Sister-chromatid exchanges (SCE) induction by inhibitors of DNA topoisomerases in cultured human lymphocytes

Genetic Toxicology

ELSEVIER

Mutation Research 368 (1996) 205-211

Sister-chromatid exchanges (SCE) induction by inhibitors of DNA topoisomerases in cultured human lymphocytes G. Ribas, N. Xamena, A. Creus, R. Marcos * Grup de Mutag~nesi, Unitat de Gen~tica, Departament de Genbtica i de Microbiologia. Unit,ersitat Autbnoma de Barcelona, Edifici Cn, 08193 Bellaterra, Barcelona, Spain

Received 28 July 1995;revised 30 January 1996;accepted 31 January 1996

Abstract

The induction of sister-chromatid exchanges (SCE) in cultured human lymphocytes by four inhibitors of DNA topoisomerases: m-amsacrine, camptothecin, etoposide and nalidixic acid has been evaluated. Although the four compounds apparently increase the frequency of SCE, the effect of nalidixic acid is weak because only a statistically significant positive response was found in one donor at the highest concentration (500 /xM). The other compounds tested act as SCE inducers in both donors, camptothecin being the most effective. In addition, the influence of these four topoisomerase inhibitors on the SCE frequency induced by MMC was also analysed. The results reveal that less than additive SCE effect was induced by the combined treatments which could suggest that the process leading to SCE induction by MMC and the four inhibitors of DNA topoisomerases are not totally independent. Keywords: Topoisomerase inhibitor; SCE assay; Human lymphocyte

I. Introduction

Sister-chromatid exchanges (SCE) are widely used as a measure of genetic damage, In spite that its exact mechanism(s) is(are) not well understood, it is generally believed that SCE represent the interchange of DNA replication products at apparently homologous loci, involving DNA breakage and reunion (Latt et al., 1981). Considering that SCE are produced at or near the DNA replication fork, several models have provided a rationale for explaining these switches. Many models have postulated the involvement of DNA topoisomerases in the rejoining

* Corresponding author. Tel.: 34-3-5812052; Fax: 34-35812387. 0165-1218/96/$15.00 © 1996 Elsevier Science B.V. PII S01 65- 12 18(96)00013-4

of DNA strand breaks that may lead to SCE formation (Liu et al., 1980; Dillehay et al., 1989). DNA topoisomerases I and II are nuclear enzymes responsible for controlling, maintaining and modifying the DNA topology during various processes including replication, recombination, transcription and chromosome partitioning during cell division. To carry out these functions, DNA topoisomerases induce transient cuts in one or both DNA strands, allowing strands to pass through the nick and then rejoining the nicked strand to the DNA. During its normal function, a covalent linkage is formed between topoisomerase and DNA, called cleavable complex, which could be a pathway to induce SCE (Pommier et al., 1985; Darroudi and Natarajan, 1989; Dillehay et al., 1989; Holden et al., 1989).

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G. Ribas et al. / Mutation Research 368 (1996) 205-211

To understand the role of topoisomerases in the SCE formation, different topoisomerase-interactive agents have been used in various studies indicating that all of them are able to induce SCE in different cultured mammalian cells (Anderson and Berger, 1994), as well as chromosome aberrations (CA) and cell death. To extend the general knowledge on the effect of topoisomerase inhibitors we report here data on the ability of one inhibitor of type I topoisomerase (camptothecin), two inhibitors of type II topoisomerase (m-amsacrine and etoposide), and one inhibitor of bacterial DNA gyrase (nalidixic acid) to induce SCE in cultured human lymphocytes. In addition, to find out if there is any relationship between the mutagen induced SCE and topoisomerase activity, the effect of such inhibitors on the activity of the very potent SCE inducer mitomycin C (MMC) has also been studied. It should be indicated that, although studies on the effects of different topoisomerase inhibitors on cultured mammalian cells are frequent, they are scarce for human lymphocyte cultures.

incubated at 37°C for 72 h and for SCE demonstration, BrdU at the concentration of 15 /zg/ml was added 24 h after the initiation of cultures. The tested compounds, topoisomerase inhibitors alone or combined with MMC, were added together with the BrdU. Throughout the experiments, all cultures were maintained in the dark to minimize photolysis of BrdU. The treatment was continued up to harvest and, 2 h prior to harvesting, 0.6 /zg/ml of Colcemid (Gibco) was added to arrest the chromosomes at metaphase. The cells were collected by centrifugation, resuspended in a pre-warmed hypotonic solution (KC1 0.075 M) for 20 rain and fixed in acetic acid/methanol (1:3, v / v ) . Air-dried preparations were made and the slides were stained with the fluorescence plus Giemsa method (Perry and Wolff, 1974). The test compounds were added together with 0.05 ml of the appropriate concentration of each compound, or the mixture, and added to cell cultures 24 h after its initiation, with the BrdU.

2.3. SCE analysis 2. Materials and methods

2.1. Chemicals m-Amsacrine, camptothecin, etoposide, nalidixic acid and mitomycin C (MMC, CAS No. 50-07-7) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 5-Bromodeoxyuridine (BrdU) was provided by Kodak (Rochester, NY, USA). All of them were dissolved in dimethyl sulphoxide (DMSO, Panreac, Barcelona, Spain), except MMC that was dissolved in bidistilled water, at a final volume in the culture of 4% of the total.

2.2. Lymphocyte cultures Lymphocyte cultures were set up by adding 0.5 ml of heparinized whole blood, from two young non-smoking healthy donors, to 4.5 ml of RPMI 1640 chromosome medium (Gibco) supplemented with 16% heat inactivated fetal calf serum (Gibco), antibiotics (penicillin and streptomycin) and Lglutamine. Lymphocytes were stimulated by 4% phytohemagglutinin (PHA, Gibco). The cultures were

A total of 50 well-spread metaphases were examined for each experimental concentration and donor on coded slides to determine the SCE frequencies. In addition, 100 metaphases per donor were also scored to determine the proportion of cells that had undergone one, two, or three or more divisions. The proliferative rate index (PRI) was calculated according to the formula PRI = (MI + 2M2 + 3M3)/N, where MI, M2 and M3 indicate those metaphases corresponding to first, second and third or subsequent divisions, and N is the total number of metaphases scored (Lamberti et al., 1983). The effects on cell cycle delay (PRI) were evaluated by using the Fisher's exact test. For the statistical evaluation of the SCE frequencies we used an analysis of variance (ANOVA) test for intergroup differences. When a significant F value was found ( p < 0.05), the Dunnet's test was used to test the differences of each dose group to the control value (Cooke et al., 1989). To analyze the interaction between the MMC and the different inhibitors we applied a multiple regression equation using the statistical package GLIM

G. Ribas et al. / Mutation Research 368 (1996) 205-211

(Baker and Nelder, 1978). For this analysis the number of SCE per cell was In transformed. For all tests the level of significance was set at 0.05.

Table 2 Frequencies o f S C E and proliferative rate indices (PRI) in cultured h u m a n l y m p h o c y t e s treated with four inhibitors of topoisomerases. D o n o r B Treatment

n

SCE _+ SE

PRI

70 50

9.26 5=0.36 10.62-+0.53

2.43 2.52

50 50 50 50

10.86 5=0.70 10.18-+0.59 10.06_+0.55 14.82_+0.59 ~

2.07 1.90 1.72 1,43

* * * *

0.5 1.0 2.0 4.0 8.0

50 50 50 50 50

11,40_+0.78 8.84_+0.60 13.00 _+0.64 12.64 _+0.76 16.94 _+0.98 *

2.06 2.01 1.86 2.04 1.94

* * * *

Etoposide

0.025 0.050 0.100 0.250 0.500

50 50 50 50 24

15.82 _+0.95 17.02_+0.85 26.38_+1.34 28.36 _+0.99 38.79_+2.75

~ * * ~ *

2.29 2.05 * 2.03 * 1.64 1.29 *

Camptothecin

0.0050 0.0075 0.0100 0.0250

50 50 50 50

28.04 _+ 1.30 29.40_+ 1.48 44.38_+ 1.71 55.76_+ 2.71

* * * *

2.13 2.00 1.95 2.09

Nalidixic acid

Tables 1 and 2 show the results obtained after the treatment with the four selected topoisomerase inhibitors, from lymphocyte cultures set-up from donors A and B, respectively. Table 3 summarizes the results of the analysis of variance for intergroup differences. As shown, the four compounds tested are able to apparently increase the SCE frequency, although with different efficiencies. Nalidixic acid only induces clear SCE increases in one donor at the highest

Table 1 Frequencies o f S C E and proliferative rate indices (PRI) in cultured h u m a n l y m p h o c y t e s treated with four inhibitors of topoisomerases. D o n o r A Treatment

Conc.[/xM]

Control D M S O 1%

n

S C E _+ SE

PR!

100 133

8.35 _+ 0.37 8.98_+0.36

2.78 2.38

25 50 100 500 1000

50 50 50 50

11.00_+0.65 10.64 5=0.48 11.38_+0.65 13.68_+0.81

2.55 2.48 2.40 2.00 1.21

m-Amsacrine

1 2 4 8

47 50 50 15

11.00+_0.59 12.04_+0.90 13.88_+0.71 16.66_+ 1.17 ~

2.46 2.30 2.19 1.91

Etoposide

0.10 0.25 0.50

50 50 21

16.10_+0.85 * 22.66_+ 1.22 * 32.86_+2,76 *

2.17 1.91 1.35

Nalidixic acid

-

1.00

Camptothecin

0.0050 0.0075 0.0100 0.0250 0.5000

Conc.[/zM]

Control D M S O 1%

3. Results and discussion

-

-

-

-

50 50 50 50 -

22.42+0.97 2 4 . 8 2 ± 1.11 32.30__+ 1.57 52.34__+ 1.73 -

1.17

* " * ~

2.38 2.35 2.48 2.35

n, n u m b e r o f metaphases scored; * p < 0.05 ( D u n n e t ' s test for S C E and Chi-square test for PRI).

207

m-Amsacrine

25 50 100 500

* * * *

n, n u m b e r of metaphases scored; * p < 0.05 ( D u n n e t ' s test for S C E and Chi-square test for PRI).

concentration tested (500 /~M), therefore this effect could he considered as weak (or marginal). This bacterial inhibitor of gyrase A does not seem very toxic in human lymphocyte cultures and, consequently, it has been possible to test concentrations as high as 500 /~M. Nevertheless, at 1000 /~M, it appeared to be toxic reducing the number of second division metaphases almost completely. Although it has been proved that nalidixic acid can interfere with topoisomerase activity in eukaryotic systems (Rusquet et al., 1984; Drlica and Franco, 1988), few studies have been conducted in mammalian cell systems. Thus, Stenchever et al. (1970) and Sumner (1992) showed that there is no clastogenic activity in cultured human lymphocytes, while Kowalczyk (1980) reported elevated levels of SCE in children treated with the compound. On the other hand, Holden et al. (1989) using the nalidixic acid

G. Ribas et a l . / Mutation Research 368 (1996) 205-211

208

analog (CP-67.015) found strong clastogenic activity in human lymphocytes and in CHO cells, although only a slight elevation of SCE. m-Amsacrine is an intercalating drug that has been reported to be active against acute myelogenous leukaemia, although its severe toxicity has limited its potential clinical use. In our experiments, mamsacrine increases the SCE frequency in a high dose-response way attaining a statistically significant response in both donors at the highest dose tested (8 /zM), but its activity is much lower than that observed with etoposide and camptothecin. In other mammalian cell systems, such as CHO cells, mamsacrine also gives a significant increase in SCE, although at much lower concentrations (Cort6s et al., 1993; Cort6s and Pifiero, 1994). Furthermore, in vivo studies also indicated a high increase of the SCE frequency in mouse bone marrow, without affecting the cell cycle (Backer et al., 1990). Recently it has

been shown that in Chinese hamster-human hybrid cell line, m-amsacrine induced megabase-pair deletions of the human chromosomes and it has been postulated that exchanges between topoisomerase II subunits could be responsible for these deletions (Shibuya et al., 1994). Etoposide is a semisynthetic podophyllotoxic derivative being very active in cancer therapy and that has no intercalating ability. Our data indicated that I p,M is completely toxic in cultured human lymphocytes and that a low dose (0.025 /xM) was high enough to induce a very significant increase in the SCE frequency. Etoposide as well as the other inhibitors, has shown to induce chromosomal structural aberrations, chromosome fragmentation and SCE in cultured mammalian cells (Tominaga et al., 1986; Darroudi and Natarajan, 1989; Mailhes et al., 1994). Camptothecin is a naturally occurring plant alka-

Table 3 Analysis of variance ( A N O V A ) for intergroup differences for the SCE results obtained with the four topoisomerase inhibitors Compound

Donor

Source o f variation

NA

A

Between Between Error Between Between Error

conc. replicates

Between Between Error Between Between Error

conc. replicates

B

m-AMSA

A

B

VP-16

A

B

CAMP

A

B

conc. replicates

conc. replicates

Between Between Error Between Between Error

conc. replicates

Between Between Error Between Between Error

conc. replicates

conc. replicates

conc. replicates

SS

DF

MS

F

p

9.19 2.67 77.91 6.37 1.11 37.43

4 5 323 4 5 240

2.297 0.534 0.241 1.592 0.222 0.156

4.301 2.214

0. l 1782 0.06723

7.174 1.423

0.027 * 0.217

14.99 1.64 68.03 16.25 2.20 58.82

4 5 288 5 6 288

3.747 0.327 0.236 3.250 0.366 0.204

11.448 1.385

59.72 1.04 61.45 46.13 2.23 27.14

3 4 245 5 6 262

19.9 l0 0.351 0.251 9.225 0.372 0.104

56.697 1.400

0.00434 * 0.2434

24.769 3.596

0.00061 * ~ * 0.00192 * *

164.64 1.11 61.92 81.90 3.2t 25.43

12 16 323 4 5 240

5.847 0.270 0.193 20.475 0.642 0.162

21.687 0.314

0.00068 * " * 0.32252

31.867 6.064

0.00094 * * * 0.00003 * ~ *

8.874 1.793

NA, nalidixic acid; m - A M S A , m-amsacrine; VP-16, etoposide; C A M P , camptothecin; Conc., concentration.

0.04338 * 0.24735 0.00967 ~ * 0.10030

209

G. Ribas et al. / Mutation Research 368 (1996) 205-211 Table 4 SCE values for joint treatments between topoisomerase inhibitors and three MMC concentrations. Donor A Treatment

Control DMSO 1% NA (500 ttM) m-AMSA (4/zM) VP-16 (0.05 /.tM) CAMP (0.005/zM)

Without MMC

MMC (0.025 /zM)

MMC (0.050/zM)

MMC (0.100 p.M)

SCE 4- SE

SCE + SE

SCE 4- SE

SCE + SE

7.38 9.02 15.16 14.14 15.56 27.74

23.62 + 0.90

30.96 + 1.25

46.00 4- 1.56

28.84 27.88 30.88 32.92

34.08 35.54 38.18 39.24

46.20 50.52 52.27 53.80

+ 0.39 4-0.41 4- 0.84 + 0.75 4- 0.86 + 1.41

4- 1.16 4- 0.99 4- 1.30 4- 1.25

4444-

1.53 1.40 1.39 1.42

4- 1.69 + 1.72 _ 1.52 4- 2.17

NA, nalidixic acid; m-AMSA, m-amsacrine; VP-16, etoposide; CAMP, camptothecin. 50 metaphases were scored for SCE.

loid from Camptotheca accuminata used for treatment of neoplasias in China. This compound is an inhibitor of topoisomerase I activity without an intercalating activity. In our experiments, camptothecin has shown to be the most potent SCE inducer since, at a concentration as low as 0.005/zM, it was able to induce very significant increases in the frequency of SCE. These results are evident in cultures from both donors, and a direct dose-response relationship was observed. In addition to its clastogenic activity (Degrassi et al., 1989; Backer et al., 1990), significant SCE increases following treatment with camptothecin has been reported in V79 Chinese hamster lung fibroblasts (Lim et al., 1986) and in CHO cells (Cort6s and Pifiero, 1994), although only when using concentrations much higher than those used in this work. It is not clearly understood how toposoimerases I and II inhibitors induce SCE, but it seems that drug-induced stabilization of topoisomerase-DNA complexes are the initial events in the pathway leading to SCE. In this context, if chemical induction of SCE involves the participation of DNA topoisomerases, the simultaneous treatment with topoisomerase inhibitors and a very strong inducer of SCE, as is MMC, would produce some kind of interaction, possibly synergistic effects. Nevertheless, the statistical analysis of the results presented in Table 4 seems to show an opposite effect, since less than an additive SCE effect was induced by the combined treatments, for all the compounds tested, as indicated in Table 5. MMC is a cross-linking agent very efficient as

SCE inducer and, due to its bulky structure, damage induced by MMC probably depends on chromatin structure. This would indicate that MMC activity would depend on any higher-order chromatin-structure dependent of topoisomerase activity (Lim et al., 1986) and therefore, modifying the topoisomerase activity would affect the SCE induction ability of

Table 5 Multiple regression analysis for the interaction between the four topoisomerase inhibitors and the three MMC concentrations used Treatment (/zM)

MMC [/xM]

13

SE

p

Effect

Nalidixic acid

0.025

-0.451

0.077

0.0001

L

0.050 0.100

-0.563 -0.668

0.083 0.077

0.0001 0.0001

L L

0.025

- 0.422

0.071

0.0001

L

0.050 0.100

- 0.448 -0.500

0.076 0.070

0.000 1 0.0001

L L

0.025

- 0.448

0.077

0.000 1

L

0.050 0.100

-0.488 -0.573

0.078 0.071

0.0001 0.0001

L L

0.025

- 0.931

0.072

0.000 1

L

0.050 0.100

- 1.019 - 1.120

0.076 0.075

0.0001 0.0001

L L

(500)

m-Amsacrine

(4)

Etoposide

(0.05)

Camptothecin

(0.005)

/3, estimated coefficient; SE, standard error; P, probability; L, less than additive.

210

G. Ribas et al. /Mutation Research 368 (1996) 205-211

M M C . In fact, the clear antagonistic effects observed in the cotreatments b e t w e e n M M C and the four topoisomerase inhibitors seem to confirm that the topoisomerases are i n v o l v e d in the ability o f M M C to induce S C E either by limiting initial d a m a g e or in response to M M C treatment. Similar results were reported in V79 cells by L i m et al. (1986), but by using pretreatment with camptothecin, m-amsacrine and V M - 2 6 and treatment with M M C , P U V A and X-rays, indicating a high reduction in the S C E frequency mainly after camptothecin pretreatment. In sum,qary, our results show that the four inhibitors o f D N A topoisomerases tested increased the frequency o f S C E in cultured human lymphocytes. As expected, m-amsacrine, camptothecin and etoposide were g o o d S C E inducers, although with different ability in relation to that observed in other cell systems. In our study camptothecin has shown to be the most efficient S C E inducer while m-amsacrine was m o r e effective in C H O cells ( P o m m i e r et al., 1988; Cortes and Pifiero, 1994). Finally, the inhibition of type I and II topoisomerases influences the induction o f e x c h a n g e s by M M C and leads to less than an additive effect, suggesting that these agents may not be acting independently to induce SCEs.

Acknowledgements This investigation has been supported in part by grants of the Spanish Ministry of Education and Science ( C I C Y T , S A F 9 4 - 0 6 9 7 ) and the G e n e r a l i t a t de C a t a l u n y a (CIRIT, GRQ93-2023). G. Ribas was supported during this w o r k by a fellowship from the U n i v e r s i t a t A u t b n o m a de B a r c e l o n a ( U A B ) . W e w o u l d like to thank G. U m b e r t and M. M c C a r t h y for their expert technical and secretarial assistance, respectively.

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