Chromosomal aberrations of don lung cells of Chinese hamster after exposure to vinblastine in vitro

Chromosomal aberrations of don lung cells of Chinese hamster after exposure to vinblastine in vitro

99 Mutation Research, 66 (1979) 99--102 © Elsevier/North-Holland Biomedical Press Short C o m m u n i c a t i o n CHROMOSOMAL A B E R R A T I O N S...

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99

Mutation Research, 66 (1979) 99--102

© Elsevier/North-Holland Biomedical Press

Short C o m m u n i c a t i o n CHROMOSOMAL A B E R R A T I O N S OF DON L U N G CELLS OF CHINESE H A M S TER A F T E R EXPOSURE TO VINBLASTINE IN V I T R O .

MICHIHARU SEGAWA1, SHINSAKU NADAMITSU2, KATSUHIKO KONDO1 and IZUMI YOSHIZAKI2 I Faculty o f Integrated Arts and Sciences, Hwoshima University, Hirosh~ma 730, 2Faculty o f Home Economws, Htroshima Women's Unzversity, Hiroshima 734 (Japan)

(Received 18 April 1978) (Revision received 27 June 1978) (Accepted 20 July 1978)

Effects o f vmblastine have been studied with special reference to prophage induction [5], carcinostatic effects [2,6--8], teratogenic and embryocidal effects [1,4,9,14], and m ode of action on dividing cells in vitro [10]. These reports often say t hat effects o f vinblastine are similar to those o f colchicine. Tests o f vinblastine effects at the concentrations of 0.001, 0.01, 0.1 and 5.0 pg/ml in vitro were made, and colchico-mitotic effects were report ed in certain ammal cells (J-96 which is a hum a n m o n o c y t i c leukemia and LLC-Hel cells whmh are h u man e m b r y o n i c connective-tissue cells) by Palmer et al. [10]. T h e y n o ted th at vinblastine-treated cultures had an increase in the n u m b e r of dividing cells as well as an increase in the n u m b e r o f metaphases when compared with u n t r e a t e d controls. This was not a stimulation of mitosis but represented an accumulation of metaphases. There was no change in the percentage o f prophases, and no post-metaphase stages including anaphases, telophases and cells th at appeared to have just completed cell division. T he absence of postmetaphase stages seems to indicate an inhibition of the spindle, which is similar to the pattern obtained with colchicine treatment. When cultures were treated for a long time, or with a higher c o n c e n t r a t i o n of the drug, more multinucleate cells and cells with lobed nuclei were f o r m e d [10]. Similar results on the s t a t h m o k m e t i c (C-mitosis) and m e r o s t a t h m o k i n e t i c (incomplete C-mitosis) effects have been observed in certain plant cells in vivo [3,12,13]. The clastogenic effects o f vinblastine, however, are poorl y r e p o r t e d in most standard references. Don lung cells of Chinese hamster were incubated for 72 h at 37°C in culture flasks with 4.5 ml of Eagle's MEM medium (Nissui), 0.5 ml of fetal calf serum (10%), 0.015 g of glutamine (Nissui) and a few drops o f 7% sodium bicarbonate to give pH 7.2, before t he y were treated with vinblastine sulfate (Exal: Shionogi and Co. ®, Japan; Eli Lilly and Co., U.S.A.) at a c o n c e n t r a t i o n of 0.0039, 0.0078 and 0.0156 pg/ml for 2, 4, 6, 8, 10, 12, 24 or 32 h. Colchicine at

100

the final co n cen tr at i on of 0.5 pg/ml was added for 2 h before fixation m order to facihtate c h r o m o s o m e analysis as a usual procedure of the experiments. The slides were prepared by the regular flame-drying m e t h o d , and then their chromosomes were stained with 6% Giemsa (Merck, Darmstadt, West Germany). T h e numbers and types of chromosomal aberrations were assessed by examining 100 metaphase cells at each concentration. The results of clastogenic effects on the Don lung cells of Chinese hamster induced by three concentrations of vinblastlne are listed m Table 1. A bout 2 h from the beginning of the treatments with the three concentrations of vinblastine, lobulated nuclei at lnterphase started to increase gradually. More metaphase cells were available for examination when treated with the lowest concentration and shortest time. The control cultures exhibited low levels of chro-

TABLE

1

MITOTIC INDICES AND FREQUENCIES OF CHROMOSOMAL ABERRATIONS AT METAPHASE PRODUCED BY TREATMENTS WITH THREE CONCENTRATIONS OF VINBLASTINE IN DON LUNG CELLS OF CHINESE HAMSTER Coneentratlon (#g/ml)

Tnne of treatment (h)

Mltotic a index (%)

Metaphases b containmg aberrahons (%)

Aberrations Rings

p e r 1 0 0 cells

Gaps

Chromatxd breaks

Isochromatxd breaks

Chromatld exchanges

Ceils with multiple aberrations (%)

Control

0

12.1

6

3

1

0

2

0

0

0.0039

2 4 6 8 10 12 24 32

11.7 10.6 77 6.6 6.1 5.1 4 7 3 1

1 7 15 8 28 15 21 30

1 1 1 0 1 1 0 1

0 3 8 6 16 12 10 21

0 2 2 1 8 2 2 6

0 1 3 2 5 6 7 12

0 0 1 0 2 0 2 2

0 0 0 1 4 6 0 12

0.0078

2 4 6 8 10 12 24 32

8.9 7.3 7.3 7.2 6 5 3.2 3.1 2.2

10 21 25 52 44 31 42 54

0 l 1 5 3 2 1 0

8 10 7 35 35 19 31 43

2 3 3 7 4 6 4 12

4 7 14 15 3 4 6 2

0 1 0 1 0 0 2 4

4 1 0 11 1 0 2 7

0.0156

2 4 6 8 10 12 24 32

11.3 5 5 5.0 4.0 3.9 2.8 2.4 1.9

12 11 25 29 35 37 25 33

2 3 3 7 6 5 2 1

6 4 22 20 33 26 16 19

10 1 1 4 9 7 3 7

2 5 4 5 8 6 4 6

2 3 I 8 4 2 0 0

10 5 6 15 25 9 0 0

a P e r c e n t a r i s i n g f r o m 1 0 0 0 cells m e a c h o b s e r v a h o n . b 100 metaphases were analyzed m each observation.

101

6oJ

~%

/\ ~

.ff

40-

L f

E 20-

/

0

16

2~1

tr+'4tnli'llt

( h<)tlr +,/

0

tll/It + ~lt

32

Fig. 1. P e r c e n t a g e s of m e t a p h a s e s w z t h c h r o m o s o m a l a b e r r a t z o n s o b t a i n e d m cell cultttres o f D o n l u n g cells o f t h e C h i n e s e h a m s t e r at v a r i o u s t z m e s o f t z e a t m e n t w z t h vlnblastzne at c o n c e n t r a t i o n s o f 0 . 0 0 3 9 ( e ) , 0 . 0 0 7 8 (o) a n d 0 . 0 1 5 6 ( ' ) # g / m l . 1 0 0 m e t a p h a s e cells w e r e s c o r e d at e a c h o b s e r v a t i o n .

mosome damage, with a mean of 6%. The numbers of chromosomal aberrations increased gradually up to 10 treated hours and then decreased slightly (Fig. 1). The reduced frequency of aberrations with increasing concentration is only reflected with regard to gaps and not to other aberrations such as rings, breaks and exchanges; the latter types of aberration are more reliable. Beside the major aberrations of chromosomes mentioned above, some other aberrations found during the course of investigation were as follows: (1) metaphase chromosomes in elongated shape with brief interspersed area lacking splralization (the same result as that shown by Prantera et al. [11]); (2) C-metaphases; (3) chromosome stickiness; (4) chromosomes completely broken; (5) endoreduplicated chromosomes more often contained multiple aberrations which might be correlated with the concentration of vinblastine and time sequence since they were induced more at higher concentrations and longer treatment times. Acknowledgement We thank Dr. W.W. Nichols, Institute for Medmal Research, Camden, N.J. (U.S.A.) for stimulating discussions. References 1 C u r y , G., a n d C. B o d e l e t , E f f e t s c o m p a r 6 s d e la v ] n c a l e u c o b l a s t m e et d e la c o l c h z c i n e sur les tzssus h ~ m a t o p o z ~ t l q u e s d e l ' e m b r y o n d e p o u l e t ~ t u d e in vivo e t m w t r o , C. R. Soe. Biol. ( P a n s ) , 163 (1969) 1601--1603. 2 Cutts, J . H . , C.T. B e e r a n d R . L . N o b l e , B i o l o g i c a l p r o p e r t i e s o f w n c a l e u k o b l a s t m e , an a l k a l o i d m V m c a r o s e a L m n . w i t h r e f e r e n c e t o its a n t z t u m o r a c t z o n , C a n c e r Res., 20 ( 1 9 6 0 ) 1 0 2 3 - - 1 0 3 1 .

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3 D e g r a e v e , N., a n d J . G l l o t - D e l h a l l e , C y t o l o g i c a l e f f e c t s o f w n b l a s t m e m p l a n t s , E x p e r l e n t t a , 28 ( 1 9 7 2 ) 581--582. 4 Ferm, V.H., Congcmtal malformatlons m hamster embryos after treatment with wnblastme and wncnstme, Semnce, 141 (1963) 426. 5 Hcmemann, B., and A.J. Howard, Inductlon of lambda-bactenophage in E~chcr~ch~a cob as a screening test for potentlal antltumor agents, APpl. Mlcroblol., 12 (1964) 234--239. 6 Hertz, R., M.B. Lipsett and R.H. Moy, Effect of wncaleukoblastme on metastatlc chonocarcmoma and related trophoblastlc tumors in women, Cancer Res., 20 (1960) 1050--1053. 7 Hodes, M.E , R.J. Rohn and W.H. Bond, Vmcaleukoblastine, I, Prehmmary chmcal studles, Cancer Rcs., 20 (1960) 1041--1049. 8 Johnson, I.S., H.F. Wright, G.H. Svoboda and J Vlantls, Antltumor pnnclples derived from VInca rosea Lmn. I. Vmcaleukoblastme and leurosme, Cancer Res., 20 (1960) 1016--1022. 9 Noble, R.L., C.T. Beer and J.H. Cutts, Role of chance observatlons m chemotherapy l"mca rosea, Ann. N.Y. Aead. Scl., 76 (1958) 882--894 10 Palmer, C.G., D. Llvengood, A.K. Warren, P.J Simpson and I.S. Johnson, The action of vmcaleukoblastlne on mltosls in vltro, Exp. Cell Res., 20 (1960) 198--201 11 Prantera, G., M. DI Castro, E. Marchettl and A. Rocchl, Effect of dastamycm A on Chinese hamster chromosomes, Exp. Cell Res., 109 (1977) 459--462. 12 Segawa, M., and K. Kondo, Effects of wnblastme on menstematlc cells of ,lllzutn cepa, I, Experlentla, in press S e g a w a , M., a n d K. K o n d o , E f f e c t s o f v l n b l a s t m e o n m e r l s t e m a t l c cells o f A l b u m c e p a , II, M e m . Fac. I n t e g r a t e d A r t s a n d Scl., H l r o s h l m a U m v . , Ser. I V , 3 ( 1 9 7 7 ) 1 - - 1 1 . 14 S e n t e m , P., L ' a c t l o n de la v m c a l e u c o b l a s t m e s u r la m l t o s e c h e z T r z t u r u s h e h , e t l c u s R a z . , C h r o m o 13

s o m a , 15 ( 1 9 6 4 ) 4 1 6 - - 4 5 6 15 W a r w i c k , O . H . , J . M . M . D a r t e a n d T . C B r o w n , S o m e b m l o g l c a l e f f e c t s o f w n c a l e u k o b l a s t l n e , an a l k a l m d in VL nca r o s e a L m n . m p a t i e n t s w i t h m a h g n a n t chsease, C a n c e r R e s . , 20 ( 1 9 6 0 ) 1 0 3 2 - - 1 0 4 0

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