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Clastogenic activity of cytosine arabinoside and 3′-deoxy-3′-fluorothymidine in ehrlich ascites tumour cells in vitro
101
Mutation Research, 40 (1976) 101--106
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
CLASTOGENIC ACTIVITY OF CYTOSINE ARABINOSIDE AND 3'-DEOXY-3'-FLUOROTHYMIDINE IN EHRLICH ASCITES TUMOUR CELLS IN VITRO
ANNA M. WOBUS Zentralinstitut fiir Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR, 4325 Gatersleben (DDR)
(Received March 17th, 1975) (Revision received November 4th, 1975) (Accepted November 28th, 1975)
Introduction 1-fl-D-arabinofuranosylcytosine (ara-C), an analogue of cytidine, inhibits the growth of mammalian cells in vitro [4,10,16], and DNA synthesis in L cells [16] and murine lymphoblasts [4]. The c o m p o u n d interferes with DNA polymerase [6,7,12] and becomes incorporated into DNA [5,17]. Ara-C is known to produce chromosomal aberrations in vivo [1] and in vitro [2,3,8,14]. 3'-deoxy-3'-fluorothymidine (3'-FTdR), a newly synthesized analogue of thymidine, equally inhibits cell multiplication and DNA synthesis of Ehrlich ascites t u m o u r cells in vitro [9]. Inhibition of DNA synthesis may be reversed by high concentrations of thymidine. Neither RNA nor protein synthesis is inhibited [9]. 3'-FTdR was phosphorylated to the 5'-triphosphate level and incorporated into DNA [11]. In experiments on the inhibition of cell multiplication of ascites cells with ara-C and 3'-FTdR, Langen and Graetz [10] showed that 3'-FTdR was much better tolerated by the cells than ara-C. This is due to a longer lag period preceding cell death. In concentrations that produce an 80% inhibition of cell multiplication within 24 h (1 X 10 -s M 3'-FTdR, 1 X 10 .6 M ara-C), the treatment time needed to achieve a 50% killing rate was about one generation time longer for 3'-FTdR than for ara-C [10]. Because of these differences our aim was to determine the clastogenic effectivity of ara-C and 3'-FTdR in Ehrlich ascites tumour cells in vitro. Furthermore, we investigated whether some differences in the aberration frequencies exist during the recovery period in correlation with the differences in cell survival.
I
cells.
24 TdR
24 h 3’-FTdR
b Cells
with
a 1 X lo-’
ma.
and
24
24 h
f. per
100
a
12
24 h
TdR
_
48 h
5.5
11.7
4.4
8.6
11.4
-
30 h
4.1
36 h
13.5
24 h
4.0
27.7
8.9
12 h
a
12TdRa
24 h 3’-FTdR
TdR
24
24 h 3’-FTdR
-
12
Controls
0.5
-
36 h 3’-FTdR
24 h 3’-FTdR
a
2.6
30 h 3’-FTdR
15.5
1.1
1.6
(%)
200
200
200
200
200
300
200
200
200
300
300
300
300
300
300
Metaphases scored
frequency
INDUCED
Mitotic
ABERRATIONS
12 h 3’-FTdR
(h)
time
CHROMATID
Recovery
OF
24 h 3’-FTdR
M TdR.
FREQUENCIES
Treatment
THE
TABLE BY
4.50
4.00
1.00
0.50
1.50
0.33
0
9.00
55.50
21.00
69.33
98.33
94.00
67.67
23.67
* 0.58
?; 3.59
f 2.96
2 2.46
2 0.61
f 0.61
f 4.18
+ 0.96
+ 0.82
+ 0.85
+ 0.50
* 0.96
+ 0.33
(%I gaps
f 2.75
(Z * S,)
(9)
excluding
aberrations
with
M 3’-FTdR
Metaphases
1 X lo-’
7.0
4.0
0.5
3.5
3.5
1.0
1.0
5.0
23.5
12.7
47.7
62.3
58.3
36.0
72.0
3.0 1.0
3.5
1.0
0.5
1.0
-
5.0
_
0.5
0.5
17.5
12.7 11.5
17.0
126.3
_
1.0
1.0
1.7
21.7
22.1 61.0
430.7
21.3
1.3
19.6
4.3
m.a.
b
THE
403.3
t
cells
OF
1.6
100
CELLS
13.3
36.6 150.2
59.0
b
per
ASCITES
26.3
g
Aberrations
IN EHRLICH
1.0
1.3
2.1
0.7
1.3
fb
MOUSE
h.s.c.
103
Material and methods Ehrlich ascites t u m o u r cells (strain "Berlin-Buch") with a stem line of 44 chromosomes were cultivated in vitro according to a modified m e t h o d of Negelein et al. [13]. Cells 5 days old were removed from the peritoneal cavity of mice under sterile conditions, gassed with 5% CO2, 1.5% 02 in N2, and incubated in a modified Parker medium [13] for different times in the presence of 1 X 10 -6 M ara-C or 1 X 10 -s M 3'-FTdR (for reasons see Results) and subsequently counted. Incubation was performed in a Warburg apparatus (V 85, B. Braun Apparatebau, Melsungen) at 35°C with horizontal agitation. If the cultivation time exceeded 24 h, cells were passaged by centrifuging and washing twice with medium. Colchicine (0.002%) was added 3 h before fixation and the production of air-dried preparations [15]. For estimation of mitotic frequency in these preparations, 2000 cells were analysed for each value in Tables I and II. To estimate the aberration frequencies, cells were analysed for chromatid breaks (b), chromatid translocations (t), multiple aberrations (m.a.), single highly shattered chromosomes (h.s.c.), and totally fragmented or pulverized chromosomes (f). Gaps were separately counted but not included in the category of true aberrations. The experimental values as reported in Tables I and II and Fig. 1 are means of three sets of experiments. Ara-C and 3'-FTdR were dissolved in sterile bidistilled water, stored at --20°C and used without filtration. Results and discussion Identical concentrations and treatment times usually provide the basis of exTABLE THE
n FREQUENCIES
EHRLICH
ASCITES
Treatment
OF CELLS
CHROMATID OF
THE
ABERRATIONS
INDUCED
BY"
1
X
10 -6
M
ARA-C
IN
MOUSE
Recovery
Mitotic
Meta-
Metaphases
time
frequency
phases
with
aberra-
(h)
(%)
scored
tions
(%)
Aberrations g
per 100
b
cells
t
m.a.
b f b
h.s.c
excluding gaps
(g)
(~ ± s m) 6 h ara-C
--
4.2
200
19.00
± 1.28
16
30
18 h ara-C
--
0.3
200
56.50
+ 1.71
45
122
2
--
--
--
1
--
3
1 1
6 h ara-C
12
12.7
200
26.00
-+ 1 . 6 3
14
30
10
1
--
6 h ara-C
18
12.8
200
17.00
± 1.29
5
22
16
--
--
1
6 h ara-C
24
5.5
200
3.50
-+ 0 . 9 6
3
8
--
--
--
200
10.00
+- 0 . 8 2
8
6
--
--
--
1
5
--
--
--
6 h ara-C
12, + CdR
a
6 h ara-C
24, + CdR
a
19.1 5.1
200
4.00
-+ 0 . 8 2
-+ 0 . 5 8
-4 --
Controls 6h
--
6.9
200
1.00
3O h
--
5.5
200
0
6h
12 CdR
a
11.4
200
1.00
-+ 0 . 5 8
6h
24 CdR
a
4.5
200
4.00
± 0.82
a 1 X 10 -5 MCdR, b See Table
I.
2 .
2 .
-4
.
.
.
. .
. .
.
1
1
--
--
--
5
1
--
--
--
104 100'
v
50,
c_
1
0
6
11 18 2'4 30 t r e a t m e n t t i m e (h)
F i g . 1. E f f e c t o f 1 × 1 0 - 6 M a r a - C ( o cites turaour ceils in vitro.
3'6
e ) a n d 1 X 1 0 -5 M 3 ' - F T d R
(c,
5 ) o n t h e s u r v i v a l o f as-
perimental comparisons of two substances. Under such conditions, however, ara-C t r e a t m e n t results in very high and 3'-FTdR in rather low aberration frequencies. For instance, a treatment for 12 h with 1 X 10 .5 M caused 93.00 i 1.00% aberrant metaphases with ara-C and only 23.67 i 2.75% with 3'-FTdR. After 30 h and treatment with 1 X 10 -s M 3'-FTdR, 94.00 i 0.61% of aberrant metaphases was obtained. Since we were interested in checking the clastogenic effectivity of the two substances under conditions that cause comparable biological effects {inhibition of cell multiplication, effect on cell survival) we used exposure times that showed an identical cell killing rate of 10--15%, i.e. a treatment of 6 h for araC and of 24 h for 3'-FTdR (Fig. 1). Additionally, the yield of aberrant metaphases was determined after different recovery times in the presence or absence of TdR and CdR, respectively (Tables I and II). With a cell killing percentage of 10--15 (Fig. 1), ara-C induced 19% and 3'F T d R 68% metaphases containing aberrations. Only much longer treatment times, i.e. 18 h with 1 X 10 -6 M ara-C and 36 h with 1 X 10 -s M 3'-FTdR, dramatically decreased the mitotic index (see Tables I and II). For these treatment times, 57% aberrant metaphases were scored after ara-C treatment but 98% aberrant metaphases were obtained after 3'-FTdR treatment. Both substances induced chromatid breaks and gaps. As compared with araC, however, 3'-FTdR treatment resulted in more breaks per chromosome. Translocations were relatively rare after this treatment, but in the course of recovery time an increase of the relative frequency of translocations occurred. During a 12 h recovery period an increase of aberrant metaphases containing translocations was observed for ara-C and, though only to a lower extent, also for 3'-FTdR. Furthermore, during the recovery period, the high frequency of breaks decreased more rapidly after treatment with 3'-FTdR than after treatm e n t with ara-C. Cultivation of ascites cells in medium containing CdR and TdR, respectively, after pretreatment with ara-C or 3'-FTdR, resulted in a decreased aberration frequency. Treatment with CdR and TdR alone induced a slight increase of the spontaneous aberration frequency. With respect to ara-C, our results confirm earlier findings [2,3,8,14]. The substance induced gaps, chromatid breaks and chromatid interchanges (the latter being observed only in the course of recovery time). 3'-FTdR showed
105 qualitatively similar effects, although some quantitative differences were also found. For the same concentration and treatment time, ara-C caused very high and 3'-FTdR rather low aberration frequencies. At a b o u t the same cell killing rate, however, 3'-FTdR induced markedly higher aberration frequencies than ara-C, indicating the survival of 3'-FTdR-treated cells although they had a much higher aberration frequency. It is interesting to note that both substances seem to act differently [7,11] and thus may also be assumed to induce aberrations and/or to influence repair processes via different routes.
Acknowledgements I wish to thank Professor Langen, Berlin-Buch and Dr. SchSneich, Gatersleben for helpful discussions and Prof. Rieger, Gatersleben for critical reading of the manuscript.
References 1 Bell, W , R . , J . J . W a n g , P.P. C a r b o n e , G. B l e c h e r a n d J.B. B l o c k , C y t o g e n e t i c a n d m o r p h o l o g i c a b n o r m a l i t i e s in h u m a n b o n e m a r r o w cells d u r i n g c y t o s i n e a r a b i n o s i d e t h e r a p y , B l o o d , 2 7 ( 1 9 6 6 ) 7 7 1 . 2 B e n e d i c t , W . F . , N. H a r r i s a n d M. K a r o n , K i n e t i c s of 1 - ~ - D - a r a b i n o f u r a n o s y l c y t o s i n e - i n d u c e d c h r o m o s o m e b r e a k s . C a n c e r Res., 3 0 ( 1 9 7 0 ) 2 4 7 7 . 3 B r e w e n , J . G . a n d N . T . C h r i s t i e , S t u d i e s o n t h e i n d u c t i o n o f c h r o m o s o m a l a b e r r a t i o n s in h u m a n l e u k o c y t e s b y c y t o s i n e a r a b i n o s i d e , E x p . Cell Res., 4 6 ( 1 9 6 7 ) 4 6 . 4 C h u , M.Y. a n d G . A . F i s c h e r , A p r o p o s e d m e c h a n i s m of a c t i o n o f 1 - f l - D - a r a b i n o f u r a n o s y l c y t o s i n e as a n i n h i b i t o r o f t h e g r o w t h o f l e u k e m i c cells, B i o c h e m . P h a r m a c o l . , 11 ( 1 9 6 2 ) 4 2 3 . 5 C h u , M,Y. a n d G . A . F i s c h e r , T h e i n c o r p o r a t i o n o f 3 H - c y t o s i n e a r a b i n o s i d e a n d its e f f e c t o n r o u t i n e l e u k e m i c cells ( L 5 1 7 8 Y), B i o c h e m . P h a x m a c o l . , 1 7 ( 1 9 6 8 ) 7 5 3 . . 6 F u r t h , J.J. a n d S.S. C o h e n , I n h i b i t i o n o f m a m m a l i a n D N A p o l y m e r a s e b y t h e 5 ' - t r i p h o s p h a t e o f 1-~D - a r a b i n o f u r a n o s y l c y t o s i n e a n d t h e 5 ' - t r i p h o s p h a t e o f 9 - ~ - D - a r a b i n o f u r a n o s y l a d e r d n e , C a n c e r Res., 28 (1968) 2061. 7 G r a h a m , F . L . a n d G . F . W h i t m o r e , S t u d i e s in m o u s e L-cells o n t h e i n c o r p o r a t i o n o f 1 - f l - D - a x a b i n o f u r a n o s y l c y t o s i n e i n t o D N A a n d o n i n h i b i t i o n of D N A p o l y m e r a s e b y 1 - ~ - D - a r a b i n o f u r a n o s y l c y t o s i n e 5'triphosphate, Cancer Res., 30 (1970) 2636. 8 K i h l m a n , B . A . , W.W. N i c h o l s a n d A. L e v a n , T h e e f f e c t o f d e o x y a d e n o s i n e a n d c y t o s i n e a r a b i n o s i d e o n t h e c h r o m o s o m e s of h u m a n l e u k o c y t e s in v i t r o , H e r e d i t a s , 5 0 ( 1 9 6 3 ) 1 3 9 . 9 L a n g e n , P., G. E t z o l d , R. H i n s c h k e a n d G. K o w o l l i k , 3 t - D e o x y - 3 t - f l u o r o t h y m i d i n e , a n e w selective inh i b i t o r o f D N A - s y n t h e s i s , A c t a Biol. Med. G e r m . , 23 ( 1 9 6 9 ) 7 5 9 . 1 0 L a n g e n , P. a n d H. G r a c t z , Cell d e a t h r e s u l t i n g f r o m i n h i b i t i o n b y 3 r - d e o x y - 3 t - f l u o r o t h y m i d i n e , c y t o sine a r a b i n o s i d e , 5 - f l u o r o - 2 ' - d e o x y u r i d i n e , a n d h y d r o x y u r e a o f D N A s y n t h e s i s in c u l t u r e d E h r i i c h asc i t e s c a r c i n o m a cells, S t u d i a B i o p h y s . , 3 1 1 3 2 ( 1 9 7 2 ) 3 5 9 . 11 L a n g e n , P., G. K o w o l l i k , G. E t z o l d , H, V e n n e r a n d H. R e i n e r t , T h e p h o s p h o r y l a t i o n o f 3 ' - d e o x y - 3 ' f l u o r o t h y m i d i n e a n d its i n c o r p o r a t i o n i n t o D N A in a cellfree s y s t e m f r o m t u m o r cells, A c t a Biol. Med. Germ., 29 (1972) 483. 1 2 Mfiller, G.W., Z.-J. Y a m a z a k i , H . H . S S g t r o p a n d R . K . Z a h n , A c t i o n o f 1 - f l - D - a x a b i n o f u r a n o s y l c y t o s i n e o n m a m m a l i a n t u m o r cells. 2. I n h i b i t i o n o f m a m m a l i a n a n d o n c o g e a i c viral p o l y m e r a s e s , E u r . J. C a n cer, 8 (1972) 421. 1 3 N e g e t e i n , E., I. L e i s t n e r , u n d L. Jifiunchen, A n w e n d u n g d e r M a n o m e t r i e z u r U n t e r s u c h u n g d e s Zellw a c h s t u m s b e i m E h r l i c h - A s c i t e s k a r z i n o m . E i n f l u s s d e r S a u e r s t o f f k o n z e n t r a t i o n a u f die Z e l l v e r m e h r u n g , A c t a Biol. M e d . G e r m . , 1 6 ( 1 9 6 6 ) 3 7 2 . 1 4 N i c h o l s , W.W. a n d W.K. H e n e e n , C h r o m o s o m a l e f f e c t s of a r a b i n o s y l c y t o s i n e in a h u m a n d i p l o i d cell strain, Hereditas, 52 (1965) 402. 1 5 S c h w a r z a c h e r , H . G . , P r ~ p a x a t i o n v o n M i t o s e c h r o m o s o m e n , in H . G . S c h w a r z a c h e r u n d U, W o l f (eds.). M e t h o d e n in d e r m e d i z i n i s c h e n C y t o g e n e t i k , S p r i n g e r B e r l i n - - H e i d e l b e r g - - N e w Y o r k , 1 9 7 0 , p. 55. 1 6 Silagi, S., M e t a b o l i s m o f 1 - ~ - D - a r a b i n o f u r a n o s y l c y t o s i n e o n L-cells, C a n c e r Res., 2 5 ( 1 9 6 5 ) 1 4 4 6 . 1 7 Z a h n , R . K . , W . E . G . Mfiller, W. F o r s t e r , A. M a i d h o f a n d R. B e y e r , A c t i o n o f 1 - f l - D - a r a b i n o f u r a n o s y l c y t o s i n e o n m a m m a l i a n t u m o r cells. 1. I n c o r p o r a t i o n i n t o D N A , E u r . J. C a n c e r , 8 ( 1 9 7 2 ) 3 9 1 .
Mutation Research, 40 (1976) 101--106
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
CLASTOGENIC ACTIVITY OF CYTOSINE ARABINOSIDE AND 3'-DEOXY-3'-FLUOROTHYMIDINE IN EHRLICH ASCITES TUMOUR CELLS IN VITRO
ANNA M. WOBUS Zentralinstitut fiir Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR, 4325 Gatersleben (DDR)
(Received March 17th, 1975) (Revision received November 4th, 1975) (Accepted November 28th, 1975)
Introduction 1-fl-D-arabinofuranosylcytosine (ara-C), an analogue of cytidine, inhibits the growth of mammalian cells in vitro [4,10,16], and DNA synthesis in L cells [16] and murine lymphoblasts [4]. The c o m p o u n d interferes with DNA polymerase [6,7,12] and becomes incorporated into DNA [5,17]. Ara-C is known to produce chromosomal aberrations in vivo [1] and in vitro [2,3,8,14]. 3'-deoxy-3'-fluorothymidine (3'-FTdR), a newly synthesized analogue of thymidine, equally inhibits cell multiplication and DNA synthesis of Ehrlich ascites t u m o u r cells in vitro [9]. Inhibition of DNA synthesis may be reversed by high concentrations of thymidine. Neither RNA nor protein synthesis is inhibited [9]. 3'-FTdR was phosphorylated to the 5'-triphosphate level and incorporated into DNA [11]. In experiments on the inhibition of cell multiplication of ascites cells with ara-C and 3'-FTdR, Langen and Graetz [10] showed that 3'-FTdR was much better tolerated by the cells than ara-C. This is due to a longer lag period preceding cell death. In concentrations that produce an 80% inhibition of cell multiplication within 24 h (1 X 10 -s M 3'-FTdR, 1 X 10 .6 M ara-C), the treatment time needed to achieve a 50% killing rate was about one generation time longer for 3'-FTdR than for ara-C [10]. Because of these differences our aim was to determine the clastogenic effectivity of ara-C and 3'-FTdR in Ehrlich ascites tumour cells in vitro. Furthermore, we investigated whether some differences in the aberration frequencies exist during the recovery period in correlation with the differences in cell survival.
I
cells.
24 TdR
24 h 3’-FTdR
b Cells
with
a 1 X lo-’
ma.
and
24
24 h
f. per
100
a
12
24 h
TdR
_
48 h
5.5
11.7
4.4
8.6
11.4
-
30 h
4.1
36 h
13.5
24 h
4.0
27.7
8.9
12 h
a
12TdRa
24 h 3’-FTdR
TdR
24
24 h 3’-FTdR
-
12
Controls
0.5
-
36 h 3’-FTdR
24 h 3’-FTdR
a
2.6
30 h 3’-FTdR
15.5
1.1
1.6
(%)
200
200
200
200
200
300
200
200
200
300
300
300
300
300
300
Metaphases scored
frequency
INDUCED
Mitotic
ABERRATIONS
12 h 3’-FTdR
(h)
time
CHROMATID
Recovery
OF
24 h 3’-FTdR
M TdR.
FREQUENCIES
Treatment
THE
TABLE BY
4.50
4.00
1.00
0.50
1.50
0.33
0
9.00
55.50
21.00
69.33
98.33
94.00
67.67
23.67
* 0.58
?; 3.59
f 2.96
2 2.46
2 0.61
f 0.61
f 4.18
+ 0.96
+ 0.82
+ 0.85
+ 0.50
* 0.96
+ 0.33
(%I gaps
f 2.75
(Z * S,)
(9)
excluding
aberrations
with
M 3’-FTdR
Metaphases
1 X lo-’
7.0
4.0
0.5
3.5
3.5
1.0
1.0
5.0
23.5
12.7
47.7
62.3
58.3
36.0
72.0
3.0 1.0
3.5
1.0
0.5
1.0
-
5.0
_
0.5
0.5
17.5
12.7 11.5
17.0
126.3
_
1.0
1.0
1.7
21.7
22.1 61.0
430.7
21.3
1.3
19.6
4.3
m.a.
b
THE
403.3
t
cells
OF
1.6
100
CELLS
13.3
36.6 150.2
59.0
b
per
ASCITES
26.3
g
Aberrations
IN EHRLICH
1.0
1.3
2.1
0.7
1.3
fb
MOUSE
h.s.c.
103
Material and methods Ehrlich ascites t u m o u r cells (strain "Berlin-Buch") with a stem line of 44 chromosomes were cultivated in vitro according to a modified m e t h o d of Negelein et al. [13]. Cells 5 days old were removed from the peritoneal cavity of mice under sterile conditions, gassed with 5% CO2, 1.5% 02 in N2, and incubated in a modified Parker medium [13] for different times in the presence of 1 X 10 -6 M ara-C or 1 X 10 -s M 3'-FTdR (for reasons see Results) and subsequently counted. Incubation was performed in a Warburg apparatus (V 85, B. Braun Apparatebau, Melsungen) at 35°C with horizontal agitation. If the cultivation time exceeded 24 h, cells were passaged by centrifuging and washing twice with medium. Colchicine (0.002%) was added 3 h before fixation and the production of air-dried preparations [15]. For estimation of mitotic frequency in these preparations, 2000 cells were analysed for each value in Tables I and II. To estimate the aberration frequencies, cells were analysed for chromatid breaks (b), chromatid translocations (t), multiple aberrations (m.a.), single highly shattered chromosomes (h.s.c.), and totally fragmented or pulverized chromosomes (f). Gaps were separately counted but not included in the category of true aberrations. The experimental values as reported in Tables I and II and Fig. 1 are means of three sets of experiments. Ara-C and 3'-FTdR were dissolved in sterile bidistilled water, stored at --20°C and used without filtration. Results and discussion Identical concentrations and treatment times usually provide the basis of exTABLE THE
n FREQUENCIES
EHRLICH
ASCITES
Treatment
OF CELLS
CHROMATID OF
THE
ABERRATIONS
INDUCED
BY"
1
X
10 -6
M
ARA-C
IN
MOUSE
Recovery
Mitotic
Meta-
Metaphases
time
frequency
phases
with
aberra-
(h)
(%)
scored
tions
(%)
Aberrations g
per 100
b
cells
t
m.a.
b f b
h.s.c
excluding gaps
(g)
(~ ± s m) 6 h ara-C
--
4.2
200
19.00
± 1.28
16
30
18 h ara-C
--
0.3
200
56.50
+ 1.71
45
122
2
--
--
--
1
--
3
1 1
6 h ara-C
12
12.7
200
26.00
-+ 1 . 6 3
14
30
10
1
--
6 h ara-C
18
12.8
200
17.00
± 1.29
5
22
16
--
--
1
6 h ara-C
24
5.5
200
3.50
-+ 0 . 9 6
3
8
--
--
--
200
10.00
+- 0 . 8 2
8
6
--
--
--
1
5
--
--
--
6 h ara-C
12, + CdR
a
6 h ara-C
24, + CdR
a
19.1 5.1
200
4.00
-+ 0 . 8 2
-+ 0 . 5 8
-4 --
Controls 6h
--
6.9
200
1.00
3O h
--
5.5
200
0
6h
12 CdR
a
11.4
200
1.00
-+ 0 . 5 8
6h
24 CdR
a
4.5
200
4.00
± 0.82
a 1 X 10 -5 MCdR, b See Table
I.
2 .
2 .
-4
.
.
.
. .
. .
.
1
1
--
--
--
5
1
--
--
--
104 100'
v
50,
c_
1
0
6
11 18 2'4 30 t r e a t m e n t t i m e (h)
F i g . 1. E f f e c t o f 1 × 1 0 - 6 M a r a - C ( o cites turaour ceils in vitro.
3'6
e ) a n d 1 X 1 0 -5 M 3 ' - F T d R
(c,
5 ) o n t h e s u r v i v a l o f as-
perimental comparisons of two substances. Under such conditions, however, ara-C t r e a t m e n t results in very high and 3'-FTdR in rather low aberration frequencies. For instance, a treatment for 12 h with 1 X 10 .5 M caused 93.00 i 1.00% aberrant metaphases with ara-C and only 23.67 i 2.75% with 3'-FTdR. After 30 h and treatment with 1 X 10 -s M 3'-FTdR, 94.00 i 0.61% of aberrant metaphases was obtained. Since we were interested in checking the clastogenic effectivity of the two substances under conditions that cause comparable biological effects {inhibition of cell multiplication, effect on cell survival) we used exposure times that showed an identical cell killing rate of 10--15%, i.e. a treatment of 6 h for araC and of 24 h for 3'-FTdR (Fig. 1). Additionally, the yield of aberrant metaphases was determined after different recovery times in the presence or absence of TdR and CdR, respectively (Tables I and II). With a cell killing percentage of 10--15 (Fig. 1), ara-C induced 19% and 3'F T d R 68% metaphases containing aberrations. Only much longer treatment times, i.e. 18 h with 1 X 10 -6 M ara-C and 36 h with 1 X 10 -s M 3'-FTdR, dramatically decreased the mitotic index (see Tables I and II). For these treatment times, 57% aberrant metaphases were scored after ara-C treatment but 98% aberrant metaphases were obtained after 3'-FTdR treatment. Both substances induced chromatid breaks and gaps. As compared with araC, however, 3'-FTdR treatment resulted in more breaks per chromosome. Translocations were relatively rare after this treatment, but in the course of recovery time an increase of the relative frequency of translocations occurred. During a 12 h recovery period an increase of aberrant metaphases containing translocations was observed for ara-C and, though only to a lower extent, also for 3'-FTdR. Furthermore, during the recovery period, the high frequency of breaks decreased more rapidly after treatment with 3'-FTdR than after treatm e n t with ara-C. Cultivation of ascites cells in medium containing CdR and TdR, respectively, after pretreatment with ara-C or 3'-FTdR, resulted in a decreased aberration frequency. Treatment with CdR and TdR alone induced a slight increase of the spontaneous aberration frequency. With respect to ara-C, our results confirm earlier findings [2,3,8,14]. The substance induced gaps, chromatid breaks and chromatid interchanges (the latter being observed only in the course of recovery time). 3'-FTdR showed
105 qualitatively similar effects, although some quantitative differences were also found. For the same concentration and treatment time, ara-C caused very high and 3'-FTdR rather low aberration frequencies. At a b o u t the same cell killing rate, however, 3'-FTdR induced markedly higher aberration frequencies than ara-C, indicating the survival of 3'-FTdR-treated cells although they had a much higher aberration frequency. It is interesting to note that both substances seem to act differently [7,11] and thus may also be assumed to induce aberrations and/or to influence repair processes via different routes.
Acknowledgements I wish to thank Professor Langen, Berlin-Buch and Dr. SchSneich, Gatersleben for helpful discussions and Prof. Rieger, Gatersleben for critical reading of the manuscript.
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