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The effect of chloramphenicol on the chromosome-breaking action of 8-ethoxycaffeine and 6-methylcoumarin in Vicia faba
Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands
615
T H E E F F E C T OF CHLORAMPHENICOL ON T H E CHROMOSOME-BREAKING ACTION OF 8 - E T H O X Y C A F F E I N E AND 6-METHYLCOUMARIN IN
VICIA FABA V I T T O R I A NUTI RONCHI AND M. B U I A T T I Regional Institute for Grain Culture, and Institute of Genetics, University of Pisa (Italy) (Received J a n u a r y 3ist, 1967)
SUMMARY
The influence of the protein synthesis inhibitor chloramphenicol (CHL) on the chromosome-breaking action of 8-ethoxycaffeine (EOC) and 6-methylcoumarin (6-MC) has been investigated in Vicia faba root meristems. CHL, when applied as a post-treatment, increased the frequency of true chromatid breaks, achromatic lesions (gaps) and subchromatid exchanges induced by EOC. When given with the mutagen, CHL failed to show any significant effect. A greater yield of subchromatid exchanges was obtained after a 6-MC treatment followed by CHL than after 6-MC alone; but less significant differences in chromatid break frequencies were observed. The possible implications of these findings for the problem of the mechanism of action of mutagens effective in the DNA-postsynthetic phase (G2) are discussed.
INTRODUCTION
Both EOC and 6-MC are most effective in inducing chromosome aberrations in cells exposed during the DNA postsynthetic phase (G,)',9,1°. A similar behaviour is shown by tetramethyluric acid and streptonigrin 7. The non-requirement of DNA synthesis for the chromosome-breaking action of EOC and 6-MC has stimulated a series of experiments on the influence of the protein synthesis inhibitor CHL on the mutagenic action of the 2 compounds. MATERIAL AND METHODS
L~teral roots of Viciafaba seedlings, grown in the dark at 19 ° i I°, were submitted to the following treatments: (I) EOC, 0.5" IO-2 M for 2 h; (2) EOC, 0.5" IO-* M in combination with 300 mg/1 of CHL for 2 h; (3) EOC, o.5.1o -3 M and I .io -* M for 2 h followed by CHL (300 mg/1) for i h; (4) 6-MC, 2. lO -3 M for 2 h; (5) 6-MC, 2. Io --a M for 2 h followed by CHL (300 rag/1 ) for I h. Abbreviations: CHL, chloramphenicol; EOC, 8-ethoxycaffeine; 6-MC, 6-methylcoumarin.
Mutation Res., 4 (1967) 615-619
616
v. NUTI RONCHI, M. BUIATTI
After treatment, the seedlings were thoroughly washed and transferred to tap water. Root tips were fixed 2, 4 and 6 h after treatment. In the CHL post-treatment, the period in CHL was considered as part of the recovery time. Anaphase and metaphase analyses for true chromatid breaks (B'), subchromatid exchanges and achromatic lesions (gaps) were carried out by using the Feulgen squash technique s. For metaphase analysis, the roots were treated for 2 h before fixation with 0.05% colchicine. RESULTS
8-Ethoxycaffeine Tables I and I I show the frequencies of chromatid breaks B' (chromatid fragments and chromatid bridges in anaphase, isolocus breaks and chromatid exchanges TABLE I FREQUENCIES
OF ABERRATIONS
SCORED
AT A N A P H A S E
AFTER
TREATMENT
WITH
0 . 5 • lO -3 M
EOC
ALONE AND FOLLOWED BY C H L POST-TREATMENT ( E O C + C H L )
Treatment
Hours of Number of Abnormal recovery cells analyzed anaphases (%)
B' per ioo cells
Subchromatid exchanges per ioo cells
Achromatic lesions per ioo cells
EOC alone EOC+CHL EOC alone EOC+CHL EOC alone EOC+CHL
2 2 4 4 6 6
5.96 19.65 9.44 22.44 1.31 11.53
47.Ol 92.77 13.38 21.39 0.66 5.I2
lO.59 30.72 42.51 81.28 3.28 32.69
151 166 127 187 152 156
F test P =
42.38 74.69 41.73 64.71 3.94 28.20 0.003
0.004
0.050
0.004
in metaphase), subchromatid exchanges (sub-chromatid breaks followed by reunion within and between chromosomes) and achromatic lesions (gaps) in cells treated with 0.5" lO-2 M EOC alone and followed b y a post-treatment with 300 rag/1 of CHL. In Tables I I I and IV, the corresponding results for treatments with I.lO -2 M EOC are presented. As m a y be seen, the post-treatment with CHL enhanced the chromosomebreaking effect of EOC at either concentration, the differences being more significant at the higher dose. Combined treatments with EOC and CHL, on the other hand, failed to yield clear results, the aberration frequencies induced by EOC alone and EOC plus CHL not being significantly different. The different kinds of aberration seem to be affected differently by the CHL post-treatment. The most striking differences were found in the frequencies of subchromatid exchanges after 2 h of recovery and in the frequencies of B' and achromatic lesions after 4 h. The non-significance of the differences in subchromatid exchange frequencies in metaphase at the lower dose of EOC can be ascribed to the difficulty in scoring such aberrations in metaphase and to the more pronounced action of CHL at the higher dose of the mutagen. The CHL post-treatment had no significant effect on the ratio bridges/fragment at anaphase when this ratio was taken as a parameter for an estimate of the amount of rejoining of broken chromosome ends.
Mutation Res., 4 (1967) 615-619
CHLORAMPHENICOL ON CHROMOSOME BREAKING IN
Vicia faba
617
TABLE II FREQUENCIES OF ABERRATIONS SCORED AT METAPHASE AFTER TREATMENT WITH 0. 5 • lO -2 M EO C FOLLOWED BY C H L POST-TREATMENT (EOC + CHL)
ALONE
AND
Treatment
Hours of recoverv
Number Abnormal of cells metaanalyzed phases (%)
Aberrations per zoo cells 1 II III I + II + chromatid isolocus chromatid I I I breaks breaks exchanges
Subchromatid Achromatic exchanges lesions per per zoo cells rOD cells
EOC alo ne EOC+CHL E O C alone EOC+CHL E O C alone EOC+CHL
2 2 4 4 6 6
162 157 18o 141 152 166
o.oo o.oo o.55 2.83 o.oo o.oo
16.66 29.93 5.55 11.34 o.oo 4.21
F test P=
41.93 52.17 3o.75 65.95 12.5o 18.1o
3.7 ° 3 .82 2.22 2.83 0.65 3.Ol
5.55 9.55 2.77 11.34 o.oo 5.42
9.25 13.37 5.54 17.oo 0.65 8.43
o.112
o.o51
29.62 26.75 32-77 93 .61 ii.IO 11.44
o.162
o.o2o
TABLE III FREQUENCIES OF ABERRATIONS SCORED AT ANAPHASE AFTER TREATMENT WITH I • I0 2 M E O C ALONE AND FOLLOWED BY C H L POST-TREATMENT ( E O C + C H L )
Treatment
Hours of Number of Abnormal recovery cells analyzed anaphases (%)
B' per zoo cells
Subchromatid exchanges per Ioo cells
Achromatic lesions per Ioo cells
E O C alone EOC+CHL EOC alone EOC+CHL E O C alone EOC+CHL
2 2 4 4 6 6
48.84 19.64 8.Ol 28.44 5.31 36.36
25.8o 177.77 6.87 41.28 2.12 1.31
48.64 23.21 27.09 66.97 9.04 33.33
217 191 262 lO9 188 132
62.67 96.42 31.29 68.8o 14.89 33.33
F test P=
o.iio
0.o03
T A B L E IV FREQUENCIES OF ABERRATIONS SCORED AT FOLLOWED BY C H L ( E O C + C H L )
METAPHASE
Treatment
Hours of recovery
Number Abnormal of cells metaanalyzed phases (%)
Aberrations per zoo cells I II III I + II + chromatid isolocus chromatid I I I breaks breaks exchanges
Subchromatid A chromatic lesions per exchanges per zoo cells zoo cells
EOC alone EOC+CHL EOC alone EOC + CHL EOC alone EOC+CHL
2 2 4 4 6 6
189 13o 115 IOI lO8 171
1.o5 o.oo o.oo 0.99 o.oo o.58
18.51 44.61 9.56 19.8o o.oo 5.26
F test P =
41.79 66.92 44.34 70.43 13.83 46.19
AFTER TREATMENT
8.99 4.61 3.47 7.92 2.77 4.67
4.23 9.23 lO.43 38.61 1.65 32.74
WITH
I4.27 13.84 13.9o 47.52 4.42 37.99 o.ooi
I" lO -2 M
o.oo3
EOC
ALONE
17.98 33.o 7 22.6o 56.43 II.II 3o.4 ° o.oo 5
6-Methylcoumarin Table V shows the frequencies of chromatid breaks (fragments and chromatid bridges), subchromatid exchanges and achromatic lesions after tl eatment with 6-MC, both alone and followed b y a CHL post-treatment. The frequencies of subchromatid exchanges and achromatic lesions were more affected b y CHL, whereas less significant differences were found between the 2 treatments as to chromatid breaks. Mutation Res., 4 (1967) 6 1 5 - 6 1 9
AND
618 TABLE
v . N U T I R O N C H I , M. B U I A T T I V
FREQUENCIES
OF A B E R R A T I O N S
ALONE AND FOLLOWED
BY" C H L
S C O R E D AT A N A P H A S E POST-TREATMENT
AFTER TREATMENT
WITH
2 • IO 3 2}I 6 - M C
(6-MC+CHL)
Treatment
Hours of Number of recovery cells analyzed
Abnormal anaphases (%)
B' per ioo cells
Subchromatid exchanges per too cells
Achromatic lesions per J:oo cells
6-MC alone 6-MC+CHL 6-MC alone 6-MC+CHL 6-MC alone 6-MC+ CHL
2 2 4 4 6 6
9.15 55.64 6.66 34.83 3.00 5.49
i. 73 5.64 3.33 7.74 1.oo 1.o9
6. i 8 76.61 2.66 26.45 i.oo 4.39
2.97 16.53 0.66 12.25 i.oo o.oo
0.026
404 248 15o 155 lOO 91
F test P --
0.002
DISCUSSION
The results obtained in the present study show a common effect of CHL on the chromosome breakage induced by EOC and 6-MC: subchromatid exchange frequencies are strikingly enhanced by both substances, and chromatid break frequencies only in EOC-treated root meristems. Moreover, in absence of CHL, 6-MC is almost ineffective in Vicia. In Allium, on the contrary, 6-MC induces a high frequency of aberrations and the effect of CHL, though significant, is less striking than in Vicia (V. NUTI RONCHI AND M. BUIATTI, unpublished results). Our results cannot be interpreted in terms of mitotic delay induced by CHL as the aberration peak occurred after 2 h recovery after treatments both with and without CHL; only a few aberrations were found after 30 rain and I h recovery. EOC is known to induce both subchromatic and chromatid breaksT, 1°essentially in G2; 6-MC, though exerting its chromosome-breaking effect mostly in G~ (all subchromatid breaks are induced in this stage), gives a low frequency of chromatid breaks during the DNA synthetic phase 9. In contrast to other G~-effective agents such as fluorodeoxyuridine 1, deoxyadenosine4,L cytosine arabinoside 6 and hydroxyurea s, EOC, X-rays, streptonigrin and 6-MC induce subchromatid exchanges and reunions. WOLFF 11 has shown that CHL, an inhibitor of protein synthesis, through inhibition of rejoining, keeps chromatid breaks open for a longer time. The effect of CHL observed in our experiments could probably be ascribed to a similar mechanism of action, limited to the G~ phase, and hence acting mainly on subchromatid exchanges. If protein synthesis is postulated to be necessary in G~ for the fast reconstitution, after breakage, of the structural integrity of chromosomes, then the action of CHL, delaying such a process, could increase the probability of subchromatid rearrangements. This hypothesis is supported by the observation that CHL is particularly active in increasing chromatid exchanges whereas it has little or no effect on isolocus break frequencies. Moreover such an effect is more striking after 4 and 6 than after 2 h recovery. This, indeed, is to be expected if CHL delays but does not suppress reunion of broken chromatid ends. The proposed explanation of our results could be of interest also in connection with the unsolved problem of the nature of achromatic lesions. Our results, showing a highly significant increase in achromatic lesions after treatments with CHL and both EOC and 6-MC, could support the hypothesis that Mutation Res., 4 (1967) 6 1 5 - 6 1 9
CHLORAMPHENICOL ON CHROMOSOME BREAKING IN
Viciafaba
619
achromatic lesions represent partial chromatid breaks (as proposed by D'AMATO AND D'AMATO-AVANZI~) which, owing to the negative effect on restitution of CHL, remain evident for a longer time. ACKNOWLEDGEMENTS
Our thanks are due to Prof. F. D'AMATO for his criticism, and to Mr. R. FIORIO for technical assistance. Part of the work was supported by the Consiglio Nazionale delle Ricerche, Centro Nazionale di Genetica (Italy). REFERENCES I BI~LL, S., AND S. WOLFF, Studies on the mechanism of the effect of fluorodeoxyuridine on chromosomes, Proc. Natl. Acad. Sci. (U.S.), 51 (1954) 195-2o2. 2 D'AMATO, F., Osservazioni critiche su alcuni metodi di impiego della reazione nucleale in citologia vegetale, Nuovo Giorn. Bot. Ital., (1946) 657-668. 3 D'AMATO, F., AND M. G. D'AMATO AVANZI, The chromosome breaking effect of coumarin derivatives in the Allium Test, Caryologia, 6 (1954) 134-149. 4 KIHLMAN, B. A., Deoxyiadenosine as an inducer of chromosomal aberrations in Vicia faba, J. Cellular Comp. Physiol., 62 (1963) 267-272. 5 KIHLMAN, B. A., The production of chromosomal aberrations by streptonigrin in Vicia faba, Mutation Res., i (1964) 54-62 6 KIHLMAN, B. A., W. W. NICHOLS AND A. LEVAN, The effect of deoxyadenosine and cytosine arabinoside on the chromosomes of human leucocytes in vitro, Hereditas, 5° (1963) 139-143. 7 K1HLMAN, B. A., AND K. G. ODMARK, Deoxyribonucleic acid synthesis and the production of chromosomal aberrations by streptonigrin, 8-ethoxycaffeine and 1,3,7,9-tetramethyluric acid, Mutation Res., 2 (1965) 494-5o 5. 8 KIHLMAN, B. A., T. ERIKSSON AND G. ODMARK,Effects of hydroxyurea on chromosomes, cell division and nucleic acid synthesis in Vicia faba, Hereditas, 55 (I966) 386-397 . 9 NoTI RONCHI, V., AND P. G. A.RCARA,in preparation. IO SCOTT, n., AND H. J. EVANS, On the non requirement for deoxyribonucleic acid synthesis in the production of chromosome aberrations by 8-ethoxycaffeine, Mutation Res., I (1964) 146156. 11 WOLFF, S., Radiation studies on the nature of chromosome breakage, Am. Naturalist, 94 (I96o) 85-93.
Mutation Res., 4 (I967) 615-619
615
T H E E F F E C T OF CHLORAMPHENICOL ON T H E CHROMOSOME-BREAKING ACTION OF 8 - E T H O X Y C A F F E I N E AND 6-METHYLCOUMARIN IN
VICIA FABA V I T T O R I A NUTI RONCHI AND M. B U I A T T I Regional Institute for Grain Culture, and Institute of Genetics, University of Pisa (Italy) (Received J a n u a r y 3ist, 1967)
SUMMARY
The influence of the protein synthesis inhibitor chloramphenicol (CHL) on the chromosome-breaking action of 8-ethoxycaffeine (EOC) and 6-methylcoumarin (6-MC) has been investigated in Vicia faba root meristems. CHL, when applied as a post-treatment, increased the frequency of true chromatid breaks, achromatic lesions (gaps) and subchromatid exchanges induced by EOC. When given with the mutagen, CHL failed to show any significant effect. A greater yield of subchromatid exchanges was obtained after a 6-MC treatment followed by CHL than after 6-MC alone; but less significant differences in chromatid break frequencies were observed. The possible implications of these findings for the problem of the mechanism of action of mutagens effective in the DNA-postsynthetic phase (G2) are discussed.
INTRODUCTION
Both EOC and 6-MC are most effective in inducing chromosome aberrations in cells exposed during the DNA postsynthetic phase (G,)',9,1°. A similar behaviour is shown by tetramethyluric acid and streptonigrin 7. The non-requirement of DNA synthesis for the chromosome-breaking action of EOC and 6-MC has stimulated a series of experiments on the influence of the protein synthesis inhibitor CHL on the mutagenic action of the 2 compounds. MATERIAL AND METHODS
L~teral roots of Viciafaba seedlings, grown in the dark at 19 ° i I°, were submitted to the following treatments: (I) EOC, 0.5" IO-2 M for 2 h; (2) EOC, 0.5" IO-* M in combination with 300 mg/1 of CHL for 2 h; (3) EOC, o.5.1o -3 M and I .io -* M for 2 h followed by CHL (300 mg/1) for i h; (4) 6-MC, 2. lO -3 M for 2 h; (5) 6-MC, 2. Io --a M for 2 h followed by CHL (300 rag/1 ) for I h. Abbreviations: CHL, chloramphenicol; EOC, 8-ethoxycaffeine; 6-MC, 6-methylcoumarin.
Mutation Res., 4 (1967) 615-619
616
v. NUTI RONCHI, M. BUIATTI
After treatment, the seedlings were thoroughly washed and transferred to tap water. Root tips were fixed 2, 4 and 6 h after treatment. In the CHL post-treatment, the period in CHL was considered as part of the recovery time. Anaphase and metaphase analyses for true chromatid breaks (B'), subchromatid exchanges and achromatic lesions (gaps) were carried out by using the Feulgen squash technique s. For metaphase analysis, the roots were treated for 2 h before fixation with 0.05% colchicine. RESULTS
8-Ethoxycaffeine Tables I and I I show the frequencies of chromatid breaks B' (chromatid fragments and chromatid bridges in anaphase, isolocus breaks and chromatid exchanges TABLE I FREQUENCIES
OF ABERRATIONS
SCORED
AT A N A P H A S E
AFTER
TREATMENT
WITH
0 . 5 • lO -3 M
EOC
ALONE AND FOLLOWED BY C H L POST-TREATMENT ( E O C + C H L )
Treatment
Hours of Number of Abnormal recovery cells analyzed anaphases (%)
B' per ioo cells
Subchromatid exchanges per ioo cells
Achromatic lesions per ioo cells
EOC alone EOC+CHL EOC alone EOC+CHL EOC alone EOC+CHL
2 2 4 4 6 6
5.96 19.65 9.44 22.44 1.31 11.53
47.Ol 92.77 13.38 21.39 0.66 5.I2
lO.59 30.72 42.51 81.28 3.28 32.69
151 166 127 187 152 156
F test P =
42.38 74.69 41.73 64.71 3.94 28.20 0.003
0.004
0.050
0.004
in metaphase), subchromatid exchanges (sub-chromatid breaks followed by reunion within and between chromosomes) and achromatic lesions (gaps) in cells treated with 0.5" lO-2 M EOC alone and followed b y a post-treatment with 300 rag/1 of CHL. In Tables I I I and IV, the corresponding results for treatments with I.lO -2 M EOC are presented. As m a y be seen, the post-treatment with CHL enhanced the chromosomebreaking effect of EOC at either concentration, the differences being more significant at the higher dose. Combined treatments with EOC and CHL, on the other hand, failed to yield clear results, the aberration frequencies induced by EOC alone and EOC plus CHL not being significantly different. The different kinds of aberration seem to be affected differently by the CHL post-treatment. The most striking differences were found in the frequencies of subchromatid exchanges after 2 h of recovery and in the frequencies of B' and achromatic lesions after 4 h. The non-significance of the differences in subchromatid exchange frequencies in metaphase at the lower dose of EOC can be ascribed to the difficulty in scoring such aberrations in metaphase and to the more pronounced action of CHL at the higher dose of the mutagen. The CHL post-treatment had no significant effect on the ratio bridges/fragment at anaphase when this ratio was taken as a parameter for an estimate of the amount of rejoining of broken chromosome ends.
Mutation Res., 4 (1967) 615-619
CHLORAMPHENICOL ON CHROMOSOME BREAKING IN
Vicia faba
617
TABLE II FREQUENCIES OF ABERRATIONS SCORED AT METAPHASE AFTER TREATMENT WITH 0. 5 • lO -2 M EO C FOLLOWED BY C H L POST-TREATMENT (EOC + CHL)
ALONE
AND
Treatment
Hours of recoverv
Number Abnormal of cells metaanalyzed phases (%)
Aberrations per zoo cells 1 II III I + II + chromatid isolocus chromatid I I I breaks breaks exchanges
Subchromatid Achromatic exchanges lesions per per zoo cells rOD cells
EOC alo ne EOC+CHL E O C alone EOC+CHL E O C alone EOC+CHL
2 2 4 4 6 6
162 157 18o 141 152 166
o.oo o.oo o.55 2.83 o.oo o.oo
16.66 29.93 5.55 11.34 o.oo 4.21
F test P=
41.93 52.17 3o.75 65.95 12.5o 18.1o
3.7 ° 3 .82 2.22 2.83 0.65 3.Ol
5.55 9.55 2.77 11.34 o.oo 5.42
9.25 13.37 5.54 17.oo 0.65 8.43
o.112
o.o51
29.62 26.75 32-77 93 .61 ii.IO 11.44
o.162
o.o2o
TABLE III FREQUENCIES OF ABERRATIONS SCORED AT ANAPHASE AFTER TREATMENT WITH I • I0 2 M E O C ALONE AND FOLLOWED BY C H L POST-TREATMENT ( E O C + C H L )
Treatment
Hours of Number of Abnormal recovery cells analyzed anaphases (%)
B' per zoo cells
Subchromatid exchanges per Ioo cells
Achromatic lesions per Ioo cells
E O C alone EOC+CHL EOC alone EOC+CHL E O C alone EOC+CHL
2 2 4 4 6 6
48.84 19.64 8.Ol 28.44 5.31 36.36
25.8o 177.77 6.87 41.28 2.12 1.31
48.64 23.21 27.09 66.97 9.04 33.33
217 191 262 lO9 188 132
62.67 96.42 31.29 68.8o 14.89 33.33
F test P=
o.iio
0.o03
T A B L E IV FREQUENCIES OF ABERRATIONS SCORED AT FOLLOWED BY C H L ( E O C + C H L )
METAPHASE
Treatment
Hours of recovery
Number Abnormal of cells metaanalyzed phases (%)
Aberrations per zoo cells I II III I + II + chromatid isolocus chromatid I I I breaks breaks exchanges
Subchromatid A chromatic lesions per exchanges per zoo cells zoo cells
EOC alone EOC+CHL EOC alone EOC + CHL EOC alone EOC+CHL
2 2 4 4 6 6
189 13o 115 IOI lO8 171
1.o5 o.oo o.oo 0.99 o.oo o.58
18.51 44.61 9.56 19.8o o.oo 5.26
F test P =
41.79 66.92 44.34 70.43 13.83 46.19
AFTER TREATMENT
8.99 4.61 3.47 7.92 2.77 4.67
4.23 9.23 lO.43 38.61 1.65 32.74
WITH
I4.27 13.84 13.9o 47.52 4.42 37.99 o.ooi
I" lO -2 M
o.oo3
EOC
ALONE
17.98 33.o 7 22.6o 56.43 II.II 3o.4 ° o.oo 5
6-Methylcoumarin Table V shows the frequencies of chromatid breaks (fragments and chromatid bridges), subchromatid exchanges and achromatic lesions after tl eatment with 6-MC, both alone and followed b y a CHL post-treatment. The frequencies of subchromatid exchanges and achromatic lesions were more affected b y CHL, whereas less significant differences were found between the 2 treatments as to chromatid breaks. Mutation Res., 4 (1967) 6 1 5 - 6 1 9
AND
618 TABLE
v . N U T I R O N C H I , M. B U I A T T I V
FREQUENCIES
OF A B E R R A T I O N S
ALONE AND FOLLOWED
BY" C H L
S C O R E D AT A N A P H A S E POST-TREATMENT
AFTER TREATMENT
WITH
2 • IO 3 2}I 6 - M C
(6-MC+CHL)
Treatment
Hours of Number of recovery cells analyzed
Abnormal anaphases (%)
B' per ioo cells
Subchromatid exchanges per too cells
Achromatic lesions per J:oo cells
6-MC alone 6-MC+CHL 6-MC alone 6-MC+CHL 6-MC alone 6-MC+ CHL
2 2 4 4 6 6
9.15 55.64 6.66 34.83 3.00 5.49
i. 73 5.64 3.33 7.74 1.oo 1.o9
6. i 8 76.61 2.66 26.45 i.oo 4.39
2.97 16.53 0.66 12.25 i.oo o.oo
0.026
404 248 15o 155 lOO 91
F test P --
0.002
DISCUSSION
The results obtained in the present study show a common effect of CHL on the chromosome breakage induced by EOC and 6-MC: subchromatid exchange frequencies are strikingly enhanced by both substances, and chromatid break frequencies only in EOC-treated root meristems. Moreover, in absence of CHL, 6-MC is almost ineffective in Vicia. In Allium, on the contrary, 6-MC induces a high frequency of aberrations and the effect of CHL, though significant, is less striking than in Vicia (V. NUTI RONCHI AND M. BUIATTI, unpublished results). Our results cannot be interpreted in terms of mitotic delay induced by CHL as the aberration peak occurred after 2 h recovery after treatments both with and without CHL; only a few aberrations were found after 30 rain and I h recovery. EOC is known to induce both subchromatic and chromatid breaksT, 1°essentially in G2; 6-MC, though exerting its chromosome-breaking effect mostly in G~ (all subchromatid breaks are induced in this stage), gives a low frequency of chromatid breaks during the DNA synthetic phase 9. In contrast to other G~-effective agents such as fluorodeoxyuridine 1, deoxyadenosine4,L cytosine arabinoside 6 and hydroxyurea s, EOC, X-rays, streptonigrin and 6-MC induce subchromatid exchanges and reunions. WOLFF 11 has shown that CHL, an inhibitor of protein synthesis, through inhibition of rejoining, keeps chromatid breaks open for a longer time. The effect of CHL observed in our experiments could probably be ascribed to a similar mechanism of action, limited to the G~ phase, and hence acting mainly on subchromatid exchanges. If protein synthesis is postulated to be necessary in G~ for the fast reconstitution, after breakage, of the structural integrity of chromosomes, then the action of CHL, delaying such a process, could increase the probability of subchromatid rearrangements. This hypothesis is supported by the observation that CHL is particularly active in increasing chromatid exchanges whereas it has little or no effect on isolocus break frequencies. Moreover such an effect is more striking after 4 and 6 than after 2 h recovery. This, indeed, is to be expected if CHL delays but does not suppress reunion of broken chromatid ends. The proposed explanation of our results could be of interest also in connection with the unsolved problem of the nature of achromatic lesions. Our results, showing a highly significant increase in achromatic lesions after treatments with CHL and both EOC and 6-MC, could support the hypothesis that Mutation Res., 4 (1967) 6 1 5 - 6 1 9
CHLORAMPHENICOL ON CHROMOSOME BREAKING IN
Viciafaba
619
achromatic lesions represent partial chromatid breaks (as proposed by D'AMATO AND D'AMATO-AVANZI~) which, owing to the negative effect on restitution of CHL, remain evident for a longer time. ACKNOWLEDGEMENTS
Our thanks are due to Prof. F. D'AMATO for his criticism, and to Mr. R. FIORIO for technical assistance. Part of the work was supported by the Consiglio Nazionale delle Ricerche, Centro Nazionale di Genetica (Italy). REFERENCES I BI~LL, S., AND S. WOLFF, Studies on the mechanism of the effect of fluorodeoxyuridine on chromosomes, Proc. Natl. Acad. Sci. (U.S.), 51 (1954) 195-2o2. 2 D'AMATO, F., Osservazioni critiche su alcuni metodi di impiego della reazione nucleale in citologia vegetale, Nuovo Giorn. Bot. Ital., (1946) 657-668. 3 D'AMATO, F., AND M. G. D'AMATO AVANZI, The chromosome breaking effect of coumarin derivatives in the Allium Test, Caryologia, 6 (1954) 134-149. 4 KIHLMAN, B. A., Deoxyiadenosine as an inducer of chromosomal aberrations in Vicia faba, J. Cellular Comp. Physiol., 62 (1963) 267-272. 5 KIHLMAN, B. A., The production of chromosomal aberrations by streptonigrin in Vicia faba, Mutation Res., i (1964) 54-62 6 KIHLMAN, B. A., W. W. NICHOLS AND A. LEVAN, The effect of deoxyadenosine and cytosine arabinoside on the chromosomes of human leucocytes in vitro, Hereditas, 5° (1963) 139-143. 7 K1HLMAN, B. A., AND K. G. ODMARK, Deoxyribonucleic acid synthesis and the production of chromosomal aberrations by streptonigrin, 8-ethoxycaffeine and 1,3,7,9-tetramethyluric acid, Mutation Res., 2 (1965) 494-5o 5. 8 KIHLMAN, B. A., T. ERIKSSON AND G. ODMARK,Effects of hydroxyurea on chromosomes, cell division and nucleic acid synthesis in Vicia faba, Hereditas, 55 (I966) 386-397 . 9 NoTI RONCHI, V., AND P. G. A.RCARA,in preparation. IO SCOTT, n., AND H. J. EVANS, On the non requirement for deoxyribonucleic acid synthesis in the production of chromosome aberrations by 8-ethoxycaffeine, Mutation Res., I (1964) 146156. 11 WOLFF, S., Radiation studies on the nature of chromosome breakage, Am. Naturalist, 94 (I96o) 85-93.
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