Mutation Research, 228 (1990) 187-192 Elsevier
187
MUT 04829
Induction of chromosome damage by benzo[ a ]pyrene, 2-aminofluorene and cyclophosphamide in the root cells of Vicia faba Nobuhiro Kanaya Department of Biology, Keio University, Yokohama223 (Japan) (Received12 May 1989) (Revision received22 August 1989) (Accepted 19 September1989)
Keywords: Sister-chromatidexchange; Chromosome aberration; Benzo[a]pyrene;2-Aminofluorene;Cyclophosphamide; Viciafaba Summary The induction of sister-chromatid exchanges (SCEs) and chromosome aberrations (CAs) by benzo[a]pyrene (BP), 2-aminofiuorene (2-AF) and cyclophosphamide (CP) in the root cells of Vicia faba was examined. BP and 2-AF induced CAs, but not SCEs. CP induced both SCEs and CAs.
It has been reported that many carcinogenic promutagens are activated by plants. The activation of promutagens in plant cells in vivo and induction of mutations or chromosome aberrations (CAs) have been reviewed by Velerninsk~, and Gichner (1988). Extracts from plant tissues have been found to activate promutagens in vitro, resulting in increased mutation in Salmonella typhimurium (Higashi et al., 1981; Plewa and Gentile, 1982; Gentile and Plewa, 1988). Takehisa et al. (1988) reported that benzo[a]pyrene (BP) and 2-aminofluorene (2-AF), wellknown carcinogenic promutagens, were not activated by extracts from Vicia faba roots (Vicia S10) and there was no increase in the frequency of sister-chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells. In contrast, cyclophosphamide (CP), another carcinogenic promutagen, was observed to be activated by Vicia S10 and the
Correspondence: Dr. Nobuhiro Kanaya, Keio University, Department of Biology,Yokohama 223 (Japan).
SCE frequency in C H O cells was increased (Takehisa et al., 1988). It would thus seem pertinent to determine whether the activation of promutagens can occur in intact roots of Vicia faba. Since no report is presently available on the induction of SCEs by BP, 2-AF or CP in the root cells of Vicia faba, an examination of this matter was made in the present study. The induction of CAs by these chemicals was also examined. Materials and methods
Cultivation of Vicia faba Seeds of Vicia faba, cultivar. Wase (Sakata Seed Co., Yokohama), were soaked in running tap water for 24 h, and allowed to germinate in moist Vermiculite for 3 - 4 days at 24 ° C. Seedlings with main roots of 3 - 6 cm length were used for the experiments. Chemicals BP (CAS No. 50-32-8, Wako Pure Chem., Osaka) and 2-AF (CAS No. 153-78-6, Aldrich,
0027-5107/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
188
Milwaukee, WI) were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chem., Osaka), and diluted with distilled water. The concentration of DMSO in each solution was less than 0.2%. CP (CAS No. 50-18-0, Shionogi, Osaka) was dissolved in distilled water. The positive controls, mitomycin C (MMC; CAS No. 50-07-7, Kyowa Hakko, Tokyo) and ethanol (CAS No. 64-17-5, Wako Pure Chem., Osaka), were dissolved in distilled water. All chemicals were prepared immediately before use.
SCE analysis The seedlings were incubated in a 5-bromodeoxyuridine (BrdUrd) solution (100 /~M BrdUrd, 0.1 /~M 5-fluorodeoxyuridine, and 5 /~M uridine dissolved in tap water) for 17 h at 2 0 ° C with aeration in the dark. This was followed by exposure to each chemical solution for 3 h at 20 ° C in the dark. After being rinsed in running tap water, the seedlings were incubated in an aerated thymidine (dThd) solution (100 # M dThd and 5 ~ M uridine dissolved in tap water) for 22 or 25 h at 20 ° C in the dark. The main roots were then cut at 1 cm length from the tips followed by immersion in 0.05% colchicine solution for 2 h at 20 o C. The root tips were then fixed in ethanol-acetic acid (3: 1) for 24 h at 4 ° C . Differential staining of sister chromatids was conducted by the methods of Kihlman and Kronborg (1975) and Schubert et al. (1979) with slight modifications. The fixed root tips were washed in tap water for 10 min and macerated with 1% pectinase solution dissolved in 0.01 M citric acid-sodium citrate buffer (pH 4.7) for 4 h at 37 ° C and then with 2% cellulase solution dissolved in the same buffer for 2 h at 37 ° C. The macerated root tips were squashed in 45% acetic acid. Coverslips were removed on dry ice and the preparations were passed through a 99.5, 85, 70, 50 and 30% graded ethanol series, distilled water and 0.5 x SSC (0.075 M NaC1 + 0.0075 M sodium citrate). This was followed by exposure to 0.01% R N a s e solution in 0.5 x SSC for 1 h at 37°C, dehydration in the ethanol series and air-drying. The preparations were then exposed to 0.0005% Hoechst 33258 solution dissolved in 0.5 x SSC for 30 min. After being rinsed in distilled water, the preparations were mounted with 0.5 × SSC, placed
on a hot plate at 60 °C, and exposed to a black light lamp (Ultra-Violet Products, Inc., U.S.A.; model XX-15) for 30-35 min at 1 cm distance. They were then rinsed in 0.5 x SSC, incubated for 1 h in 2 x S S C (0.3 M N a C I + 0 . 0 3 M sodium citrate) at 80 ° C, and stained with 4% Giemsa in 0.067 M phosphate buffer (pH 6.8) for 20 rain. The preparations were rinsed in distilled water, air-dried and mounted with Eukitt. A complete metaphase cell of Vicia faba contains 2 long metacentric chromosomes with secondary constrictions (M-chromosomes) and 10 shorter subtelocentric c h r o m o s o m e s (S-chromosomes). Since it was difficult to find a metaphase cell with a complete chromosome number in these FPG-stained preparations, cells with incomplete as well as complete chromosome numbers were analyzed. Thus at least 50 M-chromosomes and 250 S-chromosomes were analyzed for each treatment. SCEs which occurred at the secondary constrictions of M-chromosomes and at the centromeres of both M- and S-chromosomes were included in the scores. Statistical analysis was performed by Student's t-test.
Chromosome aberration analysis The seedlings were treated with each chemical solution for 3 h at room temperature (22 + 2 ° C ) in the dark. After being rinsed in running tap water, they were incubated in aerated tap water for 22 h at 20 ° C in the dark. The main roots were then cut at 1 cm length from the tips, immersed in 0.05% colchicine solution for 2 h at 2 0 ° C , and fixed in ethanol-acetic acid (3 : 1) for 24 h at 4 ° C. The root tips were rinsed in running tap water, hydrolyzed in 1 N HCI at 60 ° C for 8-10 rain, and stained by the Feulgen staining reaction. They were then squashed in 45% acetic acid, and the coverslips were removed on dry ice. The preparations were air-dried and mounted with Eukitt. At least 100 metaphase cells with complete chromosome numbers were observed for each treatment. Chromatid-type aberrations such as chromatid breaks, isochromatid breaks, and chromatid exchanges but not gaps were counted. The isochromatid breaks at the secondary constrictions of M-chromosomes were not counted. Statistical analysis was conducted by Student's t-test.
189
Results
The results for induction of SCEs by BP, 2-AF, CP, MMC and ethanol in Vicia faba are summarized in Table 1. The mean SCE frequencies among seedlings were different. The range of the SCE frequencies among the control seedlings treated with 0.1% DMSO followed by incubation in dThd solution for 22 h was 1.27-2.09/S-chromosome and 3.69-5.77/M-chromosome. The SCE value of each chromosome was pooled and the mean SCEs/chromosome are presented in Table 1. The mean SCE frequencies of the control cells were 1.59/S-chromosome and 4.40/M-chromosome. The ratio of the SCE frequency in the M-chromosome to that in the S-chromosome was 1:0.36, approximately equal to the ratio of the mean length of the M-chromosome to that of the S-chromosome (1 : 0.44). The treatment with 10 -6 M MMC, a positive control, resulted in increases in SCEs in both Sand M-chromosomes (Table 1). The SCE frequency induced by MMC was about 3 times that of the control. The range of the SCE frequen-
cies in the MMC-treated seedlings was 4.11-5.43/ S-chromosome and 9.33-13.88/M-chromosome, which was beyond that in the control seedlings. As shown in Table 1, the SCE frequency induced by 0.1 M ethanol, another positive control, was about 3 times that of the control. The SCE range among the ethanol-treated seedlings was 4.88-4.98/Schromosome and 10.17-10.82/M-chromosome. The treatment with BP (2 × 1 0 - 5 - 5 x 1 0 - 6 M) or 2-AF (10-4-2.5 × 10 -5 M) followed by incubation in dThd solution for 22 h failed to increase SCEs in either S- or M-chromosomes (Table 1). The SCE frequencies of root cells incubated for 25 h in dThd solution after treatment with BP or 2-AF were also almost the same as those of the control cells. The range of SCE frequencies in the BP-treated seedlings was 1.24-1.79/S-chromosome and 2.88-5.33/M-chromosome, and that in the 2-AF-treated seedlings was 1.40-1.60/S-chromosome and 3.85-4.41/M-chromosome, i.e., within the control level. On the other hand, CP treatment resulted in a dose-dependent increase in SCEs. The mean SCE frequencies in cells treated with 10 - 2 M CP was 2.53/S-chromosome and
TABLE 1 I N D U C T I O N OF SISTER-CHROMATID EXCHANGES BY BP, 2-AF, CP, MMC A N D E T H A N O L IN Treatment
dThd (h)
Mean SCEs/Schromosome + SEM
Observed Mchromosome number
BP ( 2 × 1 0 -5 M)
22 25 22 25 22
355 412 599 352 254
441 649 956 528 426
1.24+0.06 1.58 + 0.06 1.60+0.05 1.50 + 0.06 1.68-t-0.09
53 53 106 52 66
174 241 425 230 311
3.28+0.27 4.55 + 0.26 4.01 +0.19 4.42 + 0.31 4.71+0.29
2-AF (5 x l 0 -5 M) 2-AF (2.5 × 10 - s M)
22 25 22 22
899 430 427 358
1257 668 683 537
1.40 + 0.04 1.55 + 0.06 1.60+0.06 1.50+0.06
117 75 54 59
451 317 221 260
3.85 + 0.20 4.23 + 0.25 4.09+0.23 4.41+0.28
CP (10- 2 M) CP(10 -3 M)
22 22
356 286
902 551
2.53 + 0.08 * 1.93+0.08 **
57 53
376 299
6.60 + 0.33 * 5.64+0.28 *
MMC (10 -6 M) Ethanol (0.1 M)
22 22
410 303
1872 1503
4.57 +0.12 * 4.96 + 0.12 *
55 51
586 544
10.65 + 0.57 * 10.67 + 0.46 *
Dimethyl sulfoxide
22
1363
2173
1.59 + 0.04
236
1038
BP (10 -5 M) BP ( 5 × 1 0 -6 M) 2-AF (10 -4 M)
Observed Schromosome number
Total SCEs in S-chromosomes
Viciafaba
Total SCEs in M-chromosomes
Mean S C E s / M chromosome ± SEM
4.40 + 0.14
The root cells were incubated in a BrdUrd solution for 17 h and treated with each chemical solution for 3 h. After being rinsed in tap water, they were incubated in a dThd solution for 22 or 25 h, in a colchicine solution for 2 h and then fixed. * p
187
MUT 04829
Induction of chromosome damage by benzo[ a ]pyrene, 2-aminofluorene and cyclophosphamide in the root cells of Vicia faba Nobuhiro Kanaya Department of Biology, Keio University, Yokohama223 (Japan) (Received12 May 1989) (Revision received22 August 1989) (Accepted 19 September1989)
Keywords: Sister-chromatidexchange; Chromosome aberration; Benzo[a]pyrene;2-Aminofluorene;Cyclophosphamide; Viciafaba Summary The induction of sister-chromatid exchanges (SCEs) and chromosome aberrations (CAs) by benzo[a]pyrene (BP), 2-aminofiuorene (2-AF) and cyclophosphamide (CP) in the root cells of Vicia faba was examined. BP and 2-AF induced CAs, but not SCEs. CP induced both SCEs and CAs.
It has been reported that many carcinogenic promutagens are activated by plants. The activation of promutagens in plant cells in vivo and induction of mutations or chromosome aberrations (CAs) have been reviewed by Velerninsk~, and Gichner (1988). Extracts from plant tissues have been found to activate promutagens in vitro, resulting in increased mutation in Salmonella typhimurium (Higashi et al., 1981; Plewa and Gentile, 1982; Gentile and Plewa, 1988). Takehisa et al. (1988) reported that benzo[a]pyrene (BP) and 2-aminofluorene (2-AF), wellknown carcinogenic promutagens, were not activated by extracts from Vicia faba roots (Vicia S10) and there was no increase in the frequency of sister-chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells. In contrast, cyclophosphamide (CP), another carcinogenic promutagen, was observed to be activated by Vicia S10 and the
Correspondence: Dr. Nobuhiro Kanaya, Keio University, Department of Biology,Yokohama 223 (Japan).
SCE frequency in C H O cells was increased (Takehisa et al., 1988). It would thus seem pertinent to determine whether the activation of promutagens can occur in intact roots of Vicia faba. Since no report is presently available on the induction of SCEs by BP, 2-AF or CP in the root cells of Vicia faba, an examination of this matter was made in the present study. The induction of CAs by these chemicals was also examined. Materials and methods
Cultivation of Vicia faba Seeds of Vicia faba, cultivar. Wase (Sakata Seed Co., Yokohama), were soaked in running tap water for 24 h, and allowed to germinate in moist Vermiculite for 3 - 4 days at 24 ° C. Seedlings with main roots of 3 - 6 cm length were used for the experiments. Chemicals BP (CAS No. 50-32-8, Wako Pure Chem., Osaka) and 2-AF (CAS No. 153-78-6, Aldrich,
0027-5107/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
188
Milwaukee, WI) were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chem., Osaka), and diluted with distilled water. The concentration of DMSO in each solution was less than 0.2%. CP (CAS No. 50-18-0, Shionogi, Osaka) was dissolved in distilled water. The positive controls, mitomycin C (MMC; CAS No. 50-07-7, Kyowa Hakko, Tokyo) and ethanol (CAS No. 64-17-5, Wako Pure Chem., Osaka), were dissolved in distilled water. All chemicals were prepared immediately before use.
SCE analysis The seedlings were incubated in a 5-bromodeoxyuridine (BrdUrd) solution (100 /~M BrdUrd, 0.1 /~M 5-fluorodeoxyuridine, and 5 /~M uridine dissolved in tap water) for 17 h at 2 0 ° C with aeration in the dark. This was followed by exposure to each chemical solution for 3 h at 20 ° C in the dark. After being rinsed in running tap water, the seedlings were incubated in an aerated thymidine (dThd) solution (100 # M dThd and 5 ~ M uridine dissolved in tap water) for 22 or 25 h at 20 ° C in the dark. The main roots were then cut at 1 cm length from the tips followed by immersion in 0.05% colchicine solution for 2 h at 20 o C. The root tips were then fixed in ethanol-acetic acid (3: 1) for 24 h at 4 ° C . Differential staining of sister chromatids was conducted by the methods of Kihlman and Kronborg (1975) and Schubert et al. (1979) with slight modifications. The fixed root tips were washed in tap water for 10 min and macerated with 1% pectinase solution dissolved in 0.01 M citric acid-sodium citrate buffer (pH 4.7) for 4 h at 37 ° C and then with 2% cellulase solution dissolved in the same buffer for 2 h at 37 ° C. The macerated root tips were squashed in 45% acetic acid. Coverslips were removed on dry ice and the preparations were passed through a 99.5, 85, 70, 50 and 30% graded ethanol series, distilled water and 0.5 x SSC (0.075 M NaC1 + 0.0075 M sodium citrate). This was followed by exposure to 0.01% R N a s e solution in 0.5 x SSC for 1 h at 37°C, dehydration in the ethanol series and air-drying. The preparations were then exposed to 0.0005% Hoechst 33258 solution dissolved in 0.5 x SSC for 30 min. After being rinsed in distilled water, the preparations were mounted with 0.5 × SSC, placed
on a hot plate at 60 °C, and exposed to a black light lamp (Ultra-Violet Products, Inc., U.S.A.; model XX-15) for 30-35 min at 1 cm distance. They were then rinsed in 0.5 x SSC, incubated for 1 h in 2 x S S C (0.3 M N a C I + 0 . 0 3 M sodium citrate) at 80 ° C, and stained with 4% Giemsa in 0.067 M phosphate buffer (pH 6.8) for 20 rain. The preparations were rinsed in distilled water, air-dried and mounted with Eukitt. A complete metaphase cell of Vicia faba contains 2 long metacentric chromosomes with secondary constrictions (M-chromosomes) and 10 shorter subtelocentric c h r o m o s o m e s (S-chromosomes). Since it was difficult to find a metaphase cell with a complete chromosome number in these FPG-stained preparations, cells with incomplete as well as complete chromosome numbers were analyzed. Thus at least 50 M-chromosomes and 250 S-chromosomes were analyzed for each treatment. SCEs which occurred at the secondary constrictions of M-chromosomes and at the centromeres of both M- and S-chromosomes were included in the scores. Statistical analysis was performed by Student's t-test.
Chromosome aberration analysis The seedlings were treated with each chemical solution for 3 h at room temperature (22 + 2 ° C ) in the dark. After being rinsed in running tap water, they were incubated in aerated tap water for 22 h at 20 ° C in the dark. The main roots were then cut at 1 cm length from the tips, immersed in 0.05% colchicine solution for 2 h at 2 0 ° C , and fixed in ethanol-acetic acid (3 : 1) for 24 h at 4 ° C. The root tips were rinsed in running tap water, hydrolyzed in 1 N HCI at 60 ° C for 8-10 rain, and stained by the Feulgen staining reaction. They were then squashed in 45% acetic acid, and the coverslips were removed on dry ice. The preparations were air-dried and mounted with Eukitt. At least 100 metaphase cells with complete chromosome numbers were observed for each treatment. Chromatid-type aberrations such as chromatid breaks, isochromatid breaks, and chromatid exchanges but not gaps were counted. The isochromatid breaks at the secondary constrictions of M-chromosomes were not counted. Statistical analysis was conducted by Student's t-test.
189
Results
The results for induction of SCEs by BP, 2-AF, CP, MMC and ethanol in Vicia faba are summarized in Table 1. The mean SCE frequencies among seedlings were different. The range of the SCE frequencies among the control seedlings treated with 0.1% DMSO followed by incubation in dThd solution for 22 h was 1.27-2.09/S-chromosome and 3.69-5.77/M-chromosome. The SCE value of each chromosome was pooled and the mean SCEs/chromosome are presented in Table 1. The mean SCE frequencies of the control cells were 1.59/S-chromosome and 4.40/M-chromosome. The ratio of the SCE frequency in the M-chromosome to that in the S-chromosome was 1:0.36, approximately equal to the ratio of the mean length of the M-chromosome to that of the S-chromosome (1 : 0.44). The treatment with 10 -6 M MMC, a positive control, resulted in increases in SCEs in both Sand M-chromosomes (Table 1). The SCE frequency induced by MMC was about 3 times that of the control. The range of the SCE frequen-
cies in the MMC-treated seedlings was 4.11-5.43/ S-chromosome and 9.33-13.88/M-chromosome, which was beyond that in the control seedlings. As shown in Table 1, the SCE frequency induced by 0.1 M ethanol, another positive control, was about 3 times that of the control. The SCE range among the ethanol-treated seedlings was 4.88-4.98/Schromosome and 10.17-10.82/M-chromosome. The treatment with BP (2 × 1 0 - 5 - 5 x 1 0 - 6 M) or 2-AF (10-4-2.5 × 10 -5 M) followed by incubation in dThd solution for 22 h failed to increase SCEs in either S- or M-chromosomes (Table 1). The SCE frequencies of root cells incubated for 25 h in dThd solution after treatment with BP or 2-AF were also almost the same as those of the control cells. The range of SCE frequencies in the BP-treated seedlings was 1.24-1.79/S-chromosome and 2.88-5.33/M-chromosome, and that in the 2-AF-treated seedlings was 1.40-1.60/S-chromosome and 3.85-4.41/M-chromosome, i.e., within the control level. On the other hand, CP treatment resulted in a dose-dependent increase in SCEs. The mean SCE frequencies in cells treated with 10 - 2 M CP was 2.53/S-chromosome and
TABLE 1 I N D U C T I O N OF SISTER-CHROMATID EXCHANGES BY BP, 2-AF, CP, MMC A N D E T H A N O L IN Treatment
dThd (h)
Mean SCEs/Schromosome + SEM
Observed Mchromosome number
BP ( 2 × 1 0 -5 M)
22 25 22 25 22
355 412 599 352 254
441 649 956 528 426
1.24+0.06 1.58 + 0.06 1.60+0.05 1.50 + 0.06 1.68-t-0.09
53 53 106 52 66
174 241 425 230 311
3.28+0.27 4.55 + 0.26 4.01 +0.19 4.42 + 0.31 4.71+0.29
2-AF (5 x l 0 -5 M) 2-AF (2.5 × 10 - s M)
22 25 22 22
899 430 427 358
1257 668 683 537
1.40 + 0.04 1.55 + 0.06 1.60+0.06 1.50+0.06
117 75 54 59
451 317 221 260
3.85 + 0.20 4.23 + 0.25 4.09+0.23 4.41+0.28
CP (10- 2 M) CP(10 -3 M)
22 22
356 286
902 551
2.53 + 0.08 * 1.93+0.08 **
57 53
376 299
6.60 + 0.33 * 5.64+0.28 *
MMC (10 -6 M) Ethanol (0.1 M)
22 22
410 303
1872 1503
4.57 +0.12 * 4.96 + 0.12 *
55 51
586 544
10.65 + 0.57 * 10.67 + 0.46 *
Dimethyl sulfoxide
22
1363
2173
1.59 + 0.04
236
1038
BP (10 -5 M) BP ( 5 × 1 0 -6 M) 2-AF (10 -4 M)
Observed Schromosome number
Total SCEs in S-chromosomes
Viciafaba
Total SCEs in M-chromosomes
Mean S C E s / M chromosome ± SEM
4.40 + 0.14
The root cells were incubated in a BrdUrd solution for 17 h and treated with each chemical solution for 3 h. After being rinsed in tap water, they were incubated in a dThd solution for 22 or 25 h, in a colchicine solution for 2 h and then fixed. * p
