Mutation Researrh, 268{1992) 167-191

J67

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MUT 0510q

Induction of chromosomal aberrations by camptothecin in root-tip cells of Vicia[aba H.C. Andersson and B.A. Kihlman l)¢partment of Genetics, Uppsala Unit'ersl~'. 5-750 ~rr Uppsala (.~;ivedel,~ (Received29 October 1991) (Revision received 24 D~ccmbcr 19911 (Accepted 8 Sanuap/1992)

Keya~rd~: Campioth¢¢in; Chrornosomai abcrratko~ in 16cia[aba; 5-Fluov.,~lcoxyuridinc;Hyd~oxs'urca:DNA topoisomerase I

Summary When root-tip cells of Viciafaba were exposed during early and middle interphas¢ to camptothccin (Cpt), the aberrations obtained were exclusively of the chromatid type and tended to be localized in late replicating helerochromatic regions of the chromosomes. In these respects the clastogcnic effect of Cpt resembles that of agents that act by an S-phase-dependent mechanism. In contrast to typical S-phase-dependent agents, Cpt produced lesions capable of giving rise to aberrations only in S-phase cells that were synthesizing DNA at the time of treatment. The dependence on ongoing DNA synthcsis was suggested in autoradiographic experiments and by the fact that the clastogenic effect of Cpt was strongly suppressed by hydroxyurea, an inhibitor of DNA synthesis. After Cpt treatments, there were many more cells with 3-12 aberrations and far fewer ce!ls with 0, 1 or 2 aberrations than expected on the basis of a Poisson distribution. Cpt further differed from typical g-phase-dependent agents by being capable of inducing lesions in the G z phase that give rise to chromosomal abc~ations in the first mitosis after treatment. This effect was obtained at Cpt concentrations around 1(/~.M, whereas only 0.03 ~M Cpt was required to produce chromatid aberrations in the S phase, Results of Gz-phas¢ ¢.,q~crim¢nts with Cp! and 5-fluorodeoxyuridin¢, an inhibitor of DNA synthesis, saggcst that DNA synthesis is required for the clastogenic effect of Cpt not only during the S phase, but also during the G z phase, although the DNA syntheses involved are both quantitatively and qualitatively different.

The ¢.tkaloid camptothccin (Cpt) was isolated from the stem wood of Camptotheca ao,minata (Nyssacea¢), a tree native to south-western China and Tibet (Wall et el., 1966). Cpt effectively suppresses cell cycle progression and growth, and exhibits a strong antitumour activity in several experimental turnouts (e.g., Gallo et al., 1971; Tobey and Crissman, 1972; Li ct al., 1972; Huang

Carresp~ndence: H.C. Andcrsson, Department of Gentiles. Uppsala University, Box 7003, S-750 07 Uppsala (S~cdcnL

et al., 19"/3}. At the molecular level Cpt inhibit q both DNA and RNA synthesis, and causes flagmentatio,t of DNA (¢.g., Horwitz ¢t al., 1971; Li e* al., 1972; Horwitz and Horwi~., 1973). pc. ccntly, type I DNA topoisomerasc was identified as the cellular target of Cpt (Hsiang et at., 1985; Hsiang and Liu, 19gq;; Eng et at., 1988). The DNA topoisomerases are ubiquitous enz-/mes that are invo|v~d in essential processes such as DNA replication, recombination, chromatid segTegation and transcription. The name 'topoisomerase' alludes to their function as cata-

168 lysts of intercot,versions between ;.opological isomers of DNA. They work by producing transient breaks in the DNA molecule through which other DNA strands can pass. "/'he type I topoisomerases break and rejoin one strand of the DNA double hdix, whereas the type II isomerases catalyze the breakage and rejoining of both. Certain steps of the topoisomerase lI (tope It) reaction arc energy-dependent and require the hydrolysis of ATP, whereas the topoisomera~ 1 (tope I) reaction is independent of ATP. During each cycle of strand breakage and reunion, a covalent protein-DNA intermediate is formed. Type !I topoisomerases bind covaiently to the 5'-phosphoryl end of the broken DNA strand, whereas euka~otic type 1 topoisomerases bind to the Yphosphoryi end. The covalent protein-DNA intermediate has been termed the 'cleavable complex'. It has recently been shown that the cleavable complex is trapped or stabilized by several important antitumour drugs. By treatment with a protein denaturant such as SDS (sodium dodecyl sulphate), the stabilized cleavable complcxcs arc converted into protein-linked DNA strand breaks. Drugs that stabilize the cleavable complex in the tope I! reaction arc for instance the cpipodophyllotoxins VP-16 (etoposide) and VM.26 (teniposide) and the acridine derivative m-AMSA (amsacrine). When cells are first trca*.ed with one of these drugs and then exposed to SDS, singleand double-strand breaks are formed in which one of the two subunits of the type II homodimer is linked to the 5'-phosphoryl end of each broken DNA strand. Cpt and some of its derivatives are the only drugs known to stabilize specifically the cleavable complex in the tope I reaction, When Cpt.stabilized cleavable complexes are treated with SDS, single-strand DNA breaks are formed in which the enzyme is linked to the 3'-phosphoryl end of the broken DNA strand. The mode of action of topoi~merases and topoisomcrasc-target!ng antitumour drugs has recently been reviewed by D'Arpa and Liu (1989) and by Smith (1990). The reader is referred to these reviews for detailed information and for references to original papers. There is an extensive amino acid homology between type I1 '.opoisomerascs isolated from dif-

ferenl types of organism, but also eukaryotic type I tOlX)ig>merases have regions that are highly conserved from yeast to man (Eng ct al., 1989; Sutcliffe etal., 1989; Wyckoff et al., 1989; Agris et al., 1990). Probably, these are the regions that are important for thc structure and function of the cn~mcs. This homology between enzymes from different sources is also likely to be the reason why tope I1 inhibitors such as m-AMSA and VM-26, and the tope l inhibitor Cpt arc effective not only in mammalian culls (Zwelling, 1985; Glisson and Ross, 1987; Hsiang and Liu, 1988; D'Arpa and Liu, 1989), but also in plant cells (Fukata etal., 1986; Champoux and Aronoff, 1989; Carballo ct al., 1991) and in yeast (Eng ¢t ai., 1988; Nitiss and Wang, 1988). Although there is strong evidence that the cytotoxicity of tope I and tope 1I poi.';ons is closely related to their ability to stabilize the cleavable complex, the chain,of events leading from the stabilized cleavable complex to cell death still remains obscure. It seems reasonable to assume that the first step must be the transformation of the reversible cleavable complex to a more permanent DNA damage. The cytotoxic effect of Cpt has proved to be dependent on DNA synthe. sis, and it has been suggested that irrcversible DNA damage is produced when a trapped dear. able complex collides with a replication fork (Holm et al.: 1989; Hsiang et al., 1989; D'Arpa and Liu, 1989). Like many other andJtumour agents, the topoisomerase poisons mentioned are efficient inducers of chromosomal aberrations and sister-chromatid exchanges (Huang et al., 1973, 1983; for rcfcrcnccs, scc also Backer ct al,, 1990). The chromosomal aberrations may well bc one direct cau~ of cell death (Co~nforth and Bedford, 1987; Bedford and Cornfonh, 1987; for earlier references see Kihlman. 1977). After having studied in human lymphocytcs the clastogenic effects of VP-16, VM-26 and m. AMSA (DcSalvia et al., 1987; Andersson and Kihlman, 1989; Andersson etal., in preparation), we tried to use the same drugs for the induction of chromosomal aberrations and sister-chromatid exchanges in root tips of Viola faba, but without success. This was somewhat surprising, since in vitro studies in other laboratories have shown

that plant cells contain a t.~e II topoisomerase that is inhibited by m-AMSA and VM-26 (Fukata et ai., 1986; Carballo et al., 1991). A possible reason for our failure could be that the comparatively large drug molecules were unable to enter the root-tip cells. Perhaps there is a permeability barrier at the cell walls of the root-tip cells similar to that previously observed in yeast, Saccharomyces cerevisiae (Nitiss and Wang, 1988). Not only m-AMSA, but also Cpt was prevented from entering the yeast cells by the permeability barrier. It is cvidem from the present paper, bowever, that in root-tip cells of VicJafaba there is no permeability barrier preventing the entrance of Cpt, since these cells proved to bc cxlrcmely sensitive to the clastogcnic effect of this drug. Materials and methods

The material consisted of lateral roots of the field bean, Hcia faba, var. minor. In the majority of the experiments the cv. "Herra' (standard karyotype) was used, but studies on the localization of chromosomal aberrations were carried out with the reconstructed karyotype 'ACB' (MichaeIts and Rieger, 1971) as experimental material. The cultivation of the material has been described previously (Kihlman and Ander~-son, 1984). The treatments were carried out on 1-2 cm long lateral roots of 9-day-old seedlings. For each treatment 2-4 seedlings were used. The clastogens and/or inhibitors of DNA syn. thesis used in this study were camptothecin (Cpt` CAS No. 7689-03.4), 1,2.dihydro.3,6-pyridazinc (maleic hydrazide, MH, CAS No. 123-33-1), N. methyl-N-nitrosourea (MNU, CAS No. 684-93..5), 5-fluorodeoxyuridine (FdUrd, CAS No. 30-91-9) and hydroxyurea (HU, CAS No. 127-07-1), all from Sigma Chemical Co. In some experiments thymidine (dThd, CAS No. 50.89.5) from Fiuka AG was used to reverse the effect of FdUrd.

lion). In one ~ries of experiments, howgvcr, the stock solution was dilut@ in 0.~7 M phosphate buffer, pH 6 (see Fig. 5). The same buffer was used for preparing solutions of MH, MNU and HU. The duration of the treatment was 2 or 3 h for Cpt, 2 h for MH and MNU and 3 h for HU. After the treatments, the roots ~crc thoroughly rinsed in tap water and the seedlings allowed to recover in lap water for periods of varying duration. The water was always changed after the first hour of the recovery period. The last 3 h before fixation, roots were exposed to a 0.05% ,'~,qution of colchicinc in distilled water.

G2 experiments The I0--" M stock solution or Cpt in DMSO was diluted with the disti",u water/tap water mixture or with 0.05% colehicine to obtain the appropriate concentrations for treatment. Similarly, FdUrd and dThd were dissolved either in the water mixture or in the colchicine solution to which the roots were exposed during the last 2.5 h before fixation. As a rule, the cells were scored for chromosomal aberrations at mctaphase, and the tools were then treated the last 2.5 h before fixation with fl.t)Se~ colchicine. The colchicine treatment was omitted when the effect was to be ~ r e d as 'abnormal ana-/tclophases' (aria-/ tclophases containing chromatid fragments and/or bridges). Both the Gt/S and the G: experiments were carried out at 20°C in the dark. More detailed information on the experiments is given in the next section. Roots were fixed in methyl alcohol-glacial acetic acid, 3 : ! , and slides were prepared as Fculgen squashes as described previously (Kihlman and Andersson, 1984). After each treatment, a total of at least 100 mctaphases (or ana-/telophases) were analyzed for chromosomal aberrations by both authors independently,

Autoradiographic ~periraems GI / S experiments Cpt was dissolved in dimethyl sulphoxide (DMSO) at a concentration of 10 -2 M and stored frozen at -20°C, Samples of this stock solution were usually diluted with a mixture of distilled water and tap water (1 : 1) to obtain the appropriate concentrations for treatment (see next ~c.

Information on the duration of the period between S phase and ana-/telophase in untreated cells and on the cell cycle stage at the lime Of the Cpt treatment was obtained by the autoradiographic technique. In the former case, lateral roots of 9-day-old seedlings were exposed for 2 h to 2 pCi/ml tritiated thymidine ([3H]dThd; spce.

I 7fl

act. 5 Ci/mM) and fixed in a;cohol-acetic acid without any previous colchicine treatment at 0.5, 1, 2, 3, 4 and 5 h after exposure. In the latter ca~, lhe lateral roots were exposed for 3 h ~o 2 p.Ci/ml [3H]dThd and 0.05 p.M Cpt and fixed at 19 h after the treatment. The last 3 h before f~ation, the roots were exposed to 0.05% colchicine. In both types of experiment, Fculgen squash preparations were made on slides coated with a mixture of 1[): 1 gelatine and chrome alum (RiedeI-Ha~n AG). Cover slips were removed on dry ice and the slides were air-dried after being passed through two changes of ethyl alcohol. Autoradiographs were then made by dipping the slides in K-5 emulsion (llford Scientific Products). After an 18-day exposure in the dark, the autoradiographs were developed in Kodak D-19 developer for 5 rain, rinsed in distilled water, fixed in Kodat'tx for 5 rain, washed in running tap water for about I h, air-dried and mounted. The labelling status (labelled or unlabelled) of cells in ana-/telophase {G2 experiments) or in metaphase (Cpt-trealed cells) was determined by scoring the number of sliver grains overlying the chromo,~(.Imes.

Results

G1/S phase experiments The aberTations produced by Cpt when root-tip cells were treated in early or middle interphasc were exclusively of the chromatid ~ype, an effect characteristic of clastogenic agents that act by an S-phase-dependent mechanism {Kihlman and Natarajan, 1984). Fig. 1 illu~strates the frequencies of chromatid aberrations obtained at 20 and at 23 h after 3-h treatments with various concentrations of Cpt. For roots fixed at 23 h, the do~-effect curve is incomplete because few or no mitoses were found after treatments with 3.3 and 10 #M Cpt. Nevertheless, it is evident that the two curves have very similar shapes with two peaks, one smaller at concentrations close to 0.1 #M and a main peak at 1.0 I~M Cpt. In both cases the frequency of chromatid aberrations obtained after treatments with 100 p.M Cpl was only 1/6 of that obtained after treatments with 1 p.M Cpt. This is indeed a very unusual do,e--effect curve, all the more ~ as

Z40

0 ¢i

1o

T:~o

+~o

Fig. 1. The frequenci¢~ of chrolaalid abcrralions ~b~.alncd in loot til~ o:[ k~c/a ]'aM at 20 (o) aad .?..~{x ) h after treatn~n! with various cnnccntralions of camplothe~n.

the inhibitory ,.fffcct on mitosis was similarly affected by the concentration of Cpt. Thus, there was a much stronger mitotic inhibition at i, 3.3, and 10 #M than at 33 and 100 #M Cpt (data not shown). Finally, it should be noted in Fig. | that very low concentrations of Cpt were required to produce chromosomal aberrations when root-tip cells of Viciafaba were treated in early or middle intcrphase. The c[astogcnic effect of Cpt in Vicia faba proved to be remarkablc also in other respects. At 19 h after 3-h treatments with 0.05 p.M Cpt, chromatid aberrations were found in 419 of the 1757 metaphases scored. The.~ 419 abnormal mctaphases contained a total of 1206 aberrations. As shown in Table 1, the number of mctaphases with 0, 1 or 2 aberrations was much lower, and the numer of metaphascs with 3-12 aberrations much higher than expected on the basis of a Poisson distribution. Evidently, thc relatively few cells affected by the Cpt treatment werc usually very heavily damaged. The photomicrographs in Fig. 2 show :,uch metaphases with heavily damaged chromosomes. The aberrations consisted of isochromatid breaks (A-D), chromafid intra- and interchanges (B, C), trlradials (A,'D) and small interstitial deletions (A-C), the last type of aberration being particularly characteristic of the clastogenic effect of Cpt in root-tip cells treated in early and middle interphase. Frequendy, two or more of these small interstitial deletions were found very close to. gether. This was never observed after treatment

with alkylating agents, such as mitomycin C (data not shown). Note aim that only three chromosomes are involved in the two tdradials in Fig. 2D. one of the triradials containing the centric, the other the acentric fragment of the same chromosome. We have further studied the distribution of Cpt-induced ehromatid aberrations between and within chromnmmes. To make this po.~sible, the reconstructed karyotype ACB (Michaelis and Rieger, 1971) was used. For the purpose of mapping chromatid aberrations, the metaphas~ chromosomes of Viciafaba (standard karyotype) were divided up into 28 segments by Rigger and Michaelis (1970). Since ka~oty- : ACB resulted from the combination by cro~' .,tg of two reciprocal translocations and one pericentrie inversion,

the 28 ~gments do nut always oc~r in numer]ca~ o:der. It is evident in Fig. 3 that the Cpt-indueed aberrations were not dLstributed at random between and within chromosomes, but were clustered in cer..aia segments. Such aberration "hot six)Is' ate ~gmcnts 15, 1l, 4, 19, 23, and 26. Fig. 4 shows the position and expressivity of aberration hot spots. The expressivity of hot spots is illustrated 'as the quotient fro) from the observed freouency of involvement of a ~gment in ehromatid aberrations (z i) and the upper confidence l',mit of the value (z()) expected for random involvement ia aberrations of the same segment; f0 = zi/z0' (Ricgcr et al., 1977). In the ~pes of experiment so tar described, bean roots were fixed at 19-23 h after exposure

3. A

C

Fig. 2. Camptulh~cin-indueedchromosomalahcrrat~s at metaphasc in root tips of ~cfi~faM. Rootswere fixedat '~ h (A-C) and lq h (D) after 3-h treatmentswith 0.~ v.M Cpt. (A-C) ACB karyol)lx; (D) ~tandardImryot~.

i't2

40

36 32 28 24 20 16, 12 8. 4' 0

Chromosome segment Fig. 3. TI~ percentage of camptothccin-induced isochxomatid breaks ( I , totally 504 al~rratioas), intercalary delctioas (n, 419 aberrations) and chromatid exchanges([3,334 abenafions) in the varJolL~;chromosome segrnenlsof the slructu~ly reconstructed ACB kar~otyp¢of V,:iufa~. The aberratioes were obtained at 19-23 h after 3-h Ircatm.entswith 0.05#M camptothccin.

exposed for 3 h to Cpt and [3H]dThd and f'=ed 19 h after the end of the combined treatment. Autoradiographs were then prepared and s:ored for chromatid aberrations and silver grains ovcrlying the chromosomes.

to Cpt. Very likely, the cells scored at mctaphase after these recovery periods were in early or middle interphase at tbe time of treatment. To obtain more exact information on the cell cycle stage at the time of Cpt treatment, roots were

61

Io IH

:

:

o 116651141516171e ~.d

Chromosome segment Fig. 4. Pofiti~nand ¢xprc~skityof abet'rationhot spotsobtainedat 19-23 h atl©ra 3-b treatmentwith 4).0~p.M ¢ampto~h¢cinin the r©constru-cledACB karyotypeof Vie'in [aba, Hot spots representchromosome scf,ments in~ved in aberration formation more [requentlythanexpectedon the basisof a randomdistribution.Aberrationt~cs as in Fig. 3.

t73 TABLE. 1 THE NUMBER OF ABERRATIONS 5CORED PER ANALYZED CELL AT 19-23 h AFTER A 3-h TREATMENT WITH 0,05 ~M CAMPTOTHECIN Number of Number aberra~ioos of cells pet celt obeyed

(o)

Number ( O - E ) "~ o/cells X2-~ E cxp~aed

(E)

0 1

13.]8 183

885A 606.2

2

56

208.2

231.4 295.4 111.2

5 6

32 12 It

57.2

263.6

Y ] 757

1757.0

X ~' - 90L6

7 S 9 10 II 12

Statistics:

dr=2 Xu.

= 5.99

p ( , 1 . 901.b) < 0.001

Of the 588 metaphases analyzed, 66 were found to contain a total of 114 chromatid aberrations. Four of these aberrations were in metapha~s scored as unlabelied, whereas the remaining ll(} were found in labelled metaphases. Thus. 96.5% of the aberrations scored were in labelled cells. The results of the autoradiographic experiment suggest that Cpt produced aberrations mainly, if not exclusively, in cells that were in the S phase. Possibly, ]3HA lesions resuhing in chromadd aberrations were produced by Cpt only in cells that were involved in replication of chromosomal DNA. if this is true, inhibitors of replicatire DNA synthesis such as ilU should suppress the clastogenic effect of Cpt. That this indeed is the cast is shown in Fig. 5. The frequency of chromatid ~berradons was strongly reduced when the Cpt h~atmcnt was carried out in the presence el HU. This is in marked contrast to the clastogenic effect of other S-dependent agents. As shown in Fig. 5, the frequencics of chromatid aberrations produced

by the S-dcpcndent agunts maicic hydrazidc and N-mcthyl-N-nitrosourca werc not at all affected by the presence of HU during treatment. Similar rcsuhs were obtained when FdUrd was substituted for HU (data not shown).

G 2-phaseexperiments Dcgrassi ct at. (1989) have recently reported that Cpt induces chromatid aberrations in human lymphocytes not only during the S phase, but also during the G z phase. This was an unexpected finding since agents that act by an S-phase-dependent mechanism, as a rule, do not produce DNA lesions in the G, phase that appear as chromosomal aberrations in thc first mitosis aftcr treatment (Kihlman and Natarajan, 1984). The finding was also unexpected on the basis of what is known about the function and mode of action of tope I, the cellular target of Cpt. Tope I has been shown to be involved in DNA replication, but apparently the enzyme is not required between DNA s3rathesis and mitosis for proces~s such as the separatio- of intertwined DNA

8

!

Fi

9

I C~

C~

OJ 'J tt"¢U

D,~U

Fig. 5. The effect of 5x10 -J M ~dro~.~r=a (HU) on th,: frequencies of ehnxnatid abcrr'dfi~5 i n d ~ d by 2-h Ircatmen~ with 5 x l 0 -~ M camptothccin (Cpt), 1,5×10 4 M maletc hy'dra2tde(MID or 8 x 10-4 M N-mcthyl-N.niu~.v~uurca (MNU), HU v,a~ actdtd I h before the clastogen treatmenl and removed togelher glth the elastolells. (Cpl. MH. MNU). Roots were fixed at 18 and 2b h (CI~) or 22 and 26 h (M] [ and MNU) after lrealmenL The dart item the two fixatiun tim¢~ were pooled,

15/~M C:pt + FdUrd

15 ~ M

t~

~0

I1

0.5 ~M FdUrd

1 5 ~ M Cp!

8

-

0,5 U M FdUrd

Cpt 4

200

I00 ~uM dThd

15 IzM Cpt

7

05 ~ M

21N)

I~ ,gM CpI

6

0.5 p M FdUrd

1~) p M dThd

~

2(X)

200

2~)

300

15 ,aM Cpl + 100 tzM dThd

O5 ~ M FdUrd

5

2llO

I011p M dThd

0.5 ,aM FdUrd

2110

2'lg)

4

[0O p M dThd

200

0.5/zM FdUrd

-

3

2

I

of anai~,zcd cells

During the cob chic[he esprwre (25 h)

N0.

During a 2-h period immcdial¢ly b~forc colchicinc ¢ xpo',,'ur c

Numb,'r

Treatment

2.5

39,0

37.0

35,0

26.0

.~5

9.0

0.5

31,0

O,O

0,11

(%)

met:,phases

Abnormal

0.5

I1.0

26_5

8.5

3.5

4.0

2,~

0.5

29.0

0.0

0.0

Chromatid hrcakg

1.5

2.0

i.5

~5

4.5

4,5

1.3

O.0

2.5

0.0

0.0

Isochro. matid hrea k~,

Abcrr'alions per I00 cells

0,0

3Z5

2.5

15_5

!q,.0

16.5

2.3

0.0

2.0

0,(I

0.0

Suhchro. matid exchang~

0.5

)15

0.0

21_S

25

18.5

3.1

l),(l

1.0

D,0

0.(.I

Chromatid c~ha|tgcs

2.5 + 1.0

57.0 + O5

30÷5 + 21.5

48.11+ 6.0

33.5 + 3,5

43_5 + 3.1t

9.0 + 2.0

11..~+ ill;'

34..5 + I"/.5

[L0 + [1.5

0.0+ U.0

gap,

4-

Total ahcrralkms

CHROMOSOMAL ADERRAT1ONS OBTAINED AT M E r A P H A S E AIq'ER TREATMENTS WITlt Cpz AND FdUrd. ALONE AND IN VARIOUS COMBINATIONS. AND W|TI[ OR WiTIIOUT dThd ADDED TO ]'HE COLClilCINE SOLL-I'ION TO WHICH THE ROOTS WERE EXPOSED DURING TI IF,, b%ST 2+5 h BI:,~)RE FIXATION

TABLE 2

~7.:.

daughter molecules following replication (D'Arpa

and Liu, 1989). The unexpected finding by Dcgr&ssi and coworkers was confirmed by the results of our studies in root-tip cells of Hcia [aba. No aberrations were produced when bean roots were treated during the G2 phase with Cpt at concentrations between 0.1 and 1 .~M, i.e., concentrations that were highly effective during the S phase. Chromosomal aberrations were found, however, when cells in G 2 phase were exposed for at least 2 h to Cpt concentrations around 10 #M. The aberrations consisted of ehromatid breaks and exchanges, isochromatid breaks with sister-chro. matid union (SU) and sub-chromatid exchanges (Table 2). The last type of aberration is probably formed during prophasc and, as the name implies, involves units that appear to comprise less than the whole width of the chromatid (Fig. 6A). In anapha~, sub-chromatid exchanges are easily detected because of the characteristic side-arm bridges they give rise.to (Fig. 6B). Like chromatid interchanges, sub-chromatid exchanges appear as exchanges of the chrom(~om¢ type (e.g., diccntrics) at the second division after their production, a fact that suggesLs that the difference between thcsc two types of aberration is only apparent. In both cases the unit of exchange formation is the chromatin fibre, which in the propha~ chromatid is in a highly folded state (Kihlman, 1970). To obtain more information on the sensitivity of Vicia ,faba chrom~me~ to the clastogenie effect of Cpt during G2/prophase, roots were c~oscd for 3 h to 10 ~.M Cpt and fixed at 0, 1.5, 3.5 and 4..5 h after the treatment. The effect, scored as abnormal ana-/ielopha~s (anaphases and telopha~s containing fragments and/or bridges),is illustrated in Fig. 7. The figure also shows the abnormal ana-/telophascs found after l-h treatments with I. #M FdUrd, as well as the anv/telopha~ labelling indice~ obtained with the autoradiographic technique ~.t various times after 2-h treatments with 2 pCi/ml [ZHldThd.To expose the cells to Cpt simultaneously with [ZH]dThd proved not to be practicable, since Cpt strongly suppresses the at this stage already weak incorporation of [~HIdThd into chromosomal DNA.

e,,,.,t.x'o A

B Fig. 6. (A) Sub