Mutagenesis, Vol. 16, No. 6, 487-493,
November 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Effect of the dithiocarbamate pesticide zineb and its commercial formulation azzurro. I. Genotoxic evaluation on cultured human lymphocytes exposed in vitro
Laboratorio de Citogenética, Cátedra de Citología, Facultad de Ciencias Naturales y Museo, Calle 37 Numero 668 7mo `B' and 1 Departamento de Matemáticas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 1900 La Plata, Argentina
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Abstract |
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The in vitro cytogenetic effects exerted by the dithiocarbamate fungicide zineb and one of its commercial formulations currently used in Argentina, azzurro, were studied in whole blood human lymphocyte cultures. The genotoxicity of the fungicides was measured by analysis of the frequency of chromosomal aberrations and sister chromatid exchanges (SCEs) and cell cycle progression assays. Both zineb and azzurro activities were tested within the range 0.1–100.0 µg/ml immediately after in vitro lymphocyte stimulation. Only concentrations of 50.0 and 100.0 µg/ml zineb and azzurro induced a significant increase in SCE frequency over control values. Furthermore, this genotoxicity appears to be correlated with its cytotoxicity, measured as cell cycle kinetics, since both a significant delay in cell cycle progression and a significant reduction in proliferative rate index were only observed in those cultures treated with these fungicide concentrations. For both chemicals, a progressive dose-related inhibition of the mitotic activity of cultures was observed when increasing the fungicide concentration. Moreover, only the mitotic activity statistically differed from control values when doses of zineb or azzurro <10 µg/ml were employed. For both fungicides the mitotic index reached the minimal value at doses of 100 µg/ml. Both products induced a significant dose-dependent increase in the number of abnormal cells, chromatid-type and chromosome-type aberrations as well as in the total number of aberrations in the 0.1–100.0 µg/ml dose range. Based on these results, the evaluation of zineb as a controversial genotoxic/non-genotoxic compound for human health should be reconsidered. Instead, we demonstrate that the fungicide induces large DNA alterations and should be considered as a clastogenic mutagen.
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Introduction |
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Large amounts of pesticides are released daily into the environment and, hence, they represent a potential hazard not only to the human genetic material, but also to other living species, since their residues and derivatives are known to contaminate field crops. Several reports have demonstrated that several commonly used pesticides have genotoxic properties (IARC, 1976
, 1986, 1987, 1991).
Dithiocarbamates are agrochemicals with a variety of applications. Dithiocarbamate fungicides are currently used mainly for eradication of fungal infections on fruit plants and vegetables. These compounds have produced conflicting results in mutagenicity tests, conclusions varying according to both the test and the compound employed (IARC, 1976, 1987, 1991). Carcinogenic and teratogenic properties have been reported for ethylene thiourea, the main metabolic and degradation product of ethylene bis(dithiocarbamate) pesticides (IARC, 1987).
Among the zinc-containing ethylene bis(dithiocarbamate) pesticides, zineb [ethylene bis(dithiocarbamate)zinc] is a widely used foliar fungicide and is registered for use on a large number of fruits, vegetables and field crops, as well as on a large number of ornamental plants and for the treatment of many seeds. Zineb is also registered for use as a fungicide in paints and for mold control on fabrics, leather, linen, painted surfaces, surfaces to be painted and paper, plastic and wood surfaces (Environmental Protection Agency, 1974). Though the mutagenic activity of ethylene thiourea is well documented (IARC, 1987), very little is known about the deleterious effects of zineb. The available reported data do not allow a proper evaluation of the carcinogenicity of zineb. Plate incorporation assays with Salmonella typhimurium demonstrated a direct non-mutagenic effect of the pesticide, whereas mitotic chromosome malsegregation, gene conversion and point mutation assays with Saccharomyces cerevisiae and Bacillus subtilis gave positive results (Shiau et al., 1980; Franekic et al., 1994; Della Croce et al., 1996). Zineb exerted a high dose-related cytotoxicity in BALB/c 3T3 mouse cells in vitro, but only in the absence of an exogenous metabolizing system (Perocco et al., 1995). Trigathy et al. (1988) reported zineb as a positive genotoxic agent in somatic and germ cells of Drosophila. While Chernov and Khitsenko (1969) observed an increased incidence of lung tumors after its oral administration to C57BL mice, negative results have also been reported in both mouse strains (Innes et al., 1969) and in rats (Blackwell-Smith et al., 1953; Andrianova and Alekseev, 1970). Moreover, after its s.c. administration systematic reticulum cell sarcomas and a variety of sarcomas were observed in mice (NTIS, 1968) and rats (Andrianova and Alekseev, 1970), respectively. In humans, development of sulphemoglobinemia, hemolytic anemia and Heinz body formation have been reported in a person suffering from hypocatalasemia after contact with zineb (Pinkhas et al., 1963). Finally, an increase in the frequency of chromosomal aberrations was observed in lymphocytes of persons occupationally exposed to zineb (Pilinskaya, 1974).
In the present study the in vitro cytogenetic effects on human lymphocytes exerted by zineb and one of its commercial formulations currently used in Argentina, azzurro, were studied using the frequencies of chromosomal aberrations and sister chromatid exchanges and cell cycle progression as genetic end-points.
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Materials and methods |
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Chemicals
Zineb [ethylene bis(dithiocarbamate)zinc, CAS no. 12122-67-7] was obtained from Riedel-de Haën (Pestanal; Hannover, Germany). Azzurro (70% zineb) was kindly provided by Chemiplant (Buenos Aires, Argentina). Dimethyl sulfoxide (DMSO) was purchased from Sigma Chemical Co. (St Louis, MO).
Blood samples
Blood samples were obtained from six healthy male non-smoking volunteer donors (age 20–30 years) selected according to previously described recommendations (Bianchi et al., 1979). Samples of 20 ml of blood were drawn from each donor by venipuncture immediately prior to culturing.
Whole blood lymphocyte cultures and pesticide treatment
Whole blood cultures were set up by inoculating 1.0 ml of whole blood in 9.0 ml of culture medium [90% Ham's F10 (Gibco, Grand Island, NY), 10% fetal calf serum (Gibco), 100 U penicillin/ml (Gibco), 10 µg streptomycin/ml (Gibco) and 10 µg BrdUrd/ml (Sigma Chemical Co.)]. Immediately after seeding, zineb and azzurro, dissolved in DMSO, were diluted in culture medium such that addition of 100 µl to cultures would achieve the desired drug concentration. The final solvent concentration was <1% for all treatments. Zineb and azzurro were used at final concentrations of 0.1, 1.0, 5.0, 10.0, 25.0, 50.0 and 100.0 µg/ml. For each donor, four negative controls (two untreated and two solvent/vehicle-treated cultures) were performed and run simultaneously with 14 zineb (donors 1–3)- or azzurro-treated (donors 4–6) cultures. None of the treatments produced significant pH changes in the culture medium, even at the highest concentrations of zineb and azzurro. Immediately after pesticide treatment, 0.3 ml of phytohemagglutinin M (Gibco) was added to each culture (0 h). After treatment, cells were incubated at 37°C in a 5% CO2 atmosphere for 48 and 72 h. During the last 3 h of culture the cells were treated with 0.1 µg/ml colchicine (Sigma Chemical Co.). At the end of the culture period cells were harvested, exposed to a hypotonic solution (0.075 M KCl, 37°C, 15 min) and fixed in methanol/acetic acid (3:1). Chromosome spreads were obtained using the air drying technique. The same batches of culture medium, sera and reagents were used throughout the study.
Fluorescence-plus-Giemsa (FPG) staining for sister chromatid differentiation
Chromosome spreads were stained using the FPG technique for sister chromatid differentiation as described in detail by Larramendy and Knuutila (1990). All cytogenetic tests slides were coded and scored blind by two cytogeneticists.
Cell cycle kinetics and mitotic index
A minimum of 200 metaphase cells per sample were scored to determine the percentage of cells which had undergone one (M1), two (M2) or three or more mitoses (M3+). All those metaphases showing differential staining of sister chromatids in <25% of the chromosomal complement were considered to be in at least the fourth cell cycle. The proliferative rate index (PRI) was calculated for each experimental point according to the formula PRI = [(%M1) + 2(%M2) + 3(%M3+)]/100, which indicated the average number of times the cells had divided in the medium between incorporation of BrdUrd and harvesting (Lamberti et al., 1983). The mitotic index (MI) was determined by scoring 2000 cells for each experimental point from each donor and are expressed as number of mitoses among 1000 nuclei. Changes in MI were expressed as a factor (f) of the mean MI from treated cultures (MIt) over the mean MI from controls (MIc) (f = MIt/MIc) (Miller and Adler, 1989).
Sister chromatid exchange analysis
For the SCE assay a total of 50 well-spread diploid metaphases were scored per treatment for each donor in M2 cells. The data are expressed as the mean number of SCEs per cell ± SE.
Chromosomal aberrations
Frequencies of chromosome aberrations were scored in 100 well-spread metaphases per experimental point in M1 cells from 48 h cultures. Data for each experimental point are the combined values from two experiments run simultaneously. Aberrations were scored according to the recommendations of the International System for Human Cytogenetic Nomenclature (ISCN, 1995). Achromatic lesions smaller than the width of a chromatid and continuous with the chromosome axis were considered chromatid or isochromatid gaps, respectively, and were not included in the scoring. Chromatid and chromosome aberrations were scored separately and expressed as the number of aberrations per 100 cells.
Statistical analysis
The Kruskal–Wallis one way analysis of variance was used to compare differences among donors and treatments. The two-tailed Student's t-test was used to compare SCE frequencies between treated and control groups. A 
2 test was used for cell cycle progression, MI data and chromosomal aberration incidence. The level of significance chosen was 0.05 unless indicated otherwise.
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Results |
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Table I
shows the frequencies of SCEs obtained after treatment with the different doses of zineb and azzurro in whole blood cultures. Since no differences in SCEs were observed between untreated whole blood cultures and DMSO-treated whole blood cultures (negative controls), pooled data are presented for control cultures.
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Only concentrations of 50.0 and 100.0 µg/ml zineb induced a significant increase in SCE frequency over control values in those lymphocytes harvested 72 h after treatment (P
0.01). Furthermore, this genotoxicity appears to be correlated with its cytotoxicity, measured as cell cycle kinetics, since both a significant delay in cell cycle progression and a significant reduction in PRI were only observed in treated cultures. At 48 h after treatment either a significant decrease in the frequency of M2 and a significant increase in the frequency of M1 or a reduction in PRI were observed in those cultures treated with 50.0 and 100 µg/ml pesticide with respect to control values (Figure 1A and Table II , P 0.01). Similarly, in those cultures harvested at 72 h after treatment a significant decrease in the frequency of M3+ and a significant increase in the frequency of M2 and M1 as well as a decrease in PRI values were observed in treated cultures with respect to control values (Figure 1B and Table II , P 0.01).
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Azzurro induced a significant increase in SCE frequency over control values in those lymphocytes harvested at 72 h after treatment only when concentrations of 50.0 and 100.0 µg/ml were employed (Table I
, P 0.01). Figure 2 and Table II also demonstrate induction of a significant delay in cell cycle progression and a significant reduction in PRI in those cultures treated with these fungicide concentrations. At 48 h after treatment a significant decrease in the frequency of M2, concomitant with a significant increase in the frequency of M1 and a reduction in PRI, were observed with respect to control values (Figure 2A and Table II , P 0.01). In cultures harvested at 72 after treatment a significant decrease in the frequency of M3+ and a significant increase in the frequency of M2 and M1, as well as a reduction in PRI, were observed in cultures treated with 50.0 and 100.0 µg/ml azzurro with respect to control values (Figure 2B and Table II , P 0.01).
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The MI data for both zineb- and azzurro-treated cultures are presented in Figure 3
. For both chemicals, a progressive dose-related inhibition of mitotic activity of cultures was observed when doses ranging from 10.0 to 100.0 µg/ml were employed at 48 and 72 h after treatment (P 0.01). Overall, when 100.0 µg/ml zineb was used the mitotic activities of lymphocytes from 48 and 72 h cultures were decreased over control values (f = 1.00) by a mean f of 0.36 (range 0.31–0.42) and 0.35 (range 0.19–0.44), respectively (Table II). Similarly, after 100.0 µg/ml azzurro the decrease in mitotic activities reached mean f values of 0.22 (range 0.14–0.31) and 0.36 (range 0.31–0.43) in cultures harvested 48 and 72 h after treatment, respectively (Table II).
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Tables III and IV
show the effect of zineb and azzurro on the induction of chromosomal aberrations in lymphocytes cultures, respectively, while the average values obtained within all donors are depicted in Figure 4. Since no differences in chromosomal aberration values were observed between untreated whole blood cultures and DMSO-treated whole blood cultures (negative controls), pooled data are presented for control cultures. Both products were able to induce a significant increase in the number of chromosomal aberrations of both chromatid- and chromosome-type in the 0.1–100.0 µg/ml dose range (P 0.01). However, chromatid-type exchanges as well as chromosome-type breaks were rarely observed (Tables III and IV). Overall, zineb induced a significant dose-dependent increase in the number of abnormal cells (r = 0.84, P 0.01, donors 1–3), chromatid-type (r = 0.86, P 0.01, donors 1–3) and chromosome-type aberrations (r = 0.91, P 0.01, donors 1–3) and total number of aberrations (r = 0.89, P 0.01, donors 1–3) (Figure 4A). Similarly, a significant dose-dependent increase in the number of abnormal cells (r = 0.70, P = 0.05, donors 4–6), chromatid-type (r = 0.73, P 0.05, donors 4–6) and chromosome-type aberrations (r = 0.83, P 0.05, donors 4–6) and total number of aberrations (r = 0.79, P < 0.05, donors 4–6) was observed after azzurro treatment (Figure 4B).
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Discussion |
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In the present report the genotoxicities of zineb and the zineb-containing commercial formulation azzurro were evaluated in in vitro cultures of human lymphocytes using three different genetic end-points, namely analysis of chromosomal aberrations in first mitosis, the frequency of SCEs in second mitosis and the kinetics of cell cycle progression. The results demonstrate that both products induce a significant dose-dependent increase in the number of abnormal cells and chromatid-type and chromosome-type aberrations, as well as in the total number of aberrations, in the 0.1–100.0 µg/ml dose range. Moreover, the frequency of SCEs and cell cycle progression were altered only when doses of 50.0 and 100.0 µg/ml were employed. The results obtained demonstrate that all the assays employed were sensitive enough to detect the genotoxicity of the fungicide tested. These data support the view of many authors who have indicated the high sensitivity of the end-points we used in genotoxicity testing (WHO, 1985
) and that zineb can be considered as an inducer of structural chromosome aberrations and a weak inducer of SCEs.
In our experiments the test compounds were added to whole blood lymphocyte cultures to exploit the metabolic capability of whole blood compared with isolated lymphocytes (Ray and Altenburg, 1978; Norppa et al., 1980). With no information on the rate of metabolism of zineb and azzurro under these conditions, a long treatment time was chosen leading to a total culture time of up to 72 h. Such a culture time is not optimal for the detection of chromosomal aberrations, whose frequency may be underestimated as a consequence of the presence of second mitosis. In order to avoid this problem, a harvesting time of 48 h after treatment was selected and chromosomal aberrations in the first metaphase after in vitro stimulation were analysed using a FPG method for sister chromatid differentiation, which makes the restriction of scoring chromosomal aberrations in first mitosis only feasible. However, it is well known that the FPG methodology produces some chromosomal swelling, which can make the identification of chromosomal aberrations difficult. Hence, the frequency of chromosomal aberrations detected with this method will be lower than those obtained with other conventional staining method for chromosomal morphology, e.g. Giemsa or carbol fuchsin staining. Even if the frequencies of chromosomal aberrations we found after both fungicide treatments were underestimated with the protocol adopted, this would not modify the basic conclusion that zineb and the zineb-containing formulation azzurro were both positive in this assay.
Furthermore, no major differences in responses between pure zineb and the commercial product azzurro in inducing chromosomal damage and proliferative effects were observed. Accordingly, it can be assumed that the effects induced by azzurro can be accounted for by the zineb component of the mixture and that a non-clastogenic agent(s) other than zineb might be present in the technical formulate. Unfortunately, the identities of the components of the commercial product were not made available to us.
The molecular mechanism that gives rise to SCEs is not yet completely understood. Years ago, Painter (1980) proposed a replication model for the mechanism leading to SCEs. He suggested that during S phase double-strand breaks originate at the junctions between completely and partially duplicated replicon clusters. Accordingly, agents that induce absolute blocks to DNA fork displacement will allow more time for double-strand breaks to accrue at junctions, favouring abnormal recombination of DNA strands with the subsequent appearance of SCEs. On the other hand, agents that inhibit initiation of clusters will rarely cause SCE formation. Painter's hypothesis could furnish an explanation for the higher frequency of SCEs observed over control values if we assume that the lengthening of the generation time observed in these cells reflects a lengthening of their S period. Accordingly, the rate of DNA fork displacement in lymphocytes treated within the 50.0–100.0 µg/ml dose range of both zineb and azzurro could be slower than in control cultures and in those cultures treated with lower pesticide concentrations, resulting in an increase in SCEs. In support of the above finding is the fact that the pesticide doses that were able to induce an increase in the frequency of SCEs over control values were essentially similar to the concentrations that caused a significant delay in cell cycle kinetics.
Even though it has been demonstrated that zineb can be considered as a non-mutagenic agent in bacterial systems, this does not necessarily imply that the pesticide cannot directly damage genetic material. In bacterial systems point mutations and small alterations, but not large DNA alterations, are often detected (IARC, 1976). On the other hand, the detection of chromosomal aberrations in lymphocytes from crop workers occupationally exposed to zineb previously reported by Pilinskaya (1974) and the findings we report here on its ability to induce both chromosomal aberrations and SCEs in vitro demonstrate that zineb induces large DNA alterations and thus should be considered a clastogenic mutagen. Furthermore, considering that large amounts of this dithiocarbamate pesticide are released into the environment daily and that its residue tolerance for most raw agricultural crops has been established at 7 mg/kg (Environmental Protection Agency, 1974), caution should be taken since zineb represents a potential hazard to the genetic material.
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Acknowledgments |
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The authors thank M.Mendiburu for performing the statistical analyses. This study was supported by the National Agency of Scientific and Technological Promotion (contract grant no. BID 802/OC-AR PICT no. 01-00000-00753), the National Council of Scientific and Technological Research (CONICET), the Commission of Scientific Research of Buenos Aires Province (CIC) and the National University of La Plata, Argentina (grant no. 11/N325).
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2 To whom correspondence should be addressed. Fax: +54 221 423 3340; Email: m_larramendy{at}hotmail.com
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References |
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Received on November 11, 2000; accepted on June 19, 2001.
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