Food and Chemical Toxicology 48 (2010) 789797

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Pnar Goc Rasgele *, Fisun Kaymak

Trakya University, Faculty of Sciences and Arts, Department of Biology, 22030 Edirne, Turkey

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 19 August 2009 Accepted 11 December 2009

1. Introduction

In recent years, the widespread use of food additives has improved due to developing industry, increasing population and consumption of food. So it is essential to nd new food sources and preserve them for a long time without molding. Different methods were developed and many chemical preservatives were used for this purpose. It was reported that certain food additives, especially antimicrobial agents are genotoxic in different test systems (Njagi and Gopalan, 1982; Luca et al., 1987; Akn and Smer, 1991; ullar et al., 2001). But, there are still many food additives Renczog whose genotoxic effects are unknown. Delvocid is a food additive of which natamycin is the active substance and is used to inhibit yeast and fungi growth on cheese and sausages (EMEA, 1998). It is produced from Streptomyces natalensis.

Abbreviations: CA, chromosome aberration; CHO, Chinese hamster over; MI, mitotic index; MMC, mitomycin C; MN, micronucleus; MNNCE, micronucleated normochromatic erythrocyte; MNPCE, micronucleated polychromatic erythrocyte; NCE, normochromatic erythrocyte; PCE, polychromatic erythrocyte; RI, replication index; SCE, sister chromatid exchange; SE, standard error. * Corresponding author. Tel.: +90 284 235 28 25; fax: +90 284 235 40 10. E-mail addresses: pinargoc@hotmail.com (P.G. Rasgele), kaymakf@trakya.edu.tr (F. Kaymak). 0278-6915/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.12.007

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Keywords: Delvocid Mice bone marrow Chromosome aberration Micronucleus Sperm Testosterone Food additive

Delvocid is used as preservative in foods. The genotoxic effects of the food preservative Delvocid were evaluated using chromosome aberrations and micronucleus test in bone marrow cells and sperm head abnormality assays in mice. Blood samples were taken from mice and levels of total testosterone in serum were also determined. Delvocid was intraperitoneally (ip) injected at 200, 400 and 800 mg/kg. Delvocid did not induce chromosome aberrations but signicantly increased the number of micronucleated polychromatic erythrocytes in bone marrow and sperm head abnormalities at all concentrations and treatment periods. It also decreased MI at all concentrations for 6, 12 and 24 h treatment periods. Delvocid decreased PCE/NCE ratio at all concentrations for 48 h in female mice, for 24 and 48 h treatment periods in male mice. At the 800 mg/kg concentration, Delvocid decreased PCE/NCE ratio for 24 and 72 h in female mice. A dose dependent increase was observed in the percentage of sperm head abnormalities. The levels of serum testosterone decreased dose-dependently. The obtained results indicate that Delvocid is not clastogenic, but it is aneugenic in mice bone marrow and it is a potential germ cell mutagen in sperm cells. 2009 Elsevier Ltd. All rights reserved.

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Chromosome aberrations, micronucleus and sperm head abnormalities in mice treated with Delvocid, a food preservative

Delvocid did not induce reverse mutation in Salmonella typhimurium TA1535, TA1538, TA98 and TA100 strains and in Escherichia coli WP2uvrA and WP2 strains, and was not mutagenic in Bacillus subtilis (WHO, 2006). Cox et al. (1973) reported that natamycin is not clastogenic in male and female mice, Levinskas et al. (1966) suggested that fertility, gestation, lactation and viability indices did not differ in male and female rats receiving diets containing natamycin (WHO, 2006). Natamycin did not induce allergic reactions in 111 patients (Grupper, 1964) and 73 workers (Malten, 1967). Natamycin was found to be a non-mutagenic in Salmonella/ mammalian microsome mutation assay in S. typhimurium strains, in a mouse lymphoma mutation assay, in chromosome aberration assay with CHO cells and it is not effective on the reproductive performance in rats, and it has low acute toxicity in dogs and rabbits (EMEA, 1998). In these studies, natamycin is not mutagenic. Delvocid is a commercial form of natamycin. There is no study available on the genotoxic effects of natamycin. But the chemicals that are used as additives may disrupt the properties of substances. So, Delvocid may have different peculiarities from natamycin (Holland et al., 2002). No report is found about the genotoxic effect of Delvocid in mice. In the present study, it is aimed to investigate genotoxic effects of Delvocid by chromosome aberration, micronucleus and sperm head abnormality assays in mice.

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P.G. Rasgele, F. Kaymak / Food and Chemical Toxicology 48 (2010) 789797 2.3. Chromosome aberration assay Delvocid was dissolved in distilled water and was injected intraperitoneally to female and male mice (1012 weeks) in 6, 12 and 24 h periods. Bone marrow chromosomes were prepared according to the method of Preston et al. (1987). In order to arrest cells at metaphase, colchicine (0.01%) was injected intraperitoneally 3 h before cervical dislocation. Then, the bone marrow from a femur was ushed out in 1% sodium citrate, the suspension was centrifuged for 5 min at 1000 rpm. The cells were incubated at 37 C for 25 min with hypotonic solution (1% sodium citrate) and xed with xative (1:3 acetic acid:methanol) three times. The cells were spread on glass slides and left to dry. The slides were stained with 10% Giemsa in Srensen buffer for 10 min. One hundred well-spread metaphase were examined for each concentration and treatment period. Chromosomal aberrations were investigated at 1000 magnication. For MI, 3000 cells were scored from each animal. The gaps were not evaluated as chromosomal aberrations according to Mace et al. (1978).

Fig. 1. Chemical structure of Delvocid.

2. Materials and methods 2.1. Test chemicals Delvocid (CAS No. 7681-93-8; 50% natamycin, 50% lactose) (Fig. 1) whose effective substance is Natamycin was used as a test material. Giemsa (Cat. No. 109204, CAS No. 51811-82-6) and May Grunwald (Cat. No. 101424) were obtained from Merck. Fetal calf serum (Cat. No. N4762) and mitomycin C (Cat. No. M0503, CAS No. 50-07-7) were purchased from Sigma Aldrich. MMC was used as positive control, distilled water was used as negative control. 2.2. Animals and dose Male and female mice (Mus musculus) (812 weeks of age, with average body weight of 2025 g), were purchased from Trakya University Scientic Research Center. The animals were maintained in closely inbred colony under conventional laboratory conditions at a room temperature of 25 5 C and in 12 h dark and 12 h light cycles. Food pellets and water were provided ad libitum. Five groups were prepared for the chromosome aberration assay (ve animals each), micronucleus assay (ve animals each) and sperm head abnormality assay (three animals each). Three of these were experiment groups. One of these was the positive, and the other one was the negative control group. According to van Eeken and Wubs (1976), the LD50 of Delvocid (intraperitoneal) was found to be 1600 mg/kg bw (WHO, 2006). In the study, mice were injected with 200, 400 and 800 mg/kg bw (1/8, 1/4, 1/2 LD50, respectively) concentrations of Delvocid intraperitoneally.

Fig. 2. (a) 1 MNPCE observed after 800 mg/kg Delvocid treatment for 24 h (1000). (b) 2 MNPCE observed after 800 mg/kg Delvocid treatment for 24 h (1000).

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Fig. 3. (a) 1 MNNCE (800 mg/kg, 48 h); (b) 2 MNNCE (800 mg/kg, 24 h) (1000).

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2.4. Micronucleus assay 2.5. Sperm head abnormality assay

Female and male mice (810 weeks) were treated with the same concentrations intraperitoneally for 24, 48 and 72 h. Bone marrow smears were done according to the methods of Schmid (1975) and Aaron et al. (1989) with minor modications. The bone marrow cells were ushed out with fetal calf serum, and the suspension was centrifuged for 10 min at 2000 rpm. The pellets were spread on a slide glass and xed with methanol. The slides were stained with May Grunwald for 3 min, May Grunwald:distilled water (1:1) for 2 min, 10% Giemsa in Srensen buffer for 10 min. A total of 1000 erythrocytes were scored for each animal at a magnication of 1000. The numbers of micronucleated PCE and micronucleated NCE were counted. PCE/NCE ratio was calculated.

Male mice (1012 weeks) were intraperitoneally injected Delvocid at the same concentrations for 6, 12 and 24 h. The smears were prepared according to the methods of Wyrobek and Bruce (1975) with minor modications. The animals were sacriced by cervical dislocation. Both of the cauda epididymuses were dissected out, cut into pieces in 5 ml of physiological saline solution, ltered and smears were made. The smears were xed in methanol and were stained with 10% Giemsa in Srensen buffer for 10 min. One thousand sperms per animal were scored and sperm head abnormalities were determined.

P.G. Rasgele, F. Kaymak / Food and Chemical Toxicology 48 (2010) 789797 2.6. Testosterone measurement Mitotic index, %

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3.63 2.30** 2.66* 2.37** 2.17***

3.93 2.67** 3.00* 2.37*** 1.93***

3.17 2.10** 2.26* 2.07** 1.96**

Blood samples were taken from mice, levels of total testosterone were measured using Immulite 2000 assay at Trakya University, Medical Faculty, Center Laboratory. 2.7. Statistical analysis

Centromeric attenuation

Contraction

Isochromatid gap

Table 1 Frequency of chromosome aberrations and mitotic index in bone marrow cells of female mice induced by Delvocid.

Total abnormality (gap)

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0 15 3 3 4 2 13 2 5 3 1 17 2 4 3 Normal cell number 100 85 97 97 96 98 87 98 95 97 Total cell number 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99 83 98 96 97 Concentrations () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg Treatment periods and sex 12 h-female 24 h-female P 6 0.05. P 6 0.01. P 6 0.001. 6 h-female
* *** **

Abnormal cell number

R
1 9* 1 1 2

0 12*** 2 2 4

1 12* 1 2 3

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4. Discussion

0 0 0 0 1

0 0 0 0 1

0 0 0 0 0

Chromatid gap

0 11 1 3 1

0 3 0 1 2

1 5 1 2 1

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3. Results 3.1. Chromosome aberration assay 3.2. Micronucleus assay 3.3. Sperm head abnormality assay 3.4. Serum testosterone concentration

Variance analysis of data was done using STATISTICA AXA 7.1 computer program. Fishers exact test and X2 test were used for CA and MI, respectively. For parametric data, Kalmogorov Smirnov test was used, and the signicance between groups was determined using the one-way analysis of the variance (ANOVA), followed by a post hoc test. If ANOVA was signicant, Dunnetts test was performed (Zar, 1999). For nonparametric data, the KruskalWallis test was carried out followed by the MannWhitney U test. Doseresponse relationship was determined using Pearson correlation analysis. P < 0.05 was considered as the level of signicance.

0 5 1 0 3

0 2 0 1 1 0 0 0 0 1

0 0 0 1 0

Isochromatid break

0 0 0 0 0

0 0 1 1 0

In this study, the observed aberrations were chromatid gap, isochromatid gap, chromatid break, isochromatid break, contraction and centromeric attenuation. However, Delvocid did not induce a signicant increase of chromosome aberrations both in female and male mice at all concentrations and treatment periods when compared with the negative control (Tables 1 and 2). Delvocid signicantly decreased the MI both in male and female mice at all treatment periods when compared to the negative control (Tables 1 and 2).

0 0 0 0 0

0 0 0 0 0 1 10 1 1 1

Chromatid break

0 7 1 1 1

1 9 0 0 2

0 0 0 0 0

The results obtained were given in Tables 3 and 4. In female mice, Delvocid induced a signicant increase in the frequency of micronucleated PCE at all concentrations both in 24 and 48 h. Delvocid signicantly decreased the PCE/NCE ratio at 800 mg/kg concentration for 24 and 72 h treatments and at all the concentrations for 48 h treatment periods when compared with the negative control. In male mice, the 400 and 800 mg/kg concentrations of Delvocid signicantly increased the number of micronucleated PCE for 24 and 48 h treatment periods. The signicant reduction for the PCE/NCE was observed at all the concentrations for 24 and 48 h when compared with the control. Samples of the micronucleated PCE and NCE were given in Figs. 2 and 3.

Delvocid induced various types of abnormalities in sperm head morphology. Head abnormalities were banana shaped, hookless, amorphous and folded. All the concentrations tested caused a signicant increase in the frequency of abnormal sperms compared with the control (Table 5). The types of sperm head abnormalities were presented in Fig. 4.

Delvocid signicantly decreased the testosterone concentrations in the serum at 400 and 800 mg/kg concentrations for all the treatment periods (Table 6). Levels of testosterone decreased with the increasing concentrations at these periods (Fig. 5). This decrease was concentration-dependent.

Delvocid has been widely used in food products like cheese, sausage and salami (EMEA, 1998). The widespread use of the food

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P.G. Rasgele, F. Kaymak / Food and Chemical Toxicology 48 (2010) 789797

preservatives cause serious problems of health (Sarkaya and Solak, 2003). Therefore, it is very important to assess genotoxicity and cytotoxicity of these chemicals. Chromosome aberrations, micronucleus and sperm head abnormality assays are often used for evaluating genotoxic agents (Ieradi et al., 2003). There is no report about the effects of Delvocid and natamycin on hereditary material and sperm. In this study, Delvocid was not clastogenic at all concentrations and treatment periods. Also, according to WHO (2006), Delvocid was not mutagenic in S. typhimurium, E. coli and B. subtilis. Cox et al. (1973) indicated that neither in male nor in female mice Natamycin was clastogenic (WHO, 2006). In the studies of EMEA (1998), it was reported that natamycin was not mutagenic and induced low acute toxicity in rabbits and dogs. These statements are consistent with the results of the present study. Furthermore, some preservatives in food products also revealed negative results in genotoxicity tests. Boric acid did not induce CA and SCE in CHO (National Toxicology Program, 1987), sodium nitrite did not increase CA in mice, rats and rabbits (Luca et al., 1987), sorbic acid was not clastogenic and mutagenic in CA, SCE and Ames assays (Walker, 1990). Sodium and potassium metabisulphite were not teratogenic and mutagenic in mice, rats, hamsters and rabbits (Nair and Elmore, 2003). MI is used to indicate cytotoxicity of chemicals. A decreased MI reects the inhibition of cell cycle and effects the cell division negatively (Amorim et al., 2000). In the present study, Delvocid significantly decreased MI compared to control and caused a toxic effect. In the present test conditions, negative correlation was found between MI and concentrations and between MI and CA frequency. But the increase in the frequency of CAs was not signicant. This nding suggests that toxicity increased with the increasing of concentrations. MN assay is the most widely used short-term in vivo assay for identication of genotoxic effects (Heddle, 1973). Micronuclei are consisted of chromosome fragments or whole chromosomes which lag behind at anaphase of mitosis and are not incorporated into daughter nuclei. They form single or multiple micronuclei in the cytoplasm. The assay is based on the increase in frequency of micronucleated PCEs in bone marrow of the treated animals (EPA, 1996). We selected two types of erythrocytes (PCEs and NCEs) to evaluate the incidence of MN (Vijayalaxmi and Venu, 1999). They were easy to determine due to staining characteristics and the extent of nuclear damage during the erythropoiesis (Rabbani et al., 2005). In the present study, tested concentrations of Delvocid did not signicantly induce CAs (without gaps), but the same concentrations of Delvocid signicantly induced MN in mice bone marrow. MN may be formed from clastogenic and aneugenic effects. But, in CA assay, a clastogenic effect was not observed. So, formation of MN suggests that Delvocid might be aneugenic. In the studies done using the same food preservatives, different results have been reported by many authors. Renner and Wever (1983) indicated that sodium metabisulphite did not induce CA, SCE and MN in mice ullar et al. (2001), it is bone marrow while in the study of Renczog stated that sodium metabisulphite induced CA and SCE and decreased MI and RI at all concentrations in human lymphocytes. According to the Kayraldz and Topaktas (2007), intraperitoneal administration of sodium metabisulphite was more effective than gavage administration. The same chemical substance can reveal different results. Differences in dose levels, sampling times and way of treatment may be related to these differences in response. According to results of the present study, CA frequency increased with the increasing concentrations. So, higher concentrations of Delvocid has a probable genotoxic effect in mice bone marrow. According to many investigators (Cole et al., 1979; Salamone and Heddle, 1983), PCEs remain alive within the bone marrow in

Mitotic index %

3.97 2.40*** 2.73** 2.50** 2.26***

4.53 2.73*** 3.20** 2.53*** 2.13*** 0 5 0 0 4

Centromeric attenuation

0 6 0 0 0

Contraction

0 2 0 2 4

4.37 2.53*** 3.03** 2.96** 1.87***

Isochromatid gap

Table 2 Frequency of chromosome aberrations and mitotic index in bone marrow cells of male mice induced by Delvocid.

Total abnormality (gap)

R
Total cell number 100 100 100 100 100 Concentrations () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg Treatment periods and sex 12 h-male 6 h-male

100 100 100 100 100

() Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg

() Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg

100 100 100 100 100

P 60.05. P 6 0.01. *** P 6 0.001.

24 h-male

**

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0 13 2 2 2 1 19 5 3 6 100 87 98 98 98 99 81 96 97 94 98 80 96 97 86 2 20 4 3 15

Normal cell number

Abnormal cell number

R
2 21*** 0 3 7

0 13*** 1 1 2

0 19*** 2 3 5

AC

0 0 1 0 0

0 0 1 0 0

Chromatid gap

0 6 5 3 11

1 5 5 0 3

0 4 1 1 0

0 0 0 1 1

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Isochromatid break

0 1 0 0 0 0 9 2 3 0

Chromatid break

1 18 0 1 1

0 7 1 1 2

0 1 0 0 0

0 0 0 0 0

1 0 0 0 2

0 4 0 0 1

0 0 0 0 0

P.G. Rasgele, F. Kaymak / Food and Chemical Toxicology 48 (2010) 789797 Table 3 Micronucleus induction and numbers of PCEs and NCEs in Delvocid-exposed female mice bone marrow. Treatment periods 24 h-female Concentrations () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg Total cell number/ mice number 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 Total MNPCE% SE 6.20 2.20 56.40 6.65*** 21.20 3.72* 28.80 2.06*** 30.40 0.98*** 5.20 1.02 51.20 2.58*** 12.40 1.72* 16.00 1.79** 26.80 3.32*** 9.20 0.80 33.60 7.55*** 12.80 2.15 10.80 2.42 23.20 3.32* 1 MNPCE SE 5.40 2.04 51.60 4.91*** 21.20 3.72** 24.40 1.72*** 25.60 1.32*** 4.80 0.80 45.60 1.47*** 11.60 1.72* 14.80 2.49* 25.60 3.18*** 8.80 1.02 28.40 6.21*** 10.00 1.78 7.60 1.60 16.00 1.67* 2 MNPCE SE 0.20 0.20 4.00 1.67* 1.20 0.80 4.00 0.89* 4.80 0.49** 0.40 0.40 5.60 1.47*** 0.40 0.40 0.80 0.49 1.20 0.80 0.40 0.40 5.20 1.49* 2.80 0.80 3.20 1.02 6.00 1.41** 3 MNPCE SE 0 0.80 0.49 0 0.40 0.40 0 0 0 0.40 0.40 0.40 0.40 0 0 0 0 0 1.20 0.80 1 MNNCE SE 1.40 0.51 9.20 0.80** 3.60 1.16 4.80 1.02* 7.20 1.02** 2.40 0.40 6.40 2.04 4.00 1.09 5.20 1.02* 6.40 1.72* 4.00 0.63 7.60 2.13 5.20 1.62 5.20 0.80 5.60 1.93 2 MNNCE SE 0.20 0.20 0.40 0.40 0.40 0.40 0.40 0.40 0.80 0.49 0 1.20 0.49* 0.80 0.49 0.40 0.40 0.80 0.80 0 0.80 0.80 0.80 0.49 0.80 0.49 1.20 0.49*

793

PCE/NCE SE 1.67 0.20 0.98 0.12* 1.10 0.14 1.05 0.28 0.88 0.14* 1.90 0.19 0.90 0.08** 1.01 0.29** 0.84 0.09*** 0.99 0.10** 1.51 0.18 1.06 0.20 1.39 0.13 1.31 0.09 1.01 0.10*

48 h-female

72 h-female

MNPCE: micronucleated polychromatic erythrocyte, MNNCE: micronucleated normochromatic erythrocyte, SE: standard error. * P 6 0.05. ** P 6 0.01. *** P 6 0.001.

Table 4 Micronucleus induction and numbers of PCEs and NCEs in Delvocid-exposed male mice bone marrow. Treatment periods 24 h-male Concentrations () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg Total cell number/ mice number 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 5000/5 Total MNPCE% SE 1 MNPCE SE

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2 MNPCE SE 2.00 0.63 3.60 0.74 2.00 0.63 4.80 1.02 5.60 0.74* 19.60 0.75 37.20 3.83*** 23.60 3.54 34.00 2.28** 30.00 1.79* 16.00 2.28 37.60 2.71*** 19.20 2.58 35.20 4.32*** 34.40 1.94*** 15.20 1.36 36.80 2.87*** 19.20 1.96 16.80 0.80 19.20 1.96 17.60 0.74 32.80 4.03*** 21.20 2.93 29.20 1.74* 24.40 1.47* 14.80 1.62 34.00 1.78*** 16.80 1.85 30.40 3.18*** 28.80 1.85*** 13.60 1.16 30.80 2.57*** 15.60 1.32 13.20 1.49 16.40 1.16 1.20 0.80 3.60 1.16 2.40 0.98 4.00 0.63 5.60 0.74** 1.60 0.74 4.80 1.02* 3.60 0.74 3.20 0.80 2.80 1.02

48 h-male

72 h-male

R
5000/5 5000/5 5000/5 5000/5 5000/5

MNPCE: micronucleated polychromatic erythrocyte, MNNCE: micronucleated normochromatic erythrocyte, SE: standard error. P 6 0.05. P 6 0.01. *** P 6 0.001.
* **

between 10 and 33 h and the number of MNPCEs is increased at 6 h for aneugens and 10 h for clastogens (Cole et al., 1981; Vanderkerken et al., 1989), so, spindle poisons and clastogenic chemicals could be detected in bone marrow 24 and 48 h after the treatment (Vanparys et al., 1992). Therefore, two treatment periods (24 and 48 h) are sufcient to detect clastogens and aneugens. In the present study, we have demonstrated that Delvocid increased the number of micronucleated PCEs (for 24 and 48 h; in female mice at all concentrations, in male mice at 400 and 800 mg/kg concentrations) and decreased the PCE/NCE ratio in bone marrow of mice. It has been shown by many investigators that food preservatives are genotoxic in a variety of test systems. Citric acid ullar et al., 2008) in(Ylmaz et al., 2008) and biphenyl (Renczog creased frequency of MN in human lymphocytes. Sodium benzoate, boric acid, citric acid, potassium citrate and sodium citrate caused lu, 2007). Our nding is in formation of MN in Allium cepa (Trkog accord with the results of these studies.

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3 MNPCE SE 1 MNNCE SE 8.00 1.41 9.20 0.80 5.20 1.35 7.20 0.49 7.60 0.74 5.20 0.49 8.80 1.02* 9.20 1.35* 13.60 1.47** 17.20 1.35** 5.60 1.16 10.40 0.74* 6.00 0.63 6.40 0.74 8.40 1.16 2 MNNCE SE 3.60 0.74 4.00 4.00 0.40 0.40 4.40 1.72 5.60 0.74 2.00 0.89 1.60 0.74 0 2.00 1.26 0.80 0.80 2.00 0.63 2.00 0.00 1.60 0.40 2.40 0.74 2.00 0.00 0 0.80 0.49 0.40 0.40 0 0 0 0 0 0.80 0.80 0 0 1.20 0.49* 0 0.40 0.40 0

PCE/NCE SE 1.48 0.03 0.91 0.02*** 1.26 0.09* 0.96 0.05*** 0.86 0.05*** 1.70 0.11 0.81 0.03*** 1.00 0.05*** 0.97 0.03*** 0.81 0.03*** 1.52 0.05 0.82 0.08*** 1.35 0.10 1.23 0.02 1.26 0.11

The PCE/NCE ratio is used to obtain information about the cell cycle specic action of a positive chemical. A decrease in PCE/ NCE ratio reects a cytotoxic effect or alterations in erythropoiesis. The PCE/NCE ratio is decreased because of the cavity formation in bone marrow when there are cytotoxic effects on the cell division and/or maturation of the nucleated cells (Gollapudi et al., 1984). In addition, newly maturated NCEs remain behind the bone marrow due to the failure of release into the peripheral blood on schedule (Von Ledebur and Schmid, 1973). Generally, aneugenic substances cause MN inhibiting spindle formation. Abnormalities due to inhibition of spindle formation reect high toxicity of chemicals (Haliem, 1990). Both MI and PCE/ NCE ratio are used to monitor toxicity in CA assay and MN assay, respectively. In the present study, the tested concentrations of Delvocid did not signicantly induce CAs without gaps but the same concentrations signicantly induced MN. The PCE/NCE ratio in bone marrow of mice decreased with the increase of cells with

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Table 5 Number and mean percentage of sperm head abnormalities in control and Delvocid treated mice. Treatment periods 6h Concentrations Number of sperms examined/ number of animals 3000/3 3000/3 3000/3 3000/3 3000/3 3000/3 3000/3 3000/3 3000/3 3000/3 3000/3 3000/3 3000/3 3000/3 3000/3 Banana shaped 64 362*** 168* 227** 288*** 66 413*** 204*** 247*** 287*** 61 406*** 189*** 253*** 294*** Without hook 36 241*** 137** 153*** 170*** 45 250*** 101 152** 140** Amorphous head 33 119*** 61*** 67*** 68*** 31 117*** 37 48 92*** 37 112*** 56 84*** 98*** Bent at cephalocaudal junction 33 53 80* 99** 177*** 35 56* 89*** 117*** 89*** 40 76** 70* 138*** 98*** Total sperm abnormality 167 784*** 447*** 552*** 714*** 178 844*** 432*** 576*** 616*** 187 882*** 441*** 633*** 667*** % Abnormal sperm (mean SE) 5.56 0.20 26.13 0.68*** 14.9 0.87*** 18.4 0.11*** 23.8 1.53*** 5.93 0.18 28.13 0.03*** 14.4 1.06*** 19.2 0.77*** 20.53 1.04*** 6.23 0.32 29.4 1.05*** 14.7 1.04*** 21.1 0.65*** 22.23 2.10***

() Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg

12 h

24 h

47 276*** 117** 148*** 165***

SE: standard error. * P 6 0.05. ** P 6 0.01. *** P 6 0.001.

Treatment periods 6h

Concentrations () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg () Control (+) Control 200 mg/kg 400 mg/kg 800 mg/kg

12 h

24 h

SE: standard error. * P 6 0.05. ** P 6 0.01. *** P 6 0.001.

MN. Positive correlation was found between MI and PCE/NCE ratio in both male and female mice for 24 h treatment periods. Since it decreased the MI and the PCE/NCE ratio in bone marrow of mice, Delvocid can be accepted as a toxic agent. In sperm head abnormality assay, sperm morphology is used to investigate carcinogenic and mutagenic chemicals (Ieradi et al., 2003). This assay is a sensitive and reliable method (Giri et al., 2002). In this study, Delvocid signicantly increased the percentage of abnormal sperm at all concentrations. The dose dependent increase in the frequency of sperm head abnormalities suggests that Delvocid caused differentiation in male germ cells and it is a germ cell mutagen. Abdel Aziz et al. (1997) have reported that Eritrosin induced an increase in the abnormal sperm; Heindel et al. (1997) have found that boric acid affected reproduction and fertility in rodents negatively; and in study of Oishi (2002) propyl paraben affected the hormonal secretion and the male reproduc-

ET

R
51.00 2.30 20.00 0.00*** 45.66 2.40 38.00 1.52* 35.00 5.85**

AC
Total testosterone (SE) 56.00 6.55 22.33 1.45*** 46.66 0.88 2.33 1.45*** 20.00 0.00*** 43.70 4.07 20.00 0.00*** 40.33 0.88 30.66 0.88* 25.93 3.72**

Table 6 Effects of Delvocid on the serum testosterone hormone levels in male mice.

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tion functions in rats. Our results are in agreement with these ndings. The exact reason of the increase in the frequency of abnormal sperm is not known but there are different opinions. This increase may be related to decrease of the fertility. Induction of abnormal sperms is assumed to be a result of naturally occurring errors during the differentiation or the consequence of an abnormal chromosome (Bruce et al., 1974). In addition, Y chromosomes have an important role in determining frequency of sperm abnormalities (Krzanowska, 1976; Styrna et al., 1991). According to Topham (1980), the properties controlling sperm morphology exist on the autosomes and those agents are identied by sperm abnormality assay and they cause minor alterations in testicular DNA (Giri et al., 2002). In the study of Chauhan et al. (2000), it has been reported that the exogenous factors induce alterations in sperm morphology by point mutations (Narayana et al., 2002). Bone marrow and spermatogenic tissues are rich in terms of investigation of mitotic cells, and both bone marrow and sperm abnormality assays provide reliable results (Wang et al., 1998). According to many investigators (Hemavathi and Rahiman, 1993; Jayashree et al., 1994; Chauhan et al., 2000), several chemicals which induced cytotoxic effects and formation of MN in bone marrow cells caused abnormal sperms as well (Giri et al., 2002). In the present study, Delvocid did not increase CA frequency but it increased formation of MN. A signicant relation was found between MI which is an indicator of cytotoxicity in CA assay and percentage of abnormal sperms at all treatment periods. The same relation was also observed between PCE/NCE ratio and percentage of abnormal sperm for 24 h. The increased frequency of abnormal sperm may be from toxic effects due to aneugeni. As a result, Delvocid is considered as toxic and germ cell mutagen because of its effect on hereditary material and sperm, respectively. Desjardins (1985) and Herbert et al. (1995) have reported that testosterone controls the development of male reproductive system and spermatogenesis (Oishi, 2002). The epididymis is an important organ for sperm maturation. The functions of epididymis and sperm may be changed by lowering hormone levels. According to Ono et al. (1999), direct sperm toxicity may exist within the epididymis. In the sperm maturation process, sperm acquires structural stabilization of the head and tail (Calvin and Bed-

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Fig. 4. (a) Normal sperm; (b) banana shaped (200 mg/kg,12 h); (c) without hook (800 mg/kg, 12 h); (d) amorphous head sperm (400 mg/kg, 24 h); (e) bent at cephalocaudal junction (400 mg/kg, 6 h) (1000).

ford, 1971), its activity (Hoskins et al., 1978) and fertilizing capacity (Yanagimachi, 1988; Miller et al., 1996). In the present study, a signicantly increased percentage of abnormal sperm occured in Delvocid treated mice and Delvocid decreased testosterone levels. Consequently, in male reproductive system, the development and functioning disorder emerged. Decreases in the levels of testosterone have been reported after the treatment of rats with other food preservatives (Oishi, 2002; Jeong et al., 2005).

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In summary, the obtained results indicate that Delvocid is not clastogenic in the CA assay but an aneugenic chemical in the MN assay. In addition, Delvocid has cytotoxic effects in mice because it reduced MI and PCE/NCE ratio. Delvocid may be regarded as a potential germ cell mutagen due to its effect on sperm. For this reason, it is necessary to be careful when using these chemicals in food as preservatives, and they should not be used in excessive amounts in food industry.

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Total testosterone (ng/ml)

60 50 40 30 20 10 0 0

y = 3E-05x 2 - 0.0415x + 51.526 y = 2E-05x 2 - 0.0369x + 44.614 R2 = 0.9787 R2 = 0.9502

y = 3E-05x 2 - 0.0677x + 56.854 R2 = 0.9882

200

400

600

800

1000

Concentrations (mg/kg)
total testosterone 6 saat total testosterone 24 saat Polinom (total testosterone 24 saat) total testosterone 12 saat Polinom (total testosterone 6 saat) Polinom (total testosterone 12 saat)

Fig. 5. Relation between testosterone and concentrations of Delvocid in mice (for 6 h Pearson correlation P = 0.01; for 12 and 24 h Pearson correlation P = 0.05).

Conict of Interest The authors declare that there are no conicts of interest. References
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