Clastogenic effects of the fasciolicide drug fasinex on river buffalo lymphocyte cultures in vitro

Mutation Research 541 (2003) 115–121

Clastogenic effects of the fasciolicide drug fasinex on river buffalo lymphocyte cultures in vitro Sahar Ahmed∗ , Othman E. Othman Cell Biology Department, National Research Center, Dokki, Giza, Cairo, Egypt Received 2 April 2003; received in revised form 23 July 2003; accepted 31 July 2003

Abstract Fasinex (triclabendazole) has been reported to be an active fasciolocidal agent used in humans and in farm animals. The clastogenic effects of fasinex were tested in lymphocyte cultures of the river buffalo at three final concentrations: 25, 50 and 100 ␮g/ml. Chromosomal aberrations, sister chromatid exchanges and micronucleus formation are the three cytogenetic parameters used in this study. The results demonstrated that the number of cells with different types of chromosomal aberrations, including chromatid breaks and gaps, isochromatid breaks and gaps and polyploidy, was increased significantly in cultures treated with different doses of fasinex compared to the control. This increase was dose-dependent where there was a positive correlation between increased drug concentration and induction of chromosomal aberrations. The frequency of sister chromatid exchanges and the formation of micronuclei in all lymphocyte cultures treated with different doses of fasinex were increased significantly compared to the control; these increases were also dose-dependent. In conclusion, the three cytogenetic parameters used to evaluate the effect of fasinex revealed that the drug has a strong clastogenic effect on river buffalo lymphocytes in vitro. © 2003 Elsevier B.V. All rights reserved. Keywords: Fasinex; Chromosomal aberrations; Micronucleus; Sister chromatid exchange; River buffalo lymphocytes

1. Introduction Fascioliasis is an acute or chronic disease of the liver and bile ducts resulting in chronic digestive and nutritional disturbance, caused by Fasciola gigantica or F. hepatica. It is an endemic infectious disease in farm animals such as sheep, goat, cattle and river buffalo [1]. Infections with fascioliasis in humans is an increasingly recognised public health problem in different countries like Egypt [2,3], Chile [4] and Australia [1].

∗ Corresponding author. Fax: +20-23370931. E-mail address: [email protected] (S. Ahmed).

Fasinex (triclabendazole) has been reported to be a highly effective drug against immature and mature Fasciola in humans [2,3], sheep [5], goat [6], cattle [7], rabbit [8] and river buffalo [9]. Fasinex is reported as a benzimidazole derivative [9]. Most of the benzimidazole derivatives have clastogenic and aneugenic effects [10] and they also act as inhibitors of topoisomerase II [11]. Topoisomerase II is a gene involved in essential cellular process including chromosomal segregation at mitosis. This gene is a critical gene involved in DNA replication and maintenance of genomic stability. In this study, we evaluated the clastogenic effects of fasinex on river buffalo lymphocytes in vitro. The frequency of chromosomal aberrations, the induction

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of sister chromatid exchanges and the measurement of micronucleus formation are the three cytogenetic parameters used in this study.

2. Materials and methods 2.1. Cytotoxicity test of the drug The clastogenic effect of the drug was tested at 25, 50 and 100 ␮g/ml. These concentrations were chosen on the basis of a cytotoxicity test [12,13], an established standard assay for the detection of chemical compound genotoxicity on mammalian cell cultures in vitro. According to the cytotoxicity test procedures, we prepared eight blood lymphocyte cultures. The first is considered as a control. The other seven cultures were each treated with a different concentration of the drug: 1, 10, 100, 200 and 500 ␮g/ml, and 1 and 2 mg/ml. The mitotic index frequency (the number of dividing cells in 2000 cells) was scored in each of the eight cultures. The concentration (100 ␮g/ml) that reduced the mitotoxic index to about 50% was used as the highest dose in the cytogenetic analysis. The drug doses of 0.5 and 0.25 ␮g/ml were taken as medium and low doses, respectively.

penicillin-streptomycin (Gibco), 1% l-glutamine (Gibco) and 4% phytohaemagglutinin (Gibco). After 44 h of incubation, cytochalasin B (Sigma) was added to the cultures at a final concentration 4 ␮g/ml [15]. Twenty-four hours culture initiation, fasinex (Novartis Co., Basle, Switzerland) was added to the cultures at the three final concentrations of 25, 50 and 100 ␮g/ml. 2.3. Chromosome preparation Colchicine (Sigma) at a concentration of 100 ␮g/ml was added 1.5 h before harvesting to the cultures for analysis of chromosomal aberrations and sister chromatid exchanges. At harvest, the cells were treated with a hypotonic solution (0.075 M KCl), for 20 min. The cells were fixed three times with methanol–acetic acid (3:1). The cells were spread onto cold slides dipped in 70% ethyl alcohol. The slides were air-dried. For the micronucleus test, the hypotonic treatment was performed with distilled water-medium (4:1) and 2% foetal calf serum for 10 min. The cells were fixed twice with methanol–acetic acid (3:1). The cells were spread onto cold slides dipped in 70% ethyl alcohol. The slides were air-dried.

2.2. Culture of blood lymphocytes

2.4. Slide staining

Blood samples were taken from five healthy animals, which had no treatment during the last 3 months. For measurement of chromosomal aberrations and sister chromatid exchanges, 1 ml of whole heparinised blood was cultured at 38.5 ◦ C for 72 h in 5 ml RPMI 1640 medium (Gibco) supplemented with 20% foetal calf serum (Gibco), 0.1% penicillin-streptomycin (Gibco), 1% l-glutamine (Gibco) and 4% phytohaemagglutinin (Gibco). The blood cultures for sister chromatid exchanges (SCEs) were treated with bromodeoxyuridine (BrdU, Sigma) at a final concentration of 10 ␮g/ml, 24 h after culture initiation [14]. For the micronucleus test, buffy coat was obtained by centrifugation of the blood at 1200 rpm for 20 min or by leaving it in vertical position till it is separated into layers. The buffy coat was cultured at 38.5 ◦ C for 72 h in 5 ml RPMI 1640 medium (Gibco) supplemented with 20% foetal calf serum (Gibco), 0.1%

For chromosomal aberrations, the slides were stained with 10% Giemsa stain solution (diluted with phosphate buffer, pH 6.8) for 30 min and washed twice in phosphate buffer. The slides were air-dried and examined under light microscope. For the micronucleus test, the slides were stained with 5% Giemsa stain solution (diluted with phosphate buffer, pH 6.8) for 6 min. For sister chromatid exchanges, the fluorescence– photolysis–giemsa technique was used [16]. The slides were stained with an aqueous solution of Hoechst 33258 (50 ␮g/ml, Sigma) for 20 min and then rinsed in distilled water. On a warmer tray at 50 ◦ C, the slides were layered with McIlvaine buffer and subjected to fluorescent black-blue light at a distance of 5 cm for 55 min. The slides were stained with 4% Giemsa, diluted with 1 part of Mcllvaine buffer and 4 parts of distilled water, for 5 min.

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2.5. Scoring In each animal at the different concentrations studied, 100 metaphases were examined for chromosomal aberrations, while SCE were scored from 30s division and 1000 cells were examined for measuring the micronucleus (MN) formation. A chromatid gap was defined as an achromatic or unstained constricted region on one chromatid, the size of which is equal to or smaller than the width of the chromatid, while a chromosome gap was scored as a gap present on both chromatids, either in the same position (isogap or isolocus) or at different positions along the chromosome length. A chromatid break is an achromatic region in one chromatid larger than the width of the chromatid. It may be either aligned or unaligned with the chromatid. The displacement of the broken chromatid fragment results in a terminal deletion, while a chromosome break is observed as breaks in both chromatids. A fragment was defined as a single chromatid without an evident centromer [17,18].

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was increased at a highly significant level P < 0.001; there was a non-significant increase in the total number of chromosomal gaps. At the intermediate dose (50 ␮g/ml), the mean frequency of aberrant cells was increased at a highly significant level P < 0.001. The mean values of chromatid breaks, gaps and polyploidy were 26.5, 10.5 and 4.6%, respectively. The total number of chromosomal aberrations was significantly increased (P < 0.001), while the total number of chromosomal gaps was also significantly increased (P < 0.01). At the highest drug concentration (100 ␮g/ml), the mean frequency of aberrant cells was 46.4 ± 2.87% compared to 15 ± 0.63% in the control. This increase was statistically significant (P < 0.001). Chromatid breaks and gaps, isochromatid breaks and gaps and polyploidy were strongly increased compared with the control. The total chromosomal aberrations and gaps were increased at a highly significant level P < 0.001. 3.2. Sister chromatid exchanges

3. Results

The effect of fasinex on the frequency of sister chromatid exchanges is shown in Table 2. The number of SCEs and the means of SCEs/cell were measured. The results show that this drug increased the frequency of sister chromatid exchanges at all three tested concentrations, with different levels of significance compared with the control, varying from P < 0.05 at low dose (where the mean of SCEs/cell was 8.17 ± 1.69), to P < 0.01 at medium dose (9.13±1.71) and P < 0.001 at high dose (11.07 ± 1.76).

3.1. Analysis of chromosomal aberrations

3.3. Micronucleus formation

The mean frequencies of chromosomal aberrations were recorded in blood cultures of the river buffalo treated with fasinex at three different concentrations (Table 1). The chromosomal aberrations involved chromatid breaks (breaks, fragments and deletions), isochromatid breaks, chromatid gaps, isochromatid gaps and polyploidy. In cultures treated with the low dose (25 ␮g/ml), the mean frequency of aberrant cells was increased at a significant level P < 0.001. The mean values of chromatid breaks, gaps and polyploidy were 17.8, 8.9 and 2.2% compared to 7.4, 6.0 and 0.6% in the control. The total number of chromosomal aberrations

The numbers and the means ± SD of binucleated cells, the binucleated cells with MN and the micronuclei were recorded at each drug concentration and in the control (Table 3). The micronuclei were scored in binucleated cells. The results show that the number of binucleated cells was decreased at the significant level P < 0.01 with low dose and decreased at a highly significant level P < 0.001 with both medium and high doses. The number of binucleated cells with MN as well as the number of micronuclei was highly significantly increased (P < 0.001) in cultures treated with all tested concentrations. The only exception was the number of binucleated cells with MN in cultures

2.6. Statistical analysis The chi-square test (2 × 2 contingency table) was used for the chromosomal aberrations data analysis, while the t-test was used to evaluate the sister chromatid exchanges and micronucleus formation induced by the drug.

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Drug concentration

No. of animals

No. of scored cells

Aberrant cells % (mean ± S.D.)

Control 25 ␮g/ml 50 ␮g/ml 100 ␮g/ml

5 5 5 5

500 500 500 500

15 29 38.6 46.4

∗∗

P < 0.01. P < 0.001.

∗∗∗

± ± ± ±

0.63 2.10∗∗∗ 2.58∗∗∗ 2.87∗∗∗

Aberrations/100 cells Chromatid break

Isochromatid break

Chromatid gap

Isochromatid gap

Polyploid

7.4 17.8 26.5 34.4

0.4 1.2 2.9 6.4

6.0 8.9 10.5 13.1

0.6 0.9 1.1 1.5

0.6 2.2 4.6 9.8

Total aberrations % excluding gaps mean ± S.D. 8.4 21.2 34.0 50.6

± ± ± ±

0.49 1.58∗∗∗ 3.02∗∗∗ 3.36∗∗∗

Total gaps % mean ± S.D.

6.6 9.8 11.6 14.6

± ± ± ±

1.02 0.76 1.08∗∗ 1.36∗∗∗

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Table 1 Frequency of chromosomal aberrations induced by fasinex in lymphocyte cultures of the river buffalo

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Table 2 Effect of fasinex on sister chromatid exchanges in lymphocyte cultures of the river buffalo Drug concentration

Control 25 ␮g/ml 50 ␮g/ml 100 ␮g/ml

No. of animals

5 5 5 5

No. of examined cells

No. of SCEs

150 150 150 150

690 1225 1370 1660

SCEs/cell Range

Mean ± SD

3–8 6–11 6–13 8–15

6.2 ± 1.11 8.17 ± 1.69∗ 9.13 ± 1.71∗∗ 11.07 ± 1.76∗∗∗

∗

P < 0.05. P < 0.01. ∗∗∗ P < 0.001. ∗∗

Table 3 Effect of fasinex on frequencies of binucleated cells and micronuclei (MN) Drug concentration

No. of animals

No. of examined cells

Binucleated cells No.

Mean ± SD

No.

Mean ± SD

No.

Mean ± SD

Control 25 ␮g/ml 50 ␮g/ml 100 ␮g/ml

5 5 5 5

5000 5000 5000 5000

517 405 371 349

103.4 ± 11.29 81 ± 7.69∗∗ 74.2 ± 3.54∗∗∗ 69.8 ± 5.91∗∗∗

56 81 116 129

11.2 16.2 23.2 25.8

± ± ± ±

62 99 171 209

12.4 19.8 34.2 41.8

∗∗

Binucleated cells with MN

1.17 2.32∗∗ 2.79∗∗∗ 4.02∗∗∗

Micronuclei (MN)

± ± ± ±

1.50 2.04∗∗∗ 3.54∗∗∗ 2.93∗∗∗

P < 0.01. P < 0.001.

∗∗∗

treated with the low dose, where the level of significance was P < 0.01.

4. Discussion Fascioliasis due to F. gigantica and F. hepatica is the most prevalent disease in farm animals. In Egypt, the treatment records from veterinary hospitals revealed that the use of fasciolicide drugs is much higher in buffalo than any other animal. This may be due to the higher prevalence of the infection in the buffalo, and the greater attention paid by farmers to this species [19]. Fasinex has been reported to be an active fasciolocidal agent used in humans and in farm animals [2,3,5–9].The clastogenic effects of this drug were tested in lymphocyte cultures of the river buffalo at three final concentrations, 25, 50 and 100 ␮g/ml. The tested doses were chosen according to the cytotoxicity test [12,13]. The results of present study demonstrate that the numbers of cells with different types of chromosomal aberrations were increased significantly in cultures

treated with fasinex compared with the control. Chromatid breaks and gaps and isochromatid breaks and gaps were the structural chromosomal aberrations induced by the three different concentrations of the drug. The mean value of aberrant cells showed increases at low dose (29±2.1%), medium dose (38.6±2.58%) and high dose (46.4 ± 2.87%) compared with the control (15 ± 0.63%). These increases were dose-dependent where there was a positive correlation between increased drug concentration and induction of chromosomal aberrations. The clastogenic effect of the drug was illustrated by the increases in the total chromosomal aberrations and gaps at low dose (21.2 ± 1.58% and 9.8 ± 0.76%), medium dose (34.0 ± 3.02% and 11.6 ± 1.08%) and high dose (50.6 ± 3.36% and 14.6 ± 1.36%) compared with the control (8.4 ± 0.49% and 6.6 ± 1.02%). The high level of chromosomal aberrations resulting from exposure to the tested drug at the three different concentrations may be due to the fact that this drug is a benzimidazole derivative, which acts as an inhibitor for topoisomerase II [9]. The eukaryotic topoisomerase II gene is involved in essential cellular

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processes including chromosomal segregation at mitosis [11]. This enzyme can pass an intact DNA helix through a transient double-strand break that it generates in a separate strand. Topoisomerase II inhibitors can stabilise the cleavage complex, resulting in an increase in double-strand breaks that ultimately results in cell death [20]. The other two cytogenetic parameters studied in this work were sister chromatid exchange and micronuclei formation. Sister chromatid exchanges have been used as sensitive markers for assessing chromosome instability to determine the clastogenic effect of chemical compounds [21]. The frequencies of SCEs in cultures treated with the three tested concentrations were 8.1 ± 1.6 (low dose); 9.1 ± 1.7 (medium dose) and 11.07±1.7 (high dose) compared with 6.2±1.1 in the control. The significant level observed in our results is indicative of genomic instability and deranged DNA repair, which may account for segregation of certain genes [22]. The micronucleus test has been adopted for use in human cells to evaluate the clastogenic effects of drugs before they are commercialised [23]. Our results show that the numbers of binucleated cells were decreased to 81±7.6 at low dose, 74.2±3.5 at medium dose and 69.8 ± 5.9 at high dose, compared with 103.4 ± 4.2 in the control. This result revealed that the drug has a cytotoxic effect on the number of cell divisions. On the other hand, the numbers of binucleated cells with MN and the frequencies of micronuclei were increased significantly compared with the control (Table 3). As the micronuclei are small chromatin-containing bodies arising from chromosome fragmentation by breaks or deletion, the results of MN formation confirmed our results of chromosome aberrations indicating the clastogenic effects of fasinex. The effects of the drug fasinex reported in the present study agreed with the results of previous reports about the clastogenic and mutagenic effects of benzimidazole derivatives. The ability of benzimidazole derivatives to induce structural chromosomal aberrations and to increase the frequencies of sister chromatid exchanges as well as micronucleus formation in human peripheral lymphocytes in vitro was reported [10,24]. The cytogenetic effects of benzimidazole and its derivatives on the bone marrow of the mouse and the Chinese hamster have also been studied [25–27]. These results showed increased mi-

cronuleus formation, structural chromosomal changes and some aneugenic effects. In conclusion, the three cytogenetic parameters used to evaluate the effect of fasinex revealed that the drug has a strong clastogenic effect on river buffalo lymphocytes in vitro.

References [1] P.P. Laird, J.C. Boray, Human fascioliasis successfully treated with triclabendazole, Aust. N. Z. J. Med. 22 (1) (1992) 45–47. [2] N.A. Hammouda, S.T. El-Mansoury, M.Z. El-Azzouni, Y. El-Gohari, Therapeutic effect of triclabendazole in patients with fascioliasis in Egypt. A preliminary study, J. Egypt Soc. Parasitol. 25 (1) (1995) 137–143. [3] H. El-Karaksy, B. Hassanein, S. Okasha, B. Behairy, I. Gadallah, Human fascioliasis in Egyptian children: successful treatment with triclabendazole, Trop. Pediatr. 45 (3) (1999) 135–138. [4] W. Apt, X. Aguilera, F. Vega, C. Miranda, I. Zulantay, C. Perez, M. Gabor, P. Apt, Treatment of human chronic fascioliasis with triclabendazole: drug efficacy and serologic response, Am. J. Trop. Med. Hyg. 52 (6) (1995) 532–535. [5] L. Maes, O. Vanparijs, H. Lauwers, W. Deckers, Comparative efficacy of closantel and triclabendazole against Fasciola hepatica in experimentally infected sheep, Vet. Rec. 127 (1990) 450–452. [6] K. Wolff, J. Eckert, G. Schneiter, H. Lutz, Efficacy of triclabendazole against Fasciola hepatica in sheep and goats, Vet. Parasitol. 13 (2) (1983) 145–150. [7] D.G. Stansfield, B. Lonsdale, P.A. Lowndes, E.W. Reeves, D.M. Schofield, Field trials of triclabendazole against mixed age infections of Fasciola hepatica in sheep and cattle, Vet. Rec. 120 (19) (1987) 459–460. [8] M.H. El-Sayed, Comparative studies on the effect of bithionol, praziqantel and triclabendazole in rabbit’s fascioliasis. Part 1. Parasitological studies, J. Egypt Soc. Parasitol. 27 (1) (1997) 131–142. [9] S.N. Mahato, L.J.S. Harrison, J.A. Hammond, Efficacy of triclabendazole against Fasciola gigantica in buffaloes in eastern Nepal, Vet. Rec. 135 (1994) 579–580. [10] P. Van Hummelen, A. Elhajouji, M. Kirsch-Volders, Clastogenic and aneugenic effects of three benzimidazole derivatives in the in vitro micronucleus test using human lymphocytes, Mutagenesis 10 (1) (1995) 23–29. [11] T. Berend, A.H. John, H. Daniel, Transcriptional regulation of topoisomerase II ␣ at confluence and pharmacological modulation of expression by bis-benzimidazole drugs, Mol. Pharm. 59 (4) (2001) 699–706. [12] B.J. Dean, N. Danford, Assays for the detection of chemicallyinduced chromosome damage in cultured mammalian cells, in: S. Venitt, J.M. Parry, D. Rickwood, B.D. Hames (Eds.), Mutagenicity Testing: A Practical Approach, Oxford, Washington, DC, 1994, pp. 187–232.

S. Ahmed, O.E. Othman / Mutation Research 541 (2003) 115–121 [13] R. Julian Preston, J.R. San Sebastian, A.F. McFee, The in vitro human lymphocyte assay for assessing the clastogenicity of chemical agents, Mutat. Res. 189 (1987) 175–183. [14] L. Iannuzzi, G.P. Perucatti, G.P. Di Meo, L. Ferrara, Sister chromatid exchange in chromosomes of river buffalo (Bubalus bubalis L.), Caryologia 41 (1988) 237–244. [15] A. Sahar, E. Hala, Measurement of micronuclei by cytokinesis-block method in buffalo lymphocytes, Cytologia 65 (2000) 319–324. [16] S.A. Latt, S.E. Allen, A.B. Loom, E. Carrano, D. Falke, E. Kram, R. Schneider, R. Schreck, B. Tice, B. White Field, S. Wolff, Sister chromatid exchanges; a report of the gene-tox program, Mut. Res. 87 (1981) 17–62. [17] D. Brusick, Fundamental of Genetic Toxicology, Plenum, New York, 1980, pp. 33–34. [18] A.V. Carrano, A.I. Natarajan, Consideration for population monitoring using cytogenetic techniques, Mutat. Res. 204 (1988) 394–406. [19] H. A. Shalaby, Some epidemiological and serological studies on Fascioliasis, M.V.SC., Department of Parasitology, Faculty of Veterinary Medicine, Cairo University, Cairo, 1998. [20] D.A. Burden, N. Osheroff, Mechanism of action of eukaryotic topoisomerase II and drug targeted to the enzyme, Biochim. Biophys. Acta 1400 (1998) 139–154. [21] B.E. Anderson, E. Zeiger, M.D. Shelby, M.A. Resnick, D.K. Gulati, J.L. Ivett, K.S. Loveday, Chromosome aberration and

[22]

[23]

[24]

[25]

[26]

[27]

121

sister chromatid exchange test results with 42 chemicals, Environ. Mol. Mutag. 16 (1990) 55–137. P.G. Mc Kinnon, A taxia-te-Longiectasia an inherited disorder of ionizing radiation sensitivity in man, Hum. Genet. 75 (1987) 197–208. J.A. Heddle, M.C. Cimino, M. Hayashi, F. Romagna, M.D. Shelby, J.D. Tucker, P.H. Vanparys, J.T. Mac Gregor, Micronuclei as an index of cytogenetic damage: past, Environ. Mol. Mutag. 18 (1991) 277–284. N. Banduln, G. Obe, Mutagenicity of methyl 2-benzimidazole carbamate, diethylstilbestrol and estradiol: structural chromosomal aberrations, sister chromatid exchanges, C-mitoses, polyploidies and micronuclei, Mut. Res. 156 (3) (1985) 199–218. J.P. Seiler, The mutagenicity of benzimidazole and benzimidazole derivatives. Part VI. Cytogenetic effects of benzimidazole derivatives in the bone marrow of the mouse and the Chinese hamster, Mut. Res. 40 (4) (1976) 339– 347. A.M. Lynch, J.M. Parry, The cytochalasin-B-micronucleus/ kinetochore assay in vitro studies with 10 suspected aneugens, Mut. Res. 287 (1) (1993) 71–86. R. Barale, C. Scapoli, C. Meli, D. Casini, M. Minunni, A. Marrazzini, N. Loprieno, I. Barrai, Cytogenetic effects of benzimidazoles in mouse bone marrow, Mut. Res. 300 (1) (1993) 15–28.