Chemically induced aneuploidy: investigations into chromosome specific effects in mitosis

Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation Research 404 (1998) 191-197

Chemically induced aneuploidy: investigations into chromosome specific effects in mitosis Russell D.P. Bourner, E.M. Parry *, J.M. Parry Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK

Received 23 March 1998; accepted 30 March 1998

Abstract Genotoxicity studies of aneuploidy may potentially produce different results depending upon the chromosome selected for analysis if chromosome-specific sensitivities to chemical exposure exist. Any chromosome specificity characteristics that predispose to aneuploidy might interact with environmental exposures in additional different ways related to the mechanism of aneuploidy induction. Thus, we have undertaken an investigation of chromosome-specific effects using morphologically distinct chromosomes in a hybrid cell line. We were able to identify eight different chromosomes simultaneously by dual colour FISH analysis in controls and in cells exposed to a range of griseofulvin concentrations. Certain chromosomes were more frequently involved in aneuploidy, but no simple relationship between chromosome organisation and sensitivity emerged apart from the over-representation of the alien human chromosome. Aneuploidy was detected at higher frequencies in interphase cells compared with metaphase cells. Overall the data indicate that chemically induced aneuploidy may be detected for a variety of chromosomes and cell types using both interphase and metaphase protocols. However, the data obtained should be used with care in the hazard evaluation of chemical aneugens. 9 1998 Elsevier Science B.V. All rights reserved. Keywords: Aneuploidy; Mitosis; Aneugen; Hybrid cell; Chromosome specificity

1. Introduction Chemically induced aneuploidy may arise by a variety of mechanisms and there are various properties of individual chromosomes that may render them at increased risk of induced aneuploidy [1]. A fundamental question concerning the origins of aneuploidy is whether all chromosomes behave in the same w a y at cell division or whether chromosome-specific fac-

9 Corresponding author. Tel.: +44-1792-295388; fax: +441792-295,t47; E-mail: [email protected]

tors influence their passage through division and their susceptibility to aneuploidy. This question is relevant to both the basic comprehension o f the origins of aneuploidy and to hazard assessment following exposure to potential aneugens. If chromosome-specific sensitivities exist, then knowing their order is important and also if such sensitivities are the same to different aneugens. Thus, understanding the mechanisms causing any such chromosomespecific factors is important. Chromosome-specific induction o f aneuploidy and micronuclei in human lymphocytes by exposure to

0027 5107/98/$19.00 9 1998 Elsevier Science B.V. All rights reserved. PII: S0027-5107(98)00113-4

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metabolites of 1,3-butadiene [2] for chromosomes 12 and X has been reported, but no effect was seen for chromosomes 7 and 8. A preferential effect of colchicine for micronucleus induction of acrocentric chromosomes in Down syndrome individuals, their parents and a matched control group has been reported [3]. A similar preferential involvement of acrocentric chromosomes in micronuclei induced by vanadium salts has been observed [4]. There are a number of properties of chromosome structure which might alter the probability of normal segregation at cell division. Certain of these have been identified by experimental evidence, others can be theoretically inferred, e.g. chromosome size, centromere position, centromere size, centromeric heterochromatin, patterns of recombination and gene density [5]. There is clear evidence that chromosomes are different in their susceptibility to non-disjunction [6]. Additionally, maternal and paternal age may act differently to increase non-disjunction in a chromosome-specific manner and a stage of meiosis-specific way. The human spontaneous abortion data suggest real differences in their rates of origin [7], but no single factor can explain the large differences, nor are many of the factors such as chromosome length, recombination and gene density truly independent. To address some of these points, we have studied mitotic aneuploidy in a cell line with chromosomes of distinctive size and shape in their natural or in an alien background with and without exposure to two known aneugens, colcemid and griseofulvin [8]. The cell line used is a hybrid monochromosomal human/mouse cell line, R3-5, containing a variety of morphologically distinct chromosomes. 2. Materials and methods 2.1. Cell line and culture The R3-5 cell-line used in this study is a monochromosomal human/mouse hybrid donated by Professor R.S. Athwal, New Jersey Medical School, USA. It was developed by Athwal and Sandu [9] as a potential selectable plating test system for aneuploidy detection. R3-5 is deficient for the enzyme hypoxanthine-guanine phosphoribosyl transferase (hprt). The human chromosome 2 carries a dominant

selectable marker, Escherichia coli xanthineguanine phosphoribosyl transferase (Ecogpt), so that the cell line is hgprt - / E c o g p t + . Presence of the human chromosome allows the cells to grow in media containing mycophenolic acid and cells that have lost the human chromosome grow in the presence of 6-thioguanine. Thus the cells were cultured at 37~ in a 10% CO 2 atmosphere in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum and mycophenolic acid (70 /xg/ml) and xanthine (25 /xg/ml) (MX) to ensure maintenance of the human chromosome in the culture. As a selective plating system, this cell line can only be used to detect chromosome loss. We decided to exploit the fact that the cells contain a variety of distinct chromosomes to measure aneuploidy by measuring both chromosome loss and gain. 2.2. Aneugenic treatments R 3 - 5 cells that had been subcultured in DMEM + MX medium were harvested and resuspended in non-selective DMEM at a titre of 2 X 105 cells and distributed to 25 mm z tissue culture flasks. Flasks were gassed with 5% CO 2 and incubated at 37~ for 24 h when they were washed and refed with, fresh DMEM supplemented with appropriated graded quantities of either colcemid (Gibco) or griseofulvin (Sigma). Stock solutions were prepared in phosphate buffered saline solution (colcemid) and in dimethyl sulphoxide (DMSO) for griseofulvin. Appropriately diluted solutions of aneugen were added in 15 /xl aliquots to the cells which were incubated for a further 24 h. Solvent controls were conducted alongside chemical treatments. After 24 h incubation in the presence of the aneugen, the cells were washed and returned to normal DMEM for 24 h before being harvested for cytogenetic analysis. Colcemid (0.45 /xg/ml) was added 2 h prior to harvest. Cells were collected by trypsinisation into centrifuge tubes, treated with 75 mM KC1, fixed, distributed onto glass slides and air dried. 2.3. Fluorescence in situ hybridisation (FISH) Two classes of probe were used to specifically identify chromosomes: chromosome-specific composite DNA probes and a centromere-specific alpha

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satellite probe. Whole chromosome paint probes allow the rapid and unambiguous detection of numerical and structural aberrations in metaphase cells; the alpha-satellite probe was informative in both metaphase and interphase cells. Prior to FISH the slides were immersed in acetone for 15 min, air dried, treated with RNAse (Sigma) [100 / z g / m l in 2 • SSC, pH 7.0] for 1 h at 37~ washed three times in 2 • SSC, treated with proteinase K (Boehringer Mannheim) [0.5 / z g / m l in 2 mM CaCI2, 20 mM Tris-HCl, pH 7.4], rinsed twice in 2 • SSC and dehydrated through an ethanol seties. These steps were necessary to render chromosomal DNA accessible to the probe and to minimise background signals. Whole chromosome probes for human chromosome 2 and mouse chromosomes 3 and X were obtained from Cambio and used according to the manufacturers instructions. Pairs of chromosomes were probed simultaneously, one labelled with biotin the other with fluorescein. Hybridisation signals were detected by incubation with (i) avidin-Texas Red (Vector Labs) (diluted 1:500 in blocking reagent [0.02% w / v sodium azide/5% w / v B S A / P N

3

t3a

t3b

t3c

buffer]). (ii) biotinylated anti-avidin (Vector Labs) and mouse anti-FITC (Sigma) (each diluted 1:250 in blocking reagen0. (iii) avidin-Texas Red and rabbit anti-mouse FITC-conjugated Fab fragments (Sigma) (each diluted 1:500 in blocking reagents). All antibody incubations were performed under a plastic coverslip at 37~ for 20 min in a humidified chamber, followed by 3 • 5 min washes in 4 • SSC containing 0.05% Tween-20 (ICI). The slides were then briefly dehydrated through an ethanol series and air dried in the dark. All slides were mounted in Vectashield antifade (Vector Labs) containing 0.5 /xg/ml 4',6-diamino-2-phenylindole (DAPI) counterstain and visualised using an Olympus BH2-RFL fluorescence microscope equipped with single band pass filters for the red, blue and green spectra and a triple-band pass filter (Chromatechnology) to allow simultaneous observation of both labelled chromosomes and counterstain. Images were captured and stored via the Probemaster digital imaging system (PSI). All manipulations were performed under subdued lighting because of the light sensitivity of many of the reagents. Hybridised slides were either used immediately or stored at 4~ in the dark.

X

tX

Dicentric

Fig. I. Ideogramsof the six mouse chromosomesidentified by FISH. Green colour represents chromosome3-specificDNA, red represents chromosome X-specific DNA, blue represents DNA of unidentified origin and black represents the centromeres. Relative lengths of the chromosomesare portrayed.

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A human chromosome 2 centromere DNA probe was constructed from R3-5 DNA using oligonucleotide p r i m e r s d i r e c t e d to a c o n s e r v e d r e g i o n o f t h e a l p h a satellite m o n o m e r [10] to g e n e r a t e t h e D N A sequence by PCR [ll].The probe was labelled with b i o t i n a n d d e t e c t e d in t h e u s u a l w a y [12].

Table 2 Frequencies of induced chromosome loss in R3-5 cell line GF (/xg/ml) Chromosome 0

5

2.4. Cytogenetic analysis T w o - h u n d r e d i n t a c t w e l l - s p r e a d m e t a p h a s e cells w e r e a n a l y s e d f o r the p r e s e n c e o f t h e h u m a n a n d d i c e n t r i c m o u s e m a r k e r c h r o m o s o m e after b o t h colc e m i d a n d g r i s e o f u l v i n t r e a t m e n t s at all doses. T h e six c h r o m o s o m e s i d e n t i f i a b l e b y m o u s e 3 a n d X p a i n t p r o b e s w e r e also a n a l y s e d in t h e g r i s e o f u l v i n e x p e r i m e n t (3 • 2 0 0 cell p e r dose). A d d i t i o n a l l y , after b o t h t r e a t m e n t s , 1000 i n t e r p h a s e n u c l e i / d o s e p o i n t w e r e s c o r e d for s e g r e g a t i o n o f t h e h u m a n c h r o m o s o m e 2.

3. Results 3.1. R 3 - 5 karyotype R 3 - 5 cells w e r e c o n s t r u c t e d f r o m a h y p e r t r i p l o i d m o u s e cell line. T h e y t y p i c a l l y c o n t a i n b e t w e e n 5 0 6 0 c h r o m o s o m e s . In a d d i t i o n to t h e a l i e n h u m a n c h r o m o s o m e , t h e cell line c o n t a i n s a n u m b e r o f rearranged mouse chromosomes including one that a p p e a r s to b e d i c e n t r i c b u t is f u n c t i o n a l l y m o n o c e n tric. P r o b i n g w i t h w h o l e c h r o m o s o m e p a i n t p r o b e s for m o u s e c h r o m o s o m e s 3 a n d X r e v e a l s the pres-

10

20

Human Dicentric

X

tX 3

8 4 7 9 11 16 11 13 16 25 23 27

4 3 1 8 6 5 13 8 7 10 16 13

4 1 2 2 1 3 5 2 2 9 2 6 4 1 6 7 2 4 7 6 10 12 2 11 10 5 13 15 11 17 9 11 12 12 9 19

5 7 3 6 5 8 9 7 10 16 18 16

Table 3 Comparison of the frequencies of chromosome gain amongst eight chromosomes in R3-5 cell line GF (/xg/ml) 0

Chromosome

5

0

1

2

3

3 2 1 1 0 1 4 7

97 98 99 96 99 98 96 89

0 0 0 1 1 1 0 3

0 0 0 2 0 0 0 1

One-hundred cells analysed for each chromosome.

14 12 10 7 8 10 12 16 19 21 18 15

e n c e o f six m o r p h o l o g i c a l l y d i s t i n c t c h r o m o s o m e s . T h e p r o b e for c h r o m o s o m e 3 p a i n t s 4 d i f f e r e n t c h r o m o s o m e s , o n e c o m p l e t e l y a n d t h u s n a m e d 3 here, a l t h o u g h , in t h e a b s e n c e o f G - b a n d i n g data, w e c a n n o t b e c e r t a i n that it is a n o r m a l c h r o m o s o m e 3. T h e o t h e r three, t3a, t3b a n d t3c i n v o l v e d r e a r r a n g e ments with unpainted mouse chromosomes. The p r o b e for the X c h r o m o s o m e r e v e a l e d t w o c h r o m o s o m e s , o n e totally p a i n t e d X a n d o n e t r a n s l o c a t i o n ,

Table 1 Frequency of cells within a culture of R3-5 line with specific chromosome constitution

Human Dicentric X tX 3 t3a t3b t3c

9 5 8 7 5 5 14 9 10 13 12 15

Values given are the number of losses in 200 cells. Three samples per dose-point of griseofulvin were taken.

Chromosome Human Dicentric X tX 3

Number of copies of given chromosome

t3a t3b t3c

10

20

1 3 0 6 11 8 7 10 6 18 11 21

0 0 1 5 6 3 3 7 6 11 9 15

0 0 0 6 3 5 5 7 9 9 9 4

t3a t3b t3c

3 4 3 3 3 2 0 1 4 1 6 6 2 7 6 0 9 8 9 10 7 10 8 5 6 5 6 11 6 12 8 8 7 14 9 7

1 0 2 2 1 1 1 3 2 3 5 5

7 5 2 5 8 5 6 5 7 9 6 6

Values given are the number of gains in 200 cells. Three samples per dose-point were taken. Exposure to 0 to 20 /xg/ml of griseofulvin.

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Table 4 Frequencies of induced aneuploidy for the human and mouse dicentric chromosomes in the R3-5 cell-line following chemical exposure

marised in T a b l e 1. T h e y are all represented in m o s t cells b y one copy, thus the presence o f 2 or 0 c h r o m o s o m e s was considered as e v i d e n c e o f aneuploidy resulting f r o m c h r o m o s o m e gain or loss respectively. U s i n g two c o l o u r c h r o m o s o m e painting eight individual c h r o m o s o m e s c o u l d be identified and analysed after exposure to griseofulvin. Tables 2 and 3 show the data for c h r o m o s o m e loss and gain respectively. The c h r o m o s o m e loss data (Table 2) d e m o n strates that the c h r o m o s o m e 3 translocations b and c are unstable in control cultures. Both the h u m a n c h r o m o s o m e 2 and the m o u s e m a r k e r dicentric are also lost at relatively high frequencies. H o w e v e r , f o l l o w i n g griseofulvin exposure the h u m a n c h r o m o s o m e 2 was lost at substantially higher rates than the m o u s e c h r o m o s o m e s . A dose related increase in c h r o m o s o m e loss was seen for all c h r o m o s o m e s . The c h r o m o s o m e gain data (Table 3) d e m o n strates that c h r o m o s o m e t3c is the most unstable c h r o m o s o m e in the control culture. F o l l o w i n g e x p o sure to griseofulvin all c h r o m o s o m e s s h o w e d a dose related increase in c h r o m o s o m e gain. This effect was greatest for the h u m a n c h r o m o s o m e and smallest for the various m o u s e 3 c h r o m o s o m e s . The effect o f exposure to a range o f doses o f c o l c e m i d ( 0 - 0 . 1 / z g / m l ) and griseofulvin ( 0 - 3 0 / x g / m l ) on aneuploidy o f the h u m a n and m o u s e dicentric marker c h r o m o s o m e s is shown in T a b l e 4. Both treatments induced loss and gain o f both chrom o s o m e s in a dose d e p e n d a n t manner. H o w e v e r , the

Concentration of colcemid (/~g/ml) 0

0.0025 0.005 0.01 0.015 0.02 0.03 0.05 0.1

Loss of chromosome

Human 7 8 Dicentric 11 9

8 12

10 18

10 14

18 15

23 27

31 28

28 35

7 5

8 8

11 9

12 14

20 17

19 12

21 17

Gain of chromosome

Human Dicentric

4 4 3 7

Concentration of griseofulvin (/xg/ml) 0 1.25

2.5

5.0

7.5

10

15

20

30

7 7

9 6

7 9

11 9

16 11

25 16

37 31

1 2

6 5

8 5

7 3

13 8

18 11

29 21

Loss of chromosome

Human Dicentric

8 6 5 5

Gain of chromosome

Human Dicentric

1 0 0 1

Two-hundred diploid metaphases were examined per dose point for chromosome loss and gain. tX. T w e n t y - f i v e metaphase spreads probed with the m o u s e 3 and X paints were analysed to d e t e r m i n e the physical lengths o f these c h r o m o s o m e s . T h e average values w e r e then used to create the i d e o g r a m s shown in Fig. 1. A n analysis o f the distribution o f these c h r o m o somes, as well as the h u m a n c h r o m o s o m e is sum-

Table 5 Frequencies of induced aneuploidy for the human chromosome 2 in interphase cells of the R3-5 cell-line following exposure to colcemid and griseofulvin Concentration of colcemid (/zg/ml) Chromosome loss Chromosome gain No. cells

0

0.0025

0.005

0.01

0.015

0.02

0.03

0.05

0.1

62 41 1000

67 56 1000

76 62 1000

70 56 1000

95 65 1000

146 113 1000

181 158 1000

249 216 1000

371 255 1000

Concentration of griseofulvin (/xg/ml) 0 Chromosome loss Chromosome gain No. cells

50 40 1000

1.25 51 36 1024

2.5 61 45 1000

5.0 58 52 1000

7.5 64 47 1000

10

15

20

30

75 44 1000

109 76 1008

181 129 1000

241 193 1000

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relative frequencies of loss and gain of the human chromosome were consistently greater than those of the mouse chromosome. The data in Table 5 is an interphase analysis o f the frequencies o f aneuploidy o f the human chromosome following treatments with colcemid or griseofulvin. Clearly both chromosome loss and gain were induced by both treatments.

4. Discussion The chromosomes studied were selected for ease of recognition, all eight could be identified simultaneously by two colour FISH, and because they represented distinct chromosome classes for analysis. These are a mouse autosome, a mouse gonosome, a mouse dicentric marker behaving as a submetacentric chromosome, an X chromosome translocation of large size with an X centromere, three mouse chromosome 3 rearrangements; t3a predominantly chromosome 3 with an unknown centromere; t3b and t3c both with chromosome 3 centromeres but different amounts of unknown translocated material attached and an alien human chromosome. Clear differences in the behaviour of these chromosomes were detected in control and treated cultures. In untreated cultures the human chromosome, the mouse dicentric, t3b and t3c showed most chromosome loss and for chromosome gain t3c was the most unstable. Although dose dependant increases in aneuploidy were detected for all chromosomes, there was a marked difference in response between chromosomes. Specifically, chromosomes t3c and the human chromosome were lost more than the others after griseofulvin exposure whereas chromosome 3 and X were the most stable. The human chromosome was most commonly gained after griseofulvin exposure and t3b was the most stable.

5. Conclusions Thus, the human chromosome was the most unstable of the chromosomes tested in this cell line. This was not unexpected. A n alien chromosome might well be expected to encounter difficulties related to species differences in s p i n d l e - c h r o m o s o m e interactions.

The mouse chromosomes varied in the order of their sensitivities. The dicentric was the most unstable. The translocated chromosome 3, t3a, with a non-3-centromere was relatively stable. Much of the variation in sensitivity between the four chromosomes identified with the chromosome 3 probe appeared to be correlated with increased chromosome size or increased non-chromosome 3 material. The two chromosomes identified by painting with chromosome X were relatively stable. The translocation tX;?, which resulted in the largest chromosome studied, did not adversely affect its segregation at mitosis. Thus, we did not see enhanced aneuploidy o f the sex chromosome, this may be related to the fact that we were examining cells with single copy numbers of chromosomes. Chromosome loss always exceeded chromosome gain in both metaphase and interphase analyses. Interphase analyses appeared to detect higher levels of aneuploidy, this difference was greatest for loss. Such data should be treated with caution in this case where a reduction in copy number from 1 involved a loss o f signal.

Acknowledgements This work was supported in part by a grant from the European Union Environmental Research Programme. R.D.P.B. was supported by a U K BBSRC research studentship.

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