G2 repair and the formation of chromosomal aberrations—I. The effect of hydroxyurea and caffeine on maleic hydrazide-induced chromosome damage in Vicia faba

Environm,,n~ asd Ex~r~waml Botaay, Vol. 20, pp. 119 to 129 .~ Pergamon Press Ltd. 1980. Printed in Great Britain

0098-8472180/0501-0119 $2.00/0

R E P A I R A N D THE F O R M A T I O N OF C H R O M O S O M A L A B E R R A T I O N S - - I . T H E EFFECT OF H Y D R O X Y U R E A A N D CAFFEINE O N MALEIC H Y D R A Z I D E - I N D U C E D C H R O M O S O M E D A M A G E IN VICIA FABA G2

B. HARTLEY-ASP,* H. C. A N D E R S O N , $. STU~le.I.m ,,,,,I B. A. K I t H , M A N Department of General Genetics, University of Uppsala, S-750 07 Uppsala 7, Sweden

(Received 19 Februa(y 1979; revised 30 April 1979; revised 25 August 1979; accepted 6 September 1979) HARTLEY-AsP B., ANDERSSONH. C., STURELID S. and KaHLMANB. A. G 2 repair and the formation of chromosomal aberrations--L The effect of hydroxyurea and caffeine on maleic hydrazide-indu~ed chromosome damage in Vicia faba. ENVmONMENTAL AND EXPERIMENTAL BOTANY 20, 119--129, 1980.--The potentiating effects of caffeine and hydroxyurea (HU), alone or in combination, on maleic hydrazide (MH)-indueed chromosome damage were studied in root tips of Vicia faba. Roots were exposed to 2.5 x 1 0 - a M caffeine for 5hr immediately or delayed until 5hr a f t e r the M H treatment, and/or to 5 x 1 0 - a M HU during the last 22-t-3hr before fixation. The frequency of MH-induced chromatid aberrations was inereased by both types of posttreatment, but whereas caffeine produced an increase of all types of aberrations without markedly influencing their relative proportions, HU affected the aberration yield not only quantitatively but also qualitatively. The largest potentiation by HU was obtained for gaps, chromatid breaks and isochromatid breaks of the non-union type, whereas chromatid exchanges and isochromatid breaks with sister union were comparatively little affected. Although a particularly dramatic enhancement was obtained when the MH-treated roots were first exposed to caffeine and then to HU, the frequency of aberrations obtained after a combined MH and caffeine treatment was not increased more by HU than that obtained after MH alone. Possible molecular mechanisms involved in the action of caffeine and HU on the formation of MH-induced chromatid aberrations are discussed.

INTRODUCTION THE FIRST evidence for a connection between the formation o f c h r o m a d d aberrations and D N A r e p a i r d u r i n g G 2 was o b t a i n e d b y TAYLOR el a/. (37) These authors found that the frequency of c h r o m a t i d gaps a n d breaks prod u c e d by X - r a y s d u r i n g G 2 in root-tip cells of Vicia faba could be d r a m a t i c a l l y increased by exposing the roots to 5-fluorodeoxyuridine

( F d U r d ) . T h e m o n o p h o s p h a t e of F d U r d is an efficient and specific inhibitor of t h y m i d y l a t e synthetase, an enzyme that catalyzes the in vivo formation of thymidylic acid from deoxyuridylic acid. T h e p o t e n t i a t i n g effect of F d U r d on X r a y - i n d u c e d chromosome d a m a g e was counteracted by thymidine. T a y l o r et al. concluded that F d U r d increased the frequency of chrom a t i d gaps and breaks by preventing the r e p a i r

*Research laboratories, AB Leo, S-251 00 Helsingborg, Sweden. Reprint requests should be made to B. A. Kihlman, Department of General Genetics, P.O. Box 7003, S-750 07 Uppsala 7 (Sweden). 119

120

B. HARTLEY-ASP, H. C. ANDERSSON, S. STURELID and B. A. KIHLMAN

of X-ray-induced damage in chromosomal DNA. The observations of Taylor and his coworkers were soon confirmed and extended in our laboratory and e l s e w h e r e . ~21' 2 . " 25' 39) We found in Viola faba that other inhibitors of deoxyribonucleotide synthesis had an effect similar to that of FdUrd, and not only on X-rayinduced chromosome damage, but also on the frequency of chromatid aberrations induced by such different types of chemical mutagens as the antibiotics streptonigrin and plileomycin, the alkylating agent nitrogen mustard and the herbicide maleic hydrazide.t24.2s) Only the chromosome-damaging effect of 8-ethoxycaffeine was not affected by treatments with the inhibitors. The inhibitor used in most of these experiments was hydroxyurea (HU) which blocks the reduction of ribonucleoside diphosphatea to the corresponding deoxyribonucleoside diphosphates, a reaction catalyzed by the enzyme ribonucleotide reductase. As an inhibitor, H U acts rapidly and efficiendy and its action is reversible; H U has the fui'ther advantage of being easily available and inexpensive. Like FdUrd, H U dramatically increased the frequency of gaps, chromatid breaks and NUpd isochromadd breaks produced by the mutagenic agents. A less pronounced, but consistent and quite significant increase of the frequency of chromadd exchanges was also found. H U was most effective when applied late during the cell cycle; the largest increase was obtained when H U was added to the colchicine solution to which the roots were exposed the last 2½hr before fixation. The effectiveness of the H U treatment proved to be dependent not only on the stage of the cell cycle during exposure to the inhibitor, but also on the stage during exposure to the mutagen. As a rule, the earlier during the cell cycle the cells were exposed to the mutagen, the more effective was the subsequent HU treatment. This was only to be expected for agents such as nitrogen mustard and maleic hydrazide, since the damage produced by these agents has to be replicated before it can give rise to chromosomal aberrationsJ t1'~2) It was more surprising that the frequency of chromosomal

aberrations produced by X-rays, streptonigrin and phleomycin, which all have S-independent effects,~11'23) was most strongly increased by H U when the damage was induced during early or mid-interphase. On the basis of these findings we concluded (1) that the exposure of chromosomes to X-rays and to certain chemical mutagens produces lesions, the repair of which requires deoxyribonucleotides and (2), that this repair process takes place at a late stage of the cell cycle, probably during late G2-prophaseJ 2s) Subsequently, our findings and conclusions were confirmed in Crepis capillaris by Lucm~m et al. ~29) Although very striking and likely to have important implications for our concepts of the molecular mechanisms involved in the formation of chromosomal aberrations, the H U effect on induced chromosome dn'mage was not pursued for a decade. The main reason for this was probably that our interpretation of the H U effect appeared to be in conflict with available evidence on the effects of H U on the repair of DNA damage. Thus, H U was reported to be without inhibitory effect on repair replication and unscheduled DNA synthesis, c4's'~'31) In fact, H U has often been used to facilitate the detection of repair synthesis by reducing the background level of semi-conservative DNA synthesis. ~2'13'33'ss) However, it has recently been found that H U effectively inhibits the rejoining of the single strands that arise during the repair of u.v.-induced DNA damage in mammalian cells. ~1'7'9"t°'2°~ The inhibition by H U of the rejoining of single strand DNA breaks can be reversed by the addition of all four deoxyriboside precursors of DNA. TM 2o~ Against this background it seemed worthwhile to resume our studies of the effect of H U on induced chromosome damage. We further believed that it would be of interest to compare and combine the effect of H U with that of caffeine, another substance capable of increasing the frequencies of chromosomal aberrations produced by mutagenic agents in Vicia faba, as well as in other materials (for references see 23). In the experiments reported here, root tips of Vicia faba have been used as experimental material. It is, however, our intention to investigate this phenomenon in cultured mammalian cells.

CHROMOSOMAL ABERRATIONS IN VICIA FABA MATERIAL AND M~THODS

Lateral root-tips of Vicia faba. var minor (Weibulls ,~kerbtna Primus) were used in the experiments. The herbicide maleic hydrazide (MH), 1,2-dihydro-3,6-pyridazinedione, was used for the induction of chromosomal aberrations. The concentration of 1VIH in the different experiments varied between 5 x 10 -5 and 3.5 x I 0 - 4 M and the period of treatment between I and 2 hr. M H was dissolved in 0.007 M phosphate buffer, p H 6.0 caffeine (caff.), at a concentration of 2.5 x 1 0 - 3 M , was given as a 5-hr pulse, either immediately or delayed until 5 h r after the M H treatment. In some experiments 2.5 x 1 0 - a M caffeine was added to the 0.05% colchicine solution to which the roots were exposed during the last 2½-3hr before fixation. Hydroxyurea (HU), at a concentration of 5 x 1 0 - 3 M , was given simultaneously with colchicine 221-3 hr before fixa'tion. Caffeine was dissolved in tap water when not given together with colchicine. The concentrations of caffeine and H U , as well as the timing and duration of the treatments with these substances, were chosen, from previous experience, so that they would produce a negligible frequency of chromosomal aberrations when given alone but a maximum potentiation of MH-induced chromosome damage when given as posttreatments.t25, 26) All experiments were carried out at 20°C in the dark. At 20-23 hr after the MH-treatments, roots were exposed to 0.050/0 colchicine for 2½ - 3 hr, and fixed in methyl alcohol-glacial acetic acid, 3:1. Slides were prepared as permanent Feulgen squashes as described previously, t22) The result of each treatment was always analyzed by two persons, who scored at least 100 metaphases each. Before scoring, the slides were assigned code numbers and masked. As a measure of the degree of enhancement we used the potentiation factor, Pf, which we define as the ratio of the effect of the combined treatment (Ei+p) to the sum of the effects of the inducer ( M H ) and the potentiating agent (caff. and/or H U ) given separately (Ei+Ep); that is Pf=Ei+v/Ei+Ev. (a4) A potentiation factor of one corresponds to no potentiating effect. In the Figures the aberration yields for car-

121

feine and H U when given alone have been substracted from the effect of the combined treatment. RESULTS Table 1 shows how the frequencies of M H induced chromatid aberrations are influenced by the addition of 5 x 1 0 - 3 M I-IU to the colchicine solution used for collecting metaphases during the last 22L-3hr before fixation. It is evident that the frequency of all types of aberration scored is more or less markedly increased by the addition of H U . The largest increase was obtained for gaps, chromatid breaks and isochromatid breaks of the NU type, but there was also an increase of aberrations involving rejoining. The potentiation factors for the various types of aberration (excluding gaps) and for the total aberration frequency are given in Table 4A. On the average, the total aberration frequency was about doubled by the H U treatment. For chromatid breaks and isochromatid breaks of the N U type, a 4.5-5-fold increase was found. The data in Table 2 show the effect of caffeine alone and in combination with H U on the frequency of aberrations obtained 24 hr after 140

ml~ t7-/1 ~,-i*ea~.

[-

12o 100

60 ~

40

Chrommid breaks

Zsocnromatid breaks, SU-h~e

L~Ochrm~id breaks. NU-type

Chromatid exchanges

FIO. 1. The effect of 2.5x10-3M caffeine (carl'.) alone or in combination with 5 x 1 0 - 3 M hydroxyurea (HU) on the frequencies of chromatid-type aberrations, per 200 cells, obtained at 24 hr after 1 hr treatments with 1.5 x 10-4M maleic hydrazide (MH). The 5 hr caff. treatments were given 5 hr after the exposure to MH and the HU treatment during the last 2~ hr before fixation.

200 200 200 200 200 200 200 200 200 7.5

8.0

59.5 78.5 10.0 32.5 75.0 30.5 61.0

Abnormal metaphases (~o) 35 107 13 17 167 14 92 11 10

Gaps 7 51 3 7 66 5 42 9 5

Chromatid breaks 93 102 3 30 76 27 60 0 0

5 16 0 7 53 4 17 0 I

lsochromatid breaks SU NUp, NUd NUpd 43 65 2 24 28 17 22 0 I

4 l 0 3 7 I 6 O 0

Chromatid exchanges complete incomplete

152+35 235+107 8 + 13 71 + 17 2 3 0 + 167 54+14 147 + 92 9+ I I 7 + 10

Total aberrations +gaps

76.0+ 17.5 117.5+53.3 4.0+6.5 35.5+8.5 115.0+83.5 27.0+7.0 73.5 + 46.0 4.5+5.5 3.5+5.0

Aberraliolts +gaps per 100 cells

MH+21½hr HzO+2½hr colch MH + 5 hr calF. + 16~ hr H 2 0 + 2½hr MH + 5 hr calF. + 16~ hr H 2 0 + 2½hr 5 hr caff. + 16~ hi" H 2 0 ÷ 2½hr 5 hr caff. + 16~ hr H 2 0 + 2½hr

Treatment

co|ch colch + H U colch colch + H U

20.2 4i.3 63.0 1.0 2.5

500 300 200 200 200

,

Abnormal metaphas~ ("o)

Number of metapha~es analyzed

1.2 8.3 33.5 0,5 1.0

Chromatid breaks

12.8 43.3 45.5 0 1.5

3.4 4.7 20.0 0 0

isochromatid breaks NUp, NUd SU NUpd

3.4 24.7 31.0 0 0

I.O 6.3 14.0 0 O

Chromatid exchanges complete incomplete

Aberrations per I00 cells

21.8 87.3 I~.0 0.5 2.5

Total

Table 2. The eJ)ectoJ 2.5 x I0- 3 M ca~feim {caft.) alone or in combination with 5 x 10 3 M hydrox)'urea(HU) on thefiequentlesof ¢hwmatid-typr aberrationsprodMced by l-hr trealmenlswith 1.5 × I0- 4 M maleic hydrazide (MH). Roots were exposed to caJJ~~ a u l 3 , offer the MH-treatmt:U

l hr 3.5 x 10-* M MH+21½hr H ~ O + 2 l h r colch I hr 3.5 x t0-4 M MH +21½ hr H 2 0 + 2 ~ h r colch + HU 2~hr colch + H U 2hr 1 0 - 4 M MH + 2 0 h r H z O + 3 h r colch 2hr 10-4 M MH + 2 0 h r H 2 0 + 3 hr colch + H U 2hr 1 0 - * M M H + 2 3 h r H 2 0 + 3 h r colch 2 hr 10- 4 M M H + 23 hr H=O + 3 hr colch + H U 3hr c:olch+HU 3hr colch+ HU

Treatment

Number of metaphases analyzed

Table I. The ej~ectof post-treatmentswith 5 x 10-3 M h2drox3urea (IIU) during Ou last2½-3 hr bcJbrefixation on thefieqvcnd~s of cloomatid-12p r abenations produced bv maleic h3dvazide (MH)

Z

O

>

C3

sum

200 400

colch sum

200 400 400

sum 3 h r colch + H U

colch + H U

200

sum

5 h r carl.+ 13hr H 2 0 + 3 h r

200 400

3he care. + 13hr H ~ O + 3 h r colch

5 h r care + 10hr H a O + 3 h r colch + H U

'20(}

colch

5 h r caff,+10hr H 2 0 + 3 h r

sum

400

31

29

17

12

3

2

I

289

165 124

200

colch+HU

200

caK+13hr H20+3hr

M H + S h r H z O + S h r caff.+ 10hr H = O + 3 h r c o l c h + H U

179

76

103

45

21

24

Abnormal metaphases

Mtt+Shr HzO+5hr

200

colch

MH+Shr H20+Sbr

taft.+ 13hr H 2 0 + 3 h r

M H + S h r H z O + S h r caff.+ 10br H 2 0 + 3 h r

400

200

MH+23hr H20+3hr

eolch

200

M H + 20 hr H zO + 3 hr colch

Treatment

Number of" metaphases a.alyzed

21

20

10

10

0

0

0

288

130

158

24

6

18

16

6

10

Gaps

14

9

7

2

I

0

I

145

62

83

17

7

10

4

2

2

Chromadd breaks

0

I

0

1

1

I

0

119

54

65

125

63

62

16

0

8

F

I

2

1

I

0

0

0

96

23

73

12

7

5

4

4

0

lsochromatid breaks NUp, NUd $U NUpd

I

0

0

0

I

I

0

149

54

95

121

51

70

12

4

0

0

0

0

0

0

0

0

66

26

40

14

5

9

I

0

I

Chromatid exchanges complete incomplete

16+21

12+20

8 + 10

4+10

3+0

2+0

1+0

575+288

219+130

356+ 158

289+24

133+6

156+ 18

37 + 16

18+6

19 + l0

Total aberrations +gaps

4+5.25

3+5

0.75+0

143.75+72

72.25+0.0

9.25 + 4

Aberrations + gaps per 100 cells

'lable 3. The e{/~a q/' 2.3 x 10 3 M caffeine (cq[J~) alone or in combination with 5 x 10- ~ M I~ydrox.ymrea(HU) on thefrequencies of chromatid-t~pe aberrationsproduced by 2drr treatment with 5 x 10-s M maleic ~Tdrazide ( MH). Roots were exposed to cafJ: 5 hr after the MH-treatment

(.o

Z

Oo

© ©

o

m

C3

C

A

P f = E Ma+¢,ff +au/ (E ua +caf~+ Eno )

mEMH+caff+HU

MH in combination with caff and HU

EMH+¢aff+ Enu

MH in combination with caff=EMu+~af Hydroxyurea alone = Enu

P f = EMtl+caff/E~ln + Ecaff

MH in combination with caff=Eun+caf

Eun + E0aff

Maleic hydrazide a l o n e = E u n Caffeine alone = Ec,ff

MH in combination with HU =Eun+nu P f = E ua +tlu/Eua + Enu

Eua + Eau

Maleic hydrazide alone = Euu Hydroxyurea alone = Eau

Treatment combinations and method for determining the potentiation factor

64 230 3.4

6 27 4.5

230 5.6

271 1.2

230

62 2

5 1

41

238 1.6

159 4.4

230 0

153

36

27 14

150 3

19 17

134 7.1

19

18 1

18 3.6

3

5 0

86 5.1

17

16 1

365 1.7

216

215 1

215 6.7

32

31 1

129 i.4

95

92 3

1000 2.0

506

490 16

4~) 4.8

107

103 4

612 2.0

301

277 24

Frequencies rtE) of and potentiation factor (Pf) for various types of chromatid aberrations Isochromatid breaks Chromatid NUp, NUd Chromatid Total breaks SU NUpd exchanges aberrations

Table 4. The poteniiating effects (expressed by the potentiation factor) of caffeine (calf.) and hydroxyurea (HU) alone or in combination on the frequencies of chromatid-type aberrations produced by maleic hydrazide ( M H )

>

.>

,q

~3

©

f/3

Z

m

G'3

~q

125

CHROMOSOMAL ABERRATIONS IN VICIA FABA a 1-hr treatment with 1.5x 1 0 - * M MH. The 5-hr caffeine treatment was given immediately after the exposure to M H . Post-treatment with caffeine alone produced a 4-fold increase of the total aberration frequency. When MH-treated roots were first exposed to caffeine and then to HU, there was a 6-fold increase of the M H induced total aberration frequency. The frequency of chromatid breaks was increased 4.9 and 15.2 times by caffeine and caffeine+HU, respectively. The corresponding figures for chromatid exchanges are 7.0 and 10.2. In most of our experiments, the caffeine posttreatment was given 5 h r after instead of immediately after the M H treatment. As shown by Fig. 1 and Table 3, the results of these experiments are very similar to those reported in Table 2. The frequency of MH-induced aberrations was strongly potentiated by caffeine and the potentiation was most dramatic for the chromatid exchanges. H U , given together with colchicine during the last 22-~-3hr before fixation, produced a further increase of the M H effect and this increase was most marked for chromatid breaks and isochromatid breaks of the N U type. We attempted to determine (1) whether the potentiating effect of H U on MH-induced chromosome damage is influenced quantitatively or qualitatively by exposing the roots to caffeine between the M H and H U treatments and (2), whether it is possible to enhance the effects of M H or of M H + c a f f . by the addition of caffeine, instead of HU, to the colchicine solution to which the roots are exposed during the last 2{--3 hr before fixation. In Fig. 2 we have compared the potentiating effect of H U on the frequencies of aberrations produced by M H with (A) and without (B) a post-treatment with caffeine. The comparison was made at approximately the same aberration level, that is, the experiments were chosen in such a way that M H alone in one experiment should yield approximately the same frequency of aberrations as M H + c a f f . in the other. The results illustrated in Fig. 2 do not reveal any marked difference between the effect of H U on the frequency of chromatid aberrations produced by M H alone or by M H + caff. In Table 4 we have calculated the poten-

100

17.'1MH*coff

A

r ~ l MH *c(:lff.* HU

80 60

.~

~0~

~

20

~ : n ~t.t÷ HU

C~omotld

~ chromcl~

Iio ~lr~tid

Chrorr, et¢l

e~t~s

I:eoks

FIG. 2. The effect of 5 x 10-3M hydroxyurea (HU) on the frequencies of chromatid-type aberrations per 200 cells produced by (A) a 2-hr treatment with 5 x l0-SM maleic hydrazide (MH) in combination with a 5-hr treatment with 2 . 5 x l 0 - a M caffeine (carl'.) and (B), a 1-hr treatment with 3.5x 10-4M MH. In "A" the caff. treatment was given 5 hr after MH and the HU treatment during the last 2½hr before fixation; in "B" the HU treatment was given during the last 3 hr before fixation. tiation factors for different types of chromatid aberrations after different treatment combinations. The figures in Table 4A are based on the experimental data in Table 1 and those in Table 4B and 4C on the results presented in Fig. 1 and Table 3. I f we compare the potentiation factors in Table 4A with those in Table 4C, it is evident that a previous caffeine post-treatment had no effect on the HU-induced enhancement of the total aberration frequency, the potentiation factors in both cases being two. Possibly the data might suggest a qualitative difference in as much as exchanges tend to increase more and SU-isochromatid breaks less when roots are exposed to caffeine between the M H and H U treatments. However, since there is a considerable variation between experiments, we should not like to attach too much importance to this observation. A number of experiments was performed to determine whether it is possible to enhance the

126

B. HARTLEY-ASP, H. C. ANDERSSON, S. STURELID and B. A. KIHLMAN

as a reduction (or increase) of the percentage of cells in mitosis. In fact the mitotic index was found to be reduced by 15-30°/o by the addition of HU. However, if the potentiation of chromosome damage by H U was the result of a selective action of H U on the progression of cells from G 2 to metaphase, H U must inhibit cells with few of no aberrations more than cells with a high frequency of chromosomal aberrations. We believe this to be a rather unlikely possibility; it is more reasonable to assume that ceils with a high frequency of aberrations are DI~3~ION delayed by H U more than cells containing less The results reported here have shown that damage. Therefore, the fact that the mitotic post-treatments with both caffeine and hydro- index is somewhat reduced by the addition of xyurea strongly enhance the frequency of chro- H U can hardly be used as evidence for the H U matid aberrations induced in root tips of Vida potentiation being only an apparent one. faba by the herbicide maleic hydrazide. Post- Furthermore, it should be noted in Table 3 that treatments with caffeine caused a near five-fold the frequency of aberrations in cells fixed 23 hr increase of the MH-induced frequency of chro- after exposure to M H is not markedly different matid aberrations. The presence of H U during from that in cells fixed 26hr after treatment the last 22X--Shr before fixation doubled the with the herbicide. Thus, a 3-hr difference in frequency of aberrations produced both by M H fixation time does not change the aberration alone and by MH+caffeine. This means that frequency markedly. However, the strongest caffeine caused a five-fold increase not only of argument in favor of the idea that the H U aberrations, but also of lesions capable of being effect is on chromosomal aberrations rather converted to aberrations by H U (HU-sensitive than on cell cycle progression is the observation lesions). that the aberrations are changed not only quanA question that arises in connection with titatively but also qualitatively by H U , chroexperiments of this type is whether the poten- matid breaks and isochromatid breaks of the tiation observed is a true one and not a result of NU-type being increased much more than other an effect of the inhibitors on cell cycle pro- types of aberration. Such high frequencies of gression. I f there is such an influence of the open breaks are never found in MH-treated inhibitors, the metaphase cells fixed at a certain roots that have not been exposed to HU. time after M H alone may no~ represent the As shown by the potentiation factors given in same population as those fixed after M H +caff. Table 4, the caffeine treatments are more than or M H + H U . twice as effective as the H U treatments in For caffeine, experiments carried out in many potentiating MH-induced chromosome damage. different laboratories have clearly demonstrated But to some extent the potentiation factors may that the effect on the induced frequency of be misleading; if they had been calculated in chromosomal aberrations is a true potentiation another way the difference in effectiveness bewhich cannot be ascribed to an effect on cell tween the two substances would have been less cycle progression. The literature on caffeine marked. Thus, the potentiation factor could also potentiation has been thoroughly reviewed by be defined as the ratio of the effect of the Kihlman.(2a) combined treatment (Ei+v) minus the effect of For H U the evidence for a true potentiation the potentiating agent alone (Ep) to the effect is as follows. Until the addition of colchicine of the inducer alone (El), that is, Pf=Ei+v +_HU at 22-~-3hr before fixation the roots have -Ep/EI (compare Material and Methods). received exactly the same treatment. Any effect Since H U produces more aberrations a l o n e of H U on cell cycle progression should appear than does caffeine, this method would give effect of M H or of M H + caff. by the addition of caffeine to the colchicine solution. These experiments gave no consistent results; in some experiments a weak enhancement was found, in others no enhancement, or even a reduction. We conclude that when given during the last 2~-3 hr before fixation, caffeine does not have any potentiating effect on MH-induced chromosome damage of a magnitude comparable to that obtained with HU.

CHROMOSOMAL ABERRATIONS IN VICIA FABA larger potentiation factors for H U without markedly changing those for caffeine. Contrary to the preliminary observations reported previously by one of us, t~6) we were unable to find any consistent marked potentiation by caffeine of the MH-induced chromosome damage when the caffeine treatment was given during the last 22i--3hr before fixation. That the great majority of the aberrations obtained when M H is combined with caffeine and/or H U are the result of MH-induced DNA lesions is indicated by the fact that the distribution of aberrations between chromosomes characteristic for M H ~3°~ was not altered by the post-treatments. After all types of treatment, 4o7o0/o of the aberrations were located in a heterochromatic segment close to the centromere in the satellite arm of the M-chromosome [I. : Schubert, personal communication). Although both caffeine and H U effectively enhance the frequency of MH-induced chromatid aberrations, they are likely to act by different mechanisms. At the cytological level the two, substances are most effective during different stages of the cell cycle, caffeine during S and H U during G2-prophase. (25'26'36) Also caffeine and H U affect the various types of chromatid aberrations differently. Caffeine appears to act mostly as an amplifier of the effect produced by M H alone; only the frequency but not the relative proportions of aberrations are altered. As pointed out by SWI~TLINSKA,~35) there is, however, a tendency for the exchanges to increase more than other types of aberrations as a result of caffeine post-treatments (see Table 4B). In contrast to caffeine, HU influences the MH-effect not only quantitati~vely, but also qualitatively. As shown by Table 4A and 4C, the largest potentiation factors were obtained for chromatid breaks and for isochromatid breaks of the NU type, whereas chromatid exchanges and SU-isochromatid breaks were comparatively little affected. Caffeine and H U have in common that no type of MH-induced chromatid aberrations is ever markedly reduced by post-treatments with these substances, a fact which suggests that one type of aberration does not increase at the expense of another, but only at the expense of restitution, that is, successful repair. .

O /

127

Any suggestions for possible molecular mechanisms involved in the potentiation by caffeine and H U of induced chromosome damage must be at present highly speculative. Caffeine, at concentrations which have little effect on normal, semiconservative DNA synthesis, is an inhibitor of the gap-filling process in connection with post-replication repair of induced DNA damage. ~6'ls'2s) A slowing down of the gapfilling process would explain the increase of the frequency of exchanges, since it would provide a longer time for an interaction of gaps. If an inhibitory effect on the gap-filling process were responsible for the potentiation by caffeine of induced chromosome damage, it would explain why caffeine is most active during S, since the gap-filling process is closely linked to semiconservative DNA synthesis. Post-replication repair is also inhibited by HU, ~3'2~) but unlike caffeine, H U is an effective inhibitor of semiconservative DNA synthesis at the same concentrations that block the gap-filling process. ~14) The cytological observations suggest that caffeine and H U enhance induced chromosome damage by different mechanisms. Therefore, if an inhibitory effect on the gap-filling process in connection with post-replication repair is responsible for the enhancement by caffeine of induced chromosome damage, this mechanism is not likely to be responsible for the enhancement produced by HU. We mentioned in the Introduction that although H U does not reduce the amount of repair replication, it effectively inhibits the rejoining of single-strand breaks that arise in connection with excision repair of u.v.-induced DNA damage in mammalian cells. Against this background we would like to propose a model for the action of caffeine and H U on the formation of MH-induced chromatid aberrations. The results obtained with H U suggest that a large proportion, of the chromatid-type aberrations are formed as mistakes in connection with a repair process that operates at the very end of interphase when the extended chromatin fibers of the interphase nucleus begin to fold to form the chromatids of the metaphase chromosome. It could be argued that the PCC-technique has demonstrated that aberrations are realized much earlier during the G2 phase. (i7-19) To this can be replied that

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B. HARTLEY-ASP, H. C. ANDERSSON, S. STURELID and B. A. KIHLMAN

when the folding of the chromatin fibers is initiated by cell fusion, repair and hence misrepair and aberration formation is also initiated. It has been known since the work of EVANS and SCOTTCtt) that the D N A lesions induced by M H (like those induced by alkylating agents and by u.v.) are not capable of giving rise to chromatid aberrations before they have been replicated. Replication of D N A d a m a g e d by u.v. and by alkylating and arylalkylating agents produces gaps in the newly synthesized D N A opposite the lesions in-the parental strands. ¢32) We assume by analogy that replication of D N A damaged by M H also produces gaps in the newly synthesized DNA. Most of these gaps are rapidly filled in a n d sealed during the S-phase. No chromatid aberrations are believed to be formed in connection with this S-dependent gap-filling process. We further assume that gaps that have not been completely filled in and sealed during S will be subjected to repair when the folding of the chromatin fibers begins at the end of interphase. Failure of repair and misrepair will give rise to the various types of chromatid aberrations. We suggest that caffeine acts by increasing the proportion of gaps that remain unsealed through S and that H U interferes with the repair process during G2prophase in such a way that a greater proportion of the D N A gaps are incompletely repaired or mis-repaired. Acknowledgement--This study was supported by a grant from the Swedish Natural Science Research Council. We are greatly indebted to M^j-Bm-rr KAaLSSON and LEENA WISKARI for skilful technical assistance.

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