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Chromosomal aberrations and frequency of micronuclei in sheep subchronically exposed to the fungicide Euparen Multi (tolylfluanid)
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 64 (2006) 312–320 www.elsevier.com/locate/ecoenv
Chromosomal aberrations and frequency of micronuclei in sheep subchronically exposed to the fungicide Euparen Multi (tolylfluanid) Irena Sˇutiakova´, Nata´lia Kovalkovicˇova´, Juraj Pistl, Jaroslav Novotny´, Jaroslav Lega´th, Gabriel Kova´cˇ, Sabina Hlincˇı´ kova´, Va´clav Sˇutiak University of Veterinary Medicine, Komenske´ho 73, 041 81 Kosˇice, Slovak Republic Received 9 July 2004; received in revised form 14 April 2005; accepted 21 April 2005 Available online 6 June 2005
Abstract We analyzed chromosome aberrations, micronucleus frequency, mitotic index (MI), and nuclear division index (NDI) in peripheral lymphocytes of sheep subchronically exposed to the fungicide Euparen Multi (containing 50% tolylfluanid). Euparen Multi was administered by rumen sonde to group of Merino sheep (seven sheep/group) at 93 mg/kg body weight (1/20 LD50) daily for 28 days to assess its genotoxic effects. The frequencies of aberrant cells (ABC) in the experimental and control groups were 5.5071.38% and 2.4071.14%, respectively, and the increase in ABC in the treated group was significant (P ¼ 0:003). Significantly increased numbers of chromatid breaks (5.6771.21% against 2.4071.14%; P ¼ 0:001), chromatid gaps (10.3372.73% against 4.0071.23%; P ¼ 0:001), and chromosome gaps (1.8370.75% against 0.8070.45%; P ¼ 0:025) and exchanges (3.1771.94% against 0.2070.45%; P ¼ 0:009) were observed in exposed animals in comparison to control animals. The frequency of micronuclei (MN) was 29.4075.86 per 1000 binucleated cells in peripheral lymphocytes of sheep in the control group and 49.57719.12 per 1000 binucleated cells in the treated group. A significant increase in the frequency of MN in peripheral lymphocytes also was observed between the two groups (P ¼ 0:0477). No statistical differences in MI and NDI values were found in the groups (P ¼ 0:181 and 0.761, respectively). Thus, our results suggest that exposure to Euparen Multi may cause genome damage in somatic cells. r 2005 Elsevier Inc. All rights reserved. Keywords: Sheep; Euparen Multi (tolylfluanid); Chromosomal aberrations; Micronuclei; Mitotic index; Nuclear division index; Genotoxicity
1. Introduction At the present, there are more than 1000 chemicals classified as pesticides. Pesticides are designed to control target pests, either diseases or weeds. They represent an important group of environmental pollutants to which man and other animals are daily exposed, mainly as a consequence of their wide use in agriculture. Exposure to pesticides is likely to continue and possibly may increase in the near future, because of increased use and their persistence in the environment. Moreover, exposure to pesticides has been related to several diseases Corresponding author. Fax: +42155/6331853.
E-mail address: [email protected] (I. Sˇutiakova´). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.04.011
including cancer. Cancer is a complex disease in which cells with altered gene expression grow abnormally, invade other tissues, and disrupt their normal function (Fenech, 2002). For many years cytogenetic alterations in cultured peripheral blood lymphocytes, such as chromosomal aberrations, sister chromatid exchanges, and micronuclei (MN), have been applied as biomarkers of genotoxic exposure and early effects of genotoxic carcinogens (Norppa, 2004). In addition to genotoxicity, stimulation of cellular proliferation may contribute to the carcinogenity of chemicals (Holland et al., 2002). Pesticides are of great concern for their toxic effects and for their potential as a long-term genetic hazard. The most problematic pesticides are fungicides since about 90% of them used currently or formerly have
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been determined to be carcinogenic in experimental animals (Saunders and Harper, 1994). Recently, the genotoxicity of many antifungal agents has been investigated, including fenarimol and propamocarb (Aydemir and Bilaloglu, 2003; Poli et al., 2003), maneb and zineb (Gerber et al., 2002; Soloneski et al., 2002, 2003; Osaba et al., 2002), ferbam (Shanthi and Krishnamoorthy, 2002), carbendazim, captan, and captafol (Rahden-Staron, 2002; Goumenou and Machera, 2003), chlorothalonil (Godard et al., 1999), zinc dimethyl (Zenzen et al., 2001), and thiram (Villani et al., 1998). For this study tolylfluanid, 1,1-dichloro-N-[(dimethylamino)-sulfonyl]-1-fluoro-N-(4-methylphenyl)methanesulfenamide, was chosen because it is heavily used in agriculture and represents one of the most frequently detected pesticides in vegetables and fruit (Nowacka, 2002). Tolylfluanid has a multisite mode of action against phytophagous mites and fungal diseases caused by Botrytis, Didymella, and Drepanopeziza in strawberries, raspberries, hops, apples, ribes, vine, and tomatoes. It is safe to all beneficial organisms (bees, beneficial mites, anthocorids). Tolylfluanid is very rapidly degradated in soil and water and because it is strongly absorbed by soil particles it has no potential to migrate into deeper soil layers (The Pesticide Manual, 2000; EPA, 2002b). In animals, tolylfluanid is rapidly absorbed and hydrolyzed within 48 h to dimethylamino sulfotoluidide and then transformed to the main metabolite 4(dimethylaminosulfonylamino) benzoic acid which can be further demethylated. There is no accumulation in organs and tissues (Abbink and Weber, 1988). Subchronic and chronic toxicity studies conducted in animals showed altered liver enzyme activities and thyroid hormone levels, accompanied by an increased incidence of hyperplastic and neoplastic lesions of the thyroid (primarily adenomas) and damage of renal cortical tubules (PFPC Newsletter Special Issue, 1999). In sheep, tolylfluanid, administered orally, caused anorexia, weakness of the extremities, and loose feces (Hoffmann, 1983). The mutagenicity of tolylfluanid has not yet been firmly established. It was variably active in a variety of in vitro assays, e.g., weakly positive in the Ames test (Herbold, 1979) and positive in the forward mutation in vitro test in mouse lymphoma cells (Hoom, 1985) and in the cytogenetic assay in human lymphocytes (Herbold, 1984a, b). A weakly clastogenic effect of tolylfluanid was observed in Chinese hamster V79 cells in the presence of S9 activation (EPA, 2002a). In vivo, however, it was inactive in mammals (EPA, 2002b). To provide additional genotoxicity data, we report here our recent results on the effect of tolylfluanid in the induction of chromosome aberrations and MN as cytogenetic endpoints in subchronically exposed sheep.
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2. Materials and methods The organization of the experiment, the investigations conducted, and the related documentation complied with legislative regulations governing the protection of experimental animals of the Slovak Republic. 2.1. Animals The experiment was carried out on 12 clinically healthy 18–24-month-old female sheep (Merino breed) which had no previous exposure to pesticides or any other agents suspected of being genotoxic. There were seven sheep weighing 50.7976.52 kg in the experimental group. The control group consisted of five sheep with mean body weight (b.wt.) 49.1074.98 kg. Animals were housed, treated with antihelminthic agent Aldifal 2.5% susp. a.u.v. (Mevak a.s., Nitra, Slovak Republic) at a dose of 2 mL per 10 kg b.wt., acclimatized for 1 week before dosing, and observed before initiation of the study to ensure that they were healthy. Only animals found to be in a clinically acceptable condition were assigned to the study. During the study, food and water were offered ad libitum. Animals were housed at 8–12 1C and 70% relative humidity. Food consumption, general condition, and any other clinical symptoms were monitored daily. The reference values for ovine pulse and respiratory rate, temperature, and rumen rotations were reported by Slanina et al. (1993). Body weights were recorded weekly. 2.2. Tested pesticide Experimental animals were exposed to Euparen Multi manufactured by Bayer AG, containing 50% tolylfluanid. 2.3. Dose and exposure The tested pesticide preparation was freshly prepared each day and administered by rumen sonde at a dose of 93 mg/kg b.wt. (46.5 mg active ingredient/kg b.wt.; 1/20 LD50) daily for 28 days. The dose was calculated on the basis of the mean LD50 value of tolylfluanid for sheep and its purity as reported by Hoffmann (1983), i.e., 937.57312.5 mg/kg b.wt. (purity 99.2%). 2.4. Cytogenetic assays 2.4.1. Lymphocyte cultures For cytogenetic analysis blood samples were taken from the jugular vein (vena jugularis) into glass tubes containing heparin (100 IU/mL blood). Aliquots of 0.4–0.5 mL of whole blood were cultured at 37.5 1C in 6 mL Panserin 701 chromosome medium supplemented with fetal calf serum, phytohemagglutinin, and
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(PAN Systems GMbH Biotechnologische Produkte, Germany). Penicillin G (100 IU/mL), streptomycin (100 mg/mL), and 7.5% NaHCO3 (PAN Systems GMbH Biotechnologische Produkte) were added to the cultivation medium.
2.5. Cell proliferation assays
2.4.2. Chromosomal aberration analysis Conventional cytogenetic culturing was carried out for 72 h according to the recommendations of Lioi et al. (2004) and Sˇivikova´ and Kalina (1990). These authors observed that in ruminants a longer time of cultivation is more optimal for the detection of chromosomal aberrations than the standard 48 h procedure. Colchicine (10 mg/mL) was added to the medium 1 h before the end of incubation. The cultures were hypotonized with a 0.035 M KCl solution for 30 min and subsequently fixed in 1:3 (v/v) methanol:acetic acid mixture. The slides were stained with 10% conventional hypotonic Giemsa in pH 6.8 phosphate buffer for 5–7 min. For each donor, 100 well-spread metaphases containing 2n ¼ 54, XX chromosomes were scored for chromosomal aberrations. The metaphase cells were evaluated (magnification 1000 ) from each animal. Structural chromosomal aberrations were classified according to Savage (1975). The identification of the individual chromosome pairs was carried out according to the standardized karyotyping of the domestic sheep (Di Berardino et al., 2001). Chromosome-type and chromatid-type aberrations were scored and classified as breaks, gaps, and exchanges. Chromatid breaks were distinguished from gaps (achromatic lesions) when the centric piece was displaced with respect to the chromosome axis or the size of the discontinuity exceeded the width of the chromatid. Exchanges were classified as stable (translocation, when detection was possible) and unstable (dicentric, ring, fragments). Images were taken by imaging microscopy (Nikon) using a CCD-100 camera system (Mitsubishi). The images were processed using the Animal and Photostyler software system.
2.5.1. Mitotic index In the chromosomal aberration study, MI was evaluated by counting at least 1000 cells per treatment and dividing the number of dividing cells (metaphases) by the total number of cells (Preston et al., 1987).
L-glutamine
2.4.3. Micronucleus analysis Lymphocyte cultures were incubated at 37 1C for 72 h. Cytokinesis was blocked by cytochalasin B (Sigma, USA; diluted in dimethyl sulfoxide) which was added at 44 h after culture initiation to achieve a final concentration of 6 mg/mL. The slides were stained with 10% Giemsa–Romanowski in So˜rensen phosphate buffer, pH 6.8, for 3–6 min. Using a Nikon microscope the coded slides were scored blinded at magnifications of 400 and 1000 . The induction of MN was evaluated by scoring a total of 1000 binucleated cells per donor and concentration. MN were identified using well-known criteria (Coutryman and Heddle, 1976; Surralle´s and Natarajan, 1997).
Effects on the cellular proliferation rate were estimated by calculating mitotic index (MI) and nuclear division index (NDI).
2.5.2. Nuclear division index In the micronucleus study, a minimum of 500 lymphocytes was scored to evaluate the percentage of cells with one, two, three, and four or more nuclei. An NDI was calculated according to Eastmond and Tucker (1987) as NDI ¼ [M1+(2M2+3M3+4M4)]/N, where M1–M4 represent the number of cells with one to four nuclei, respectively, and N is the number of cells scored. 2.6. Statistical analysis The comparison of the exposed and control groups was performed by applying the t-test. The Sigma Stat program (Statistical software, Jandel Scientific) was employed for statistical evaluation of the results.
3. Results No overt signs of toxicity were observed during the study. Occasional deceleration of rumen rotations (6.0071.15/5 min) toward the lower limit of reference values (6–16/5 min), sharp breathing sounds, cough, and respiratory rates (30.43714.70/min) at the upper limit of the physiological norm (12–30/min) were observed in animals of the exposed group. These events may have arisen from an irritant effect of the preparation itself or by irritation caused by the frequent application of the sonde. Body weight was not markedly affected by treatment. The mean body weight for animals in the experimental group was 50.7976.52 kg at the beginning and 47.3877.16 kg at the end of treatment. Tables 1 and 3 report the mean values of the cytogenetic parameters analyzed for both groups before exposure to tolylfluanid. The results of the chromosomal aberration analysis for both the exposed and the control groups after 28-day exposure to tolylfluanid are reported in Table 2. The exposed group of animals exhibited a significantly higher frequency of genetic damage (5.5071.38% aberrant cells (ABC)) compared to control animals (2.4071.14% ABC; P ¼ 0:003). As shown in Table 2
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Table 1 Chromosomal aberration frequency in sheep peripheral lymphocytes before subchronic 28-day exposure to Euparen Multi (tolylfluanid) Group
Experimental (n ¼ 7)
Number of metaphase cells analyzed
Chromosomal aberrations B1
B2
G2
G2
Exchanges
No. ABC
(1) (2) (3) (4) (5) (6) (7)
2 2 3 1 2 4 3
0 0 1 0 0 2 0
5 3 2 2 0 6 2
3 0 0 0 0 3 1
3 1 0 0 0 1 1
2 2 3 1 2 3 2
2.43 0.98 0.37 0.42
0.430 0.79 0.3 0.45
2.86 2.04 0.77 0.048
1.00 1.41 0.54 0.57
0.86 1.07 0.40 0.64
2 4 2 2 5
1 0 1 0 2
6 5 7 4 4
2 1 1 1 2
1 1 0 1
3.00 1.41 0.63
0.80 0.84 0.37
5.20 1.30 0.58
1.40 0.55 0.25
0.60 0.55 0.25
100 100 100 100 100 100 100
Statistical mean value 7SD SEM P Control (n ¼ 5)
Statistical mean value 7SD SEM
(8) (9) (10) (11) (12)
100 100 100 100 100
ABC % B/C
0.0286
G/C
MI
0.0386
1.760 1.821 1.791 1.930 1.851 1.682 1.78
2.14 0.69 0.26 0.23
1.802 0.078 0.029 0.149 0.038
4 2 2 4 2.80 1.10 0.49
0.066
1.940 1.871 1.902 1.915 1.740 1.873 0.079 0.035
ABC, aberrant cells; B1, G1, chromatid breaks, gaps; B2, G2, isochromatid breaks, gaps; B/C, number of breaks per cell; G/C, number of gaps per cell; MI, mitotic index; SEM, standard error mean; SD, standard deviation.
and Fig. 1, in the experimental group the frequencies of chromatid aberrations (P ¼0.001), chromosome gaps (P ¼ 0:025), and exchanges (P ¼ 0:009) were significantly higher than those in the control group. Centromere extension of metacentric chromosomes (10%), endoreduplication (1%), and aneuploidy (3%) were also observed in the exposed group. No statistical differences in MI values were found in the groups (Table 2; P ¼ 0:181). The frequency of MN and their distribution for groups examined after 28-day exposure to tolylfluanid are shown in Table 4. The mean value of the total number of MN observed in binucleated peripheral blood lymphocytes of the exposed group was significantly higher than that found in the control group (49.57719.12 against 29.4075.86 MN/1000 binucleated cells; P ¼ 0:0477). No significant reduction of the NDI in exposed animals was observed (Table 4; P ¼ 0:761).
4. Discussion The extensive use of pesticides raises many problems due to their toxicity in non-target organisms, persistence, and combined effects with other agrochemicals and environmental factors. In the past, risk assessment was limited to the danger to human health, but currently
ecological risks are also taken into account. In this regard, further studies are required to determine the mechanism of action of pesticides, including how they interfere with metabolism, the genetic damage that they induce, and their detoxification mechanisms in domestic animals (Lioi et al., 1998). About two-thirds to fourfifths of food animals are exposed to toxic substances (Bo¨hmer et al., 1991) that can be important risk factors to human health (Cristaldi et al., 2004). Free-living grazing animals are among the animal groups immediately exposed to and most affected by the harmful influence of pesticides. We have used sheep as a model animal source of peripheral blood leukocytes because they are similar to wild ruminants and are a better indicator of environmental exposure than rodents kept strictly under laboratory conditions. Cytogenetic biomarkers have been widely used in the genotoxicity assessment of environmental mutagens under in vitro and in vivo conditions. Cytogenetic biomarkers reflect the cellular effects of genotoxic carcinogens, and different cellular processes may be involved in genetic instability in the early or late stages of cancer. The most notable types of instability of the genome in cancer cells involve chromosome rearrangements, and aneuploidy (Murnane, 1996). Although chromosome analysis is not as specific as molecular studies monitoring effects on single genes, it monitors the entire genome.
2.40 1.14 0.51
0.60 0.55 0.25
0 1 0 1 1
0.83 0.41 0.17 0.438
1 1 1 0 1 1
B2
4.00 1.23 0.55
5 4 5 2 4
10.33 2.73 1.12 0.001
14 12 12 9 8 7
G1
0.80 0.45 0.20
1 1 0 1 1
1.83 0.75 0.31 0.025
3 2 1 2 1 2
G2
0.20 0.45 0.20
0 0 1 0 0
3.17 1.94 0.79 0.009
5 3 2 1 2 6
Exchanges
2 4 1 2 3
8 5 6 5 4 5
No. ABC
2.40 1.14 0.51
5.50 1.38 0.56 0.003
ABC %
0.030
0.065
B/C
0.048
0.121
G/C
1.87 0.059 0.026
1.88 1.91 1.85 1.93 1.78
1.77 0.14 0.06 0.181
1.65 1.77 1.89 1.94 1.58 1.81
MI
ABC, aberrant cells; B1, G1, chromatid breaks, gaps; B2, G2, isochromatid breaks, gaps; B/C, number of breaks per cell; G/C, number of gaps per cell; MI, mitotic index; SEM, standard error mean; SD, standard deviation. Experimental sheep no. 3 was not included in statistical analysis because of low mitotic index.
Statistical mean value 7SD SEM
2 3 1 2 4
Control (n ¼ 5)
100 100 100 100 100
5.67 1.21 0.49 0.001
8 6 5 5 5 5
B1
Chromosomal aberrations
Statistical mean value 7SD SEM P
(8) (9) (10) (11) (12)
(1) (2) (4) (5) (6) (7)
Experimental (n ¼ 6)
100 100 100 100 100 100
Number of metaphase cells analyzed
Group
Table 2 Chromosomal aberration frequency in sheep peripheral lymphocytes after daily 28-day exposure to Euparen Multi (tolylfluanid)
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Fig. 1. Sheep chromosomes with different types of aberrations. (a–d) Chromatid breaks, gaps, and exchanges.
In the present study, the possible genotoxic effect of tolylfluanid was assessed by chromosomal aberration analysis and micronucleus assay. Chromosomal aberration analysis revealed an increased number of structural chromosomal aberrations in sheep after subchronic oral exposure to this fungicide. The aberrations induced by tolylfluanid were mainly of the chromatid type. Chromosome breaking agents (clastogens) can be classified as S-dependent and -independent agents (Natarajan, 2002). Most chemicals are S-dependent clastogens, which induce chromatid-type aberrations that are largely unstable and wherein only symmetrical exchanges would be expected to be stable (Kirkland, 1998). However, the chromatid type of aberrations may be converted to the chromosome type, e.g., translocations that persist in organisms for a longer time. Cells with chromosome aberrations may go on to become cancerous, and
chromosome deletions and translocations are observed in most cancer cells (Mitelman et al., 1997; Obe et al., 2002). Stable chromosomal damage is important as an initiating carcinogenic event, but ‘‘unstable’’ damage is important in inducing the loss of heterozygosity during the promotion and progression stages of cancer (Kirkland, 1998; Bonassi and Au, 2002). Chromosome aberrations may indicate alterations in cell homeostasis that are important in genome instability (i.e., interference with DNA synthesis or condensation; Galloway, 1994). The highest frequency of gaps observed in the experimental group was of the chromatid type. It is generally proposed that gaps should not be included in chromosomal aberration frequencies for statistical analysis. However, since some of the gaps could be deletions, it is appropriate to analyze the data excluding
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Table 3 Micronucleus frequencies and distributions in peripheral sheep lymphocytes before subchronic exposure to Euparen Multi (tolylfluanid) Group
Experimental (n ¼ 7)
No. of analyzed cells
(1) (2) (3) (4) (5) (6) (7)
1000 1000 1000 1000 1000 1000 1000
Statistical mean value 7SD SEM P Control (n ¼ 5)
(8) (9) (10) (11) (12)
1000 1000 1000 1000 1000
Statistical mean value 7SD SEM
No. of MN
MN/cell
% MN cells
18 29 23 43 25 19 31
0.018 0.029 0.022 0.043 0.024 0.019 0.029
1.80 2.89 2.20 4.25 2.43 1.89 2.93
26.86 8.57 3.24 0.48
0.026 0.009 0.003 0.4996
2.63 0.84 0.32 0.5103
25 32 23 33 38
0.025 0.031 0.023 0.030 0.038
2.49 3.12 2.29 2.96 3.77
30.20 6.14 2.75
0.029 0.006 0.003
2.93 0.58 0.26
Micronucleus distribution 0
1
2
3
Cells with MN
986 972 978 960 976 981 969
11 27 21 37 23 19 31
2 1 1 3 1 0 0
1 0 0 0 0 0 0
14 28 22 40 24 19 31
NDI
1.114 1.087 1.132 1.183 1.171 1.163 1.169 1.15 0.04 0.01 0.74
976 968 977 968 965
23 32 23 31 32
1 0 0 1 3
0 0 0 0 0
24 32 23 32 35
1.147 1.113 1.108 1.258 1.149 1.16 0.06 0.03
MN, micronuclei; MN/cells, micronuclei per cells; % MN cells, percentage of micronucleated cells; SEM, standard error mean; SD, standard deviation; NDI, nuclear division index.
Table 4 Micronucleus frequencies and distributions in peripheral sheep lymphocytes after subchronic exposure to Euparen Multi (tolylfluanid) Group
Experimental (n ¼ 7)
No. of analyzed cells
(1) (2) (3) (4) (5) (6) (7)
1000 1000 1000 1000 1000 1000 1000
Statistical mean value 7SD SEM P Control (n ¼ 5)
Statistical mean value 7SD SEM
(8) (9) (10) (11) (12)
1000 1000 1000 1000 1000
No. of MN
MN/cell
% MN cells
50 49 40 90 32 49 37
0.050 0.049 0.040 0.090 0.032 0.049 0.037
5.00 4.90 4.00 9.00 3.20 4.90 3.70
49.57 19.12 7.23 0.0477
0.050 0.019 0.007 0.0477
4.957 1.91 0.72 0.0477
25 31 23 30 38
0.025 0.031 0.023 0.030 0.038
2.50 3.10 2.30 3.00 3.80
29.40 5.86 2.62
0.029 0.006 0.003
2.94 0.58 0.26
Micronucleus distribution 0
1
2
3
Cells with MN
951 952 961 917 969 955 964
48 47 38 76 30 41 35
1 1 1 7 1 4 1
0 0 0 0 0 0 0
49 48 39 83 31 45 36
NDI
1.179 1.063 1.108 1.097 1.092 1.091 1.161 1.113 0.043 0.016 0.761
976 969 977 971 965
23 31 23 28 32
1 0 0 1 3
0 0 0 0 0
24 31 23 29 35
1.144 1.084 1.102 1.127 1.141 1.120 0.026 0.012
MN, micronuclei; MN/cells, micronuclei per cells; % MN cells, percentage of micronucleated cells; SEM, standard error mean; SD, standard deviation; NDI, nuclear division index.
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and including gaps (Preston et al., 1987). According to Brogger (1982), gaps are a sensitive indicator of exposure to genotoxic drugs and serve mainly at low doses as a ‘‘guard’’ parameter. Forni (1992) studied the reference values of chromosome aberrations in human peripheral blood lymphocytes as an indicator in genotoxicity studies. This author reported great variability arising from differences in methodological procedures and conditions among laboratories and from intrinsic individual variability. Forni (1992) recommended that the results of chromosome aberration studies be compared with the control group in the same laboratory using the same methodological procedures and conditions. Therefore, we reported the main cytogenetic parameters of both groups of sheep before our experiment. An increased number of chromosomal aberrations in the exposed group occurred simultaneously with the increase in MN. According to Savage (1988) the observed frequencies of MN are often lower than the frequencies of the chromosomal aberrations seen at the first post-treatment metaphase, because not all fragments necessarily form visible MN. Formation of MN and chromosome aberrations are two important cytogenetic endpoints considered to be biomarkers of early biological effects from exposure to mutagenic agents (Bonassi and Au, 2002). Over the past several years the cytokinesis-block micronucleus assay has evolved into a comprehensive method for measuring chromosome breakage, chromosome loss, non-disjuction, necrosis, apoptosis, and cytostasis (Fenech and Crott, 2002). MN are thought to arise from both clastogenic (chromosome breakage) and aneugenic (chromosome lagging and spindle) effects, while structural chromosome aberrations are thought to arise from chromosome breakage and exchange. Cellular proliferation rate is a phenomenon that may be causally associated with the induction of chromosomal damage for a number of compounds which are nonDNA-reactive and thus are threshold in vitro clastogens (Mu¨ller and Kasper, 2000). MI gives direct information of toxic effect on metaphases. Although depression of MI may be the consequence of cell death, it is more often the result of cell cycle delay (Kuroda et al., 1992). The other parameter of cellular proliferation rate that has been studied is NDI which was used for the evaluation of the effect of chemicals on cell division (Eastmond and Tucker, 1987). In our study, MI and NDI were not significantly decreased in exposed animals in comparison with controls. Lymphocyte proliferation is an extremely complex system and is influenced by many factors (Snyder and Walle, 1991). The data on tolylfluanid genotoxicity in animals are limited. Here we report, for the first time to our knowledge, an induction of structural chromosomal aberrations and MN by this fungicide in sheep
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lymphocytes after subchronic oral exposure. Our results suggest that this potential genetic hazard requires examination from the viewpoint of both human and other animal health.
5. Conclusion Our results demonstrate that the fungicide tolylfluanid induces a significant increase in chromosomal aberrations and micronucleated cells. We can conclude that there is a genotoxic effect of this fungicide in sheep. More studies are needed to assess its genotoxicity in other domestic and wild animals, including under in vitro conditions. The findings of this study stress the necessity for the implementation of safety measures both during and after pesticide application to reduce the health hazards of pesticides in humans and other animals.
Acknowledgments This work was supported by VEGA Grant No. 1/ 1362/04 and No. 1/0606/03 and the National Reference Laboratory for Pesticides University of Veterinary Medicine in Kosˇ ice. The authors thank Mr. J. Chabala for his linguistic correction and adaptation of our manuscript to native English language.
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Poli, P., de Mello, M.A., Buschini, A., de Castro, V.L.S.S., Restivo, F.M., Rossi, C., Zucchi, T.M.A.D., 2003. Mutat. Res./Genet. Toxicol. Environ. Mutagen. 540, 57–66. Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, A.F., Shelby, M., 1987. Mutat. Res. 189, 157–165. Rahden-Staron, I., 2002. Mutat. Res./Genet. Toxicol. Environ. Mutagen. 518, 205–213. Saunders, D.S., Harper, C., 1994. Pesticides. Principles and Methods of Toxicology. Raven Press, New York, pp. 386–415. Savage, J.R.K., 1975. J. Med. Genet. 12, 103–122. Savage, J.R.K., 1988. Mutat. Res. 207, 33–36. Shanthi, R., Krishnamoorthy, M., 2002. Teratogen. Carcinogen. Mutagen. 22, 451–459. Sˇivikova´, K., Kalina, I., 1990. Biologia (Bratislava) 45 (11), 899–906. Slanina, L’., Dvorˇ a´k, R., Bartko, P., Hana´k, J., Hofı´ rek, B., Lehocky´, J., Zendulka, I., 1993. Veterina´rna klinicka´ diagnostika vnu´torny´ch choroˆb. Prı´ roda, Bratislava, pp. 41–217. Snyder, C.A., Walle, C.D., 1991. J. Toxicol. Environ. Health 34, 127–139. Soloneski, S., Gonzalez, M., Piaggio, E., Reigosa, M.A., Larramendy, M.L., 2002. Mutat Res./Genet. Toxicol. Environ. Mutagen. 514, 201–212. Soloneski, S., Reigosa, M.A., Larramendy, M.L., 2003. Mutagenesis 18, 505–510. Surralle´s, J., Natarajan, A.T., 1997. Mutat. Res. 392, 165–174. The Pesticide Manual, 2000. A world compendium. In: Tomlin, C. D. S. (Ed.), Version 2.2, 12th ed. The British Crop Protection Council. Villani, P., Andreoli, C., Crebelli, R., Pacchierotti, F., Zijno, A., Carere, A., 1998. Food Chem. Toxicol. 36 (3), 155–164. Zenzen, V., Fauth, E., Zankl, H., Janzowski, C., Eisenbrand, G., 2001. Mutat. Res./Genet. Toxicol. Environ. Mutagen. 497, 89–99.
Ecotoxicology and Environmental Safety 64 (2006) 312–320 www.elsevier.com/locate/ecoenv
Chromosomal aberrations and frequency of micronuclei in sheep subchronically exposed to the fungicide Euparen Multi (tolylfluanid) Irena Sˇutiakova´, Nata´lia Kovalkovicˇova´, Juraj Pistl, Jaroslav Novotny´, Jaroslav Lega´th, Gabriel Kova´cˇ, Sabina Hlincˇı´ kova´, Va´clav Sˇutiak University of Veterinary Medicine, Komenske´ho 73, 041 81 Kosˇice, Slovak Republic Received 9 July 2004; received in revised form 14 April 2005; accepted 21 April 2005 Available online 6 June 2005
Abstract We analyzed chromosome aberrations, micronucleus frequency, mitotic index (MI), and nuclear division index (NDI) in peripheral lymphocytes of sheep subchronically exposed to the fungicide Euparen Multi (containing 50% tolylfluanid). Euparen Multi was administered by rumen sonde to group of Merino sheep (seven sheep/group) at 93 mg/kg body weight (1/20 LD50) daily for 28 days to assess its genotoxic effects. The frequencies of aberrant cells (ABC) in the experimental and control groups were 5.5071.38% and 2.4071.14%, respectively, and the increase in ABC in the treated group was significant (P ¼ 0:003). Significantly increased numbers of chromatid breaks (5.6771.21% against 2.4071.14%; P ¼ 0:001), chromatid gaps (10.3372.73% against 4.0071.23%; P ¼ 0:001), and chromosome gaps (1.8370.75% against 0.8070.45%; P ¼ 0:025) and exchanges (3.1771.94% against 0.2070.45%; P ¼ 0:009) were observed in exposed animals in comparison to control animals. The frequency of micronuclei (MN) was 29.4075.86 per 1000 binucleated cells in peripheral lymphocytes of sheep in the control group and 49.57719.12 per 1000 binucleated cells in the treated group. A significant increase in the frequency of MN in peripheral lymphocytes also was observed between the two groups (P ¼ 0:0477). No statistical differences in MI and NDI values were found in the groups (P ¼ 0:181 and 0.761, respectively). Thus, our results suggest that exposure to Euparen Multi may cause genome damage in somatic cells. r 2005 Elsevier Inc. All rights reserved. Keywords: Sheep; Euparen Multi (tolylfluanid); Chromosomal aberrations; Micronuclei; Mitotic index; Nuclear division index; Genotoxicity
1. Introduction At the present, there are more than 1000 chemicals classified as pesticides. Pesticides are designed to control target pests, either diseases or weeds. They represent an important group of environmental pollutants to which man and other animals are daily exposed, mainly as a consequence of their wide use in agriculture. Exposure to pesticides is likely to continue and possibly may increase in the near future, because of increased use and their persistence in the environment. Moreover, exposure to pesticides has been related to several diseases Corresponding author. Fax: +42155/6331853.
E-mail address: [email protected] (I. Sˇutiakova´). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.04.011
including cancer. Cancer is a complex disease in which cells with altered gene expression grow abnormally, invade other tissues, and disrupt their normal function (Fenech, 2002). For many years cytogenetic alterations in cultured peripheral blood lymphocytes, such as chromosomal aberrations, sister chromatid exchanges, and micronuclei (MN), have been applied as biomarkers of genotoxic exposure and early effects of genotoxic carcinogens (Norppa, 2004). In addition to genotoxicity, stimulation of cellular proliferation may contribute to the carcinogenity of chemicals (Holland et al., 2002). Pesticides are of great concern for their toxic effects and for their potential as a long-term genetic hazard. The most problematic pesticides are fungicides since about 90% of them used currently or formerly have
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been determined to be carcinogenic in experimental animals (Saunders and Harper, 1994). Recently, the genotoxicity of many antifungal agents has been investigated, including fenarimol and propamocarb (Aydemir and Bilaloglu, 2003; Poli et al., 2003), maneb and zineb (Gerber et al., 2002; Soloneski et al., 2002, 2003; Osaba et al., 2002), ferbam (Shanthi and Krishnamoorthy, 2002), carbendazim, captan, and captafol (Rahden-Staron, 2002; Goumenou and Machera, 2003), chlorothalonil (Godard et al., 1999), zinc dimethyl (Zenzen et al., 2001), and thiram (Villani et al., 1998). For this study tolylfluanid, 1,1-dichloro-N-[(dimethylamino)-sulfonyl]-1-fluoro-N-(4-methylphenyl)methanesulfenamide, was chosen because it is heavily used in agriculture and represents one of the most frequently detected pesticides in vegetables and fruit (Nowacka, 2002). Tolylfluanid has a multisite mode of action against phytophagous mites and fungal diseases caused by Botrytis, Didymella, and Drepanopeziza in strawberries, raspberries, hops, apples, ribes, vine, and tomatoes. It is safe to all beneficial organisms (bees, beneficial mites, anthocorids). Tolylfluanid is very rapidly degradated in soil and water and because it is strongly absorbed by soil particles it has no potential to migrate into deeper soil layers (The Pesticide Manual, 2000; EPA, 2002b). In animals, tolylfluanid is rapidly absorbed and hydrolyzed within 48 h to dimethylamino sulfotoluidide and then transformed to the main metabolite 4(dimethylaminosulfonylamino) benzoic acid which can be further demethylated. There is no accumulation in organs and tissues (Abbink and Weber, 1988). Subchronic and chronic toxicity studies conducted in animals showed altered liver enzyme activities and thyroid hormone levels, accompanied by an increased incidence of hyperplastic and neoplastic lesions of the thyroid (primarily adenomas) and damage of renal cortical tubules (PFPC Newsletter Special Issue, 1999). In sheep, tolylfluanid, administered orally, caused anorexia, weakness of the extremities, and loose feces (Hoffmann, 1983). The mutagenicity of tolylfluanid has not yet been firmly established. It was variably active in a variety of in vitro assays, e.g., weakly positive in the Ames test (Herbold, 1979) and positive in the forward mutation in vitro test in mouse lymphoma cells (Hoom, 1985) and in the cytogenetic assay in human lymphocytes (Herbold, 1984a, b). A weakly clastogenic effect of tolylfluanid was observed in Chinese hamster V79 cells in the presence of S9 activation (EPA, 2002a). In vivo, however, it was inactive in mammals (EPA, 2002b). To provide additional genotoxicity data, we report here our recent results on the effect of tolylfluanid in the induction of chromosome aberrations and MN as cytogenetic endpoints in subchronically exposed sheep.
313
2. Materials and methods The organization of the experiment, the investigations conducted, and the related documentation complied with legislative regulations governing the protection of experimental animals of the Slovak Republic. 2.1. Animals The experiment was carried out on 12 clinically healthy 18–24-month-old female sheep (Merino breed) which had no previous exposure to pesticides or any other agents suspected of being genotoxic. There were seven sheep weighing 50.7976.52 kg in the experimental group. The control group consisted of five sheep with mean body weight (b.wt.) 49.1074.98 kg. Animals were housed, treated with antihelminthic agent Aldifal 2.5% susp. a.u.v. (Mevak a.s., Nitra, Slovak Republic) at a dose of 2 mL per 10 kg b.wt., acclimatized for 1 week before dosing, and observed before initiation of the study to ensure that they were healthy. Only animals found to be in a clinically acceptable condition were assigned to the study. During the study, food and water were offered ad libitum. Animals were housed at 8–12 1C and 70% relative humidity. Food consumption, general condition, and any other clinical symptoms were monitored daily. The reference values for ovine pulse and respiratory rate, temperature, and rumen rotations were reported by Slanina et al. (1993). Body weights were recorded weekly. 2.2. Tested pesticide Experimental animals were exposed to Euparen Multi manufactured by Bayer AG, containing 50% tolylfluanid. 2.3. Dose and exposure The tested pesticide preparation was freshly prepared each day and administered by rumen sonde at a dose of 93 mg/kg b.wt. (46.5 mg active ingredient/kg b.wt.; 1/20 LD50) daily for 28 days. The dose was calculated on the basis of the mean LD50 value of tolylfluanid for sheep and its purity as reported by Hoffmann (1983), i.e., 937.57312.5 mg/kg b.wt. (purity 99.2%). 2.4. Cytogenetic assays 2.4.1. Lymphocyte cultures For cytogenetic analysis blood samples were taken from the jugular vein (vena jugularis) into glass tubes containing heparin (100 IU/mL blood). Aliquots of 0.4–0.5 mL of whole blood were cultured at 37.5 1C in 6 mL Panserin 701 chromosome medium supplemented with fetal calf serum, phytohemagglutinin, and
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(PAN Systems GMbH Biotechnologische Produkte, Germany). Penicillin G (100 IU/mL), streptomycin (100 mg/mL), and 7.5% NaHCO3 (PAN Systems GMbH Biotechnologische Produkte) were added to the cultivation medium.
2.5. Cell proliferation assays
2.4.2. Chromosomal aberration analysis Conventional cytogenetic culturing was carried out for 72 h according to the recommendations of Lioi et al. (2004) and Sˇivikova´ and Kalina (1990). These authors observed that in ruminants a longer time of cultivation is more optimal for the detection of chromosomal aberrations than the standard 48 h procedure. Colchicine (10 mg/mL) was added to the medium 1 h before the end of incubation. The cultures were hypotonized with a 0.035 M KCl solution for 30 min and subsequently fixed in 1:3 (v/v) methanol:acetic acid mixture. The slides were stained with 10% conventional hypotonic Giemsa in pH 6.8 phosphate buffer for 5–7 min. For each donor, 100 well-spread metaphases containing 2n ¼ 54, XX chromosomes were scored for chromosomal aberrations. The metaphase cells were evaluated (magnification 1000 ) from each animal. Structural chromosomal aberrations were classified according to Savage (1975). The identification of the individual chromosome pairs was carried out according to the standardized karyotyping of the domestic sheep (Di Berardino et al., 2001). Chromosome-type and chromatid-type aberrations were scored and classified as breaks, gaps, and exchanges. Chromatid breaks were distinguished from gaps (achromatic lesions) when the centric piece was displaced with respect to the chromosome axis or the size of the discontinuity exceeded the width of the chromatid. Exchanges were classified as stable (translocation, when detection was possible) and unstable (dicentric, ring, fragments). Images were taken by imaging microscopy (Nikon) using a CCD-100 camera system (Mitsubishi). The images were processed using the Animal and Photostyler software system.
2.5.1. Mitotic index In the chromosomal aberration study, MI was evaluated by counting at least 1000 cells per treatment and dividing the number of dividing cells (metaphases) by the total number of cells (Preston et al., 1987).
L-glutamine
2.4.3. Micronucleus analysis Lymphocyte cultures were incubated at 37 1C for 72 h. Cytokinesis was blocked by cytochalasin B (Sigma, USA; diluted in dimethyl sulfoxide) which was added at 44 h after culture initiation to achieve a final concentration of 6 mg/mL. The slides were stained with 10% Giemsa–Romanowski in So˜rensen phosphate buffer, pH 6.8, for 3–6 min. Using a Nikon microscope the coded slides were scored blinded at magnifications of 400 and 1000 . The induction of MN was evaluated by scoring a total of 1000 binucleated cells per donor and concentration. MN were identified using well-known criteria (Coutryman and Heddle, 1976; Surralle´s and Natarajan, 1997).
Effects on the cellular proliferation rate were estimated by calculating mitotic index (MI) and nuclear division index (NDI).
2.5.2. Nuclear division index In the micronucleus study, a minimum of 500 lymphocytes was scored to evaluate the percentage of cells with one, two, three, and four or more nuclei. An NDI was calculated according to Eastmond and Tucker (1987) as NDI ¼ [M1+(2M2+3M3+4M4)]/N, where M1–M4 represent the number of cells with one to four nuclei, respectively, and N is the number of cells scored. 2.6. Statistical analysis The comparison of the exposed and control groups was performed by applying the t-test. The Sigma Stat program (Statistical software, Jandel Scientific) was employed for statistical evaluation of the results.
3. Results No overt signs of toxicity were observed during the study. Occasional deceleration of rumen rotations (6.0071.15/5 min) toward the lower limit of reference values (6–16/5 min), sharp breathing sounds, cough, and respiratory rates (30.43714.70/min) at the upper limit of the physiological norm (12–30/min) were observed in animals of the exposed group. These events may have arisen from an irritant effect of the preparation itself or by irritation caused by the frequent application of the sonde. Body weight was not markedly affected by treatment. The mean body weight for animals in the experimental group was 50.7976.52 kg at the beginning and 47.3877.16 kg at the end of treatment. Tables 1 and 3 report the mean values of the cytogenetic parameters analyzed for both groups before exposure to tolylfluanid. The results of the chromosomal aberration analysis for both the exposed and the control groups after 28-day exposure to tolylfluanid are reported in Table 2. The exposed group of animals exhibited a significantly higher frequency of genetic damage (5.5071.38% aberrant cells (ABC)) compared to control animals (2.4071.14% ABC; P ¼ 0:003). As shown in Table 2
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Table 1 Chromosomal aberration frequency in sheep peripheral lymphocytes before subchronic 28-day exposure to Euparen Multi (tolylfluanid) Group
Experimental (n ¼ 7)
Number of metaphase cells analyzed
Chromosomal aberrations B1
B2
G2
G2
Exchanges
No. ABC
(1) (2) (3) (4) (5) (6) (7)
2 2 3 1 2 4 3
0 0 1 0 0 2 0
5 3 2 2 0 6 2
3 0 0 0 0 3 1
3 1 0 0 0 1 1
2 2 3 1 2 3 2
2.43 0.98 0.37 0.42
0.430 0.79 0.3 0.45
2.86 2.04 0.77 0.048
1.00 1.41 0.54 0.57
0.86 1.07 0.40 0.64
2 4 2 2 5
1 0 1 0 2
6 5 7 4 4
2 1 1 1 2
1 1 0 1
3.00 1.41 0.63
0.80 0.84 0.37
5.20 1.30 0.58
1.40 0.55 0.25
0.60 0.55 0.25
100 100 100 100 100 100 100
Statistical mean value 7SD SEM P Control (n ¼ 5)
Statistical mean value 7SD SEM
(8) (9) (10) (11) (12)
100 100 100 100 100
ABC % B/C
0.0286
G/C
MI
0.0386
1.760 1.821 1.791 1.930 1.851 1.682 1.78
2.14 0.69 0.26 0.23
1.802 0.078 0.029 0.149 0.038
4 2 2 4 2.80 1.10 0.49
0.066
1.940 1.871 1.902 1.915 1.740 1.873 0.079 0.035
ABC, aberrant cells; B1, G1, chromatid breaks, gaps; B2, G2, isochromatid breaks, gaps; B/C, number of breaks per cell; G/C, number of gaps per cell; MI, mitotic index; SEM, standard error mean; SD, standard deviation.
and Fig. 1, in the experimental group the frequencies of chromatid aberrations (P ¼0.001), chromosome gaps (P ¼ 0:025), and exchanges (P ¼ 0:009) were significantly higher than those in the control group. Centromere extension of metacentric chromosomes (10%), endoreduplication (1%), and aneuploidy (3%) were also observed in the exposed group. No statistical differences in MI values were found in the groups (Table 2; P ¼ 0:181). The frequency of MN and their distribution for groups examined after 28-day exposure to tolylfluanid are shown in Table 4. The mean value of the total number of MN observed in binucleated peripheral blood lymphocytes of the exposed group was significantly higher than that found in the control group (49.57719.12 against 29.4075.86 MN/1000 binucleated cells; P ¼ 0:0477). No significant reduction of the NDI in exposed animals was observed (Table 4; P ¼ 0:761).
4. Discussion The extensive use of pesticides raises many problems due to their toxicity in non-target organisms, persistence, and combined effects with other agrochemicals and environmental factors. In the past, risk assessment was limited to the danger to human health, but currently
ecological risks are also taken into account. In this regard, further studies are required to determine the mechanism of action of pesticides, including how they interfere with metabolism, the genetic damage that they induce, and their detoxification mechanisms in domestic animals (Lioi et al., 1998). About two-thirds to fourfifths of food animals are exposed to toxic substances (Bo¨hmer et al., 1991) that can be important risk factors to human health (Cristaldi et al., 2004). Free-living grazing animals are among the animal groups immediately exposed to and most affected by the harmful influence of pesticides. We have used sheep as a model animal source of peripheral blood leukocytes because they are similar to wild ruminants and are a better indicator of environmental exposure than rodents kept strictly under laboratory conditions. Cytogenetic biomarkers have been widely used in the genotoxicity assessment of environmental mutagens under in vitro and in vivo conditions. Cytogenetic biomarkers reflect the cellular effects of genotoxic carcinogens, and different cellular processes may be involved in genetic instability in the early or late stages of cancer. The most notable types of instability of the genome in cancer cells involve chromosome rearrangements, and aneuploidy (Murnane, 1996). Although chromosome analysis is not as specific as molecular studies monitoring effects on single genes, it monitors the entire genome.
2.40 1.14 0.51
0.60 0.55 0.25
0 1 0 1 1
0.83 0.41 0.17 0.438
1 1 1 0 1 1
B2
4.00 1.23 0.55
5 4 5 2 4
10.33 2.73 1.12 0.001
14 12 12 9 8 7
G1
0.80 0.45 0.20
1 1 0 1 1
1.83 0.75 0.31 0.025
3 2 1 2 1 2
G2
0.20 0.45 0.20
0 0 1 0 0
3.17 1.94 0.79 0.009
5 3 2 1 2 6
Exchanges
2 4 1 2 3
8 5 6 5 4 5
No. ABC
2.40 1.14 0.51
5.50 1.38 0.56 0.003
ABC %
0.030
0.065
B/C
0.048
0.121
G/C
1.87 0.059 0.026
1.88 1.91 1.85 1.93 1.78
1.77 0.14 0.06 0.181
1.65 1.77 1.89 1.94 1.58 1.81
MI
ABC, aberrant cells; B1, G1, chromatid breaks, gaps; B2, G2, isochromatid breaks, gaps; B/C, number of breaks per cell; G/C, number of gaps per cell; MI, mitotic index; SEM, standard error mean; SD, standard deviation. Experimental sheep no. 3 was not included in statistical analysis because of low mitotic index.
Statistical mean value 7SD SEM
2 3 1 2 4
Control (n ¼ 5)
100 100 100 100 100
5.67 1.21 0.49 0.001
8 6 5 5 5 5
B1
Chromosomal aberrations
Statistical mean value 7SD SEM P
(8) (9) (10) (11) (12)
(1) (2) (4) (5) (6) (7)
Experimental (n ¼ 6)
100 100 100 100 100 100
Number of metaphase cells analyzed
Group
Table 2 Chromosomal aberration frequency in sheep peripheral lymphocytes after daily 28-day exposure to Euparen Multi (tolylfluanid)
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Fig. 1. Sheep chromosomes with different types of aberrations. (a–d) Chromatid breaks, gaps, and exchanges.
In the present study, the possible genotoxic effect of tolylfluanid was assessed by chromosomal aberration analysis and micronucleus assay. Chromosomal aberration analysis revealed an increased number of structural chromosomal aberrations in sheep after subchronic oral exposure to this fungicide. The aberrations induced by tolylfluanid were mainly of the chromatid type. Chromosome breaking agents (clastogens) can be classified as S-dependent and -independent agents (Natarajan, 2002). Most chemicals are S-dependent clastogens, which induce chromatid-type aberrations that are largely unstable and wherein only symmetrical exchanges would be expected to be stable (Kirkland, 1998). However, the chromatid type of aberrations may be converted to the chromosome type, e.g., translocations that persist in organisms for a longer time. Cells with chromosome aberrations may go on to become cancerous, and
chromosome deletions and translocations are observed in most cancer cells (Mitelman et al., 1997; Obe et al., 2002). Stable chromosomal damage is important as an initiating carcinogenic event, but ‘‘unstable’’ damage is important in inducing the loss of heterozygosity during the promotion and progression stages of cancer (Kirkland, 1998; Bonassi and Au, 2002). Chromosome aberrations may indicate alterations in cell homeostasis that are important in genome instability (i.e., interference with DNA synthesis or condensation; Galloway, 1994). The highest frequency of gaps observed in the experimental group was of the chromatid type. It is generally proposed that gaps should not be included in chromosomal aberration frequencies for statistical analysis. However, since some of the gaps could be deletions, it is appropriate to analyze the data excluding
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Table 3 Micronucleus frequencies and distributions in peripheral sheep lymphocytes before subchronic exposure to Euparen Multi (tolylfluanid) Group
Experimental (n ¼ 7)
No. of analyzed cells
(1) (2) (3) (4) (5) (6) (7)
1000 1000 1000 1000 1000 1000 1000
Statistical mean value 7SD SEM P Control (n ¼ 5)
(8) (9) (10) (11) (12)
1000 1000 1000 1000 1000
Statistical mean value 7SD SEM
No. of MN
MN/cell
% MN cells
18 29 23 43 25 19 31
0.018 0.029 0.022 0.043 0.024 0.019 0.029
1.80 2.89 2.20 4.25 2.43 1.89 2.93
26.86 8.57 3.24 0.48
0.026 0.009 0.003 0.4996
2.63 0.84 0.32 0.5103
25 32 23 33 38
0.025 0.031 0.023 0.030 0.038
2.49 3.12 2.29 2.96 3.77
30.20 6.14 2.75
0.029 0.006 0.003
2.93 0.58 0.26
Micronucleus distribution 0
1
2
3
Cells with MN
986 972 978 960 976 981 969
11 27 21 37 23 19 31
2 1 1 3 1 0 0
1 0 0 0 0 0 0
14 28 22 40 24 19 31
NDI
1.114 1.087 1.132 1.183 1.171 1.163 1.169 1.15 0.04 0.01 0.74
976 968 977 968 965
23 32 23 31 32
1 0 0 1 3
0 0 0 0 0
24 32 23 32 35
1.147 1.113 1.108 1.258 1.149 1.16 0.06 0.03
MN, micronuclei; MN/cells, micronuclei per cells; % MN cells, percentage of micronucleated cells; SEM, standard error mean; SD, standard deviation; NDI, nuclear division index.
Table 4 Micronucleus frequencies and distributions in peripheral sheep lymphocytes after subchronic exposure to Euparen Multi (tolylfluanid) Group
Experimental (n ¼ 7)
No. of analyzed cells
(1) (2) (3) (4) (5) (6) (7)
1000 1000 1000 1000 1000 1000 1000
Statistical mean value 7SD SEM P Control (n ¼ 5)
Statistical mean value 7SD SEM
(8) (9) (10) (11) (12)
1000 1000 1000 1000 1000
No. of MN
MN/cell
% MN cells
50 49 40 90 32 49 37
0.050 0.049 0.040 0.090 0.032 0.049 0.037
5.00 4.90 4.00 9.00 3.20 4.90 3.70
49.57 19.12 7.23 0.0477
0.050 0.019 0.007 0.0477
4.957 1.91 0.72 0.0477
25 31 23 30 38
0.025 0.031 0.023 0.030 0.038
2.50 3.10 2.30 3.00 3.80
29.40 5.86 2.62
0.029 0.006 0.003
2.94 0.58 0.26
Micronucleus distribution 0
1
2
3
Cells with MN
951 952 961 917 969 955 964
48 47 38 76 30 41 35
1 1 1 7 1 4 1
0 0 0 0 0 0 0
49 48 39 83 31 45 36
NDI
1.179 1.063 1.108 1.097 1.092 1.091 1.161 1.113 0.043 0.016 0.761
976 969 977 971 965
23 31 23 28 32
1 0 0 1 3
0 0 0 0 0
24 31 23 29 35
1.144 1.084 1.102 1.127 1.141 1.120 0.026 0.012
MN, micronuclei; MN/cells, micronuclei per cells; % MN cells, percentage of micronucleated cells; SEM, standard error mean; SD, standard deviation; NDI, nuclear division index.
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and including gaps (Preston et al., 1987). According to Brogger (1982), gaps are a sensitive indicator of exposure to genotoxic drugs and serve mainly at low doses as a ‘‘guard’’ parameter. Forni (1992) studied the reference values of chromosome aberrations in human peripheral blood lymphocytes as an indicator in genotoxicity studies. This author reported great variability arising from differences in methodological procedures and conditions among laboratories and from intrinsic individual variability. Forni (1992) recommended that the results of chromosome aberration studies be compared with the control group in the same laboratory using the same methodological procedures and conditions. Therefore, we reported the main cytogenetic parameters of both groups of sheep before our experiment. An increased number of chromosomal aberrations in the exposed group occurred simultaneously with the increase in MN. According to Savage (1988) the observed frequencies of MN are often lower than the frequencies of the chromosomal aberrations seen at the first post-treatment metaphase, because not all fragments necessarily form visible MN. Formation of MN and chromosome aberrations are two important cytogenetic endpoints considered to be biomarkers of early biological effects from exposure to mutagenic agents (Bonassi and Au, 2002). Over the past several years the cytokinesis-block micronucleus assay has evolved into a comprehensive method for measuring chromosome breakage, chromosome loss, non-disjuction, necrosis, apoptosis, and cytostasis (Fenech and Crott, 2002). MN are thought to arise from both clastogenic (chromosome breakage) and aneugenic (chromosome lagging and spindle) effects, while structural chromosome aberrations are thought to arise from chromosome breakage and exchange. Cellular proliferation rate is a phenomenon that may be causally associated with the induction of chromosomal damage for a number of compounds which are nonDNA-reactive and thus are threshold in vitro clastogens (Mu¨ller and Kasper, 2000). MI gives direct information of toxic effect on metaphases. Although depression of MI may be the consequence of cell death, it is more often the result of cell cycle delay (Kuroda et al., 1992). The other parameter of cellular proliferation rate that has been studied is NDI which was used for the evaluation of the effect of chemicals on cell division (Eastmond and Tucker, 1987). In our study, MI and NDI were not significantly decreased in exposed animals in comparison with controls. Lymphocyte proliferation is an extremely complex system and is influenced by many factors (Snyder and Walle, 1991). The data on tolylfluanid genotoxicity in animals are limited. Here we report, for the first time to our knowledge, an induction of structural chromosomal aberrations and MN by this fungicide in sheep
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lymphocytes after subchronic oral exposure. Our results suggest that this potential genetic hazard requires examination from the viewpoint of both human and other animal health.
5. Conclusion Our results demonstrate that the fungicide tolylfluanid induces a significant increase in chromosomal aberrations and micronucleated cells. We can conclude that there is a genotoxic effect of this fungicide in sheep. More studies are needed to assess its genotoxicity in other domestic and wild animals, including under in vitro conditions. The findings of this study stress the necessity for the implementation of safety measures both during and after pesticide application to reduce the health hazards of pesticides in humans and other animals.
Acknowledgments This work was supported by VEGA Grant No. 1/ 1362/04 and No. 1/0606/03 and the National Reference Laboratory for Pesticides University of Veterinary Medicine in Kosˇ ice. The authors thank Mr. J. Chabala for his linguistic correction and adaptation of our manuscript to native English language.
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