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The investigation of the genotoxic effects of fenarimol and propamocarb in mouse bone marrow in vivo
Toxicology Letters 147 (2004) 73–78
The investigation of the genotoxic effects of fenarimol and propamocarb in mouse bone marrow in vivo Nilüfer Aydemir∗ , Rahmi Bilalo˘glu Department of Biology, Faculty of Arts and Science, University of Uludag, 16059 Görükle, Bursa, Turkey Received 16 June 2003; received in revised form 21 October 2003; accepted 23 October 2003
Abstract In this study, we aimed to evaluate the genotoxic effects of fungicides fenarimol and propamocarb which are used to protect crops from fungi. For this reason, bone-marrow micronucleus and chromosome aberration tests were carried out in Swiss albino mice. Mice were injected with four different doses of fenarimol and propamocarb intraperitoneally; 50, 100, 200 and 400 mg/kg b.w. Fenarimol did not induce any significant increase in micronucleated erythrocytes after 24, 36, and 48 h treatment but it decreased the ratio of polychromatic/normochromatic erythrocytes at all dose groups and sampling intervals. Fenarimol did not increase the number of chromosome aberrations significantly, but it reduced the mitotic index at the higher doses (P < 0.05). Propamocarb did not increase the frequency of micronucleated erythrocytes, but decreased the polychromatic/normochromatic erythrocytes ratio at all sampling intervals. Propamocarb increased only gaps in total chromosome aberrations, but when gaps were excluded, there were no significant differences in total aberrations between the control and dose groups (P > 0.05). Propamocarb also reduced the mitotic index compared with the negative control group (P < 0.001). Contributing these results, we can suggest that fenarimol and propamocarb are non-genotoxic in mouse bone marrow in vivo but have cytotoxic effects. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Fungicides; Propamocarb; Fenarimol; Micronucleus; Chromosome aberrations; Mouse; Bone marrow
1. Introduction Pesticides are chemicals used to control agricultural pests. Their widespread application with undesirable side effects represents a potential risk to human and environmental health. Unintended exposure to pesticides can occur from environmental residues after application (Albert et al., 1992). ∗ Corresponding author. Tel.: +90-224-4429258; fax: +90-224-4428136. E-mail address: [email protected] (N. Aydemir).
Fungicides also are very important and widely used pesticides. The fungicide fenarimol belongs to the chemical class of halogenated pesticides (which also include chemicals such as DDT) and known as tumor promoters and hepatocarcinogens in rodents (Flodstrom et al., 1990). Fenarimol is a pyrimidine carbinol fungicide with activity against several Ascomycetes, Basidiomycetes and Fungi imperfecti (Paolini et al., 1996a). Toxicological data on fenarimol are controversial and incomplete. Fenarimol did not induce unscheduled DNA synthesis in primary rat hepatocytes (Bellicampi et al., 1980). It did not
0378-4274/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2003.10.015
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N. Aydemir, R. Bilalo˘glu / Toxicology Letters 147 (2004) 73–78
induce chromosomal aberrations in rodents and gave negative results in mutagenicity tests with bacteria (EPA, 1985). On the other hand, fenarimol showed positive results in the fluorometric unwinding test assessing the induction of DNA single strand breaks in rat hepatocytes in vivo (Grilli et al., 1991). The other fungicide propamocarb is widely used in the greenhouse-based production of vegetables and fruits in Turkey and Southern Europe. Propamocarb is a carbamated fungicide and used particularly against Aphanomyces, Phytophtora and Phytium (Fernandez-Alba et al., 2001). The mutagenic properties of various carbamated compounds have been investigated (Vasudev and Krishnamurthy, 1994). However, there is no report on the genotoxic effects of propamocarb in any mutagenicity test systems. In this study we decided to detect the clastogenicity and aneugenicity of propamocarb and the suspected mutagen, fenarimol, by metaphase analyses and micronucleus tests in Swiss albino mice bone marrow in vivo.
2. Materials and methods 2.1. Test animals Swiss albino mice were obtained from the Laboratory Animal Center of Uludag University (Bursa, Turkey). They were 8–10-week-old and housed in plastic cages with a bedding of wood shavings. Animals weighed 25–30 g and were fed with fresh standart pellet and given tap water ad libitum. All mice were kept under constant environmental conditions with a 12–12 h light–dark cycle. This study has been approved by the Ethical Committee on Animal Experiments of Uludag University. 2.2. Test chemicals Fenarimol (2,4-dichloro-pyrimidin-5-yl benzydil alcohol, Cas no: 60168-88-9) was obtained from Dow Elanco Europe Laboratoires and the purity of this compound is 99.4%. Propamocarb (propyl-3-dimethylamino-propyl carbamate, Cas no: 24579-73-5), a liquid compound that is an active substance of Previcur-N producted by Schering Agrochemicals. The purity of this fungicide
is 72.2% and it was obtained from the Uludag University, Agricultural Faculty. Other chemicals, Hanks balanced salt solution (HBSS), fetal calf serum (FCS) and ethyl methane sulfonate (EMS) was purchased from Sigma–Aldrich Chemical Company. 2.3. Experimental protocol The intraperitoneal (i.p.) route of application was used in all experiments. Corn oil solvent was used for fenarimol and it was administered in a single doses of 50, 100, 200 and 400 mg/kg b.w., suspensioned in corn oil. These doses were selected according to the LD50 of fenarimol for mice and this was 600 mg/kg b.w. (Paolini et al., 1996b). Control mice were injected with only corn oil. The fungicide propamocarb (Previcur-N) was diluted in sterile distilled water. The LD50 of propamocarb was 1600 mg/kg for mice and we injected mice in single doses of 50, 100, 200 and 400 mg/kg b.w. per mouse. The negative control mice were injected with distilled water. For two fungicides, the highest dose was tolerated by the animals with minimal toxic symptoms, such as tremor and loss of appetite. However, the animals recovered within 1 h of the treatment. EMS was used as a positive control and given in a single dose of 300 mg/kg b.w. per mouse. 2.4. Procedures For metaphase analysis, negative control group included 12 mice. Positive control and all dose groups consisted of eight mice. The sampling time was 24 and 2 h before sacrifice, mice were injected with 4 mg/kg colchicine. Bone marrow metaphase slides were prepared according to the Adler (1984). The bone-marrow cells were collected from both femura by flushing in Hank’s balanced salt solution and cells were centrifuged at 1000 rpm for 5 min. After centrifugation, bone-marrow cells were treated with KCl (0.56%) and incubated at room temperature for 10 min. Following incubation, the material was centrifuged. The supernatant was discharged and the cell pellet was fixed with cold 1:3 (v/v) acetic acid–methanol. Fixation was repeated twice with an interval of 10 min and last fixation step was done for 30 min at 4 ◦ C. After
N. Aydemir, R. Bilalo˘glu / Toxicology Letters 147 (2004) 73–78
the centrifugation, pellet was resuspended with fresh fixative and two or three drops of the fixed material were dropped on a clean slide and placed on to slide warmer to dry. Slides were stained in 5% Giemsa for 5 min. The scoring and classification of aberrations were done as described by Preston et al. (1987). Structural and numerical chromosomal aberrations (CAs) were scored in 50 metaphases for each mouse. Gaps were both included and excluded from the total number of CAs. The mitotic index was also recorded. In the micronucleus test, all dose groups contained six mice. Negative control included 12, and the positive control included 4 mice respectively. The sampling intervals were 24, 36 and 48 h and the micronucleus test was performed according to the Adler (1984). For each sampling interval, the animals were killed and the femurs of each animal were dissected out. The bone marrow was flushed with fetal calf serum and the suspension was centrifuged. The supernatant was discharged and a few drops of fetal calf serum was added. The pellet was mixed and dropped on to clean slide. The slides were air dried for 12 h and stained with a combination of May Grünwald–Giemsa. Total of 2000 polychromatic erythrocytes (PCEs) per mouse were analyzed for the presence or absence of micronuclei. The frequency of micronucleated normochromatic erythrocytes (MNNCEs) was also recorded. The ratio of PCEs per normochromatic erythrocytes (NCEs) was also calculated to determine the cytotoxic effects of chemicals. Data were analyzed using the non-parametric Kruskal–Wallis and Tukey type multiple comparisons tests. All statistical analysis were done by the Statistica for Windows programme.
3. Results The results for the micronucleus tests and metaphase analysis are given in Tables 1 and 2 for fenarimol and propamocarb. Fenarimol did not increase the frequency of MNPCEs and MNNCEs compared with the negative control and at all treatment periods (24, 36 and 48 h). However, it decreased the PCE/NCE ratio for 100, 200 and 400 mg/kg dose groups compared with the control, at the 24 and 48 h treatment times (P < 0.001). Also, fenarimol significantly reduced the PCE/NCE
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ratio for 36 h, at the 200 and 400 mg/kg dose groups (P < 0.001). Fenarimol did not induce a significant increase in the frequency of chromosomal aberrations. It affected the mitotic index at the 200 and 400 mg/kg dose group (6.22 and 6.43%; P < 0.05) compared with the negative control. There was no significant elevation with the fungicides propamocarb in the frequency of MNPCEs and MNNCEs for the 24, 36 and 48 h sampling intervals (Table 1). Propamocarb decreased the PCE/NCE ratio significantly for 24, 36 and 48 h at the 100, 200 and 400 mg/kg dose groups compared with the negative control (P < 0.001). Propamocarb significantly increased the frequency of total aberrations including gaps only at the 400 mg/kg dose group compared with the negative control group (P < 0.01). Propamocarb did not increase the frequency of total aberrations when gaps were excluded. Propamocarb reduced the mitotic index in both the 200 and 400 mg/kg dose groups (P < 0.001).
4. Discussion In this study, we have demonstrated that two fungicides fenarimol and propamocarb did not show any genotoxic effect in mouse bone marrow with two short-term tests. However, both fungicides were found to be cytotoxic. In our study fenarimol did not induce any micronucleated erythrocytes and chromosomal aberrations. Cantelli-Forti et al. (1993) and Paolini et al. (1996a) showed that fenarimol increased the number of micronucleated erythrocytes in Swiss albino CD-1 mice at doses between 37.5 and 300 mg/kg for 24 h. Although these results contradict our findings, there are other studies that agree with our data. When fenarimol was given to Swiss albino mice at doses of 50 and 100 mg/kg, it did not increase the number of micronuclei in spleen and bone marrow at different treatment times (Tyrkiel and Ludwicki, 1992). Also Tyrkiel et al. (1996) gave 200 mg/kg fenarimol for 21 days to mice and they did not find any micronuclei in bone marrow or peripheral blood erythrocytes. It has been reported in the studies of Cantelli-Forti et al.(1993) and Paolini et al. (1996a) that
76
Treatments
Dose (mg/kg)
n
MNPCE/ 1000 PCE
MNNCE/ 1000 NCE
Corn oil fenarimol
0 50 100 200 400
D.W. propamocarb
EMS
12 6 6 6 6
24 h 1.83 1.91 2.00 2.16 2.00
± ± ± ± ±
1.02 0.97 0.89 1.03 1.09
0.70 0.50 0.58 0.83 0.75
± ± ± ± ±
0.54 0.44 0.49 0.51 0.52
1.01 0.94 0.84 0.60 0.56
± ± ± ± ±
0 50 100 200 400
12 6 6 6 6
1.62 2.00 2.08 2.00 1.83
± ± ± ± ±
0.85 1.00 1.06 1.14 0.81
0.66 0.75 0.83 0.41 0.58
± ± ± ± ±
0.49 0.88 0.68 0.37 0.58
1.00 0.98 0.77 0.59 0.57
± ± ± ± ±
300
4
11.0 ± 2.94
0.16 ± 0.18
PCE/NCE
MNPCE/ 1000 PCE
MNNCE/ 1000 NCE
0.02 0.05 0.06∗∗∗ 0.05∗∗∗ 0.05∗∗∗
36 h 1.79 2.08 2.00 2.16 1.83
± ± ± ± ±
0.81 0.97 1.14 1.12 0.87
0.70 0.66 0.83 0.91 0.83
± ± ± ± ±
0.54 0.60 0.40 0.37 0.81
1.00 0.90 0.95 0.59 0.52
± ± ± ± ±
0.06 0.04 0.09∗∗∗ 0.09∗∗∗ 0.09∗∗∗
1.70 1.83 2.00 1.91 1.66
± ± ± ± ±
0.89 0.81 0.89 0.80 0.75
0.62 0.58 0.83 0.75 0.66
± ± ± ± ±
0.48 0.49 0.51 0.52 0.31
1.00 0.97 0.69 0.57 0.55
± ± ± ± ±
0.30 ± 0.10
13.0 ± 4.70
0.00 ± 0.00
PCE/NCE
MNPCE/ 1000 PCE
MNNCE/ 1000 NCE
0.10 0.05 0.07 0.06∗∗∗ 0.04∗∗∗
48 h 1.58 1.66 1.83 1.75 1.75
± ± ± ± ±
0.99 0.87 1.03 1.12 0.93
0.66 0.75 0.66 0.83 0.75
± ± ± ± ±
0.38 0.52 0.40 0.25 0.41
0.98 0.83 0.59 0.64 0.48
± ± ± ± ±
0.05 0.05 0.06∗∗∗ 0.06∗∗∗ 0.02∗∗∗
0.06 0.02 0.10∗∗∗ 0.07∗∗∗ 0.06∗∗∗
1.58 1.66 1.83 1.75 1.50
± ± ± ± ±
0.82 0.60 0.98 1.03 0.63
0.66 0.50 0.75 0.58 0.50
± ± ± ± ±
0.53 0.44 0.52 0.37 0.54
0.97 0.89 0.69 0.57 0.56
± ± ± ± ±
0.05 0.08 0.10∗∗∗ 0.06∗∗∗ 0.06∗∗∗
0.28 ± 0.10
13.25 ± 2.90
0.00 ± 0.00
PCE/NCE
0.25 ± 0.30
2000 PCEs were analyzed per animal. Abbreviations: n, number of animals per group; h, sampling interval (hour); D.W., distilled water, MNPCE, micronucleated polychromatic erythrocytes; MNNCE, micronucleated normochromatic erythrocytes; PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; EMS, ethyl methanesulfonate-positive control. All data are presented as mean ± standard deviation. ∗∗∗ P < 0.001; significant when compared with the negative control.
N. Aydemir, R. Bilalo˘glu / Toxicology Letters 147 (2004) 73–78
Table 1 The Frequency of MNPCE, MNNCE and PCE/NCE ratios in mice bone marrow injected with fenarimol and propamocarb at 24, 36 and 48 h intervals
N. Aydemir, R. Bilalo˘glu / Toxicology Letters 147 (2004) 73–78
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Table 2 Mitotic index, distribution of the different types of chromosomal aberrations observed in swiss-albino mice bone-marrow cells Treatments
Dose (mg/kg)
n
MI (M ± S.D., %)
Gapsa C
IC
Breaksa
Acentric fragmentsa
C
IC
C
IC
TA + Gapsb (M ± S.D.)
TA-Gapsb (M ± S.D.)
Corn oil fenarimol
0 50 100 200 400
10 8 8 8 8
7.82 7.27 7.40 6.22 6.43
± ± ± ± ±
0.45 0.49 0.41 0.90∗ 0.94∗
4 3 3 4 3
2 0 2 3 2
0 0 0 1 0
0 0 0 0 0
2 0 1 0 0
0 0 0 0 0
0.80 0.37 0.75 1.00 0.62
± ± ± ± ±
0.42 0.50 0.40 1.30 0.50
0.10 0.00 0.13 0.13 0.00
± ± ± ± ±
0.31 0.00 0.30 0.30 0.00
D.W. propamocarb
0 50 100 200 400
10 8 8 8 8
7.52 5.85 5.45 5.22 4.10
± ± ± ± ±
0.65 0.52 0.68 0.57∗∗∗ 0.56∗∗∗
4 3 7 5 4
3 1 3 4 10
1 0 1 0 1
0 0 0 0 0
1 1 2 1 2
0 0 1 0 0
0.90 0.62 1.75 1.25 2.12
± ± ± ± ±
0.70 0.50 1.30 1.00 0.90∗∗
0.20 0.13 0.50 0.13 0.38
± ± ± ± ±
0.40 0.30 1.41 0.30 0.51
EMS
300
8
5
9
13
4
6
3
5.00 ± 2.97
6.73 ± 0.71
3.25 ± 2.18
Fifty metaphases were analyzed per animal. Abbreviations: n, number of animals per group; MI, mitotic index; M ± S.D., mean ± standard deviation; C, chromatid type; IC, isochromatid type; TA, total aberrations; D.W., distilled water. a Number of aberrations in total analyzed cells. b Number of aberrations/number of cells analyzed. ∗ P < 0.05; significant when compared with the negative control. ∗∗ P < 0.01; significant when compared with the negative control. ∗∗∗ P < 0.001; significant when compared with the negative control.
fenarimol may act as an aneugen, rather than a clastogen. However, there is no evidence or report on the aneugenic effect of fenarimol such as the presence of centromeric heterochromatin in micronuclei induced by this fungicide. Moreover fenarimol induced different cytochrome P450 enzymes (CYP) in various tissues of Sprague–Dawley rats. This indicates the cotoxic–cocarcinogenic potential of fenarimol and it may act as a tissue specific tumor promoting agent (Paolini et al., 1996b). It is suggested in this study that the CYP induction may have a direct role in the development of malignancy and this is particularly important for non-genotoxic carcinogens such as many pesticides (Paolini et al., 1996b). In general, CYP enzyme inducers have been known to be non-genotoxic hepatocarcinogens such as DDT and related compounds (Kostka et al., 2000). It is known that fenarimol belongs to the chemical class of halogenated pesticides which also include chemicals such as DDT (Paolini et al., 1996a). Fenarimol seems to act as a hepatocarcinogen and this type of chemicals may give negative results in short-term mutagenicity tests (Paolini et al., 1996a). It is shown in our study that fenarimol strongly reduced PCE/NCE ratio and affected mitotic index at the higher dose
groups and these results probably indicate that this fungicide leads to cell death in bone marrow and then reduce mitotic index. We have found in our study that propamocarb used as technical grade compound may be cytotoxic because it either reduced PCE/NCE ratio or affected the mitotic index in bone marrow. It did not induce any micronuclei in mice bone marrow. Propamocarb significantly increased the total chromosomal aberrations including gaps in mice bone marrow. However, when we exclude gaps from the total aberrations, there was no statistical significancy between the control and dose groups. It is generally assumed that gaps are sites of despiralization in the metaphase chromosome that render the DNA non-visible under light microscopy (IPCS, 1985). It has also proposed that an achromatic lesion may actually be a single-strand break in the DNA double helix as a result of incomplete excision repair and, thus, it may represent a point of possible instability (IPCS, 1985). However, the causes of the gap formation is unknown. We, therefore, reported the gaps together with and separately from true chromosomal aberrations. Regarding the mutagenicity and toxicity studies, there are no reports dealing with the metabolic
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pathway, degradation and metabolic products and also genotoxic effects of propamocarb. It is only known to have toxic effects to the environment (Fernandez-Alba et al., 2001). In our study, we have shown the cytotoxic effect of propamocarb on mammalian cells and further in vivo and in vitro test systems need to be used to determine its clastogenic and aneugenic properties. In conclusion, fenarimol and technical grade compound propamocarb did not show any aneugenic and clastogenic activity in mouse bone-marrow cells under our experimental conditions.
References Adler, I.D., 1984. Cytogenetic tests in mammals. In: Venitt S., Parry, J. (Eds.), Mutagenicity Testing: A Practical Approach, IRL Press, Oxford, pp. 273–306. Albert, R., Baker, S., Doull, S., Butler, G., Nelson, N., Peakall, D., Pimentel, D., Tardiff, R.G., 1992. Introduction, general conclusion and recommendations. In: Tardiff, R.G. (Ed.), Methods to assess adverse effects of pesticides on non-target organisms. Wiley, New York, pp. 3–13. Bellicampi, D., Gualandi, G., La Monica, E., Poley, C., Morpurgo, G.D., 1980. Membrane-damaging agents cause mitotic non-disjunction in Aspergillus nidulans. Mutat. Res. 79, 169–172. Cantelli-Forti, G., Paolini, M., Hrelia, P., 1993. Multiple end point procedure to evaluate risk from pesticides. J. Env. Health Perspect. 101 (Suppl. 3), 15–20. Environmental Protection Agency. EPA, 1985. Pesticide fact sheet “fenarimol”. Toxicol. Chem. 207(1), 1–9. Fernandez-Alba, A.R., Guil, L.H., Lopez, G.D., Chisti, Y., 2001. Toxicity of pesticides in wastewater: a comparative assessment of rapid bioassays. Anal. Chim. Acta 426, 289–301.
Flodstrom, S., Hemming, H., Warngard, L., Ahlborg, V.G., 1990. Promotion of altered hepatic foci development in rat liver cytochrome P450 enzyme induction and inhibition of cell–cell communication by DDT and some structurally related organohalogen pesticides. Carcinogenesis 11, 1413–1418. Grilli, S., Ancora, G., Rani, P., Valenti, A.P., Mazzullo, M., Colacci, A.M., 1991. In vivo unwinding fluorometric assay as evidence of the damage induced by fenarimol and DNOC in rat liver DNA. J. Toxicol. Environ. Health 34, 485–494. IPCS, 1985. Guide to short-term tests for detecting mutagenic and carcinogenic chemicals, vol. 208. Environmental Health Criteria, World Health Organization, Geneva, 51, pp. 102–103. Kostka, G., Palut, D., Kopec-Szlezak, J., Ludwicki, J.K., 2000. Early hepatic changes in rats induced by permethrin in comparison with DDT. Toxicology 142, 135–143. Paolini, M., Mesirca, R., Pozzetti, L., Maffei, F., Vigagni, F., Hrelia, P., Cantelli-Forti, G., 1996a. Genetic and non-genetic biomarkers related to carcinogenesis in evaluating toxicological risk from fenarimol. Mutat. Res. 368, 27–39. Paolini, M., Mesirca, R., Pozzetti, L., Sapone, A., Cantelli-Forti, G., 1996b. Molecular non-genetic biomarkers related to fenarimol cocarcinogenesis: organ and sex specific CYP induction in rat. Cancer Lett. 101, 171–178. Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, A.F., Shelby, M., 1987. Mammalian in vivo cytogenetic assays: analysis of chromosome aberrations in bone-marrow cells. Mutat. Res. 189, 157–165. Tyrkiel, E., Ludwicki, J.K., 1992. The influence of DDT analogues (nuarimol and fenarimol) on the frequency of occurence of micronuclei in erythrocytes of bone marrow and spleen in mice. Rocz. Panstw. Zakl. Hig. 43 (3–4), 325–331. Tyrkiel, E., Wiadrowska, B., Ludwicki, J.K., 1996. Induction of micronuclei in erythrocytes of bone marrow and peripheral blood in laboratory mice following acute and subchronic exposure to DDT (fenarimol and nuarimol). Rocz. Panstw. Zakl. Hig. 47 (2), 151–158. Vasudev, V., Krishnamurthy, N.B., 1994. In vivo cytogenetic analyses of the carbamate pesticides Dithane M-45 and Baygon in mice. Mutat. Res. 323, 133–135.
The investigation of the genotoxic effects of fenarimol and propamocarb in mouse bone marrow in vivo Nilüfer Aydemir∗ , Rahmi Bilalo˘glu Department of Biology, Faculty of Arts and Science, University of Uludag, 16059 Görükle, Bursa, Turkey Received 16 June 2003; received in revised form 21 October 2003; accepted 23 October 2003
Abstract In this study, we aimed to evaluate the genotoxic effects of fungicides fenarimol and propamocarb which are used to protect crops from fungi. For this reason, bone-marrow micronucleus and chromosome aberration tests were carried out in Swiss albino mice. Mice were injected with four different doses of fenarimol and propamocarb intraperitoneally; 50, 100, 200 and 400 mg/kg b.w. Fenarimol did not induce any significant increase in micronucleated erythrocytes after 24, 36, and 48 h treatment but it decreased the ratio of polychromatic/normochromatic erythrocytes at all dose groups and sampling intervals. Fenarimol did not increase the number of chromosome aberrations significantly, but it reduced the mitotic index at the higher doses (P < 0.05). Propamocarb did not increase the frequency of micronucleated erythrocytes, but decreased the polychromatic/normochromatic erythrocytes ratio at all sampling intervals. Propamocarb increased only gaps in total chromosome aberrations, but when gaps were excluded, there were no significant differences in total aberrations between the control and dose groups (P > 0.05). Propamocarb also reduced the mitotic index compared with the negative control group (P < 0.001). Contributing these results, we can suggest that fenarimol and propamocarb are non-genotoxic in mouse bone marrow in vivo but have cytotoxic effects. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Fungicides; Propamocarb; Fenarimol; Micronucleus; Chromosome aberrations; Mouse; Bone marrow
1. Introduction Pesticides are chemicals used to control agricultural pests. Their widespread application with undesirable side effects represents a potential risk to human and environmental health. Unintended exposure to pesticides can occur from environmental residues after application (Albert et al., 1992). ∗ Corresponding author. Tel.: +90-224-4429258; fax: +90-224-4428136. E-mail address: [email protected] (N. Aydemir).
Fungicides also are very important and widely used pesticides. The fungicide fenarimol belongs to the chemical class of halogenated pesticides (which also include chemicals such as DDT) and known as tumor promoters and hepatocarcinogens in rodents (Flodstrom et al., 1990). Fenarimol is a pyrimidine carbinol fungicide with activity against several Ascomycetes, Basidiomycetes and Fungi imperfecti (Paolini et al., 1996a). Toxicological data on fenarimol are controversial and incomplete. Fenarimol did not induce unscheduled DNA synthesis in primary rat hepatocytes (Bellicampi et al., 1980). It did not
0378-4274/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2003.10.015
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N. Aydemir, R. Bilalo˘glu / Toxicology Letters 147 (2004) 73–78
induce chromosomal aberrations in rodents and gave negative results in mutagenicity tests with bacteria (EPA, 1985). On the other hand, fenarimol showed positive results in the fluorometric unwinding test assessing the induction of DNA single strand breaks in rat hepatocytes in vivo (Grilli et al., 1991). The other fungicide propamocarb is widely used in the greenhouse-based production of vegetables and fruits in Turkey and Southern Europe. Propamocarb is a carbamated fungicide and used particularly against Aphanomyces, Phytophtora and Phytium (Fernandez-Alba et al., 2001). The mutagenic properties of various carbamated compounds have been investigated (Vasudev and Krishnamurthy, 1994). However, there is no report on the genotoxic effects of propamocarb in any mutagenicity test systems. In this study we decided to detect the clastogenicity and aneugenicity of propamocarb and the suspected mutagen, fenarimol, by metaphase analyses and micronucleus tests in Swiss albino mice bone marrow in vivo.
2. Materials and methods 2.1. Test animals Swiss albino mice were obtained from the Laboratory Animal Center of Uludag University (Bursa, Turkey). They were 8–10-week-old and housed in plastic cages with a bedding of wood shavings. Animals weighed 25–30 g and were fed with fresh standart pellet and given tap water ad libitum. All mice were kept under constant environmental conditions with a 12–12 h light–dark cycle. This study has been approved by the Ethical Committee on Animal Experiments of Uludag University. 2.2. Test chemicals Fenarimol (2,4-dichloro-pyrimidin-5-yl benzydil alcohol, Cas no: 60168-88-9) was obtained from Dow Elanco Europe Laboratoires and the purity of this compound is 99.4%. Propamocarb (propyl-3-dimethylamino-propyl carbamate, Cas no: 24579-73-5), a liquid compound that is an active substance of Previcur-N producted by Schering Agrochemicals. The purity of this fungicide
is 72.2% and it was obtained from the Uludag University, Agricultural Faculty. Other chemicals, Hanks balanced salt solution (HBSS), fetal calf serum (FCS) and ethyl methane sulfonate (EMS) was purchased from Sigma–Aldrich Chemical Company. 2.3. Experimental protocol The intraperitoneal (i.p.) route of application was used in all experiments. Corn oil solvent was used for fenarimol and it was administered in a single doses of 50, 100, 200 and 400 mg/kg b.w., suspensioned in corn oil. These doses were selected according to the LD50 of fenarimol for mice and this was 600 mg/kg b.w. (Paolini et al., 1996b). Control mice were injected with only corn oil. The fungicide propamocarb (Previcur-N) was diluted in sterile distilled water. The LD50 of propamocarb was 1600 mg/kg for mice and we injected mice in single doses of 50, 100, 200 and 400 mg/kg b.w. per mouse. The negative control mice were injected with distilled water. For two fungicides, the highest dose was tolerated by the animals with minimal toxic symptoms, such as tremor and loss of appetite. However, the animals recovered within 1 h of the treatment. EMS was used as a positive control and given in a single dose of 300 mg/kg b.w. per mouse. 2.4. Procedures For metaphase analysis, negative control group included 12 mice. Positive control and all dose groups consisted of eight mice. The sampling time was 24 and 2 h before sacrifice, mice were injected with 4 mg/kg colchicine. Bone marrow metaphase slides were prepared according to the Adler (1984). The bone-marrow cells were collected from both femura by flushing in Hank’s balanced salt solution and cells were centrifuged at 1000 rpm for 5 min. After centrifugation, bone-marrow cells were treated with KCl (0.56%) and incubated at room temperature for 10 min. Following incubation, the material was centrifuged. The supernatant was discharged and the cell pellet was fixed with cold 1:3 (v/v) acetic acid–methanol. Fixation was repeated twice with an interval of 10 min and last fixation step was done for 30 min at 4 ◦ C. After
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the centrifugation, pellet was resuspended with fresh fixative and two or three drops of the fixed material were dropped on a clean slide and placed on to slide warmer to dry. Slides were stained in 5% Giemsa for 5 min. The scoring and classification of aberrations were done as described by Preston et al. (1987). Structural and numerical chromosomal aberrations (CAs) were scored in 50 metaphases for each mouse. Gaps were both included and excluded from the total number of CAs. The mitotic index was also recorded. In the micronucleus test, all dose groups contained six mice. Negative control included 12, and the positive control included 4 mice respectively. The sampling intervals were 24, 36 and 48 h and the micronucleus test was performed according to the Adler (1984). For each sampling interval, the animals were killed and the femurs of each animal were dissected out. The bone marrow was flushed with fetal calf serum and the suspension was centrifuged. The supernatant was discharged and a few drops of fetal calf serum was added. The pellet was mixed and dropped on to clean slide. The slides were air dried for 12 h and stained with a combination of May Grünwald–Giemsa. Total of 2000 polychromatic erythrocytes (PCEs) per mouse were analyzed for the presence or absence of micronuclei. The frequency of micronucleated normochromatic erythrocytes (MNNCEs) was also recorded. The ratio of PCEs per normochromatic erythrocytes (NCEs) was also calculated to determine the cytotoxic effects of chemicals. Data were analyzed using the non-parametric Kruskal–Wallis and Tukey type multiple comparisons tests. All statistical analysis were done by the Statistica for Windows programme.
3. Results The results for the micronucleus tests and metaphase analysis are given in Tables 1 and 2 for fenarimol and propamocarb. Fenarimol did not increase the frequency of MNPCEs and MNNCEs compared with the negative control and at all treatment periods (24, 36 and 48 h). However, it decreased the PCE/NCE ratio for 100, 200 and 400 mg/kg dose groups compared with the control, at the 24 and 48 h treatment times (P < 0.001). Also, fenarimol significantly reduced the PCE/NCE
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ratio for 36 h, at the 200 and 400 mg/kg dose groups (P < 0.001). Fenarimol did not induce a significant increase in the frequency of chromosomal aberrations. It affected the mitotic index at the 200 and 400 mg/kg dose group (6.22 and 6.43%; P < 0.05) compared with the negative control. There was no significant elevation with the fungicides propamocarb in the frequency of MNPCEs and MNNCEs for the 24, 36 and 48 h sampling intervals (Table 1). Propamocarb decreased the PCE/NCE ratio significantly for 24, 36 and 48 h at the 100, 200 and 400 mg/kg dose groups compared with the negative control (P < 0.001). Propamocarb significantly increased the frequency of total aberrations including gaps only at the 400 mg/kg dose group compared with the negative control group (P < 0.01). Propamocarb did not increase the frequency of total aberrations when gaps were excluded. Propamocarb reduced the mitotic index in both the 200 and 400 mg/kg dose groups (P < 0.001).
4. Discussion In this study, we have demonstrated that two fungicides fenarimol and propamocarb did not show any genotoxic effect in mouse bone marrow with two short-term tests. However, both fungicides were found to be cytotoxic. In our study fenarimol did not induce any micronucleated erythrocytes and chromosomal aberrations. Cantelli-Forti et al. (1993) and Paolini et al. (1996a) showed that fenarimol increased the number of micronucleated erythrocytes in Swiss albino CD-1 mice at doses between 37.5 and 300 mg/kg for 24 h. Although these results contradict our findings, there are other studies that agree with our data. When fenarimol was given to Swiss albino mice at doses of 50 and 100 mg/kg, it did not increase the number of micronuclei in spleen and bone marrow at different treatment times (Tyrkiel and Ludwicki, 1992). Also Tyrkiel et al. (1996) gave 200 mg/kg fenarimol for 21 days to mice and they did not find any micronuclei in bone marrow or peripheral blood erythrocytes. It has been reported in the studies of Cantelli-Forti et al.(1993) and Paolini et al. (1996a) that
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Treatments
Dose (mg/kg)
n
MNPCE/ 1000 PCE
MNNCE/ 1000 NCE
Corn oil fenarimol
0 50 100 200 400
D.W. propamocarb
EMS
12 6 6 6 6
24 h 1.83 1.91 2.00 2.16 2.00
± ± ± ± ±
1.02 0.97 0.89 1.03 1.09
0.70 0.50 0.58 0.83 0.75
± ± ± ± ±
0.54 0.44 0.49 0.51 0.52
1.01 0.94 0.84 0.60 0.56
± ± ± ± ±
0 50 100 200 400
12 6 6 6 6
1.62 2.00 2.08 2.00 1.83
± ± ± ± ±
0.85 1.00 1.06 1.14 0.81
0.66 0.75 0.83 0.41 0.58
± ± ± ± ±
0.49 0.88 0.68 0.37 0.58
1.00 0.98 0.77 0.59 0.57
± ± ± ± ±
300
4
11.0 ± 2.94
0.16 ± 0.18
PCE/NCE
MNPCE/ 1000 PCE
MNNCE/ 1000 NCE
0.02 0.05 0.06∗∗∗ 0.05∗∗∗ 0.05∗∗∗
36 h 1.79 2.08 2.00 2.16 1.83
± ± ± ± ±
0.81 0.97 1.14 1.12 0.87
0.70 0.66 0.83 0.91 0.83
± ± ± ± ±
0.54 0.60 0.40 0.37 0.81
1.00 0.90 0.95 0.59 0.52
± ± ± ± ±
0.06 0.04 0.09∗∗∗ 0.09∗∗∗ 0.09∗∗∗
1.70 1.83 2.00 1.91 1.66
± ± ± ± ±
0.89 0.81 0.89 0.80 0.75
0.62 0.58 0.83 0.75 0.66
± ± ± ± ±
0.48 0.49 0.51 0.52 0.31
1.00 0.97 0.69 0.57 0.55
± ± ± ± ±
0.30 ± 0.10
13.0 ± 4.70
0.00 ± 0.00
PCE/NCE
MNPCE/ 1000 PCE
MNNCE/ 1000 NCE
0.10 0.05 0.07 0.06∗∗∗ 0.04∗∗∗
48 h 1.58 1.66 1.83 1.75 1.75
± ± ± ± ±
0.99 0.87 1.03 1.12 0.93
0.66 0.75 0.66 0.83 0.75
± ± ± ± ±
0.38 0.52 0.40 0.25 0.41
0.98 0.83 0.59 0.64 0.48
± ± ± ± ±
0.05 0.05 0.06∗∗∗ 0.06∗∗∗ 0.02∗∗∗
0.06 0.02 0.10∗∗∗ 0.07∗∗∗ 0.06∗∗∗
1.58 1.66 1.83 1.75 1.50
± ± ± ± ±
0.82 0.60 0.98 1.03 0.63
0.66 0.50 0.75 0.58 0.50
± ± ± ± ±
0.53 0.44 0.52 0.37 0.54
0.97 0.89 0.69 0.57 0.56
± ± ± ± ±
0.05 0.08 0.10∗∗∗ 0.06∗∗∗ 0.06∗∗∗
0.28 ± 0.10
13.25 ± 2.90
0.00 ± 0.00
PCE/NCE
0.25 ± 0.30
2000 PCEs were analyzed per animal. Abbreviations: n, number of animals per group; h, sampling interval (hour); D.W., distilled water, MNPCE, micronucleated polychromatic erythrocytes; MNNCE, micronucleated normochromatic erythrocytes; PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; EMS, ethyl methanesulfonate-positive control. All data are presented as mean ± standard deviation. ∗∗∗ P < 0.001; significant when compared with the negative control.
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Table 1 The Frequency of MNPCE, MNNCE and PCE/NCE ratios in mice bone marrow injected with fenarimol and propamocarb at 24, 36 and 48 h intervals
N. Aydemir, R. Bilalo˘glu / Toxicology Letters 147 (2004) 73–78
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Table 2 Mitotic index, distribution of the different types of chromosomal aberrations observed in swiss-albino mice bone-marrow cells Treatments
Dose (mg/kg)
n
MI (M ± S.D., %)
Gapsa C
IC
Breaksa
Acentric fragmentsa
C
IC
C
IC
TA + Gapsb (M ± S.D.)
TA-Gapsb (M ± S.D.)
Corn oil fenarimol
0 50 100 200 400
10 8 8 8 8
7.82 7.27 7.40 6.22 6.43
± ± ± ± ±
0.45 0.49 0.41 0.90∗ 0.94∗
4 3 3 4 3
2 0 2 3 2
0 0 0 1 0
0 0 0 0 0
2 0 1 0 0
0 0 0 0 0
0.80 0.37 0.75 1.00 0.62
± ± ± ± ±
0.42 0.50 0.40 1.30 0.50
0.10 0.00 0.13 0.13 0.00
± ± ± ± ±
0.31 0.00 0.30 0.30 0.00
D.W. propamocarb
0 50 100 200 400
10 8 8 8 8
7.52 5.85 5.45 5.22 4.10
± ± ± ± ±
0.65 0.52 0.68 0.57∗∗∗ 0.56∗∗∗
4 3 7 5 4
3 1 3 4 10
1 0 1 0 1
0 0 0 0 0
1 1 2 1 2
0 0 1 0 0
0.90 0.62 1.75 1.25 2.12
± ± ± ± ±
0.70 0.50 1.30 1.00 0.90∗∗
0.20 0.13 0.50 0.13 0.38
± ± ± ± ±
0.40 0.30 1.41 0.30 0.51
EMS
300
8
5
9
13
4
6
3
5.00 ± 2.97
6.73 ± 0.71
3.25 ± 2.18
Fifty metaphases were analyzed per animal. Abbreviations: n, number of animals per group; MI, mitotic index; M ± S.D., mean ± standard deviation; C, chromatid type; IC, isochromatid type; TA, total aberrations; D.W., distilled water. a Number of aberrations in total analyzed cells. b Number of aberrations/number of cells analyzed. ∗ P < 0.05; significant when compared with the negative control. ∗∗ P < 0.01; significant when compared with the negative control. ∗∗∗ P < 0.001; significant when compared with the negative control.
fenarimol may act as an aneugen, rather than a clastogen. However, there is no evidence or report on the aneugenic effect of fenarimol such as the presence of centromeric heterochromatin in micronuclei induced by this fungicide. Moreover fenarimol induced different cytochrome P450 enzymes (CYP) in various tissues of Sprague–Dawley rats. This indicates the cotoxic–cocarcinogenic potential of fenarimol and it may act as a tissue specific tumor promoting agent (Paolini et al., 1996b). It is suggested in this study that the CYP induction may have a direct role in the development of malignancy and this is particularly important for non-genotoxic carcinogens such as many pesticides (Paolini et al., 1996b). In general, CYP enzyme inducers have been known to be non-genotoxic hepatocarcinogens such as DDT and related compounds (Kostka et al., 2000). It is known that fenarimol belongs to the chemical class of halogenated pesticides which also include chemicals such as DDT (Paolini et al., 1996a). Fenarimol seems to act as a hepatocarcinogen and this type of chemicals may give negative results in short-term mutagenicity tests (Paolini et al., 1996a). It is shown in our study that fenarimol strongly reduced PCE/NCE ratio and affected mitotic index at the higher dose
groups and these results probably indicate that this fungicide leads to cell death in bone marrow and then reduce mitotic index. We have found in our study that propamocarb used as technical grade compound may be cytotoxic because it either reduced PCE/NCE ratio or affected the mitotic index in bone marrow. It did not induce any micronuclei in mice bone marrow. Propamocarb significantly increased the total chromosomal aberrations including gaps in mice bone marrow. However, when we exclude gaps from the total aberrations, there was no statistical significancy between the control and dose groups. It is generally assumed that gaps are sites of despiralization in the metaphase chromosome that render the DNA non-visible under light microscopy (IPCS, 1985). It has also proposed that an achromatic lesion may actually be a single-strand break in the DNA double helix as a result of incomplete excision repair and, thus, it may represent a point of possible instability (IPCS, 1985). However, the causes of the gap formation is unknown. We, therefore, reported the gaps together with and separately from true chromosomal aberrations. Regarding the mutagenicity and toxicity studies, there are no reports dealing with the metabolic
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pathway, degradation and metabolic products and also genotoxic effects of propamocarb. It is only known to have toxic effects to the environment (Fernandez-Alba et al., 2001). In our study, we have shown the cytotoxic effect of propamocarb on mammalian cells and further in vivo and in vitro test systems need to be used to determine its clastogenic and aneugenic properties. In conclusion, fenarimol and technical grade compound propamocarb did not show any aneugenic and clastogenic activity in mouse bone-marrow cells under our experimental conditions.
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