effects of spiramycin on chromosome aberration and

C 2001 The Japan Mendel Society Cytologia 66: 395-401, 2001

Effects of Spiramycin on Chromosome Aberration and

Micronucleus Formation in Rat Bone Marrow Cells

Hasan Basri hal and Mehmet Topaktas21 Cukurova University

, Institute of Natural and Applied Science, 01330-Adana-Turkey2 Cukurova University

, Faculty of Arts and Sciences, Department of Biology, 01330-Adana-Turkey

Accepted October 10, 2001

Summary The aim of this study was to investigate potential clastogenicity/genotoxicity of the an-tibiotic spiramycin in bone marrow cells of rats (Rattus norvegicus var. albinos). In the study, spi-ramycin induced the chromosome aberations at all concentrations (100, 200, 400 mg/kg bwt. (body weight)/d oral) in 21 d when compared with the solvent control group (sunflower seed oil). In addi-tion, spiramycin induced the chromosomal aberration (CA) at only 200 mg/kg bwt. intraperitoneal

(i.p.) in 12 h and 100 and 400 mg/kg bwt. i.p. in 24 h when compared with the control group. Spi-ramycin induced the micronucleated polichromatic erythrocytes (MNPCE) only at 200 mg/kg bwt. i.p. in 12 h and 100 mg/kg bwt. i.p. in 24 h. However, there was no significant difference in the for-mation of micronuclei at the other concentrations. Key words Antibiotic, Spiramycin, Chromosome aberration, Micronucleus, Rat.

Spiramycin, is a macrolide antibiotic, isolated from Streptomyces ambiofaciens which was found in soil near Peronne (France) by Pinnert-Sindico at 1954 (Kernbaum 1982). Spiramycin is ac-tive against almost all Streptococci (not belonging to the group D), Meningococci, Bordotella per-tussis, Actinomyces israelii, Corinobacteria, Bacteroides melaninogenicus, Legionella pneumophil-ia and Mycoplasma. In addition, it is active against most Gonococci, Listeria and Clostridia (Kern-baum 1982). Spiramycin is useful when toxoplasmosis occurs during pregnancy (Kernbaum 1982). Spiramycin, at a daily dose of 2 to 3 g (30 to 50 mg/kg bwt.) in courses of 3 to 4 weeks separated by 2 weeks intervals decreased the frequency certain and probable fetal abnormalities that occurred by toxoplasmosis in pregnant women (according to Desmonts and Courvereur 1974 based on Kern-baum 1982). In addition, Kernbaum (1982) suggested that spiramycin is probably one of the most effective antibiotic against Chlamidia. tla and Topakta§ (1999) reported that spiramycin (100 mg/kg bwt./d) induced the CA in bone marrow cells of rats when oral treatment for 7 d. However, acetyl-spiramycin which is a derivative of spiramycin was not mutagenic for Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 strains (Guo and Yuan 1985). Erythromycin, is a macrolide antibiotic induced the CA in human lymphocytes (Agiiloglu and Ortakaya 1992). A new macrolide antibiotic, miporamicin has no effect on the induction of gene mutation, but has a weak clastogenicity which is detectable only by in vitro test (Sono et al. 1989). The other macrolide an-tibiotic, miokamycin was not mutagenic against S. typhimurium strains TA98, TA100, TA1535 and

TA1538 both with and without S9 mix, and also did not induce the dominant lethal mutations in mice (Hirano and Takeda 1981).

It was reported that many groups of antibiotic induced the CAs, SCEs and micronuclei in both in vitro and in vivo test systems, and also have a mutagenic effect against various strains of S. ty-

phimurium (Neu et al. 1965, Rasko et al. 1979, Meiying et al. 1982, Mavournin et al. 1990, Miller 1991, Rao et al. 1991, Risley and Pohorenec 1991, Sbrana et al. 1991, Aboul-Ela 1995, Holmalahti

et al. 1996). A macrolide antibiotic spiramycin, which is used as the test substance in this study, has fre-

quently been recommended in curing various infections. However, the genotoxic effects of spi-

396 Hasan Basri Ila and Mehmet Topakta§ Cytologia 66

ramycin was not investigated within the rec-ommended curing time (21 d). For this reason, the aim of this study was to investigate effects of the antibiotic spiramycin on chromosome aberrations and micronuclei formation in bone marrow cells of rats (Rattus norvegicus var. al-binos).

Material and methods

Albino rats (Rattus narvegicus var. albi-nos, 2n =42) were used as the test animals in this study. Six rats (3 females, 3 males, 12-16 weeks old) were used for each treatment and control group. Spiramycin was used as the testsubstance (Fig. 1) (Spiramycin is an effective ingredient of antibiotic Rovamycine). The sunflower

seed oil and dimethy sulfoxide (DMSO) were used as the solvent of spiramycin. Spiramycin was

obtained from Eczacibai Rhone Poulenc Firm (Turkey). Test animals were procured from cukuro-

va University Medical Experimental Research Centre (TIBDAM).

Administering the test substance on animals and preparing slides from bone marrow

Dose-levels of spiramycin were determined such as therapeutic regimes. 3 different concentra-

tions of spiramycin (100, 200, 400 mg/kg) were applied to the rats in treatment groups as 2 ways.

First, sub-chronic doese of spiramycin were given to rats per oral as daily dosage in their food for

21 d, and second, it was applied intraperitoneal for 12 and 24 h in the acute experiment. The solvent

control animals were treated with solvent substances (sunflower seed oil, DMSO) as the same

amount of the treated groups.

Slide preparation for investgating the CA

First of all, spiramycin was dissolved in sunflower seed oil, and then, 3 different concentrations

(100, 200, 400 mg/kg) were given to rats as daily dosage in their food for long term (21 d) treat-

ment. For short term treatment (12, 24 h), antibiotic was dissolved in DMSO and injected to rats in-

traperitoneal. In order to arrest mitosis, colchicine (3 mg/kg bwt.) was injected intraperitoneal 2 h

before the animals were sacrificed by cervical dislocation. Later on, both femurs were stripped from

muscles. One of the femurs was used for CA investigation, another one was used to investigate

MNPCE. The bone marrow was aspirated in 4 ml of 0.9% NaC1 (37•Ž). Next, the suspension was

centrifuged for 15 min at 1100 rpm, and the bone marrow pellet was resuspended in hypotonic solu-

tion (0.4% KC1). After that, cells were incubated in this solution at 37•Ž for 35 min. Bone marrow

cells suspension was then centrifuged (15 min, 1100 rpm), and the cells were resuspended in cold

fixative (1 : 3 glacial acetic acid : methanol). The cells were fixed for 20 min at the room temperature

then centrifuged. This process was repeated 2 times with the above fixative. After wards, cells were

spread on cold glass slides and air dried. Finally one-day-old slides were stained with Giemsa (5%

in Sorensen buffer) and mounted with Entellan.

Slide preparation for investigating the MNPCE

The bone marrow in 1 femur was used for this preparation. Bone marrow was aspirated in 2 ml

foetal bovine (37•Ž). Next, homogeneous bone marrow solution was spread on dried glass slides

and dried at the room temperature for about 4-6 h. Finally, slides were stained with May-Gruen-

wald/Giemsa (according to Schmid's) (Schmid 1975) method.

CAS registry No: 8025-81-8

Sigma No: S-9132

Fig. I. The formula of spiramycin.

2001 Effects of Spiramycin on Chromosome Aberration in Rat Bone Marrow Cells 397

Slides investigations One hundred well spread metaphases per animal were examined at X1000 magnification for

the occurrence of different types of chromosomal aberration (CA). Mitotic index (MI) was also de-termined by scoring a total of 3000 cells from each animal. The number of abnormal cells per ani-mal was determined, and the mean frequency of abnormal cells was calculated for each group. The number of CA per cell was also calculated. One thousand PCE were observed to investigate mi-cronucleus formation. Furthermore, polychromatic erythrocytes/normochromatic erythrocytes

(PCEs/NCEs) ratio was determined in 1000 cells.

Statistical significance The main percentage of the abnormal cells, CA/cell and MI in treated group were compared

with the obtained results from control groups. The significance between abnormal cells, CA/cell and MIs of treated groups and control groups was compared by t-test.

The significance of obtained result from micronucleated PCE and PCE/NCE ratio was analysed by anova. The t-test was also used if there was a significant difference between the groups.

Results

The mean percentage of abnormal cell, CA/cell and MI in rats bone marrow cells administered with spiramycin for 21 d were compared with control groups (Table 1).

The mean percentage of abnormal cell and CA/cell were significantly increased when com-

pared with solvent control group (sunflower seed oil). In addition, MI was significantly decreased compared to the solvent control group (Table 1).

Various chromosome abnormalities were formed by spiramycin (Fig. 2a, b). In the short time treatment, the percentage of cell with CA increased at 200 mg/kg bwt. in 12 h

and 100 and 400 mg/kg bwt. dose in 24 h compared to the control group (Table 2). CA/cell was sig-nificant at only one concentration (200 mg/kg bwt. in 12 h) of spiramycin, but other concentrations were not significant (Table 2). Generally, MI significantly decreased compared to the control group

(Table 2). The means of micronucleated polychromatic erythrocytes (MNPCE) and the ratios of

PCE/NCE were shown in Table 3. The micronucleated PCE (MNPCE) (Fig. 2c) only increased at 200 mg/kg bwt. in 12 h and 100 mg/kg bwt. in 24 h compared to the control group. At the same con-centrations and treatment times, the mean of MNPCE of the treatment group was found higher than the mean of MNPCE of the solvent control however, there was no statistical significance (Table 3). CA was also induced at the same concentration. These results show that there is a relation between

Table 1. The mean percentage of abnormal cell, CA/cell and MI* in bone marrow cells of rats

administered with spiramycin for 21 d

* 600 cells were calculated for each group. **B', Chromatid break; B", chromosome break; F, fragment; SU, sister

union; DC, dicentric chromosome; P, polyploid cell; T, translocation; CE, chromatid exchange. a, Significant from control

group; b, significant from solvent control group (sunflower seed oil). 1, p•…0.05; 2, p•…0.01; 3, p•…0.005; 4, p•…0.001.

398 Hasan Basri I la and Mehmet Topakta§ Cytologia 66

Fig. 2. Dicentric chromosome (a) (400 mg/kg/d, 21 d) x2000, chromatid break (b) (200 mg/kg, 24 h) X 1500, micronucleated polichromatic erythrocytes (c) (200 mg/kg, 12 h) X2000.

Table 2. The mean percentage of abnormal cell, CA/cell and MI* in bone marrow cells of

rats administered with spiramycin for 12 and 24 h

* 600 cells were calculated for each group . ** B', Chromatid break; B", chromosome break; F, fragment; SU, sister

union; DC, dicentric chromosome; P, polyploid cell; T, translocation; CE, chromatid exchange; inv., invertion; R, ring chro-

mosome. a, Significant from control group; b, significant from solvent control group (DMSO). 1, p5_0.05; 2, p_0.01; 3,

p•…0.005; 4, p•…0.001.


CA and MN formation.

Discussion

In the present study, CA in the bone marrow cells of rats was induced compared to the solvent control group at 21 d oral (recommended curing time of spiramycin), but the difference compared to the control group was not significant. On the other hand, results from 200 mg/kg bwt., 12 h and 100 and 400 mg/kg bwt., 24 h experiments were significant but other doses (100 mg/kg bwt., 400 mg/kg bwt. at 12 h and 200 mg/kg bwt. at 24 h per i.p.) were not significant. However, CA was induced at the recommended curing dose of spiramycin (Protein biosynthesis inhibitor) (100 mg/kg bwt./d) for 7 d (ila and Topakta§ 1999). For example, acetyl-spiramycin which is a derivative of spiramycin was not mutagenic for Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 strains (Guo and Yuan 1985). Erythromycin, is a macrolide antibiotic induced the CA in human lymphocytes (Aguloglu and Ortakaya 1992). A new macrolide antibiotic, miporamicin has no effect on the induction of gene mutation, but has a weak clastogenicity which is detectable only by in vitro test (Sono et al. 1989). The other macrolide antibiotic, miokamycin was not mutagenic against S. typhimurium strains TA98, TA100, TA1535 and TA1538 both with and without S9 mix, and also did not induce the dominant lethal mutations in mice (Hirano and Takeda 1981). As shown, protein synthesis inhibiting antibiotics including macrolide have weak genotoxic effect. Various researchers have found different results by macrolide group antibiotics including spiramycin. Furthermore some of protein synthesis inhibitor antibiotics except macrolide group also have shown different results. For example, antibiotic streptomycin (Protein synthesis inhibitor) has caused meiotic abnormalities in barley (Bilge et al. 1977) and Lathyrus sativus (Kumar and Sinha 1991). Griseofulvin (Protein synthesis inhibitor) has caused various meiotic abnormalities in female mouse germ cells (Marchet-ti et al. 1992). Tiveron et al. (1992) have determined that griseofulvin induced meiotic arrest and aneuploid cells in mouse. Oxytetracycline (Protein synthesis inhibitor) was not mutagenic for Sal-monella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 strains (Guo and Yuan 1985). Same researchers found that gentamycin (Protein synthesis inhibitor) was also not mutagenic for Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 strains. Chen et al. (1996) have researched for 1555 A-to-G substitution in the 12S mitochondrial ribosomal RNA gene by

gentamicin. None of the subjects (10 subjects) demonstrated this specific mutation. Hartman et al. (1995) reported that tetracycline (Protein synthesis inhibitor) inhibited the DNA synthesis and caused to unscheduled DNA synthesis in fibroblast of Wilson Disease patients. Cycloheximide

Table 3. Mean percentages of MNPCE and ratios of PCE/NCE in rat bone marrow cells after

12 and 24 h treatment with spiramycin

* 6000 PCE was counted for each group . **MNPCE, mikronucleated polichromatic erythrocyte; ***NCE, nor-

mochromatic erythrocyte. a, Significant from control group; b, significant from solvent control group (DMSO). 1, p•…0.05;

2,p•…0.01; 3, p•…0.005; 4, p•…_0.001.

400 Hasan Basri I la and Mehmet Topakta§ Cytologia 66

(Protein synthesis inhibitor) has not induced frameshift mutation and point mutation without meta-bolic activation in Salmonella typhimurium, but it has been observed that cycloheximide has caused various genetic damage in Allium cepa root tip cells and mouse bone marrow cells (Basic-Zani-novic et al. 1991). Wilmer and Bloom (1991) found that cyclophosphamide (Protein synthesis in-hibitor) has increased SCE in B lymphocytes of chicken embryos about 10.4 fold compared with control. As shown above, some antibiotics have genotoxic effect some of them have not. Each an-tibiotic have separately investigated to decide their genotoxicity.

Antibiotic spiramycin has not increased MNPCE dose-dependent relation in rat bone marrow cells. However, 200 mg/kg bwt., 12 h and 100 mg/kg bwt., 24 h dosages increased MNPCE in rat bone marrow cells compared to the control group. Many researchers have investigated whether an-tibiotics have induced the formation of MNPCE. They have found that some antibiotics have in-creased MNPCE while some others have not. For example, tetracycline (Protein synthesis inhibitor) caused to formation of micronuclei in mouse bone marrow cells (Sylianco et al. 1987, Villasenor

1995), chloramphenicol in rat bone marrow cells (Martelli et al. 1991), aureofungin in mouse bone marrow cells (Mujumdar et al. 1994) have not induced MN formation. As shown above, some an-tibiotics have induced MN formaton although some others have not. According to Tables 2 and 3, we can state that there is a correlation between CA and MN formation in PCE (200 mg/kg bwt., 12 h, 100 mg/kg bwt., 24 h dosages). As consequence, it can be stated that CA inducer substances can also induce the MN formation. In addition, PCE/NCE ratio has not changed at short term (12, 24 h) treatment by spiramycin.

MI has decreased at the long term (21 d) treatment compared to the solvent control group in this study (Table 1). MI has decreased 7 d treatment and sacrificed after 12 h the last treatment com-

pared to the solvent control (Ila and Topakta5 1999). This result is similar with our present study. However, MI was not influenced compared to the solvent control group in short term (12, 24 h). Gentamicin has decreased MI dose and time dependent manner (Agilloglu and Ortakaya 1994). This difference arise from different application of test substances. Because we applied in vivo route whereas gentamicin was applied in vitro. In addition, gentamicin was bound 30S subunit of bacteria ribosomes however, spiramycin was bound 50S subunit of bacteria ribosomes.

Finally, genotoxicity of spiramycin must be investigated another genotoxicity tests too.

Acknowledgements

This study was supported by c.U. Research Fund, FBE.99.D2.

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effects of spiramycin on chromosome aberration and

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