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Differential effects of aneugens and clastogens on incidences of multinucleated cells and of micronucleate cells in Chinese hamster lung (V79) cell line in vitro
Mutation Research 413 Ž1998. 39–45
Differential effects of aneugens and clastogens on incidences of multinucleated cells and of micronucleate cells in Chinese hamster lung žV79 / cell line in vitro Yun-gang Liu ) , Zhong-liang Wu, Jia-kun Chen Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou 510182, China Received 21 August 1997; revised 13 January 1998; accepted 13 January 1998
Abstract Two aneugens, vinblastine Ž0.025–0.4 m grml. and colchicine Ž0.05–0.4 m grml., and two clastogens, mitomycin C Ž0.05–0.4 m grml. and cyclophosphamide Ž1–16 m grml. were applied respectively to the micronucleus test in Chinese hamster lung ŽV79. cells in vitro, and the frequency of multinucleated cells ŽFmu. and that of micronucleate cells ŽFmi. in each group were observed. The results showed that at relatively high concentrations, all four agents increased both Fmu and Fmi, but the ratios of Fmu to Fmi in groups of the two aneugens Žaverage of 2.2, 2.8, respectively. were much Ž10–30 folds. higher than that in groups of the two clastogens Ž0.09, 0.20.. The difference between aneugens and clastogens in the above ratio was much more remarkable than that in areas of micronuclei Žonly 1.6–2.5 folds for the latter.. At relatively low concentrations, the two clastogens increased only Fmi Žbut not Fmu., while the two aneugens increased only Fmu Žbut not Fmi.. This indicates that the induction of multinuclei by aneugens may be more sensitive than by clastogens, and the induction of micronuclei by clastogens may be more sensitive than by aneugens. So, it is possible for the ratio of Fmu to Fmi to become a simple and sensitive Žthough indirect. index for distinguishing aneugens from clastogens. Further studies with other mutagens and Žor. other cell types will be needed to confirm the deduction. As no difference in frequency of polyploid cells was observed between control group and each treatment, the multinucleation does not seem related to endoreduplication of chromosomes. q 1998 Elsevier Science B.V. Keywords: Aneugen; Clastogen; Multinucleus; Micronucleus
1. Introduction The micronucleus ŽMN. test was primarily developed for the detection of chromosome breakage w1,2x, an effect induced by clastogens. Afterwards, it was shown that the micronucleus test also detects segre) Corresponding author. Tel.: q86-20-81340186; fax: q86-2081360294.
gation errors of chromosome, activity of aneugens. The methods to distinguish aneugens from clastogens in micronucleus test have been: Ža. based on the fact that the average areas or diameters of MNs induced by aneugens are significantly larger or longer than that by clastogens, the two kinds of mutagens can be distinguished by measuring the size of MNs w3,4x; Žb. with the antikinetochore antibody fluorescent staining method, it is possible to determine
1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 1 2 - 6
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Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45
kinetochore-containing MN, which is supposed to represent whole chromatid or chromosome. If most of the MNs induced by an agent are with positive staining, it may be classified as an aneugen, otherwise it may be regarded as a clastogen w5,6x. It has been reported that asbestos Žincluding chrysotile and crocidolite. induced much higher frequencies of multinucleated cells ŽFmu. than that of micronucleate cells ŽFmi. in Syrian hamster embryo ŽSHE. cells w7x, cultured human bronchial epithelial cells w8x, and Chinese hamster lung ŽV79. cells w9x. As asbestos has been proven very aneugenic w6,10x, the above experimental results spur us to presume that the higher sensitivity of cultured cells to multinucleation than to micronucleation might be a characteristic effect of aneugens. In order to verify this hypothesis, two aneugens Žvinblastine ŽVin. and colchicine ŽCol.. and two clastogens Žmitomycin C ŽMMC. and cyclophosphamide ŽCP.. were applied to micronucleus test in Chinese hamster lung ŽV79. cells in vitro, Fmu and Fmi in each group were observed and compared. Meanwhile, the diameter distribution of MNs in each treatment was assayed, and the extent of difference between aneugens and clastogens in areas of MNs was compared with that in ratios of Fmu to Fmi.
2. Material and methods 2.1. Cell line and culture V79 cells with an average cell-cycle time in culture of 12 h were used in the study. These cells maintain a high viability upon subculture and are free of mycoplasma contamination. V79 cells were grown exponentially in minimum essential medium ŽGibco. supplemented with 10% fetal bovine serum, 100 IU penicillin G, 100 m grml streptomycin. Cells were maintained in culture flasks at 378C in a humidified atmosphere containing 5% CO 2 and were subcultured by treatment with 0.25% trypsin in phosphate buffered saline ŽPBS.. 2.2. Chemicals Vinblastine Žsulfate salt, CAS-143-67-9., cyclophosphamide ŽCAS-50-18-0. and colchicine ŽCAS-
68797-31-9. were products of Sigma Chemical, St. Louis ŽUSA.; Mitomycin C ŽCAS-50-07-7. was purchased from Kyowa Hakko Kogyo, Tokyo ŽJapan., all of the above agents were dissolved in distilled water. 2.3. Mutagen treatments V79 cells were inoculated to 15 cm2 culture flasks in a density of 3 = 10 5rflask, then cultured for 24 h. After that, Vin, Col, MMC and CP were added to the cultures respectively at various concentrations. S 9 mix Žrat liver microsome mixture, induced by Aroclor 1254. was added simultaneously with CP at a concentration of 5% ŽV:V.. Two duplicate culture flasks for each treatment. After being cultured for 6 h, the medium was replaced with fresh medium. The cells were harvested after expression time of 18 h. 2.4. Cell harÕesting of micronucleus test At the end of the treatment, the cultures were washed twice with PBS and trypsinized at 378C. Protease activity was stopped by adding culture medium and the cells were centrifuged at 100 = g for 8 min. The supernatant was removed, the cells were resuspended in 1 ml 0.075 molrl KCl and incubated at 378C for 5 min, then added with 1 ml methanol:acetic acid Ž3:1. and centrifuged as above. The supernatant was removed and the cells were fixed in methanol:acetic acid Ž3:1. for 10 min, then centrifuged as above. Most of the supernatant was removed and about 0.2 ml remained. The cells were resuspended and the cell suspension was spread on cold Žy208C. slides by dropping. After being dried, the slides were stained with Giemsa stain Ž1:10, in phosphate buffer, pH 6.8.. 2.5. Multinucleated cells and micronucleate cells scoring All slides were coded prior to scoring to avoid bias. A total of 2000 cells were scored in each treatment. One trained person scored all slides using the following criteria: Ž1. only morphologically integrated cells were scored, Ž2. cells containing overlapping nuclei were not scored, only cells with two
Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45
or more separate nuclei were regarded as multinucleated cells, Ž3. micronucleus must be round or oval, with the same colour, 1r20–1r5 size as the main nucleus, and clearly distinct from it, Ž4. cells with both multinuclei and MNŽs. were scored as both multinucleated cells and micronucleate cells.
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treatment were analysed with x 2 test. The comparison of distribution of micronuclei diameters between aneugens and clastogens was performed by rank sum test ŽNemenyi method..
3. Results 2.6. Measurement of micronuclei diameter and calculation of the area The diameters of 100 round MNs randomly observed in each treatment were measured Žexcluding oval MN which occurred occasionally for the convenience of calculation of area. with an eyepiece micrometer quantified by an objective micrometer, in grades of 0, 1 m m, 2 m m, 3 m m, and 4 m m. The median diameter of MN in each treatment was referred to the 50th percentile of the diameter dimensions, and the ratio of MN area induced by one mutagen to that by another is calculated as ratio of the two Žmedian radium. 2 . 2.7. Mitotic index and polyploid cells scoring V79 cells were seeded and cultivated on sterile glass slides at a density of 20,000rcm2 in glass dishes Ž60-mm diameter., three slides and 5-ml medium in each dish, two dishes for each treatment. After 24 h of precultivation, cells were exposed to each mutagen at series of concentrations for 6 h, in CP groups, S 9 mix was added as described above. Then the medium containing mutagens was removed, cells were incubated with fresh medium for a further 18 h. Later procedure Žhypotonic treatment, fixation and staining. was analogous to that in micronucleus test, except for its being in situ Žin order to minimize lesions of cells during slides preparation which may cause loss of chromosomes. and fixed for two times. For each treatment, 2000 cells and 200 morphologically integrated metaphases were examined for estimation of mitotic index ŽMI. and frequency of polyploid cells, respectively. Cells with more than 33 chromosomes were termed polyploidy w11x. 2.8. Statistical analyses The comparisons of Fmi, Fmu, MI, and frequencies of polyploid cells between control and each
As shown in Table 1, each of the four mutagens increased Fmu and Fmi in a concentration-dependent manner, however, with different sensitivity between aneugens and clastogens. At relatively low concentrations, aneugens ŽVin and Col. induced only multinuclei, and clastogens ŽMMC and CP. induced only MNs. At relatively high concentrations where both multinuclei and MNs were induced, the ratios of Fmu to Fmi varied greatly between aneugens and clastogens, i.e., the ratios in Vin and Col groups were 10–30 folds higher than in MMC and CP groups. The above results indicate that under the treatments of Vin and Col, the induction of multinuclei is obviously more sensitive than that of MNs, on the other hand, under the treatments of MMC and CP the induction of MNs is much more sensitive than that of Vin and Col. It was noticed that of the observed multinucleated or micronucleated cells in each treatment, about 4–10% were cells with both MN and multinuclei, and in control groups no such cells were observed Ždata not shown.. Most of the multinucleated cells in each treatment were binucleated cells, tetranucleated cells were much fewer, while trinucleated cells were the fewest. As shown in Table 2, the distributions of MN diameter in various treatments varied significantly, in both Col and Vin treatments, the dimensions of MN diameter were longer than that in CP or MMC groups Ž P - 0.01.. The ratios of MN areas between aneugens and clastogens were 1.6–2.5. This result was analogous with other reports observed in mice bone marrow in vivo w4,12x. Obviously, the ratios of MN areas between aneugens and clastogens was much Ž4–12 folds. weaker than ratios of Fmu to Fmi, which suggested that the latter index has much greater power to distinguish aneugens from clastogens than the former. In each treatment, no tremendous cell toxicity was microscopically observed even up to the highest concentration of each mutagen by a rough estimation
Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45
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Table 1 Effects of vinblastine, colchicine, mitomycin C, and cyclophosphamide on frequency of multinucleated or micronucleate cells in V79 cell line Treatmentrconcentration Ž m grml.
Frequency of multinucleated cells ‰ ŽA.
Negative control Vinblastine 0.025 0.05 0.1 0.2 0.4
Frequency of micronucleate cells ‰ ŽB.
3.0
4.5
ArB 0.67
11.5)) 20.0)) 39.5)) 50.0)) 68.0))
5.0 9.0 17.5)) 24.5)) 31.0))
2.30 2.22 2.26 2.04 2.19
13.5)) 25.5)) 50.0)) 105.0))
5.5 12.0)) 16.5)) 30.0))
2.45 2.13 3.03 3.50
Mitomycin C 0.05 0.1 0.2 0.4
3.0 5.0 7.5) 10.0))
35.5)) 54.5)) 77.0)) 125.5))
0.08 0.09 0.10 0.08
Cyclophosphamide 0 ŽS 9 . 1 2 4 8 16
2.5 3.5 3.5 5.5 8.0)) 10.5))
4.0 12.5)) 22.0)) 29.0)) 40.5)) 61.5))
0.63 0.28 0.16 0.19 0.20 0.17
Colchicine 0.05 0.1 0.2 0.4
The cells were treated as above for 6 h Žcyclophosphamide groups were with simultaneous S 9 mix activating system treatment, S 9 mix was prepared from Aroclor 1254-induced rat liver., 24 h after being inoculated, and then continuously cultured for 18 h. 2000 cells were scored in each group. ) P - 0.05, )) P - 0.01, with cyclophosphamide groups compared with S 9 group, and other treatments compared with negative control.
Table 2 Distribution of micronuclei diameters in each mutagen treatment Treatment a
CP MMC b Vinb Col b
Distribution of MN diameter Ž%. 0-
1 m m-
2 m m-
3 m m-
4 m m-
21 25 15 5
33 17 12 20
28 33 37 27
9 18 25 34
8 5 11 14
Median of MN diameter Ž m m.
Ratio of MN areac
1.84 2.23 2.85)) 2.93))
1.0 1.5 2.4 2.5
MN: micronucleus; CP: cyclophosphamide; MMC: mitomycin C; Vin: vinblastine; Col: colchicine. a At a concentration of 16 m grml. b At a concentration of 0.4 m grml. c Area was calculated according to median radium. A total of 100 micronuclei were measured for diameter distribution in each treatment. )) P - 0.01, compared with either MMC or CP group, by Nemenyi’s rank sum test.
Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45
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Table 3 Mitotic index and percentages of polyploid V79 cells after 6-h treatment with each mutagen and 18-h further culture Mutagen and concentration Ž m grml.
Mitotic index Ž%.
Negative control Vinblastine sulfate 0.1 0.4 0.8
2.0
3.0
1.8 2.6 4.2))
3.0 3.5 4.5
Colchicine 0.1 0.2 0.4
1.7 2.0 3.6))
4.0 2.5 2.0
Mitomycin C 0.1 0.2 0.4
0.8)) 0.3)) 0.2))
Cyclophosphamide 0ŽS 9 control. 2 8 16
2.2 1.9 1.6 1.5
Percentage of polyploid cells
ya ya ya
3.0 2.0 2.5 3.5
See legend of Table 1 for experimental condition. In each treatment, 2000 cells and 200 metaphases were observed for scoring of mitotic index and frequency of polyploid cells respectively Žonly cells and metaphases with morphological integrity were counted.. a Because of inhibited mitosis, metaphases in mitomycin C treatments were too seldom to be scored. )) P - 0.01, compared with control.
on both the cell densities just before cells harvesting and the integrity of cells on slides. As shown in Table 3, Vin and Col increased MI Ž P - 0.01. at relatively high concentrations Žat least up to 0.8 and 0.4 m grml, respectively., and a clear concentration-dependent decrease in MI in MMC treatment was observed. But CP induced no statistical change in MI. The percentage of polyploid cells in all groups ranged 2.0–4.5%, without any statistical difference among them.
4. Discussion It has been known that the cytogenetic effect induced by MMC and CP is mainly breakage of DNA, which results in clastogenesis. MMC both alkylates and cross-links DNA and these lead to breakage of DNA w13x. CP, after metabolic activation, becomes a chloroethyl alkylating agent that has genetic effect similar to MMC w14x. As to Vin and Col, both have been reported to inhibit the assembly
of microtubules in vitro, and when spindle microtubules are affected during mitosis, aneuploidy may happen w15x. Aneugens generally produce micronuclei containing whole chromosomes or centric fragments and clastogens produce micronuclei containing predominantly acentric fragments, and repeated reports have demonstrated that the micronuclei induced by either Vin or Col were predominantly kinetochore positive, while that induced by MMC and CP were mainly kinetochore negative w5,16,17x. The present study indicated that the induction of multinucleated cells by Vin and Col in V79 cells was obviously more sensitive than that of micronucleate cells, on the contrary, the induction of micronucleate cells by MMC and CP was much more sensitive than that of multinucleated cells Žas shown in Table 1.. Why did aneugens such as Vin and Col induce multinuclei more easily than micronuclei? It has been known that post mitotic cytokinesis depends upon functions of cytoskeleton system which includes microtubules, microfilaments, and some other components. When mitosis is completed, the spindle
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Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45
microtubules are dismissed to take part in cellular microtubules network which is soon involved in the mechanism of cytokinesis w18x. So, if cellular microtubules are depolymerized or changed, both mitotic spindle and cellular microtubules network could be affected, which may lead to aneuploidy after mitosis and formation of binucleated or multinucleated cell as a result of failure in cytokinesis. The formation of binucleated cells might be due to failure in cytokinesis after a successful mitosis, and when cytokinesis is stopped two times with mitosis not fully disturbed, a tetranucleated cell thus might form. Fusion of two or more cells with cellular lesion could not be entirely excluded from the cause of multinucleation, however, based on the observation that trinucleated cells were much fewer than either binucleated or tetranucleated cells in each treatment Ždata not shown., this possibility must be little important in the mechanism of multinucleation. After all, the concentration related effects of aneugens on post-mitosis microtubules, cytokinesis, and their actual contribution to multinuclei induction remain to be further investigated. An important problem is whether other aneugens and clastogens show effects similar to the present results, and whether the prior induction of multinuclei to MNs by aneugens is a constant characteristic of other cell types. Chrysotile, another variety of aneugen, induced binucleated cells with 7–8 folds higher frequency than micronucleate cells in SHE cells 24 h after being treated with it, and the frequencies of binucleated cells in various doses were all parallel with that of aneuploid cells w7x. In cultured human bronchial epithelial cells, chrysotile showed similar effects as above, and interestingly, crocidolite Žalso very aneugenic. did not increase Fmi, however, it increased Fmu actually Žthough in slight intensity. w8x. In V79 cells, which is much more sensitive than human cells to genetic toxicity, crocidolite showed similar results to the present study w9x. The above findings in asbestos provide effective support to the above deduction, even though further studies on other aneugens and clastogens with various cell types will be necessary to draw a still more solid conclusion. The magnitude of difference between aneugens and clastogens in ratio of Fmu to Fmi was much greater than that in MN areas Žshown in Tables 1 and
2., so, if further studies on other aneugens and clastogens Žor. with other cell types show results similar to that in the present study, the index postulated in this study–ratio of Fmu to Fmi will be a simple and powerful tool for identifying mutagens as aneugens or clastogens. Especially because it needs no complicated instruments, and both multinuclei and micronuclei are more easily observed than cellular chromosomes, it may be suitable for preliminary scanning of genetic toxicities induced by large quantities of chemicals. The concentration-dependent decrease of MI induced by MMC Žshown in Table 3. accorded with its known effect: inhibition of mitosis, and the slight increase of mitotic index by Vin and Col in relatively high concentrations could be interpreted as their ability to induce mitotic arrest w15x. The absence of induction of polyploid cells in each treatment indicated that the increased multinucleated cells were not relative to increased DNA duplication within nucleus, i.e., endoreduplication. In conclusion, the present study indicates that observation on both micronucleate and multinucleated cells in micronucleus test in vitro may be hopeful to become a simple and effective method to distinguish aneugens from clastogens, and it calls for further studies to fully verify this deduction. Acknowledgements We gratefully acknowledge Prof. Shou-qi Li for his valuable advice on part of the study plan. References w1x J.A. Heddle, A rapid in vivo test for chromosome damage, Mutation Res. 18 Ž1973. 191–197. w2x W. Schmid, The micronucleus test, Mutation Res. 31 Ž1975. 9–15. w3x K.I. Yamamoto, Y. Kikuchi, A comparison of diameters of micronuclei induced by clastogens and by spindle poisons, Mutation Res. 71 Ž1980. 127–131. w4x P. Vanprays, F. Vermeiren, M. Sysmans, R. Temmerman, The micronucleus assay as a test for the detection of aneugenic activities, Mutation Res. 244 Ž1990. 95–103. w5x G. Krishna, R. Fiedler, J.C. Theiss, Simultaneous analysis of chromosome damage and aneuploidy in cytokinesis-blocked V79 Chinese hamster lung cells using an antikinetochore antibody, Mutation Res. 282 Ž1992. 79–88.
Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45 w6x E. Dopp, J. Saedler, H. Stopper, D.G. Weiss, D. Schiffmann, Mitotic disturbance and micronucleus induction in Syrian hamster embryo fibroblast cells caused by asbestos fibers, Environ. Health Perspect. 103 Ž1995. 268–271. w7x M. Oshimura, T.W. Hesterburg, T. Tsutsui, J.C. Barrett, Correlation of asbestos induced cytogenetic effects with cell transformation of Syrian hamster embryo cells in culture, Cancer Res. 44 Ž1984. 5017–5022. w8x Y. Kodama, C.J. Boreiko, S.C. Maness, Cytotoxic and cytogenetic effects of asbestos on human bronchial epithelial cells in culture, Carcinogenesis 14 Ž1993. 691–697. w9x L. Yungang, L. Yuqing, Z. Gu, P. Wenzhen, W. Yang, L. Shouqi, D. Qinan, Z. Chenglie, Induction of micronuclei and multinuclei in vitro by crocidolite, Chin. J. Pharmacol. Toxicol. 11 Ž1997. 63–66. w10x M. Oshimura, T.W. Hesterburg, J.C. Barrett, An early, nonrandom karyotypic change in immortal Syrian hamster cell lines transformed by asbestos: trisomy of chromosome 11, Cancer Genet. Cytogenet. 22 Ž1986. 225–237. w11x G. Motykiewicz, W. Hadnagy, N.H. Seemayer, J. Szeliga, A. Tkocz, M. Chorazy, Influence of airborne suspended matter on mitotic cell division, Mutation Res. 260 Ž1991. 195–202. w12x H. Tinwell, J. Ashby, Micronucleus morphology as a means
w13x
w14x
w15x
w16x
w17x
w18x
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to distinguish aneugens and clastogens in mouse bone marrow micronucleus assay, Mutagenesis 6 Ž1991. 193–198. S. Kerpel-Fronius, J. Verwey, M. Stuurman, B. Kanyar, P. Lelieveld, H.M. Pinedo, Pharmacokinetics and toxicity of mitomycin C in rodents, given alone, in combination, or after induction of microsomal drug metabolism, Cancer Chemother. Pharmacol. 22 Ž1988. 104–108. M. Colvin, B.A. Chabner, Alkylating agents, in: B.A. Chabner, J.M. Collins ŽEds.., Cancer Chemotherapy: Principles and Practices, J.B. Lippencott, Philadelphia, 1990, pp. 276– 313. M. Wallin, B. Friden, M. Billger, Studies of the interaction of chemicals with microtubules assembly in vitro can be used as an assay for detection of cytotoxic chemicals and possible inducers of aneuploidy, Mutation Res. 201 Ž1988. 303–311. D.A. Eastmond, J.D. Tucker, Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody, Environ. Mol. Mutagen. 13 Ž1989. 34–43. D. Baoquan, L. Shouqi, D. Qinan, N. Zhuyao, Immunofluorescent staining method in micronucleus assay for detecting aneugens, Chin. J. Pharmacol. Toxicol. 9 Ž1995. 146–148. P. Dustin, Microtubules, Springer-Verlag, Berlin, 1984.
Differential effects of aneugens and clastogens on incidences of multinucleated cells and of micronucleate cells in Chinese hamster lung žV79 / cell line in vitro Yun-gang Liu ) , Zhong-liang Wu, Jia-kun Chen Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou 510182, China Received 21 August 1997; revised 13 January 1998; accepted 13 January 1998
Abstract Two aneugens, vinblastine Ž0.025–0.4 m grml. and colchicine Ž0.05–0.4 m grml., and two clastogens, mitomycin C Ž0.05–0.4 m grml. and cyclophosphamide Ž1–16 m grml. were applied respectively to the micronucleus test in Chinese hamster lung ŽV79. cells in vitro, and the frequency of multinucleated cells ŽFmu. and that of micronucleate cells ŽFmi. in each group were observed. The results showed that at relatively high concentrations, all four agents increased both Fmu and Fmi, but the ratios of Fmu to Fmi in groups of the two aneugens Žaverage of 2.2, 2.8, respectively. were much Ž10–30 folds. higher than that in groups of the two clastogens Ž0.09, 0.20.. The difference between aneugens and clastogens in the above ratio was much more remarkable than that in areas of micronuclei Žonly 1.6–2.5 folds for the latter.. At relatively low concentrations, the two clastogens increased only Fmi Žbut not Fmu., while the two aneugens increased only Fmu Žbut not Fmi.. This indicates that the induction of multinuclei by aneugens may be more sensitive than by clastogens, and the induction of micronuclei by clastogens may be more sensitive than by aneugens. So, it is possible for the ratio of Fmu to Fmi to become a simple and sensitive Žthough indirect. index for distinguishing aneugens from clastogens. Further studies with other mutagens and Žor. other cell types will be needed to confirm the deduction. As no difference in frequency of polyploid cells was observed between control group and each treatment, the multinucleation does not seem related to endoreduplication of chromosomes. q 1998 Elsevier Science B.V. Keywords: Aneugen; Clastogen; Multinucleus; Micronucleus
1. Introduction The micronucleus ŽMN. test was primarily developed for the detection of chromosome breakage w1,2x, an effect induced by clastogens. Afterwards, it was shown that the micronucleus test also detects segre) Corresponding author. Tel.: q86-20-81340186; fax: q86-2081360294.
gation errors of chromosome, activity of aneugens. The methods to distinguish aneugens from clastogens in micronucleus test have been: Ža. based on the fact that the average areas or diameters of MNs induced by aneugens are significantly larger or longer than that by clastogens, the two kinds of mutagens can be distinguished by measuring the size of MNs w3,4x; Žb. with the antikinetochore antibody fluorescent staining method, it is possible to determine
1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 1 2 - 6
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Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45
kinetochore-containing MN, which is supposed to represent whole chromatid or chromosome. If most of the MNs induced by an agent are with positive staining, it may be classified as an aneugen, otherwise it may be regarded as a clastogen w5,6x. It has been reported that asbestos Žincluding chrysotile and crocidolite. induced much higher frequencies of multinucleated cells ŽFmu. than that of micronucleate cells ŽFmi. in Syrian hamster embryo ŽSHE. cells w7x, cultured human bronchial epithelial cells w8x, and Chinese hamster lung ŽV79. cells w9x. As asbestos has been proven very aneugenic w6,10x, the above experimental results spur us to presume that the higher sensitivity of cultured cells to multinucleation than to micronucleation might be a characteristic effect of aneugens. In order to verify this hypothesis, two aneugens Žvinblastine ŽVin. and colchicine ŽCol.. and two clastogens Žmitomycin C ŽMMC. and cyclophosphamide ŽCP.. were applied to micronucleus test in Chinese hamster lung ŽV79. cells in vitro, Fmu and Fmi in each group were observed and compared. Meanwhile, the diameter distribution of MNs in each treatment was assayed, and the extent of difference between aneugens and clastogens in areas of MNs was compared with that in ratios of Fmu to Fmi.
2. Material and methods 2.1. Cell line and culture V79 cells with an average cell-cycle time in culture of 12 h were used in the study. These cells maintain a high viability upon subculture and are free of mycoplasma contamination. V79 cells were grown exponentially in minimum essential medium ŽGibco. supplemented with 10% fetal bovine serum, 100 IU penicillin G, 100 m grml streptomycin. Cells were maintained in culture flasks at 378C in a humidified atmosphere containing 5% CO 2 and were subcultured by treatment with 0.25% trypsin in phosphate buffered saline ŽPBS.. 2.2. Chemicals Vinblastine Žsulfate salt, CAS-143-67-9., cyclophosphamide ŽCAS-50-18-0. and colchicine ŽCAS-
68797-31-9. were products of Sigma Chemical, St. Louis ŽUSA.; Mitomycin C ŽCAS-50-07-7. was purchased from Kyowa Hakko Kogyo, Tokyo ŽJapan., all of the above agents were dissolved in distilled water. 2.3. Mutagen treatments V79 cells were inoculated to 15 cm2 culture flasks in a density of 3 = 10 5rflask, then cultured for 24 h. After that, Vin, Col, MMC and CP were added to the cultures respectively at various concentrations. S 9 mix Žrat liver microsome mixture, induced by Aroclor 1254. was added simultaneously with CP at a concentration of 5% ŽV:V.. Two duplicate culture flasks for each treatment. After being cultured for 6 h, the medium was replaced with fresh medium. The cells were harvested after expression time of 18 h. 2.4. Cell harÕesting of micronucleus test At the end of the treatment, the cultures were washed twice with PBS and trypsinized at 378C. Protease activity was stopped by adding culture medium and the cells were centrifuged at 100 = g for 8 min. The supernatant was removed, the cells were resuspended in 1 ml 0.075 molrl KCl and incubated at 378C for 5 min, then added with 1 ml methanol:acetic acid Ž3:1. and centrifuged as above. The supernatant was removed and the cells were fixed in methanol:acetic acid Ž3:1. for 10 min, then centrifuged as above. Most of the supernatant was removed and about 0.2 ml remained. The cells were resuspended and the cell suspension was spread on cold Žy208C. slides by dropping. After being dried, the slides were stained with Giemsa stain Ž1:10, in phosphate buffer, pH 6.8.. 2.5. Multinucleated cells and micronucleate cells scoring All slides were coded prior to scoring to avoid bias. A total of 2000 cells were scored in each treatment. One trained person scored all slides using the following criteria: Ž1. only morphologically integrated cells were scored, Ž2. cells containing overlapping nuclei were not scored, only cells with two
Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45
or more separate nuclei were regarded as multinucleated cells, Ž3. micronucleus must be round or oval, with the same colour, 1r20–1r5 size as the main nucleus, and clearly distinct from it, Ž4. cells with both multinuclei and MNŽs. were scored as both multinucleated cells and micronucleate cells.
41
treatment were analysed with x 2 test. The comparison of distribution of micronuclei diameters between aneugens and clastogens was performed by rank sum test ŽNemenyi method..
3. Results 2.6. Measurement of micronuclei diameter and calculation of the area The diameters of 100 round MNs randomly observed in each treatment were measured Žexcluding oval MN which occurred occasionally for the convenience of calculation of area. with an eyepiece micrometer quantified by an objective micrometer, in grades of 0, 1 m m, 2 m m, 3 m m, and 4 m m. The median diameter of MN in each treatment was referred to the 50th percentile of the diameter dimensions, and the ratio of MN area induced by one mutagen to that by another is calculated as ratio of the two Žmedian radium. 2 . 2.7. Mitotic index and polyploid cells scoring V79 cells were seeded and cultivated on sterile glass slides at a density of 20,000rcm2 in glass dishes Ž60-mm diameter., three slides and 5-ml medium in each dish, two dishes for each treatment. After 24 h of precultivation, cells were exposed to each mutagen at series of concentrations for 6 h, in CP groups, S 9 mix was added as described above. Then the medium containing mutagens was removed, cells were incubated with fresh medium for a further 18 h. Later procedure Žhypotonic treatment, fixation and staining. was analogous to that in micronucleus test, except for its being in situ Žin order to minimize lesions of cells during slides preparation which may cause loss of chromosomes. and fixed for two times. For each treatment, 2000 cells and 200 morphologically integrated metaphases were examined for estimation of mitotic index ŽMI. and frequency of polyploid cells, respectively. Cells with more than 33 chromosomes were termed polyploidy w11x. 2.8. Statistical analyses The comparisons of Fmi, Fmu, MI, and frequencies of polyploid cells between control and each
As shown in Table 1, each of the four mutagens increased Fmu and Fmi in a concentration-dependent manner, however, with different sensitivity between aneugens and clastogens. At relatively low concentrations, aneugens ŽVin and Col. induced only multinuclei, and clastogens ŽMMC and CP. induced only MNs. At relatively high concentrations where both multinuclei and MNs were induced, the ratios of Fmu to Fmi varied greatly between aneugens and clastogens, i.e., the ratios in Vin and Col groups were 10–30 folds higher than in MMC and CP groups. The above results indicate that under the treatments of Vin and Col, the induction of multinuclei is obviously more sensitive than that of MNs, on the other hand, under the treatments of MMC and CP the induction of MNs is much more sensitive than that of Vin and Col. It was noticed that of the observed multinucleated or micronucleated cells in each treatment, about 4–10% were cells with both MN and multinuclei, and in control groups no such cells were observed Ždata not shown.. Most of the multinucleated cells in each treatment were binucleated cells, tetranucleated cells were much fewer, while trinucleated cells were the fewest. As shown in Table 2, the distributions of MN diameter in various treatments varied significantly, in both Col and Vin treatments, the dimensions of MN diameter were longer than that in CP or MMC groups Ž P - 0.01.. The ratios of MN areas between aneugens and clastogens were 1.6–2.5. This result was analogous with other reports observed in mice bone marrow in vivo w4,12x. Obviously, the ratios of MN areas between aneugens and clastogens was much Ž4–12 folds. weaker than ratios of Fmu to Fmi, which suggested that the latter index has much greater power to distinguish aneugens from clastogens than the former. In each treatment, no tremendous cell toxicity was microscopically observed even up to the highest concentration of each mutagen by a rough estimation
Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45
42
Table 1 Effects of vinblastine, colchicine, mitomycin C, and cyclophosphamide on frequency of multinucleated or micronucleate cells in V79 cell line Treatmentrconcentration Ž m grml.
Frequency of multinucleated cells ‰ ŽA.
Negative control Vinblastine 0.025 0.05 0.1 0.2 0.4
Frequency of micronucleate cells ‰ ŽB.
3.0
4.5
ArB 0.67
11.5)) 20.0)) 39.5)) 50.0)) 68.0))
5.0 9.0 17.5)) 24.5)) 31.0))
2.30 2.22 2.26 2.04 2.19
13.5)) 25.5)) 50.0)) 105.0))
5.5 12.0)) 16.5)) 30.0))
2.45 2.13 3.03 3.50
Mitomycin C 0.05 0.1 0.2 0.4
3.0 5.0 7.5) 10.0))
35.5)) 54.5)) 77.0)) 125.5))
0.08 0.09 0.10 0.08
Cyclophosphamide 0 ŽS 9 . 1 2 4 8 16
2.5 3.5 3.5 5.5 8.0)) 10.5))
4.0 12.5)) 22.0)) 29.0)) 40.5)) 61.5))
0.63 0.28 0.16 0.19 0.20 0.17
Colchicine 0.05 0.1 0.2 0.4
The cells were treated as above for 6 h Žcyclophosphamide groups were with simultaneous S 9 mix activating system treatment, S 9 mix was prepared from Aroclor 1254-induced rat liver., 24 h after being inoculated, and then continuously cultured for 18 h. 2000 cells were scored in each group. ) P - 0.05, )) P - 0.01, with cyclophosphamide groups compared with S 9 group, and other treatments compared with negative control.
Table 2 Distribution of micronuclei diameters in each mutagen treatment Treatment a
CP MMC b Vinb Col b
Distribution of MN diameter Ž%. 0-
1 m m-
2 m m-
3 m m-
4 m m-
21 25 15 5
33 17 12 20
28 33 37 27
9 18 25 34
8 5 11 14
Median of MN diameter Ž m m.
Ratio of MN areac
1.84 2.23 2.85)) 2.93))
1.0 1.5 2.4 2.5
MN: micronucleus; CP: cyclophosphamide; MMC: mitomycin C; Vin: vinblastine; Col: colchicine. a At a concentration of 16 m grml. b At a concentration of 0.4 m grml. c Area was calculated according to median radium. A total of 100 micronuclei were measured for diameter distribution in each treatment. )) P - 0.01, compared with either MMC or CP group, by Nemenyi’s rank sum test.
Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45
43
Table 3 Mitotic index and percentages of polyploid V79 cells after 6-h treatment with each mutagen and 18-h further culture Mutagen and concentration Ž m grml.
Mitotic index Ž%.
Negative control Vinblastine sulfate 0.1 0.4 0.8
2.0
3.0
1.8 2.6 4.2))
3.0 3.5 4.5
Colchicine 0.1 0.2 0.4
1.7 2.0 3.6))
4.0 2.5 2.0
Mitomycin C 0.1 0.2 0.4
0.8)) 0.3)) 0.2))
Cyclophosphamide 0ŽS 9 control. 2 8 16
2.2 1.9 1.6 1.5
Percentage of polyploid cells
ya ya ya
3.0 2.0 2.5 3.5
See legend of Table 1 for experimental condition. In each treatment, 2000 cells and 200 metaphases were observed for scoring of mitotic index and frequency of polyploid cells respectively Žonly cells and metaphases with morphological integrity were counted.. a Because of inhibited mitosis, metaphases in mitomycin C treatments were too seldom to be scored. )) P - 0.01, compared with control.
on both the cell densities just before cells harvesting and the integrity of cells on slides. As shown in Table 3, Vin and Col increased MI Ž P - 0.01. at relatively high concentrations Žat least up to 0.8 and 0.4 m grml, respectively., and a clear concentration-dependent decrease in MI in MMC treatment was observed. But CP induced no statistical change in MI. The percentage of polyploid cells in all groups ranged 2.0–4.5%, without any statistical difference among them.
4. Discussion It has been known that the cytogenetic effect induced by MMC and CP is mainly breakage of DNA, which results in clastogenesis. MMC both alkylates and cross-links DNA and these lead to breakage of DNA w13x. CP, after metabolic activation, becomes a chloroethyl alkylating agent that has genetic effect similar to MMC w14x. As to Vin and Col, both have been reported to inhibit the assembly
of microtubules in vitro, and when spindle microtubules are affected during mitosis, aneuploidy may happen w15x. Aneugens generally produce micronuclei containing whole chromosomes or centric fragments and clastogens produce micronuclei containing predominantly acentric fragments, and repeated reports have demonstrated that the micronuclei induced by either Vin or Col were predominantly kinetochore positive, while that induced by MMC and CP were mainly kinetochore negative w5,16,17x. The present study indicated that the induction of multinucleated cells by Vin and Col in V79 cells was obviously more sensitive than that of micronucleate cells, on the contrary, the induction of micronucleate cells by MMC and CP was much more sensitive than that of multinucleated cells Žas shown in Table 1.. Why did aneugens such as Vin and Col induce multinuclei more easily than micronuclei? It has been known that post mitotic cytokinesis depends upon functions of cytoskeleton system which includes microtubules, microfilaments, and some other components. When mitosis is completed, the spindle
44
Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45
microtubules are dismissed to take part in cellular microtubules network which is soon involved in the mechanism of cytokinesis w18x. So, if cellular microtubules are depolymerized or changed, both mitotic spindle and cellular microtubules network could be affected, which may lead to aneuploidy after mitosis and formation of binucleated or multinucleated cell as a result of failure in cytokinesis. The formation of binucleated cells might be due to failure in cytokinesis after a successful mitosis, and when cytokinesis is stopped two times with mitosis not fully disturbed, a tetranucleated cell thus might form. Fusion of two or more cells with cellular lesion could not be entirely excluded from the cause of multinucleation, however, based on the observation that trinucleated cells were much fewer than either binucleated or tetranucleated cells in each treatment Ždata not shown., this possibility must be little important in the mechanism of multinucleation. After all, the concentration related effects of aneugens on post-mitosis microtubules, cytokinesis, and their actual contribution to multinuclei induction remain to be further investigated. An important problem is whether other aneugens and clastogens show effects similar to the present results, and whether the prior induction of multinuclei to MNs by aneugens is a constant characteristic of other cell types. Chrysotile, another variety of aneugen, induced binucleated cells with 7–8 folds higher frequency than micronucleate cells in SHE cells 24 h after being treated with it, and the frequencies of binucleated cells in various doses were all parallel with that of aneuploid cells w7x. In cultured human bronchial epithelial cells, chrysotile showed similar effects as above, and interestingly, crocidolite Žalso very aneugenic. did not increase Fmi, however, it increased Fmu actually Žthough in slight intensity. w8x. In V79 cells, which is much more sensitive than human cells to genetic toxicity, crocidolite showed similar results to the present study w9x. The above findings in asbestos provide effective support to the above deduction, even though further studies on other aneugens and clastogens with various cell types will be necessary to draw a still more solid conclusion. The magnitude of difference between aneugens and clastogens in ratio of Fmu to Fmi was much greater than that in MN areas Žshown in Tables 1 and
2., so, if further studies on other aneugens and clastogens Žor. with other cell types show results similar to that in the present study, the index postulated in this study–ratio of Fmu to Fmi will be a simple and powerful tool for identifying mutagens as aneugens or clastogens. Especially because it needs no complicated instruments, and both multinuclei and micronuclei are more easily observed than cellular chromosomes, it may be suitable for preliminary scanning of genetic toxicities induced by large quantities of chemicals. The concentration-dependent decrease of MI induced by MMC Žshown in Table 3. accorded with its known effect: inhibition of mitosis, and the slight increase of mitotic index by Vin and Col in relatively high concentrations could be interpreted as their ability to induce mitotic arrest w15x. The absence of induction of polyploid cells in each treatment indicated that the increased multinucleated cells were not relative to increased DNA duplication within nucleus, i.e., endoreduplication. In conclusion, the present study indicates that observation on both micronucleate and multinucleated cells in micronucleus test in vitro may be hopeful to become a simple and effective method to distinguish aneugens from clastogens, and it calls for further studies to fully verify this deduction. Acknowledgements We gratefully acknowledge Prof. Shou-qi Li for his valuable advice on part of the study plan. References w1x J.A. Heddle, A rapid in vivo test for chromosome damage, Mutation Res. 18 Ž1973. 191–197. w2x W. Schmid, The micronucleus test, Mutation Res. 31 Ž1975. 9–15. w3x K.I. Yamamoto, Y. Kikuchi, A comparison of diameters of micronuclei induced by clastogens and by spindle poisons, Mutation Res. 71 Ž1980. 127–131. w4x P. Vanprays, F. Vermeiren, M. Sysmans, R. Temmerman, The micronucleus assay as a test for the detection of aneugenic activities, Mutation Res. 244 Ž1990. 95–103. w5x G. Krishna, R. Fiedler, J.C. Theiss, Simultaneous analysis of chromosome damage and aneuploidy in cytokinesis-blocked V79 Chinese hamster lung cells using an antikinetochore antibody, Mutation Res. 282 Ž1992. 79–88.
Y.-g. Liu et al.r Mutation Research 413 (1998) 39–45 w6x E. Dopp, J. Saedler, H. Stopper, D.G. Weiss, D. Schiffmann, Mitotic disturbance and micronucleus induction in Syrian hamster embryo fibroblast cells caused by asbestos fibers, Environ. Health Perspect. 103 Ž1995. 268–271. w7x M. Oshimura, T.W. Hesterburg, T. Tsutsui, J.C. Barrett, Correlation of asbestos induced cytogenetic effects with cell transformation of Syrian hamster embryo cells in culture, Cancer Res. 44 Ž1984. 5017–5022. w8x Y. Kodama, C.J. Boreiko, S.C. Maness, Cytotoxic and cytogenetic effects of asbestos on human bronchial epithelial cells in culture, Carcinogenesis 14 Ž1993. 691–697. w9x L. Yungang, L. Yuqing, Z. Gu, P. Wenzhen, W. Yang, L. Shouqi, D. Qinan, Z. Chenglie, Induction of micronuclei and multinuclei in vitro by crocidolite, Chin. J. Pharmacol. Toxicol. 11 Ž1997. 63–66. w10x M. Oshimura, T.W. Hesterburg, J.C. Barrett, An early, nonrandom karyotypic change in immortal Syrian hamster cell lines transformed by asbestos: trisomy of chromosome 11, Cancer Genet. Cytogenet. 22 Ž1986. 225–237. w11x G. Motykiewicz, W. Hadnagy, N.H. Seemayer, J. Szeliga, A. Tkocz, M. Chorazy, Influence of airborne suspended matter on mitotic cell division, Mutation Res. 260 Ž1991. 195–202. w12x H. Tinwell, J. Ashby, Micronucleus morphology as a means
w13x
w14x
w15x
w16x
w17x
w18x
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to distinguish aneugens and clastogens in mouse bone marrow micronucleus assay, Mutagenesis 6 Ž1991. 193–198. S. Kerpel-Fronius, J. Verwey, M. Stuurman, B. Kanyar, P. Lelieveld, H.M. Pinedo, Pharmacokinetics and toxicity of mitomycin C in rodents, given alone, in combination, or after induction of microsomal drug metabolism, Cancer Chemother. Pharmacol. 22 Ž1988. 104–108. M. Colvin, B.A. Chabner, Alkylating agents, in: B.A. Chabner, J.M. Collins ŽEds.., Cancer Chemotherapy: Principles and Practices, J.B. Lippencott, Philadelphia, 1990, pp. 276– 313. M. Wallin, B. Friden, M. Billger, Studies of the interaction of chemicals with microtubules assembly in vitro can be used as an assay for detection of cytotoxic chemicals and possible inducers of aneuploidy, Mutation Res. 201 Ž1988. 303–311. D.A. Eastmond, J.D. Tucker, Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody, Environ. Mol. Mutagen. 13 Ž1989. 34–43. D. Baoquan, L. Shouqi, D. Qinan, N. Zhuyao, Immunofluorescent staining method in micronucleus assay for detecting aneugens, Chin. J. Pharmacol. Toxicol. 9 Ž1995. 146–148. P. Dustin, Microtubules, Springer-Verlag, Berlin, 1984.