig. 5, show that the effectiveness of MH in inducing SCE increases with increasing
a
Fig. 2. Experimental protocol for monitoring the second S-phase time when cells were kept in BrdUrd for two cell cycles (a) and when additional treatments with MH were performed at different times throughout both cycles (b). Asterisks show the times of the pulse-treatment with 3H-Thd and horizontal bars represent the times of the MH treatment.
60
,00t
/°\
90t
¢,\
801
70 . 60
o
o
141312 11
/
9 8 o. to
6
$
24
18
15
10
5
Time before the end of the culture (h)
Fig. 3. Percentage of labeled metaphases obtained at the second mitosis after cells replicated in BrdUrd for two cell cycles and were pulsed with 3H-Thd alone (O) or with 3H-Thd followed by 100 btM MH ($) at different times throughout both cycles.
concentrations of MH and that within each concentration this efficiency varies according to the phase of the cell cycle in which the treatment is performed. Since MH interferes with normal DNA replication (Evans and Scott, 1964) it is to be expected that cells in S phase were the most affected by this herbicide. This is what we found with 3/~M MH for which the maximum SCE yield was obtained when the treatment was carried out upon replicating cells (see Fig. 3). When the treatment was carried out before the S phase (h 18), no increase of SCE compared to the control values was observed at the second mitosis, probably because the induced lesions could be efficiently repaired before replication. These results are similar to the ones obtained by Latt and Loveday (1978)
Fig. 4. Experimental protocol for studying SCE induction by MH at different times of the cell cycle. In every case, BrdUrd was present in the culture medium for two complete cell cycles and treatments with MH for 1 h (horizontal bars) were performed at different times throughout both cycles.
2/,,
18
t5
10
5
2
Time before the end of the culture (h)
Fig. 5. Effectiveness of a treatment with varying concentrations of MH in increasing the SCE yield at different times of the cell cycleL The abscissa represents the time (h) before the end of culture (h 0) at which treatments with MH were performed, and we considered h 0 to be the end of the BrdUrd treatment prior to metaphase accumulation with colchicine. The concentrations of MH used were 100 ~M (O), 10 /~M ([3) and 3 #M (zx). The shaded area shows the range of SCE control values. Range bars represent 95% confidence limits of the mean.
using 8-methoxypsoralen plus near-UV light in CHO cells and by Schvartzman and Guti6rrez (1980) exposing BrdUrd-substituted root-tip cells of A Ilium cepa to visible light, and it appears to be a general response of the cells to moderate DNA damage. As the concentration of MH increased, the SCE values gradually increased as well, suggesting a higher frequency of lesions in the DNA molecule. But the most striking fact was that the summit of the curve was also displaced to the left, and with 100 ttM MH it appeared inside the non-replicative periods before the last S phase (h 18). In this case, when more time for repair was available (h 24) the SCE value obtained was lower. At this time of the treatment (h 24) the level of DNA damage should be the same as at any other time, but the number of the non-repaired lesions which reached the replicating fork was apparently lower, thereby giving a lower number of SCE.
61
her skillful technical assistance. This research was supported by a fellowship from the "Caja de Ahorros y Monte de Piedad de Madrid" and by a grant from the "Comisi6n Asesora para la Investigaci6n Cientifica y T6cnica" and by the "Acuerdo de Cooperaci6n Cientifica CSIC (Spain)-Universidad de Chile (Chile)".
~3
3
10 30 1,48 concentration()JM)
100
Fig. 6. Dose-respon~ of SCE in meristem cells treated with
MH. SCE values at the time of highest sensitivity in the cycle (h 18 for 100/~M and h 10 for 10 #M, 3/~M and 1/tM) are represented as increments over the control SCE yield per chromosome (3.5).
Dose-response of SCE Sasaki (1982) demonstrated a linear dose-response for SCE produced by several alkylating agents, suggesting a single-hit mechanism for this event. In Fig. 6 we have plotted the highest SCE values obtained at every concentration of MH. Our data conform to a linear function of dose, indicating that most of the SCE produced by MH are single-hit events, originating from single lesions. This fact indicates that MH induces SCE in a similar way to that of alkylating agents.
Conclusions DNA damage caused by MH in plant cells can be accurately measured by SCE induction. By means of this test we have demonstrated a differential sensitivity of plant cells to MH throughout their division cycle. The maximum sensitivity is obtained during DNA replication, as it was clearly seen using short-time incubations in MH at low concentrations. Higher doses of MH suggest that SCE induction might be dependent on the time available for repair before the induced lesions are reached by the replicating forks. Lastly, SCE produced by MH are single-hit events, indicating that MH behaves as an alkylating agent.
Acknowledgements The authors are indebted to Prof. J.F. L6pezShez for his advice and constructive comments on the manuscript. We also thank M.L. Martinez for
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