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Chromosomal aberrations induced by dialkylating agents in allium cepa root-tips and their relation to the mitotic cycle and DNA synthesis
Radiation
Botany,
1968,
Vol.
8, pp. 489 to 497.
Pergamon
Press.
Printed
in Great
Britain.
CHROMOSOMAL ABERRATIONS INDUCED BY DIALKYLATING AGENTS IN ALLIUM CEPA ROOT-TIPS AND THEIR RELATION TO THE MITOTIC CYCLE AND DNA SYNTHESIS R. Department
of Genetics,
University
(Received
MATAGNE of Liege,
16
15, rue
Forgeur,
Liege,
Belgique
July 1968)
Abstract-The alkylating agents r.-diepoxybutane and the products of in viho transformation of L-threitol-1, 4-bismethanesulfonate induce the same kinds of chromosomal abnormalities in Allium cepa root-tip cells. The aberrations (chromatid type) are observed 6-8 hr after the beginning of treatment : the effect is delayed. The proportion of some types of aberrations varies with the sampling time; in particular, the relative frequency of chromatid interchanges increases with time. From 8 to 16 hr after treatment, most aberrations are localized in (or near) heterochromatic segments but the localization is not so high at later times. After treatment, the mitotic cycle, mainly the DNA synthesis phase (S) is protracted. The aberrations are induced when the chemical is given during the S and the G, period. G, and mitosis are completely insensitive to the chemical. The localization of aberrations in specific chromosome segments for cells treated at the end of S period is interpreted in relation to the asynchronous replication of DNA in heterochromatin. R&u&-Des agents d’alkylation : le L-diepoxybutane et les produits de transformation in vitro du L-thrtitol-1, 4-bismethanesulfonate induisent les memes types d’anomalies chromosomiques dans les cellules de meristtmes radiculaires d’dllium cepa. Les aberrations (appartenant h la classe chromatidiquc) ssnt observees 6-8 hr aprts le debut du traitement : il y a effet retard& La proportion de certains types d’aberrations varie en fonction du moment ou les racines sont prelevees; ainsi, la frequence relative des interchangements chromatidiques augmente avec le temps. De la 8eme a la 16eme heure qui suit le traitement, la plupart des aberrations sont localisees dans les segments heterochromatiques ou dans leur voisinage; le degrt de localisation diminue au tours des prelevements ulttrieurs. Le traitement a pour effet d’allonger la duree du cycle mitotique et plus particulierement la ptriode de synthtse du DNA (S). 0 n observe des aberrations dans les cellules qui ont ete traittes pendant les periodes S et G,. La phase G, et la mitose sont complttement insensibles a l’action du toxique. La localisation des lesions dans certains segments specifiques du genome dans le cas des cellules traittes en fin de ptriode S peut s’interpreter sur la base de la duplication asynchrone du DNA dans l’htttrochromatine. ‘Zusammenfassung-Die alkylierenden Age&en r.-Diepoxybutan und die in vitro Transformation von r.-Threitol1,4-bismethylsulfonat induzieren bei Zellen spitzen von Allium cepa die gleiche Art von chromosomalen Anomalien. Die (Chromatidentyp) werden 6-8 Std. nach Beginn der Behandlung beobachtet: ist verzogert. Der Anteil einigerTypen von Aberrationen variiertje nachzeitpunktder insbesondere nimmt die relative Haufigkeit des Chromatidenaustausches mit 8 bis 16 Std. nach der Behandlung sind die meisten Aberrationen in (oder 489
Produkte der der WurzelAberrationen die Wirkung Au&se; der Zeit zu. nahe) hetero-
R. MATAGNE
490
chromatischen Segmenten lokalisiert; die Lokalisationsrate ist aber zu spateren Zeiten nicht so hoch. Nach der Behandlung dauert der mitotische Zyklus, in der Hauptsache die Phase der DNSSynthese (S), linger. Die Aberrationen werden wihrend dcr S- und der G, -Periode induziert. G, -Periode und Mitose sind den Chemikalien gegentiber vollig unempfindlich. Die Lokalisierung von Aberrationen in bestimmten Chromosomensegmenten wird fur Zellen, die gegen Ende der S-Phase behandelt wurden. internretiert in ihrem Verhlltnis zur asynchronen Replikation der DNS im Heterochromatin.
INTRODUCTION IN THE root me&em of Vi&z, alkylating induce
chromatid
aberrations
which
agents appear
8-12 hr after treatment: the effect is delayed in comparison with radiations so they were first considered as acting only in the early part of interphase.c61 20* w Many authors have attempted to relate the nuclear DNA synthesis (S period) with the production of aberrations. Taking into account their estimate of G,, S and G, in Vicia root-tips, HOWARD and PELSO) interpreted REVELL’S results(22)
with
diepoxypropylether
in
Vi&
on
the view that only G, cells were sensitive to the chemical. KIHLMAN(~~) and RIECER(~~) suggested that chemicals with ‘delayed effect’ could induce chromatid aberrations only when the cell is in S period at the time of treatment. These authors gave no information about a possible mitotic delay due to treatment. Autoradiographic experiments recently performed in V~CLZ(~J 4l 7-g) and in human cells cultured in vitro have allowed a precise estimate of the period of mitotic cycle sensitive to certain chemical agents. After treatment with alkylating agents in conjunction with 3H-thymidine, it was shown that chromatid aberrations are mainly induced at the S period, phase of DNA replication. G, is not sensitive to chemical attack. The sites within the chromosome complement at which the aberrations are produced, have also been largely investigated. In Vi&, chromatid aberrations are generally localized within the nucleolar constriction and the heterochromatic segments although the proportion of localized aberrations can vary with the alkylating agent usedt2t 119 1% 24) and the experimental conditions.07n 20) The localization or the types of aberrations also change with the stage of S period at which the cell is treated.(% 71 8) The
variable distribution of breaks in Vinu has been interpreted in relation to the asynchronous replication of DNA within the different chromosome segments.c4) The aim of the present work is to relate chromosomal abnormalities induced by two dialkylating agents with the mitotic cycle in Allium cepu root-tips. The parameter values of the mitotic cycle as well as the pattern of DNA replication have been previously determined in the same material.(r6) To evaluate the induced mitotic delay and the sensitivity of the different phases of the cycle, an experiment combining alkylating agent and 3H-thymidine was carried out. MATERIALS AND METHODS Bulbs of Al&m cepu var. Stuttgart (small size variety) were cultivated on aerated tap water at 21 f0.2”C. After 3 days of culture, the roots were treated for 1 hr with a 0.1 mM (8.6 mg/l) of L-diepoxybutane (L-DEB) :
/O\
CH,-&H--CH
-
cH,
lo/ Another dialkylating agent, bismethanesulfonate (L-TBMS) some experiments : OH CH,-SO,-0-CH,-AH-CH-CH,
L-DEB L-threitol-1, was used
4in
I OH
-0--SO,-CH, L-TBMS For L-TBMS, roots were treated either with a fresh solution in distilled water (pH 5.5) or a solution of the compound completely degraded (pH 5.5-6). In this case, the chemical transformation was performed in such conditions that most of L-TBMS is probably converted into L-DEB (experimental details were given pre-
CHROMOSOMAL
ABERRATIONS
INDUCED
viously).trs) Contrariwise, in a fresh solution of L-TBMS kept at 21 “C and pH 5.5 for 1 hr, no measurable amount of the compound is transformed. After treatment, the roots were carefully washed and transferred to tap water. Roots were fixed (Carnoy) at various time elapsing after treatment and prepared as Feulgen squashes. For metaphase analysis root-tips were dipped (l-2 hr) in Colchicine (0.5 g/l) before fixation. In Figures and Tables, the fixation times are expressed as times from beginning of treatment. In a labelling experiment, roots were immersed (1 hr) in a solution (2 &/ml) of 3Hthymidine (C.E.N. Mol; s.a. 3.5 C/mM) used alone or in mixture with ‘transformed’ TBMS 0.1 mM. The autoradiographic procedure has been previously described. (le)
RESULTS 1. Egkcts of L-DEB First anaphase aberrations are observed 6 hr after beginning of treatment: the effect is delayed. The aberration rate increases and a maximum is reached after 26 hr (Fig. 1). Mitotic index in treated meristems drops from the 8th hr after treatment and remains lower than in control until about 20 hr. For the
B 2 0” 9d la;: E P s .< m-
low
mitotic
index
20 FIXATION
FIG. 1. Treatment:
30 TIME
(h 1
L-DEB, 0.1 mM/l hr. Percentage of abnormal anaphases (with fragments, microfragments or dicentric bridges) in relation to fixation time (200-300 anaphases analyzed per fixation). Confidence limits (95 per cent) are given for each point.
BY DIALKYLATING
AGENTS
491
same period, the percentage of aberrations does not vary much. The metaphase aberrations observed during the 32 hr post-treatment belong to the chromatid class: chromatid breaks (B’), sister-reunions (SR), chromatid exchanges (inter- and intra-), chromatid gaps (G’), minutes (< 0.5 CL). Achromatic lesions of sister-chromatids at the same level (isochromatidgaps : G”) are frequently observed (see Table 1). In Allium, these gaps cannot be confused with negatively heteropyknotic segments of heterochromatin as described in TIicinfaba (no intercalary heterochromatin occurs in onion chromosomes). These G” have to be considered separately from breaks with no sister-reunion (B”) since the chromosome is not actually broken. Moreover, B” have never been observed in our slides whereas G” are frequent. If it is assumed that alkylating agents induce only aberrations of the chromatid class, it should be considered that B” are true chromosome breaks (in agreement with REVELL@~)) and not isochromatid breaks (NUpd or non-union proximal and distal) according to the interpretation of CATCHESIDE et al.(l) Some metaphase aberrations are presented in Fig. 2 (a-d). Abnormal metaphases observed for the 32 hr following treatment arise from cells which perform their first division after treatment. In all cases, the deficient chromosomes and complementary acentric fragments occur in the same cell. Moreover, mitotic cells containing micronuclei are never observed. The mitotic cycle-mean duration 23 hr in control roots(16) -is almost certainly protracted after treatment with L-DEB. It can be deduced from Table 1 that the frequency of each type of aberrations increases for the 32 hr post-treatment. However, this increase is different for some types: SR’s occur with a constant relative frequency in the various sampling times whereas the proportion of interchanges increases in later samples (Fig. 3). The induction of interchanges is then favoured when cells are in the earlier part of the sensitive period at the time of treatment. The localization of breaks in the chromosome complement has also been investigated. If DEB induced breaks are preferentially localized
R. MATAGNE
492 Table
Fixation time, hr 8 12 16 24 28 32
Total metaphases checked
1. .Numbers
G”
69 69 100 85 90 100
2 4 9 4 7 17
SR
G’
2 8 9 12 15 24
FIXATION
3. Treatment: L-DEB, SR and tetraradials (per relation to fixation time. cent) are given FIG.
of aberrations
B
1 0 0 7 8 2
TIME
after treatment
Intrachanges
1 1 4 1 1 4
( h 1
0.1 mM/l hr. Numbers of hundred aberrations) in Confidence limits (95 per for each point.
in heterochromatic region as in the case of ViGiafaba,(30) one should find a high number of aberrations in segments near centromeres (C,), Table 2.
Fixation time, hr 8 12 16 24 28 32
Treatment
Total metaphases checked 69 69 100 85 90 100
: L-DEB (0.1 mM/l
Total No. of localized aberrations 6 14 24 26 35 58
(100) (100) (100) (100) (100) (100)
with
L-DEB (0.1 mM/l
hr)
Interchanges Tetraradials Triradials
0 0 0 1 3 8
0 1 1 1 0 3
0 0 1 0 1 2
Min
Total
0 3 4 6 9 5
6 17 28 32 44 65
telomeres (tel) and secondary constrictions (C,), since in A&urn, these are the regions which are heterochromatic.(12-14127) In Table 2, aberrations are classified according to their localization within the chromosome complement. Aberrations like minutes, impossible to localize with accuracy have been ignored. Most aberrations observed at the first sampling times occur in (or near) heterochromatic regions. In these, the frequency of aberrations does not vary much from the 12th to the 32nd hr. Contrariwise, the frequency of aberrations induced in euchromatin increases with the time of sampling. As shown by the relative percentages of aberrations in the different segments, the degree of localization in heterochromatin is high at the beginning and decreases later on: the ratio, No. of aberrations in heterochromatin/ No. of aberrations in euchromatin, varies from 5.2 to 0.6.
hr). Number of aberrations of the genome
Cl 1 (16.7) 3 (21.4) 2 (8.3) 3 (11.5) 3 ( 8.6) 5 ( 8.6)
Numbers in parentheses are relative percentages of aberrations
localized
in the dzyerent
No. of aberrations Heterochromatic C, 0 1 2 1 0 2
(0) (7.1) (8.3) (3.8) (0) (3.4)
segments
in segments tel 4 6 12 7 8 14
(66.7) (42.9) (50.0) (26.9) (22.9) (24.1)
(for 100 aberrations).
Euchromatic 1 4 8 15 24 37
(16.7) (28.6) (33.3) (57.7) (68.5) (63.8)
FIG. 2. Aberrations induced after treatment with L-DEB (0.1 mM/l hr) : A. Chromatid break (B’) in telomeric region. x 1750. B. Sister-reunion in the centric fragment (SRC,) in the segment adjacent to centromere. x 1500. C. Chromatid intrachange. x 1600. D. Three dicentric triradials with exchange in distal regions, tetraradial and SRCoC,. x 1550. R.B. f.p. 492
CHROMOSOMAL Table
Fixation time, hr
3. .Numbers
Total metaphases checked
2 8 II 19 24 30
2. E$ects
100 100 100 152 171 150
of
L-TBMS
ABERRATIONS of chromafid
aberrations
INDUCED after treatment
BY DIALKYLATING
AGENTS
L-TBMS
with ‘tramformed’
493
(0.1 mM/l
hr)
Min
Total
Interchanges G”
SR
G’
B’
Intrachanges Tetraradials
0 5 3 15 15 25
0 1 8 18 29 29
0 1 2 6 9 8
and its products
qf
0 0 0 0 4 2
0 0 1 1 5 2
Triradials
0 0 0 1 2 3
0 0 0 1 1 3
0 2 2 10 9 11
0 9 16 52 74 83
Ira~ufoormation
After treatments (1 hr) with fresh solutions of L-TBMS at various concentrations (up to 50 no significant level of chromosomal n-W, aberrations is ever reached, which agrees with previous results in barley.(r6) When L-TBMS is chemically transformed in vitro (See Materials and Methods), and subsequently used in root treatment (0.1 mM/l hr) chromosome breakage is induced. The types of aberrations and their percentages at different sampling times (deduced from Table 3) are comparable with those obtained with L-DEB. As for L-DEB, the aberrations induced by transformed L-TBMS are preferentially localized in heterochromatic segments at early sampling times : the ratio : No. aberrations in heterochromatin/No. aberrations in euchromatin is equal to six after 8 hr, but drops to 1.9 and 0.9 after 19 hr and 30 hr, respectively. Sensitivity of mitotic cycle To relate the induction of chromosomal aberrations with the different phases of mitotic cycle, root-tips were treated by transformed L-TBMS and sH-thymidine. The variation of percentages of labelled prophases (p) and anaphases + telophases (a + t) was analyzed for the 44 hr post-treatment (Fig. 4). The parameters of mitotic cycle were determined from the labelling curves according to the method previously described.(16J The comparison of the values of mitotic cycle parameters in control and treated root-tips (Table 4) cultivated under the same conditions 3.
t 0
6
16
24 FIXATION
32
40
TIME
( h )
FIG. 4. Labelling index curves showing the relationship between the percentage of labelled mitotic phases (solid line : prophases ; broken line : anaphases + telophases) and the time of fixation after beginning of combined treatment (3H-thymidine : 2 PC/ml and transformed L-TBMS : 0.1 mM/l hr) (300 phases observed at each fixation time).
Table 4. Mitotic cycle parameters Ytransfbrmed’ L-TMBS 0.1 mM/l
Mitotic cycle (T) Mitosis (M) Gl s G3
in control(l’J and treated hr) root-tibs at 2l’C.
Control, hr
Treated, hr
23 4 3.3 12 3.6
35.6 4.1 5.5 22 4
494
R. MATAGNE
indicates that the chemical treatment strikingly increases the duration of mitotic cycle T and more precisely the time of DNA synthesis. No significant difference was found between the duration of G, and mitosis in control and treated roots. As the value of G, is a biased estimate (G,=I-S-Gs-M), it is difficult to conclude that this phase is significantly modified by the treatment. These results indicate that metaphases observed during the 32 hr following treatment are at their first posttreatment mitosis, as suggested above. In this experiment, abnormal anaphases are observed from the 8th hour after treatment (alkylatingagent+SH-thymidine).Theseaberrations have probably been induced by the alkylating agent since in roots immersed for 1 hr in a 3H-thymidine solution at the same concentration (2 @/ml), such abnormalities are never detected. The question arises whether the lengthening of mitotic cycle is identical for cells with aberrations and for apparently undamaged cells. The variation of percentage of labelled anaphases in the two cellular populations : anaphases without visible chromosomal abnormalities and aberrant ones, is presented in Fig. 5. Since anaphase aberrations do not occur during the first 8 hours after treatment, it is not possible to draw the labelling curve of abnormal anaphases for that period. It can be concluded from Fig. 5 that the two cell populations progress through the mitotic cycle and more particularly through the S phase, at the same rate. The percentages of abnormal anaphases after the combined treatment 3H-thymidine ‘transformed’ L-TBMS have been recorded during the 35 hr following the treatment (solid line in Fig. 6). The abnormal anaphases labelled (broken line) and non labelled (dotted line) have been distinguished. It can be deduced from the curve of abnormal anaphases (solid line) that beginning of mitosis (M) and G, are insensitive to the chemical. The first aberrations are observed in cells which were at the end of S at the time of treatment. The percentage of abnormal anaphases increases and reaches a maximum for the cells treated at the beginning of S.
20OL 0
!
' 6
I 24
16
FIXATION
I 32
I 40
TIME
FIG. 5. Combined treatment: SH-thymidine formed L-TBMS (as in Fig. 4). Relationship
( h 1
+ trans-
between percentage of labelled anaphases and time of fixation after beginning of combined treatment. Open circles: No. of labelled undamaged an@hases/ 100 undamaged anaphases. Closed circles : No. of labelled damaged anaphases/ 100 damaged anaphases.
6
16
24 FIXATION
32 TIME ( h)
FIG. 6. Combined treatment: SH-thymidine + transformed L-TBMS (as in Fig. 4). Relationship between percentage of aberrant anaphases and fixation time after beginning of combined treatment. solid line: labelled + unlabelled anaphases with aberrations. broken line: labelled anaphases with aberrations. dotted line: unlabelled anaphases with aberrations,
CHROMOSOMAL
ABERRATIONS
INDUCED
From the 24th hr after treatment, unlabelled abnormal anaphases (dotted line) are observed and their percentage increases up to 34 hr. These cells were then in G, or previous mitosis at the time of treatment, as confirmed in Fig. 6.
DISCUSSION The analysis of chromosomal aberrations observed at different times during 30-35 hr after the beginning of treatment indicates that L-DEB and ‘transformed’ L-TBMS produce very comparable effects when utilized at the same dose. The analogy confirms the expectation(15) according to which L-TBMS is quantitatively transformed into L-DEB in our in vitro conditions. When L-TBMS is given to root-tip cells in such a way that no measurable transformation can occur during the treatment (i.e. in acid solution for a short time), no chromosomal aberration is induced : L-TBMS is inactive on chromosomes as suggested earlier.(r5) The aberrations, all of the chromatid class, are observed only about 6-8 hr after treatment and their percentage increases during the posttreatment period : the compounds reproduce the ‘delayed effect’ described in I%in for alkylating agents or other chemicals (see introduction). The proportion of some types of aberration varies with the sampling time. This is particularly the case for chromatid interchanges : the relative frequency of these exchanges is increasing with the recovery period. A similar observation was made in Vin’a after treatment with several chemicals.(4~7~s~20) Chromatid interchanges are then more frequently produced when the cell is treated at an early stage of the sensitive phase, for both Allium and Vi& If one assumes that the aberrations are actually produced at the time of DNA replication and cannot be induced after it (cJ infia), the probability to obtain an interchange involving two chromosomal segments is the highest when all segments are synthesizing DNA at the same time, i.e. at the beginning of the S period in Allium. ~0) The sensitivity of the different phases of the mitotic cycle was also investigated. All the analyzed cells were in the first mitosis (T,)
BY DIALKYLATING
AGENTS
495
after treatment. The results indicate that the G, period and prophase are not sensitive to the alkylating agents : their duration is not modified and no chromatid aberration is induced during these periods. On the other hand, the duration of DNA synthesis period (S) is considerably increased and aberrations are induced during this period, with a maximum for cells which were at the beginning of S at the time of treatment. Similar sensitivity of S was demonstrated in Vicia after treatment with maleic hydrazide,c4) diethylsulfate,(Q) nitrosomethyl and -ethyl urethane,(71s) nitrogen mustard.(6) From the 24th to the 35th hr after treatment (Fig. 6)) the percentage of abnormal unlabelled anaphases is increasing: these aberrant cells were then in G, at the time of treatment. All the aberrations are of the chromatid class; chromosome aberrations have never been observed which is in contrast with radiations.(2g) The same situation was found in Vicia after treatment with maleic hydrazidec4) and nitrogen mustard.c5) The fact that chromosome aberrations have never been detected indicates that the observed aberrations (of chromatid class) are not effectively induced during G,. A possible explanation(4) could be that during G,, the chemical is in some way bound to the chromosome and this lesion would result in a breakage at the time of DNA synthesis. Moreover, from personal unpublished data, it can be concluded that, for aberrations to be produced in the second (T,) mitosis, treatment in mitosis, G, or S is also highly efficient. This result fully agrees with those obtained with Myleran(ls) and nitrogen mustardc5) on other plant materials. The present work also indicates that aberrations appearing at first (induced at the end of S) are mainly localized in heterochromatic regions of the chromosome complement and that the proportion of localized aberrations is not so high at later times by the fact that the frequency of aberrations in euchromatin is increasing. Since at the end of S, the DNA replication is mainly restricted to hcterochromatin in Allium chromosonles,(l”) it could be concluded that chromosome is sensitive to chemical action, only at the time of DNA replication. It could then be considered that the localization of aberrations (for cells treated in S) indicates
R. MATAGNE
496 with a great precision in the chromosome
the time segments.
of DNA
synthesis
Acknowledgements-The chemicals were generously supplied by Dr P. W. FEIT, from Leo Pharmaceutical Products, Copenhagen. I am grateful to Prof. J. MOUT~CHEN (Genetics Department, Liege) for critical reading of the manuscript.
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25. RISER
R. and tidenaberrationen methanesulfonat auf Primarwurzeln 8,230-243.
MICHAELIS A. (1960) Chromanach Einwirkung von Athyl(Methansulfonsaurelthylester) von Vicia faba L. KulturpfZanze
CHROMOSOMAL 26.
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INDUCED
SCOTT D. (1968) The additive effect of X-rays and maleic hydrazide in inducing chromosomal aberrations at different stages of the mitotic cycle in Vicia faba. Mutation Res. 5, 65-92. SHARMA A. K. (1951) Trichloracetic acid and Feulgen staining. Nature 167, 441-442. TSUENG-HSXNG C. and ELEQUIN F. T. (1967) Induction of chromosome aberrations in cultured human cells by ethylenimine and its relation to cell cycle. Mutation Res. 4, 8339.
29.
30.
BY DIALKYLATING
AGENTS
497
WOLFF S. and LUIPPOLD H. E. (1964) Chromosome splitting as revealed by combined X-ray and labeling experiments. &!I!/. Cell Res. 34, 548-556. ZI?K J. and SWIETLIASKA Z. (1966) Localized chromosome breakage induced in Vicia faba with dispoxybutane. Symp. on the Mutational process. pp. 391-395. Prague, 1965.
Botany,
1968,
Vol.
8, pp. 489 to 497.
Pergamon
Press.
Printed
in Great
Britain.
CHROMOSOMAL ABERRATIONS INDUCED BY DIALKYLATING AGENTS IN ALLIUM CEPA ROOT-TIPS AND THEIR RELATION TO THE MITOTIC CYCLE AND DNA SYNTHESIS R. Department
of Genetics,
University
(Received
MATAGNE of Liege,
16
15, rue
Forgeur,
Liege,
Belgique
July 1968)
Abstract-The alkylating agents r.-diepoxybutane and the products of in viho transformation of L-threitol-1, 4-bismethanesulfonate induce the same kinds of chromosomal abnormalities in Allium cepa root-tip cells. The aberrations (chromatid type) are observed 6-8 hr after the beginning of treatment : the effect is delayed. The proportion of some types of aberrations varies with the sampling time; in particular, the relative frequency of chromatid interchanges increases with time. From 8 to 16 hr after treatment, most aberrations are localized in (or near) heterochromatic segments but the localization is not so high at later times. After treatment, the mitotic cycle, mainly the DNA synthesis phase (S) is protracted. The aberrations are induced when the chemical is given during the S and the G, period. G, and mitosis are completely insensitive to the chemical. The localization of aberrations in specific chromosome segments for cells treated at the end of S period is interpreted in relation to the asynchronous replication of DNA in heterochromatin. R&u&-Des agents d’alkylation : le L-diepoxybutane et les produits de transformation in vitro du L-thrtitol-1, 4-bismethanesulfonate induisent les memes types d’anomalies chromosomiques dans les cellules de meristtmes radiculaires d’dllium cepa. Les aberrations (appartenant h la classe chromatidiquc) ssnt observees 6-8 hr aprts le debut du traitement : il y a effet retard& La proportion de certains types d’aberrations varie en fonction du moment ou les racines sont prelevees; ainsi, la frequence relative des interchangements chromatidiques augmente avec le temps. De la 8eme a la 16eme heure qui suit le traitement, la plupart des aberrations sont localisees dans les segments heterochromatiques ou dans leur voisinage; le degrt de localisation diminue au tours des prelevements ulttrieurs. Le traitement a pour effet d’allonger la duree du cycle mitotique et plus particulierement la ptriode de synthtse du DNA (S). 0 n observe des aberrations dans les cellules qui ont ete traittes pendant les periodes S et G,. La phase G, et la mitose sont complttement insensibles a l’action du toxique. La localisation des lesions dans certains segments specifiques du genome dans le cas des cellules traittes en fin de ptriode S peut s’interpreter sur la base de la duplication asynchrone du DNA dans l’htttrochromatine. ‘Zusammenfassung-Die alkylierenden Age&en r.-Diepoxybutan und die in vitro Transformation von r.-Threitol1,4-bismethylsulfonat induzieren bei Zellen spitzen von Allium cepa die gleiche Art von chromosomalen Anomalien. Die (Chromatidentyp) werden 6-8 Std. nach Beginn der Behandlung beobachtet: ist verzogert. Der Anteil einigerTypen von Aberrationen variiertje nachzeitpunktder insbesondere nimmt die relative Haufigkeit des Chromatidenaustausches mit 8 bis 16 Std. nach der Behandlung sind die meisten Aberrationen in (oder 489
Produkte der der WurzelAberrationen die Wirkung Au&se; der Zeit zu. nahe) hetero-
R. MATAGNE
490
chromatischen Segmenten lokalisiert; die Lokalisationsrate ist aber zu spateren Zeiten nicht so hoch. Nach der Behandlung dauert der mitotische Zyklus, in der Hauptsache die Phase der DNSSynthese (S), linger. Die Aberrationen werden wihrend dcr S- und der G, -Periode induziert. G, -Periode und Mitose sind den Chemikalien gegentiber vollig unempfindlich. Die Lokalisierung von Aberrationen in bestimmten Chromosomensegmenten wird fur Zellen, die gegen Ende der S-Phase behandelt wurden. internretiert in ihrem Verhlltnis zur asynchronen Replikation der DNS im Heterochromatin.
INTRODUCTION IN THE root me&em of Vi&z, alkylating induce
chromatid
aberrations
which
agents appear
8-12 hr after treatment: the effect is delayed in comparison with radiations so they were first considered as acting only in the early part of interphase.c61 20* w Many authors have attempted to relate the nuclear DNA synthesis (S period) with the production of aberrations. Taking into account their estimate of G,, S and G, in Vicia root-tips, HOWARD and PELSO) interpreted REVELL’S results(22)
with
diepoxypropylether
in
Vi&
on
the view that only G, cells were sensitive to the chemical. KIHLMAN(~~) and RIECER(~~) suggested that chemicals with ‘delayed effect’ could induce chromatid aberrations only when the cell is in S period at the time of treatment. These authors gave no information about a possible mitotic delay due to treatment. Autoradiographic experiments recently performed in V~CLZ(~J 4l 7-g) and in human cells cultured in vitro have allowed a precise estimate of the period of mitotic cycle sensitive to certain chemical agents. After treatment with alkylating agents in conjunction with 3H-thymidine, it was shown that chromatid aberrations are mainly induced at the S period, phase of DNA replication. G, is not sensitive to chemical attack. The sites within the chromosome complement at which the aberrations are produced, have also been largely investigated. In Vi&, chromatid aberrations are generally localized within the nucleolar constriction and the heterochromatic segments although the proportion of localized aberrations can vary with the alkylating agent usedt2t 119 1% 24) and the experimental conditions.07n 20) The localization or the types of aberrations also change with the stage of S period at which the cell is treated.(% 71 8) The
variable distribution of breaks in Vinu has been interpreted in relation to the asynchronous replication of DNA within the different chromosome segments.c4) The aim of the present work is to relate chromosomal abnormalities induced by two dialkylating agents with the mitotic cycle in Allium cepu root-tips. The parameter values of the mitotic cycle as well as the pattern of DNA replication have been previously determined in the same material.(r6) To evaluate the induced mitotic delay and the sensitivity of the different phases of the cycle, an experiment combining alkylating agent and 3H-thymidine was carried out. MATERIALS AND METHODS Bulbs of Al&m cepu var. Stuttgart (small size variety) were cultivated on aerated tap water at 21 f0.2”C. After 3 days of culture, the roots were treated for 1 hr with a 0.1 mM (8.6 mg/l) of L-diepoxybutane (L-DEB) :
/O\
CH,-&H--CH
-
cH,
lo/ Another dialkylating agent, bismethanesulfonate (L-TBMS) some experiments : OH CH,-SO,-0-CH,-AH-CH-CH,
L-DEB L-threitol-1, was used
4in
I OH
-0--SO,-CH, L-TBMS For L-TBMS, roots were treated either with a fresh solution in distilled water (pH 5.5) or a solution of the compound completely degraded (pH 5.5-6). In this case, the chemical transformation was performed in such conditions that most of L-TBMS is probably converted into L-DEB (experimental details were given pre-
CHROMOSOMAL
ABERRATIONS
INDUCED
viously).trs) Contrariwise, in a fresh solution of L-TBMS kept at 21 “C and pH 5.5 for 1 hr, no measurable amount of the compound is transformed. After treatment, the roots were carefully washed and transferred to tap water. Roots were fixed (Carnoy) at various time elapsing after treatment and prepared as Feulgen squashes. For metaphase analysis root-tips were dipped (l-2 hr) in Colchicine (0.5 g/l) before fixation. In Figures and Tables, the fixation times are expressed as times from beginning of treatment. In a labelling experiment, roots were immersed (1 hr) in a solution (2 &/ml) of 3Hthymidine (C.E.N. Mol; s.a. 3.5 C/mM) used alone or in mixture with ‘transformed’ TBMS 0.1 mM. The autoradiographic procedure has been previously described. (le)
RESULTS 1. Egkcts of L-DEB First anaphase aberrations are observed 6 hr after beginning of treatment: the effect is delayed. The aberration rate increases and a maximum is reached after 26 hr (Fig. 1). Mitotic index in treated meristems drops from the 8th hr after treatment and remains lower than in control until about 20 hr. For the
B 2 0” 9d la;: E P s .< m-
low
mitotic
index
20 FIXATION
FIG. 1. Treatment:
30 TIME
(h 1
L-DEB, 0.1 mM/l hr. Percentage of abnormal anaphases (with fragments, microfragments or dicentric bridges) in relation to fixation time (200-300 anaphases analyzed per fixation). Confidence limits (95 per cent) are given for each point.
BY DIALKYLATING
AGENTS
491
same period, the percentage of aberrations does not vary much. The metaphase aberrations observed during the 32 hr post-treatment belong to the chromatid class: chromatid breaks (B’), sister-reunions (SR), chromatid exchanges (inter- and intra-), chromatid gaps (G’), minutes (< 0.5 CL). Achromatic lesions of sister-chromatids at the same level (isochromatidgaps : G”) are frequently observed (see Table 1). In Allium, these gaps cannot be confused with negatively heteropyknotic segments of heterochromatin as described in TIicinfaba (no intercalary heterochromatin occurs in onion chromosomes). These G” have to be considered separately from breaks with no sister-reunion (B”) since the chromosome is not actually broken. Moreover, B” have never been observed in our slides whereas G” are frequent. If it is assumed that alkylating agents induce only aberrations of the chromatid class, it should be considered that B” are true chromosome breaks (in agreement with REVELL@~)) and not isochromatid breaks (NUpd or non-union proximal and distal) according to the interpretation of CATCHESIDE et al.(l) Some metaphase aberrations are presented in Fig. 2 (a-d). Abnormal metaphases observed for the 32 hr following treatment arise from cells which perform their first division after treatment. In all cases, the deficient chromosomes and complementary acentric fragments occur in the same cell. Moreover, mitotic cells containing micronuclei are never observed. The mitotic cycle-mean duration 23 hr in control roots(16) -is almost certainly protracted after treatment with L-DEB. It can be deduced from Table 1 that the frequency of each type of aberrations increases for the 32 hr post-treatment. However, this increase is different for some types: SR’s occur with a constant relative frequency in the various sampling times whereas the proportion of interchanges increases in later samples (Fig. 3). The induction of interchanges is then favoured when cells are in the earlier part of the sensitive period at the time of treatment. The localization of breaks in the chromosome complement has also been investigated. If DEB induced breaks are preferentially localized
R. MATAGNE
492 Table
Fixation time, hr 8 12 16 24 28 32
Total metaphases checked
1. .Numbers
G”
69 69 100 85 90 100
2 4 9 4 7 17
SR
G’
2 8 9 12 15 24
FIXATION
3. Treatment: L-DEB, SR and tetraradials (per relation to fixation time. cent) are given FIG.
of aberrations
B
1 0 0 7 8 2
TIME
after treatment
Intrachanges
1 1 4 1 1 4
( h 1
0.1 mM/l hr. Numbers of hundred aberrations) in Confidence limits (95 per for each point.
in heterochromatic region as in the case of ViGiafaba,(30) one should find a high number of aberrations in segments near centromeres (C,), Table 2.
Fixation time, hr 8 12 16 24 28 32
Treatment
Total metaphases checked 69 69 100 85 90 100
: L-DEB (0.1 mM/l
Total No. of localized aberrations 6 14 24 26 35 58
(100) (100) (100) (100) (100) (100)
with
L-DEB (0.1 mM/l
hr)
Interchanges Tetraradials Triradials
0 0 0 1 3 8
0 1 1 1 0 3
0 0 1 0 1 2
Min
Total
0 3 4 6 9 5
6 17 28 32 44 65
telomeres (tel) and secondary constrictions (C,), since in A&urn, these are the regions which are heterochromatic.(12-14127) In Table 2, aberrations are classified according to their localization within the chromosome complement. Aberrations like minutes, impossible to localize with accuracy have been ignored. Most aberrations observed at the first sampling times occur in (or near) heterochromatic regions. In these, the frequency of aberrations does not vary much from the 12th to the 32nd hr. Contrariwise, the frequency of aberrations induced in euchromatin increases with the time of sampling. As shown by the relative percentages of aberrations in the different segments, the degree of localization in heterochromatin is high at the beginning and decreases later on: the ratio, No. of aberrations in heterochromatin/ No. of aberrations in euchromatin, varies from 5.2 to 0.6.
hr). Number of aberrations of the genome
Cl 1 (16.7) 3 (21.4) 2 (8.3) 3 (11.5) 3 ( 8.6) 5 ( 8.6)
Numbers in parentheses are relative percentages of aberrations
localized
in the dzyerent
No. of aberrations Heterochromatic C, 0 1 2 1 0 2
(0) (7.1) (8.3) (3.8) (0) (3.4)
segments
in segments tel 4 6 12 7 8 14
(66.7) (42.9) (50.0) (26.9) (22.9) (24.1)
(for 100 aberrations).
Euchromatic 1 4 8 15 24 37
(16.7) (28.6) (33.3) (57.7) (68.5) (63.8)
FIG. 2. Aberrations induced after treatment with L-DEB (0.1 mM/l hr) : A. Chromatid break (B’) in telomeric region. x 1750. B. Sister-reunion in the centric fragment (SRC,) in the segment adjacent to centromere. x 1500. C. Chromatid intrachange. x 1600. D. Three dicentric triradials with exchange in distal regions, tetraradial and SRCoC,. x 1550. R.B. f.p. 492
CHROMOSOMAL Table
Fixation time, hr
3. .Numbers
Total metaphases checked
2 8 II 19 24 30
2. E$ects
100 100 100 152 171 150
of
L-TBMS
ABERRATIONS of chromafid
aberrations
INDUCED after treatment
BY DIALKYLATING
AGENTS
L-TBMS
with ‘tramformed’
493
(0.1 mM/l
hr)
Min
Total
Interchanges G”
SR
G’
B’
Intrachanges Tetraradials
0 5 3 15 15 25
0 1 8 18 29 29
0 1 2 6 9 8
and its products
qf
0 0 0 0 4 2
0 0 1 1 5 2
Triradials
0 0 0 1 2 3
0 0 0 1 1 3
0 2 2 10 9 11
0 9 16 52 74 83
Ira~ufoormation
After treatments (1 hr) with fresh solutions of L-TBMS at various concentrations (up to 50 no significant level of chromosomal n-W, aberrations is ever reached, which agrees with previous results in barley.(r6) When L-TBMS is chemically transformed in vitro (See Materials and Methods), and subsequently used in root treatment (0.1 mM/l hr) chromosome breakage is induced. The types of aberrations and their percentages at different sampling times (deduced from Table 3) are comparable with those obtained with L-DEB. As for L-DEB, the aberrations induced by transformed L-TBMS are preferentially localized in heterochromatic segments at early sampling times : the ratio : No. aberrations in heterochromatin/No. aberrations in euchromatin is equal to six after 8 hr, but drops to 1.9 and 0.9 after 19 hr and 30 hr, respectively. Sensitivity of mitotic cycle To relate the induction of chromosomal aberrations with the different phases of mitotic cycle, root-tips were treated by transformed L-TBMS and sH-thymidine. The variation of percentages of labelled prophases (p) and anaphases + telophases (a + t) was analyzed for the 44 hr post-treatment (Fig. 4). The parameters of mitotic cycle were determined from the labelling curves according to the method previously described.(16J The comparison of the values of mitotic cycle parameters in control and treated root-tips (Table 4) cultivated under the same conditions 3.
t 0
6
16
24 FIXATION
32
40
TIME
( h )
FIG. 4. Labelling index curves showing the relationship between the percentage of labelled mitotic phases (solid line : prophases ; broken line : anaphases + telophases) and the time of fixation after beginning of combined treatment (3H-thymidine : 2 PC/ml and transformed L-TBMS : 0.1 mM/l hr) (300 phases observed at each fixation time).
Table 4. Mitotic cycle parameters Ytransfbrmed’ L-TMBS 0.1 mM/l
Mitotic cycle (T) Mitosis (M) Gl s G3
in control(l’J and treated hr) root-tibs at 2l’C.
Control, hr
Treated, hr
23 4 3.3 12 3.6
35.6 4.1 5.5 22 4
494
R. MATAGNE
indicates that the chemical treatment strikingly increases the duration of mitotic cycle T and more precisely the time of DNA synthesis. No significant difference was found between the duration of G, and mitosis in control and treated roots. As the value of G, is a biased estimate (G,=I-S-Gs-M), it is difficult to conclude that this phase is significantly modified by the treatment. These results indicate that metaphases observed during the 32 hr following treatment are at their first posttreatment mitosis, as suggested above. In this experiment, abnormal anaphases are observed from the 8th hour after treatment (alkylatingagent+SH-thymidine).Theseaberrations have probably been induced by the alkylating agent since in roots immersed for 1 hr in a 3H-thymidine solution at the same concentration (2 @/ml), such abnormalities are never detected. The question arises whether the lengthening of mitotic cycle is identical for cells with aberrations and for apparently undamaged cells. The variation of percentage of labelled anaphases in the two cellular populations : anaphases without visible chromosomal abnormalities and aberrant ones, is presented in Fig. 5. Since anaphase aberrations do not occur during the first 8 hours after treatment, it is not possible to draw the labelling curve of abnormal anaphases for that period. It can be concluded from Fig. 5 that the two cell populations progress through the mitotic cycle and more particularly through the S phase, at the same rate. The percentages of abnormal anaphases after the combined treatment 3H-thymidine ‘transformed’ L-TBMS have been recorded during the 35 hr following the treatment (solid line in Fig. 6). The abnormal anaphases labelled (broken line) and non labelled (dotted line) have been distinguished. It can be deduced from the curve of abnormal anaphases (solid line) that beginning of mitosis (M) and G, are insensitive to the chemical. The first aberrations are observed in cells which were at the end of S at the time of treatment. The percentage of abnormal anaphases increases and reaches a maximum for the cells treated at the beginning of S.
20OL 0
!
' 6
I 24
16
FIXATION
I 32
I 40
TIME
FIG. 5. Combined treatment: SH-thymidine formed L-TBMS (as in Fig. 4). Relationship
( h 1
+ trans-
between percentage of labelled anaphases and time of fixation after beginning of combined treatment. Open circles: No. of labelled undamaged an@hases/ 100 undamaged anaphases. Closed circles : No. of labelled damaged anaphases/ 100 damaged anaphases.
6
16
24 FIXATION
32 TIME ( h)
FIG. 6. Combined treatment: SH-thymidine + transformed L-TBMS (as in Fig. 4). Relationship between percentage of aberrant anaphases and fixation time after beginning of combined treatment. solid line: labelled + unlabelled anaphases with aberrations. broken line: labelled anaphases with aberrations. dotted line: unlabelled anaphases with aberrations,
CHROMOSOMAL
ABERRATIONS
INDUCED
From the 24th hr after treatment, unlabelled abnormal anaphases (dotted line) are observed and their percentage increases up to 34 hr. These cells were then in G, or previous mitosis at the time of treatment, as confirmed in Fig. 6.
DISCUSSION The analysis of chromosomal aberrations observed at different times during 30-35 hr after the beginning of treatment indicates that L-DEB and ‘transformed’ L-TBMS produce very comparable effects when utilized at the same dose. The analogy confirms the expectation(15) according to which L-TBMS is quantitatively transformed into L-DEB in our in vitro conditions. When L-TBMS is given to root-tip cells in such a way that no measurable transformation can occur during the treatment (i.e. in acid solution for a short time), no chromosomal aberration is induced : L-TBMS is inactive on chromosomes as suggested earlier.(r5) The aberrations, all of the chromatid class, are observed only about 6-8 hr after treatment and their percentage increases during the posttreatment period : the compounds reproduce the ‘delayed effect’ described in I%in for alkylating agents or other chemicals (see introduction). The proportion of some types of aberration varies with the sampling time. This is particularly the case for chromatid interchanges : the relative frequency of these exchanges is increasing with the recovery period. A similar observation was made in Vin’a after treatment with several chemicals.(4~7~s~20) Chromatid interchanges are then more frequently produced when the cell is treated at an early stage of the sensitive phase, for both Allium and Vi& If one assumes that the aberrations are actually produced at the time of DNA replication and cannot be induced after it (cJ infia), the probability to obtain an interchange involving two chromosomal segments is the highest when all segments are synthesizing DNA at the same time, i.e. at the beginning of the S period in Allium. ~0) The sensitivity of the different phases of the mitotic cycle was also investigated. All the analyzed cells were in the first mitosis (T,)
BY DIALKYLATING
AGENTS
495
after treatment. The results indicate that the G, period and prophase are not sensitive to the alkylating agents : their duration is not modified and no chromatid aberration is induced during these periods. On the other hand, the duration of DNA synthesis period (S) is considerably increased and aberrations are induced during this period, with a maximum for cells which were at the beginning of S at the time of treatment. Similar sensitivity of S was demonstrated in Vicia after treatment with maleic hydrazide,c4) diethylsulfate,(Q) nitrosomethyl and -ethyl urethane,(71s) nitrogen mustard.(6) From the 24th to the 35th hr after treatment (Fig. 6)) the percentage of abnormal unlabelled anaphases is increasing: these aberrant cells were then in G, at the time of treatment. All the aberrations are of the chromatid class; chromosome aberrations have never been observed which is in contrast with radiations.(2g) The same situation was found in Vicia after treatment with maleic hydrazidec4) and nitrogen mustard.c5) The fact that chromosome aberrations have never been detected indicates that the observed aberrations (of chromatid class) are not effectively induced during G,. A possible explanation(4) could be that during G,, the chemical is in some way bound to the chromosome and this lesion would result in a breakage at the time of DNA synthesis. Moreover, from personal unpublished data, it can be concluded that, for aberrations to be produced in the second (T,) mitosis, treatment in mitosis, G, or S is also highly efficient. This result fully agrees with those obtained with Myleran(ls) and nitrogen mustardc5) on other plant materials. The present work also indicates that aberrations appearing at first (induced at the end of S) are mainly localized in heterochromatic regions of the chromosome complement and that the proportion of localized aberrations is not so high at later times by the fact that the frequency of aberrations in euchromatin is increasing. Since at the end of S, the DNA replication is mainly restricted to hcterochromatin in Allium chromosonles,(l”) it could be concluded that chromosome is sensitive to chemical action, only at the time of DNA replication. It could then be considered that the localization of aberrations (for cells treated in S) indicates
R. MATAGNE
496 with a great precision in the chromosome
the time segments.
of DNA
synthesis
Acknowledgements-The chemicals were generously supplied by Dr P. W. FEIT, from Leo Pharmaceutical Products, Copenhagen. I am grateful to Prof. J. MOUT~CHEN (Genetics Department, Liege) for critical reading of the manuscript.
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32,4494X38. 14. MFJCINEN Y. (1963) The mitotic cycle in Allium cepa, with special reference to the diurnal periodicity and to the seedling aberrations. Ann. Botan. Sot. zoo. Botan. Fennicae Vanamo 34, 1-61. R. (1967) Activity of L-threitol-1, 15. MATACNE 4-bismethanesulfonate on barley chromosomes in relation to chemical transformation during treatment. Mutation Res. 4, 621-630. R. (1968) Duration of mitotic cycle 16. MATACNE and patterns of DNA replication in chromosome of Allium cepa. Caryologia (in press). hydrazide 17. MCLEISH J. (1953) The action ofmaleic in Vicia. Heredity 6 (suppl.) 125-147. 18. MOUTSCHEN J. (1964) Les effets cytogenttiques des composes alkane sulfonate d’alkyl. Me’m. SOG. Roy. Sci. Liege 11, l-296. 19. MOUTSCHEN-DAHMEN J. and M. (1958) L’action du Myleran (dimethanesulfonyloxybutane) sur les chromosomes chez Hordeum sativum et Vicia faba. Hereditas 44, 18-36. comparison 20. OCKEY C. H. (1957) A quantitative between the cytotoxic effects produced by proflavine, acetylethyleneimine and triethylene melamine on root-tips of Vicia faba. 3. Genet. 55, 525-549. 21.
22. 23.
24.
OCICEY C. H. (1960) Chromatid aberrations induced by ethyleneimines. Erwin-Bauer-Gediichtni.r-Vorlesungen I. Abh. dt. Akad. Wiss., Berlin, Kl. Med. 1, 47-53. REVELL S. H. (1953) Chromosome breakage by X-rays and radiomimetic substances in Vicia. Heredity 6 (suppl.), 107-124. REVELL S. H. (1961) An attempt at continuous metaphase estimation of chromatid and chromosome aberration frequencies in broad bean root me&em cells in the period 2-23 hr after 50 r of X-rays. Proc. Symp. Effects of ionizing radiations on see&, pp. 229-240. IAEA, Vienna. RIECER R. (1965) On the experimental induction of chromosomal aberrations in plants. In Induction of mutations and the mutation process. (Edited by VELEMINSKY J. and GICHNER T.) pp. 85-100. Proc. Symp. Prague, 1963.
25. RISER
R. and tidenaberrationen methanesulfonat auf Primarwurzeln 8,230-243.
MICHAELIS A. (1960) Chromanach Einwirkung von Athyl(Methansulfonsaurelthylester) von Vicia faba L. KulturpfZanze
CHROMOSOMAL 26.
27. 28.
ABERRATIONS
INDUCED
SCOTT D. (1968) The additive effect of X-rays and maleic hydrazide in inducing chromosomal aberrations at different stages of the mitotic cycle in Vicia faba. Mutation Res. 5, 65-92. SHARMA A. K. (1951) Trichloracetic acid and Feulgen staining. Nature 167, 441-442. TSUENG-HSXNG C. and ELEQUIN F. T. (1967) Induction of chromosome aberrations in cultured human cells by ethylenimine and its relation to cell cycle. Mutation Res. 4, 8339.
29.
30.
BY DIALKYLATING
AGENTS
497
WOLFF S. and LUIPPOLD H. E. (1964) Chromosome splitting as revealed by combined X-ray and labeling experiments. &!I!/. Cell Res. 34, 548-556. ZI?K J. and SWIETLIASKA Z. (1966) Localized chromosome breakage induced in Vicia faba with dispoxybutane. Symp. on the Mutational process. pp. 391-395. Prague, 1965.