Assessment of the ability of Imazaquin herbicide to induce chromosomal aberrations in vitro in cultured Chinese hamster ovary cells and micronuclei in vivo in mice

Assessment of the ability of Imazaquin herbicide to induce chromosomal aberrations in vitro in cultured Chinese hamster ovary cells and micronuclei in vivo in mice

Food and Chemical Toxicology 42 (2004) 1245–1249 Assessment of the ability of Imazaquin herbicide to induce chrom...

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Food and Chemical Toxicology 42 (2004) 1245–1249

Assessment of the ability of Imazaquin herbicide to induce chromosomal aberrations in vitro in cultured Chinese hamster ovary cells and micronuclei in vivo in mice R. Losi-Guembarovski a


lus , F.V. Santos a, F.L. Dias b, R.G. Frederico a, I.M.S. Co


Departamento de Biologia Geral, Centro de Ci^encias, Biologicas, Universidade Estadual de Londrina (UEL), CEP 86051.990 Londrina, Parana, Brazil b Departamento de Ci^encias Biologicas, Faculdade de Medicina do Tri^angulo Mineiro (FMTM), Uberaba, Minas Gerais, Brazil Received 8 June 2003; accepted 2 March 2004

Abstract The agricultural chemicals marketed to increase food production may not only combat pests and weeds but also present toxic properties and cause genetic damage to the fauna and flora. The Imazaquin herbicide (Scepter 70 DG-Cyanamid) has been widely used in soybean fields in Parana (Brazil), but information on its genotoxicity is scarce. Thus, in vivo and in vitro studies were carried out to assess the possible clastogenic effect of this herbicide on eukaryote cells. In the in vitro studies, the Chinese hamster ovarian cell lines CHO-K1 (wild) and CHO xrs-5 (mutant) were treated at the three phases of the cell cycle (G1, S and G2) for chromosome aberration (CA) analysis. The in vivo assessment was carried out by the micronucleus test (MN) on Swiss mice (Mus musculus) bone marrow cells. The herbicide did not induce a significant increase in the CA frequency in any of the treatments. No statistically significant differences were observed in the MN frequencies among the groups treated with the herbicide and the negative control. From the test system used in this study, we can conclude that the Imazaquin herbicide did not act as a clastogenic agent either in vitro or in vivo. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Imazaquin (Scepter 70 DG) herbicide; CHO cells; Mice

1. Introduction Agronomic practices used in recent years have made the agrarian sector depend heavily on agrochemicals such as herbicides, which are compounds designed to control the development of undesirable plants that may interfere with the growing of commercial crops (Blair et al., 1990; Biradar and Rayburn, 1995). The herbicides have undoubtedly increased crop yields and reduced post-harvest losses, but at the same time they may have negative environmental consequences (Al-Saleh, 1994; Biradar and Rayburn, 1995). Besides the direct occupational exposure of workers during the formulation, manipulation and application of pesticides, human population may also be exposed to them indirectly due to the persistence of their residues in the soil and in


Corresponding author. Tel./fax: +55-43-3342-2284. E-mail address: [email protected] (R. Losi-Guembarovski).

0278-6915/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2004.03.001

animal and plant tissues (Grover and Malhi, 1985). This has led to an increased concern for the possible threat to human health posed by exposure to these chemicals (Moretti et al., 2002). Although the impact of currently used pesticides may not be as dramatic as those used in past decades because of their reduced half-life and bioaccumulative potentials, the genotoxic effect of these compounds may be considered a potential risk (Clements et al., 1997). Many pesticides have been shown to produce genetic damage, and according to Garret et al. (1986), such genotoxic effects are of special concern due to the irreversible nature of the process. Different authors have reported the genotoxic effects of herbicides in vivo (Th€ orn et al., 2000; Madrigal-Bujaidar et al., 2001; Amer and Aly, 2001) and in vitro (Douglas et al., 1981; Ribas et al., 1996; Sinha et al., 1998). However, other studies have reported that herbicides do not have genotoxic activity in different genetic systems (Ruiz and Marzin, 1997; Charles et al., 1999). Considering these contradictory results there is a need to allocate more investigations


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in order to evaluate the potential genotoxic effects of such compounds. The Imazaquin herbicide (Scepter 70 DG-Cyanamid) is widely used in soybean fields in Paran a, Brazil. As there is not enough data available concerning its potential genotoxic effects, the present study was conducted to characterize the possible clastogenic action using the micronucleus test in vivo in mice and chromosomal aberrations in vitro on Chinese hamster CHOK1 (wild) and CHO xrs-5 (deficient in DNA double strand break repair) ovarian cell lines.

2. Materials and methods 2.1. Herbicide The herbicide Imazaquin (2-[4,5-dihydro-4-1-methyl4-(1-metiletil)5-oxo-1H-imidazol-2-ilo]-3-quinolinacarboxilic acid) is a selective herbicide from the imidazolinone chemical class. In the present study the commercially available herbicide named Scepter 70 DG (Cyanamid) was used in the form of water-dispersible granules, containing 70% of the herbicide active ingredient (700 g/ kg). Its solubility is 60 ppm at 25 °C and the density 1.040 g/cm3 at 20 °C. This herbicide is recommended for wide leaf weed control in soybean and has been used in several regions of Brazil since 1981 (Garcia et al., 1984; Frenhani et al., 1984; Almeida et al., 1984). Based on the employed concentration in the farming, we dissolved it in water and performed pilot tests in vivo and in vitro to determine the doses and concentrations to be used. 2.2. Cell lines Chinese hamster ovary cell lines, CHO-K1 (wild-type) and xrs-5 (deficient in DNA double strand break repair) were kindly provided by Prof. A. T. Natarajan (University of Leiden, The Netherlands) and were repassed by Prof. Dra. Elza T. Sakamoto-Hojo (FFCLRP-USP) to the UEL Mutagenesis Laboratory. These cells were previously described by Jeggo et al. (1982) and Jeggo (1985). 2.3. In vivo assessment The maximum tolerated dose to Scepter was obtained by preliminary toxicity tests using three animals/dose. The micronucleus test (according to Schmid, 1975 modified) was used on bone marrow cells from five groups consisting of ten Swiss line (Mus musculus) mice (five males and five females). The animals came from the Central Animal House at UEL and were submitted to single acute treatments (intraperitonial injection–ip) with three concentrations of the herbicide: 1.25, 2.50

and 3.75 g/kg body weight corresponding, respectively, to 25%, 50% and 75% of the LD50 previously determined in our laboratory in pilot experiments. The positive and negative control groups received, respectively, cyclophosphamide injections (10 mg/kg bw) and saline solution. The animals were sacrificed after 24 h of treatment by cervical displacement and the bone marrow was collected by washing the femurs with 1-mL fetal bovine serum (Cultilab-Brazil). The material was processed and, when the smears had been made, the slides were stained with Giemsa diluted in phosphate buffer (5%) for eight minutes, then washed in distilled water and dried in the air. Two thousand polychromatic erythrocytes were analyzed per animal for the in vivo test by noting the MN frequencies and applying the analysis of variance (ANOVA) statistical test and the Student NewmanKeuls test (SNK). 2.4. In vitro assessment Chinese hamster CHO-K1 (wild) and CHO xrs-5 (deficient in DNA double strand break repair) ovarian cell lines were used. The cells were maintained as monolayers growing at 37 °C in 25 cm2 flasks (Corning) containing HAM-F10 (Sigma) + DEM (Dulbecco’s modified Eagle’s medium; Sigma) (1:1) culture medium supplemented with 10% fetal calf serum (Cultilab), 2.38 mg/mL Hepes (Sigma), plus 0.01 mg/mL streptomycin and 0.005 mg/mL penicillin. All the experiments with the cell lines CHO-K1 and xrs-5 were carried at out between 3rd and 5th culture passage after thawing; 106 cells/flask were seeded, incubated for one cycle (14 h) and then submitted to treatments. Since the concentration equivalent to 13200 lg/ mL culture medium proved to be highly cytotoxic, we established the final concentrations of 1320, 132, 13.2 and 1.32 lg/mL culture medium as the test doses. Thus, the cultures were treated with these four concentrations of the herbicide. The positive control was treated with mitomycin-C (MMC) at the final concentration of 1.6 lg/mL culture medium. The duration of the cell cycle in these cells varies from 12 to 14 h (Preston et al., 1981). Thus, CHO-K1 and xrs5 cells were treated with the herbicide 14 h after thawing and then harvested at different recovery times to analyze cells treated at G1, S or G2 phases of the cell cycle. The cell cultures (CHO-K1 and xrs-5) were treated with the Scepter herbicide for 1 h in serum-free medium during the phases G1 and S and for 2 h in the phase G2 of the cell cycle. After this, the cells were washed with PBS and incubated in complete medium for an additional period of 11 and 6 h respectively in the G1 and S phases, until harvesting. In the G2 phase of the cell cycle, the cells received treatment with the herbicide or

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with MMC and were not washed because of the short period of time between the treatment and fixing. Therefore, in this phase the cultures received the herbicide or MMC during the last two hours of the cycle. The fixing times after the beginning of treatments, eleven, six and two hours were defined to guarantee that fixed cells would be respectively in early G1/S, middle S and late G2 phases of the cell cycle at the moment they received the treatment. Untreated controls were handled identically, with the same changes in medium. Each experiment was repeated two times. Cell harvesting have been performed as follows: lines CHO-K1 and xrs-5 received, respectively, 100 or 30 ll of Demecolcine (Sigma) and the harvesting of the cultures was done 2 or 1:30 h after the treatment with Demecolcine to block cells at metaphase stage during mitosis. Because CHO xrs-5 presents another mutation characterized by a higher chromatin condensation, different harvesting conditions have to be employed. Therefore, in the G2 phase of the cell cycle the K1 cells received simultaneous treatment with the herbicide or with MMC and Demecolcine (Sigma) while the xrs-5 line received Demecolcine 30 min later. After the Demecolcine time, the culture medium was removed from the two lines and placed in a centrifuge tube. The cells were washed in PBS buffer solution and a solution of 0.2% trypsin plus 0.02% versene (0.5 mL) was used to detach the cells from the inner surface of the flask. The material was re-suspended and centrifuged for 5 min at 800 rpm and about 10 mL of cold hypotonic solution was added (sodium citrate at 1%). The cells were fixed with Carnoy (3 parts methanol:1 part acetic acid) and the metaphase spreads stained with Giemsa diluted in phosphate buffer (5%) for 10 min. Only well spread metaphases presenting 21 ± 1 chromosomes were analyzed. A total of 200 metaphases (100 in each experiment) were analyzed per treatment to assess the in vitro chromosome aberrations (CA). The data obtained, including gaps, were submitted to the ANOVA statistical test for repeated means, followed by the Tukey test. The two cell lines used were compared for CA induction in the different treatments by the Student t-test.

3. Results Chromosome aberrations were scored by identifying dicentrics, rings and acentric fragments (chromosomal breaks), chromatid breaks, gaps and exchanges. The chromosomal-type aberrations were included, but most of the observed aberrations after herbicide treatment were the chromatid type (gaps and breaks). After the MMC treatment both types of abnormalities, chromosomal and chromatid, were observed. The results observed in the present study indicate that the Imazaquin compound did not induce a significant


increase in the CA frequency in any of the treatments carried out either for CHO-K1 or CHO xrs-5. Thus, no clastogenic effect produced by the herbicide was evidenced. However, as it was expected because of the mutant phenotype, higher frequencies of CA were detected for every dose of the herbicide treatment at G1 and S phase of CHO xrs-5 cells, even in negative controls. The statistical analysis for the total MN frequencies observed in this work in mice bone marrow cells after treatments with the herbicide (25%, 50% and 75% LD50 ) and their respective controls showed that the results obtained for the groups treated were not significantly different from those observed in the negative control group. The results obtained for the comparative data for males and females did not differ statistically either.

4. Discussion The possible deleterious effects and damage to the environment of many agricultural chemicals are already known, and there are many studies in the literature (Bolognesi et al., 1993; Vigreux et al., 1998; Pe~ na et al., 1999; Tennant et al., 2001; Ateeq et al., 2002) that show their mutagenic, carcinogenic and teratogenic potential. In this study, the Imazaquin herbicide, widely used in soybean fields in Northern Parana (Brazil), was assessed for its clastogenic potential in Chinese hamster ovary cells (CHO) in vitro and for its capacity to induce micronucleus in in vivo mice bone marrow cells. The CHO cell cultures were treated at the different phases of the cycle (G1, S and G2) because, according to Preston et al. (1981), the cells must be exposed to the drug at each of the three phases of the cell cycle in order to assess the capacity of a system to detect chromosome aberrations induced by a compound that may present a different specificity in the cell cycle phases. In our study, for the in vitro test, the herbicide was not clastogenic in the different treatments carried out compared to the negative control, because it was not able to induce a significant increase in the number of chromosome aberrations. Similar studies were not found in the literature using the herbicide in question, but herbicides such as 2,4-dichlorophenoxyacetic acid (Holland et al., 2002; Gollapudi et al., 1999); 4-(2,4dichlorophenoxy) butyric acid (Charles et al., 2000) and Terbutryn (Moretti et al., 2002) also failed to produce any genotoxicity in the experimental in vitro systems used. There are studies with Chinese hamster cells submitted to treatments with other herbicides, such as those of Nicotera et al. (1985), Sinha et al. (1998) and Peitl et al. (1996) with the agricultural chemicals Paraquat, Fluchloralin and Nuvacron, respectively, which presented genotoxicity when tested on fibroblast cultures and


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ovary cell cultures. The herbicides Atrazine and Bentazzon (Biradar and Rayburn, 1995); Diallate and Triallate (Douglas et al., 1981) and Alachlor (Ribas et al., 1996) and Maleic Hydrazide (Meschini et al., 1988; Ribas et al., 1996) also showed mutagenic effects in in vitro mammalian cell cultures. It is well known that cells at the different stages of the cell cycle suffer a delay in the cell cycle time after treatment with clastogenic agents, and that this delay allows the DNA to be repaired and influences the final result in the production of chromosome aberrations (Palitti, 1998). Thus the existence of repair deficient cell lines has contributed to a better understanding of the importance of this DNA mechanism that follows the treatments of cells with chemical and physical agents (Darroudi and Natarajan, 1985). Naturally, the mutant xrs-5 line presents a higher CA frequency than that observed for the wild line and it was also observed in the negative control group in the present study. Furthermore, the statistical comparison among the frequencies obtained for the two cell lines at the different phases of the cell cycle in this study indicated that the mutant line was more sensitive to CA formation in the G1 and S phases in all the treatments carried out (including the negative control) than the line with repair capacity. It is known that many compounds can produce negative in vitro results and positive in vivo results because of their indirect action and consequent need for metabolic activation. Furthermore, the possibility that many of these positive results may not be relevant in terms of human exposure (Kirkland, 1998) should be taken into account. For this reason, besides the in vitro tests, the Imazaquin herbicide was also tested for its capacity to induce micronucleus in vivo in mice bone marrow cells. No statistically significant differences were detected between the MN frequencies observed for the different treatments with herbicide and the negative control. The comparative analyses between males and females in each treatment and among the negative control groups and those treated with different solutions containing the herbicide (25%, 50% and 75% LD50 ) did not show significant differences either. This means that there was probably no hormonal influence in the study carried out and that the Scepter herbicide did not act as an indirect mutagenic agent in the in vivo system used. Our results are in line with those observed by Paiva and Takahashi (1996) who reported that the herbicide Ametrine did not present clastogenic potential in human lymphocyte cultures or in Wistar rats bone marrow cells. Some other papers did not observe genotoxicity of herbicides in vivo either, such as those reported by Kligerman et al. (2000), Tennant et al. (2001) and Holland et al. (2002). However, other agricultural chemicals have been presenting significant genotoxic effects in vivo: Propoxur, Methomyl and Aldicarb (Wei

et al., 1997); Nuvacron (Peitl et al., 1996); Paraquat (Pe~ na et al., 1999); 2,4-dichlorophenoxy acetic acid (Amer and Aly, 2001; Madrigal-Bujaidar et al., 2001) and pentachlorophenol (Ateeq et al., 2002). In the present study the in vivo micronucleus experiment confirmed that the Imazaquin herbicide has no clastogenic effect as it was show with the in vitro trial. The data obtained in vivo further indicated that the metabolism of this compound does not generate any clastogenic product to the organism. Thus, based on the experimental evidences shown in this study, we conclude that under the present conditions the Imazaquin herbicide does not produce direct or indirect clastogenic activity in the eukaryotic cells tested.

Acknowledgements We would like to thank Mr. Dario P. Tormena for his technical assistance and Prof. Cassio Egıdio Cavenaghi Prete, Director of the School Farm at Londrina State University, for supplying the Scepter 70 DG herbicide. This study was funded by UEL and CNPq-RHAE-Meio Ambiente.

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