Chromosomal aberrations in vitro induced by aneugens

Mutation Research 379 Ž1997. 83–93

Chromosomal aberrations in vitro induced by aneugens Peter Arni ) , Thomas Hertner NoÕartis Crop Protection AG, P.O. Box, CH-4002 Basel, Switzerland Received 2 January 1997; revised 24 April 1997; accepted 29 April 1997

Abstract Various aneugens were reported to induce structural chromosomal aberrations beside their influence on cell division and their aneugenic potential To assess, whether a relationship between disturbance of cell division and clastogenic potential exists, CHO cells were treated with the well-known aneugens colcemid, colchicine and vincristine and investigated for the induction of structural chromosomal aberrations, polyploid cells and alterations in mitotic index. At low and intermediate concentrations, all compounds induced polyploidy and an increase in mitotic index, but no structural aberrations at all. However, at high concentrations, colcemid and colchicine both induced numerous structural chromosomal aberrations in diploid cells. Colchicine was also clastogenic in tetraploid cells. Vincristine did not induce structural chromosomal aberrations in diploid cells, but in tetraploid cells. The clastogenic effects showed a clear-cut threshold with all three compounds. Furthermore, it was found that the tetraploid condition in CHO cells is generally accompanied by an increase in structural chromosomal aberrations, in vehicle controls as well as in cultures treated with the aneugens. Nevertheless, this study demonstrates that for the three aneugenic compounds tested, no direct relationship between compound induced disturbance of cell cycle and compound induced structural chromosomal aberration incidence exists. q 1997 Elsevier Science B.V. Keywords: Aneugen; Structural chromosomal aberration; Polyploidy; Mitotic index; Colcemid; Colchicine; Vincristine

1. Introduction Compounds which damage DNA are considered to act without threshold, i.e. no concentration can be defined below which no effect occurs. Mechanisms which lead to numerical chromosomal aberrations, on the other hand, may exhibit a characteristic dose–response pattern and include a threshold level w7–9x. This differentiation has an impact on risk assessment, since compounds which act without a threshold are treated differently from those for which )

Corresponding author.

a threshold can be established. For the latter, doses which do not represent a health hazard, are calculated on the basis of an experimentally determined ‘no effect level’. Such a procedure is currently not accepted for chemicals for which no threshold can be defined: the extrapolation of a ‘ virtual safe dose’ is based on a linear dose–response curve and generally results in a much lower value than a threshold-based calculation. Induction of chromosomal aberrations was observed with various aneuploidy-inducing chemicals Že.g. Gebhart et al. w1x, Segawa et al. w2x, KirschVolders and Parry w3x with vincristine, Galloway et

0027-5107r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 1 1 1 - 5

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P. Arni, T. Hertnerr Mutation Research 379 (1997) 83–93

al. w4x with colchicine, Matsuoka et al. w5x with various compounds, Armstrong et al. w6x with 2,4,6trichlorophenol.. For most of these compounds it is not clear by which mechanisms the clastogenic effects are induced: Gebhart et al. w1x conclude from the type of aberrations observed with vincristine that they occurred rather as a consequence of the cell division-disturbing property of the compound than as a result of direct DNA damage. The clastogenic effects observed by Matsuoka et al. w5x with colcemid and vincristine are very weak and do not show a clear concentration dependency. Colcemid and vinblastine do not induce gene mutations w10x. Colchicine was negative in a mouse lymphoma assay in our laboratory Žunpublished. and all three compounds also did not induce mutations in bacteria w11x. Therefore these compounds may not damage DNA directly, but it could be speculated that the structural chromosomal aberrations induced are a consequence of toxicity on spindle andror kinetochores as proposed by Tinwell and Ashby w9x. The objective of this study was to investigate this hypothesis. Since conflicting results concerning the induction of structural chromosomal aberrations by aneugens are found in the literature, it was also of interest to clarify the situation for some model compounds. Cytogenetic experiments were performed with the well-known aneugens, colcemid, colchicine, and vincristine. The biological effects of these compounds ¨ w12x and Parry and were reviewed, e.g. by Onfelt Sors w13x. Since colcemid and colchicine were expected to reveal similar effects, some experimental parts were performed with one of these compounds only. The experiments were conducted according to a cytogenetic study protocol generally used in routine testing, which included a 1.5 cell cycle treatment and a prolonged treatment, i.e. 1.5 cell cycles plus 24 h. In some experiments the cells were treated for 3 h, followed by a recovery period of 15 or 39 h.

2. Materials and methods 2.1. Cell line The cell line CCL 61 ŽChinese hamster ovary cells, CHO. obtained from the American Type Cul-

ture Collection ŽATCC., Rockville, MD, USA and cloned in our laboratory was used. The cells were maintained in culture medium consisting of nutrient mixture F-12 supplemented with 10% foetal calf serum q penicillinrstreptomycin 100 Urmlr100 mgrml ŽGibco AG, Basle, Switzerland. in 75 cm2 tissue-culture Žplastic. flasks. The cultures were incubated at 378C in a humidified atmosphere containing 5% CO 2 . The cells were passaged twice weekly. Subculturing was performed using a 0.25% trypsin solution. 1.0 to 2.0 = 10 5 cells were seeded into 20 ml of culture medium. Under these conditions, the duration of a normal cell cycle was 12–13 h. The cell cultures were periodically checked for mycoplasma contamination. The CCL 61 clone used has a stable modal chromosome number of 19 Žs‘2n’.. 2.2. Chemicals Colcemid, CAS 477-30-5 and colchicine, CAS 64-86-8 were obtained from Fluka AG, Buchs, Switzerland. Vincristine, CAS 2068-78-2, was purchased from Sigma AG, Buchs, Switzerland. Colcemid and colchicine were dissolved in bidistilled water. In some experiments, the compounds were dissolved in culture medium. With vincristine, 0.5% methanol was used as solvent. In one experiment, the substance was dissolved in bidistilled water. Colcemid and colchicine were tested at concentrations up to 1000 and 5000 mgrml. For technical reasons Žamount of test material available per vial. the highest concentration used with vincristine was 100 mgrml. 2.3. Cytogenetic test A series of glass slides in quadruple culture dishes ŽQuadriperm, Heraus ¨ . was seeded with Chinese hamster ovary cells at a density of at least 1 = 10 4 cellsrml Ž18-h experiments. or 4 = 10 3 cellsrml Ž42-h experiments.. The preincubation time before treatment was about 24 h. The substances were added in the vehicle 1:100 to the cells in culture medium, when an organic solvent was used, or 10:100 when water or medium were applied. A negative control was set in each experiment, supplemented with the respective volume of the vehicle.

P. Arni, T. Hertnerr Mutation Research 379 (1997) 83–93

Quadruplicate cultures were prepared for each group in each assay. To ensure analysis of first post-treatment mitoses, a major sampling time of 1.5 times cell cycle was selected. This corresponds to about 18 h for the CHO cells used. An additional sampling time was chosen 24 h after the first one, i.e. 42 h. Treatment was performed throughout the whole period. In some experiments, the cells were treated for 3 h and harvested 15 h after end of treatment. In one experiment with vincristine, the cells were treated for 3 h and harvested after a 39-h recovery period. Two hours prior to harvesting, the cultures were treated with colcemid 0.4 mgrml to arrest cells in metaphase. The experiment was terminated by hypotonic treatment Ž0.075 M KCl solution. of the cells, followed by fixation Žmethanolracetic acid, 3:1.. Slides were air-dried and stained with orcein. 2.4. Independent experiments Various independent experiments were performed with all compounds, using different treatment times and concentration ranges as shown in Table 1. Results of representative experiments were selected for presentation. 2.5. Scoring of the slides Prior to analysis, the selected slides were coded, as were the cultures treated with the vehicle alone as well as the cultures treated with the test chemicals. One or two hundred well-spread metaphase figures with 19–21 centromeres Ž‘2n’ cells from two cultures Ž50 or 100 metaphases per replicate culture. in the vehicle control and in the treated groups were scored for structural chromosomal aberrations. In addition, where possible, the number of aberrations was also assessed in ‘4n’ cells. The slides were examined for the following structural aberrations Žfor description of aberrations see w14x.: Ø chromatid and chromosome deletions Žincluding breaks, deletions and fragments.; Ø chromatid exchanges Žincluding triradials, quadriradials, end-fusions, acentric rings.; Ø chromosome exchanges Žincluding dicentrics, polycentrics, centric and acentric rings.;

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Table 1 Independent cytogenetic experiments performed Test compound

Exposure time

Concentrations Žmgrml.

Colcemid

18 h 18 h 18 h 18 h 42 h 42 h 42 h 42 h 42 h 18 h 18 h 18 h 3 hq15 h recovery 42 h 42 h 18 h 18 h 3 hq15 h recovery 42 h 3 hq39 h recovery

0.25, 0.5, 1 0.25, 0.5, 1 1.25, 2.5, 5 62.5, 125, 250, 500, 1000 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.025, 0.05, 0.1 0.025, 0.05, 0.1 0.025, 0.05, 0.1 31.25, 62.5, 125, 500, 1000 62.5, 125, 250, 500, 1000 1000, 2500, 5000 1000, 2500, 5000 500, 1000, 2000

Colchicine

Vincristine

62.5, 125, 250, 500 62.5, 125, 50, 500, 1000 1, 2, 4, 8 6.25, 12.5, 25, 50, 100 1, 2, 4, 8 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8

Ø multiple aberrations, i.e. metaphases containing more than 10 aberrations of different types or more than 5 aberrations of one particular type Žexcluding gaps.; Gaps Žachromatic chromatid and chromosome lesions. were recorded, but not included in the evaluation. As a measure for effects of the compounds on the cells, the mitotic index for a total of 1000 or 2000 cells was determined in the 18-h experiments, and the percentage of polyploid metaphases in a total 200 metaphases was assessed in the 18- and 42-h experiments. In cultures where there were obviously around 100% polyploid cells, the percentage of polyploidy was estimated Ž( 100%.. 2.6. Statistics The evaluated numbers of specific aberrations were subjected to statistical analysis. A one-sided x 2-test for trend ŽCochran–Armitage. taking the cell

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P. Arni, T. Hertnerr Mutation Research 379 (1997) 83–93

as experimental unit was performed w15x. This test allows the determination of a threshold value: for each concentration, a trend calculation which includes all lower concentrations and the negative control is performed. The calculation determines, at which concentration an increase in trend begins. The calculated p-values are given in the tables. The level of significance was set at p F 0.01.

Table 2 Number of structural aberrations Žexcl. gaps. in negative controls of ‘2n’ and ‘4n’ cells

2.7. Toxicity testing

3.2. Colcemid

With colcemid and colchicine, toxicity was determined after treatment with a concentration of 1000 mgrml of the test chemicals, in comparison with a vehicle control Žwater.. The cells were cultured and treated with the test chemicals as described above for the cytogenetic test. Cell number and cloning efficiency were measured: 18 h after beginning of treatment the chemicals were removed and the cell layer was washed with PBS. The cells were suspended by trypsinisation, pelleted, resuspended in fresh medium and counted with a haemocytometer. One hundred cells in 3 ml fresh medium were seeded in each of six 9.6-cm 2 compartments to determine the cloning efficiency. After 7 or 8 days the cultures were fixed and stained with Giemsa and the surviving colonies determined by eye.

3.2.1. 18-h treatment Concentrations from 0.25 to 1000 mgrml were tested. After treatment for 1.5 cell cycles Ž18 h. colcemid induced an increase in the mitotic index at concentrations of 0.25 mgrml and above. High concentrations Ž62.5–1000 mgrml. again led to a reduction ŽTable 3.. The obviously biologically very active low concentrations did not induce any chromatid or chromosomal aberrations in the 18-h assay. However, at high concentrations, i.e. 250–1000 mgrml, a significant increase in structural chromatid and chromosome aberrations occurred ŽFig. 1, Table 4.. The number of polyploid metaphases was not increased.

Cells

Number of cells scored

% of cells with aberrations Žmean"SD.

‘2n’ ‘4n’

124 510 418

1.7"1.3 14.6"19.2

3.2.2. 42-h treatment The concentrations tested ranged from 0.025 to 1000 mgrml. Induction of approx. 100% tetraploid

2.8. Historical controls Control data from the laboratories’ routine studies were used to establish a historical basis of the frequency of aberrations in ‘2n’ and ‘4n’ cells of vehicle-treated cultures.

3. Results 3.1. Historical control data A compilation of the number of chromosomal aberrations in ‘4n’ cells of negative controls from routine experiments performed by the laboratory revealed that the frequency is considerably enhanced in comparison to that in ‘2n’ cells. For a total of 124 510 ‘2n’ cells scored, the mean aberration frequency was 1.7 " 1.3%. For 418 ‘4n’ cells, the mean incidence of aberration was 14.6 " 19.2% ŽTable 2..

Fig. 1. % of metaphases with structural chromosomal aberrations induced by colcemid.

P. Arni, T. Hertnerr Mutation Research 379 (1997) 83–93

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Table 3 Mitotic index with colcemid after 18 h of treatment Ž1.5 cell cycles. in ‘2n’ cells Concentration Žmgrml.

Mitotic index Ž% of control.

0

0.25

0.5

1

1.25

2.5

5

62.5

125

250

500

1000

100

144

154

236

157

186

192

126

106

55

21

5

Table 4 Induction of structural aberrations Žexcl. gaps. induced by colcemid after 18 h of treatment in ‘2n’ cells Concentration Žmgrml.

Number of metaphases scored % Metaphases with aberrations Žtotal. p-value for increasing trend Chromatid deletions Chromosome deletions Chromatid exchanges Chromosome exchanges Multiple aberrations Ž) 5. % Polyploid cells

a

0

62.5

125

250

500

1000

200 0.5

200 1.5 0.308 0.5 1 0 0.5 0 6

200 3 0.025 1.5 1.5 0 0 0 9

200 5 0.003 3 1 1 0 0 6

200 20 0.000 16 1 2 1 0 3

50 48 0.000 40 6 6 0 2 3

0 0 0 0.5 0 6

a

This value may be lower than the sum of metaphases with aberrations, since one metaphase may show more than one type of aberration Žstatistical significance: p F 0.01..

cells already occurred at the dose of 0.025 mgrml. Structural chromatid and chromosomal aberrations were scored in tetraploid cells and revealed no increase at any concentration when compared with the historical control on tetraploid cells. Comparison of

the numbers of tetraploid metaphases with structural chromosomal aberrations with that of the diploid concurrent negative control revealed a slight increase, which showed, however, no concentration dependency at all. Concentrations above 125 mgrml

Table 5 Induction of structural aberrations Žexcl. gaps. induced by colcemid after 42 h of treatment in ‘4n’ cells Concentration Žmgrml. 0 Number of metaphases scored % Metaphases with aberrations Žtotal. p-value for increasing trend Chromatid deletions Chromosome deletions Chromatid exchanges Chromosome exchanges Multiple aberrations Ž) 5. % Polyploid cells a

d

b

100 2 1 1 0 0 0 3

0

c

100 15

a

0.025

0.05

0.1

0.2

0.4

31.25

62.5

125

250

100 7 0.086 1 0 3 4 0 ( 100

100 7 0.057 0 0 0 7 0 ( 100

100 2 0.500 0 0 0 2 0 ( 100

100 9 0.104 3 2 0 4 0 ( 100

100 7 0.089 0 1 0 7 0 ( 100

100 3 0.336 0 0 0 3 0 ( 100

100 5 0.391 2 3 0 1 0 ( 100

100 2 0.705 1 1 0 0 0 ( 100

50 4 0.729 4 0 0 0 ( 100

For 500 and 1000 mgrml: no scorable cells available due to growth inhibition. Concurrent negative control, only ‘2n’ cells available. c Historical ‘4n’ cell negative control. d This value may be lower than the sum of metaphases with aberrations, since one metaphase may show more than one type of aberration. Statistical comparison was made with the historical ‘4n’ cell negative control values Žsignificance: p F 0.01.. b

P. Arni, T. Hertnerr Mutation Research 379 (1997) 83–93

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Table 6 Mitotic index with colchicine after 18 h of treatment Ž1.5 cell cycles. in ‘2n’ cells Concentration Žmgrml. 0

62.5 125 250 500 1000 2500 5000

Mitotic index 100 322 Ž% of control.

ND 307 252 181

14

1

ND, not determined.

revealed growth inhibiting effects. At 500 and 1000 mgrml, no scorable metaphases were available ŽFig. 1, Table 5.. 3.3. Colchicine Since colcemid in the concentration range of 0.025 to 125 mgrml did not induce structural chromosomal aberrations, concentrations below 62.5 mgrml were not assessed in the experiments with colchicine. 3.3.1. 18-h treatment Concentrations of 62.5–5000 mgrml were tested. After 18 h treatment, an increased mitotic index was observed up to 1000 mgrml. Higher concentrations again revealed a decrease ŽTable 6.. The negative control revealed 2% of metaphases with structural aberrations, at 250 mgrml 1% and at 500 mgrml 7% of metaphases with structural chromatid and chromosomal aberrations were found. This value increased to 48% at 1000 mgrml. Higher concentrations revealed no scorable metaphases. All types of

Fig. 2. % of metaphases with structural chromosomal aberrations induced by colchicine.

aberrations were found. The number of polyploid metaphases was not enhanced ŽFig. 2, Table 7.. 3.3.2. 3-h treatmentr 15-h recoÕery In this experiment, concentrations of 500–2000 mgrml were used. When the cultures were treated for only 3 h and harvested 15 h after termination of treatment Žtotal period of 18 h, i.e. 1.5 cell cycles., no increase in structural aberrations Žnegative control 3% metaphases with structural aberrations, 500, 1000 and 2000 mgrml each 4%. and no induction of tetraploid cells were observed ŽFig. 2.. However, an

Table 7 Induction of structural aberrations Žexcl. gaps. induced by colchicine after 18 h of treatment in ‘2n’ cells Concentration Žmgrml.

Number of metaphases scored % Metaphases with aberrations Žtotal. p-value for increasing trend Chromatid deletions Chromosome deletions Chromatid exchanges Chromosome exchanges With multiple aberrations Ž) 5. % Polyploid metaphases a

a

0

62.5

125

250

500

1000

100 2

100 3 0.500 1 1 0 1 0 4

100 1 0.693 0 1 0 0 0 5

100 1 0.803 0 1 0 0 0 2

100 7 0.063 2 1 4 0 0 9

100 48 0.000 32 4 10 2 9 2

0 1 0 1 0 2

This value may be lower than the sum of metaphases with aberrations, since one metaphase may show more than one type of aberration Žstatistical significance: p F 0.01..

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Table 9 Mitotic index with vincristine after 18 h of treatment Ž1.5 cell cycles. in ‘2n’ cells Concentration Žmgrml. 0

0.5 1

2

4

6.25 12.5 25

50

100

Mitotic index 100 164 300 300 172 293 248 273 398 211 Ž% of control.

enhancement in mitotic index was seen Ždata not shown.. 3.3.3. 42-h treatment In these experiments, a concentration range of 62.5–1000 mgrml was tested. Induction of tetraploid cells was observed at all concentrations. A high incidence of structural chromosomal aberrations of all different types was found at the concentration of 1000 mgrml. At this concentration, the number of aberrations per cell was considerably higher than in the 18-h experiment. Lower concentrations revealed values which are within the range of the historical negative control of tetraploid cells and there were no concentration-dependent variations ŽFig. 2, Table 8..

Fig. 3. % of metaphases with structural chromosomal aberrations induced by vincristine.

trations ŽTable 9.. At the highest concentration tested Ž100 mgrml., there again was a slight decrease. No induction of structural chromosomal aberrations and polyploid metaphases were found ŽFig. 3, Table 10..

3.4. Vincristine

3.4.2. 3-h treatmentr 15-h recoÕery Concentrations of 1–8 mgrml were used. No increase in the number of metaphases with structural chromosomal aberrations was observed Ždata not shown..

3.4.1. 18-h treatment Concentrations of 1–100 mgrml were applied. An increase in mitotic index was found at all concen-

Table 8 Induction of structural aberrations Žexcl. gaps. induced by colchicine after 42 h of treatment in ‘4n’ cells Concentration Žmgrml. 0 Number of metaphases scored % Metaphases with aberrations Žtotal. p-value for increasing trend With chromatid deletions Chromosome deletions Chromatid exchanges Chromosome exchanges With multiple aberrations Ž) 5. % Polyploid metaphases a

a

b

100 3 0 2 0 1 0 3

0

c

15

62.5

125

250

500

1000

100 9 0.949 0 0 0 9 2 ( 100

100 6 0.995 0 1 0 5 0 ( 100

100 10 0.984 1 4 1 6 0 ( 100

100 14 0.865 3 2 2 8 4 ( 100

100 83 0.000 43 15 26 7 29 ( 100

This value may be lower than the sum of metaphases with aberrations, since one metaphase may show more than one type of aberration. Statistical comparison was made with the historical ‘4n’ cell negative control values Žsignificance: p F 0.01.. b Concurrent negative control, only ‘2n’ cells available. c Historical ‘4n’ cell negative control.

P. Arni, T. Hertnerr Mutation Research 379 (1997) 83–93

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Table 10 Induction of structural aberrations Žexcl. gaps. induced by vincristine after 18 h of treatment in ‘2n’ cells Concentration Žmgrml.

Number of metaphases scored % Metaphases with aberrations Žtotal. p-value for increasing trend With chromatid deletions Chromosome deletions Chromatid exchanges Chromosome exchanges With multiple aberrations Ž) 5. % Polyploid metaphases

a

0

6.25

12.5

25

50

100

100 1

100 1 0.761 0 0 1 0 0 2

100 1 0.500 1 0 0 0 0 2

100 2 0.273 1 1 0 0 0 4

100 3 0.104 3 0 0 0 0 6

100 0 0.417 0 0 0 0 0 1

1 0 0 1 0 0

a

This value may be lower than the sum of metaphases with aberrations, since one metaphase may show more than one type of aberration Žstatistical significance: p F 0.01..

3.4.3. 42-h treatment In this experiment, concentrations of 0.0625–8 mgrml were tested. An increase in tetraploid cells was already observed at the lowest concentration. No marked increase in the number of structural aberrations was detected when the aberration rate in ‘4n’ cells was compared to the historical 4n cell control ŽTable 11., with the exception of the highest concentration, where an increase in the number of metaphases with structural aberrations was seen, which was, however, just not statistically significant. These aberrations were mainly in the form of chromosomal deletions and exchanges Ždicentric chromo-

somes., but there was no increase in chromatid aberrations. 3.4.4. 3-h treatmentr 39-h recoÕery This experiment was performed with concentrations 1–8 mgrml and no increase in metaphases with structural chromosomal aberrations was observed in ‘2n’ cells Ždata not shown.. 3.5. Toxicity testing 3.5.1. Colcemid The treatment for 18 h at the concentration of 1000 mgrml revealed a reduction in cell number to

Table 11 Induction of structural aberrations Žexcl. gaps. induced by vincristine after 42 h of treatment in ‘4n’ cells Concentration Žmgrml. 0 Number of metaphases scored % Metaphases with aberrations Žtotal. p-value for increasing trend With chromatid deletions Chromosome deletions Chromatid exchanges Chromosome exchanges With multiple aberrations Ž) 5. % Polyploid metaphases a

a

b

100 6 0 3 0 3 0 3

0

c

15

0.125

0.25

0.5

1

2

4

8

40 5 0.973 0 3 0 3 0 85

72 10 0.934 3 7 0 1 1 ( 100

96 17 0.534 1 5 0 11 1 ( 100

100 12 0.655 1 7 0 5 0 ( 100

100 16 0.444 3 8 0 7 0 ( 100

50 12 0.519 0 4 0 8 0 ( 100

100 23 0.069 5 12 1 8 0 ( 100

This value may be lower than the sum of metaphases with aberrations, since one metaphase may show more than one type of aberration. Statistical comparison was made with the historical ‘4n’ cell negative control values Žsignificance: p F 0.01.. b Concurrent negative control, only ‘2n’ cells available. c Historical ‘4n’ cell negative control.

P. Arni, T. Hertnerr Mutation Research 379 (1997) 83–93

Fig. 4. Cell number and cloning efficiency after treatment with 1000 mgrml colchicine or colcemid for 18 h.

26% of the control value. The cloning efficiency was reduced to 48% ŽFig. 4.. 3.5.2. Colchicine Treatment in the concentration of 1000 mgrml for 18 h resulted in a reduction in cell number to 24% of the control value. The cloning efficiency revealed a reduction to 71% in comparison with the control ŽFig. 4..

4. Discussion After 42 h of treatment, with all three compounds at most concentrations used, in the ‘4n’ cells scored, an elevation in the percentage of metaphases with structural chromosomal aberrations occurred when a comparison was made with the respective concurrent ‘2n’ negative control. This increase was variable and showed no concentration dependency at all. The negative control data of the laboratory routine experiments was found to show a considerably higher mean percentage of metaphases with aberrations in ‘4n’ cells Ž14.6%. than in ‘2n’ cells Ž1.7%.. The doubled chromosome number may be responsible for part of this increase. A further reason may be found in a longer cell cycle of tetraploid cells and thereby in a lengthening of the phases sensitive to aberration induction by factors present in the cell’s normal environment. A comparison of the percentage of aberrations in ‘4n’ metaphases with that of a concur-

91

rent control of ‘2n’ cells therefore seems not to be appropriate. The aberration frequency in ‘4n’ metaphases should be compared with that of a ‘4n’ cell control. With colcemid and colchicine at concentrations up to 125 mgrml, no induction of structural chromosomal aberrations occurred, either in the 18- or in the 42-h experiments. The same was true for vincristine up to 100 mgrml in the 18-h experiment and up to 4 mgrml in 42-h experiments. These concentrations, however, were highly effective on the cell division process. Matsuoka et al. w5x reported slight increases in structural aberrations when cells were treated with 0.05 and 1.0 mgrml of colcemid or vincristine. It is not clear whether these aberrations were detected in diploid andror polyploid cells. At least for the 42-h exposure time, where apparently 60–100% polyploid cells were found, the increase in aberrations may be the result of the ‘4n’ cell condition rather than that of a direct DNA-damaging effect. Danford w16x did not observe an induction of structural chromosomal aberrations in Chinese hamster liver fibroblasts at concentrations of 0.002–0.02 mgrml of colcemid. Lafi and Parry w17x reported an increase in structural chromosomal aberrations when LuC1 cells Žderived from Chinese hamster lung. were treated with 0.04 mgrml of colcemid. This effect could not be confirmed in our experiments on CHO cells. With colchicine and colcemid, additional experiments at high concentrations were performed. As indicated earlier by Galloway et al. w4x, colchicine induces chromosomal aberrations at these concentrations mainly in the form of deletions but not complex aberration figures. In contrast to the findings of Galloway et al. w4x all kinds of chromatid and chromosomal aberrations were found, including chromatid and chromosome exchanges figures ŽTable 7.. Even higher rates of metaphases with aberrations were observed after 42 h of treatment. When the cells were treated for 3 h only and fixed after a 15-h recovery period Žtotal of 1.5 cell cycles., no induction of aberrations was seen. With colcemid, after 18 h of treatment, similar effects as with colchicine were observed. However, after 42 h of treatment no aberrations were registered, because at 500 and 1000 mgrml, no scorable cells were found due to a growth-inhibiting effect. This effect is much stronger

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P. Arni, T. Hertnerr Mutation Research 379 (1997) 83–93

with colcemid than with colchicine. With vincristine, structural aberrations were detected in tetraploid cells only, above 4 mgrml. Segawa et al. w2x observed a time-dependent increase in structural chromosomal aberrations in Don lung cells of Chinese hamster, when they treated the cultures with vinblastine for 8, 16, 24 or 32 h. There was a time-dependent increase in aberrations, but no clear concentration dependency. Gebhart et al. w1x reported the induction of structural chromosomal aberrations after treatment of human lymphocytes with concentrations of 1.25–5 mgrml. There was no clear correlation with concentration or duration of treatment. Complex aberration figures practically did not occur. The authors attributed the induction of structural aberrations to the ‘aging’ of metaphases under vincristine influence, rather than to a direct effect of the compound on DNA structure. After 18 h of treatment with all compounds, the mitotic index was increased and induction of polyploidy was observed in the 42-h experiments already at low concentrations. No induction of polyploidy was seen in the 18-h experiments. This is somewhat unexpected, since Galloway et al. w18x consider a 1.5 cell cycle treatment as sufficient for the detection of polyploidy inducing chemicals. Preliminary studies performed in our laboratory on the CHO cell clone used with colchicine showed that after a 24-h treatment, induction of tetraploid cells and endoreduplication occurs. Since all three compounds seem to induce some cell cycle delay, the treatment for an exact 1.5 cell cycle may be just a little too short for the detection of polyploidy induction. The lack of aberrations at concentrations below 250 mgrml and the sharp increase at 500 or 1000 mgrml with colcemid and colchicine indicate a threshold mechanism for the induction of chromosomal aberrations. This can particularly be seen in the experiments with colchicine ŽTables 7 and 8., where the lower doses do not show any increasing trend at all in the percentage of aberrations. The same seems to be true for vincristine, which induced aberrations in tetraploid cells at 8 mgrml only. Although the value of 23% did not result in a statistically significantly increased trend, this value is considered to be biologically relevant. In vivo colchicine and vincristine induced micronuclei and polyploidy Žfor review see w19x. beside

other effects on mitosis and meiosis. However, both compounds did not induce structural chromosomal aberrations in studies reported by Xu and Adler w20x Žcolchicine. and Manca w21x Žvincristine.. On the other hand, Marazzini et al. w22x found no increase in structural chromosomal aberrations in mouse bone marrow after treatment of the animals with vinblastine, but with colchicine, an effect was seen. SatyaPrakash et al. w23x found an elevation in structural chromosomal aberrations in mouse bone marrow of mice treated with colcemid or vinblastine. They attribute the clastogenic effect to indirect mechanisms as active metabolites of the compounds, interference with DNA synthesis and cytoplasmic endonucleases. The latter two mechanisms may also explain the clastogenic activity observed at high concentrations in vitro in our experiments. In summary, published data reveal a rather nonuniform picture concerning the induction of structural chromosomal aberrations in vitro at low concentrations of colcemid, colchicine and vincristine. In our experiments on CHO cells at low concentrations, which were highly effective on cell division processes, no structural aberrations at all were found. There is thus no indication of a relationship between the property of the compounds to induce effects on mitosis and a clastogenic activity at these concentrations.

Acknowledgements The authors are grateful to Mr. P. Christen for advice in statistical evaluation and to Mrs. B. Blankstein, Mrs. L. Haidacher, Mrs. M. Hegedus, ¨ Mrs. H. Imhof and Mrs. M. Muller for valuable ¨ technical assistance.

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