Mutation Research, 192 (1987) 277-282 277 Elsevier

MTRL 068

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and

chlorodeoxyuridine-substituted chromosomes

F e l i p e C o r t 6 s 1,2, W i l l i a m F . M o r g a n 1 a n d S h e l d o n W o l f f l

1Laboratoo, of Radiobiology and Environmental Health, University of Cali[brnia, San Francisco. CA 94143 (U.S.A.) and 2 Departamento de Citologia e Histologia Vegetal y Animal, Facultad de Biologia, 41012 Sevilla (Spare)

(Accepted 31 July 1987)

Keywords: Sister-chromatid exchange frequency; Thymidine, exogenous; Chinese hamster ovary cells; BrdUrd; CldUrd.

Summary

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister- chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this con- troversy, we have carried out a series of experiments to examine the effect of dThd on C H O cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on C H O cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addi- tion of 100 #M dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations f rom 100/~M to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subse- quent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.

In recent years, it has been reported that cytogenetic damage can result not only from the direct interaction of chemical and physical agents

Correspondence: Dr. Felipe Cort6s, Laboratory of Radio- biology and Environmental Health, University of California, San Francisco, CA 94143 (U.S.A.).

with DNA (reviewed in Littlefield, 1982; Takehisa, 1982) but also f rom perturbations in DNA- precursor pools (Davidson et al., 1980; Perry, 1983; Suzuki and Yosida, 1983; Kaufman, 1986, 1987). Most of the work on pool imbalance has been carried out with thymidine (dThd). Although this nucleoside is found in normal human serum at

0165-7992/87/$ 03.50 (~.~ 1987 Elsevier Science Publishers B.V. (Biomedical Division)

278

concentrations ranging from 0.1 to 1.16 #M (Holden et al., 1980) and is omnipresent in mam- malian sera, abnormal concentrations are known to provoke various biological effects, both in vitro and in vivo (Barclay et al., 1982; Meuth, 1984; Meuth and Nalbantoglu, 1986; Clode et al., 1986). For instance, thymidylate deprivation has been shown to cause 'thymineless death' and to be highly recombinagenic but apparently not mutagenic (Barclay et al., 1982; Ayusawa et al., 1986). Lack of dThd also results in the accumula- tion of DNA-strand breaks (Ayusawa et al., 1983) and leads to increased chromosome breakage (Hori et al., 1984) and sister-chromatid exchanges (SCEs) (Kato, 1980; Hori et al., 1984). Excess dThd, on the other hand, may lead to inhibition of DNA synthesis (Bjursell and Reichard, 1973) and has clear mutagenic effects (Bradley and Sharkey, 1978; Davidson and Kaufman, 1978).

The results reported by various investigators on the effect of exogenous dThd on SCE frequency in cultured Chinese hamster ovary (CHO) cells have been conflicting. In one type of experiment, dThd is supplied simultaneously with 5-bromodeoxy- uridine (BrdUrd) for the two cell cycles needed for differential staining of sister chromatids. With this protocol, Davidson et al. (1980) found a small but not significant decrease in the yield of SCEs, over the concentration range 1-360 ~tM dThd. Perry (1983), on the other hand, reported a dose- dependent increase in the frequency of SCEs in- duced by dThd over the concentration range 10-100 #M.

In a second type of experiment, in which cells are cultured for one cell cycle in BrdUrd followed by a second cycle with only dThd, Wilmer and Natarajan (1981) reported that dThd did not in- duce SCEs even at extremely high concentrations (9 mM). In contrast, Suzuki and Yosida (1983) found that SCEs were induced by increasing con- centrations of dThd in the culture medium during replication of the BrdUrd-substituted DNA, i.e., during the second cell cycle. In a variation of this experiment, Kaufman (1986, 1987) grew CHO cells for 4-5 days in BrdUrd to fully substitute the DNA and then grew them for two replication cycles with

dThd but without BrdUrd. In these experiments, dThd increased SCE frequency in a dose- dependent manner.

The experiments described in the present report were designed to resolve these conflicting results by testing the effect of excess dThd on the frequency of SCEs in CHO cells with BrdUrd-substituted or chlorodeoxyuridine (CldUrd)-substituted chromo- somes. The experiments show that dThd can either suppress or enhance the frequency of SCEs, depending on the dose used and the cell cycle dur- ing which it is present in the culture medium.

Materials and methods

Chinese hamster ovary cells were cultured as monolayers in McCoy's 5A medium containing fetal bovine serum 00%), L-glutamine (2 mM), penicillin (50 units/ml), and streptomycin (50 /~g/ml). To examine the effect of dThd on SCE fre- quency, cells were cultured for two replication cycles (26 h) with either of the halogenated nucleosides BrdUrd or CldUrd. Thymidine was added for the first, second, or both cell cycles. Col- cemid (2 × 10-7 M, final concentration) was add- ed for the final 2 h of cell culture.

To determine in which cell cycle dThd affected SCE frequency, we used the twins and singles method (Taylor, 1958; Wolff and Perry, 1975). Twin and single SCEs are analyzed after the initial- ly diploid cells are made tetraploid by the addition of colcemid. Colcemid (10 -7 M final concentra- tion) was added at the beginning of the culture period and the cells were cultured for two replica- tion cycles with CldUrd. Thymidine was added for either the first cycle, the second cycle, or both cell cycles. In the resulting tetraploid cells, SCEs that formed in the first cycle of replication were present in both daughter chromosomes and recorded as twins (two SCEs), whereas SCEs formed in the sec- ond cycle were present in only one of the daughter chromosomes and recorded as singles (one SCE). If SCEs occur at an equal frequency in both suc- cessive cell cycles, the ratio of singles to twins would be 2.0. The analysis of twins and singles was restricted to the large acrocentric chromosome,

which in te t raploid cells is present on ly in

duplicate. This was done to c i rcumvent the prob-

lem of 'false twins ' (Heddle, 1969) that can result

f rom scoring chromosomes that are present in

quadrupl ica te .

Flasks were gently shaken to dislodge the mitotic

cells, which were then collected by cent r i fugat ion ,

t reated with 0.075 M KC1 for 4 min, fixed in two

changes of methanol :ace t ic acid (3:1), and drop-

ped onto glass microscope slides. Differential

s ta ining of chromosomes subst i tuted with

ha logenated nucleosides was carried out by a slight

modi f ica t ion of the f luorescence-plus-Giemsa

technique of Perry and Wol f f (1974). Slides were

s tained with Hoechst 33258 dye (5 ~g /ml final con-

cent ra t ion) for 20 min, moun ted in 0.067 M

Sorensen ' s buffer (pH 6.8), and exposed on a 56°C

hot plate to 365-nm black light f rom two Sylvania

F15T8 BLB fluorescent tubes at a dose flux of

abou t 15 j / m 2 / s e c . For BrdUrd-subs t i tu ted

chromosomes , black light exposure was 4-8 min;

for CldUrd-subs t i tu ted chromosomes , 40-60 min.

Coded slides were then stained with 3% Giemsa

(Gur r ' s R66 in Sorensen ' s buffer) for 5-10 rain. 50

second-divis ion metaphase cells were scored for

each t rea tment .

R e s u l t s a n d d i s c u s s i o n

When cells were grown in the presence of

BrdUrd (20-100 ~M) or CldUrd (1-30 #M) for two

cell cycles wi thout dThd, a dose-dependent in-

crease in the frequency of SCEs was observed

(Tables 1 and 2). These results are in agreement

with previous reports f rom this and other

laborator ies (Wolf f and Perry, 1974; Davidson et

al., 1980; Perry, 1983; Hear t le in et al., 1983). Our

results also indicate that CldUrd is a much more

effective inducer of SCEs than BrdUrd, as has

been demons t ra ted previously (DuFra in and Gar-

rand, 1981). Thus, for example, in cells cul tured

with 1 ~M CldUrd , the yield of SCEs was nearly

twice that found in cells grown with 100 ~M

BrdUrd , and a 20 ~M CldUrd t rea tment resulted in

6 times more SCEs than did a 20 ~M BrdUrd

t rea tment .

279

TABLE 1

EFFECT OF 100 #M THYMIDINE ON SCE FREQUENCY IN CELLS WHEN PRESENT FOR TWO CYCLES SIMUL- TANEOUSLY WITH BROMODEOXYURIDINE

Treatment Number of Mean SCEs SCEs/ per chromo- number of some _+ S.E. chromosomes

20 ~M BrdUrd 435/1038 0.42 ± 0.02 20 ~M BrdUrd + dThd 345/1057 0.33 _+ 0.02 a 50 ~M BrdUrd 530/1039 0.51 _+ 0.02 50 #M BrdUrd + dThd 410/1044 0.39 _+ 0.02 a

100 ~M BrdUrd 559/1047 0.53 +_ 0.02 100 #M BrdUrd + dThd 498/1050 0.47 +_ 0.02

a Significantly different from treatments lacking dThd (p<0.001; Student's t-test).

TABLE 2

EFFECT OF 100 #M THYMIDINE ON SCE FREQUENCY IN CELLS WHEN PRESENT FOR TWO CYCLES SIMUL- TANEOUSLY WITH CHLORODEOXYURIDINE

Treatment Number of Mean SCEs SCEs/ per chromo- number of some _+ S.E. chromosomes

1 #M CldUrd 1044/1050 0.99 _+ 0.03 1 ~M CldUrd + dThd a _ a

5 #M CldUrd 1712/1050 1.63 _+ 0.04 5 ~M CldUrd + dThd 341/1050 0.32 +_ 0.02 b

10 #M CldUrd 2311/1050 2.20 _+ 0.05 10 #M CldUrd + dThd 529/1045 0.50 + 0 . 0 2 b

20 #M CldUrd 2670/1050 2.54 _+ 0.05 20 I~M CldUrd + dThd 809/1050 0.77 ± 0.03 b 30 #M CldUrd 2737/1050 2.60 ± 0.05 30 ~M CldUrd + dThd 942/1051 0.89 ± 0.03 b

a No differential staining. h Significantly different from treatments lacking dThd (p<O.OOl; Student's t-test).

When cells were treated s imul taneous ly with 100

~M dThd and various concent ra t ions of BrdUrd

for two cell cycles, a consistent decrease in the

yield of SCEs was observed (Table 1). A small but

not significant decrease was also observed by

Davidson et al. (1980). In contrast , Perry (1983)

reported that the addi t ion of dThd (10-100 ~M) to

the culture med ium conta in ing BrdUrd caused a

dose-dependent increase in the frequency of SCEs,

which approximate ly doubled at 100 ~M dThd.

280

When CHO cells are treated with BrdUrd at a con- centration below 100 #M, the SCE rates are usually proportional to the amount of BrdUrd substituted for dThd in the chromosomes (Mazrimas and Stetka, 1978; Heartlein et al., 1983). On this basis, it could be expected that the presence of exogenous dThd, which competes with BrdUrd for incorpora- tion into DNA, would lead to a reduction in the amount of BrdUrd incorporation and, subsequent- ly, to a reduced yield of SCEs.

The reduction in the yield of SCEs was much more apparent when the same experiment was per- formed with CldUrd (Table 2). The lower the con- centration of CldUrd, the greater the relative decrease in the frequency of SCEs found when dThd was present (5.0-, 4.4-, 3.3- and 2.9-fold decreases for 5, 10, 20 and 30/zM CldUrd, respec- tively), which was to be expected from the decreas- ed incorporation of CldUrd when both nucleosides were present. No differential staining was observed in cells treated with 1 #M CldUrd and 100 ~M dThd.

Our previous results (Escalza et al., 1985), as well as those reported by other groups (Natarajan et al., 1981; Heartlein et al., 1983; O'Neill et al., 1983; Shiraishi et al., 1983; Suzuki and Yosida, 1983; Stetka and Spahn, 1984), suggested that, during the replication of halogen-substituted parental DNA, most SCEs are induced in the sec- ond cell cycle. To test this possibility further and to determine whether the decreased yield of SCEs observed in the presence of dThd was the result of

T A B L E 3

E F F E C T O F 100 tzM T H Y M 1 D I N E O N S C E F R E Q U E N C Y

IN C E L L S C U L T U R E D F O R T W O C E L L C Y C L E S W I T H 10

# M C H L O R O D E O X Y U R I D I N E

T h y m i d i n e p re sen t N u m b e r o f S C E s / M e a n SCEs

n u m b e r o f per c h r o m o s o m e F i r s t S e c o n d

c h r o m o s o m e s ± S .E . cell cycle cell cycle

- - 1 9 7 1 / 1 0 4 2 1.89 ± 0 .4

+ + 7 3 8 / 1 0 4 6 0 .70 ± 0 .03 a

+ - 7 0 9 / 1 0 4 2 0 .68 ± 0 .03 a

- + 1 8 4 1 / 1 0 4 6 1.76 ± 0 .04

a S ign i f i c an t l y d i f f e r e n t f r o m t r e a t m e n t s in w h i c h t h y m i d i n e

was n o t p re sen t in e i the r cell cycle 6 o < 0 . 0 0 1 ; S t u d e n t ' s t-test).

a reduction in the amount of CldUrd incorporated during the first cell cycle, cells were exposed to 10 #M CldUrd for two cell cycles and simultaneously to 100/zM dThd for the first, or both cell cycles (Table 3). Addition of dThd either for both cell cycles or only for the first cell cycle dramatically reduced the number of CldUrd-induced SCEs. When dThd was present for the second cycle only, no effect on CldUrd-induced SCEs was observed. These results indicate that dThd must be present during the first cell cycle to suppress most of the SCEs induced by CldUrd.

This conclusion was confirmed when single and twin SCEs were analyzed in the large acrocentric chromosome pairs of colcemid-induced tetraploid ceils exposed to 5 #M CldUrd for two cell cycles and simultaneously to 100 #M dThd for the first, second, or both cell cycles. When dThd was pres- ent for either the first cell cycle or both cell cycles, the SCE frequency decreased (Table 4). There was approximately a 2-fold reduction of SCEs in the first cycle (recorded as twins) and a dramatic 4-fold reduction in the second cycle (recorded as singles). It is of interest that when dThd was pre- sent in the first or both cell cycles, nearly equal numbers of SCEs per chromosome were induced in each cell cycle (resulting in a singles:twins ratio of approximately 2.0) (Table 4). Equal numbers of SCEs were not induced in each cell cycle when dThd was not present at all or when it was present

T A B L E 4

E F F E C T O F 100 # M T H Y M I D I N E O N T H E F R E Q U E N C Y

O F T W I N A N D S I N G L E SCEs IN T H E L A R G E A C R O -

C E N T R I C C H R O M O S O M E P A I R O F T E T R A P L O I D C H O

C E L L S C U L T U R E D F O R T W O C E L L C Y C L E S W I T H 5 # M

C H L O R O D E O X Y U R I D I N E a

T h y m i d i n e p re sen t N u m b e r o f N u m b e r o f S ing les : twins

Firs t S e c o n d twin s ingle r a t i o

cell cycle cell cycle SCEs b SCEs c

- - 53 252 4.75

+ + 29 55 1.89

+ - 31 71 2 .29

- + 52 256 4 .92

a 100 cells per e x p e r i m e n t were a n a l y z e d .

b Ind i ca t e s SCEs i n d u c e d in the f i rs t cell cycle .

c Ind ica t e s S C E s i n d u c e d in the s econd cell cycle .

TABLE 5

SCE FREQUENCY IN CELLS C U L T U R E D W I T H 20 #M

B R O M O D E O X Y U R I D I N E FOR THE FIRST CELL CYCLE AND W I T H VARIOUS C O N C E N T R A T I O N S OF THYMID-

INE FOR THE SECOND CELL CYCLE

dThd in Number of SCEs/ Mean SCEs second number of per chromo-

cell cycle chromosomes some +_ S.E.

0 a 377/1047 0.36 _+ 0.02

100 #M 437/1051 0.41 +_ 0.02

500 #M 465/1046 0.44 + 0.02 1 mM 456/1045 0.43 + 0.02

3 mM 602/1052 0.57 _+ 0.02 b

5 mM 932/1050 0.88 _+ 0.03 b

9 m M 1210/1050 1.15 + 0.03 b

a BrdUrd was present during the second cell cycle only when

dThd was not added to cultures.

b Significantly different f rom treatments lacking dThd (p<O.O01; Student 's t-test).

for only the second cell cycle. Instead, we observed a disproportionate number of SCEs induced in the second cell cycle, when the DNA replicated on a template containing CldUrd. These observations are in agreement with the data in Table 3 and sug- gest that, in the presence of dThd, less CldUrd was incorporated in the first cell cycle, leading to a reduction in SCEs induced, especially in the second cell cycle.

Under other conditions, the addition of ex- ogenous dThd can increase SCE frequency. When cells were grown in the presence of BrdUrd during the first cell cycle only, and then in various concen- trations of dThd during the second cell cycle, in- creased frequencies of SCEs were observed (Table 5). A small but not significant increase in SCEs was observed at dThd concentrations below 3 mM, but the SCE frequency was significantly increased when dThd concentrations of 3 mM and higher were used. These results are in agreement with those of Suzuki and Yosida (1983) and Kaufman (1986, 1987) but in contrast to those reported by Wilmer and Natarajan (1981).

Our results show that exogenous dThd can either suppress or stimulate the occurrence of SCEs, depending on the experimental protocol. When cells were cultured simultaneously with a halo-

281

genated nucleoside and exogenous dThd, reduced numbers of SCEs were observed. This is presum- ably because dThd competes with ha[ogenated precursors of DNA, resulting in a reduced incor- poration of BrdUrd or CldUrd into DNA. The data from the twins and singles experiment in- dicate that this reduced incorporation must occur in the first cell cycle, reducing the number of SCEs arising in both cell cycles but having a more pro- nounced effect in the second cycle during replica- tion of template DNA containing the halogen. When increasing concentrations of dThd were add- ed in the second cell cycle after BrdUrd incorpora- tion during the first cell cycle, there was an increase in SCE frequency. This suggests that replication of halogen-substituted DNA leads to increased SCE formation when a d N T P pool im- balance is created by very high levels of dThd.

Acknowledgements

This work was supported by the Office of Health and Environmental Research of the U.S. Department of Energy, contract No. DE- AC03-76-SF01012, and by a grant (PB85-0345) f rom the Comisi6n Asesora de Investigaci6n Cien- tifica y T&nica (Spain). We thank S. Brekhus for secretarial assistance and M. McKenney for editing the manuscript.

References

Ayusawa, D., K. Shimizu, H. Koyama, K. Takeishi and T.

Seno (1983) Accumulat ion of DNA strand breaks during thymineless death in thymidylate synthase-negative mutants

o f mouse FM3A cells, J. Biol. Chem. , 258, 12448-12454.

Ayusawa, D., H. Koyama, K. Shimizu, S. Kaneda, K. Takeishi and T. Seno (1986) Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transfer-

red genetic marker in mouse FM3A cells, Mol. Cell. Biol., 6, 3463-3469.

Barclay, B.J., B.A. Kunz, J .G. Little and R.H. Haynes (1982) Genetic and biochemical consequences of thymidylate stress, Can. J. Biochem., 60, 172-194.

Bjursell, G., and P. Reichard (1973) Effects of thymidine on

deoxyribonucleoside tr iphosphate pools and deoxyribo- nucleic acid synthesis in Chinese hamster ovary cells, J. Biol. Chem. , 248, 3904-3909.

282

Bradley, M.O., and N.A. Sharkey (1978) Mutagenicity of thymidine to cultured Chinese hamster cells, Nature (Lon- don), 274, 607-608.

Clode, S.A., D. Anderson and S.D. Gangolli (1986) Studies with unbalanced DNA precursor pools in vivo, in: C. Ramel, B. Lambert and J. Magnusson (Eds.), Genetic Toxicology of Environmental Chemicals, Liss, New York. pp. 551-561.

Davidson, R.L., and E.R. Kaufman (1978) Bromodeoxyuridine mutagenesis in mammalian cells is stimulated by thymidine and suppressed by deoxycytidine, Nature (London), 276, 722-723.

Davidson, R.L., E.R. Kaufman, C.P. Dougherty, A.M. Ouellette, C.M. DiFolco and S.A. Latt (1980) Induction of sister chromatid exchanges by BUdR is largely independent of the BUdR content of DNA, Nature (London), 284, 74-76.

DuFrain, R.J., and T.J. Garrand (1981) The influence of incor- porated halogenated analogues of thymidine on the sister- chromatid exchange frequency in human lymphocytes, Mutation Res., 91, 233-238.

Escalza, P., F. Cort6s and J.B. Schvartzman (1985) Induction of sister-chromatid exchanges (SCEs) by 5-fluorodeoxy- uridine, The role of 5-bromodeoxyuridine incorporated into parental DNA, Mutation Res., 151, 77-82.

Heartlein, M.W., J.P. O'Neill and R.J. Preston (1983) SCE in- duction is proportional to substitution in DNA for thymidine by CldU and BrdU, Mutation Res., 107, 103-109.

Heddle, J.A. (1969) Influence of false twins on the ratios of twin and single sister chromatid exchanges, J. Theor. Biol., 22, 151-162.

Holden, L., A.V. Hoffbrand and M.H.N. Tattersall (1980) Thymidine concentrations in human sera: Variations in pa- tients with leukaemia and megaloblastic anaemia, Eur. J. Cancer, 16, 115-121.

Hori, T., D. Ayusawa, K. Shimizu, H. Koyama and T. Seno (1984) Chromosome breakage induced by thymidylate stress in thymidylate synthase-negative mutants of mouse FM3A cells, Cancer Res., 44, 703-709.

Kato, H. (1980) Evidence that the replication point is the site of sister chromatid exchange, Cancer Genet. Cytogenet., 2, 69-77.

Kaufman, E.R. (1986) Induction of sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA under conditions of nucleotide-pool imbalance, Mutation Res., 163, 41-50.

Kaufman, E.R. (1987) Uncoupling of the induction of muta- tions and sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA, Mutation Res., 176, 133-141.

Littlefield, L.G. (1982) Effects of DNA-damaging agents on SCE, in: A.A. Sandberg (Ed.), Sister Chromatid Exchange, Liss, New York, pp. 355-394.

Mazrimas, J.A., and D.G. Stetka (1978) Direct evidence for the role of incorporated BUdR in the induction of sister chromatid exchanges, Exp. Cell Res., 117, 23-30.

Meuth, M. (1984) The genetic consequences of nucleotide precursor pool imbalance in mammalian cells, Mutation Res., 126, 107-112.

Meuth, M., and J. Nalbantoglu (1986) Molecular analysis of mutations induced by deoxyribonucleotide pool imbalances in Chinese hamster ovary cells, in: C. Ramel, B. Lambert and J. Magnusson (Eds.), Genetic Toxicology of Environmental Chemicals, Part A: Basic Principles and Mechanisms of Ac- tion, Liss, New York, pp. 525-532.

Natarajan, A.T., I. Czuk~is and A.A. van Zeeland (1981) Con- tribution of incorporated 5-bromodeoxyuridine in DNA to the frequencies of sister-chromatid exchanges induced by in- hibitors of poly-(ADP-ribose)-polymerase, Mutation Res., 84, 125-132.

O'Neill, J.P., M.W. Heartlein and R.J. Preston (1983) Sister- chromatid exchanges and gene mutations are induced by the replication of 5-bromo- and 5-chloro-deoxyuridine substituted DNA, Mutation Res., 109, 259-270.

Perry, P.E. (1983) Induction of sister-chromatid exchanges (SCEs) by thymidine and the potentiation of mutagen- induced SCEs in Chinese hamster ovary cells, Mutation Res., 109, 219-229.

Perry, P., and S. Wolff (1974) New Giemsa method for the dif- ferential staining of sister chromatids, Nature (London), 251, 156-158.

Shiraishi, Y., T.H. Yosida and A.A. Sandberg (1983) Analyses of bromodeoxyuridine-associated sister chromatid exchanges (SCEs) in Bloom syndrome based on cell fusion: Single and twin SCEs in endoreduplication, Proc. Natl. Acad. Sci. (U.S.A.), 80, 4369-4373.

Stetka, D.G., and M.C. Spahn (1984) SCEs are induced by replication of BrdU-substituted DNA templates, but not by incorporation of BrdU into nascent DNA, Mutation Res., 140, 33-42.

Suzuki, H., and T.H. Yosida (1983) Frequency of sister- chromatid exchanges depending on the amount of 5-bromodeoxyuridine incorporated into parental DNA, Mutation Res., 111, 277-282.

Takehisa, S. (1982) Induction of sister chromatid exchanges by chemical agents, in: S. Wolff (Ed.), Sister Chromatid Ex- change, Wiley, New York, pp. 87-147.

Taylor, J.H. (1958) Sister chromatid exchanges in tritium- labeled chromosomes, Genetics, 43, 515-529.

Wilmer, J.W.G.M., and A.T. Natarajan (1981) Induction of sister-chromatid exchanges and chromosome aberrations by y-irradiated nucleic acid constituents in CHO cells, Mutation Res., 88, 99-107.

Wolff, S., and P. Perry (1974) Differential Giemsa staining of sister chromatids and the study of sister chromatid exchanges without autoradiography, Chromosoma, 48, 341-353.

Wolff, S., and P. Perry (1975) Insights on chromosome struc- ture from sister chromatid exchange ratios and the lack of both isolabelling and heterolabelling as determined by the FPG technique, Exp. Cell Res., 93, 23-30.

Communicated by R.B. Painter