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Induction of chromosome aberrations in cultured human cells by ethylenimine and its relation to cell cycle
Mutation Reseamh Elsevier Publishing Company, Amsterdam Printed in The Netherlands
83
I N D U C T I O N OF CHROMOSOME A B E R R A T I O N S IN C U L T U R E D HUMAN CELLS BY E T H Y L E N I M I N E AND ITS R E L A T I O N TO CELL CYCLE TSUENG-HSING CHANG* AND FLORA T. ELEQUIN The Wistar Institute of Anatomy and Biology, Philadelphia, Penna. (U.S.A.) (Received June I3th, 1966)
SUMMARY H u m a n cells (WI-38, passage 22 and leukocyte cultures) were treated in vitro with ethylenimine solution of various concentrations for various lengths of time before fixation. The findings are as follows: (I) Ethylenimine was cytotoxic to the cells at the concentration of io -2 M or higher. (2) Only chromatid gaps, breaks, and exchanges were recovered. (3) Cells treated for 3.5 h before fixation with ethylenimine showed little damage; only 7% of the cells showed aberrations. This is not significantly higher than the control value of 4%. On the other hand, 29% of the cells had aberrations when treated similarly for 8 h. This suggests that aberrations are induced primarily in the S-period. (4) This was confirmed b y autoradiography b y treating leukocyte cultures for 8 h with ethylenimine and [SHlthymidine. 56 cells out of ioo had aberrations. Of these 56 cells all but 4 were labeled; (5) A significantly high number of aberrations were still found 7 days after the treatment. This might be similar to the delayed action found in Drosophila, although one cannot rule out the possibility that the chemical or its active product persisted in the cytoplasm after the actual treatment. The question of the sensitivity of G1 cells to ethylenimine remains unresolved.
INTRODUCTION Among the mutagenic alkylating agents are ethylenimine (or aziridine) and its derivatives. Ethylenimine has been shown to cause mutation, for example, in Drosolhhila 1, in Neurost~ora t~, and in wheat n, and induce chromosome aberrations in root tops of Allium cepa, mouse embryonic skin cultures, and Crocker mouse Sarcoma I88'. A derivative of ethylenimine, TEM also has similar effects in Drosophila",~,*L in Neurospora "s and in cultured human leukocytes I o. Of particular interest is a * Present address: Metabolism and Radiation Research Laboratory, State University Station, Fargo, N. Dak. (U.S.A.). Abbreviation : TEM, 2,4,6-triethyleneimino-i,3,5-triazine.
Mutation Res., 4 (I967) 83-89
84
TSUENG-HS1NG CHANG, FLORA T. ELEQUIN
recent finding 23 that m a n y ethylenimine compounds (for example, apholate and tepa) have sterilizing effect on insects and that this sterilizing property m a y be successfully used in controlling insect pests. Thus, studies of these compounds have genetic, health, as well as economic importance. Generally 3 effects of biological alkylating agents are recognized2°: (z) cytostatic--the inhibition of mitosis, (2) cytotoxic--metabolic disturbances resulting in socalled superfragmentation of chromosomeslL and (3) mutagenic--the induction of mutations, chromosome breaks, and rearrangements. The distinction between (I) and (2) m a y be merely quantitative rather than qualitative. As the concentration of the chemical is increased the effect shifts from cytostatic to cytotoxic. RINALDINP9 reported that an aromatic nitrogen mustard (p-amino-N,N-di-2-chloroethylaniline) shows cytotoxic and mutagenic effects at a high concentration, but a cytostatic effect at low concentrations in cultures of chick osteoblasts. Chromosome breaks are considered the major cause of cell death a but the importance of metabolic disturbance, which results in superfragmentation (or cytotoxic effect), is also stressed TM. In systems where a correlation of action of chemicals to the cell cycle is possible, it has been shown that different stages of the cell cycle have marked differences in their susceptibility to chemicals or to a physical factor such as ionizing radiations. For instance, EVANS AND SCOTT7 showed that maleic hydrazide induces chromatid aberrations exclusively in the S-period. Cytosine arabinoside 5 and 8-ethoxycaffeine ~2 are effective in inducing aberrations primarily in the G~-period. Nitrogen mustard produces aberrations only during the S-period. The kinetics or mode of action of chemicals m a y be sought in the comparative study of various compounds which carry the same functional group but have different carrier moieties. In this paper the induction of chromosome aberrations in cultured human cells by ethylenimine is studied. It is hoped that this will serve as a basis for comparison with our later planned studies using some derivatives of ethylenimine. Briefly, the results of this study show that all recovered chromosome aberrations are of the chromatid type and that they are induced almost exclusively in the S-period of the cell cycle. A high percentage of aberrations was detected more than 2 passages after the treatment of these cells with various concentrations of ethylenimine. These aberrations m a y have been the result of a delayed effect which is characteristic of chemical mutagens. MATERIALS AND METHODS
2 kinds of culture were used. One was a culture of human-embryonic lung fibroblasts (WI-38, x x x v l I I , passage 22) and the other was short-term leukocyte cultures from a normal male. WI-38 X X X V I I I cells. The cells were maintained in Eagle's basal medium (in Eagle's BSS with IO% calf serum) and subcultured (i : 2 split) every 3 or 4 days. The medium also contained penicillin and streptomycin (ioo units and ioo/~g/ml, respectively). On the 2nd day after the 22nd subculture, the cells were subjected to the following treatments: (!) IO-* M ethylenimine* for 8 h before fixation, (2) IO-* M ethylen* Courtesy of The Dow Chemical Company, Midland, Mich.
Mutation Res., 4 (1967) 83-89
INDUCTION OF CHROMOSOMEABERRATIONSIN CULTURED HUMANCELLS
85
imine for 3.5 h before fixation, (3) IO-*, IO-*, IO-5, and IO-6 M ethylenimine for I h and fixed at I-, 7-, Io-, and I4-day post-treatment. For the treatment of cells with ethylenimine a certain volume of ethylenimine solution (in phosphate-buffer solution) was added to the culture to achieve a desired final concentration. In (3) above, the cells were washed 4 times with culture medium after the chemical treatment. Leukocyte culture. The cultures were set up following the technique of MOORHEAD et al. ~' with some modifications. The medium used was: IOO ml TC I99, 2 5 m l calf serum, 0.5 ml phytohemagglutinin M, and 1.2 ml penicillin and streptomycin solution (ioooo units and ioooo #g/ml, respectively). Leukocyte suspension was obtained by gravitational separation. To each Falcon plastic bottle (30 ml) containing 4.5 ml of medium, 0. 4 ml of leukocyte suspension was added. For chemical treatment, o.I ml of ethylenimine solution was added to the culture to obtain a final concentration (10 -4 M), These cultures were treated with: (I) [aH]thymidine (2/zC/ml, spec. activity 3 C/mmole, Schwarz BioResearch, Inc., Orangeburg, N.Y.) for 8 h before fixation at 72 h after starting the culture, (2) IO-~ M ethylenimine and [~H]thymidine (2 ~uC/ml) for 8 h before fixation at 72 h. The slides from leukocyte culture were stained with acetoorcein (2% in 65% acetic acid), scored for chromosome aberrations and then coated with Kodak NTB-2 emulsion for autoradiography. The emulsion was exposed for 7 days, developed in D- I I for 2 rain, rinsed in water, fixed in Kodak acid fixer for 2 min, and mounted in Permount. In all instances colcemid (0.05 #g/ml) was added to the cultures 3.5 h before fixation. The chromosome preparations were made by the ignition-drying technique of:MoORHEAD AND NOWELL16. RESULTS AND DISCUSSION The results are summarized in Tables I, II and III. Only the cells treated with IO-* M, 6r lower, of ethylenimine solution survived. The cells did not survive the treatment with IO-* M of ethylenimine even for I h. Under the conditions of the experiment, only chromatid gaps, breaks and exchanges were recovered. This is in agreement with the observation that only chromatid aberrations are induced by alkylating agents**. The chromosome aberrations can easily be detected by the presence of dicentrics, rings, and large double fragments 14. In the cells treated with IO-* M ethylenimine for 3.5 h, 7% of the cells had gaps and/ or breaks, with IO total gaps and breaks. This is not significantly different from the control value of 3.9%. However, in the series treated with lO -4 M solution for 8 h, 29% of the cells showed gaps and/or breaks, significantly higher than the control. Since the duration of the G, period in human cells is estimated as being 3.5 h~ to 4.0 h .1, these results suggest that ethylenimine induces damage in the S-period almost exclusively, and that it does very little damage to cells in G2. The data presented in Table III on autoradiography of cultured leukocytes confirm this. In 72-h leukocyte cultures, 56% of the cells were damaged (Table II) after treatment for 8 h with ethylenimine (IO-* M) and [*H]thymidine. Of these 56 abnormal metaphases all but 4 showed labels in their autoradiograms (Table III and Fig. I). Mutation Res., 4 (I967) 83-89
86
TSUENG-HSING CHANG, FLORA
T.
ELEQUIN
TABLE 1 FREQUENCIES OF INDUCED CHROMATID ABERRATIONS AFTER TREATMENT OF W I - 3 8 CELLS WITH ETHYLENIMINE
Cells with exchanges and o~ breaks (/o)
(PASSAGE 22)
Ethvlenimine treatment
Passage Total cells Cells with gaps checked and breaks (%)
Total gaps Total and breaks exchanges
Control a
22
lO 3
4 (3.9)
o
4
o
22
lOO
7 (7 .°)
o
lo
o
22
lO 4
3 ° (28.8)
o
37
o
22 24 25 26
lOO IOO ioo ioo
31 14 8 9
lO (IO.O) o o o
61 16 8 9
It o o o
22 24 25
1OO lOO IiO
35 (35 '°) 24 (24.0) 13 (I1.8)
2 (2.0) I (I.O) o
44 31 14
2 I o
IO 4 M a
3.5 h 10-4 ]///a
8 h lO--a M b
lO
,i
I day 7 days 1o d a y s 14 d a y s Mb I {lay, 7 days 1o d a y s
(31.o) (14.o) (8.o) (9.o)
I 0 5 llqb
I day
22
loo
2o (2o.o)
0
23
o
7 days
24
too
6 (6.o)
o
7
o
IO d a y s
25
lOO
5 (5 .0 )
o
5
o
22 24
lOO IOO
7 (7 .°) 4 (4 .°)
I (i.o)
o
8 4
I o
IO ~ M I~
I clay' 7 days
a Cells were fixed a t t h e end of t r e a t m e n t p e r i o d ; Colcemid (0.05 t ,g/ ml ) for 3.5 h before fixation. 13 Cells were t r e a t e d for I h; a n d s a m p l e s fixed a t i, 7, IO, a n d 14 da ys , p o s t - t r e a t m e n t ; Colcemid ( 0 . 0 5 / , g / m l ) for 3-5 h before fixation. TABLE
11
CHROMAT1D ABERRATIONS IN 7 2 - h HUMAN-LEUCOCYTE CULTURES AFTER TREATMENT WITH ETHYLENIMINE AND [ S H ] T H Y M I D I N E FOR 8 h
Treatment
Number of cells
io 4 M Ethylenimine + [~H]Thymidine (2/,C/ml) 8 h
ioo
Cells with breaks Cells with Total breaks and gaps ( ° / ) exchanges (%) and gaps
Total exchanges
54 (54 .°)
2
2 (2.0)
114
TABLE III CHANGE IN PERCENTAGES OF LABELED METAPHASES AFTER TREATMENT WITH E T H Y L E N I M I N E FOR 8 h
Treatment
[SH]Thymidine (2/,C/ml) + ethylenimine 8 h [ a H ] T h y m i d i n e (2/zC/ml) 8 h
Total % labeled metaphase metaphase
Labeled mitosis abnormal normal
Unlabeled mitosis abnormal normal
ioo 539
52
4
85.0 68.8
33 371
EVANS AND SCOTT 7 f o u n d t h a t m a l e i c h y d r a z i d e
ii i 68
induces chromatid
aberrations
e x c l u s i v e l y i n t h e S - p e r i o d . T h e s a m e w a s a l s o s h o w n f o r n i t r o g e n m u s t a r d ~2. OSTERTAG et al. 18 h a v e a l s o r e p o r t e d fore or during
DNA
synthesis.
arabinoside 5 cause damage
Mutation Res., 4 (1967) 83-89
that caffeine induces aberrations On the other
i n G2 c e l l s .
hand,
o n l y if i t is a d d e d b e -
8 - e t h o x y c a f f e i n e 22 a n d
cytosine
INDUCTION OF CHROMOSOME ABERRATIONS IN CULTURED HUMAN CELLS
87
Fig. I. A u t o r a d i o g r a p h of a cell from leukocyte c u l t u r e t r e a t e d with e t h y l e n i m i n e a n d [SH]thymidine for 8 h. N o t e a c h r o m a t i d e x c h a n g e b e t w e e n 2 c h r o m o s o m e s of 6-12 group.
When chromosomes in G1 cells are damaged by ionizing radiations, chromosometype aberrations are found 6. WOLFF AND LUIPPOLD2s showed that in Vicia faba the chromosome reacts to X-rays as though functionally single during most of the G1; but as though double during the S and G2 phases. The transition from "single" to "double" occurs in the late G~. Whereas in radiation experiments, the recovery of chromosome-type aberrations can be taken as an indication of damage in G1 ceils, the same cannot be said for experiments using alkylating agents because these chemicals have not been shown to induce chromosome-type aberrations. In our experiments when the cells were treated with ethylenimine for i h and fixed at various time afterward (see below), no aberration of the chromosome-type was found. Unfortunately under the experimental conditions, the question of the sensitivity of Ga cells to ethylenimine cannot be answered unequivocally. That answer can only be obtained either from experiments in which the time sequence of the appearance of aberlations is accurately correlated to the full cell cycle, or from experiments in which the cells are fixed at close time intervals after the treatment at least until the aPFearance of the 2nd metaphases. In the I-h treatment series (Table I), the frequencies of chromatid aberrations were high for the first 2 samples (i.e. xst and 7th day post-treatment). Thus, in the xo-3 M series, 3I% of the xst day sample had gaps and/or breaks and another xo% of the cells had exchanges and breaks (orgaps). At the 7th day no exchange was recovered, but the frequency of breaks and gaps (14%) was still higher than normal, whereas by the xoth and x4th day it dropped to 8 and 9% respectively, i.e. not significantly higher than control. In the xo-* M series, the picture is similar to that of IO-s M series. The frequencies of chromatid aberrations and exchanges were quite high; the combined frequenMutation Res., 4 (I967) 83-89
88
TSUENG-HSING CHANG, FLORA T. ELEQUIN
cies of breaks and exchanges were 37% and 25°/o, respectively, for the Ist and 7th day post-treatment samples. The damage in the I o t h day post-treatment sample showed 13% of the cells with gaps and breaks, significantly higher than the control. As the concentration of the chemical decreases, the damage lessens as expected. In the IO-s M series, only the Ist day sample has a singnificantly higher chromatidgap frequency than the control. For the IO-~ M series, the frequencies of induced abnormality are quite low and not singificantly higher than the control. One of the characteristics of chemical mutagens is the delayed expression of induced mutations 2. In Drosophila melanogaster males treated with ethylenimine, the delayed lethals are detected in the progeny of apparently normal F2 females 1. As pointed out by AUERBACH2 it is difficult to compare the actions of mutagens between to different species, especially so when an in vitro system from human tissue is compared with Drosophila. In an in vitro system one should be always aware of the possibility that the chemical or its active product m a y persist for 2 or 3 cell cycles in the cytoplasm of cells which are actively dividing. Nevertheless our results show that chromarid aberrations and exchanges were still detected in cells fixed 7 days after the treatment with IO-3 and lO -4 M solution of ethylenimine. These cells were harvested 2 subcultures (1:2 split) after the treatment. They were probably in their 2nd or 3rd post-treatment mitoses, assuming, of course, that any delay in mitosis caused by ethylenimine did not last as long as 7 days. These results might be parallel to the finding in Drosophila. Further experiments will be needed to clarify this question. The percentage of labeled leukocyte metaphases increased under the influence of ethylenimine (Table III) This increase m a y be explained by the slow-down in the completion of DNA synthesis of cells in the late S-period. In these cultures the metaphases accumulated mark the cells which were 4.5-8 h away from mitosis when [aH~thymidine and/or ethylenimine were added. Since Gz lasts approximately 4 h, the first of the accumulated metaphases were at the late S-period or early G~ when the chemicals were added. If ethylenimine delays the completion of DNA synthesis, the delay m a y enable more of the cells in the late S-period to be scored as "labeled". This " e x t r a " label would be reflected in the increased percentage of labeled metaphases treated with ethylenimine. Indeed there is some evidence that nitrogen mustard 15 and apholate 1~ hamper DNA synthesis. ACKNOWLEDGEMENTS
The authors are grateful to Dr. PAUL S. MOORHEADfor reading the manuscript and to Miss PAULA MANCINELLI AND Mrs. MARCIA LEBOWlTZ for their technical assistance. One of the authors (T. H. CHANG) was supported by a U.S. Public Health Service Training Grant TI-CA-5163. REFERENCES I ALEXANDER, M. L., AND E. GLANGES, Genetic d a m a g e i n d u c e d b y e t h y l e n i m i n e , Proc. Natl.
Acad. Sci. (U.S.), 53 (1965) 282-288. 2 AUERBACH, C., P r o b l e m s in chemical m u t a g e n e s i s , Cold Spring Harbor Symp. Quant. Biol., i6 (1951) 199-213. 3 AUERBACH, C., M u t a g e n i c effects of a l k y l a t i n g agents, Ann. N. Y. A cad. Sci., 63 (I958) 731-736. 4 BIESELE, J. J., F. S. PHILIPS, J. B. THIERSCH, J. H. BURCHENAL, S. M. BUCKLEY AND C. C. STOCK, C h r o m o s o m e a b e r r a t i o n s a n d t u m o r inhibition b y n i t r o g e n m u s t a r d s : t h e h y p o t h e s i s of cross-linking alkylation, Nature, i 6 6 (i95o) i i 1 2 - i i 13. Mutation Res., 4 (1967) 83-89
INDUCTION OF CHROMOSOME ABERRATIONS IN CULTURED HUMAN CELLS
89
5 BREW~N, J. G., The induction of chromatid lesions by cytosine arabinoside in post-DNAsynthetic human leukocytes, Cytogenetics, 4 (I965) 28-36. 6 EVANS, H. J., Chromosome aberrations induced by ionizing radiations, lntern. Rev. Cytol., 13 (1962) 221-321. 7 EVANS, H. J., aND D. SCOTT, Influence of DNA synthesis on the production of chromatid aberrations by X-rays and maleic hydrazide in Vicia faba, Genetics, 49 (I964) 17-38. 8 FAHMV, O. G., AND M. J. FAHMV, Cytogenetic analysis of the action of carcinogens and tumor inhibitors in Drosophila ~nelanogaster, II. The mechanism of induction of dominant lethals by z,4,6-tri-(ethyleneimino)-I,3,5,-triazine, .]. Genet., 52 (1954) 6o3-619. 9 FAHMV, O. G., AND M. J. FaHMY, Cytogenetic analysis of the action of carcinogens and tumor inhibitors in Drosophila melanogaster, IV. The cell stage during spermatogenesis and the induction of intra- and inter-genie mutations by z,4,6-tri(ethyleneimino)-l,3,5-triazine, J. Genet., 53 (1955) 563-584 • io HAMPEL, K. E., AriD H. GERHARTZ, Strukturanomalien der Chromosomen menschlicher Leukozyten in vitro dutch Tiraethylenmelamin, ExptL Cell Res., 37 (I965) 251-258. II KHVOSTOVA,V. V., V. S. MOZHAEVA, N. S. AIGAES AND S. A. VALEVA, Mutants induced by ionizing radiations and ethyleneimine in winter wheat, Mutation Res., 2 (i965) 339-344. i2 KILGORE, W. W., AND R. R. PAINTER, Effect of the chemosterilant apholate on the synthesis of cellular components in developing housefly eggs, Biochem. ]., 92 (1964) 353-357. 13 KOLLER, P. C., Comparative effects of alkylating agents in cellular morphology, Ann. N . Y . Acad. Sci., 63 (I958) 783-8Ol. 14 LEA, D. E., Actions of Radiations on Living Cells, 2nd ed., Cambridge University Press, Cambridge, England, 1955, p. 416. 15 LEvis, A. G., G. A. DANIELI AND E. PlCClNNI, Nucleic acid synthesis and the mitotic cycle in mammalian cells treated with nitrogen mustard in culture, Nature, 207 (1965) 6o8-61o. 16 MOORHEAD, P.S., AND P. C. NOWELL, Chromosome cytology, in Methods in Medical Research, Vol. IO, Yearbook Medical Publishers, Chicago, Ill., 1964, p. 31o-322. 17 MOOR~EAD, P. S., P. C. NOW~LL, W. J. MF.LLMAr~,D. M. BArTIPS AND D. A. HUNGERFORD, Chromosome preparations of leukocytes cultured from peripheral blood, Exptl. Cell Res., 20 (196o) 613-616. 18 OST~RTAG, W., E. DUISBERG AND M. STURMANN, The mutagenic activity of caffeine in man, Mutation Res., 2 (1965) 293-296. 19 RIN.~LDINI, L. M., The antimitotic action of an aromatic nitrogen mustard on tissue cultures, Brit. J. Cancer, 6 (1952) 186-I96. 20 Ross, w . C. J., Biological Alhylating Agents, Butterworths, London, p. 232. 21 SCHMID, W., DNA replication patterns of human chromosomes, Cytogenetics, 2 (1963) 175-193. 22 ScoTT, D., AND H. J. EvAr~s, On the non-requirement for deoxyribonucleic acid synthesis in the production of chromosome aberrations by 8-ethoxycaffeine, Mutation Res., i (1964) i46-156. 23 SMITH, C. N., G. C. LABRECQUE AND A. B. BORKOVEC,Insect chemosterilants, Ann. Rev. Entotool., 9 (1964) 269-284. 24 SNYDER, L. A., AND I. I. OSTER, A comparison of genetic changes induced by a monofunctional and a polyfunctional alkylating agent in Drosophila melonogaster, Mutation Res., I (1964) 437445. 2 5 WESTERGAARD, M., Chemical mutagenesis in relation to the concept of the gene, Experientia, I3 (1957) 224-234. 26 WOLFF, S., AND H. E. LUIPPOLD, Chromosome splitting as revealed by combined X-ray and labeling experiments, Exptl. Cell Res., 34 (I964) 548-556.
Mutation Res., 4 (1967) 83-89
83
I N D U C T I O N OF CHROMOSOME A B E R R A T I O N S IN C U L T U R E D HUMAN CELLS BY E T H Y L E N I M I N E AND ITS R E L A T I O N TO CELL CYCLE TSUENG-HSING CHANG* AND FLORA T. ELEQUIN The Wistar Institute of Anatomy and Biology, Philadelphia, Penna. (U.S.A.) (Received June I3th, 1966)
SUMMARY H u m a n cells (WI-38, passage 22 and leukocyte cultures) were treated in vitro with ethylenimine solution of various concentrations for various lengths of time before fixation. The findings are as follows: (I) Ethylenimine was cytotoxic to the cells at the concentration of io -2 M or higher. (2) Only chromatid gaps, breaks, and exchanges were recovered. (3) Cells treated for 3.5 h before fixation with ethylenimine showed little damage; only 7% of the cells showed aberrations. This is not significantly higher than the control value of 4%. On the other hand, 29% of the cells had aberrations when treated similarly for 8 h. This suggests that aberrations are induced primarily in the S-period. (4) This was confirmed b y autoradiography b y treating leukocyte cultures for 8 h with ethylenimine and [SHlthymidine. 56 cells out of ioo had aberrations. Of these 56 cells all but 4 were labeled; (5) A significantly high number of aberrations were still found 7 days after the treatment. This might be similar to the delayed action found in Drosophila, although one cannot rule out the possibility that the chemical or its active product persisted in the cytoplasm after the actual treatment. The question of the sensitivity of G1 cells to ethylenimine remains unresolved.
INTRODUCTION Among the mutagenic alkylating agents are ethylenimine (or aziridine) and its derivatives. Ethylenimine has been shown to cause mutation, for example, in Drosolhhila 1, in Neurost~ora t~, and in wheat n, and induce chromosome aberrations in root tops of Allium cepa, mouse embryonic skin cultures, and Crocker mouse Sarcoma I88'. A derivative of ethylenimine, TEM also has similar effects in Drosophila",~,*L in Neurospora "s and in cultured human leukocytes I o. Of particular interest is a * Present address: Metabolism and Radiation Research Laboratory, State University Station, Fargo, N. Dak. (U.S.A.). Abbreviation : TEM, 2,4,6-triethyleneimino-i,3,5-triazine.
Mutation Res., 4 (I967) 83-89
84
TSUENG-HS1NG CHANG, FLORA T. ELEQUIN
recent finding 23 that m a n y ethylenimine compounds (for example, apholate and tepa) have sterilizing effect on insects and that this sterilizing property m a y be successfully used in controlling insect pests. Thus, studies of these compounds have genetic, health, as well as economic importance. Generally 3 effects of biological alkylating agents are recognized2°: (z) cytostatic--the inhibition of mitosis, (2) cytotoxic--metabolic disturbances resulting in socalled superfragmentation of chromosomeslL and (3) mutagenic--the induction of mutations, chromosome breaks, and rearrangements. The distinction between (I) and (2) m a y be merely quantitative rather than qualitative. As the concentration of the chemical is increased the effect shifts from cytostatic to cytotoxic. RINALDINP9 reported that an aromatic nitrogen mustard (p-amino-N,N-di-2-chloroethylaniline) shows cytotoxic and mutagenic effects at a high concentration, but a cytostatic effect at low concentrations in cultures of chick osteoblasts. Chromosome breaks are considered the major cause of cell death a but the importance of metabolic disturbance, which results in superfragmentation (or cytotoxic effect), is also stressed TM. In systems where a correlation of action of chemicals to the cell cycle is possible, it has been shown that different stages of the cell cycle have marked differences in their susceptibility to chemicals or to a physical factor such as ionizing radiations. For instance, EVANS AND SCOTT7 showed that maleic hydrazide induces chromatid aberrations exclusively in the S-period. Cytosine arabinoside 5 and 8-ethoxycaffeine ~2 are effective in inducing aberrations primarily in the G~-period. Nitrogen mustard produces aberrations only during the S-period. The kinetics or mode of action of chemicals m a y be sought in the comparative study of various compounds which carry the same functional group but have different carrier moieties. In this paper the induction of chromosome aberrations in cultured human cells by ethylenimine is studied. It is hoped that this will serve as a basis for comparison with our later planned studies using some derivatives of ethylenimine. Briefly, the results of this study show that all recovered chromosome aberrations are of the chromatid type and that they are induced almost exclusively in the S-period of the cell cycle. A high percentage of aberrations was detected more than 2 passages after the treatment of these cells with various concentrations of ethylenimine. These aberrations m a y have been the result of a delayed effect which is characteristic of chemical mutagens. MATERIALS AND METHODS
2 kinds of culture were used. One was a culture of human-embryonic lung fibroblasts (WI-38, x x x v l I I , passage 22) and the other was short-term leukocyte cultures from a normal male. WI-38 X X X V I I I cells. The cells were maintained in Eagle's basal medium (in Eagle's BSS with IO% calf serum) and subcultured (i : 2 split) every 3 or 4 days. The medium also contained penicillin and streptomycin (ioo units and ioo/~g/ml, respectively). On the 2nd day after the 22nd subculture, the cells were subjected to the following treatments: (!) IO-* M ethylenimine* for 8 h before fixation, (2) IO-* M ethylen* Courtesy of The Dow Chemical Company, Midland, Mich.
Mutation Res., 4 (1967) 83-89
INDUCTION OF CHROMOSOMEABERRATIONSIN CULTURED HUMANCELLS
85
imine for 3.5 h before fixation, (3) IO-*, IO-*, IO-5, and IO-6 M ethylenimine for I h and fixed at I-, 7-, Io-, and I4-day post-treatment. For the treatment of cells with ethylenimine a certain volume of ethylenimine solution (in phosphate-buffer solution) was added to the culture to achieve a desired final concentration. In (3) above, the cells were washed 4 times with culture medium after the chemical treatment. Leukocyte culture. The cultures were set up following the technique of MOORHEAD et al. ~' with some modifications. The medium used was: IOO ml TC I99, 2 5 m l calf serum, 0.5 ml phytohemagglutinin M, and 1.2 ml penicillin and streptomycin solution (ioooo units and ioooo #g/ml, respectively). Leukocyte suspension was obtained by gravitational separation. To each Falcon plastic bottle (30 ml) containing 4.5 ml of medium, 0. 4 ml of leukocyte suspension was added. For chemical treatment, o.I ml of ethylenimine solution was added to the culture to obtain a final concentration (10 -4 M), These cultures were treated with: (I) [aH]thymidine (2/zC/ml, spec. activity 3 C/mmole, Schwarz BioResearch, Inc., Orangeburg, N.Y.) for 8 h before fixation at 72 h after starting the culture, (2) IO-~ M ethylenimine and [~H]thymidine (2 ~uC/ml) for 8 h before fixation at 72 h. The slides from leukocyte culture were stained with acetoorcein (2% in 65% acetic acid), scored for chromosome aberrations and then coated with Kodak NTB-2 emulsion for autoradiography. The emulsion was exposed for 7 days, developed in D- I I for 2 rain, rinsed in water, fixed in Kodak acid fixer for 2 min, and mounted in Permount. In all instances colcemid (0.05 #g/ml) was added to the cultures 3.5 h before fixation. The chromosome preparations were made by the ignition-drying technique of:MoORHEAD AND NOWELL16. RESULTS AND DISCUSSION The results are summarized in Tables I, II and III. Only the cells treated with IO-* M, 6r lower, of ethylenimine solution survived. The cells did not survive the treatment with IO-* M of ethylenimine even for I h. Under the conditions of the experiment, only chromatid gaps, breaks and exchanges were recovered. This is in agreement with the observation that only chromatid aberrations are induced by alkylating agents**. The chromosome aberrations can easily be detected by the presence of dicentrics, rings, and large double fragments 14. In the cells treated with IO-* M ethylenimine for 3.5 h, 7% of the cells had gaps and/ or breaks, with IO total gaps and breaks. This is not significantly different from the control value of 3.9%. However, in the series treated with lO -4 M solution for 8 h, 29% of the cells showed gaps and/or breaks, significantly higher than the control. Since the duration of the G, period in human cells is estimated as being 3.5 h~ to 4.0 h .1, these results suggest that ethylenimine induces damage in the S-period almost exclusively, and that it does very little damage to cells in G2. The data presented in Table III on autoradiography of cultured leukocytes confirm this. In 72-h leukocyte cultures, 56% of the cells were damaged (Table II) after treatment for 8 h with ethylenimine (IO-* M) and [*H]thymidine. Of these 56 abnormal metaphases all but 4 showed labels in their autoradiograms (Table III and Fig. I). Mutation Res., 4 (I967) 83-89
86
TSUENG-HSING CHANG, FLORA
T.
ELEQUIN
TABLE 1 FREQUENCIES OF INDUCED CHROMATID ABERRATIONS AFTER TREATMENT OF W I - 3 8 CELLS WITH ETHYLENIMINE
Cells with exchanges and o~ breaks (/o)
(PASSAGE 22)
Ethvlenimine treatment
Passage Total cells Cells with gaps checked and breaks (%)
Total gaps Total and breaks exchanges
Control a
22
lO 3
4 (3.9)
o
4
o
22
lOO
7 (7 .°)
o
lo
o
22
lO 4
3 ° (28.8)
o
37
o
22 24 25 26
lOO IOO ioo ioo
31 14 8 9
lO (IO.O) o o o
61 16 8 9
It o o o
22 24 25
1OO lOO IiO
35 (35 '°) 24 (24.0) 13 (I1.8)
2 (2.0) I (I.O) o
44 31 14
2 I o
IO 4 M a
3.5 h 10-4 ]///a
8 h lO--a M b
lO
,i
I day 7 days 1o d a y s 14 d a y s Mb I {lay, 7 days 1o d a y s
(31.o) (14.o) (8.o) (9.o)
I 0 5 llqb
I day
22
loo
2o (2o.o)
0
23
o
7 days
24
too
6 (6.o)
o
7
o
IO d a y s
25
lOO
5 (5 .0 )
o
5
o
22 24
lOO IOO
7 (7 .°) 4 (4 .°)
I (i.o)
o
8 4
I o
IO ~ M I~
I clay' 7 days
a Cells were fixed a t t h e end of t r e a t m e n t p e r i o d ; Colcemid (0.05 t ,g/ ml ) for 3.5 h before fixation. 13 Cells were t r e a t e d for I h; a n d s a m p l e s fixed a t i, 7, IO, a n d 14 da ys , p o s t - t r e a t m e n t ; Colcemid ( 0 . 0 5 / , g / m l ) for 3-5 h before fixation. TABLE
11
CHROMAT1D ABERRATIONS IN 7 2 - h HUMAN-LEUCOCYTE CULTURES AFTER TREATMENT WITH ETHYLENIMINE AND [ S H ] T H Y M I D I N E FOR 8 h
Treatment
Number of cells
io 4 M Ethylenimine + [~H]Thymidine (2/,C/ml) 8 h
ioo
Cells with breaks Cells with Total breaks and gaps ( ° / ) exchanges (%) and gaps
Total exchanges
54 (54 .°)
2
2 (2.0)
114
TABLE III CHANGE IN PERCENTAGES OF LABELED METAPHASES AFTER TREATMENT WITH E T H Y L E N I M I N E FOR 8 h
Treatment
[SH]Thymidine (2/,C/ml) + ethylenimine 8 h [ a H ] T h y m i d i n e (2/zC/ml) 8 h
Total % labeled metaphase metaphase
Labeled mitosis abnormal normal
Unlabeled mitosis abnormal normal
ioo 539
52
4
85.0 68.8
33 371
EVANS AND SCOTT 7 f o u n d t h a t m a l e i c h y d r a z i d e
ii i 68
induces chromatid
aberrations
e x c l u s i v e l y i n t h e S - p e r i o d . T h e s a m e w a s a l s o s h o w n f o r n i t r o g e n m u s t a r d ~2. OSTERTAG et al. 18 h a v e a l s o r e p o r t e d fore or during
DNA
synthesis.
arabinoside 5 cause damage
Mutation Res., 4 (1967) 83-89
that caffeine induces aberrations On the other
i n G2 c e l l s .
hand,
o n l y if i t is a d d e d b e -
8 - e t h o x y c a f f e i n e 22 a n d
cytosine
INDUCTION OF CHROMOSOME ABERRATIONS IN CULTURED HUMAN CELLS
87
Fig. I. A u t o r a d i o g r a p h of a cell from leukocyte c u l t u r e t r e a t e d with e t h y l e n i m i n e a n d [SH]thymidine for 8 h. N o t e a c h r o m a t i d e x c h a n g e b e t w e e n 2 c h r o m o s o m e s of 6-12 group.
When chromosomes in G1 cells are damaged by ionizing radiations, chromosometype aberrations are found 6. WOLFF AND LUIPPOLD2s showed that in Vicia faba the chromosome reacts to X-rays as though functionally single during most of the G1; but as though double during the S and G2 phases. The transition from "single" to "double" occurs in the late G~. Whereas in radiation experiments, the recovery of chromosome-type aberrations can be taken as an indication of damage in G1 ceils, the same cannot be said for experiments using alkylating agents because these chemicals have not been shown to induce chromosome-type aberrations. In our experiments when the cells were treated with ethylenimine for i h and fixed at various time afterward (see below), no aberration of the chromosome-type was found. Unfortunately under the experimental conditions, the question of the sensitivity of Ga cells to ethylenimine cannot be answered unequivocally. That answer can only be obtained either from experiments in which the time sequence of the appearance of aberlations is accurately correlated to the full cell cycle, or from experiments in which the cells are fixed at close time intervals after the treatment at least until the aPFearance of the 2nd metaphases. In the I-h treatment series (Table I), the frequencies of chromatid aberrations were high for the first 2 samples (i.e. xst and 7th day post-treatment). Thus, in the xo-3 M series, 3I% of the xst day sample had gaps and/or breaks and another xo% of the cells had exchanges and breaks (orgaps). At the 7th day no exchange was recovered, but the frequency of breaks and gaps (14%) was still higher than normal, whereas by the xoth and x4th day it dropped to 8 and 9% respectively, i.e. not significantly higher than control. In the xo-* M series, the picture is similar to that of IO-s M series. The frequencies of chromatid aberrations and exchanges were quite high; the combined frequenMutation Res., 4 (I967) 83-89
88
TSUENG-HSING CHANG, FLORA T. ELEQUIN
cies of breaks and exchanges were 37% and 25°/o, respectively, for the Ist and 7th day post-treatment samples. The damage in the I o t h day post-treatment sample showed 13% of the cells with gaps and breaks, significantly higher than the control. As the concentration of the chemical decreases, the damage lessens as expected. In the IO-s M series, only the Ist day sample has a singnificantly higher chromatidgap frequency than the control. For the IO-~ M series, the frequencies of induced abnormality are quite low and not singificantly higher than the control. One of the characteristics of chemical mutagens is the delayed expression of induced mutations 2. In Drosophila melanogaster males treated with ethylenimine, the delayed lethals are detected in the progeny of apparently normal F2 females 1. As pointed out by AUERBACH2 it is difficult to compare the actions of mutagens between to different species, especially so when an in vitro system from human tissue is compared with Drosophila. In an in vitro system one should be always aware of the possibility that the chemical or its active product m a y persist for 2 or 3 cell cycles in the cytoplasm of cells which are actively dividing. Nevertheless our results show that chromarid aberrations and exchanges were still detected in cells fixed 7 days after the treatment with IO-3 and lO -4 M solution of ethylenimine. These cells were harvested 2 subcultures (1:2 split) after the treatment. They were probably in their 2nd or 3rd post-treatment mitoses, assuming, of course, that any delay in mitosis caused by ethylenimine did not last as long as 7 days. These results might be parallel to the finding in Drosophila. Further experiments will be needed to clarify this question. The percentage of labeled leukocyte metaphases increased under the influence of ethylenimine (Table III) This increase m a y be explained by the slow-down in the completion of DNA synthesis of cells in the late S-period. In these cultures the metaphases accumulated mark the cells which were 4.5-8 h away from mitosis when [aH~thymidine and/or ethylenimine were added. Since Gz lasts approximately 4 h, the first of the accumulated metaphases were at the late S-period or early G~ when the chemicals were added. If ethylenimine delays the completion of DNA synthesis, the delay m a y enable more of the cells in the late S-period to be scored as "labeled". This " e x t r a " label would be reflected in the increased percentage of labeled metaphases treated with ethylenimine. Indeed there is some evidence that nitrogen mustard 15 and apholate 1~ hamper DNA synthesis. ACKNOWLEDGEMENTS
The authors are grateful to Dr. PAUL S. MOORHEADfor reading the manuscript and to Miss PAULA MANCINELLI AND Mrs. MARCIA LEBOWlTZ for their technical assistance. One of the authors (T. H. CHANG) was supported by a U.S. Public Health Service Training Grant TI-CA-5163. REFERENCES I ALEXANDER, M. L., AND E. GLANGES, Genetic d a m a g e i n d u c e d b y e t h y l e n i m i n e , Proc. Natl.
Acad. Sci. (U.S.), 53 (1965) 282-288. 2 AUERBACH, C., P r o b l e m s in chemical m u t a g e n e s i s , Cold Spring Harbor Symp. Quant. Biol., i6 (1951) 199-213. 3 AUERBACH, C., M u t a g e n i c effects of a l k y l a t i n g agents, Ann. N. Y. A cad. Sci., 63 (I958) 731-736. 4 BIESELE, J. J., F. S. PHILIPS, J. B. THIERSCH, J. H. BURCHENAL, S. M. BUCKLEY AND C. C. STOCK, C h r o m o s o m e a b e r r a t i o n s a n d t u m o r inhibition b y n i t r o g e n m u s t a r d s : t h e h y p o t h e s i s of cross-linking alkylation, Nature, i 6 6 (i95o) i i 1 2 - i i 13. Mutation Res., 4 (1967) 83-89
INDUCTION OF CHROMOSOME ABERRATIONS IN CULTURED HUMAN CELLS
89
5 BREW~N, J. G., The induction of chromatid lesions by cytosine arabinoside in post-DNAsynthetic human leukocytes, Cytogenetics, 4 (I965) 28-36. 6 EVANS, H. J., Chromosome aberrations induced by ionizing radiations, lntern. Rev. Cytol., 13 (1962) 221-321. 7 EVANS, H. J., aND D. SCOTT, Influence of DNA synthesis on the production of chromatid aberrations by X-rays and maleic hydrazide in Vicia faba, Genetics, 49 (I964) 17-38. 8 FAHMV, O. G., AND M. J. FAHMV, Cytogenetic analysis of the action of carcinogens and tumor inhibitors in Drosophila ~nelanogaster, II. The mechanism of induction of dominant lethals by z,4,6-tri-(ethyleneimino)-I,3,5,-triazine, .]. Genet., 52 (1954) 6o3-619. 9 FAHMV, O. G., AND M. J. FaHMY, Cytogenetic analysis of the action of carcinogens and tumor inhibitors in Drosophila melanogaster, IV. The cell stage during spermatogenesis and the induction of intra- and inter-genie mutations by z,4,6-tri(ethyleneimino)-l,3,5-triazine, J. Genet., 53 (1955) 563-584 • io HAMPEL, K. E., AriD H. GERHARTZ, Strukturanomalien der Chromosomen menschlicher Leukozyten in vitro dutch Tiraethylenmelamin, ExptL Cell Res., 37 (I965) 251-258. II KHVOSTOVA,V. V., V. S. MOZHAEVA, N. S. AIGAES AND S. A. VALEVA, Mutants induced by ionizing radiations and ethyleneimine in winter wheat, Mutation Res., 2 (i965) 339-344. i2 KILGORE, W. W., AND R. R. PAINTER, Effect of the chemosterilant apholate on the synthesis of cellular components in developing housefly eggs, Biochem. ]., 92 (1964) 353-357. 13 KOLLER, P. C., Comparative effects of alkylating agents in cellular morphology, Ann. N . Y . Acad. Sci., 63 (I958) 783-8Ol. 14 LEA, D. E., Actions of Radiations on Living Cells, 2nd ed., Cambridge University Press, Cambridge, England, 1955, p. 416. 15 LEvis, A. G., G. A. DANIELI AND E. PlCClNNI, Nucleic acid synthesis and the mitotic cycle in mammalian cells treated with nitrogen mustard in culture, Nature, 207 (1965) 6o8-61o. 16 MOORHEAD, P.S., AND P. C. NOWELL, Chromosome cytology, in Methods in Medical Research, Vol. IO, Yearbook Medical Publishers, Chicago, Ill., 1964, p. 31o-322. 17 MOOR~EAD, P. S., P. C. NOW~LL, W. J. MF.LLMAr~,D. M. BArTIPS AND D. A. HUNGERFORD, Chromosome preparations of leukocytes cultured from peripheral blood, Exptl. Cell Res., 20 (196o) 613-616. 18 OST~RTAG, W., E. DUISBERG AND M. STURMANN, The mutagenic activity of caffeine in man, Mutation Res., 2 (1965) 293-296. 19 RIN.~LDINI, L. M., The antimitotic action of an aromatic nitrogen mustard on tissue cultures, Brit. J. Cancer, 6 (1952) 186-I96. 20 Ross, w . C. J., Biological Alhylating Agents, Butterworths, London, p. 232. 21 SCHMID, W., DNA replication patterns of human chromosomes, Cytogenetics, 2 (1963) 175-193. 22 ScoTT, D., AND H. J. EvAr~s, On the non-requirement for deoxyribonucleic acid synthesis in the production of chromosome aberrations by 8-ethoxycaffeine, Mutation Res., i (1964) i46-156. 23 SMITH, C. N., G. C. LABRECQUE AND A. B. BORKOVEC,Insect chemosterilants, Ann. Rev. Entotool., 9 (1964) 269-284. 24 SNYDER, L. A., AND I. I. OSTER, A comparison of genetic changes induced by a monofunctional and a polyfunctional alkylating agent in Drosophila melonogaster, Mutation Res., I (1964) 437445. 2 5 WESTERGAARD, M., Chemical mutagenesis in relation to the concept of the gene, Experientia, I3 (1957) 224-234. 26 WOLFF, S., AND H. E. LUIPPOLD, Chromosome splitting as revealed by combined X-ray and labeling experiments, Exptl. Cell Res., 34 (I964) 548-556.
Mutation Res., 4 (1967) 83-89